Diversity of Cacao Pathogens and Impact on Yield and Global Production

*Dele Adeniyi*

## **Abstract**

Cacao, *Theobroma cacao* L., an important cash crop in foreign exchange earnings and also a major income source for many smallholder farmers in growing ecologies of West Africa. Global cocoa production has been rising fairly steadily over the years by increasing production in growing countries with most of the production taking place in areas of high pathogen biodiversity. Thus, the sustainability of the cocoa economy is under threat as diseases of various statuses now constitute the most serious constraint to production. Most important among these is the black pod disease caused by *Phytophthora* genus with annual losses of 30–90% of the crop. This economically important pathogen is very diverse in nature and varied across growing countries including species such as *palmivora*, *megakarya*, *capsici* and *citrophthora* distinguished based on chromosome number, sporangial characteristics and pedicel length. World losses of 20–25% in cacao production are due to black pod disease, an estimate of 700,000 metric tons on global scale reducing global cocoa production. High cacao loss to diseases is a prime factor limiting production; consequently, significant effort is required to deal with problems associated with disease control to ensure a sustainable cacao. The effective and sustainable management of black pod disease requires integrated approach encompassing different control measures.

**Keywords:** *Phytophthora*, pathogen, diversity, yield, production, management

## **1. Introduction**

Cacao, *Theobroma cacao*, is a major cash crop in the tropics and the source of chocolate, one of the world's most popular foods. In addition, cacao-based agroforestry systems provide a promising means to address the challenges of deforestation and create a habitat for biodiversity while simultaneously providing a profitable crop for agricultural communities [1]. Cocoa is mainly grown by smallholder farmers in West Africa and around the world where favourable tropical environments occur. The farmers plant their cocoa traditionally at random under thinned forest and/or plantain as shade crop. Moreover, when grown in traditional form under thinned, forest shade, cacao affords sustainable benefits not only to the farmer but also to the environment [2]. This low-input cultivation system uses the forest soil fertility and the existing shade.

## **2. Cocoa production status**

The West Africa region has some 6 million hectares of cocoa and provides around 70% of the total world production. In recent time, Côte d'Ivoire, Ghana, Nigeria and Cameroon have been rated as top cocoa-producing countries and the production from around 2,000,000 metric tons to around 3,000,000 metric tons in 10 years plus [3]. The world cocoa production is around 4.3 million tons, and almost 71% of it produced in a relatively small region of West Africa which comprised of Cote d'Ivoire, Ghana, Nigeria and Cameroon with 56, 29, 8 and 7% productions, respectively [3].

However, the average yields remain low, and this could be attributed to many factors ranging from pests and diseases to old and moribund farms and extensive cultivation methods, among others. Steady growths in cocoa production have been reported in Nigeria; production increase from 165,000 metric tons in 1999–2000 to 250,000 metric tons in 2013–2014 has been linked to high grower prices and government support to a limited extent through the 2011 Cocoa Transformation Action Plan [4]. The total harvested area in Nigeria was reported as 640,000 hectares with the average yield of about 400 kg per hectare.

The Cocoa Transformation Action Plan of the Federal Government of Nigeria envisaged improving cocoa situation and rising production to 500,000 metric tons by 2015; however, the yield improvement constrains were required to be better managed. The crop is seriously affected by the impact of diseases and the lowyielding potential of most plantations due to genetic and management reasons [5]. The sustainability in cocoa is currently under an increasing threat as both coevolved and new-encounter diseases of various statuses now constitute the most serious constraint to cacao production [6, 7] (**Figure 1**).

**Figure 1.**

*Shows the status of cocoa production in growing countries of West Africa. (Sources: [8–10]).*

## **3. Pathogen and disease distribution**

The cacao trees in the absence of disease infestation and good farm management provide improved productivity to the maximum of the potential of the crop, under ideal field condition (**Figure 2**).

**45**

in Brazil.

**Figure 2.**

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

However, many factors including poor farm management can introduce diseases or reawaken the inoculum from their resting stage for infection. *Phytophthora* pod rot, commonly called "black pod", is the most economically important disease of cocoa. Four species of *Phytophthora* are mainly responsible for this disease, *P. palmivora*, *P. megakarya*, *P. capsici* and P. *citrophthora*, while four additional species of *Phytophthora* have also been isolated from cacao, *P. katsurae*, *P. arecae*, *P. nicotianae* and *P. megasperma* [11], but no economic impact of these has been reported. *Phytophthora palmivora* is the most common, being present in most of the cacao-growing countries around the world, causing yield losses of 20–30% and tree deaths of 10% annually. *Phytophthora megakarya* occurs only in West and Central Africa countries but considered to be the most virulent among other species on cacao. *Phytophthora capsici* is widespread in Central and South America and prevalent

The estimate of genetic diversity and structure of *Phytophthora* isolates from Ghana, Togo, Nigeria, Cameroon, Gabon and Sao Tome using the isozyme and RAPD markers [12] separated the isolates into two different genetic groups, with one located in Central Africa and the other in West Africa. The two centres of major diversity are located in Cameroon and on the Cameroon/Nigeria border region. This distribution however coincides with two major biogeographical domains reflecting an ancient evolution of *P. megakarya.* A lower genotypic diversity was also found in isolates from Ghana, Togo and Nigeria when compared with isolates form Gabon and Sao Tome. Again, four intermediate marker patterns were observed which correspond to isolates near the border between Nigeria and Cameroon and assumed it is a genetic exchange between the Central and West African groups. Black pod disease incidence in the field is influenced by environmental conditions. Numerous studies have established the role of climatic factors on incidence of black pod disease, caused by *Phytophthora* spp. [13, 14]. Rainfall, high relative humidity and low temperature are known to create favourable humid conditions for the development of the disease. [13] showed that in Ghana, black pod disease developed when the relative humidity, particularly within the day, remained above 80% under the cocoa canopy and that the rate of disease development was influenced by the frequency and amount of rainfall. [14] also reported a significant positive correlation between rainfall when assessed after 1-week lag, and *P. megakarya* pod rot incidence in Cameroon, and emphasised the role of rainfall in the disease epidemics [14]. Further, the time and/or period for black pod peak infection in Ghana varied

annually and also with location depending on the rainfall [15].

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

*Healthy cocoa trees at varied stages (A, B and C) of maturity.*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

The West Africa region has some 6 million hectares of cocoa and provides around 70% of the total world production. In recent time, Côte d'Ivoire, Ghana, Nigeria and Cameroon have been rated as top cocoa-producing countries and the production from around 2,000,000 metric tons to around 3,000,000 metric tons in 10 years plus [3]. The world cocoa production is around 4.3 million tons, and almost 71% of it produced in a relatively small region of West Africa which comprised of Cote d'Ivoire, Ghana, Nigeria and Cameroon with 56, 29, 8 and 7% productions,

However, the average yields remain low, and this could be attributed to many factors ranging from pests and diseases to old and moribund farms and extensive cultivation methods, among others. Steady growths in cocoa production have been reported in Nigeria; production increase from 165,000 metric tons in 1999–2000 to 250,000 metric tons in 2013–2014 has been linked to high grower prices and government support to a limited extent through the 2011 Cocoa Transformation Action Plan [4]. The total harvested area in Nigeria was reported as 640,000 hectares with

The Cocoa Transformation Action Plan of the Federal Government of Nigeria envisaged improving cocoa situation and rising production to 500,000 metric tons by 2015; however, the yield improvement constrains were required to be better managed. The crop is seriously affected by the impact of diseases and the lowyielding potential of most plantations due to genetic and management reasons [5]. The sustainability in cocoa is currently under an increasing threat as both coevolved and new-encounter diseases of various statuses now constitute the most serious

The cacao trees in the absence of disease infestation and good farm management provide improved productivity to the maximum of the potential of the crop, under

*Shows the status of cocoa production in growing countries of West Africa. (Sources: [8–10]).*

**2. Cocoa production status**

the average yield of about 400 kg per hectare.

constraint to cacao production [6, 7] (**Figure 1**).

**3. Pathogen and disease distribution**

ideal field condition (**Figure 2**).

respectively [3].

**44**

**Figure 1.**

**Figure 2.** *Healthy cocoa trees at varied stages (A, B and C) of maturity.*

However, many factors including poor farm management can introduce diseases or reawaken the inoculum from their resting stage for infection. *Phytophthora* pod rot, commonly called "black pod", is the most economically important disease of cocoa. Four species of *Phytophthora* are mainly responsible for this disease, *P. palmivora*, *P. megakarya*, *P. capsici* and P. *citrophthora*, while four additional species of *Phytophthora* have also been isolated from cacao, *P. katsurae*, *P. arecae*, *P. nicotianae* and *P. megasperma* [11], but no economic impact of these has been reported. *Phytophthora palmivora* is the most common, being present in most of the cacao-growing countries around the world, causing yield losses of 20–30% and tree deaths of 10% annually. *Phytophthora megakarya* occurs only in West and Central Africa countries but considered to be the most virulent among other species on cacao. *Phytophthora capsici* is widespread in Central and South America and prevalent in Brazil.

The estimate of genetic diversity and structure of *Phytophthora* isolates from Ghana, Togo, Nigeria, Cameroon, Gabon and Sao Tome using the isozyme and RAPD markers [12] separated the isolates into two different genetic groups, with one located in Central Africa and the other in West Africa. The two centres of major diversity are located in Cameroon and on the Cameroon/Nigeria border region. This distribution however coincides with two major biogeographical domains reflecting an ancient evolution of *P. megakarya.* A lower genotypic diversity was also found in isolates from Ghana, Togo and Nigeria when compared with isolates form Gabon and Sao Tome. Again, four intermediate marker patterns were observed which correspond to isolates near the border between Nigeria and Cameroon and assumed it is a genetic exchange between the Central and West African groups. Black pod disease incidence in the field is influenced by environmental conditions. Numerous studies have established the role of climatic factors on incidence of black pod disease, caused by *Phytophthora* spp. [13, 14]. Rainfall, high relative humidity and low temperature are known to create favourable humid conditions for the development of the disease. [13] showed that in Ghana, black pod disease developed when the relative humidity, particularly within the day, remained above 80% under the cocoa canopy and that the rate of disease development was influenced by the frequency and amount of rainfall. [14] also reported a significant positive correlation between rainfall when assessed after 1-week lag, and *P. megakarya* pod rot incidence in Cameroon, and emphasised the role of rainfall in the disease epidemics [14]. Further, the time and/or period for black pod peak infection in Ghana varied annually and also with location depending on the rainfall [15].

In Ghana, it is known that primary infections usually occur around June, but the peak of *P. megakarya* black pod disease generally occurs between August and October [16, 17]. Such information on periods for attaining disease infection peaks is useful in forecasting the pattern of disease development, and it is an important tool for disease management since conditions immediately preceding the infection peaks must be favourable for disease development. The black pod disease situation in Nigeria is similar to that of Ghana and depends on growing ecologies, pattern of rainfall, high humidity and farmers' management practices. The disease inoculum can remain in the soil for a long time, the spores are brought back to viability at onset of rain and other conditions are suitable. Thus, rain splashes, infected tools and equipment and poor farm management, among others, contribute to the spread of the pathogen in the field.

#### **3.1 Expression of black pod and** *Phytophthora* **on cacao**

*Phytophthora* pathogens are ubiquitous and so cause economic loss to a greater or lesser extent in all cocoa-producing countries but most especially in those with prolonged periods of high relative humidity at, or near to, saturation levels. It infects every part of cacao plants at different developmental stages [18] and under wet and humid atmospheric conditions. *Phytophthora palmivora* is present in most of the cacao-growing countries around the globe and has a broad host range [19]. *Phytophthora megakarya* occurs only in the countries of West and Central Africa and is considered a significant pathogen only on cacao. Black pod was originally thought to be caused by a single species, *P. palmivora*, but increased knowledge have shown otherwise over the past few decades and that each continent has a complex of species of *Phytophthora* which can induce black pod symptoms in cacao. Thus, the main pathogen especially in Nigeria is *P. megakarya*, which was thought to be a variant form of *P. palmivora* but was first identified taxonomically as a species [20].

Under nursery operation, seedling infection leads to blight and root rot, while infections of stem, chupons and branches cause cankers [21, 22]. Infection of the pod leads to black pod which can occur at any stages of pod development, and all parts of the pod are also susceptible to infection [22]. However, immature pods of 10 and 20 weeks have the highest disease incidence when the dynamics of pod production and black pod disease were evaluated in relation to environmental factor impact, chemical fungicide and biological control [14]. Infected immature pods are rendered useless, while infection of ripe pods reduces the bean quality [4].

The black pod caused by all *Phytophthora* species is developed by an initial symptom showing appearance of a small translucent spot on cocoa pods [22], appearing around 2–3 days after infection, then turns brown, eventually darkens and the spot covers the entire pod between 7 and 14 days under humid conditions. Whitish spores are produced 3–5 days after the appearance of the first symptom. These species attack pods of all sizes and are harboured in the roots of cocoa during the dry season making it very hard to control [23].

Black pod symptoms due to *P. megakarya* are, however, characterised by multiple lesions which spread fast and coalesce (**Figure 3**) showing abundant bloom of white zoosporangia on the lesion except for about a centimetre from the advancing margin of the lesions (arrowed). Pods at every stage of development may be infected, and infection may start from the proximal, distal or lateral (**Figure 4A–C**) portion of the pod, and extreme cases of black pod could also affect pods at different stages of development.

Cacao fruits can become infected at all stages from setting to maturity. Observations in Nigeria suggest that the relative frequencies of different infection sites may be affected by fruit length. It was found that the mean length of distally

**47**

**Figure 4.**

**Figure 3.** *Coalescing lesions.*

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

infected fruits tended to be less than the mean length of either laterally or proximally infected fruits. These observations can be interpreted as indicating that distal infections tend to occur on relatively shorter and younger fruits, as compared with laterally and proximally infected fruits [24]. It was suggested that proximal infection might be favoured through moisture being retained in the annular depression where the stalk is inserted and at the distal ends of young fruits [25]. However, in Nigeria, the annular depression at the base may not necessarily be favourable for

infection as compared with the distal end of the pod.

*Sites of pod infection in black pod disease. A, proximal; B, distal; and C, lateral.*

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

*Diversity of Cacao Pathogens and Impact on Yield and Global Production DOI: http://dx.doi.org/10.5772/intechopen.81993*

**Figure 3.** *Coalescing lesions.*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

**3.1 Expression of black pod and** *Phytophthora* **on cacao**

of the pathogen in the field.

In Ghana, it is known that primary infections usually occur around June, but the peak of *P. megakarya* black pod disease generally occurs between August and October [16, 17]. Such information on periods for attaining disease infection peaks is useful in forecasting the pattern of disease development, and it is an important tool for disease management since conditions immediately preceding the infection peaks must be favourable for disease development. The black pod disease situation in Nigeria is similar to that of Ghana and depends on growing ecologies, pattern of rainfall, high humidity and farmers' management practices. The disease inoculum can remain in the soil for a long time, the spores are brought back to viability at onset of rain and other conditions are suitable. Thus, rain splashes, infected tools and equipment and poor farm management, among others, contribute to the spread

*Phytophthora* pathogens are ubiquitous and so cause economic loss to a greater or lesser extent in all cocoa-producing countries but most especially in those with prolonged periods of high relative humidity at, or near to, saturation levels. It infects every part of cacao plants at different developmental stages [18] and under wet and humid atmospheric conditions. *Phytophthora palmivora* is present in most of the cacao-growing countries around the globe and has a broad host range [19]. *Phytophthora megakarya* occurs only in the countries of West and Central Africa and is considered a significant pathogen only on cacao. Black pod was originally thought to be caused by a single species, *P. palmivora*, but increased knowledge have shown otherwise over the past few decades and that each continent has a complex of species of *Phytophthora* which can induce black pod symptoms in cacao. Thus, the main pathogen especially in Nigeria is *P. megakarya*, which was thought to be a variant form of *P. palmivora* but was first identified taxonomically as a species [20].

Under nursery operation, seedling infection leads to blight and root rot, while infections of stem, chupons and branches cause cankers [21, 22]. Infection of the pod leads to black pod which can occur at any stages of pod development, and all parts of the pod are also susceptible to infection [22]. However, immature pods of 10 and 20 weeks have the highest disease incidence when the dynamics of pod production and black pod disease were evaluated in relation to environmental factor impact, chemical fungicide and biological control [14]. Infected immature pods are

Black pod symptoms due to *P. megakarya* are, however, characterised by multiple lesions which spread fast and coalesce (**Figure 3**) showing abundant bloom of white zoosporangia on the lesion except for about a centimetre from the advancing margin of the lesions (arrowed). Pods at every stage of development may be infected, and infection may start from the proximal, distal or lateral (**Figure 4A–C**) portion of the pod, and extreme cases of black pod could also affect pods at different stages

Cacao fruits can become infected at all stages from setting to maturity. Observations in Nigeria suggest that the relative frequencies of different infection sites may be affected by fruit length. It was found that the mean length of distally

rendered useless, while infection of ripe pods reduces the bean quality [4]. The black pod caused by all *Phytophthora* species is developed by an initial symptom showing appearance of a small translucent spot on cocoa pods [22], appearing around 2–3 days after infection, then turns brown, eventually darkens and the spot covers the entire pod between 7 and 14 days under humid conditions. Whitish spores are produced 3–5 days after the appearance of the first symptom. These species attack pods of all sizes and are harboured in the roots of cocoa during

the dry season making it very hard to control [23].

**46**

of development.

**Figure 4.** *Sites of pod infection in black pod disease. A, proximal; B, distal; and C, lateral.*

infected fruits tended to be less than the mean length of either laterally or proximally infected fruits. These observations can be interpreted as indicating that distal infections tend to occur on relatively shorter and younger fruits, as compared with laterally and proximally infected fruits [24]. It was suggested that proximal infection might be favoured through moisture being retained in the annular depression where the stalk is inserted and at the distal ends of young fruits [25]. However, in Nigeria, the annular depression at the base may not necessarily be favourable for infection as compared with the distal end of the pod.

**Figure 5.** *Densely sporulating cacao pods indicating the presence of Phytophthora megakarya.*

The predominance of *P. megakarya* on cacao in Nigeria started in the 1980s, alongside Cameroon, Equatorial Guinea, Gabon and Togo [22]. Recent studies showed that *P. palmivora* is no longer routinely isolated from cacao in Nigeria and Cameroon [12, 26, 27]; however, the displacement of *P. palmivora* by *P. megakarya* from cacao in these countries remains unclear [28]. However *P. megakarya* continues to be the major actual and potential threat to cacao in West Africa [16]. The much denser sporulation exhibited by *P. megakarya* on the surface of cacao pod (**Figure 5A–C**) indicates greater virulence of this species than *P. palmivora* and such significantly increases inoculum loads of *P. megakarya* [7, 29].

## **4. Characteristic and genomic diversity of** *Phytophthora* **species**

Five major diseases of cacao (*Theobroma cacao* L.), *Phytophthora* pod rot (black pod), witches broom, swollen shoot virus, vascular streak dieback and monilia pod rot, account for over 40% annual loss of cocoa [30]. Correct identification of plant pathogens is critical and fundamental to population genetics, epidemiological studies and the development of disease control strategies. Due to the similarity in growth patterns of *Oomycetes* including *Phytophthora* species and fungi, *Oomycetes* were previously considered as a class within the fungi. Fundamental differences between *Oomycetes* and fungi have been established [31–33], and the two are now known to be taxonomically distinct in spite of their common infection strategy [34]. As a result of the initial consideration of *Oomycetes* as a class within the fungi, [35] reported that researchers have for several decades pursued a wrong track in addressing the menace caused by *Phytophthora infestans.* For example, chitin was earlier reported as a minor component of oomycete cell walls and, therefore, insensitive to chitin synthase inhibitors, but it is now known to be an important component of hyphal tips in *Oomycetes* [36]. Isolations of *P. palmivora* from diseased cacao pods in Nigeria have been found to be "typical" in culture [37] with respect to

**49**

passing *P. palmivora*.

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

general characters including early production of sporangia. *Phytophthora palmivora* tends to have a more rapid growth rate than *P. megakarya* in culture, possibly contributing to its ability to cause accelerated necrosis in mechanically wounded cacao tissues compared to *P. megakarya* [38]. There have been no indications of important local variations with respect to the characters of this pathogen nor as regards the nature of the black pod rot infection. Variations in dimensions of sporangia were in

*Phytophthora palmivora* was first considered the only causal agent of black pod disease. However, a reclassification of some of the isolates previously described as *P. palmivora* into distinct species was suggested [39, 40]. Classification of species within the genus *Phytophthora* has progressed through the use of several criteria, including morphological dataset of colony, sporangium and oogonium characteristics; the presence or absence of chlamydospores and hyphal swellings, physiology [20, 41], isozyme patterns [42]; and lately the combined use of molecular markers and morphological characteristics [43]. Consequently, based on the size and number of chromosomes, they introduced the S- and L-type designations, which represented isolates having comparatively smaller chromosomes with *n* = 9–12 and isolates having large chromosomes with *n* = 5, respectively. However, the earlier work of [20] and recently [28] has established the variation in genetic characteristics of *Phytophthora* species commonly associated with cacao in Nigeria, and *P. megakarya* was found as the most virulent of

Consequently, the species were reclassified into three types: chromosome number, sporangial characteristics and pedicel length [20]. The S-type was regarded as *P. palmivora* sensu Butler (MF1) with 9–12 small chromosomes, papillate sporangia varying from near spherical to ovate-elongate shape and a short pedicel (2–5 μm) and being worldwide in distribution. The L-type was reclassified as *P. megakarya* (MF3), with five to six large chromosomes, papillate near spherical sporangia shape and pedicel of medium length (10–30 μm) and found only in West and Central Africa. Thus, the name "megakarya" is derived from the relatively large (mega) chromosomes. The third group was classified as *P. capsici* (MF4), with characteristics similar to *P. capsici* from black pepper [44, 45], and had longer pedicel (20–150 μm). The MF2, however, remains a variant of *P. palmivora.* The occurrence of hybridization is an important phenomenon in *Phytophthora*, given that hybridization may result in genetic variation that will adapt to new hosts or environments. Further confusion in the *P. palmivora* complex can occur due to heterothallic mating behaviour of the species. Sexual reproduction in *P. megakarya* and *P. palmivora* results in the production of oospores, and this requires the two opposite mating types, A1 and A2. Mating types in *P. megakarya* and *P. palmivora* show a curious imbalance, with A1 predominating in *P. megakarya* and A2 in *P. palmivora* [20]. This imbalance in mating types might favour hybridization between species, but not sexual reproduction within species. In spite of the two species coexisting on cocoa fields, no hybrids have been observed. The differences in chromosome numbers between *P. megakarya* and *P. palmivora* may also hinder hybridization and, hence, the rare occurrence of oospores in nature. *Phytophthora megakarya* was first described as a new *Phytophthora* species on *cacao* in West Africa based on chromosome number, sporangial characteristics and pedicel length. *Phytophthora megakarya* is indigenous to West and Central Africa, and it has become the main yield-limiting factor for cocoa production in affected areas [17] and rapidly sur-

In a susceptible cacao genotype, mechanical wounding may not be required for infection establishment in *P. megakarya* [38]. The genome size of *Phytophthora* 

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

respect to age and nature of substratum.

the species.

#### *Diversity of Cacao Pathogens and Impact on Yield and Global Production DOI: http://dx.doi.org/10.5772/intechopen.81993*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

The predominance of *P. megakarya* on cacao in Nigeria started in the 1980s, alongside Cameroon, Equatorial Guinea, Gabon and Togo [22]. Recent studies showed that *P. palmivora* is no longer routinely isolated from cacao in Nigeria and Cameroon [12, 26, 27]; however, the displacement of *P. palmivora* by *P. megakarya* from cacao in these countries remains unclear [28]. However *P. megakarya* continues to be the major actual and potential threat to cacao in West Africa [16]. The much denser sporulation exhibited by *P. megakarya* on the surface of cacao pod (**Figure 5A–C**) indicates greater virulence of this species than *P. palmivora* and

such significantly increases inoculum loads of *P. megakarya* [7, 29].

*Densely sporulating cacao pods indicating the presence of Phytophthora megakarya.*

**4. Characteristic and genomic diversity of** *Phytophthora* **species**

Five major diseases of cacao (*Theobroma cacao* L.), *Phytophthora* pod rot (black pod), witches broom, swollen shoot virus, vascular streak dieback and monilia pod rot, account for over 40% annual loss of cocoa [30]. Correct identification of plant pathogens is critical and fundamental to population genetics, epidemiological studies and the development of disease control strategies. Due to the similarity in growth patterns of *Oomycetes* including *Phytophthora* species and fungi, *Oomycetes* were previously considered as a class within the fungi. Fundamental differences between *Oomycetes* and fungi have been established [31–33], and the two are now known to be taxonomically distinct in spite of their common infection strategy [34]. As a result of the initial consideration of *Oomycetes* as a class within the fungi, [35] reported that researchers have for several decades pursued a wrong track in addressing the menace caused by *Phytophthora infestans.* For example, chitin was earlier reported as a minor component of oomycete cell walls and, therefore, insensitive to chitin synthase inhibitors, but it is now known to be an important component of hyphal tips in *Oomycetes* [36]. Isolations of *P. palmivora* from diseased cacao pods in Nigeria have been found to be "typical" in culture [37] with respect to

**48**

**Figure 5.**

general characters including early production of sporangia. *Phytophthora palmivora* tends to have a more rapid growth rate than *P. megakarya* in culture, possibly contributing to its ability to cause accelerated necrosis in mechanically wounded cacao tissues compared to *P. megakarya* [38]. There have been no indications of important local variations with respect to the characters of this pathogen nor as regards the nature of the black pod rot infection. Variations in dimensions of sporangia were in respect to age and nature of substratum.

*Phytophthora palmivora* was first considered the only causal agent of black pod disease. However, a reclassification of some of the isolates previously described as *P. palmivora* into distinct species was suggested [39, 40]. Classification of species within the genus *Phytophthora* has progressed through the use of several criteria, including morphological dataset of colony, sporangium and oogonium characteristics; the presence or absence of chlamydospores and hyphal swellings, physiology [20, 41], isozyme patterns [42]; and lately the combined use of molecular markers and morphological characteristics [43]. Consequently, based on the size and number of chromosomes, they introduced the S- and L-type designations, which represented isolates having comparatively smaller chromosomes with *n* = 9–12 and isolates having large chromosomes with *n* = 5, respectively. However, the earlier work of [20] and recently [28] has established the variation in genetic characteristics of *Phytophthora* species commonly associated with cacao in Nigeria, and *P. megakarya* was found as the most virulent of the species.

Consequently, the species were reclassified into three types: chromosome number, sporangial characteristics and pedicel length [20]. The S-type was regarded as *P. palmivora* sensu Butler (MF1) with 9–12 small chromosomes, papillate sporangia varying from near spherical to ovate-elongate shape and a short pedicel (2–5 μm) and being worldwide in distribution. The L-type was reclassified as *P. megakarya* (MF3), with five to six large chromosomes, papillate near spherical sporangia shape and pedicel of medium length (10–30 μm) and found only in West and Central Africa. Thus, the name "megakarya" is derived from the relatively large (mega) chromosomes. The third group was classified as *P. capsici* (MF4), with characteristics similar to *P. capsici* from black pepper [44, 45], and had longer pedicel (20–150 μm). The MF2, however, remains a variant of *P. palmivora.* The occurrence of hybridization is an important phenomenon in *Phytophthora*, given that hybridization may result in genetic variation that will adapt to new hosts or environments. Further confusion in the *P. palmivora* complex can occur due to heterothallic mating behaviour of the species. Sexual reproduction in *P. megakarya* and *P. palmivora* results in the production of oospores, and this requires the two opposite mating types, A1 and A2. Mating types in *P. megakarya* and *P. palmivora* show a curious imbalance, with A1 predominating in *P. megakarya* and A2 in *P. palmivora* [20]. This imbalance in mating types might favour hybridization between species, but not sexual reproduction within species. In spite of the two species coexisting on cocoa fields, no hybrids have been observed. The differences in chromosome numbers between *P. megakarya* and *P. palmivora* may also hinder hybridization and, hence, the rare occurrence of oospores in nature. *Phytophthora megakarya* was first described as a new *Phytophthora* species on *cacao* in West Africa based on chromosome number, sporangial characteristics and pedicel length. *Phytophthora megakarya* is indigenous to West and Central Africa, and it has become the main yield-limiting factor for cocoa production in affected areas [17] and rapidly surpassing *P. palmivora*.

In a susceptible cacao genotype, mechanical wounding may not be required for infection establishment in *P. megakarya* [38]. The genome size of *Phytophthora*  *megakarya* and *P. palmivora* was estimated at 126.88 and 151.23 Mb, respectively and number of genes 42,036 and 44,327, respectively [46]. *Phytophthora palmivora* appeared to have gone through whole genome duplication and subsequent gene diversification which expanded its genetic capacity for nutrient acquisition and breakdown of complex structures like the cell walls. This capacity may have influenced *P. palmivora* vigorous growth and broad host range, even without extended co-evolution with cacao. *Phytophthora megakarya* on the other hand has undergone amplification of specific gene families, some of which are clearly virulence-related like RxLRs, CRNs, elicitins and NPPs [46]. During *Phytophthora* infection, appressoria release effectors even before penetrations that enter host cells in an attempt to suppress pathogen-associated molecular pattern (PAMP) triggered immunity [47]. Under the conditions of high and frequent rainfall in Cameroon, *P. megakarya* can cause yield losses of up to 100% when no control measures are taken [48]. In Ghana, losses ranging between 60 and 100% have been reported [15].

Some other species of *Phytophthora* have been reported on *cacao* and such include *P. botryosa*, causing cacao pod rot in Malaysia, and *P. citrophthora* in Bahia, Brazil [49, 50]. *P. capsici*, *P. citrophthora* and *P. heveae* were reported in Mexico [51], *P. katsurae* in Côte d'Ivoire [52] and *P. megasperma* in Venezuela [45]. Apart from the cosmopolitan *P. palmivora*, the other species have only been recorded in certain countries or geographical location/regions. However, factors responsible for the geographical separation of the *Phytophthora* species are yet to be elucidated, but it is possible that lack of intensive surveys, coupled with isolation of isolates from the same location, from a few plant species and on a narrow range of media could be responsible for this observation. Another possibility is that these species occur rarely on cacao; nonetheless, more investigations are required to ascertain these claims.

### **5. Impact of** *Phytophthora* **on cacao yield and bean quality**

Major economic losses in cocoa production are caused by pests and diseases, particularly in the many small and isolated farms that lack adequate control measure across West Africa region. These species cause mean annual pod losses of about 40% and even higher in parts of Ghana and Côte d'Ivoire [17, 53]. The highest incidence of black pod disease is found in the shaded cocoa in Cameroon. World losses in cacao production due to black pod disease caused by various species of *Phytophthora* have been estimated to cause about 450,000 metric tons [7]. This disease probably accounts for 20–25% of the expected crop and making it the biggest constraint to production. **Figure 6** shows the usual harvesting exercise/activities in cocoa farms where both mature healthy cocoa pods and the disease black pods (arrowed) are usually lumped together on farmer's field and processed.

Losses due to black pod can be especially severe in West and Central Africa, an area that contributes 60–70% to the worldwide cacao production [7]. In Africa, it can cause 30–90% annual crop loss for farmers, and, thus, it poses a severe threat to the cacao industry and to producers. Part of the contributory factors which is major in limiting productivity in cacao is related to the practices of farmers. Apart from the practice indicated in **Figure 6**, the observation of piles of cocoa pod husks on different locations on farmers' fields serves as the sources of inoculum of the pathogen of black pod disease. The spores of *Phytophthora* species on infected cocoa pods are usually left on the field after extraction of the beans (**Figure 7**) and are reactivated under suitable conditions to infect fresh cocoa pods.

**51**

**Figure 6.**

**Figure 7.**

*Cocoa pod husk pile on farmer's field.*

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

In the year 2012, Ghana lost more than 200,000 tonnes of cacao beans (25% of the annual output) due to black pod, costing the nation \$230 million (Ghana Cocoa Board report). Until the mid-1980s, only *P. palmivora* was present in all the cacao-growing regions of Ghana, causing limited crop losses of 4.9–19% [54]. After 1985, black pod became a major disease in Ghana and was attributed to the emergence of *P. megakarya* as a pathogen of cacao [55]; various reports from Ghana indicated a rapid spread of *P. megakarya* to various cacao districts by the late 1990s causing 60–100% crop losses [17].

The *Phytophthora* species affects different parts of the cacao, but infection of the pod is the major economic loss as pods or cherelles may be infected at any parts on the surface. Observation of the disease indicates a firm, spreading, chocolate-brown lesion which eventually covers the whole pod. The beans inside the pod may remain undamaged for several days after initial infection of the husk, but in advanced infections, *Phytophthora* invades the internal pod tissues and causes discoloration and shrivelling of the cocoa beans (**Figure 8A–C**), thus tampering with the muci-

lage colouration (**Figure 9**) and affecting quality of the cocoa bean.

**5.1 Impact of** *Phytophthora* **on cocoa beans**

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

*Harvested cocoa pods on farmer's field with black pod (arrowed).*

*Diversity of Cacao Pathogens and Impact on Yield and Global Production DOI: http://dx.doi.org/10.5772/intechopen.81993*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

*megakarya* and *P. palmivora* was estimated at 126.88 and 151.23 Mb, respectively and number of genes 42,036 and 44,327, respectively [46]. *Phytophthora palmivora* appeared to have gone through whole genome duplication and subsequent gene diversification which expanded its genetic capacity for nutrient acquisition and breakdown of complex structures like the cell walls. This capacity may have influenced *P. palmivora* vigorous growth and broad host range, even without extended co-evolution with cacao. *Phytophthora megakarya* on the other hand has undergone amplification of specific gene families, some of which are clearly virulence-related like RxLRs, CRNs, elicitins and NPPs [46]. During *Phytophthora* infection, appressoria release effectors even before penetrations that enter host cells in an attempt to suppress pathogen-associated molecular pattern (PAMP) triggered immunity [47]. Under the conditions of high and frequent rainfall in Cameroon, *P. megakarya* can cause yield losses of up to 100% when no control measures are taken [48]. In Ghana, losses ranging between 60 and 100% have

Some other species of *Phytophthora* have been reported on *cacao* and such include *P. botryosa*, causing cacao pod rot in Malaysia, and *P. citrophthora* in Bahia, Brazil [49, 50]. *P. capsici*, *P. citrophthora* and *P. heveae* were reported in Mexico [51], *P. katsurae* in Côte d'Ivoire [52] and *P. megasperma* in Venezuela [45]. Apart from the cosmopolitan *P. palmivora*, the other species have only been recorded in certain countries or geographical location/regions. However, factors responsible for the geographical separation of the *Phytophthora* species are yet to be elucidated, but it is possible that lack of intensive surveys, coupled with isolation of isolates from the same location, from a few plant species and on a narrow range of media could be responsible for this observation. Another possibility is that these species occur rarely on cacao; nonetheless, more investigations are required to ascertain these

**5. Impact of** *Phytophthora* **on cacao yield and bean quality**

(arrowed) are usually lumped together on farmer's field and processed.

reactivated under suitable conditions to infect fresh cocoa pods.

Major economic losses in cocoa production are caused by pests and diseases, particularly in the many small and isolated farms that lack adequate control measure across West Africa region. These species cause mean annual pod losses of about 40% and even higher in parts of Ghana and Côte d'Ivoire [17, 53]. The highest incidence of black pod disease is found in the shaded cocoa in Cameroon. World losses in cacao production due to black pod disease caused by various species of *Phytophthora* have been estimated to cause about 450,000 metric tons [7]. This disease probably accounts for 20–25% of the expected crop and making it the biggest constraint to production. **Figure 6** shows the usual harvesting exercise/activities in cocoa farms where both mature healthy cocoa pods and the disease black pods

Losses due to black pod can be especially severe in West and Central Africa, an area that contributes 60–70% to the worldwide cacao production [7]. In Africa, it can cause 30–90% annual crop loss for farmers, and, thus, it poses a severe threat to the cacao industry and to producers. Part of the contributory factors which is major in limiting productivity in cacao is related to the practices of farmers. Apart from the practice indicated in **Figure 6**, the observation of piles of cocoa pod husks on different locations on farmers' fields serves as the sources of inoculum of the pathogen of black pod disease. The spores of *Phytophthora* species on infected cocoa pods are usually left on the field after extraction of the beans (**Figure 7**) and are

**50**

been reported [15].

claims.

**Figure 6.** *Harvested cocoa pods on farmer's field with black pod (arrowed).*

**Figure 7.** *Cocoa pod husk pile on farmer's field.*

In the year 2012, Ghana lost more than 200,000 tonnes of cacao beans (25% of the annual output) due to black pod, costing the nation \$230 million (Ghana Cocoa Board report). Until the mid-1980s, only *P. palmivora* was present in all the cacao-growing regions of Ghana, causing limited crop losses of 4.9–19% [54]. After 1985, black pod became a major disease in Ghana and was attributed to the emergence of *P. megakarya* as a pathogen of cacao [55]; various reports from Ghana indicated a rapid spread of *P. megakarya* to various cacao districts by the late 1990s causing 60–100% crop losses [17].

## **5.1 Impact of** *Phytophthora* **on cocoa beans**

The *Phytophthora* species affects different parts of the cacao, but infection of the pod is the major economic loss as pods or cherelles may be infected at any parts on the surface. Observation of the disease indicates a firm, spreading, chocolate-brown lesion which eventually covers the whole pod. The beans inside the pod may remain undamaged for several days after initial infection of the husk, but in advanced infections, *Phytophthora* invades the internal pod tissues and causes discoloration and shrivelling of the cocoa beans (**Figure 8A–C**), thus tampering with the mucilage colouration (**Figure 9**) and affecting quality of the cocoa bean.

#### **Figure 8.**

*Status of black pod disease on cocoa bean. A: Ripe and healthy cocoa pod with quality mucilage and beans. B: Ripe but diseased cocoa pod with infected beans. C: Unripe but diseased cocoa pod with infected beans.*

**Figure 9.** *Effect of black pod disease on cocoa mucilage and beans in ripe cocoa pod.*

## **6. Management strategies for** *Phytophthora* **species**

*Phytophthora* can persist in soil and debris for several years making the control of black pod difficult [56]. Also, since susceptible pods may be present on the trees most of the year, the pathogen may always be present in the canopy, ready to cause major epidemics when environmental conditions become favourable for sporulation and dispersal [29]. Although much research has been published over the past few decades on black pod disease, sustainable management strategies that are applicable to smallholder farms are still lacking in most producing countries. Crop losses and cost of controlling *Phytophthora* (black pod) diseases constitute a significant financial burden on agricultural enterprises and have serious socioeconomic and environmental consequences wherever these pathogens are found. Neglect of cocoa

**53**

cocoa varieties.

**6.1 Cultural practices to combat black pod disease**

regular spraying of fungicides is required.

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

farms infected with *P. megakarya*, cultivation of crops other than *T. cacao* in infected areas [16] and establishment of *T. cacao* in *P. megakarya*-free forest areas have significant impacts on the economies of the cocoa-producing countries in West Africa.

*Phytophthora megakarya* has spread within the West and Central African subregions, and it is still in its invasive phase. The spread of this pathogen from one location to another in Ghana has been linked with the movement of planting materials [16, 57, 58]. The faster communication, travel and trade links and the relatively free movement of people and commodities all over the world pose a serious risk of introducing *P. megakarya* to other cacao-growing regions. On the other hand, there is a risk of introduction of other major cocoa diseases from other cocoa-producing areas into West and Central Africa [59]. This will have negative impact on world cocoa production. A devastating impact on the world's cocoa supply is eminent and extremely serious in social, economic and environmental problems. Such pathogen introduction can also be experienced within a growing country from the region of high incidence to low one. To minimise such risks, preventive measures and effective testing procedures and exchange of materials through intermediate quarantine facilities must be enforced. Consequently, there is an urgent need for effective and sustainable control of *P. megakarya*/black pod disease. The effective and sustainable management of this disease requires integrated approach of several methods, including quarantine, cultural, chemical and biological control and use of resistant

Activities to combat the menace of yield losses and decline in cocoa production resulting from black pod disease incidence in cocoa-growing communities are enormous. Cultural control practices that promote crop growth, inhibit and obstruct pathogen establishment, growth and development is one of the first approaches in plant disease control. Cultural practices are not only essential for increasing yield but also for providing the right environment for efficient performance of fungicides [60]. For the small holdings, low-input and low-income cocoa farmer use of cultural practices remains the least expensive disease control option for managing black pod disease. However, frequent harvesting saves partly infected mature pods, removal of infected pods, and reduces sources of sporangial inoculum. The regular and timely removal of infected pods and reduction in the shade to increase the humidity which in turn reduce pod losses however, additional chemical control by

In Nigeria, frequent removal of diseased pods complemented sprayed programmes in controlling *P. megakarya*, but, often, excessive tree heights hampered the effectiveness of the technique [61]. Similarly, in Togo, *P. megakarya* diseased pod removal was recommended as part of a package to reduce disease incidence [62]. In Cameroon, inoculum levels were successfully reduced by the pruning and weekly removal of pods but only in concert with spraying [63]. Another cultural method occasionally recommended is the removal or spraying of pod husk piles where they occur on farms (**Figure 7**). It is known that these pod husk piles serve as disease foci on *P. megakarya* farms. In Nigeria and Sao Tome, burying of husks was recommended, but its limited effectiveness and expense caused this option to be dropped [64]. However, in Ghana the husks are burnt into potash and used in the production of soap. Cultural practices on cacao farms are labour intensive and inadequate when applied alone for *P. megakarya* control. They need to be supplemented with other control methods, such as spraying of fungicides to reduce losses on farms [58, 65–67]. Most farmers, however, are unable to adopt this technology because of

It also has effects on biodiversity and functioning of the natural ecosystems.

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

#### *Diversity of Cacao Pathogens and Impact on Yield and Global Production DOI: http://dx.doi.org/10.5772/intechopen.81993*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

**6. Management strategies for** *Phytophthora* **species**

*Effect of black pod disease on cocoa mucilage and beans in ripe cocoa pod.*

*Phytophthora* can persist in soil and debris for several years making the control of black pod difficult [56]. Also, since susceptible pods may be present on the trees most of the year, the pathogen may always be present in the canopy, ready to cause major epidemics when environmental conditions become favourable for sporulation and dispersal [29]. Although much research has been published over the past few decades on black pod disease, sustainable management strategies that are applicable to smallholder farms are still lacking in most producing countries. Crop losses and cost of controlling *Phytophthora* (black pod) diseases constitute a significant financial burden on agricultural enterprises and have serious socioeconomic and environmental consequences wherever these pathogens are found. Neglect of cocoa

*Status of black pod disease on cocoa bean. A: Ripe and healthy cocoa pod with quality mucilage and beans. B: Ripe but diseased cocoa pod with infected beans. C: Unripe but diseased cocoa pod with infected beans.*

**52**

**Figure 8.**

**Figure 9.**

farms infected with *P. megakarya*, cultivation of crops other than *T. cacao* in infected areas [16] and establishment of *T. cacao* in *P. megakarya*-free forest areas have significant impacts on the economies of the cocoa-producing countries in West Africa. It also has effects on biodiversity and functioning of the natural ecosystems.

*Phytophthora megakarya* has spread within the West and Central African subregions, and it is still in its invasive phase. The spread of this pathogen from one location to another in Ghana has been linked with the movement of planting materials [16, 57, 58]. The faster communication, travel and trade links and the relatively free movement of people and commodities all over the world pose a serious risk of introducing *P. megakarya* to other cacao-growing regions. On the other hand, there is a risk of introduction of other major cocoa diseases from other cocoa-producing areas into West and Central Africa [59]. This will have negative impact on world cocoa production. A devastating impact on the world's cocoa supply is eminent and extremely serious in social, economic and environmental problems. Such pathogen introduction can also be experienced within a growing country from the region of high incidence to low one. To minimise such risks, preventive measures and effective testing procedures and exchange of materials through intermediate quarantine facilities must be enforced. Consequently, there is an urgent need for effective and sustainable control of *P. megakarya*/black pod disease. The effective and sustainable management of this disease requires integrated approach of several methods, including quarantine, cultural, chemical and biological control and use of resistant cocoa varieties.

## **6.1 Cultural practices to combat black pod disease**

Activities to combat the menace of yield losses and decline in cocoa production resulting from black pod disease incidence in cocoa-growing communities are enormous. Cultural control practices that promote crop growth, inhibit and obstruct pathogen establishment, growth and development is one of the first approaches in plant disease control. Cultural practices are not only essential for increasing yield but also for providing the right environment for efficient performance of fungicides [60]. For the small holdings, low-input and low-income cocoa farmer use of cultural practices remains the least expensive disease control option for managing black pod disease. However, frequent harvesting saves partly infected mature pods, removal of infected pods, and reduces sources of sporangial inoculum. The regular and timely removal of infected pods and reduction in the shade to increase the humidity which in turn reduce pod losses however, additional chemical control by regular spraying of fungicides is required.

In Nigeria, frequent removal of diseased pods complemented sprayed programmes in controlling *P. megakarya*, but, often, excessive tree heights hampered the effectiveness of the technique [61]. Similarly, in Togo, *P. megakarya* diseased pod removal was recommended as part of a package to reduce disease incidence [62]. In Cameroon, inoculum levels were successfully reduced by the pruning and weekly removal of pods but only in concert with spraying [63]. Another cultural method occasionally recommended is the removal or spraying of pod husk piles where they occur on farms (**Figure 7**). It is known that these pod husk piles serve as disease foci on *P. megakarya* farms. In Nigeria and Sao Tome, burying of husks was recommended, but its limited effectiveness and expense caused this option to be dropped [64]. However, in Ghana the husks are burnt into potash and used in the production of soap. Cultural practices on cacao farms are labour intensive and inadequate when applied alone for *P. megakarya* control. They need to be supplemented with other control methods, such as spraying of fungicides to reduce losses on farms [58, 65–67]. Most farmers, however, are unable to adopt this technology because of

the high costs of the fungicides and application problems. In practice little fungicide is used [17, 68]. However, removal of black pods from the soil surface would be a simple strategy to reduce inoculum spread by ants, as well as by flying vectors [69]. Reduction in canopy humidity and consequent sporulation can be achieved by pruning and appropriate tree spacing to increase aeration. Maintenance of leaf litter or mulches to prevent soil inoculum of *P. megakarya* reaching pods has been suggested [70], while leaf litter was found to reduce pod infection from soil inoculum [71]. The spread of the disease from infected pods can be reduced by frequent harvesting to lessen the danger of spread of disease from infected pods. Black pod disease is also managed by regular pruning to remove infected chupons and increase air circulation. Other measures, such as the removal of infected pods and husk piles, may have some effect on inoculum levels.

## **6.2 Chemical strategies to combat black pod disease**

Fungicides have been used to control *Phytophthora* pod rot of cocoa for over a century, and several experiments on different chemical control measures have been conducted in all cocoa-growing countries. The history of the development of fungicides on cocoa has been extensively reviewed [72–74]. Recommendations adopted in the different countries are based on local factors, such as species of pathogen, climatic conditions, cocoa variety, planting density and social and economic considerations [64]. Traditionally, expensive copper-based fungicides (or systemic) have been applied frequently (sometimes every 10 days) in areas of high infection. Without prophylaxis, the losses could reach 100% in areas of continuous high humidity and high disease incidence, although the disease has a normal range of 5–90%. In order to limit yield loss to black pod disease in Ghana, three preventive rounds of copper fungicide were applied during the rainy season under a national spray programme in *P. megakarya* prevalent areas. This however puts immense pressure on resource-poor farmers in the form of reduced farmgate prices, leading to ecological, socioeconomic and possibly political instability. In Nigeria, many fungicides of varied active ingredients are used by farmers across growing ecologies. The Cocoa Research Institute of Nigeria has the national mandate to screen *in vitro* and *in vivo* such fungicides among other pesticides prior to being used on cacao in Nigeria. Many of the active ingredients (product of different agrochemical companies) that have undergone a 3-consecutive-year field trials include but not limited to copper hydroxide, cuprous oxide + metalaxyl-M, cuprous oxide and metalaxyl-M + copper-1-oxide and recently are copper-1-oxide + metalaxyl, mandipropamid + mefenoxam, initium + dimethomorph and pyraclostrobin + dimethomorph + ametoctradin. The relative effectiveness of certain treatments and inconsistencies in results between countries and locations depends on the different combinations of these factors. For example, while fungicides are applied at two weekly intervals in Cameroon to control black pod disease, due to the relatively high and frequent rainfall, fungicides are applied at three to four weekly intervals in Ghana [16], and spray interval of 3 weeks is also advised in Nigeria. The reason for the difference among countries has to do with the amount and frequency of rainfall.

In West Africa, protectant fungicides that are mainly "fixed" copper compounds, e.g., copper hydroxides and copper oxides, or systemic fungicides containing copper and metalaxyl as mixtures are routinely sprayed onto pods with leveroperated knapsack sprayers for *Phytophthora* pod rot control. These fixed copper compounds are finely divided molecules that are readily mixed and easy to apply at low volumes. This is in contrast to earlier products such as the Bordeaux mixture, which had to be applied in relatively large volumes. These copper fungicides form a

**55**

by *P. megakarya*.

**7. Conclusion**

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

chemical barrier on the surface of the pod and guard against infection [58, 75]. The spraying of copper and metalaxyl mixtures is to take advantage of multisite action of the different active ingredients and to reduce the possible build-up of metalaxyl resistance in *Phytophthora* species on cocoa. Furthermore, it must be emphasised that correct dosage of fungicides, timing of initial application in relation to the epidemic, frequency and target of application are all critical factors to ensure suc-

Many natural substances, including plant extracts and bioactive compounds produced by microorganisms, have been tried to control *Phytophthora* on cacao [76, 77]. Rosemary (*Rosmarinus officinalis*) and lavender (*Lavandula officinalis*) leaf extracts when supplemented to agar plates at different dilutions were found to inhibit germination of *P. capsici*, *P. megakarya* and *P. palmivora* zoospores. Rosemary extracts, containing caffeic acid, rosmarinic acid or derivatives, thereof,

reduced necrosis of cacao leaf discs caused by *P. megakarya* zoospores [77]. Another promising class of natural microbial compounds with activity against *Phytophthora* species is the cyclic lipopeptides (CLPs) [78–81]. Studies showed that massetolide A (massA) produced by *P. fluorescens* strain SS101 caused zoospore lysis through induction of pores, reduced sporangium formation and increased branching and swelling of hyphae of *P. infestans* [78, 82]. MassA also induced systemic resistance in tomato plants and reduced the number and expansion of late blight lesions on tomato caused by *P. infestans* [83, 84]. Given that hyphae, sporangia and zoospores are important sources of inoculum and play major role in cacao black pod epidemic, there is the need to investigate if CLPs or CLP-producing microorganisms can be exploited for the management of black pod disease caused

*Phytophthora megakarya* infestation of cacao is a threat to the economies of growing countries in West Africa. This diverse pathogen is spreading fast in the subregion, displacing the original populations of the less severe *P. palmivora*. The mechanisms for this shift in population composition of the black pod disease complex remain unknown, although the possibility of further spread to other cacao-producing regions is a great concern. The available and fast-emerging genomic and genetic information on oomycete pathogens and their hosts, including *Theobroma cacao*, should be utilised and explored for the development of new sustainable management practices for *Phytophthora* species. Current methods of control through routine spraying of inorganic fungicides are expensive, hazardous and environmentally unfriendly. Programmes of integrated pest management with precise fungicide application which give less residue in the cocoa beans, combined with field sanitation and proper farm management, should be encouraged in all

areas where *Phytophthora* species cause significant losses on cocoa.

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

cessful and economic chemical control.

**6.3 Alternative practices to combat black pod disease**

chemical barrier on the surface of the pod and guard against infection [58, 75]. The spraying of copper and metalaxyl mixtures is to take advantage of multisite action of the different active ingredients and to reduce the possible build-up of metalaxyl resistance in *Phytophthora* species on cocoa. Furthermore, it must be emphasised that correct dosage of fungicides, timing of initial application in relation to the epidemic, frequency and target of application are all critical factors to ensure successful and economic chemical control.

## **6.3 Alternative practices to combat black pod disease**

Many natural substances, including plant extracts and bioactive compounds produced by microorganisms, have been tried to control *Phytophthora* on cacao [76, 77]. Rosemary (*Rosmarinus officinalis*) and lavender (*Lavandula officinalis*) leaf extracts when supplemented to agar plates at different dilutions were found to inhibit germination of *P. capsici*, *P. megakarya* and *P. palmivora* zoospores. Rosemary extracts, containing caffeic acid, rosmarinic acid or derivatives, thereof, reduced necrosis of cacao leaf discs caused by *P. megakarya* zoospores [77]. Another promising class of natural microbial compounds with activity against *Phytophthora* species is the cyclic lipopeptides (CLPs) [78–81]. Studies showed that massetolide A (massA) produced by *P. fluorescens* strain SS101 caused zoospore lysis through induction of pores, reduced sporangium formation and increased branching and swelling of hyphae of *P. infestans* [78, 82]. MassA also induced systemic resistance in tomato plants and reduced the number and expansion of late blight lesions on tomato caused by *P. infestans* [83, 84]. Given that hyphae, sporangia and zoospores are important sources of inoculum and play major role in cacao black pod epidemic, there is the need to investigate if CLPs or CLP-producing microorganisms can be exploited for the management of black pod disease caused by *P. megakarya*.

## **7. Conclusion**

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

piles, may have some effect on inoculum levels.

**6.2 Chemical strategies to combat black pod disease**

the high costs of the fungicides and application problems. In practice little fungicide is used [17, 68]. However, removal of black pods from the soil surface would be a simple strategy to reduce inoculum spread by ants, as well as by flying vectors [69]. Reduction in canopy humidity and consequent sporulation can be achieved by pruning and appropriate tree spacing to increase aeration. Maintenance of leaf litter or mulches to prevent soil inoculum of *P. megakarya* reaching pods has been suggested [70], while leaf litter was found to reduce pod infection from soil inoculum [71]. The spread of the disease from infected pods can be reduced by frequent harvesting to lessen the danger of spread of disease from infected pods. Black pod disease is also managed by regular pruning to remove infected chupons and increase air circulation. Other measures, such as the removal of infected pods and husk

Fungicides have been used to control *Phytophthora* pod rot of cocoa for over a century, and several experiments on different chemical control measures have been conducted in all cocoa-growing countries. The history of the development of fungicides on cocoa has been extensively reviewed [72–74]. Recommendations adopted in the different countries are based on local factors, such as species of pathogen, climatic conditions, cocoa variety, planting density and social and economic considerations [64]. Traditionally, expensive copper-based fungicides (or systemic) have been applied frequently (sometimes every 10 days) in areas of high infection. Without prophylaxis, the losses could reach 100% in areas of continuous high humidity and high disease incidence, although the disease has a normal range of 5–90%. In order to limit yield loss to black pod disease in Ghana, three preventive rounds of copper fungicide were applied during the rainy season under a national spray programme in *P. megakarya* prevalent areas. This however puts immense pressure on resource-poor farmers in the form of reduced farmgate prices, leading to ecological, socioeconomic and possibly political instability. In Nigeria, many fungicides of varied active ingredients are used by farmers across growing ecologies. The Cocoa Research Institute of Nigeria has the national mandate to screen *in vitro* and *in vivo* such fungicides among other pesticides prior to being used on cacao in Nigeria. Many of the active ingredients (product of different agrochemical companies) that have undergone a 3-consecutive-year field trials include but not limited to copper hydroxide, cuprous oxide + metalaxyl-M, cuprous oxide and metalaxyl-M + copper-1-oxide and recently are copper-1-oxide + metalaxyl, mandipropamid + mefenoxam, initium + dimethomorph and pyraclostrobin + dimethomorph + ametoctradin. The relative effectiveness of certain treatments and inconsistencies in results between countries and locations depends on the different combinations of these factors. For example, while fungicides are applied at two weekly intervals in Cameroon to control black pod disease, due to the relatively high and frequent rainfall, fungicides are applied at three to four weekly intervals in Ghana [16], and spray interval of 3 weeks is also advised in Nigeria. The reason for the difference among countries has to do with the amount

In West Africa, protectant fungicides that are mainly "fixed" copper compounds,

e.g., copper hydroxides and copper oxides, or systemic fungicides containing copper and metalaxyl as mixtures are routinely sprayed onto pods with leveroperated knapsack sprayers for *Phytophthora* pod rot control. These fixed copper compounds are finely divided molecules that are readily mixed and easy to apply at low volumes. This is in contrast to earlier products such as the Bordeaux mixture, which had to be applied in relatively large volumes. These copper fungicides form a

**54**

and frequency of rainfall.

*Phytophthora megakarya* infestation of cacao is a threat to the economies of growing countries in West Africa. This diverse pathogen is spreading fast in the subregion, displacing the original populations of the less severe *P. palmivora*. The mechanisms for this shift in population composition of the black pod disease complex remain unknown, although the possibility of further spread to other cacao-producing regions is a great concern. The available and fast-emerging genomic and genetic information on oomycete pathogens and their hosts, including *Theobroma cacao*, should be utilised and explored for the development of new sustainable management practices for *Phytophthora* species. Current methods of control through routine spraying of inorganic fungicides are expensive, hazardous and environmentally unfriendly. Programmes of integrated pest management with precise fungicide application which give less residue in the cocoa beans, combined with field sanitation and proper farm management, should be encouraged in all areas where *Phytophthora* species cause significant losses on cocoa.

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

## **Author details**

Dele Adeniyi Cocoa Research Institute of Nigeria (CRIN), Ibadan, Nigeria

\*Address all correspondence to: modeleadeniyi@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**57**

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

[9] ICCO. Production of Cocoa Beans, Quarterly Bulletin of Cocoa Statistics, London. 2006, XXXI, XXXIX (2013), XL (2014). 2015. Available at: http://www. icco.org/ [Accessed: March 18, 2015]

[10] ICCO. Production of cocoa beans, Quarterly Bulletin of Cocoa Statistics, London, 2017, XLIII (2017). 2017. Available from: http://www.icco.org

[11] McMahon P, Purwantara A, Drenth A, Guest D. *Phytophthora* on cocoa. In: Drenth A, editor. Diversity and Management of *Phytophthora* in Southeast Asia. Australia: Australian Centre for International Agricultural

Research; 2004. pp. 104-115

Science. 1973;**6**:93-102

Control. 2008;**44**:149-159

2000a;**33**:135-142

[12] Nyasse S, Grivet L, Risterucci AM, Blaha G, Berry D, Lanaud C. Diversity of *Phytophthora megakarya* in Central and West Africa revealed by isozyme and RAPD markers. Mycological Research. 1999a;**103**:1225-1234

[13] Dakwa JT. The relationship between Black Pod Incidence and the Weather in Ghana. Ghana Journal of Agricultural

[14] Deberdt P, Mfegue CV, Tondje PR, Bon MC, Ducamp M, Hurard C. Impact of environmental factors, chemical fungicide and biological control on cacao pod production dynamics and black pod disease (*Phytophthora megakarya*) in Cameroon. Biological

[15] Dakwa J. A serious outbreak of black pod disease in a marginal area of Ghana. In: Proceedings of the 10th International Cocoa Research Conference (Santo Domingo); 1987. pp. 447-451

[16] Opoku IY, Appiah AA, Akrofi AY. *Phytophthora megakarya*: A potential threat to the cocoa industry in Ghana. Ghana Journal of Agricultural Science.

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

[1] Perfecto I, Rice RA, Greenberg R, Van der Voort ME. Shade coffee: A disappearing refuge for biodiversity.

[2] Evans HC. Invasive neotropical pathogens of tree crops. In: Watling R, Frankland J, Ainsworth M, Isaac S, Robinson C, editors. Tropical Mycology. Vol. 2 Micromycetes. Wallingford, UK: CABI Publishing; 2002. pp. 83-112

[3] Marius W, Quist-Wessel Foluke PM. Cocoa production in West Africa, a review and analysis of recent developments. NJAS–Wageningen

[4] Nzeka UM. Nigeria hikes target on cocoa production. USDA Foreign ServiceGain Report, Lagos. 2014:8

[5] Phillips-Mora W, Castillo J, Arciniegas A, Mata A, Sánchez A, Leandro M, et al. Overcoming the main limiting factors of cacao production in Central America through the use of improved clones developed at Catie. In: Conference: 16th International Cocoa Research Conference; Bali, Indonesia;

[6] Gotsch N. Cocoa crop protection: An expert forecast on future progress, research priorities and policy with the help of the Delphi survey. Crop

[7] Bowers JH, Bailey BA, Hebbar PK, Sanogo S, Lumsden RD. The impact of plant diseases on world chocolate production. Plant Health Progress. 2001. DOI: 10.1094/PHP-2001-0709-

Production and Consumption, Cocoa Growers'Bulletin, 40. Birmingham: Cadbury Ltd; 2000. 1988, 45 (1992), 49

Protection. 1997;**16**:227-233

01-RV. Published online

(1995), 52 (2000)

[8] Lass RA, editor. Review of

September 2009

Bioscience. 1996;**46**:598-608

Journal of Life Sciences. 2015;**74-75**(2015):1-7

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*Diversity of Cacao Pathogens and Impact on Yield and Global Production DOI: http://dx.doi.org/10.5772/intechopen.81993*

## **References**

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**56**

**Author details**

Dele Adeniyi

provided the original work is properly cited.

Cocoa Research Institute of Nigeria (CRIN), Ibadan, Nigeria

\*Address all correspondence to: modeleadeniyi@gmail.com

[1] Perfecto I, Rice RA, Greenberg R, Van der Voort ME. Shade coffee: A disappearing refuge for biodiversity. Bioscience. 1996;**46**:598-608

[2] Evans HC. Invasive neotropical pathogens of tree crops. In: Watling R, Frankland J, Ainsworth M, Isaac S, Robinson C, editors. Tropical Mycology. Vol. 2 Micromycetes. Wallingford, UK: CABI Publishing; 2002. pp. 83-112

[3] Marius W, Quist-Wessel Foluke PM. Cocoa production in West Africa, a review and analysis of recent developments. NJAS–Wageningen Journal of Life Sciences. 2015;**74-75**(2015):1-7

[4] Nzeka UM. Nigeria hikes target on cocoa production. USDA Foreign ServiceGain Report, Lagos. 2014:8

[5] Phillips-Mora W, Castillo J, Arciniegas A, Mata A, Sánchez A, Leandro M, et al. Overcoming the main limiting factors of cacao production in Central America through the use of improved clones developed at Catie. In: Conference: 16th International Cocoa Research Conference; Bali, Indonesia; September 2009

[6] Gotsch N. Cocoa crop protection: An expert forecast on future progress, research priorities and policy with the help of the Delphi survey. Crop Protection. 1997;**16**:227-233

[7] Bowers JH, Bailey BA, Hebbar PK, Sanogo S, Lumsden RD. The impact of plant diseases on world chocolate production. Plant Health Progress. 2001. DOI: 10.1094/PHP-2001-0709- 01-RV. Published online

[8] Lass RA, editor. Review of Production and Consumption, Cocoa Growers'Bulletin, 40. Birmingham: Cadbury Ltd; 2000. 1988, 45 (1992), 49 (1995), 52 (2000)

[9] ICCO. Production of Cocoa Beans, Quarterly Bulletin of Cocoa Statistics, London. 2006, XXXI, XXXIX (2013), XL (2014). 2015. Available at: http://www. icco.org/ [Accessed: March 18, 2015]

[10] ICCO. Production of cocoa beans, Quarterly Bulletin of Cocoa Statistics, London, 2017, XLIII (2017). 2017. Available from: http://www.icco.org

[11] McMahon P, Purwantara A, Drenth A, Guest D. *Phytophthora* on cocoa. In: Drenth A, editor. Diversity and Management of *Phytophthora* in Southeast Asia. Australia: Australian Centre for International Agricultural Research; 2004. pp. 104-115

[12] Nyasse S, Grivet L, Risterucci AM, Blaha G, Berry D, Lanaud C. Diversity of *Phytophthora megakarya* in Central and West Africa revealed by isozyme and RAPD markers. Mycological Research. 1999a;**103**:1225-1234

[13] Dakwa JT. The relationship between Black Pod Incidence and the Weather in Ghana. Ghana Journal of Agricultural Science. 1973;**6**:93-102

[14] Deberdt P, Mfegue CV, Tondje PR, Bon MC, Ducamp M, Hurard C. Impact of environmental factors, chemical fungicide and biological control on cacao pod production dynamics and black pod disease (*Phytophthora megakarya*) in Cameroon. Biological Control. 2008;**44**:149-159

[15] Dakwa J. A serious outbreak of black pod disease in a marginal area of Ghana. In: Proceedings of the 10th International Cocoa Research Conference (Santo Domingo); 1987. pp. 447-451

[16] Opoku IY, Appiah AA, Akrofi AY. *Phytophthora megakarya*: A potential threat to the cocoa industry in Ghana. Ghana Journal of Agricultural Science. 2000a;**33**:135-142

[17] Opoku IY, Appiah AA, Akrofi AY, Owusu GK. *Phytophthora megakarya*: A potential threat to the cocoa industry in Ghana? Ghana Journal of Agricultural Science. 2000b;**33**(2000):237-248

[18] Dade HA. Economic significance of cocoa pod disease and factors determining their incidence and control. Bulletin. Gold Coast Department of Agriculture GoldCst. 1927;**6**:I-58

[19] Ndubuaku T, Asogwa E. Strategies for the control of pests and diseases for sustainable cocoa production in Nigeria. African Scientist. 2006;**7**:209-216

[20] Djocgoue P, Boudjeko T, Mbouobda H, Nankeu D, El Hadrami I, Omokolo N. Heritability of phenols in the resistance of Theobroma cacao against Phytophthora megakarya, the causal agent of black pod disease. Journal of Phytopathology. 2007;**155**:519-525. DOI: 10.1111/j.1439-0434.2007. 01268.x

[21] Ali SS, Shao J, Lary DJ, Strem MD, Meinhardt LW, Bailey BA. *Phytophthora megakarya* and *P. palmivora*, causal agents of Black Pod Rot, induce similar plant defense responses late during infection of susceptible Cacao Pods. Frontiers in Plant Science. 2017;**8**:169. DOI: 10.3389/fpls.2017.00169

[22] Evans HC, Prior C. Cocoa pod diseases: Causal agents and control. Outlook on Agriculture. 1987;**16**:35-41

[23] Flood J, Keane P, Sulistyowati E, Padi B, Guest D, Holmes K. Cocoa under attack. In: Flood J, Murphy R, editors. Cocoa Futures. London: CABI BioSciences/The Commodities Press; 2004. p. 164

[24] Benson DM. In: Erwin DC, Ribeiro OK, editors. *Phytophthora* Diseases Worldwide. St. Paul, MN, USA: APS Press; 1997. p. 592

[25] Judelson HS, Blanco FA. The spores of *Phytophthora* weapons of the plant

destroyer. Nature Reviews Microbilogy. 2005;**3**:47-58

[26] Fry W. *Phytophthora infestans*: The plant (and R gene) destroyer. Molecular Plant Patholology. 2008;**9**:385-402

[27] Latijnhowers M, de Wit PGJM, Govers F. Oomycetes and fungi similar weaponry to attack plants. Trends in Microbiology. 2003;**11**(10):462-469

[28] Govers F. Misclassification of pest as 'fungus' puts vital research on wrong track. Nature (London). 2001;**411**:633

[29] Guerriero G, Avino M, Zhou Q , Fugelstad J, Clergeot P-H, Bulone V l. Chitin synthases from *Saprolegnia* are involved in tip growth and represent a potential target for anti-Oomycete drugs. 2010. DOI: 10.1371/journal. at.1001070

[30] Ashby SF. Strains and taxonomy of *Phytophthora palmivora* Butler *(P. faberi* Maubl.). Transactions of the British Mycological Society. 1929;**14**:18-38

[31] Ali SS, Amoako-Attah I, Bailey RA, Strem MD, Schmidt M, Akrofi AY, et al. PCR-based identification of cacao black pod causal agents and identification of biological factors possibly contributing to *Phytophthora megakarya*'s field dominance in West Africa. Plant Pathology. 2016;**65**:1095-1108. DOI: 10.1111/ppa.12496

[32] Sansome E, Brasier CM, Griffin MJ. Chromosome size differences in *Phytophthora palmivora,* a pathogen of cocoa. Nature. 1975;**255**:704-705

[33] Sansome E, Brasier CM, Sansome FW. Further cytological studies on the L-type and S-type of *Phytophthora* from cocoa. Transactions of the British Mycological Society. 1979;**73**:293-302

[34] Waterhouse GM. Key to the species of *Phytophthora* de Bary. Mycoclogical Papers. 1963;**92**:1-22

**59**

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

[42] Campêlo AMFL, Luz EDMN. Etiologia de podrido-parda du cacueiro, nos Estados da Bahia e Espirito Santo, Brasil. Fitopatologia Brasileira.

[43] Kellam MK, Zentmeyer GA. Isolation of *Phytophthora citrophthora* from cocoa in Brazil. Phytopathology.

[45] Liyanage NIS, Wheeler BEJ.

Pathology. 1989;**38**:627-629

April 2013, Accra; 2013

p. 263

pp. 447-451

Society; 1983

[44] Lozano TZE, Romero CS. Estudio taxanomico de aislamientos de *Phytophthora* patogenos de cacao. Agrociencia. 1984;**56**:176-182

*Phytophthora katsurae* from cocoa. Plant

[46] N'Guessan KF. Major pests and diseases, situations and damage assessment, protocols in Côte d'Ivoire. In: Presentation at Regional Workshop on Integrated Management of Cocoa Pests and Pathogens in Africa 15-18

[47] Dakwa J. Nationwide Black Pod Survey: Joint CRIG/Cocoa Production Division Project. Annual Report 1976/77-1978/79. Ghana: Cocoa Research Institute of Ghana; 1984.

[48] Dakwa J. A serious outbreak of black pod disease in a marginal area of Ghana. In: Proceedings of the 10th International Cocoa Research Conference, 1987, Santo Domingo, Dominican Republic. Lagos, Nigeria: Cocoa Producers' Alliance; 1988.

[49] Erwin DC, Bartnicki-Garcia S, Tsao PH, editors. Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology. St. Paul, MN: American Phytopathological

[50] Opoku IY, Akrofi AY, Appiah AA. The spread of Phytophthora megakarya on cocoa in Ghana. In: Proceedings of

1981;**6**:313-321

1981;**71**:230

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

[35] Oudemans P, Coffey MD. Isozyme comparison within and among worldwide sources of three

morphologically distinct species of *Phytophthora*. Mycological Research.

[36] Kroon LPNM, Brouwer H, de Cock AWAM, Govers F. The genus *Phytophthora* anno 2012. Phytopathology. 2012;**102**:348-364

Botany. 1979;**56**:1730-1738

[38] Zentmeyer GA. Taxonomic relationships and distribution of Phytophthora causing black pod of cocoa. In: Proceedings of the 10th International Cocoa Research Conference, 17th–23rd May, Santo Domingo, Dominican Republic; 1988.

[39] Ali SS, Shao J, Lary DJ, Kronmiller B, Shen D, Strem MD, et al. *Phytophthora megakarya* and *P. palmivora*, closely related causal agents of cacao black pod rot, underwent increases in genome sizes and gene numbers by different mechanisms. Genome, Biology and Evolution. 2017;**9**(3):536-557

[40] Giraldo MC, Valent B. Filamentous plant pathogen effectors in action. Nature Reviews Microbiology.

[41] Despre'aux D, Cambony D, Cle'ment D, Nyasse´ S, and Partiot M. Etude de la pourriture brune des cabosses du cacaoyer au Cameroun: De'finition de nouvelles me'thodes de lutte. In: Cocoa Producers' Alliance, editor. Proceedings of the 10th International Cocoa Research Conference, Cocoa Producers Alliance, 1987. Santo Domingo, Dominican Republic; 1988. pp. 407-412

[37] Kaosiri T, Zentmeyer GA, Erwin DC. Stalk length as a taxonomic criterion for *Phytophthora palmivora* isolates from cacao. Canadian Journal of

1991;**95**:19-30

pp. 391-395

2013;**11**:800-814

*Diversity of Cacao Pathogens and Impact on Yield and Global Production DOI: http://dx.doi.org/10.5772/intechopen.81993*

[35] Oudemans P, Coffey MD. Isozyme comparison within and among worldwide sources of three morphologically distinct species of *Phytophthora*. Mycological Research. 1991;**95**:19-30

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

destroyer. Nature Reviews Microbilogy.

[26] Fry W. *Phytophthora infestans*: The plant (and R gene) destroyer. Molecular Plant Patholology. 2008;**9**:385-402

[27] Latijnhowers M, de Wit PGJM, Govers F. Oomycetes and fungi similar weaponry to attack plants. Trends in Microbiology. 2003;**11**(10):462-469

[28] Govers F. Misclassification of pest as 'fungus' puts vital research on wrong track. Nature (London). 2001;**411**:633

[29] Guerriero G, Avino M, Zhou Q , Fugelstad J, Clergeot P-H, Bulone V l. Chitin synthases from *Saprolegnia* are involved in tip growth and represent a potential target for anti-Oomycete drugs. 2010. DOI: 10.1371/journal.

[30] Ashby SF. Strains and taxonomy of *Phytophthora palmivora* Butler *(P. faberi* Maubl.). Transactions of the British Mycological Society. 1929;**14**:18-38

[31] Ali SS, Amoako-Attah I, Bailey RA, Strem MD, Schmidt M, Akrofi AY, et al. PCR-based identification of cacao black pod causal agents and identification of biological factors possibly contributing to *Phytophthora megakarya*'s field dominance in West Africa. Plant Pathology. 2016;**65**:1095-1108. DOI:

[32] Sansome E, Brasier CM, Griffin MJ.

[33] Sansome E, Brasier CM, Sansome FW. Further cytological studies on the L-type and S-type of *Phytophthora* from cocoa. Transactions of the British Mycological Society. 1979;**73**:293-302

[34] Waterhouse GM. Key to the species of *Phytophthora* de Bary. Mycoclogical

Chromosome size differences in *Phytophthora palmivora,* a pathogen of cocoa. Nature. 1975;**255**:704-705

2005;**3**:47-58

at.1001070

10.1111/ppa.12496

Papers. 1963;**92**:1-22

[17] Opoku IY, Appiah AA, Akrofi AY, Owusu GK. *Phytophthora megakarya*: A potential threat to the cocoa industry in Ghana? Ghana Journal of Agricultural Science. 2000b;**33**(2000):237-248

[18] Dade HA. Economic significance of cocoa pod disease and factors

determining their incidence and control. Bulletin. Gold Coast Department of Agriculture GoldCst. 1927;**6**:I-58

[19] Ndubuaku T, Asogwa E. Strategies for the control of pests and diseases for sustainable cocoa production in Nigeria. African Scientist. 2006;**7**:209-216

[20] Djocgoue P, Boudjeko T, Mbouobda H, Nankeu D, El Hadrami I, Omokolo N. Heritability of phenols in the resistance of Theobroma cacao against Phytophthora megakarya, the causal agent of black pod disease. Journal of Phytopathology. 2007;**155**:519-525. DOI: 10.1111/j.1439-0434.2007. 01268.x

[21] Ali SS, Shao J, Lary DJ, Strem MD, Meinhardt LW, Bailey BA. *Phytophthora megakarya* and *P. palmivora*, causal agents of Black Pod Rot, induce similar plant defense responses late during infection of susceptible Cacao Pods. Frontiers in Plant Science. 2017;**8**:169.

DOI: 10.3389/fpls.2017.00169

[22] Evans HC, Prior C. Cocoa pod diseases: Causal agents and control. Outlook on Agriculture. 1987;**16**:35-41

[23] Flood J, Keane P, Sulistyowati E, Padi B, Guest D, Holmes K. Cocoa under attack. In: Flood J, Murphy R, editors. Cocoa Futures. London: CABI BioSciences/The Commodities Press;

[24] Benson DM. In: Erwin DC, Ribeiro OK, editors. *Phytophthora* Diseases Worldwide. St. Paul, MN, USA: APS

[25] Judelson HS, Blanco FA. The spores of *Phytophthora* weapons of the plant

**58**

2004. p. 164

Press; 1997. p. 592

[36] Kroon LPNM, Brouwer H, de Cock AWAM, Govers F. The genus *Phytophthora* anno 2012. Phytopathology. 2012;**102**:348-364

[37] Kaosiri T, Zentmeyer GA, Erwin DC. Stalk length as a taxonomic criterion for *Phytophthora palmivora* isolates from cacao. Canadian Journal of Botany. 1979;**56**:1730-1738

[38] Zentmeyer GA. Taxonomic relationships and distribution of Phytophthora causing black pod of cocoa. In: Proceedings of the 10th International Cocoa Research Conference, 17th–23rd May, Santo Domingo, Dominican Republic; 1988. pp. 391-395

[39] Ali SS, Shao J, Lary DJ, Kronmiller B, Shen D, Strem MD, et al. *Phytophthora megakarya* and *P. palmivora*, closely related causal agents of cacao black pod rot, underwent increases in genome sizes and gene numbers by different mechanisms. Genome, Biology and Evolution. 2017;**9**(3):536-557

[40] Giraldo MC, Valent B. Filamentous plant pathogen effectors in action. Nature Reviews Microbiology. 2013;**11**:800-814

[41] Despre'aux D, Cambony D, Cle'ment D, Nyasse´ S, and Partiot M. Etude de la pourriture brune des cabosses du cacaoyer au Cameroun: De'finition de nouvelles me'thodes de lutte. In: Cocoa Producers' Alliance, editor. Proceedings of the 10th International Cocoa Research Conference, Cocoa Producers Alliance, 1987. Santo Domingo, Dominican Republic; 1988. pp. 407-412

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[44] Lozano TZE, Romero CS. Estudio taxanomico de aislamientos de *Phytophthora* patogenos de cacao. Agrociencia. 1984;**56**:176-182

[45] Liyanage NIS, Wheeler BEJ. *Phytophthora katsurae* from cocoa. Plant Pathology. 1989;**38**:627-629

[46] N'Guessan KF. Major pests and diseases, situations and damage assessment, protocols in Côte d'Ivoire. In: Presentation at Regional Workshop on Integrated Management of Cocoa Pests and Pathogens in Africa 15-18 April 2013, Accra; 2013

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[49] Erwin DC, Bartnicki-Garcia S, Tsao PH, editors. Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology. St. Paul, MN: American Phytopathological Society; 1983

[50] Opoku IY, Akrofi AY, Appiah AA. The spread of Phytophthora megakarya on cocoa in Ghana. In: Proceedings of

the 1st International Cocoa Pests and Diseases Seminar, Accra, Ghana, 6-10th November, 1995; 1997

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[58] Opoku IY, Akrofi AY, Appiah AA. Assessment of sanitation and fungicide application directed at cocoa tree trunks for the control of *Phytophthora* black pod infections in pods growing in the canopy. European Journal of Plant Pathology. 2007a;**117**:167-175

[59] Appiah AA. Variability in *Phytophthora* species causing black pod diseases of cocoa (*Theobroma cacao* L.) and their implication for assessment of host resistance screening [PhD Thesis]. UK: University of London; 2001. 200pp

[60] McHau GR, Coffey MD. Isozyme diversity in *Phytophthora palmivora*: Evidence for a south East Asian centre of origin. Mycological Research. 1994;**98**:1035-1043. DOI: 10.1016/ S0953-7562(09)80430-9

[61] Brasier CM, Griffin MJ. Taxonomy of *Phytophthora palmivora* of cocoa. Transactions of the British Mycological Society. 1979;**72**:111-143

[62] Brasier CM, Griffin MJ, Ward MR, Idowu OL, Taylor B, Adedoyin SF. Epidemiology of *Phytophthora* on cocoa in Nigeria Final Report of the International Cocoa Black Pod Research Project. *Phytopathological Papers*. Kew, Surrey, England: Commonwealth Mycological Institute; 1981. 188 pp

[63] Guest D. Black pod: Diverse pathogens with a global impact on cocoa yield. Phytopathology. 2007;**97**:1650-1653

[64] Bloomfield EM, Lass RA. Impact of structural adjustment and adoption of technology on competitiveness of major Cocoa producing countries. Working Paper No. 69. OCDE/ GD(92)120; 1992. 23p

[65] Thorold CA. Observations on blackpod disease *(Phytophthora palmivora)* of cacao in Nigeria. Transactions of the British Mycological Society. 1955;**38**(4):435-452

**61**

*Diversity of Cacao Pathogens and Impact on Yield and Global Production*

Memoria. 6. Semana Científica. Turrialba (Costa Rica). 2004:11-12

Science. 2005;**143**:11-25

Tobago, October, 1998; 1999

[76] Awuah RT. *In vivo* use of extracts from *Ocimum gratissimum* and *Cymbopogon citratus* against *Phytophthora palmivora* causing black pod disease of cocoa. Annals of Applied

Biology. 1994;**124**:173-178

2006;**115**:377-338

2003;**69**:7161-7172

2006;**19**:699-710

[77] Widmer TL, Laurent N. Plant extracts containing caffeic acid and rosmarinic acid inhibit zoospore germination of *Phytophthora* s. pathogenic to *Theobroma cacao*. European Journal of Plant Pathology.

[78] de Souza JT, de Boer M, de Waard P, vanBeek TA, Raaijmakers JM. Biochemical, genetic, and zoosporicidal properties of cyclic lipopeptide surfactants produced by *Pseudomonas fluorescens*. Applied and Environmental Microbiology.

[79] Raaijmakers JM, de Bruijn I, de Kock MJD. Cyclic lipopeptide production by plant-associated *Pseudomonas*. Diversity, activity, biosynthesis, and regulation. Molecular Plant-Microbe Interactions.

[80] Raaijmakers JM, de Bruijn I,

Nybroe O, Ongena M. Natural functions of lipopeptides from *Bacillus* and *Pseudomonas* more than surfactants

[74] Russell PE. A century of fungicide evolution. Journal of Agricultural

[75] Shripat C. Control of black pod disease of cocoa by single application of a copper fungicide: A preliminary report. Empowering farmers through agricultural research, Volumes 3 and 4. In: Proceedings of Research Division, Ministry of Agriculture, Land and Marine Resources Research Seminar Series held at Centeno, Trinidad and

*DOI: http://dx.doi.org/10.5772/intechopen.81993*

[66] Ndoumbe-Nkeng M, Cilas C, Nyemb E, Nyasse S, Bieysse D, Flori A, et al. Impact of removing diseased pods on cocoa black pod caused by *Phytophthora* megakarya and on cocoa production in Cameroon. Crop

Protection. 2004;**23**:415-424

2007b;**2**:601-604

pp. 303-319

Bulletin. 1973;**20**:10-16

Bulletin. 1984;**35**:5-22

London; 1994*.* 369pp

2003;**109**:953-961

epidemiology and control of *Phytophthora megakarya* on cocoa in West Africa [PhD Thesis]. University of

[67] Opoku IY, Assuah MK, Aneani F. Management of black pod disease of cocoa with reduced number of fungicide application and crop sanitation. African Journal of Agricultural Research.

[68] Mpika J, Kebe IB, N'Guessan KF. Isolation and identification of indigenous microorganisms of cocoa farms in Côte d'Ivoire and assessment of their antagonistic effects vis-à-vis *Phytophthora palmivora,* the causal agent of black pod disease. In: Grillo O, editor. Biodiversity Loss in a Changing Planet. Vol. 2011. Rijeka, Croatia: Intech; 2011.

[69] Evans HC. New developments in black pod epidemiology. Cocoa Growers'

[70] Gregory PH, Griffin MJ, Madddison AC, Ward MR. Cocoa black pod: A reinterpretation. Cocoa Growers'

[71] Luterbacher MC. The identification,

[72] Hidalgo E, Bateman R, Krauss U, ten Hoopen M, Martínez A. A field investigation into delivery systems for agents to control *Moniliophthora roreri*. European Journal of Plant Pathology.

[73] Bateman R, Hidalgo E, García J, Hoopen GT, Krauss U, Adonijah V, et al. Rational fungicide use in cocoa: Improving agents and application techniques. Semana Científica 2004.

*Diversity of Cacao Pathogens and Impact on Yield and Global Production DOI: http://dx.doi.org/10.5772/intechopen.81993*

[66] Ndoumbe-Nkeng M, Cilas C, Nyemb E, Nyasse S, Bieysse D, Flori A, et al. Impact of removing diseased pods on cocoa black pod caused by *Phytophthora* megakarya and on cocoa production in Cameroon. Crop Protection. 2004;**23**:415-424

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

[58] Opoku IY, Akrofi AY, Appiah AA. Assessment of sanitation and fungicide application directed at cocoa tree trunks for the control of *Phytophthora* black pod infections in pods growing in the canopy. European Journal of Plant Pathology. 2007a;**117**:167-175

[59] Appiah AA. Variability in

*Phytophthora* species causing black pod diseases of cocoa (*Theobroma cacao* L.) and their implication for assessment of host resistance screening [PhD Thesis]. UK: University of London; 2001. 200pp

[60] McHau GR, Coffey MD. Isozyme diversity in *Phytophthora palmivora*: Evidence for a south East Asian centre of origin. Mycological Research. 1994;**98**:1035-1043. DOI: 10.1016/

[61] Brasier CM, Griffin MJ. Taxonomy of *Phytophthora palmivora* of cocoa. Transactions of the British Mycological

[62] Brasier CM, Griffin MJ, Ward MR, Idowu OL, Taylor B, Adedoyin SF. Epidemiology of *Phytophthora* on cocoa in Nigeria Final Report of the International Cocoa Black Pod Research Project. *Phytopathological Papers*. Kew, Surrey, England: Commonwealth Mycological Institute; 1981. 188 pp

[63] Guest D. Black pod: Diverse pathogens with a global impact on cocoa yield. Phytopathology.

[64] Bloomfield EM, Lass RA. Impact of structural adjustment and adoption of technology on competitiveness of major Cocoa producing countries. Working Paper No. 69. OCDE/

[65] Thorold CA. Observations on blackpod disease *(Phytophthora palmivora)* of cacao in Nigeria. Transactions of the British Mycological Society.

2007;**97**:1650-1653

GD(92)120; 1992. 23p

1955;**38**(4):435-452

S0953-7562(09)80430-9

Society. 1979;**72**:111-143

the 1st International Cocoa Pests and Diseases Seminar, Accra, Ghana, 6-10th

[51] Akrofi AY, Appiah AA, Opoku IY. Management of *Phytophthora* pod rot disease on cocoa farms in Ghana. Crop

[52] End MJ, Daymond AJ, Hadley P, editors. Technical guidelines for the safe movement of cacao germplasm (Revised from the FAO/IPGRI Technical Guidelines No. 20). Global Cacao Genetic Resources Network (CacaoNet), Biodiversity International, Montpellier,

[53] Akrofi AY, Opoku IY, Appiah AA. On farm farmer-managed trials to control black pod disease caused by Phytophthora megakarya in Ghana. In: Proceedings First International Cocoa Pests and Diseases Seminar, Accra, Ghana, 6-10 November, 1995; 1997

[54] Maddison AC, Idowu OL. The epidemic on sprayed cocoa at Owena. In: Gregory PH, Maddison AC, editors. Epidemiology of *Phytophthora* on cocoa in Nigeria. Phytopathological Paper No. 25. Kew, UK: Commonwealth Mycological Institute; 1981. pp. 163-172

[55] Djiekpor EK, Partiot M, Lucas P. The cacao black pod disease due to *Phytophthora* sp in Togo -Determination of species responsible. Cafe Cacao Thé.

[56] Tondje PR, Berry D, Bakala J, Ebandan S. Interêt de diverses pratiques

culturales dans la lutte contre la pourriture brune des cabosses dûe à Phytophthora sp au Cameroun. 11e Conference. Internationalesur la recherche cacaoyère. Yamoussoukro-Côte d'Ivoire, 18-24 Juillet 1993; 1993.

[57] Wood GAR, Lass RA. Cocoa. In: Tropical Agricultural Series. 4th ed. UK: Longman Group Ltd; 1985. p. 620

1982;**26**(2):97-108

November, 1995; 1997

France; 2010

Protection. 2003;**22**:469-477

**60**

pp. 175-183

[67] Opoku IY, Assuah MK, Aneani F. Management of black pod disease of cocoa with reduced number of fungicide application and crop sanitation. African Journal of Agricultural Research. 2007b;**2**:601-604

[68] Mpika J, Kebe IB, N'Guessan KF. Isolation and identification of indigenous microorganisms of cocoa farms in Côte d'Ivoire and assessment of their antagonistic effects vis-à-vis *Phytophthora palmivora,* the causal agent of black pod disease. In: Grillo O, editor. Biodiversity Loss in a Changing Planet. Vol. 2011. Rijeka, Croatia: Intech; 2011. pp. 303-319

[69] Evans HC. New developments in black pod epidemiology. Cocoa Growers' Bulletin. 1973;**20**:10-16

[70] Gregory PH, Griffin MJ, Madddison AC, Ward MR. Cocoa black pod: A reinterpretation. Cocoa Growers' Bulletin. 1984;**35**:5-22

[71] Luterbacher MC. The identification, epidemiology and control of *Phytophthora megakarya* on cocoa in West Africa [PhD Thesis]. University of London; 1994*.* 369pp

[72] Hidalgo E, Bateman R, Krauss U, ten Hoopen M, Martínez A. A field investigation into delivery systems for agents to control *Moniliophthora roreri*. European Journal of Plant Pathology. 2003;**109**:953-961

[73] Bateman R, Hidalgo E, García J, Hoopen GT, Krauss U, Adonijah V, et al. Rational fungicide use in cocoa: Improving agents and application techniques. Semana Científica 2004.

Memoria. 6. Semana Científica. Turrialba (Costa Rica). 2004:11-12

[74] Russell PE. A century of fungicide evolution. Journal of Agricultural Science. 2005;**143**:11-25

[75] Shripat C. Control of black pod disease of cocoa by single application of a copper fungicide: A preliminary report. Empowering farmers through agricultural research, Volumes 3 and 4. In: Proceedings of Research Division, Ministry of Agriculture, Land and Marine Resources Research Seminar Series held at Centeno, Trinidad and Tobago, October, 1998; 1999

[76] Awuah RT. *In vivo* use of extracts from *Ocimum gratissimum* and *Cymbopogon citratus* against *Phytophthora palmivora* causing black pod disease of cocoa. Annals of Applied Biology. 1994;**124**:173-178

[77] Widmer TL, Laurent N. Plant extracts containing caffeic acid and rosmarinic acid inhibit zoospore germination of *Phytophthora* s. pathogenic to *Theobroma cacao*. European Journal of Plant Pathology. 2006;**115**:377-338

[78] de Souza JT, de Boer M, de Waard P, vanBeek TA, Raaijmakers JM. Biochemical, genetic, and zoosporicidal properties of cyclic lipopeptide surfactants produced by *Pseudomonas fluorescens*. Applied and Environmental Microbiology. 2003;**69**:7161-7172

[79] Raaijmakers JM, de Bruijn I, de Kock MJD. Cyclic lipopeptide production by plant-associated *Pseudomonas*. Diversity, activity, biosynthesis, and regulation. Molecular Plant-Microbe Interactions. 2006;**19**:699-710

[80] Raaijmakers JM, de Bruijn I, Nybroe O, Ongena M. Natural functions of lipopeptides from *Bacillus* and *Pseudomonas* more than surfactants

and antibiotics. FEMS Microbiology Reviews. 2010;**34**(6):1037-1062

[81] Tran HT, Ficke A, Asiimwe T, Hofte M, Raaijmakers JM. Role of the cyclic lipopeptide massetolide A in biological control of *Phytophthora infestans* and in colonization of tomato plants by *Pseudomonas fluorescens*. New Phytologist. 2007;**175**:731-742

[82] de Bruijn I, Kock MJD, Raaijmakers JM. Comparative genomics and regulation of cyclic lipopeptide synthesis in antagonistic *Pseudomonas fluorescens*. Bulletin OILB/SROP. 2007;**30**(6):113

[83] van de Mortel JE, Tran H, Govers F, Raaijmakers JM. Cellular responses of the late blight pathogen *Phytophthora infestans* to cyclic lipopeptide surfactants and their dependence on G proteins. Applied and Environmental Microbiology. 2000;**75**:4950-4957

[84] Tran HT, Raaijmakers JM. Frequency, diversity and biocontrol activity of surfactant-producing *Pseudomonas* species in Vietnam. Bulletin OILB/SROP. 2007;**30**(6):369

**63**

Section 3

Cocoa Value Added

and By-Products for

Consumption

Section 3

Cocoa Value Added and By-Products for Consumption

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

and antibiotics. FEMS Microbiology Reviews. 2010;**34**(6):1037-1062

[81] Tran HT, Ficke A, Asiimwe T, Hofte M, Raaijmakers JM. Role of the cyclic lipopeptide massetolide A in biological control of *Phytophthora infestans* and in colonization of tomato plants by *Pseudomonas fluorescens*. New

Phytologist. 2007;**175**:731-742

JM. Comparative genomics and regulation of cyclic lipopeptide synthesis in antagonistic *Pseudomonas fluorescens*. Bulletin OILB/SROP.

*infestans* to cyclic lipopeptide

[84] Tran HT, Raaijmakers JM. Frequency, diversity and biocontrol activity of surfactant-producing *Pseudomonas* species in Vietnam. Bulletin OILB/SROP. 2007;**30**(6):369

2007;**30**(6):113

[82] de Bruijn I, Kock MJD, Raaijmakers

[83] van de Mortel JE, Tran H, Govers F, Raaijmakers JM. Cellular responses of the late blight pathogen *Phytophthora* 

surfactants and their dependence on G proteins. Applied and Environmental Microbiology. 2000;**75**:4950-4957

**62**

**65**

**Chapter 4**

**Abstract**

lining erosion.

**1. Introduction**

prevention of certain diseases.

Perspective

*and Isaac J. Asiedu-Gyekye*

Unsweetened Natural Cocoa

*Kwame Benoit N'guessan Banga, Seth K. Amponsah* 

*Lovia Allotey-Babington, Awo Afi Kwapong,* 

Powder: A Potent Nutraceutical in

Unsweetened natural cocoa powder is a pulverized high-grade powder of compressed solid blocks which remains after extraction and removal of the cocoa butter. The authors determined the elementary composition of UNCP, investigated its effect on nitric oxide levels, toxicity, and its protective effect on the heart, kidney, and liver during simultaneous administration with high dose (HD) artemether/ lumefantrine (A/L). Macro- and microelements in UNCP were analyzed with energy dispersive x-ray fluorescence spectroscopy (EDXRF). Adult male guinea pigs were administered various doses of UNCP alone and also simultaneously with A/L. Phytochemical analysis of UNCP showed the presence of saponins, flavonoids, tannins, cardiac glycosides, and 38 macro- and microelements. Histopathological analysis showed no toxic effect on the heart, liver, kidney, lungs, testis, and spleen. Administration of various doses of UNCP increased white blood cell counts and lymphocyte count (*p* > 0.05) compared with the controls. Additionally, UNCP and A/L combination caused an increase in nitric oxide levels when compared with the control group and restores some hematological disorders induced by the 3-day HD A/L administration. Even though UNCP appears to be relatively safe, care should be taken due to the high content of copper element to avoid the possibility of intestinal

**Keywords:** unsweetened cocoa powder, artemether-lumefantrine, malaria,

Nutraceuticals, a word coined by DeFelice [1], can be described as any nontoxic

Natural products have been a good source of drug leads from time immemorial for as long as history has been recorded. The most common among these natural products are plants, which have been exploited for their medicinal purposes [2–4]. Interestingly, many consider plant products to be devoid of adverse effects; thus, the demand for herbal remedies has been on the increase in industrialized

food extract scientifically proven to have health benefits for the treatment and

*Theobroma cacao*, nutraceutical, dark chocolate

## **Chapter 4**

## Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective

*Lovia Allotey-Babington, Awo Afi Kwapong, Kwame Benoit N'guessan Banga, Seth K. Amponsah and Isaac J. Asiedu-Gyekye*

## **Abstract**

Unsweetened natural cocoa powder is a pulverized high-grade powder of compressed solid blocks which remains after extraction and removal of the cocoa butter. The authors determined the elementary composition of UNCP, investigated its effect on nitric oxide levels, toxicity, and its protective effect on the heart, kidney, and liver during simultaneous administration with high dose (HD) artemether/ lumefantrine (A/L). Macro- and microelements in UNCP were analyzed with energy dispersive x-ray fluorescence spectroscopy (EDXRF). Adult male guinea pigs were administered various doses of UNCP alone and also simultaneously with A/L. Phytochemical analysis of UNCP showed the presence of saponins, flavonoids, tannins, cardiac glycosides, and 38 macro- and microelements. Histopathological analysis showed no toxic effect on the heart, liver, kidney, lungs, testis, and spleen. Administration of various doses of UNCP increased white blood cell counts and lymphocyte count (*p* > 0.05) compared with the controls. Additionally, UNCP and A/L combination caused an increase in nitric oxide levels when compared with the control group and restores some hematological disorders induced by the 3-day HD A/L administration. Even though UNCP appears to be relatively safe, care should be taken due to the high content of copper element to avoid the possibility of intestinal lining erosion.

**Keywords:** unsweetened cocoa powder, artemether-lumefantrine, malaria, *Theobroma cacao*, nutraceutical, dark chocolate

## **1. Introduction**

Nutraceuticals, a word coined by DeFelice [1], can be described as any nontoxic food extract scientifically proven to have health benefits for the treatment and prevention of certain diseases.

Natural products have been a good source of drug leads from time immemorial for as long as history has been recorded. The most common among these natural products are plants, which have been exploited for their medicinal purposes [2–4]. Interestingly, many consider plant products to be devoid of adverse effects; thus, the demand for herbal remedies has been on the increase in industrialized

countries as it has been with developing countries [5]. Currently, extensive research is being carried out on natural products including plants from the rain forests, among other places for their potential medicinal value as well as potential toxic effect [6].

One such plant that has generated a lot of interest is *Theobroma cacao,* belonging to the class *Equisetopsida* and family *Malvaceae*. For some decades now, Ghana and Cote D'voire, two neighboring countries in West Africa, have been the world's leading producers of cocoa. Seeds from the fruit of *Theobroma cacao* are used to make cocoa powder and chocolate. Cocoa seeds (or the powder) are major nutraceuticals in Ghana and most parts of Africa. As food, it is consumed by most indigenous people in the raw state (the bean or the pod), as dark chocolate, and as a beverage prepared with powder obtained from cocoa beans. The cocoa powder is prepared by the removal of cocoa butter from the beans by fermentation, which is then followed by the following processes: drying and bagging, winnowing, roasting, grinding, and pressing. After the final process, solid blocks of compressed cocoa are obtained (press cake), which are pulverized into a powder to produce a high-grade cocoa powder. Most of the marketed natural cocoa powder in Ghana is sweetened, even though it has been proven that regular intake of unsweetened natural cocoa powder as a beverage has immense health benefits. Pharmacologically, cocoa is known to exert antioxidant [7], anti-inflammatory [8], antimalarial [2, 3], and anti-asthmatic properties [8, 9].

### **1.1 Composition of unsweetened cocoa powder**

The chemical composition of cocoa powder is well documented. Cocoa contains water-soluble polyphenols (also called flavanols), which include catechins, epicatechin, procyanidins, anthocyanins, and leukoanthocyanins [10]. The antioxidant properties of cocoa are partly ascribed to its structural characteristics, and this is the basis of its role as a free-radical scavenger [10]. Quantitative analysis of unsweetened natural cocoa powder (UNCP) has shown that flavanol contents do not change during different manufacturing processes [11, 12]. Several polyphenols such as 14 N-phenylpropenoyl-L-amino acids, N-[4′-hydroxy-(E)-cinnamoyl]-Ltryptophan, and N-[4′-hydroxy-3′-methoxy- (E)-cinnamoyl]-L-tyrosine have also been found to be present in cocoa powder [13–15].

### *1.1.1 Phytochemical analysis*

A study conducted to ascertain the phytochemical components of unsweetened cocoa powder in Ghana used various pharmacopoeias tests to confirm the presence of the various components. To test for saponins, about 0.5 g of the UNCP was added to water in a test tube and shaken to observe foam formation. To test for the presence of tannins, about 0.5 g of UNCP was dissolved in 80% of aqueous methanol (10 mL). Freshly prepared iron III chloride solution was added and observed for a color change. The presence of alkaloids was confirmed by performing the Dragendorff 's, Mayer's, and Wagner's reagent tests as previously described [16].

To identify flavonoids in the UNCP, about 0.1 g of the sample was added to 80% ethanol and filtered. Subsequently, magnesium turnings were added to the filtrate, followed by concentrated HCl. A color change was observed within 10 min. A test for cardiac glycosides was conducted by dissolving about 0.5 g of UNCP in chloroform (2 mL) in a test tube, after which concentrated sulfuric acid was carefully added down the side of the test tube to form a lower layer. This result obtained

**67**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

confirmed work by Oracz et al. [11], Andres-Lacueva et al. [12], and Stark et al. [17] that the flavanol contents of cocoa does not change during different manufacturing

Besides the phytochemical components, micro- and macro elements have been shown to be present in most plant products. The most toxic of these are the heavy metals. These have the potential to interfere with the availability of secondary metabolites [18]. For the safety of consumers, the World Health Organization states maximum permissible levels of heavy metals in raw plant

To establish the elemental components of marketed UNCP, energy dispersive X-ray fluorescence method was used. Briefly, samples of the powder were well dried and pelleted using Licowax C micropowder PM-Hoechstwax as binder. Simultaneous measurements of the elemental components were measured with a SPECTRO X-Lab 2000 spectrometer (Geological Survey Department, Accra, Ghana) as previously described [20]. The results revealed a total of 38 elements, of

Rarely are toxicological studies conducted on nutraceuticals. This could be because such products are generally termed "natural" and perceived to be devoid of any adverse effects. Interestingly, cardiotoxic and teratogenic potentials have been reported for cocoa [21, 22]. Against this background, a study was conducted to elucidate the safety of cocoa powder in Sprague Dawley (SD) rats. The experimental procedure for this study was approved by the departmental ethical and protocol review committee and the Noguchi Memorial Institute for Medical Research, University of Ghana. Institutional Animal Care and Use Committee also approved the protocol for the study. The study was conducted in accordance with interna-

The design of the experiment was such that the rats were randomly assigned to either the experimental group or the control group. There were 20 rats in all, 10 in each group. All rats had access to water and food except for a 12-hour fasting period before the administration of the unsweetened natural cocoa powder. The experimental group of rats received UNCP at a dose of 2000 mg/kg, while the control group received an equal volume of distilled water only. After the study period, effects of UNCP on selected organs, as well as on biochemical indices of blood, are

The effect of UNCP on the body and relative weight of organs was determined as described previously [20]. Selected organs, the lungs, heart, liver, spleen, kidney, intestines, and testis, were excised. The organs were then placed in ice-cold saline to wash off blood. They were trimmed of fat, connective tissues, blot dried, and then weighed on a balance. The organ-to-body weight index, OBI, was determined as the ratio of organ weight and the body weight of the animal before sacrifice × 100. Body weight of rats was also taken dosing, a week after dosing on day 7 and before

*1.2.1 The effect of UNCP on the body and relative weight of organs*

sacrificing them on day 14 (**Tables 2** and **3**).

), arsenic (1 mg kg<sup>−</sup><sup>1</sup>

), and lead

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

*1.1.2 Micro- and macro-elemental composition*

materials for only cadmium (0.3 mg kg<sup>−</sup><sup>1</sup>

which 12 were macro, and are listed in **Table 1**.

**1.2 Toxicity of unsweetened cocoa powder**

)[19].

tional ethical guidelines.

described below.

processes.

(10 mg kg<sup>−</sup><sup>1</sup>

confirmed work by Oracz et al. [11], Andres-Lacueva et al. [12], and Stark et al. [17] that the flavanol contents of cocoa does not change during different manufacturing processes.

## *1.1.2 Micro- and macro-elemental composition*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

toxic effect [6].

properties [8, 9].

**1.1 Composition of unsweetened cocoa powder**

been found to be present in cocoa powder [13–15].

*1.1.1 Phytochemical analysis*

described [16].

countries as it has been with developing countries [5]. Currently, extensive research is being carried out on natural products including plants from the rain forests, among other places for their potential medicinal value as well as potential

One such plant that has generated a lot of interest is *Theobroma cacao,* belonging to the class *Equisetopsida* and family *Malvaceae*. For some decades now, Ghana and Cote D'voire, two neighboring countries in West Africa, have been the world's leading producers of cocoa. Seeds from the fruit of *Theobroma cacao* are used to make cocoa powder and chocolate. Cocoa seeds (or the powder) are major nutraceuticals in Ghana and most parts of Africa. As food, it is consumed by most indigenous people in the raw state (the bean or the pod), as dark chocolate, and as a beverage prepared with powder obtained from cocoa beans. The cocoa powder is prepared by the removal of cocoa butter from the beans by fermentation, which is then followed by the following processes: drying and bagging, winnowing, roasting, grinding, and pressing. After the final process, solid blocks of compressed cocoa are obtained (press cake), which are pulverized into a powder to produce a high-grade cocoa powder. Most of the marketed natural cocoa powder in Ghana is sweetened, even though it has been proven that regular intake of unsweetened natural cocoa powder as a beverage has immense health benefits. Pharmacologically, cocoa is known to exert antioxidant [7], anti-inflammatory [8], antimalarial [2, 3], and anti-asthmatic

The chemical composition of cocoa powder is well documented. Cocoa contains water-soluble polyphenols (also called flavanols), which include catechins, epicatechin, procyanidins, anthocyanins, and leukoanthocyanins [10]. The antioxidant properties of cocoa are partly ascribed to its structural characteristics, and this is the basis of its role as a free-radical scavenger [10]. Quantitative analysis of unsweetened natural cocoa powder (UNCP) has shown that flavanol contents do not change during different manufacturing processes [11, 12]. Several polyphenols such as 14 N-phenylpropenoyl-L-amino acids, N-[4′-hydroxy-(E)-cinnamoyl]-Ltryptophan, and N-[4′-hydroxy-3′-methoxy- (E)-cinnamoyl]-L-tyrosine have also

A study conducted to ascertain the phytochemical components of unsweetened cocoa powder in Ghana used various pharmacopoeias tests to confirm the presence of the various components. To test for saponins, about 0.5 g of the UNCP was added to water in a test tube and shaken to observe foam formation. To test for the presence of tannins, about 0.5 g of UNCP was dissolved in 80% of aqueous methanol (10 mL). Freshly prepared iron III chloride solution was added and observed for a color change. The presence of alkaloids was confirmed by performing the Dragendorff 's, Mayer's, and Wagner's reagent tests as previously

To identify flavonoids in the UNCP, about 0.1 g of the sample was added to 80% ethanol and filtered. Subsequently, magnesium turnings were added to the filtrate, followed by concentrated HCl. A color change was observed within 10 min. A test for cardiac glycosides was conducted by dissolving about 0.5 g of UNCP in chloroform (2 mL) in a test tube, after which concentrated sulfuric acid was carefully added down the side of the test tube to form a lower layer. This result obtained

**66**

Besides the phytochemical components, micro- and macro elements have been shown to be present in most plant products. The most toxic of these are the heavy metals. These have the potential to interfere with the availability of secondary metabolites [18]. For the safety of consumers, the World Health Organization states maximum permissible levels of heavy metals in raw plant materials for only cadmium (0.3 mg kg<sup>−</sup><sup>1</sup> ), arsenic (1 mg kg<sup>−</sup><sup>1</sup> ), and lead (10 mg kg<sup>−</sup><sup>1</sup> )[19].

To establish the elemental components of marketed UNCP, energy dispersive X-ray fluorescence method was used. Briefly, samples of the powder were well dried and pelleted using Licowax C micropowder PM-Hoechstwax as binder. Simultaneous measurements of the elemental components were measured with a SPECTRO X-Lab 2000 spectrometer (Geological Survey Department, Accra, Ghana) as previously described [20]. The results revealed a total of 38 elements, of which 12 were macro, and are listed in **Table 1**.

## **1.2 Toxicity of unsweetened cocoa powder**

Rarely are toxicological studies conducted on nutraceuticals. This could be because such products are generally termed "natural" and perceived to be devoid of any adverse effects. Interestingly, cardiotoxic and teratogenic potentials have been reported for cocoa [21, 22]. Against this background, a study was conducted to elucidate the safety of cocoa powder in Sprague Dawley (SD) rats. The experimental procedure for this study was approved by the departmental ethical and protocol review committee and the Noguchi Memorial Institute for Medical Research, University of Ghana. Institutional Animal Care and Use Committee also approved the protocol for the study. The study was conducted in accordance with international ethical guidelines.

The design of the experiment was such that the rats were randomly assigned to either the experimental group or the control group. There were 20 rats in all, 10 in each group. All rats had access to water and food except for a 12-hour fasting period before the administration of the unsweetened natural cocoa powder. The experimental group of rats received UNCP at a dose of 2000 mg/kg, while the control group received an equal volume of distilled water only. After the study period, effects of UNCP on selected organs, as well as on biochemical indices of blood, are described below.

## *1.2.1 The effect of UNCP on the body and relative weight of organs*

The effect of UNCP on the body and relative weight of organs was determined as described previously [20]. Selected organs, the lungs, heart, liver, spleen, kidney, intestines, and testis, were excised. The organs were then placed in ice-cold saline to wash off blood. They were trimmed of fat, connective tissues, blot dried, and then weighed on a balance. The organ-to-body weight index, OBI, was determined as the ratio of organ weight and the body weight of the animal before sacrifice × 100. Body weight of rats was also taken dosing, a week after dosing on day 7 and before sacrificing them on day 14 (**Tables 2** and **3**).


#### **Table 1.**

*Mean and standard deviation (SD) of measured elements (mg/4000 mg).*

#### *1.2.2 Histopathology examination*

The histopathology examination was performed as previously reported by Asiedu-Gyekye et al. [20] The lungs, heart, liver, spleen, small intestinal organs, and testis were placed in a 10% buffered formaldehyde solution for 24 h. Tissue samples from the organs were paraffin embedded and sectioned at 5 m thickness. The sectioned tissues were stained with hematoxylin and eosin (H&E) and evaluated under a light microscope (Olympus BX 51TF) for histological changes.

#### *1.2.3 The effect of UNCP on hematological parameters*

The effect of UNCP on hematological parameters was studied as described previously by Asiedu-Gyekye et al. [20]. Blood (2 mL) from euthanized SD rats was drawn out by cardiac puncture. This was then transferred into ethylene diamine tetra acetic acid (EDTA) test tubes. An automated hematology analyzer was used for the evaluation. Peripheral blood smear was used to examine the nature of blood cells.

**69**

weighs 60.70 kg.

**Table 2.**

**Table 3.**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

*1.2.4 The effect of UNCP on serum biochemistry*

should not be envisaged always as contaminants.

The effect of UNCP on serum biochemistry was studied as described previously

Lead and arsenic limits permissible by the WHO are 0.00016 and 0.0010 mg/ kg, respectively [23]. Heavy metals determined in UNCP were Pb—0.0036 mg and As—0.002 mg corresponding to 0.0002 and 0.0001 mg/kg, respectively, which are far below WHO guidelines. There was a high content of copper (0.2984 mg per 4 g UNCP) observed which should be of concern especially when high doses of UNCP are consumed. This is due to the fact that copper has been shown to play a role in the pathogenesis of Wilson's syndrome and liver damage [16, 24], while the high content of chromium could have beneficial effect in the management of diabetes mellitus and cardiovascular disorders [18, 25, 26]. Thus, the relationship between these elements, nutrition, and medicine observed is indicative of the fact that micro- and macro-elements of herbal products does influence certain body processes and hence

Body weight changes between the control group and the experimental group were observed on day 1, day 7, and day 14. However, with respect to dosing, they were found not to be statistically significant ( > 0.05). Body weight of the SD rats decreased by 8.3 and 22.2% ( < 0.05) in the 2nd week after dosing for the control and test groups, respectively. Although the decrease in body weight with the control animals and the test animals is consistent with the corresponding decrease in their food and water intake, that for the test animals might partly be due to the ability of UNCP to react with nutrients in the body including stored fat, carbohydrate, and protein.

by Asiedu-Gyekye et al. [20]. Blood (1 mL) of sacrificed rats was collected via cardiac puncture. The blood sample was allowed to stand for a while, this was then centrifuged at 4000 rpm for 15 minutes using a Wiperfuge centrifuge. The serum was then collected for the measurement of the biochemical parameters (**Table 4**). Herbal medicines are thought to have no side effects or the potential to cause harm due to their natural origins and are often considered as healthy food supplements and not drugs. Additionally, most herbs used for medicinal purposes lack specific instructions concerning dose, frequency, and route of administration. Evaluating UNCP as nutraceutical, the assumption was that the average African

*Changes in organ weight of SD rats after administration of unsweetened natural cocoa powder solution.*

*Changes in body weight of SD rats after administration of unsweetened natural cocoa powder solution.*

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*


#### **Table 2.**

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

**68**

**Table 1.**

*1.2.2 Histopathology examination*

*1.2.3 The effect of UNCP on hematological parameters*

*Mean and standard deviation (SD) of measured elements (mg/4000 mg).*

The histopathology examination was performed as previously reported by Asiedu-Gyekye et al. [20] The lungs, heart, liver, spleen, small intestinal organs, and testis were placed in a 10% buffered formaldehyde solution for 24 h. Tissue samples from the organs were paraffin embedded and sectioned at 5 m thickness. The sectioned tissues were stained with hematoxylin and eosin (H&E) and evaluated under a light microscope (Olympus BX 51TF) for histological changes.

The effect of UNCP on hematological parameters was studied as described previously by Asiedu-Gyekye et al. [20]. Blood (2 mL) from euthanized SD rats was drawn out by cardiac puncture. This was then transferred into ethylene diamine tetra acetic acid (EDTA) test tubes. An automated hematology analyzer was used for the evaluation. Peripheral blood smear was used to examine the nature of blood cells.

*Changes in body weight of SD rats after administration of unsweetened natural cocoa powder solution.*


#### **Table 3.**

*Changes in organ weight of SD rats after administration of unsweetened natural cocoa powder solution.*

#### *1.2.4 The effect of UNCP on serum biochemistry*

The effect of UNCP on serum biochemistry was studied as described previously by Asiedu-Gyekye et al. [20]. Blood (1 mL) of sacrificed rats was collected via cardiac puncture. The blood sample was allowed to stand for a while, this was then centrifuged at 4000 rpm for 15 minutes using a Wiperfuge centrifuge. The serum was then collected for the measurement of the biochemical parameters (**Table 4**).

Herbal medicines are thought to have no side effects or the potential to cause harm due to their natural origins and are often considered as healthy food supplements and not drugs. Additionally, most herbs used for medicinal purposes lack specific instructions concerning dose, frequency, and route of administration. Evaluating UNCP as nutraceutical, the assumption was that the average African weighs 60.70 kg.

Lead and arsenic limits permissible by the WHO are 0.00016 and 0.0010 mg/ kg, respectively [23]. Heavy metals determined in UNCP were Pb—0.0036 mg and As—0.002 mg corresponding to 0.0002 and 0.0001 mg/kg, respectively, which are far below WHO guidelines. There was a high content of copper (0.2984 mg per 4 g UNCP) observed which should be of concern especially when high doses of UNCP are consumed. This is due to the fact that copper has been shown to play a role in the pathogenesis of Wilson's syndrome and liver damage [16, 24], while the high content of chromium could have beneficial effect in the management of diabetes mellitus and cardiovascular disorders [18, 25, 26]. Thus, the relationship between these elements, nutrition, and medicine observed is indicative of the fact that micro- and macro-elements of herbal products does influence certain body processes and hence should not be envisaged always as contaminants.

Body weight changes between the control group and the experimental group were observed on day 1, day 7, and day 14. However, with respect to dosing, they were found not to be statistically significant ( > 0.05). Body weight of the SD rats decreased by 8.3 and 22.2% ( < 0.05) in the 2nd week after dosing for the control and test groups, respectively. Although the decrease in body weight with the control animals and the test animals is consistent with the corresponding decrease in their food and water intake, that for the test animals might partly be due to the ability of UNCP to react with nutrients in the body including stored fat, carbohydrate, and protein.


#### **Table 4.**

*Changes in serum biochemistry of SD rats after administration of unsweetened natural cocoa powder solution.*

A reduction of 10.90% ( > 0.05) in the organ weight was observed in the test group as compared to the control group. These variations in the relative organ weight of the control and experimental groups of SD rats were not significantly different.

High-density lipoproteins (HLD) increased slightly with a value <0.05, a decrease in the level of triglycerides and low density lipoprotein (LDL) cholesterol of the UNCP group ( > 0.05) was observed. In comparison with that of the control, the cholesterol levels remained relatively unchanged. This is consistent with the speculation that UNCP possess lipid lowering abilities.

Hematological results (**Table 5**) revealed a decrease (28.44%, > 0.05) in the level of platelet in the UNCP group in comparison with the controls. Polyphenols in cocoa have been found to reduce platelet count. Neutrophil and lymphocyte polymorph of white blood cells showed a slight decrease of 8.02 and 18.73%, respectively ( > 0.05).

Histopathology evaluation of the organs studied; liver, kidney, heart, lungs, spleen, and the testis of the animals that received UNCP showed no toxic effect as compared to that of the control group. UNCP solution therefore is not likely to have toxic effects on the kidney when administered in a single oral high dose of 2000 mg/kg. Notable changes were observed on the small intestines in the form of erosions of the mucosal lining of the villi (**Figure 1**). These effects were, however,

**71**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

not observed in the control animals those that received equivalent volumes of the vehicle (distilled water). This could be due to the high concentration of proanthocyanidins contained in the 2000 mg/kg dose of UNCP (approximately 2.5 g in man). Proanthocyanidins have been found to instigate the destruction of the mucosal

*Changes in hematological indices in rats after receiving 2000 mg/kg bwt of UNCP.*

In conclusion, the aqueous solution of unsweetened natural cocoa powder administered at the single oral high dose of 2000 mg/kg appears to be relatively safe in male SD rats. Caution should however be taken when using UNCP especially in high quantities or amounts since it is capable of causing considerable damage to the

Since UNCP is widely consumed by individuals in their reproductive period of life, it is worth investigating the generative toxicity, genotoxic, and aspects of the reproductive toxicity potential of UNCP in both male and female white wistar rats. As a preliminary study, the genotoxic potential of UNCP was assessed using the DNA comet method. DNA breaks in the rectal epithelium, liver, bone marrow, and kidney of the mice were quantified and compared with the negative control (2% starch). Pre-implantation loss, viable fetuses, corpora lutea and post-implantation

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

lining of the gastrointestinal tract.

mucosal lining of the small intestines.

*1.2.6 Reproductive toxicity*

*1.2.5 Conclusion*

**Table 5.**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*


#### **Table 5.**

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

A reduction of 10.90% ( > 0.05) in the organ weight was observed in the test group as compared to the control group. These variations in the relative organ weight of the control and experimental groups of SD rats were not significantly

*Changes in serum biochemistry of SD rats after administration of unsweetened natural cocoa powder solution.*

High-density lipoproteins (HLD) increased slightly with a value <0.05, a decrease in the level of triglycerides and low density lipoprotein (LDL) cholesterol of the UNCP group ( > 0.05) was observed. In comparison with that of the control, the cholesterol levels remained relatively unchanged. This is consistent with the

Hematological results (**Table 5**) revealed a decrease (28.44%, > 0.05) in the level of platelet in the UNCP group in comparison with the controls. Polyphenols in cocoa have been found to reduce platelet count. Neutrophil and lymphocyte polymorph of white blood cells showed a slight decrease of 8.02 and 18.73%, respec-

Histopathology evaluation of the organs studied; liver, kidney, heart, lungs, spleen, and the testis of the animals that received UNCP showed no toxic effect as compared to that of the control group. UNCP solution therefore is not likely to have toxic effects on the kidney when administered in a single oral high dose of 2000 mg/kg. Notable changes were observed on the small intestines in the form of erosions of the mucosal lining of the villi (**Figure 1**). These effects were, however,

speculation that UNCP possess lipid lowering abilities.

**70**

different.

**Table 4.**

tively ( > 0.05).

*Changes in hematological indices in rats after receiving 2000 mg/kg bwt of UNCP.*

not observed in the control animals those that received equivalent volumes of the vehicle (distilled water). This could be due to the high concentration of proanthocyanidins contained in the 2000 mg/kg dose of UNCP (approximately 2.5 g in man). Proanthocyanidins have been found to instigate the destruction of the mucosal lining of the gastrointestinal tract.

### *1.2.5 Conclusion*

In conclusion, the aqueous solution of unsweetened natural cocoa powder administered at the single oral high dose of 2000 mg/kg appears to be relatively safe in male SD rats. Caution should however be taken when using UNCP especially in high quantities or amounts since it is capable of causing considerable damage to the mucosal lining of the small intestines.

#### *1.2.6 Reproductive toxicity*

Since UNCP is widely consumed by individuals in their reproductive period of life, it is worth investigating the generative toxicity, genotoxic, and aspects of the reproductive toxicity potential of UNCP in both male and female white wistar rats. As a preliminary study, the genotoxic potential of UNCP was assessed using the DNA comet method. DNA breaks in the rectal epithelium, liver, bone marrow, and kidney of the mice were quantified and compared with the negative control (2% starch). Pre-implantation loss, viable fetuses, corpora lutea and post-implantation

**Figure 1.**

*H&E stained sections of the small intestine of 20× magnification. (a) Small intestine sections of control male SD rates showing villi (1) goblet cells (2) basement membrane with no proliferation (4). (b) Small intestine sections of the group treated with UNCP showing moderate changes and erosion of the mucosal lining of the villi (3).*

loss, and implantation sites/number of implants were assessed. Results show that UNCP does not exhibit any reproductive toxicity potential.

## **1.3 Organoprotective effect of unsweetened cocoa powder against high-dose artemether-lumefantrin-induced effects**

Artemisinin and its derivatives, derived from *Artemisia annua* (**sweet** wormwood), have impressive parasiticidal properties *in vivo* and *in vitro* [27, 28]. Despite the efficacy of artemisinin in reducing the malaria parasite load in the body, monotherapy was discouraged in an attempt to delay or prevent the development of drug resistance. Artemisinin-based combination therapy (ACT) was recommended by the WHO for use in the treatment of malaria after resistance was developed to the quinine derivatives used at the time [29, 30]. Increased therapeutic efficacy was reported to be associated with the use of the combination. Treatment failure to these therapies is suspected, and in recent years, some countries have considered increasing the dose of the A/L in treatment in order to arrest the issue of resistance [31], but increase in dose implies that there will be increased side effects, adverse reactions, and hepatotoxic effects [32]. In fact, there are already concerns about frequent usage of A/L on some organ systems [28]. Considering the fact that, so far, A/L is one of the most effective combination therapies, the issue of drug-induced hepatotoxicity needs to be addressed. Another effect of A/L is its effect on nitric oxide levels, where it has been found to reduce nitric oxide levels. However, other studies show that A/L increase nitric oxide levels as a compensatory mechanism in cases of reduced nitric oxide levels [28]. Additionally, some studies of artemether in rats have shown changes in the hematological profile of the rats [33]. It is therefore suspected that artemether may aggravate anemia in malaria patients. In another study, A/L was found to reduce red blood cell count (RBC), HGB, and packed cell volume (PCV) in patients taking treatment [34].

Ghana is the second producer of cocoa in the world. It is therefore not surprising that many consume cocoa beverage at one time or the other during the day. It is also one of the endemic regions for malaria. Thus, a high possibility of consuming a cocoa product while being treated for malaria using any of the ACTs is

**73**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

therapies used more frequently for treating malaria in Ghana.

reduces platelet aggregation, and improves lipid profile [25, 35].

given distilled water (vehicle control group [VCG]).

Group 1: Control (distilled water only)/vehicle control (CTRL). Group 2: 75 mg/kg A/L(last 3 days)/negative control(COARTEM). Group 3: Cocoa 300 mg/kg (14 days) + 75 mg/kg A/L (last 3 days) (300). Group 4: Cocoa 900 mg/kg (14 days) + 75 mg/kg A/L (last 3 days) (900). Group 5: Cocoa 1500 mg/kg (14 days) + 75 mg/kg A/L (last 3 days) (1500). The guinea pigs were, thus, observed daily for a total period of 14 days.

The value of km factors (i.e., body weight, kg/surface area, m2

ferred into EDTA-2 k test tubes for immediate analysis.

Conversion of animal doses to HED was based on basal surface area. HEDs were calculated according to a study by Reagan-Shaw et al. [24] and Asiedu-Gyekye et al.

Human equivalent dose (HED) (mg/kg) = Animal dose (mg/kg) × (Human km/

pigs to be 37 and 8, respectively, and an average weight of Ghanaian to be 70.0 kg. After day 14, the guinea pigs were euthanized with 50 mg/kg chloroform by exsanguination, and 2 ml of blood was sampled by cardiac puncture and trans-

) for adult and guinea

The dosage regimen was as follows:

recommended by WHO. Artemether/lumefantrine (A/L) is one of the combination

It is interesting to note that anecdotal reports of the ability of cocoa to prevent malaria exist. Following these reports, regular intake of cocoa powder as a beverage has been shown to be associated with reduction in the incidence of episodic malaria and its use as diet-mediated malaria prophylaxis [3]. The antiplasmodial activity of different fractions especially the nonpolar solvent fractions have also been confirmed. Thus, simultaneous consumption of cocoa beverage during antimalarial treatment with A/L is expected to have dual benefits such as rapid clearance of the malaria parasites.

Regular intake of unsweetened natural cocoa powder as a beverage has immense health benefits including both cardiovascular and neurodegenerative disorders,

Determination of the major elemental composition and toxicity of unsweetened

cocoa powder was conducted. Another set of projects were aimed at assessing whether UNCP will worsen or is able to prevent some common cardiovascular and renal side effects associated with the use of A/L, its effect on nitric oxide levels and to assess its hepatoprotective potential against A/L-induced liver toxicity during their simultaneous ingestion in male guinea pigs, and finally to investigate the effect of UNCP on the hematological parameters and NO levels during high-dose (HD)

The methods used in the assessment of the parameters listed above are as described previously by Asiedu-Gyekye et al. [26] as follows: 30 adult male guinea pigs weighing between 300 and 350 g were purchased from the Animal Experimentation Department of the Noguchi Memorial Institute for Medical Research, University of Ghana, Legon. The guinea pigs were acclimatized to laboratory environment (20–23°C) with a 12 h light-darkness cycle for 7 days prior to experimentation. The guinea pigs had access to standard laboratory diet and water *ad libitum*. The adult male guinea pigs were randomly assigned to five groups with each group containing six guinea pigs, 500 mg/kg per body weight, respectively, for 14 days, with two other groups serving as controls. One control group (negative control group [NCG]) received 75 mg/kg body weight A/L, and the other group was

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

A/L administration.

[26] using the formula:

Human km).

#### *Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

loss, and implantation sites/number of implants were assessed. Results show that

*H&E stained sections of the small intestine of 20× magnification. (a) Small intestine sections of control male SD rates showing villi (1) goblet cells (2) basement membrane with no proliferation (4). (b) Small intestine sections of the group treated with UNCP showing moderate changes and erosion of the mucosal lining of the* 

**1.3 Organoprotective effect of unsweetened cocoa powder against high-dose** 

Artemisinin and its derivatives, derived from *Artemisia annua* (**sweet** wormwood), have impressive parasiticidal properties *in vivo* and *in vitro* [27, 28]. Despite the efficacy of artemisinin in reducing the malaria parasite load in the body,

monotherapy was discouraged in an attempt to delay or prevent the development of drug resistance. Artemisinin-based combination therapy (ACT) was recommended by the WHO for use in the treatment of malaria after resistance was developed to the quinine derivatives used at the time [29, 30]. Increased therapeutic efficacy was reported to be associated with the use of the combination. Treatment failure to these therapies is suspected, and in recent years, some countries have considered increasing the dose of the A/L in treatment in order to arrest the issue of resistance [31], but increase in dose implies that there will be increased side effects, adverse reactions, and hepatotoxic effects [32]. In fact, there are already concerns about frequent usage of A/L on some organ systems [28]. Considering the fact that, so far, A/L is one of the most effective combination therapies, the issue of drug-induced hepatotoxicity needs to be addressed. Another effect of A/L is its effect on nitric oxide levels, where it has been found to reduce nitric oxide levels. However, other studies show that A/L increase nitric oxide levels as a compensatory mechanism in cases of reduced nitric oxide levels [28]. Additionally, some studies of artemether in rats have shown changes in the hematological profile of the rats [33]. It is therefore suspected that artemether may aggravate anemia in malaria patients. In another study, A/L was found to reduce red blood cell count (RBC), HGB, and packed cell

Ghana is the second producer of cocoa in the world. It is therefore not surprising that many consume cocoa beverage at one time or the other during the day. It is also one of the endemic regions for malaria. Thus, a high possibility of consuming a cocoa product while being treated for malaria using any of the ACTs is

UNCP does not exhibit any reproductive toxicity potential.

**artemether-lumefantrin-induced effects**

volume (PCV) in patients taking treatment [34].

**72**

**Figure 1.**

*villi (3).*

recommended by WHO. Artemether/lumefantrine (A/L) is one of the combination therapies used more frequently for treating malaria in Ghana.

It is interesting to note that anecdotal reports of the ability of cocoa to prevent malaria exist. Following these reports, regular intake of cocoa powder as a beverage has been shown to be associated with reduction in the incidence of episodic malaria and its use as diet-mediated malaria prophylaxis [3]. The antiplasmodial activity of different fractions especially the nonpolar solvent fractions have also been confirmed. Thus, simultaneous consumption of cocoa beverage during antimalarial treatment with A/L is expected to have dual benefits such as rapid clearance of the malaria parasites.

Regular intake of unsweetened natural cocoa powder as a beverage has immense health benefits including both cardiovascular and neurodegenerative disorders, reduces platelet aggregation, and improves lipid profile [25, 35].

Determination of the major elemental composition and toxicity of unsweetened cocoa powder was conducted. Another set of projects were aimed at assessing whether UNCP will worsen or is able to prevent some common cardiovascular and renal side effects associated with the use of A/L, its effect on nitric oxide levels and to assess its hepatoprotective potential against A/L-induced liver toxicity during their simultaneous ingestion in male guinea pigs, and finally to investigate the effect of UNCP on the hematological parameters and NO levels during high-dose (HD) A/L administration.

The methods used in the assessment of the parameters listed above are as described previously by Asiedu-Gyekye et al. [26] as follows: 30 adult male guinea pigs weighing between 300 and 350 g were purchased from the Animal Experimentation Department of the Noguchi Memorial Institute for Medical Research, University of Ghana, Legon. The guinea pigs were acclimatized to laboratory environment (20–23°C) with a 12 h light-darkness cycle for 7 days prior to experimentation. The guinea pigs had access to standard laboratory diet and water *ad libitum*. The adult male guinea pigs were randomly assigned to five groups with each group containing six guinea pigs, 500 mg/kg per body weight, respectively, for 14 days, with two other groups serving as controls. One control group (negative control group [NCG]) received 75 mg/kg body weight A/L, and the other group was given distilled water (vehicle control group [VCG]).

The dosage regimen was as follows:

Group 1: Control (distilled water only)/vehicle control (CTRL). Group 2: 75 mg/kg A/L(last 3 days)/negative control(COARTEM). Group 3: Cocoa 300 mg/kg (14 days) + 75 mg/kg A/L (last 3 days) (300). Group 4: Cocoa 900 mg/kg (14 days) + 75 mg/kg A/L (last 3 days) (900). Group 5: Cocoa 1500 mg/kg (14 days) + 75 mg/kg A/L (last 3 days) (1500). The guinea pigs were, thus, observed daily for a total period of 14 days.

Conversion of animal doses to HED was based on basal surface area. HEDs were calculated according to a study by Reagan-Shaw et al. [24] and Asiedu-Gyekye et al. [26] using the formula:

Human equivalent dose (HED) (mg/kg) = Animal dose (mg/kg) × (Human km/ Human km).

The value of km factors (i.e., body weight, kg/surface area, m2 ) for adult and guinea pigs to be 37 and 8, respectively, and an average weight of Ghanaian to be 70.0 kg.

After day 14, the guinea pigs were euthanized with 50 mg/kg chloroform by exsanguination, and 2 ml of blood was sampled by cardiac puncture and transferred into EDTA-2 k test tubes for immediate analysis.

*1.3.1 Potential of unsweetened natural cocoa powder to attenuate high-dose artemether-lumefantrine-induced hepatotoxicity in nonmalarious guinea pigs*

#### *1.3.1.1 Biochemical assays*

Blood samples were collected from the descending aorta and aliquoted into EDTA-2K tubes and plain tubes, respectively, at the end of the dosing period. This was done after euthanization of the animals under ether anesthesia. The EDTA blood was immediately analyzed for hematological parameters using the SYSMEX Hematology Autoanalyzer (Kobe, Japan), while sera prepared from blood in plain tubes were used for biochemical examinations including clinical chemistry measurements such as alkaline phosphatase (ALP), alanine aminotransferase (ALT) or glutamic pyruvic transaminase (SGPT) levels, serum glutamic oxaloacetic transaminase (SGOT) or aspartate transaminase, and gamma glutamyl transpeptidase (GGT). These were measured as liver function tests (LFT), which gives an indication of the state of the liver.

Nitric oxide levels were also measured using the Griess Reagent System. The total nitric oxide kit by R&D Systems was used in this study, and the assay reported previously by Bryan and Grisham [36] was employed for this purpose. In this assay, nitrate is converted to nitrite by nitrate reductase after which colorimetric detection of nitrite as an azo dye is carried out. The Griess reaction involves a two-step diazotization reaction, acidified NO2 <sup>−</sup> to produce a nitrosating agent, which then reacts with sulfanilic acid to produce the diazonium ion. The diazonium then couples to *N*-(1-naphthyl) ethylenediamine to form azo-derivatives, which are chromophoric. These azo-derivatives are absorbed within the range of 540–570 nm.

Elevation of serum and plasma enzymes such as alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate transferase (AST) has been shown to be reliable markers of acute hepatocellular damage. This occurs due to hepatocyte membrane distortion leading to membrane leakage of the hepatocyte cytosolic contents. AST is abundant in the cardiac muscles, skeletal muscles, kidneys than in the liver; thus, ALT is the most reliable marker.

This study revealed that A/L increased ALT, AST, GGT, and bilirubin levels but witnessed a reduction in albumin and total protein levels indicating the presence of hepatotoxicity (**Figures 2**–**4**). The increases in AST and ALT were found to be dose dependent.

Usually in patients with hepatotoxicity, ALT levels increase by more than three times the upper limit of normal, ALP levels also increase by more than twice the upper limit or total bilirubin more than twice when associated with increased ALP or ALT. It is important to mention that liver damage could manifest with either predominately initial alanine transferase elevation (hepatocellular) or initial alkaline phosphatase rise (cholestatic). The two are, however, not mutually exclusive.

The synthetic function of the liver can be assessed by the levels of total protein and albumins. Administration of A/L leads to a decrease in total protein and albumin levels supporting the hepatotoxic effects of A/L (**Figures 5**–**7**).

UNCP significantly increased the levels of total proteins, which further supports its hepatoprotective effect. Elevation of albumin levels after administration of UNCP was significant. For the first time, this study reports the hepatoprotective effect of UNCP against A/L-induced hepatic damage in guinea pigs.

#### *1.3.1.2 Histopathological studies*

Guinea pigs were euthanized, and their livers were swiftly excised and washed with 0.9% saline. The livers were stored in 10% neutral buffered formaldehyde.

**75**

**Figure 3.**

*compared with the A/L group.*

**Figure 2.**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

The liver tissues were then cut and sectioned using a microtone into 2 m thick liver slices and stained with hematoxylin-eosin for examination. The stained tissues were observed with an Olympus microscope (BX-51) and photographed by INFINITY 4

 *means p < 0.05 when* 

*Changes in ALP levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration of UNCP followed by a 3-day A/L administration. The differences among the means were* 

*Changes in ALT levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc analysis, where \*\* means p < 0.001 when compared with the control* 

*(distilled water) and ++p < 0.001 when compared with the A/L group.*

USB Scientific Camera (Lumenera Corporation, Otawa, Canada).

*analyzed using one-way ANOVA followed by Newman-Keuls post hoc analysis, where <sup>+</sup>*

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*

#### **Figure 2.**

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

*1.3.1 Potential of unsweetened natural cocoa powder to attenuate high-dose* 

*1.3.1.1 Biochemical assays*

tization reaction, acidified NO2

*1.3.1.2 Histopathological studies*

the liver; thus, ALT is the most reliable marker.

*artemether-lumefantrine-induced hepatotoxicity in nonmalarious guinea pigs*

Blood samples were collected from the descending aorta and aliquoted into EDTA-2K tubes and plain tubes, respectively, at the end of the dosing period. This was done after euthanization of the animals under ether anesthesia. The EDTA blood was immediately analyzed for hematological parameters using the SYSMEX Hematology Autoanalyzer (Kobe, Japan), while sera prepared from blood in plain tubes were used for biochemical examinations including clinical chemistry measurements such as alkaline phosphatase (ALP), alanine aminotransferase (ALT) or glutamic pyruvic transaminase (SGPT) levels, serum glutamic oxaloacetic transaminase (SGOT) or aspartate transaminase, and gamma glutamyl transpeptidase (GGT). These were measured as liver function tests (LFT), which gives an indication of the state of the liver. Nitric oxide levels were also measured using the Griess Reagent System. The total nitric oxide kit by R&D Systems was used in this study, and the assay reported previously by Bryan and Grisham [36] was employed for this purpose. In this assay, nitrate is converted to nitrite by nitrate reductase after which colorimetric detection of nitrite as an azo dye is carried out. The Griess reaction involves a two-step diazo-

with sulfanilic acid to produce the diazonium ion. The diazonium then couples to *N*-(1-naphthyl) ethylenediamine to form azo-derivatives, which are chromophoric.

Elevation of serum and plasma enzymes such as alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate transferase (AST) has been shown to be reliable markers of acute hepatocellular damage. This occurs due to hepatocyte membrane distortion leading to membrane leakage of the hepatocyte cytosolic contents. AST is abundant in the cardiac muscles, skeletal muscles, kidneys than in

This study revealed that A/L increased ALT, AST, GGT, and bilirubin levels but witnessed a reduction in albumin and total protein levels indicating the presence of hepatotoxicity (**Figures 2**–**4**). The increases in AST and ALT were found to be dose

Usually in patients with hepatotoxicity, ALT levels increase by more than three times the upper limit of normal, ALP levels also increase by more than twice the upper limit or total bilirubin more than twice when associated with increased ALP or ALT. It is important to mention that liver damage could manifest with either predominately initial alanine transferase elevation (hepatocellular) or initial alkaline phosphatase rise (cholestatic). The two are, however, not mutually exclusive.

The synthetic function of the liver can be assessed by the levels of total protein and albumins. Administration of A/L leads to a decrease in total protein and albu-

UNCP significantly increased the levels of total proteins, which further supports its hepatoprotective effect. Elevation of albumin levels after administration of UNCP was significant. For the first time, this study reports the hepatoprotective

Guinea pigs were euthanized, and their livers were swiftly excised and washed with 0.9% saline. The livers were stored in 10% neutral buffered formaldehyde.

min levels supporting the hepatotoxic effects of A/L (**Figures 5**–**7**).

effect of UNCP against A/L-induced hepatic damage in guinea pigs.

These azo-derivatives are absorbed within the range of 540–570 nm.

<sup>−</sup> to produce a nitrosating agent, which then reacts

**74**

dependent.

*Changes in ALT levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc analysis, where \*\* means p < 0.001 when compared with the control (distilled water) and ++p < 0.001 when compared with the A/L group.*

#### **Figure 3.**

*Changes in ALP levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way ANOVA followed by Newman-Keuls post hoc analysis, where + means p < 0.05 when compared with the A/L group.*

The liver tissues were then cut and sectioned using a microtone into 2 m thick liver slices and stained with hematoxylin-eosin for examination. The stained tissues were observed with an Olympus microscope (BX-51) and photographed by INFINITY 4 USB Scientific Camera (Lumenera Corporation, Otawa, Canada).

#### **Figure 4.**

*Changes in AST levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way ANOVA followed by Newman-Keuls post hoc analysis, where \*means p < 0.05, \*\* means p < 0.001 when compared with the control (distilled water) and ++ p < 0.001 when compared with the A/L group.*

#### **Figure 5.**

*Changes in albumin levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way ANOVA followed by Newman-Keuls post hoc analysis.*

The hepatoprotective effect of UNCP was further confirmed in the histological data presented above, where very insignificant abnormalities were observed in the group that received UNCP before A/L. Damaged liver tissues in animals that received A/L alone was observed, which is evidenced by disturbed (necrotic)

**77**

damage).

**Figure 7.**

*Newman-Keuls post hoc analysis.*

**Figure 6.**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

liver parenchyma (NeLP), a highly congested and dilated central vein (CCV), and lymphocytic infiltration (LYM) in all animals (**Figure 8(b)**). Administration of UNCP before A/L reduced the extent of liver damage evidenced by the undisturbed liver parenchyma with an uncongested but dilated central vein (mild liver

*Changes in total bilirubin levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way ANOVA followed by* 

*Changes in total protein levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The difference among the means were analyzed using one-way ANOVA followed by Newman-Keuls post hoc anlaysis, where \* means p < 0.05, \*\* means p < 0.001 when compared with the control* 

*(distilled water) and ++p < 0.001 when compared with A/L group.*

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*

#### **Figure 6.**

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

*Changes in AST levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way ANOVA followed by Newman-Keuls post hoc analysis, where \*means p < 0.05, \*\* means p < 0.001 when compared with the control (distilled* 

*water) and ++ p < 0.001 when compared with the A/L group.*

The hepatoprotective effect of UNCP was further confirmed in the histological data presented above, where very insignificant abnormalities were observed in the group that received UNCP before A/L. Damaged liver tissues in animals that received A/L alone was observed, which is evidenced by disturbed (necrotic)

*Changes in albumin levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way ANOVA followed by Newman-*

**76**

**Figure 5.**

*Keuls post hoc analysis.*

**Figure 4.**

*Changes in total protein levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The difference among the means were analyzed using one-way ANOVA followed by Newman-Keuls post hoc anlaysis, where \* means p < 0.05, \*\* means p < 0.001 when compared with the control (distilled water) and ++p < 0.001 when compared with A/L group.*

#### **Figure 7.**

*Changes in total bilirubin levels of guinea pigs during a 14-day administration of UNCP followed by a 3-day A/L administration. The differences among the means were analyzed using one-way ANOVA followed by Newman-Keuls post hoc analysis.*

liver parenchyma (NeLP), a highly congested and dilated central vein (CCV), and lymphocytic infiltration (LYM) in all animals (**Figure 8(b)**). Administration of UNCP before A/L reduced the extent of liver damage evidenced by the undisturbed liver parenchyma with an uncongested but dilated central vein (mild liver damage).

## *1.3.1.3 Conclusion*

Unsweetened natural cocoa powder has hepatoprotective potential during highdose A/L administration. The simultaneous consumption of UNCP and A/L is not likely to result in liver injury or dysfunction. However, care must however be taken during high daily consumption due to the high copper content.

## *1.3.2 Cardio and renal toxicity*

Artemether-lumefantrine is one of the fixed-dose combination therapies recommended by the WHO for the treatment of malaria falciparum in Africa. Administration of this medication, however, generates free radicals that have the potential of causing cellular damage and other organ toxicity, characteristic being cardio-hepato- and renal toxicity.

It is speculated that simultaneous consumption of natural cocoa during antimalarial treatment with A/L is expected to rapidly clear the malaria parasites as well as ameliorate the A/L-induced toxic injury to heart and kidneys. Thus, the consumption of natural antioxidants such as found in cocoa could be beneficial in rectifying such damage in humans. The study also aimed at assessing whether UNCP will worsen or is able to prevent some common cardiovascular and renal side effects associated with the use of A/L. To establish the protective effect on the heart and

#### **Figure 8.**

*Microtome sections of liver from guinea pigs that received (a) only a 3-day HD A/L administration (75 mg/kg/bwt) showing disturbed (necrotic) liver parenchyma (NeLP), a highly congested and dilated central vein (CCV), and lymphocytic infilteration (LYM). (b) Distilled water, control showing undisturbed liver parenchyma (NoLP) with uncongested central veins (UCV). Note the regularity of liver cell plates, microcirculatory zones, and sinusoids. (c) A 14-day UNCP administration (300 mg/kg/bwt) followed by a 3-day A/L administration (75 mg/kg/bwt) showing undisturbed liver parenchyma with uncongested but dilated central vein (DCV). (d) A 14-day UNCP administration (900 mg/kg/bwt) followed by a 3-day A/L administration (75 mg/kg/bwt) showing undisturbed liver parenchyma with uncongested but dilated central vein (DCV). (e) A 14-day UNCP administration (1500 mg/kg/bwt) followed by a 3-day A/L administration (75 mg/kg/bwt) showing undisturbed liver parenchyma with uncongested but dilated central vein (DCV) (H&E stain ×40).*

**79**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

Autoanalyzer (Vital Scientific BV, Version 04, Netherlands).

1500 mg/ kg UNCP (1.478 ± 0.487 mmol/L) (*p* < 0.05) [16].

kidney against (A/L) administration, various disease markers were assayed after concomitant administration of cocoa and artemether/lumefantrine (A/L). The biochemical assays were performed as reported previously by Asiedu-Gyekye [16]. Blood samples collected in plain gel tubes were allowed to clot. It was then centrifuged at 3000 rpm for 15 min, sera removed and stored at −20°C. The sera were analyzed for biochemical parameters such as the total cholesterol, triglycerides, high density lipoproteins (HDL), low density lipoproteins, very low density lipoproteins (VLDL), creatinine, urea, blood electrolytes, creatine kinase (CK), and aspartate transferase [35, 37]. Measurements were carried out on the Selectra Junior

Euthanized guinea pigs were dissected, their hearts and kidneys were removed.

In the medium and high UNCP dose groups, there was a decrease in the mean serum levels of low density lipoprotein by 11.6 and 10.6% (*p* < 0.05), respectively, in comparison with the negative control group (NCG) (0.662 ± 0.269 mmol/L)[16]. A dose-dependent increment was observed for both VLDL and triglycerides upon

Groups, which received 900 mg/kg UNCP, showed 12.9% (*p* < 0.05) increase in the mean serum levels of high density lipoprotein (HDL) in comparison with the

The levels of coronary risk was high in animals that received 1500 mg/kg UNCP

Significant changes in serum levels of total cholesterol, low density lipoproteins cholesterol, and triglycerides were not observed after administration of both A/L and UNCP. It must however be noted that lipid profile including cholesterol in general takes considerable time to show significant changes even with cholesterol lowering agents. The authors conclude that the 14-day administration of cocoa may

Asiedu-Gyekye's group observed a significant difference in the mean levels of CK in VCG (598.0 ± 382.425 μmol/L) and NCG (1039.0 ± 749.494 μmol/L) [16]. The groups that received 300 mg/kg UNCP, 900 mg/kg UNCP, and 1500 mg/kg UNCP had their CK as follows: 552.2 ± 399.968 μmol/L, 318.5 ± 122.516 μmol/L, and 366.8 ± 174.921 μmol/L, respectively (**Figure 8**). The LD, MD, and HD cocoa groups hence reduced the creatine levels by 46.9, 69.3, and 64.7%, respectively (*p* < 0.05). Creatine kinase (CK) or creatine phosphokinase is a marker of damaged tissue. Myocardial injury is often associated with an increase in CK levels. In this study, it was observed that animals that received 900 mg/kg bwt + A/L had 69.3% reduction

exhibited high levels of coronary risk (11.778 ± 1.167), but those on 900 mg/kg UNCP showed low levels (8.470 ± 2.624) in comparison with the NCG (9.08 ± 2.894,

not have been long enough to produce significant changes expected.

administration of UNCP [16]. The triglyceride changes were as follows: controls (1.075 ± 0.360 mmol/L), A/L-administered group (0.966 ± 0.619 mmol/L), 300 mg/ kg UNCP (0.980 ± 0.391 mmol/L), 900 mg/kg UNCP (1.208 ± 0.317 mmol/L),

The tissues were kept in 10% buffered formalin. The tissues were embedded in paraffin wax and sectioned at 4 μm thickness, stained with hematoxylin-eosin. The light microscope was employed for the histological studies of the study animals as well as two control groups. A total of 30 photomicrographs at a magnification

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

of ×40 were used for each group.

NCG 0.148 ± 0.046, (*p* < 0.05) [16].

*p* < 0.05) [16].

*1.3.2.2 Creatine kinase (CK)*

*1.3.2.1 Biochemical assays*

#### *Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*

kidney against (A/L) administration, various disease markers were assayed after concomitant administration of cocoa and artemether/lumefantrine (A/L).

The biochemical assays were performed as reported previously by Asiedu-Gyekye [16]. Blood samples collected in plain gel tubes were allowed to clot. It was then centrifuged at 3000 rpm for 15 min, sera removed and stored at −20°C. The sera were analyzed for biochemical parameters such as the total cholesterol, triglycerides, high density lipoproteins (HDL), low density lipoproteins, very low density lipoproteins (VLDL), creatinine, urea, blood electrolytes, creatine kinase (CK), and aspartate transferase [35, 37]. Measurements were carried out on the Selectra Junior Autoanalyzer (Vital Scientific BV, Version 04, Netherlands).

Euthanized guinea pigs were dissected, their hearts and kidneys were removed. The tissues were kept in 10% buffered formalin. The tissues were embedded in paraffin wax and sectioned at 4 μm thickness, stained with hematoxylin-eosin. The light microscope was employed for the histological studies of the study animals as well as two control groups. A total of 30 photomicrographs at a magnification of ×40 were used for each group.

### *1.3.2.1 Biochemical assays*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

during high daily consumption due to the high copper content.

Unsweetened natural cocoa powder has hepatoprotective potential during highdose A/L administration. The simultaneous consumption of UNCP and A/L is not likely to result in liver injury or dysfunction. However, care must however be taken

Artemether-lumefantrine is one of the fixed-dose combination therapies recommended by the WHO for the treatment of malaria falciparum in Africa. Administration of this medication, however, generates free radicals that have the potential of causing cellular damage and other organ toxicity, characteristic being

*Microtome sections of liver from guinea pigs that received (a) only a 3-day HD A/L administration (75 mg/kg/bwt) showing disturbed (necrotic) liver parenchyma (NeLP), a highly congested and dilated central vein (CCV), and lymphocytic infilteration (LYM). (b) Distilled water, control showing undisturbed liver parenchyma (NoLP) with uncongested central veins (UCV). Note the regularity of liver cell plates, microcirculatory zones, and sinusoids. (c) A 14-day UNCP administration (300 mg/kg/bwt) followed by a 3-day A/L administration (75 mg/kg/bwt) showing undisturbed liver parenchyma with uncongested but dilated central vein (DCV). (d) A 14-day UNCP administration (900 mg/kg/bwt) followed by a 3-day A/L administration (75 mg/kg/bwt) showing undisturbed liver parenchyma with uncongested but dilated central vein (DCV). (e) A 14-day UNCP administration (1500 mg/kg/bwt) followed by a 3-day A/L administration (75 mg/kg/bwt) showing undisturbed liver parenchyma with uncongested but dilated central vein (DCV)* 

It is speculated that simultaneous consumption of natural cocoa during antimalarial treatment with A/L is expected to rapidly clear the malaria parasites as well as ameliorate the A/L-induced toxic injury to heart and kidneys. Thus, the consumption of natural antioxidants such as found in cocoa could be beneficial in rectifying such damage in humans. The study also aimed at assessing whether UNCP will worsen or is able to prevent some common cardiovascular and renal side effects associated with the use of A/L. To establish the protective effect on the heart and

*1.3.1.3 Conclusion*

*1.3.2 Cardio and renal toxicity*

cardio-hepato- and renal toxicity.

**78**

*(H&E stain ×40).*

**Figure 8.**

In the medium and high UNCP dose groups, there was a decrease in the mean serum levels of low density lipoprotein by 11.6 and 10.6% (*p* < 0.05), respectively, in comparison with the negative control group (NCG) (0.662 ± 0.269 mmol/L)[16].

A dose-dependent increment was observed for both VLDL and triglycerides upon administration of UNCP [16]. The triglyceride changes were as follows: controls (1.075 ± 0.360 mmol/L), A/L-administered group (0.966 ± 0.619 mmol/L), 300 mg/ kg UNCP (0.980 ± 0.391 mmol/L), 900 mg/kg UNCP (1.208 ± 0.317 mmol/L), 1500 mg/ kg UNCP (1.478 ± 0.487 mmol/L) (*p* < 0.05) [16].

Groups, which received 900 mg/kg UNCP, showed 12.9% (*p* < 0.05) increase in the mean serum levels of high density lipoprotein (HDL) in comparison with the NCG 0.148 ± 0.046, (*p* < 0.05) [16].

The levels of coronary risk was high in animals that received 1500 mg/kg UNCP exhibited high levels of coronary risk (11.778 ± 1.167), but those on 900 mg/kg UNCP showed low levels (8.470 ± 2.624) in comparison with the NCG (9.08 ± 2.894, *p* < 0.05) [16].

Significant changes in serum levels of total cholesterol, low density lipoproteins cholesterol, and triglycerides were not observed after administration of both A/L and UNCP. It must however be noted that lipid profile including cholesterol in general takes considerable time to show significant changes even with cholesterol lowering agents. The authors conclude that the 14-day administration of cocoa may not have been long enough to produce significant changes expected.

#### *1.3.2.2 Creatine kinase (CK)*

Asiedu-Gyekye's group observed a significant difference in the mean levels of CK in VCG (598.0 ± 382.425 μmol/L) and NCG (1039.0 ± 749.494 μmol/L) [16]. The groups that received 300 mg/kg UNCP, 900 mg/kg UNCP, and 1500 mg/kg UNCP had their CK as follows: 552.2 ± 399.968 μmol/L, 318.5 ± 122.516 μmol/L, and 366.8 ± 174.921 μmol/L, respectively (**Figure 8**). The LD, MD, and HD cocoa groups hence reduced the creatine levels by 46.9, 69.3, and 64.7%, respectively (*p* < 0.05).

Creatine kinase (CK) or creatine phosphokinase is a marker of damaged tissue. Myocardial injury is often associated with an increase in CK levels. In this study, it was observed that animals that received 900 mg/kg bwt + A/L had 69.3% reduction in serum CK showing the greatest mitigating activity against coartem toxicity. CK plays a crucial role in the conversion of creatine to phosphocreatine and adenosine diphosphate; thus, it might also protect or enhance myocardial bioenergetics.

#### *1.3.2.3 Renal function test*

Asiedu-Gyekye's group reported a reduction in urea by 53% in 1500 mg/kg when compared with the VCG (*p* < 0.05) [16]. Those of groups 3 and 4 were reduced by 14 and 10.64% in comparison with the VCG.

For the creatinine levels, significantly increments was observed; a 24.08% increase in NCG compared with VCG and decrease of 21.27, 17.54, and 11.05% in groups 3, 4, and 5, respectively, when compared with group 1 (*p* < 0.05) [16] .

The sodium, potassium, and chloride levels remained relatively unchanged in all study groups when compared with the control that received distilled water only [16].

The 1500 mg/kg group showed a significant reduction in urea levels as compared to the Coartem® only group. Creatinine levels decreased in all the groups compared with the control group. The authors attribute the observed effects to the antioxidant and nephroprotective effects of cocoa. Guinea pigs that received only the 75 mg/kg Coartem® group showed high levels of renal damage.

#### *1.3.2.4 Histopathological examination*

Aspartate transferase, one of the enzymes mentioned earlier in this chapter, is distributed mostly in the heart followed by the liver and skeletal muscles. Elevated serum aspartate transferase values are hence indicative of cellular injury and may present in myocardial disease, shock, hypoxia, among others. The group administered with of distilled water +A/L showed a significantly increased serum levels of aspartate transferase. The reverse (significant reduction) was observed in all animals administered unsweetened natural cocoa powder extract.

These observations are corroborated by histopathological examination of the myocardial tissues of the guinea pigs (figure not shown), where tissue sections from animals that received only A/L 75 mg/kg showed evidence of inflammation and degeneration of the myocardial tissue. Normal cardiac tissue structures of myocardial tissue were observed in animals administered UNCP extract except those of animals that received 900 mg/kg UNCP where there was a single case observed with suspected ongoing tissue necrosis at the initial stages. Similar observations of the cardioprotective effect have also been made by other researchers.

Photomicrographs of myocardial tissues of animals from the different experimental groups revealed patchy areas of congestion, edema, extensive nuclear, and tissue degeneration leading to loss of microstructure of myocardial tissues for animals receiving 75 mg/kg A/L [16]. Animals that received 300 mg/kg UNCP and the control group retained the normal branching of myocardial cells characteristics [16].

Coronary risk ratio is an important indicator of cardiovascular health. Assessment of it showed that groups on 1500 mg/kg cocoa +A/L were at a higher risk compared with the control groups [16]. This observation corroborates the findings that although cocoa possesses many health benefits, high level intake could be deleterious, an effect believed to be caused by (−) epigallocatechin-3-gallate.

#### *1.3.2.5 Conclusion*

Unsweetened Natural Cocoa Powder showed renoprotective and cardioprotective potential during high-dose A/L administration [16]. It therefore suggests that

**81**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

simultaneous ingestion of A/L and UNCP may be beneficial to the heart and kidney. However, regular intake of large quantities of UNCP could be deleterious to health

Hematological parameters are one of the vital indices monitored during malaria treatment. Thus, in this study, the effect of UNCP on the hematological parameters

The results obtained from this study showed that A/L administration decreased

Asiedu-Gyekye's group reported a reduction of 31.87% in the WBC count of the NCG (Coartem®) in comparison with the vehicle control group(*p* > 0.05) [26]. Administration of 300, 900, and 1500 mg/kg body weight of UNCP restored the WBC levels during concomitant administration with A/L (*p =* 0.1158)[26]. Prophylactic administration of UNCP with A/L at doses of 300, 900, and 1500 mg/kg body weight restored the decreased levels of the RBC count by 4.17,

Administration of UNCP at doses of 300, 900, and 1500 mg/kg body weight restored not just the WBC levels during concomitant administration with A/L but also other hematological parameters like the hemoglobin levels, red blood cell

Nitric oxide (NO) is known to have hepatoprotective and cardioprotective effects. Upon concomitant administration of A/L and UNCP at doses of 300, 900 and 1500 mg/kg, respectively. The NO observed by Asiedu-Gyekye's group was dose dependent, with 300 mg/kg of UNCP exhibiting the highest increase of 149.71% in NO (*p < 0.05*), 900 mg/kg gave a 34.25% (*p < 0.05*), and the 1500 mg/kg dose of UNCP showed 4.88% increment in NO (*p <* 0.05) [26]. The observed moderate nitric oxide increases that are beneficial could be attributed to the flavonoid content

UNCP restored some hematological disorders induced by high-dose A/L in guinea pigs by causing a significant increase in lymphocyte and platelet (PLT) levels at a dose of 1500 mg/kg. There was also an increase in NO with different doses of UNCP administration as a sequel to A/L dosing, which suggests that the combination (A/L and UNCP) is safe and advantageous. This study indicates the health benefits of daily ingestion of UNCP to prevent deleterious effects of A/L for the management of malaria.

*Theobroma cacao* has a lot of potential. UNCP was also investigated on its ability to help manage bronchial asthma. Anecdotal reports indicated that regular

and NO levels during high-dose (HD) A/L administration was investigated. The nitrite concentration in the plasma was measured as an index of NO levels by Griess reagent system (South Africa) according to the manufacturer's

the levels of white blood cell(WBC) count, lymphocyte count, hemoglobin (HGB), Red blood cell (RBC) count, and platelet counts in the group that received Coartem® (negative control group). Normally, these reduced indices imply bone marrow depression, autoimmune hemolytic anemia, systemic lupus, or severe

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

because of the high content of copper.

5.55, and 12.55%, respectively (*p* > 0.05).

of the unsweetened natural cocoa. Reference to this work is: [26].

*1.3.3.2 Anti-asthma potential*

*1.3.3.1 Conclusion*

counts, platelet counts, and all other parameters measured.

instruction.

hemorrhage.

*1.3.3 Hematological changes and nitric oxide levels*

simultaneous ingestion of A/L and UNCP may be beneficial to the heart and kidney. However, regular intake of large quantities of UNCP could be deleterious to health because of the high content of copper.

## *1.3.3 Hematological changes and nitric oxide levels*

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

*1.3.2.3 Renal function test*

14 and 10.64% in comparison with the VCG.

Coartem® group showed high levels of renal damage.

animals administered unsweetened natural cocoa powder extract.

cardioprotective effect have also been made by other researchers.

*1.3.2.4 Histopathological examination*

in serum CK showing the greatest mitigating activity against coartem toxicity. CK plays a crucial role in the conversion of creatine to phosphocreatine and adenosine diphosphate; thus, it might also protect or enhance myocardial bioenergetics.

Asiedu-Gyekye's group reported a reduction in urea by 53% in 1500 mg/kg when compared with the VCG (*p* < 0.05) [16]. Those of groups 3 and 4 were reduced by

The sodium, potassium, and chloride levels remained relatively unchanged in all study groups when compared with the control that received distilled water only [16]. The 1500 mg/kg group showed a significant reduction in urea levels as compared to the Coartem® only group. Creatinine levels decreased in all the groups compared with the control group. The authors attribute the observed effects to the antioxidant and nephroprotective effects of cocoa. Guinea pigs that received only the 75 mg/kg

Aspartate transferase, one of the enzymes mentioned earlier in this chapter, is distributed mostly in the heart followed by the liver and skeletal muscles. Elevated serum aspartate transferase values are hence indicative of cellular injury and may present in myocardial disease, shock, hypoxia, among others. The group administered with of distilled water +A/L showed a significantly increased serum levels of aspartate transferase. The reverse (significant reduction) was observed in all

These observations are corroborated by histopathological examination of the myocardial tissues of the guinea pigs (figure not shown), where tissue sections from animals that received only A/L 75 mg/kg showed evidence of inflammation and degeneration of the myocardial tissue. Normal cardiac tissue structures of myocardial tissue were observed in animals administered UNCP extract except those of animals that received 900 mg/kg UNCP where there was a single case observed with suspected ongoing tissue necrosis at the initial stages. Similar observations of the

Photomicrographs of myocardial tissues of animals from the different experimental groups revealed patchy areas of congestion, edema, extensive nuclear, and tissue degeneration leading to loss of microstructure of myocardial tissues for animals receiving 75 mg/kg A/L [16]. Animals that received 300 mg/kg UNCP and the control group retained the normal branching of myocardial cells characteristics [16]. Coronary risk ratio is an important indicator of cardiovascular health. Assessment of it showed that groups on 1500 mg/kg cocoa +A/L were at a higher risk compared with the control groups [16]. This observation corroborates the findings that although cocoa possesses many health benefits, high level intake could be deleterious, an effect believed to be caused by (−) epigallocatechin-3-gallate.

Unsweetened Natural Cocoa Powder showed renoprotective and cardioprotective potential during high-dose A/L administration [16]. It therefore suggests that

For the creatinine levels, significantly increments was observed; a 24.08% increase in NCG compared with VCG and decrease of 21.27, 17.54, and 11.05% in groups 3, 4, and 5, respectively, when compared with group 1 (*p* < 0.05) [16] .

**80**

*1.3.2.5 Conclusion*

Hematological parameters are one of the vital indices monitored during malaria treatment. Thus, in this study, the effect of UNCP on the hematological parameters and NO levels during high-dose (HD) A/L administration was investigated.

The nitrite concentration in the plasma was measured as an index of NO levels by Griess reagent system (South Africa) according to the manufacturer's instruction.

The results obtained from this study showed that A/L administration decreased the levels of white blood cell(WBC) count, lymphocyte count, hemoglobin (HGB), Red blood cell (RBC) count, and platelet counts in the group that received Coartem® (negative control group). Normally, these reduced indices imply bone marrow depression, autoimmune hemolytic anemia, systemic lupus, or severe hemorrhage.

Asiedu-Gyekye's group reported a reduction of 31.87% in the WBC count of the NCG (Coartem®) in comparison with the vehicle control group(*p* > 0.05) [26]. Administration of 300, 900, and 1500 mg/kg body weight of UNCP restored the WBC levels during concomitant administration with A/L (*p =* 0.1158)[26].

Prophylactic administration of UNCP with A/L at doses of 300, 900, and 1500 mg/kg body weight restored the decreased levels of the RBC count by 4.17, 5.55, and 12.55%, respectively (*p* > 0.05).

Administration of UNCP at doses of 300, 900, and 1500 mg/kg body weight restored not just the WBC levels during concomitant administration with A/L but also other hematological parameters like the hemoglobin levels, red blood cell counts, platelet counts, and all other parameters measured.

Nitric oxide (NO) is known to have hepatoprotective and cardioprotective effects. Upon concomitant administration of A/L and UNCP at doses of 300, 900 and 1500 mg/kg, respectively. The NO observed by Asiedu-Gyekye's group was dose dependent, with 300 mg/kg of UNCP exhibiting the highest increase of 149.71% in NO (*p < 0.05*), 900 mg/kg gave a 34.25% (*p < 0.05*), and the 1500 mg/kg dose of UNCP showed 4.88% increment in NO (*p <* 0.05) [26]. The observed moderate nitric oxide increases that are beneficial could be attributed to the flavonoid content of the unsweetened natural cocoa.

Reference to this work is: [26].

## *1.3.3.1 Conclusion*

UNCP restored some hematological disorders induced by high-dose A/L in guinea pigs by causing a significant increase in lymphocyte and platelet (PLT) levels at a dose of 1500 mg/kg. There was also an increase in NO with different doses of UNCP administration as a sequel to A/L dosing, which suggests that the combination (A/L and UNCP) is safe and advantageous. This study indicates the health benefits of daily ingestion of UNCP to prevent deleterious effects of A/L for the management of malaria.

## *1.3.3.2 Anti-asthma potential*

*Theobroma cacao* has a lot of potential. UNCP was also investigated on its ability to help manage bronchial asthma. Anecdotal reports indicated that regular consumption of UNCP was accompanied by prevention and reduction of asthmatic episodes. Bronchial asthma is prevalent in Ghana and West Africa. Due to its phytochemical composition and the presence of theobromine and theophylline, there could be a high possibility of a bronchodilatatory effect, the experiment was carried on in guinea pigs with prednisone and the drug for comparison. This study was a bronchial asthma model induced via the introduction of ovalbumin sensitization. The results showed a reduction in the brochoconstriction, inflammatory responses, and eosinophilia infiltration [9].

## *1.3.3.3 In vitro antimalarial activity of natural cocoa powder*

Anecdotal reports from Ghana suggest that a daily intake of a beverage of natural unsweetened cocoa powder could protect an individual against *Plasmodium falciparum* malaria. However, as at then, there was no known scientific report linking the consumption of cocoa or its products to the reduction in malaria incidence. Thus, a research conducted to determine this antiplasmodial activity and elucidate possible mechanisms of this activity. An *in vitro* inhibitory studies of extracts and fractions of cocoa powder against *P. falciparum* revealed that the nonpolar extract (chloroform, ethyl acetate, and petroleum ether) had better antiplasmodial activity than the polar extract [2]. The chloroform extract was the most active, with 50 and 90% inhibition concentration at 48.3 ± 0.9 and 41.7 ± 7.8 μg/mL, respectively. The ring-stage of *P. falciparum* treated with chloroform of natural cocoa powder showed a decline in growth. These results suggested that natural cocoa powder has measurable direct inhibitory activity against *P. falciparum*.

These studies attest to the additional future health benefits of unsweetened natural cocoa powder when consumed on daily basis especially its organoprotective role and also in attenuating high-dose artemether-lumefantrine organ effects. The antimalarial potential of cocoa is very promising especially in Africa where malaria is endemic, thus could be very beneficial in the management of uncomplicated malaria with very limited adverse effects on the major organs and reproductive system.

## **Author details**

Lovia Allotey-Babington1 , Awo Afi Kwapong1 , Kwame Benoit N'guessan Banga2 , Seth K. Amponsah2 and Isaac J. Asiedu-Gyekye2 \*

1 Department of Pharmaceutics and Microbiology, University of Ghana School of Pharmacy, Accra, Ghana

2 Department of Pharmacology and Toxicology, University of Ghana School of Pharmacy, Accra, Ghana

\*Address all correspondence to: ijasiedu-gyekye@ug.edu.gh

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**83**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

[10] Stanley TH et al. Analysis of cocoa proanthocyanidins using reversed phase high-performance liquid chromatography and electrochemical detection: Application to studies on the effect of alkaline processing. Journal of Agricultural and Food Chemistry.

[11] Oracz J, Zyzelewicz D, Nebesny E. The content of polyphenolic compounds in cocoa beans (*Theobroma cacao* L.), depending on variety, growing region, and processing operations: A review. Critical Reviews in Food Science and Nutrition. 2015;**55**(9):1176-1192

[12] Andres-Lacueva C et al. Flavanol and flavonol contents of cocoa powder products: Influence of the manufacturing process. Journal of Agricultural and Food Chemistry.

[13] Brunetto MadR et al. Determination of theobromine, theophylline and caffeine in cocoa samples by a highperformance liquid chromatographic method with on-line sample cleanup in a switching-column system. Food Chemistry. 2007;**100**(2):459-467

[14] Belščak A et al. Comparative study of commercially available cocoa products in terms of their bioactive composition. Food Research International. 2009;**42**(5):707-716

[15] Robbins RJ et al. Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography– fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples. Journal of Chromatography A. 2009;**1216**(24):4831-4840

[16] Asiedu-Gyekye IJ et al. A dietary strategy for the management of

2015;**63**(25):5970-5975

2008;**56**(9):3111-3117

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

[1] DeFelice SL. The nutraceutical revolution: Its impact on food industry R&D. Trends in Food Science & Technology. 1995;**6**(2):59-61

[2] Amponsah SK et al. In vitro activity of extract and fractions of natural cocoa powder on *Plasmodium falciparum*. Journal of Medicinal Food.

[3] Addai FK. Natural cocoa as diet-mediated antimalarial prophylaxis. Medical Hypotheses.

[4] Mishra BB, Tiwari VK. Natural products: An evolving role in future drug discovery. European Journal of Medicinal Chemistry.

2012;**15**(5):476-482

**References**

2010;**74**(5):825-830

2011;**46**(10):4769-4807

[5] Abere TA, Okoto PE,

Medicine. 2010;**10**(1):71

Agoreyo FO. Antidiarrhoea and toxicological evaluation of the leaf extract of *Dissotis rotundifolia triana* (Melastomataceae). BMC Complementary and Alternative

[6] Kumar G et al. Hepatoprotective activity of *Trianthema portulacastrum* L. against paracetamol and thioacetamide intoxication in albino rats. Journal of Ethnopharmacology. 2004;**92**(1):37-40

[7] Othman A et al. Antioxidant capacity and phenolic content of cocoa beans. Food Chemistry.

[8] Pan MH, S LC, and Ho CT. Antiinflammatory activity of natural dietary flavonoids. Food & Function, 2010. **1**(2042-650X (Electronic)): p. 15-31

[9] Awortwe C et al. Unsweetened natural cocoa has anti-asthmatic potential. International Journal of Immunopathology and Pharmacology.

2007;**100**(4):1523-1530

2014;**27**(2):203-212

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*

## **References**

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

*1.3.3.3 In vitro antimalarial activity of natural cocoa powder*

able direct inhibitory activity against *P. falciparum*.

and eosinophilia infiltration [9].

consumption of UNCP was accompanied by prevention and reduction of asthmatic episodes. Bronchial asthma is prevalent in Ghana and West Africa. Due to its phytochemical composition and the presence of theobromine and theophylline, there could be a high possibility of a bronchodilatatory effect, the experiment was carried on in guinea pigs with prednisone and the drug for comparison. This study was a bronchial asthma model induced via the introduction of ovalbumin sensitization. The results showed a reduction in the brochoconstriction, inflammatory responses,

Anecdotal reports from Ghana suggest that a daily intake of a beverage of natural unsweetened cocoa powder could protect an individual against *Plasmodium falciparum* malaria. However, as at then, there was no known scientific report linking the consumption of cocoa or its products to the reduction in malaria incidence. Thus, a research conducted to determine this antiplasmodial activity and elucidate possible mechanisms of this activity. An *in vitro* inhibitory studies of extracts and fractions of cocoa powder against *P. falciparum* revealed that the nonpolar extract (chloroform, ethyl acetate, and petroleum ether) had better antiplasmodial activity than the polar extract [2]. The chloroform extract was the most active, with 50 and 90% inhibition concentration at 48.3 ± 0.9 and 41.7 ± 7.8 μg/mL, respectively. The ring-stage of *P. falciparum* treated with chloroform of natural cocoa powder showed a decline in growth. These results suggested that natural cocoa powder has measur-

These studies attest to the additional future health benefits of unsweetened natural cocoa powder when consumed on daily basis especially its organoprotective role and also in attenuating high-dose artemether-lumefantrine organ effects. The antimalarial potential of cocoa is very promising especially in Africa where malaria is endemic, thus could be very beneficial in the management of uncomplicated malaria with very limited adverse effects on the major organs and reproductive

, Awo Afi Kwapong1

1 Department of Pharmaceutics and Microbiology, University of Ghana School of

2 Department of Pharmacology and Toxicology, University of Ghana School of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*

and Isaac J. Asiedu-Gyekye2

\*Address all correspondence to: ijasiedu-gyekye@ug.edu.gh

provided the original work is properly cited.

, Kwame Benoit N'guessan Banga2

,

**82**

system.

**Author details**

Seth K. Amponsah2

Lovia Allotey-Babington1

Pharmacy, Accra, Ghana

Pharmacy, Accra, Ghana

[1] DeFelice SL. The nutraceutical revolution: Its impact on food industry R&D. Trends in Food Science & Technology. 1995;**6**(2):59-61

[2] Amponsah SK et al. In vitro activity of extract and fractions of natural cocoa powder on *Plasmodium falciparum*. Journal of Medicinal Food. 2012;**15**(5):476-482

[3] Addai FK. Natural cocoa as diet-mediated antimalarial prophylaxis. Medical Hypotheses. 2010;**74**(5):825-830

[4] Mishra BB, Tiwari VK. Natural products: An evolving role in future drug discovery. European Journal of Medicinal Chemistry. 2011;**46**(10):4769-4807

[5] Abere TA, Okoto PE, Agoreyo FO. Antidiarrhoea and toxicological evaluation of the leaf extract of *Dissotis rotundifolia triana* (Melastomataceae). BMC Complementary and Alternative Medicine. 2010;**10**(1):71

[6] Kumar G et al. Hepatoprotective activity of *Trianthema portulacastrum* L. against paracetamol and thioacetamide intoxication in albino rats. Journal of Ethnopharmacology. 2004;**92**(1):37-40

[7] Othman A et al. Antioxidant capacity and phenolic content of cocoa beans. Food Chemistry. 2007;**100**(4):1523-1530

[8] Pan MH, S LC, and Ho CT. Antiinflammatory activity of natural dietary flavonoids. Food & Function, 2010. **1**(2042-650X (Electronic)): p. 15-31

[9] Awortwe C et al. Unsweetened natural cocoa has anti-asthmatic potential. International Journal of Immunopathology and Pharmacology. 2014;**27**(2):203-212

[10] Stanley TH et al. Analysis of cocoa proanthocyanidins using reversed phase high-performance liquid chromatography and electrochemical detection: Application to studies on the effect of alkaline processing. Journal of Agricultural and Food Chemistry. 2015;**63**(25):5970-5975

[11] Oracz J, Zyzelewicz D, Nebesny E. The content of polyphenolic compounds in cocoa beans (*Theobroma cacao* L.), depending on variety, growing region, and processing operations: A review. Critical Reviews in Food Science and Nutrition. 2015;**55**(9):1176-1192

[12] Andres-Lacueva C et al. Flavanol and flavonol contents of cocoa powder products: Influence of the manufacturing process. Journal of Agricultural and Food Chemistry. 2008;**56**(9):3111-3117

[13] Brunetto MadR et al. Determination of theobromine, theophylline and caffeine in cocoa samples by a highperformance liquid chromatographic method with on-line sample cleanup in a switching-column system. Food Chemistry. 2007;**100**(2):459-467

[14] Belščak A et al. Comparative study of commercially available cocoa products in terms of their bioactive composition. Food Research International. 2009;**42**(5):707-716

[15] Robbins RJ et al. Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography– fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples. Journal of Chromatography A. 2009;**1216**(24):4831-4840

[16] Asiedu-Gyekye IJ et al. A dietary strategy for the management of

artemether-lumefantrine-induced cardiovascular and renal toxicity. BMC Complementary and Alternative Medicine. 2016;**16**(1):348

[17] Stark T, Justus H, Hofmann T. Quantitative analysis of N-phenylpropenoyl-l-amino acids in roasted coffee and cocoa powder by means of a stable isotope dilution assay. Journal of Agricultural and Food Chemistry. 2006;**54**(8):2859-2867

[18] Ronen E. Micro-elements in agriculture. Practical Hydroponics and Greenhouses. 2016;**164**:35-44

[19] Carvalho ML et al. Study of heavy metals and other elements in macrophyte algae using energydispersive x-ray fluorescence. Environmental Toxicology and Chemistry. 1997;**16**(4):807-812

[20] Asiedu-Gyekye IJ et al. Unsweetened natural cocoa powder has the potential to attenuate high dose artemether-lumefantrine-induced hepatotoxicity in non-malarious guinea pigs. Evidence-based Complementary and Alternative Medicine: Ecam. 2016;**2016**:7387286-7387286

[21] Tarka SM, Cornish HH. The toxicology of cocoa and methylxanthines: A review of the literature. CRC Critical Reviews in Toxicology. 1982;**9**(4):275-312

[22] Tarka SM Jr et al. Chronic toxicity/ carcinogenicity studies of cocoa powder in rats. Food and Chemical Toxicology. 1991;**29**(1):7-19

[23] Fraga CG et al. Cocoa flavanols: Effects on vascular nitric oxide and blood pressure. Journal of Clinical Biochemistry and Nutrition. 2010;**48**(1):63-67

[24] Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. The FASEB Journal. 2007;**22**(3):659-661

[25] Ford ES. Serum copper concentration and coronary heart disease among US adults. American Journal of Epidemiology. 2000;**151**(12):1182-1188

[26] Asiedu-Gyekye IJ et al. Hematological changes and nitric oxide levels accompanying highdose artemether-lumefantrine administration in male guinea pigs: Effect of unsweetened natural cocoa powder. Journal of Intercultural Ethnopharmacology. 2016;**5**(4):350-357

[27] Dondorp AM et al. Artemisinin Resistance in *Plasmodium falciparum* Malaria. New England Journal of Medicine. 2009;**361**(5):455-467

[28] Phyo AP et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: A longitudinal study. The Lancet. 2012;**379**(9830):1960-1966

[29] Brice BK et al. In Vitro Susceptibility of *Plasmodium falciparum* Isolates from Abidjan, Cote d'Ivoire, to Artemisinin, Chloroquine, Dihydroartemisinin and Pyronaridine. (1821-6404 (Print))

[30] Antimalarial Drug Combination Therapy. Report of a WHO Technical Consultation. 2001. p. 36

[31] Group, W.A.R.N.W.A.D.I.S. The effect of dose on the antimalarial efficacy of artemether–lumefantrine: A systematic review and pooled analysis of individual patient data. The Lancet Infectious Diseases. 2015;**15**(6):692-702

[32] Owumi SE et al. Toxicity associated with repeated administration of artemether–lumefantrine in rats. Environmental Toxicology. 2015;**30**(3):301-307

[33] Osonuga IO et al. Effect of artemether on hematological parameters of healthy and uninfected adult Wistar rats. Asian Pacific Journal of Tropical Biomedicine. 2012;**2**(6):493-495

**85**

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective*

*DOI: http://dx.doi.org/10.5772/intechopen.88145*

Akpan O. Effects of chloroquine and coartem on haematological parameters in rats. African Journal of Biomedical

[35] Grassi D et al. Blood pressure is reduced and insulin sensitivity increased in glucose-intolerant, hypertensive subjects after 15 days of consuming high-polyphenol dark chocolate. The Journal of Nutrition.

[36] Bryan NS, Grisham MB. Methods to detect nitric oxide and its metabolites in biological samples. Free Radical Biology

& Medicine. 2007;**43**(5):645-657

[37] Badrie N et al. Cocoa agronomy, quality, nutritional, and health aspects. Critical Reviews in Food Science and Nutrition. 2015;**55**(5):620-659

[34] Ofem O, Essien NM,

Research. 2013;**16**:39-46

2008;**138**(9):1671-1676

*Unsweetened Natural Cocoa Powder: A Potent Nutraceutical in Perspective DOI: http://dx.doi.org/10.5772/intechopen.88145*

[34] Ofem O, Essien NM, Akpan O. Effects of chloroquine and coartem on haematological parameters in rats. African Journal of Biomedical Research. 2013;**16**:39-46

Theobroma cacao *- Deploying Science for Sustainability of Global Cocoa Economy*

[25] Ford ES. Serum copper

[26] Asiedu-Gyekye IJ et al. Hematological changes and nitric oxide levels accompanying highdose artemether-lumefantrine administration in male guinea pigs: Effect of unsweetened natural cocoa powder. Journal of Intercultural Ethnopharmacology. 2016;**5**(4):350-357

[27] Dondorp AM et al. Artemisinin Resistance in *Plasmodium falciparum* Malaria. New England Journal of Medicine. 2009;**361**(5):455-467

[28] Phyo AP et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: A longitudinal study. The Lancet. 2012;**379**(9830):1960-1966

[29] Brice BK et al. In Vitro Susceptibility of *Plasmodium falciparum* Isolates from Abidjan, Cote d'Ivoire, to Artemisinin, Chloroquine, Dihydroartemisinin and Pyronaridine. (1821-6404 (Print))

[30] Antimalarial Drug Combination Therapy. Report of a WHO Technical

[31] Group, W.A.R.N.W.A.D.I.S. The effect of dose on the antimalarial efficacy of artemether–lumefantrine: A systematic review and pooled analysis of individual patient data. The Lancet Infectious Diseases. 2015;**15**(6):692-702

[32] Owumi SE et al. Toxicity associated

artemether on hematological parameters of healthy and uninfected adult Wistar rats. Asian Pacific Journal of Tropical Biomedicine. 2012;**2**(6):493-495

with repeated administration of artemether–lumefantrine in rats. Environmental Toxicology.

[33] Osonuga IO et al. Effect of

2015;**30**(3):301-307

Consultation. 2001. p. 36

concentration and coronary heart disease among US adults. American Journal of Epidemiology. 2000;**151**(12):1182-1188

artemether-lumefantrine-induced cardiovascular and renal toxicity. BMC Complementary and Alternative

[17] Stark T, Justus H, Hofmann T.

N-phenylpropenoyl-l-amino acids in roasted coffee and cocoa powder by means of a stable isotope dilution assay. Journal of Agricultural and Food Chemistry. 2006;**54**(8):2859-2867

[18] Ronen E. Micro-elements in agriculture. Practical Hydroponics and

Greenhouses. 2016;**164**:35-44

[20] Asiedu-Gyekye IJ et al.

Unsweetened natural cocoa powder has the potential to attenuate high dose artemether-lumefantrine-induced hepatotoxicity in non-malarious guinea pigs. Evidence-based Complementary and Alternative Medicine: Ecam. 2016;**2016**:7387286-7387286

[21] Tarka SM, Cornish HH. The

1991;**29**(1):7-19

2010;**48**(1):63-67

2007;**22**(3):659-661

toxicology of cocoa and methylxanthines: A review of the literature. CRC Critical Reviews in Toxicology. 1982;**9**(4):275-312

[22] Tarka SM Jr et al. Chronic toxicity/ carcinogenicity studies of cocoa powder in rats. Food and Chemical Toxicology.

[23] Fraga CG et al. Cocoa flavanols: Effects on vascular nitric oxide and blood pressure. Journal of Clinical Biochemistry and Nutrition.

[24] Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. The FASEB Journal.

[19] Carvalho ML et al. Study of heavy metals and other elements in macrophyte algae using energydispersive x-ray fluorescence. Environmental Toxicology and Chemistry. 1997;**16**(4):807-812

Medicine. 2016;**16**(1):348

Quantitative analysis of

**84**

[35] Grassi D et al. Blood pressure is reduced and insulin sensitivity increased in glucose-intolerant, hypertensive subjects after 15 days of consuming high-polyphenol dark chocolate. The Journal of Nutrition. 2008;**138**(9):1671-1676

[36] Bryan NS, Grisham MB. Methods to detect nitric oxide and its metabolites in biological samples. Free Radical Biology & Medicine. 2007;**43**(5):645-657

[37] Badrie N et al. Cocoa agronomy, quality, nutritional, and health aspects. Critical Reviews in Food Science and Nutrition. 2015;**55**(5):620-659

**87**

**1. Introduction**

**Chapter 5**

*and Sunday Elaigwu*

**Abstract**

Exploration of Cocoa (*Theobroma* 

*cacao*) By-Products as Valuable

Feeds and Feeding Systems

*Olayinka John Makinde, Sunday A. Okunade,* 

Potential Resources in Livestock

*Emmanuel Opoola, Akeem Babatunde Sikiru, Solomon O. Ajide* 

High cost of feeds and feeding management remain unresolved challenges facing livestock production globally specifically in developing countries. More than half of the production cost is associated with feeds and feeding alone; hence, it becomes imperative for livestock production science to explore lesser known or poorly exploited resources for use in animal feeds and feeding systems to reduce cost and increase productivity. One of such strategies is the use of alternative or nonconventional feed resources. Cocoa by-products have been reported as one of such nonconventional feed resources that can replace expensive and competitive conventional feed resources in livestock diets. Cocoa bean meal, cocoa bean shells, and cocoa pod husks are all potential but unexploited nutritive resources that can be considered as animal feed materials. Although their use is severely restricted by antinutritional factor (ANF) theobromine, which is toxic to livestock, there exist modern nutritional technologies capable of being applied to improve application of these resources in livestock feeding systems. Therefore, this chapter presents cocoa by-products as potential tropical feed resources in animal feeds and feeding systems with a view to providing solution to waste management problems associated with cocoa processing factories while increasing animal productivity and reducing cost of animal production.

**Keywords:** cocoa by-products, environmental pollution, feeds, livestock, utilization

With current rising costs of conventional feed ingredients, animal nutritionists have advocated for the use of agro-industrial by-products as unconventional feedstuffs because they are cheaper and available in large quantities in producing countries. Cocoa pod husk, cocoa bean shell and cocoa bean meal form over 70% (w/w) of a whole matured fruit of cocoa (*Theobroma cacao* L.), and these are the major agroindustrial by-products from cocoa processing industries [1]. These by-products have continued to gain interest of researchers toward converting them to valuable uses such as in production of animal feeds—a critical contribution to improved food security.

## **Chapter 5**
