**2.** *Capsicum* **seeds as a source of bioactive compounds**

Like many fruits and plants, peppers are an excellent nutritional source. As aforementioned, it has an abundance of minerals, vitamins, aminoacids, carotenoids as also phytochemicals like phenolic compounds or polyunsaturated fatty acids (PUFAs). Likewise, capsaicins are unique to the genus *Capsicum*, being responsible of the pungency of many pepper species. Capsaicinoids have demonstrated to induce a vast range of bioactivities, such as anti-inflammatory, anticancer analgesic, antimicrobial, hypotensive and induce lower adipogenesis or lower body temperature by isolated action or in synergy with other compounds [21–25].

Different bioactive compounds have been isolated and extracted from *Capsicum* spp. fruits and seeds and its concentrations vary among species. More information on reported compounds studied may be found in (**Table 4**).

#### **2.1 Phenolic compounds**

nutritional value is acknowledge as well, since it is rich in vitamins (A, C, B6, E), carotenoids (β-carotene), flavonoids, oils, oleoresins and alkaloids [19]. Therefore, the compounds that can be found in this genre are carbohydrates (accounting for approximately 85% of dry weight), polyphenols (0.5% of dry weight) and important molecules such as capsaicinoids, carotenoids and vitamins [20]. Given these facts, peppers are considered a good source of most essential nutrients [14].

*Different* Capsicum *spp. and their proximate chemical composition (g/100 g) [14–18].*

**Common name State Carbohydrates Protein Fat Capsaicinoids Fiber Ash** Daechan D Nd 13.88 20.64 Nd 54.68 3.72 Mixed D Nd 14.01 24.09 Nd 50.26 3.26 Sandia D Nd 14.95 23.07 Nd 58.34 3.22 R-Naky D Nd 14.36 23.57 Nd 60.19 3.57 New Mexico 6 D Nd 14.79 21.95 Nd 60.61 3.48 LB-25 D Nd 14.87 25.06 Nd 52.98 3.29

The capsaicinoids content, which depends on the variety and maturation stage, will determinate the pungency. *C. chinense* and *Capsicum annuum* var. *aviculare* contain larger amounts of capsaicin and dihydrocapsaicin (ratio of 2:1), while some varieties of *Capsicum annuum* var*. annuum* showed an average proportion of 1:1. These variations could be attributed to environment, genetics and extraction methodologies [15]. Even though in some cases peppers are sought to be spicy, this can also be a limitation. This is the case, for example, in obtaining dyes from this raw material in the food industry. In this case, extraction methods need to be improved to prepare non-pungent oleoresins from pungent *Capsicum* fruits. This is achieved by the selective removal of capsaicinoids which allows the exploration of a large

Capsaicinoids are the characteristic pungent compounds of the *Capsicum* genus. They include capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin. Capsaicin is the most abundant. As an example, capsa-

The characteristic red color of many peppers is determined by the presence of different carotenoids. Actually, more than 50 different carotenoids can be found in this kind of material. Some of them are capsanthin, capsorubin, and cryptocapsin which give brilliant red color (ripe fruits) or β-carotene, violaxanthin, zeaxanthin and β-cryptoxanthin which give yellow–orange color [3]. Nevertheless, the color will depend on the state of maturity. For example, jalapeño (*C. annuum*) has a green

icin constitutes together with dihydrocapsaicin approximately 90% of total

color, and when it is mature it presents an intense red color [7]. Therefore, depending on the degree of maturity, the physicochemical parameters, flavor and mineral composition differs. For example, fat, protein, ascorbic acid (vitamin C), soluble solid content and titratable acidity will increase during ripening [14]. Using jalapeño as an example to study composition, its main component is water, then carbohydrates (5.3%), fiber (2.3%), protein (1.2%), fat (0.1%) and minerals, being the most important potassium (340 mg per 100 g of fresh product). It also has calcium (25 mg per 100 g of fresh product), magnesium (25 mg per 100 g of fresh product), sodium (7 mg per 100 g of fresh product), iron (2 mg per 100 g of fresh product) and zinc (0.3 mg per 100 g of fresh product). As for vitamins, the most important are ascorbic acid, retinol and folic acid (72, 20 and 23 mg per 100 g

number of pungent varieties with good oleoresin yielding [20].

capsaicinoids content of chili peppers [15].

*F, fresh; D, dried; Nd, not described.*

**Table 3.**

Capsicum

**128**

Phenolic compounds, also referred as phenolics, are secondary metabolites that may be found in a wide spectrum of plant species. They are synthesized as a result of adaptation to biotic and abiotic stress through the phenylpropanoid pathway playing an important role in plant development, because they act as a defensive mechanism that eases plant growth against harsh conditions [33, 34]. Based on recorded knowledge, phenolic compounds exhibit numerous potential health benefits that are already well described in scientific literature and are currently a major current focus of nutritional and pharmacological research [35, 36]. Phenolic acids and flavonoids are the main phenolic phytochemicals found in peppers. Likewise, capsaicinoids are synthetized in the same biochemical pathway and exhibit some similar properties such as antioxidative activity [34].

The yield of phenolic compounds recovered from an extraction can be very different, depending largely on the extraction method, the conservation of vegetal material and maturity state [37]. Generally, fresh raw material preserves the highest quantities of phenolic compounds [16]. Several factors contribute to yielding disparity, such as heterogeneous genotypes, growing and harvesting conditions of the samples [38]. Some of the most prominent non-capsaicinoid phenolic compounds because of their valuable health benefits are phenolic acids like gallic acid, caffeic acid, chlorogenic acid or ellagic acid (**Figure 2**). Flavones are another important group of phenolic compounds, being some of the most prominent kaempferol, quercetin, luteolin or rutin (**Figure 3**) [26]. These phenolics have demonstrated great health benefits and many of them are commercially available in purified products extracted from other plant species [39, 40]. Even so, their concentration differs among species but not much among varieties [26]. Other phenolic compounds are reviewed in the next paragraphs as in the case of some carotenoids or vitamins.

Regarding capsaicinois, they are synthesized naturally in the placenta of pepper fruits by enzymatic transformation of vanillylamine, the phenolic portion of the molecule, which confers this alkaloid its antioxidant capacity [41]. The seeds are not the primary source of capsaicinoids but they may absorb them because they are in close proximity to the placenta, which is the richest capsaicin fraction [42]. Their presence in the seed and the high concentrations they achieve, has been observed to


**Table 4.**

Capsicum *seeds bioactive compounds.*

rise the riper the pepper is [43]. It is also confirmed that fresh seeds yield more capsaicin than dry seeds, which suggests that surface capsaicinoids are sensitive to heat and/or oxidation [29]. "Hot" pepper cultivars attribute their pungency to high levels of capsaicinoids whereas non-pungent or "sweet" peppers (e.g., bell pepper) have very low capsaicinoids quantities [44].

Capsinoids exhibit biological activities similar to capsaicin due to being capsaicinoids

*Chemical structure of the main flavonoids found in pepper: (A) luteolin; (B) quercetin; (C) kaempferol; and*

*Chemical structure of some main phenolic acids found in peppers: (A) gallic acid; (B) ellagic acid; (C) caffeic*

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

Capsaicinoids and capsinoids manifest many promising therapeutic properties such as apoptosis induction, antioxidation, anticancer (cytostatic), or immunomodulation,

Besides, it is worth mentioning that the well-known flavone chrysoeriol (**Figure 4**), present in a multitude of vegetables and at least in *C. annuum* and *C. frutescens* fruit and seeds, has been determined to inhibit the growth of several

analogs, except that they display much lower pungency in comparison [45].

making them attractive targets for pharmaceutical research [24, 47].

microorganisms at very low quantities [31].

**Figure 2.**

**Figure 3.**

*(D) rutin.*

**131**

*acid and (D) chlorogenic acid.*

Capsinoids are other non-pungent capsaicin analogues noticeably found in *C. baccatum* var. *praetermissum* and the sweet cultivar CH-19 of *C. annuum* [45, 46]. Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties… DOI: http://dx.doi.org/10.5772/intechopen.91785*

#### **Figure 2.**

*Chemical structure of some main phenolic acids found in peppers: (A) gallic acid; (B) ellagic acid; (C) caffeic acid and (D) chlorogenic acid.*

#### **Figure 3.**

*Chemical structure of the main flavonoids found in pepper: (A) luteolin; (B) quercetin; (C) kaempferol; and (D) rutin.*

Capsinoids exhibit biological activities similar to capsaicin due to being capsaicinoids analogs, except that they display much lower pungency in comparison [45]. Capsaicinoids and capsinoids manifest many promising therapeutic properties such as apoptosis induction, antioxidation, anticancer (cytostatic), or immunomodulation, making them attractive targets for pharmaceutical research [24, 47].

Besides, it is worth mentioning that the well-known flavone chrysoeriol (**Figure 4**), present in a multitude of vegetables and at least in *C. annuum* and *C. frutescens* fruit and seeds, has been determined to inhibit the growth of several microorganisms at very low quantities [31].

rise the riper the pepper is [43]. It is also confirmed that fresh seeds yield more capsaicin than dry seeds, which suggests that surface capsaicinoids are sensitive to heat and/or oxidation [29]. "Hot" pepper cultivars attribute their pungency to high levels of capsaicinoids whereas non-pungent or "sweet" peppers (e.g., bell pepper)

Capsinoids are other non-pungent capsaicin analogues noticeably found in *C. baccatum* var. *praetermissum* and the sweet cultivar CH-19 of *C. annuum* [45, 46].

have very low capsaicinoids quantities [44].

**Species and varieties**

Capsicum

*C. annuum*

*C. annuum*

*C. annuum*

*C. annuum*

*C. annuum*

*C. annuum*

*C. annuum* var. *annuum*

*C. baccatum*

**Table 4.**

**130**

*Nd, not described.*

**Bioactive compounds Major phenolic compounds**

4-Hydroxybenzoic acid, vanillic acid, caffeic acid, *p*coumaric acid, sinapic acid, ferulic acid, ellagic acid, naringein

Elagic acid, gallic acid, chlorogenic acid, caffeic acid, salicylic acid, rutin

Caffeic acid, gallic acid, ferulic acid, rutin, capsaicin, dihydrocapsaicin

Nd Ascorbic

Gallic acid, caffeic acid, ferulic acid, chlorogenic acid, cathechin, epicathechin capsaicin, dihydrocapsaicin

Caffeic acid, cinnamic acid, coumaric acid, capsaicin, vanillin

Capsicum *seeds bioactive compounds.*

Tannins, flavonoids, coumarins, polyphenols

acid

Ascorbic acid

**Vitamins Pigments Volatiles PUFAs Peptides Ref.**

Nd Nd Nd Nd Nd [26]

Nd Nd Nd Nd Nd [21]

Nd Linoleic,

palmitic, oleic acids

Oleic, palmitic, linoleic, elaidic acids

Nd Nd Nd Nd [30]

Nd [27]

[28]

glucose Specific lectin

alanine

acids

[31]

[32]

Nd [29]

Tocopherols Capsanthin,

zeaxanthin, capsorubin, lutein, βcarotene, cryptocapsin

Nd Nd Nd Nd Nd Mannose/

Nd Betulin, βsitosterol

Nd Nd Nd Nd Phenyl-

Nd Nd Saponins Nd Amino

**2.5 Volatile compounds**

adecanoate, hexanal or isopulegol [62].

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

presenting a high antifungal activity [60].

positive results against some fungi [28, 64].

**Figure 5.**

**133**

*Chemical structure of CAY-1. Adapted from [60].*

**2.6 Other minor compounds**

Volatile compounds in *Capsicum* spp. have been studied because of the importance they have in many plants as pollinator attractors and their influence in odor and flavor, which are key features of any edible vegetable [59, 60]. Some of the most valuable are hexyl isopentanoate, hexyl isobutanoate, and β-ionone [61]. Extracts of *C. annuum* have also showed high levels of 4-methoxyphenol, ethylhex-

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

There are a few other minor and less studied compounds amid the different pepper species, because research has focused almost entirely on finding and studying phytochemicals of metabolic importance. In regard to these bioactive compounds, *C. annuum* or *C. frutescens* saponins have showed antimicrobial activity against several opportunistic and pathogenic fungi genres [63]. Saponins are triterpene glycosides with the ability to disrupt the cell membrane and wall of fungal cells during cell proliferation, which leads to cell lysis [27] As such, saponins do not have effect on non-germinated fungi, but they inhibit its growth. Although there are several present in pepper tissue, CAY-1 has been well-studied (**Figure 5**),

Another of these minor chemicals are lectins, a group of proteins with glycoside agglutination properties. Some lectins have been described in *C. frutescens* and *C. annuum* seeds and have been studied as possible antimicrobials and have yielded

**Figure 4.** *Chemical structure of chrysoeriol.*

#### **2.2 Fatty acids**

Peppers are fruits rich in polyunsaturated fatty acids (PUFAs), and their seeds show even greater concentration per gram [42]. The main fatty acids are indeed PUFAs [48]. These PUFAs, which are in the whole fruit and seeds, are mainly linoleic acid, palmitic acid α-linolenic acid and stearic acid [16, 48]. Moreover, peppers appear to have very low levels of saturated fatty acids. Linoleic acid has the highest concentration (≈70%) and the other fatty acids show much lower levels [48, 49]. PUFAs and specifically linoleic acid and α-linolenic acid, are recognized as essential fatty acids and are precursors of other important fatty acids in metabolism like arachidonic acid and eicosapentanoic acid or prostaglandins that in whole contribute to normal physiological performance [50].

#### **2.3 Pigments**

Chlorophylls and carotenoids constitute another group of valuable pepper nutrients that affect its color, but they also have important antioxidative, antiinflammatory effects and promote immune response [22, 51, 52]. Zeaxanthin, βcarotene, violaxanthin, lutein and β-cryptoxanthin are the pigments with highest concentration among *Capsicum* spp. [37, 43]. Capsanthin is another red colored pigment mostly present in red bell pepper (*C. annum*) [53]. They all show strong antioxidative properties and increase their levels the more mature the pepper is [44].

β-carotene and β-cryptoxanthin for instance, possess the added value of being able to be converted to vitamin A [54]. Vitamin A plays a vital role in disease prevention and development [54].

#### **2.4 Vitamins**

As it has been mentioned, peppers contain several vitamins like ascorbic acid and tocopherols like α-tocopherol, γ-tocopherol and δ-tocopherol, are all isomers of vitamin E [55, 56]. These essential vitamins contribute to the normal metabolism, promote immune response and also have antioxidative bioactivity [52, 57]. Because of this, ascorbic acid is an essential vitamin that is also used as a natural food preserver [52]. Indeed, peppers contain levels of vitamin C corresponding with those found in many citrus fruits and other vegetables considered good sources of this vitamin [14]. Tocopherols are well-known lipophilic antioxidants and appear to exert a vital part in the normal T lymphocyte maturation, lower age-related increase in tissue inflammation and lower interleukin production [55, 58]. This makes vitamin E an important modulator to an orderly and better inflammatory reaction, among other health benefits [58].

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties… DOI: http://dx.doi.org/10.5772/intechopen.91785*

### **2.5 Volatile compounds**

Volatile compounds in *Capsicum* spp. have been studied because of the importance they have in many plants as pollinator attractors and their influence in odor and flavor, which are key features of any edible vegetable [59, 60]. Some of the most valuable are hexyl isopentanoate, hexyl isobutanoate, and β-ionone [61]. Extracts of *C. annuum* have also showed high levels of 4-methoxyphenol, ethylhexadecanoate, hexanal or isopulegol [62].

#### **2.6 Other minor compounds**

**2.2 Fatty acids**

*Chemical structure of chrysoeriol.*

**Figure 4.**

Capsicum

**2.3 Pigments**

**2.4 Vitamins**

**132**

prevention and development [54].

among other health benefits [58].

Peppers are fruits rich in polyunsaturated fatty acids (PUFAs), and their seeds show even greater concentration per gram [42]. The main fatty acids are indeed PUFAs [48]. These PUFAs, which are in the whole fruit and seeds, are mainly linoleic acid, palmitic acid α-linolenic acid and stearic acid [16, 48]. Moreover, peppers appear to have very low levels of saturated fatty acids. Linoleic acid has the highest concentration (≈70%) and the other fatty acids show much lower levels [48, 49]. PUFAs and specifically linoleic acid and α-linolenic acid, are recognized as essential fatty acids and are precursors of other important fatty acids in metabolism like arachidonic acid and eicosapentanoic acid or prostaglandins that in whole

Chlorophylls and carotenoids constitute another group of valuable pepper nutri-

As it has been mentioned, peppers contain several vitamins like ascorbic acid and tocopherols like α-tocopherol, γ-tocopherol and δ-tocopherol, are all isomers of vitamin E [55, 56]. These essential vitamins contribute to the normal metabolism, promote immune response and also have antioxidative bioactivity [52, 57]. Because of this, ascorbic acid is an essential vitamin that is also used as a natural food preserver [52]. Indeed, peppers contain levels of vitamin C corresponding with those found in many citrus fruits and other vegetables considered good sources of this vitamin [14]. Tocopherols are well-known lipophilic antioxidants and appear to exert a vital part in the normal T lymphocyte maturation, lower age-related increase in tissue inflammation and lower interleukin production [55, 58]. This makes vitamin E an important modulator to an orderly and better inflammatory reaction,

ents that affect its color, but they also have important antioxidative, antiinflammatory effects and promote immune response [22, 51, 52]. Zeaxanthin, βcarotene, violaxanthin, lutein and β-cryptoxanthin are the pigments with highest concentration among *Capsicum* spp. [37, 43]. Capsanthin is another red colored pigment mostly present in red bell pepper (*C. annum*) [53]. They all show strong antioxidative properties and increase their levels the more mature the pepper is [44]. β-carotene and β-cryptoxanthin for instance, possess the added value of being able to be converted to vitamin A [54]. Vitamin A plays a vital role in disease

contribute to normal physiological performance [50].

There are a few other minor and less studied compounds amid the different pepper species, because research has focused almost entirely on finding and studying phytochemicals of metabolic importance. In regard to these bioactive compounds, *C. annuum* or *C. frutescens* saponins have showed antimicrobial activity against several opportunistic and pathogenic fungi genres [63]. Saponins are triterpene glycosides with the ability to disrupt the cell membrane and wall of fungal cells during cell proliferation, which leads to cell lysis [27] As such, saponins do not have effect on non-germinated fungi, but they inhibit its growth. Although there are several present in pepper tissue, CAY-1 has been well-studied (**Figure 5**), presenting a high antifungal activity [60].

Another of these minor chemicals are lectins, a group of proteins with glycoside agglutination properties. Some lectins have been described in *C. frutescens* and *C. annuum* seeds and have been studied as possible antimicrobials and have yielded positive results against some fungi [28, 64].

**Figure 5.** *Chemical structure of CAY-1. Adapted from [60].*

### **3. Bioactivities of the raw material**

In recent years, nutraceutical and therapeutic research has focused its view towards both exotic and domestic fruits and vegetables as a source of phytochemicals with the ability to induce beneficial bioactivities [65]. These natural chemicals have been and are already used as a main source of therapeutics in traditional and modern medicine, reaching one third of the total production of therapeutics [66]. Furthermore, processing waste by-products of fruits and vegetables are being researched as a viable source of phytochemicals that would be affordable and reduce economic and ecological impacts of wasted by-products or taking produce out of the food market [67, 68]. Thus, fractions that are not employed in nourishment such as the placenta, seeds or leaves of many species can prove to be a valuable resource instead of end as waste or fertilizer, which is the most common use for vegetable non-edible parts [61].

Taking into account the aforementioned compounds present in the many different species of the genus *Capsicum* and different regional cultivars, as in the case of *C. annuum*, many biological activities are expected to be found in direct or indirect physiological response to the presence of these substances. It is widely known that capasaicinoids, capsinoids, saponins, many phenolic compounds and its valuable nutrients are behind the beneficial effects of this vegetable, which has been traditionally employed as a source of medicinal remedies all across South America, Asia and Africa [63]. The intensity and type of activity may vary depending on the concentration of these compounds and will differ in each species and cultivar of *Capsicum*, the bioavailability of each compound, the ripening state and from which fraction of the vegetable the extracts are obtained [43, 44].

These bioactivities have been described in scientific reports through tissue culture and both animal and human test research as antioxidant, antimicrobial, antiinflammatory, anticancer, analgesic or even antidiabetic [21, 22, 34]. Considering this with the fact that *Capsicum* spp. is widely grown around the world, it makes it to be considered an excellent source of new therapeutics and dietary supplements that could lead to a healthier well-being. Indeed, many extracts of pepper fruits and seeds with a varying purification degree are marketed and can be purchased [69].

Some of the most recognized bioactivities found by chemicals in the whole pepper and its seeds will be reviewed and a brief compendium of the bioactivity research can be found in **Table 5**.

#### **3.1 Antioxidant**

Oxidative stress is caused as a result of the presence of reactive oxygen species (ROS) which may be produced in oxidative metabolism and exposure to the environment [33]. The term ROS englobes the molecules superoxide radical (O2 <sup>2</sup>), the hydroxyl radical (OH) and hydrogen peroxide (H2O2). They are produced by the sequential reduction of molecular oxygen in various metabolic reactions [76]. O2 2 is the most unstable form but it may dismutate to H2O2 by the action of endogenous superoxide dismutase (SOD) enzyme reaction or non-enzymatically [76]. The effect of oxidative stress mainly translates into changes in the rate of metabolic reduction reactions and an increased rate of DNA mutations and cell mitosis. They are also main signals indicating cell death, which in turn triggers inflammation via the pro-inflammatory factor nitric oxide (NO), which is released by macrophages [33, 76]. Since these are both cause and result of cancer development, it is considered that high levels of ROS are detrimental to health [40, 65, 76]. All living aerobic

organisms have developed defense mechanisms against oxidative stress through the synthesis of reductive biochemicals or enzymes such as SOD. The most important antioxidative biochemicals are phenolic compounds, which are prominently found

*Properties and mechanisms of bioactive compounds and* Capsicum *spp. extracts evaluation.*

**Activity Bioactives Species Type of**

Capsaicin *C. annuum*, *C.*

*C. annuum*, *C. baccatum* var. *pendulum*, *C. chinense*, *C. frutescens*

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

*C. annuum* var. *acunimatum*

*baccatum*, *C. chinense*, *C. frutescens*

Phenolic extracts *C. annuum* In vitro Gia. *S. aureus*,

CAY-1 *C. frutescens* In vitro Gia. *A. flavus*,

Lectins *C. annum* In vitro Gia. *A. flavus*,

Capsaicin Nd In vitro Inhibition of

Capsaicin Nd In vitro Inhibition of Ikα and Ikβ

Capsaicin Nd In vitro Inhibition of adipose

*-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt scavenging methods; ORAC, oxygen radical*

*Nd, not described; Gia., growth inhibition against; DPPH, 2,2-diphenyl-1picrylhydrazyl scavenging method; ABTS,*

*C. annuum* In vitro Gia. *E. faecalis*,

*C. frutescens* In vitro Gia *E. faecalis*, *B. subtillis*,

*C. baccatum* In vitro Gia *S. epidermidis*,

dihydrocapsaicin

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

Total phenolic compounds

Total polyphenol content, capsaicins

Capsaicinoids, chrysoeriol

Residual aqueous extract

Antiinflammatory

*2,2*<sup>0</sup>

**Table 5.**

**135**

*absorbance capacity.*

Jalapeño extracts *C. annuum* var.

*annuum*

Antioxidant Capsaicin,

Antimicrobial Capsicosides A, G, D

**study**

Phenolic extracts *C. annuum* In vitro DPPH, ferrus chelating [39] Seed oil *C. annuum* In vitro DPPH, ABTS [42]

*C. annuum* In vitro DPPH, ABTS, ORAC [26]

**Test results Ref.**

In vitro DPPH, ABTS [68]

In vitro Gia. various yeasts [32]

[31]

[70]

[60]

[28]

[21]

[31]

[72]

[73]

[74]

[75]

In vitro Gia. *Clostridium tetani* & *C. sporogenes*

> *L. monocytogenes*, *S. typhimurium*, *B. cereus*

*A. fumigatus*, *A. parasiticus*, *A. niger*, *C. albicans*, *P. carinii*

*F. graminearum*

*S. aureus*

*P. aeruginosa*, *E. coli*,

In vitro Gia. *L. monocytogenes* [71]

*S. aureus*, *P. aeruginosa*, *E. coli*, *C. albicans*

*P. aeruginosa* biofilm

tissue inflammation (interleukin 8, c-Jun)

inflammatory transcription factor NF-κB and AP-1

via NF-κB


Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties… DOI: http://dx.doi.org/10.5772/intechopen.91785*

*Nd, not described; Gia., growth inhibition against; DPPH, 2,2-diphenyl-1picrylhydrazyl scavenging method; ABTS, 2,2*<sup>0</sup> *-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt scavenging methods; ORAC, oxygen radical absorbance capacity.*

#### **Table 5.**

**3. Bioactivities of the raw material**

Capsicum

use for vegetable non-edible parts [61].

extracts are obtained [43, 44].

research can be found in **Table 5**.

**3.1 Antioxidant**

**134**

In recent years, nutraceutical and therapeutic research has focused its view towards both exotic and domestic fruits and vegetables as a source of phytochemicals with the ability to induce beneficial bioactivities [65]. These natural chemicals have been and are already used as a main source of therapeutics in traditional and modern medicine, reaching one third of the total production of therapeutics [66]. Furthermore, processing waste by-products of fruits and vegetables are being researched as a viable source of phytochemicals that would be affordable and reduce economic and ecological impacts of wasted by-products or taking produce out of the food market [67, 68]. Thus, fractions that are not employed in nourishment such as the placenta, seeds or leaves of many species can prove to be a valuable resource instead of end as waste or fertilizer, which is the most common

Taking into account the aforementioned compounds present in the many different species of the genus *Capsicum* and different regional cultivars, as in the case of *C. annuum*, many biological activities are expected to be found in direct or indirect physiological response to the presence of these substances. It is widely known that capasaicinoids, capsinoids, saponins, many phenolic compounds and its valuable nutrients are behind the beneficial effects of this vegetable, which has been traditionally employed as a source of medicinal remedies all across South America, Asia and Africa [63]. The intensity and type of activity may vary depending on the concentration of these compounds and will differ in each species and cultivar of *Capsicum*, the bioavailability of each compound, the ripening state and from which fraction of the vegetable the

These bioactivities have been described in scientific reports through tissue culture and both animal and human test research as antioxidant, antimicrobial, antiinflammatory, anticancer, analgesic or even antidiabetic [21, 22, 34]. Considering this with the fact that *Capsicum* spp. is widely grown around the world, it makes it to be considered an excellent source of new therapeutics and dietary supplements that could lead to a healthier well-being. Indeed, many extracts of pepper fruits and seeds with a varying purification degree are marketed and can be purchased [69]. Some of the most recognized bioactivities found by chemicals in the whole pepper and its seeds will be reviewed and a brief compendium of the bioactivity

Oxidative stress is caused as a result of the presence of reactive oxygen species (ROS) which may be produced in oxidative metabolism and exposure to the environment [33]. The term ROS englobes the molecules superoxide radical (O2

hydroxyl radical (OH) and hydrogen peroxide (H2O2). They are produced by the sequential reduction of molecular oxygen in various metabolic reactions [76]. O2

is the most unstable form but it may dismutate to H2O2 by the action of endogenous superoxide dismutase (SOD) enzyme reaction or non-enzymatically [76]. The effect of oxidative stress mainly translates into changes in the rate of metabolic reduction reactions and an increased rate of DNA mutations and cell mitosis. They are also main signals indicating cell death, which in turn triggers inflammation via the pro-inflammatory factor nitric oxide (NO), which is released by macrophages [33, 76]. Since these are both cause and result of cancer development, it is considered that high levels of ROS are detrimental to health [40, 65, 76]. All living aerobic

<sup>2</sup>), the

2

*Properties and mechanisms of bioactive compounds and* Capsicum *spp. extracts evaluation.*

organisms have developed defense mechanisms against oxidative stress through the synthesis of reductive biochemicals or enzymes such as SOD. The most important antioxidative biochemicals are phenolic compounds, which are prominently found

in plants [52, 66]. These antioxidants present in peppers, mainly identified as the mentioned phenolic compounds, vitamins and pigments, have the potential to reduce biological oxidative stress and thus preventing the incidence of many related diseases, but also to further food preservation by inhibiting the oxidative metabolism of decomposing fungi and bacteria [33, 35, 38]. Phenolic compounds are the principal antioxidants in nature and generally show the greatest antioxidative capacities [65]. However, high concentrations of few phenolic compounds showed pro-oxidant effects due to a concentration imbalance between ROS and the phenols, which is why it is important to maintain intake of different phenolic compounds in order to benefit from their antioxidant properties [52]. Thus, antioxidants have a wide extent of applications, and many natural and synthetic antioxidants have been used by the food industry in order to better preserve raw or precooked products that would otherwise have a much shorter shelf-life [22].

*Listeria monocytogenes* [21, 32, 38, 79]. It has been reported that it can also reduce the virulence of pathogenic infections by *Vibrio cholerae* or *L. monocytogenes*, by inhibiting pro-inflammatory factors or toxin production [79–81]. There are few *in vivo* studies of these effects, though there are some reports of poultry showing less hepatic damage from *Salmonella enteritidis* when given feed supplemented with capsaicinoids, which could be related to its antioxidative activity [24].

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

Capsaicinoids appear to inhibit, fungi, and both Gram positive and negative bacterial growth in several studies. In all of them, capsaicin and/or dihydrocapsaicin showed to stop or slow colony development in a significant degree [24, 35]. Some interesting results of antimicrobial tests with capsaicinoids reported that it inhibits the growth of *Escherichia coli*, *L. monocytogenes*, *Bacillus subtillis* and many strains of

Even though capsaicinoids possess these interesting antimicrobial properties, the positive correlation between antimicrobial activities, concentration and its pungency and potential irritation effects make them less suitable when it comes to applying this compound as a food preserver [46]. Thus, it is interesting to study the effects on this area of alternative chemicals found in pepper fruit and seeds. CAY-1,

CAY-1 is found in *C. frutescens* and a study demonstrated to produce cell membrane lysis in fungi like *Aspergillus flavus*, *Aspergillus niger*, *Fusarium solani*, *Fusarium moniliforme*, *Pneumocystis carinii* or *Cansida albicans* [60]. It is ineffective though against bacteria though, probably because of the different type of cell membrane and wall [60]. It has also been suggested to combine this saponin with certain metallic elements such as silver in order to produce antimicrobial surfactants [82]. Other saponins isolated from *C. annuum* seeds, like capsicosides, which are furostanol saponins, have showed similar effects on growth inhibition against

Chrysoeriol is another potent antimicrobial compound present in a variety of plants as well as in peppers [83]. Several studies have presented that chrysoeriol is capable of greatly inhibit the growth of *Enterococcus faecalis*, *Klebsiella pneumoniae*, *S. aureus*, *L. monocytogenes*, *B. subtillis* or *C. albicans* [31, 84]. Given the very low concentrations at which it exerts its antimicrobial effect, it may be an optimal food

Certain lectins isolated from peppers have showed antifungal properties, inhibiting growth of some common opportunistic fungi like *Aspergillus flavus* and *Fusarium moniliforme* by limiting surface adherence. However, its inhibitory action

tides from *C. baccatum* var. *pendulum* on biofilms made by *Staphylococcus*

Furthermore, a recent study analyzed the antiadhesive capacity of natural pep-

*epidermidis* [64]. Considering that biofilms are a source of significant virulence, new studies on this field present extremely interesting. Altogether, these findings may contribute to develop new uses for natural antimicrobials, which would be an application of vital priority, given the global concern on the appearance of new-

Although an anti-inflammatory action is carried indirectly by the antioxidant capacity of other compounds to scavenge ROS and inhibit NO production, it is known that capsaicins and capsiates directly induce an anti-inflammatory effect through the activation of the transient receptor potential vanilloid 1 receptor (TRPV1) [24, 86]. TRPV1 is an ion channel receptor located in several glia that gives sensations of heat, but is also relevant in pain perception and induces this sensation,

chrysoeriol and lectins have been previously mentioned as such.

preserver against many opportunistic and nosocomial microbes.

is not as effective across all species of these genres [28, 85].

born antibiotic resistant microorganisms [31, 84].

**3.3 Anti-inflammatory**

**137**

*Staphylococcus aureus* [21].

yeasts [27].

The antioxidative effect of the aforementioned phenolic compounds essentially works by scavenging free superoxide and hydroxyl radicals and thus preventing high levels of ROS, NO and oxidation of sensitive biomolecules like proteins or lipids (**Figure 6**) [36, 40]. This results in a better physiological performance, immunomodulation and DNA mutation protection. Furthermore, the decrease of oxidative stress results in better cardiovascular health. This is due to the fact that low-density lipoprotein (LDL), as the main cholesterol carrier in the circulatory system, is susceptible to oxidation by ROS [58]. Oxidized LDL presence has been found to be cause of atherosclerosis, a vascular ailment caused by the formation of plaques inside the arteries that may weaken blood flow and lead to cardiovascular diseases [77]. On top of that, pepper and its seeds are described to be one of the richest vegetables in phenolic compounds [29]. Then, it should be taken into account the antioxidant properties of the many phenolic acids, capsaicinoids, vitamins and pigments present among the *Capsicum* genus and the fact that these exploitable compounds are available not only in the fruit, but also in the non-edible fractions [39, 57, 78].

## **3.2 Antimicrobial**

Pepper seed extracts and selected pigments, phenols, capsaicins and capsiates have been tested against the most common microorganisms present in foods and/or potential pathogens. The major antimicrobial effect of capsaicinoids has been found to be against common opportunistic and pathogenic fungi like *Fusarium* spp., *Aspergillus* spp. and yeasts like *Saccharomyces cerevisiae* or *Candida albicans*, but also against certain bacteria like *Streptococcus* spp., *Helycobacter pylori*, *Pseudomonas aeruginosa* or

#### **Figure 6.**

*Scheme of radical scavenging performed by gallic acid as an example of phenolic compounds. Adapted from [36].*

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties… DOI: http://dx.doi.org/10.5772/intechopen.91785*

*Listeria monocytogenes* [21, 32, 38, 79]. It has been reported that it can also reduce the virulence of pathogenic infections by *Vibrio cholerae* or *L. monocytogenes*, by inhibiting pro-inflammatory factors or toxin production [79–81]. There are few *in vivo* studies of these effects, though there are some reports of poultry showing less hepatic damage from *Salmonella enteritidis* when given feed supplemented with capsaicinoids, which could be related to its antioxidative activity [24].

Capsaicinoids appear to inhibit, fungi, and both Gram positive and negative bacterial growth in several studies. In all of them, capsaicin and/or dihydrocapsaicin showed to stop or slow colony development in a significant degree [24, 35]. Some interesting results of antimicrobial tests with capsaicinoids reported that it inhibits the growth of *Escherichia coli*, *L. monocytogenes*, *Bacillus subtillis* and many strains of *Staphylococcus aureus* [21].

Even though capsaicinoids possess these interesting antimicrobial properties, the positive correlation between antimicrobial activities, concentration and its pungency and potential irritation effects make them less suitable when it comes to applying this compound as a food preserver [46]. Thus, it is interesting to study the effects on this area of alternative chemicals found in pepper fruit and seeds. CAY-1, chrysoeriol and lectins have been previously mentioned as such.

CAY-1 is found in *C. frutescens* and a study demonstrated to produce cell membrane lysis in fungi like *Aspergillus flavus*, *Aspergillus niger*, *Fusarium solani*, *Fusarium moniliforme*, *Pneumocystis carinii* or *Cansida albicans* [60]. It is ineffective though against bacteria though, probably because of the different type of cell membrane and wall [60]. It has also been suggested to combine this saponin with certain metallic elements such as silver in order to produce antimicrobial surfactants [82]. Other saponins isolated from *C. annuum* seeds, like capsicosides, which are furostanol saponins, have showed similar effects on growth inhibition against yeasts [27].

Chrysoeriol is another potent antimicrobial compound present in a variety of plants as well as in peppers [83]. Several studies have presented that chrysoeriol is capable of greatly inhibit the growth of *Enterococcus faecalis*, *Klebsiella pneumoniae*, *S. aureus*, *L. monocytogenes*, *B. subtillis* or *C. albicans* [31, 84]. Given the very low concentrations at which it exerts its antimicrobial effect, it may be an optimal food preserver against many opportunistic and nosocomial microbes.

Certain lectins isolated from peppers have showed antifungal properties, inhibiting growth of some common opportunistic fungi like *Aspergillus flavus* and *Fusarium moniliforme* by limiting surface adherence. However, its inhibitory action is not as effective across all species of these genres [28, 85].

Furthermore, a recent study analyzed the antiadhesive capacity of natural peptides from *C. baccatum* var. *pendulum* on biofilms made by *Staphylococcus epidermidis* [64]. Considering that biofilms are a source of significant virulence, new studies on this field present extremely interesting. Altogether, these findings may contribute to develop new uses for natural antimicrobials, which would be an application of vital priority, given the global concern on the appearance of newborn antibiotic resistant microorganisms [31, 84].

#### **3.3 Anti-inflammatory**

Although an anti-inflammatory action is carried indirectly by the antioxidant capacity of other compounds to scavenge ROS and inhibit NO production, it is known that capsaicins and capsiates directly induce an anti-inflammatory effect through the activation of the transient receptor potential vanilloid 1 receptor (TRPV1) [24, 86]. TRPV1 is an ion channel receptor located in several glia that gives sensations of heat, but is also relevant in pain perception and induces this sensation,

in plants [52, 66]. These antioxidants present in peppers, mainly identified as the mentioned phenolic compounds, vitamins and pigments, have the potential to reduce biological oxidative stress and thus preventing the incidence of many related diseases, but also to further food preservation by inhibiting the oxidative metabolism of decomposing fungi and bacteria [33, 35, 38]. Phenolic compounds are the principal antioxidants in nature and generally show the greatest antioxidative capacities [65]. However, high concentrations of few phenolic compounds showed pro-oxidant effects due to a concentration imbalance between ROS and the phenols, which is why it is important to maintain intake of different phenolic compounds in order to benefit from their antioxidant properties [52]. Thus, antioxidants have a wide extent of applications, and many natural and synthetic antioxidants have been used by the food industry in order to better preserve raw or precooked products that

The antioxidative effect of the aforementioned phenolic compounds essentially works by scavenging free superoxide and hydroxyl radicals and thus preventing high levels of ROS, NO and oxidation of sensitive biomolecules like proteins or lipids (**Figure 6**) [36, 40]. This results in a better physiological performance, immunomodulation and DNA mutation protection. Furthermore, the decrease of oxidative stress results in better cardiovascular health. This is due to the fact that low-density lipoprotein (LDL), as the main cholesterol carrier in the circulatory system, is susceptible to oxidation by ROS [58]. Oxidized LDL presence has been found to be cause of atherosclerosis, a vascular ailment caused by the formation of plaques inside the arteries that may weaken blood flow and lead to cardiovascular diseases [77]. On top of that, pepper and its seeds are described to be one of the richest vegetables in phenolic compounds [29]. Then, it should be taken into account the antioxidant properties of the many phenolic acids, capsaicinoids, vitamins and pigments present among the *Capsicum* genus and the fact that these exploitable compounds are avail-

able not only in the fruit, but also in the non-edible fractions [39, 57, 78].

Pepper seed extracts and selected pigments, phenols, capsaicins and capsiates have been tested against the most common microorganisms present in foods and/or potential pathogens. The major antimicrobial effect of capsaicinoids has been found to be against common opportunistic and pathogenic fungi like *Fusarium* spp., *Aspergillus* spp. and yeasts like *Saccharomyces cerevisiae* or *Candida albicans*, but also against certain bacteria like *Streptococcus* spp., *Helycobacter pylori*, *Pseudomonas aeruginosa* or

*Scheme of radical scavenging performed by gallic acid as an example of phenolic compounds. Adapted*

would otherwise have a much shorter shelf-life [22].

**3.2 Antimicrobial**

Capsicum

**Figure 6.**

*from [36].*

**136**

as well as inflammation [87]. By activating this receptor, capsaicins cause the wellknown pungency sensation, as well as irritation in high enough concentrations. However, after activation by capsaicinoids, the excited neurons become resistant to further stimuli [87]. Hence, after a brief burning sensation, capsaicin may act as a local analgesic in neuropathic pain [84].

**Compound Species Extraction Medium Yield Ref. Type Method** Pigments *Capsicum* spp. Emergent SFE — 6% [90]

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

Carotenoids *C. annuum* Green EAE Viscozyme L 87% [95]

Phenolics *C. annuum* Emergent SFE — 84% [93]

Capsaicinoids *Capsicum* spp. Green MAE Acetone (30%) 0.48 mg/g [97]

*C. annuum* Green MAE Acetone-water — [91]

*C. annuum* Green MAE None 230 ppm [98]

*C. annuum* Green EAE Viscozyme L 88.8% [96]

*C. frutescens* Green MAE Acetone (pure) 5.3 mg/g [104]

*C. chinense* Green UAE Acetone (100%) 0.31 mg/g [109]

*C. baccatum* Green UAE Methanol (100%) 50% [112]

*C. annuum* Green EAE Viscozyme L 6% [101]

*C. frutescens* Emergent SFE — 0.3% [108]

Antioxidants *C. baccatum* Green UAE Methanol (100%) 27% [112] OLEORESIN *C. baccatum* Green UAE Methanol (100%) 26% [112]

Other phytochemicals *Capsicum* spp. Green UAE Methanol (80%) — [114]

*Bioactive compounds obtained from* Capsicum *spp. extracted with different extraction methods.*

**Table 6.**

**139**

Emergent SFE — 8.6% [92]

Emergent SFE — 710 μg/g [103]

Emergent SFE — 0.5% [111]

Emergent SFE — 8.2% [102]

Emergent SFE — 17.4% [92]

— 93% [93] — 7.2% [94]

Pectinase 80% [95] Cellulase Low [95] Viscozyme L 78% [96]

— 100% [94]

UAE None 200 ppm [97]

n-hexane (100%) — [99] Methanol (100%) 100% [100]

Viscozyme L 22% [101] Celluclast 20% [101]

> — — [102] — 93% [93] — 710 μg/g [103]

Ethanol (99.5%) — [105]

Methanol (100%) 100% [100] Acetone (100%) 3.92 mg/g [107]

Methanol (100%) 2.88 mg/g [110]

— 5.2% [108]

— 7.4% [94] — — [113]

UAE Ethanol (95%) 87.4% [106]

Research on capsaicinoids has showed that these molecules can also decrease inflammation of adipose tissue linked to obesity [73, 75]. It seems that capsaicin is able to inhibit or at least partially decrease the production of pro-inflammatory signals like interleukin 8 (IL-8), nuclear factor kappa-light-chain-enhancer of activated B cells (NF\kappaB) or active protein 1 (AP-1) [74, 80]. This antiinflammatory effect has the potential to make inflamed tissue less prone to tumor development as well as reduce the infection caused inflammation [25].

Still, the pungency of capsaicins poses a setback for using them without potentially hazardous secondary effects. That is why capsinoids may be a good alternative to induce similar effects to capsaicin, even though they are not found in so many pepper species [41, 45].

Although the importance of researching capsaicinoids bioactivities in pepper is due to its unique presence in this genre, the also present phenolic compounds, vitamins and pigments also bear significant anti-inflammatory properties [25, 88, 89]. Indeed, the antioxidative properties of phenolic compounds could be a key factor in reducing age-related extended tissue inflammation [25, 58]. Nonetheless, further research assessing the suitability of using as anti-inflammatory drug these phytochemicals, and specifically capsaicin, is needed.

### **4. Extraction systems for bioactive compounds from** *Capsicum* **spp.**

The bioactive compounds obtained from nature are secondary metabolites produced by the organisms. The concentrations of each type of compound are very variable, so in order to have enough quantities, the development of new and advanced technologies is needed.

Although there are several extraction methodologies, there is a demand for more appropriate and standardized extraction strategies. For choosing one method over another, it must be taken into account the quantitative and qualitative characteristics of the compound of interest. In addition, to improve the efficiency of a method it is important that the nature of the source, the different parameters of the method and the possible interaction are taken into account. Experiments carried out with peppers can be seen in **Table 6**.

#### **4.1 Conventional extraction systems**

#### *4.1.1 Maceration*

Maceration (MA) is a type of solid–liquid extraction, in which the solid would be peppers. This raw material is characterized by having several compounds soluble in the liquid phase. The type of molecules extracted will depend on two factors: the type of starting material and the type of solvent used.

In order to carry out this process, the sample is suspended in the solvent at the desired temperature during the chosen time while stirring. Once the process has been carried out, the sample is centrifuged and the supernatant, which is where the compounds of interest are found, is filtered. Therefore, the process of extraction consists of two stages. On the first (washing) there is a rapid transfer of the compounds from the surface of the solid to the solvent. On the second one (transfer), the matter passes from the interior to the exterior of the solid by diffusion


#### Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties… DOI: http://dx.doi.org/10.5772/intechopen.91785*

#### **Table 6.**

*Bioactive compounds obtained from* Capsicum *spp. extracted with different extraction methods.*

as well as inflammation [87]. By activating this receptor, capsaicins cause the wellknown pungency sensation, as well as irritation in high enough concentrations. However, after activation by capsaicinoids, the excited neurons become resistant to further stimuli [87]. Hence, after a brief burning sensation, capsaicin may act as a

Research on capsaicinoids has showed that these molecules can also decrease inflammation of adipose tissue linked to obesity [73, 75]. It seems that capsaicin is able to inhibit or at least partially decrease the production of pro-inflammatory signals like interleukin 8 (IL-8), nuclear factor kappa-light-chain-enhancer of acti-

inflammatory effect has the potential to make inflamed tissue less prone to tumor

Still, the pungency of capsaicins poses a setback for using them without potentially hazardous secondary effects. That is why capsinoids may be a good alternative to induce similar effects to capsaicin, even though they are not found in so many

Although the importance of researching capsaicinoids bioactivities in pepper is due to its unique presence in this genre, the also present phenolic compounds, vitamins and pigments also bear significant anti-inflammatory properties

[25, 88, 89]. Indeed, the antioxidative properties of phenolic compounds could be a

Nonetheless, further research assessing the suitability of using as anti-inflammatory

The bioactive compounds obtained from nature are secondary metabolites produced by the organisms. The concentrations of each type of compound are very variable, so in order to have enough quantities, the development of new and

Although there are several extraction methodologies, there is a demand for more appropriate and standardized extraction strategies. For choosing one method over another, it must be taken into account the quantitative and qualitative characteristics of the compound of interest. In addition, to improve the efficiency of a method it is important that the nature of the source, the different parameters of the method and the possible interaction are taken into account. Experiments carried out with

Maceration (MA) is a type of solid–liquid extraction, in which the solid would be peppers. This raw material is characterized by having several compounds soluble in the liquid phase. The type of molecules extracted will depend on two factors: the

In order to carry out this process, the sample is suspended in the solvent at the desired temperature during the chosen time while stirring. Once the process has been carried out, the sample is centrifuged and the supernatant, which is where the compounds of interest are found, is filtered. Therefore, the process of extraction consists of two stages. On the first (washing) there is a rapid transfer of the compounds from the surface of the solid to the solvent. On the second one (transfer), the matter passes from the interior to the exterior of the solid by diffusion

**4. Extraction systems for bioactive compounds from** *Capsicum* **spp.**

key factor in reducing age-related extended tissue inflammation [25, 58].

drug these phytochemicals, and specifically capsaicin, is needed.

vated B cells (NF\kappaB) or active protein 1 (AP-1) [74, 80]. This anti-

development as well as reduce the infection caused inflammation [25].

local analgesic in neuropathic pain [84].

pepper species [41, 45].

Capsicum

advanced technologies is needed.

peppers can be seen in **Table 6**.

*4.1.1 Maceration*

**138**

**4.1 Conventional extraction systems**

type of starting material and the type of solvent used.

being this step the limiting stage. This method is used, for example, for the isolation of phenolic compounds of vegetable origin for their subsequent use in the food, pharmaceutical and cosmetic industries [115].

In order to improve these conventional extraction techniques, other methods have been developed (ultrasound assisted extraction, pressurized hot water extraction, negative pressure cavitation-assisted extraction and pulsed electric) to partner with them and solve some of the inopportuneness of traditional techniques. In this

This group includes several techniques with the aim of not only preventing pollution but also reducing sample preparation costs by, for example, lowering

Microwave-assisted extraction (MAE) is based on the heating of solvents that are in contact with solid samples with the use of microwaves. This allows the partition of compounds of interest from the sample into the solvent. Microwave energy accelerates a great variety of chemical reactions as well as the extraction of organic compounds from different matrices [98]. During the process, electromagnetic energy is transformed into calorific energy by two mechanisms: ionic conduction and dipole rotation [119]. It is a method that could be an alternative for avoiding thermal degradation and oxidation, with no influence on cell integrity and shape. However, due to thermal stress and localized high pressures, cell rupture is more rapid than with another techniques, which is an inconvenience, for example,

Some of the advantages of this technique are simplicity, effectiveness, low processing time, low solvent consumption and energy, no generation of secondary waste and can be used for larger volumes [120]. Another advantage of this method is that it produces a uniform heating, so extraction is simultaneous regardless of the area where the compound is [121]. By using this method, lipids, pigments, carbohydrates, vitamins and proteins can be extracted [122]. Moreover, it is characterized by having a superior extraction rate of volatile compounds in *Capsicum* spp. The selectivity and efficiency of MAE largely depends on the dielectric constant of the extraction solvent mixture, which defines its chemical polarity and thus what compounds will be extracted [91]. It can be expected that by using water as a single solvent, the quantity of capsaicinoids extracted decrease due to its negligible polarity [109]. Therefore, the parameters to take into account in this type of extraction

Several experiments can be seen in a summarized form in **Table 7**, which shows the variation between the values of the parameters among different experiments with solvents of different polarity. It is worth highlighting the values obtained for capsaicinoids using ethanol [105] as solvents since it is the one with the highest

In every extraction process there is a critical step which is the degradation of cell walls and membranes. Ultrasound assisted extraction (UAE) makes this possible by applying pressure waves that are transmitted across a medium as compression and expansion (rarefaction) cycles, finding an area with maximum pressure in the compression phase and one with minimum pressure in the rarefaction phase. This pressure difference makes the cavitation phenomenon possible, which breaks the

are time, power, temperature and type of solvent and its ratios.

way the extraction efficiency is substantially improved.

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

**4.2 Green extraction techniques**

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

*4.2.1 Microwave-assisted extraction*

in the extraction of volatile oils [91].

*4.2.2 Ultrasound assisted extraction*

yield.

**141**

solvents consumption.

Water can be used as an extraction solvent, but it comes with the drawback is that it principally extracts the hydrophilic compounds present in vegetable materials. Several studies showed that water is not a very suitable solvent because its high polarity does not allow the extraction of capsaicinoids that are non-polar. However, it has the advantages or being safe, cheap and the simplest form to obtain essential oils. This technique can also be done with other solvents, being the ratio of solvent another parameter to take into account. Polarity is the reason why the most common solvents used are methanol, ethanol, water or a mixture of them [3].

Another parameter to optimize will be temperature. This makes it possible to differentiate two types of MA: cold maceration and heat maceration. Maceration with or without stirring, mild heating or heating under reflux are also possible variations to the method.

It is a quite old simple method with the inconvenience of long extraction times and large amounts of sample and solvents. Additionally, high temperatures can destroy thermolabile compounds such as phenolic compounds. In a study in which the effect of MA and ultrasound was related, it was detected that they have similar extraction yield of oleoresin; nevertheless, MA needs longer extraction times with the consequent loss of quality due to their higher times of exposure to high temperatures. It also was observed that n-hexane was a better solvent than ethanol [116].

## *4.1.2 Heat assisted extraction*

This procedure is also a type of solid-liquid extraction in which the extraction is usually carried out in thermostatic and sealed water baths so that the solvent does not evaporate. The determining parameters are extraction time, temperature and the proportion of solvent that is used as well as the solid-liquid ratio. Due to the application of high temperatures (as far as 100°C), it is not the best extraction process for this type of matrix because certain compounds present in peppers (vitamins, phenolic compounds, etc.) are degraded by heat.

#### *4.1.3 Cold pressure extraction*

Cold pressure extraction is one of the oldest techniques of extraction for obtaining oils. It consists of mechanical pressing with the absence of heating. By using this method, little to no heat is generated, however it gives low yields. This technique was applied to Capia pepper seed, resulting in a lower extraction yield compared to traditional Soxhlet extraction with hexane. Moreover, the final oil was unpopular among consumers [117].

#### *4.1.4 Organic solvent extraction*

Organic solvent extraction (OSE) allows the extraction of many compounds (oils, fats and proteins). Normally, after the extraction process another step is done. It consists of concentrating the extract by removing the solvent under atmospheric or reduced pressure. OSE is the most extensively used technique to obtain oleoresins from peppers. These oleoresins are. Usually used as color additives [118]. In this method, the most determining limitation will be the solvent properties (polarity). When choosing the solvent, it must also be taken into account if the legislation allows its use. In the EU is regulated in Commission Regulation N° 231/2012.

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties… DOI: http://dx.doi.org/10.5772/intechopen.91785*

In order to improve these conventional extraction techniques, other methods have been developed (ultrasound assisted extraction, pressurized hot water extraction, negative pressure cavitation-assisted extraction and pulsed electric) to partner with them and solve some of the inopportuneness of traditional techniques. In this way the extraction efficiency is substantially improved.

#### **4.2 Green extraction techniques**

being this step the limiting stage. This method is used, for example, for the isolation of phenolic compounds of vegetable origin for their subsequent use in the food,

Water can be used as an extraction solvent, but it comes with the drawback is that it principally extracts the hydrophilic compounds present in vegetable materials. Several studies showed that water is not a very suitable solvent because its high polarity does not allow the extraction of capsaicinoids that are non-polar. However, it has the advantages or being safe, cheap and the simplest form to obtain essential oils. This technique can also be done with other solvents, being the ratio of solvent another parameter to take into account. Polarity is the reason why the most common solvents used are methanol, ethanol, water or a mixture of them [3]. Another parameter to optimize will be temperature. This makes it possible to differentiate two types of MA: cold maceration and heat maceration. Maceration with or without stirring, mild heating or heating under reflux are also possible

It is a quite old simple method with the inconvenience of long extraction times and large amounts of sample and solvents. Additionally, high temperatures can destroy thermolabile compounds such as phenolic compounds. In a study in which the effect of MA and ultrasound was related, it was detected that they have similar extraction yield of oleoresin; nevertheless, MA needs longer extraction times with the consequent loss of quality due to their higher times of exposure to high temperatures. It also was observed that n-hexane was a better solvent than

This procedure is also a type of solid-liquid extraction in which the extraction is usually carried out in thermostatic and sealed water baths so that the solvent does not evaporate. The determining parameters are extraction time, temperature and the proportion of solvent that is used as well as the solid-liquid ratio. Due to the application of high temperatures (as far as 100°C), it is not the best extraction process for this type of matrix because certain compounds present in peppers

Cold pressure extraction is one of the oldest techniques of extraction for obtaining oils. It consists of mechanical pressing with the absence of heating. By using this method, little to no heat is generated, however it gives low yields. This technique was applied to Capia pepper seed, resulting in a lower extraction yield compared to traditional Soxhlet extraction with hexane. Moreover, the final oil was

Organic solvent extraction (OSE) allows the extraction of many compounds (oils, fats and proteins). Normally, after the extraction process another step is done. It consists of concentrating the extract by removing the solvent under atmospheric or reduced pressure. OSE is the most extensively used technique to obtain oleoresins from peppers. These oleoresins are. Usually used as color additives [118]. In this method, the most determining limitation will be the solvent properties (polarity). When choosing the solvent, it must also be taken into account if the legislation allows its use. In the EU is regulated in Commission Regulation N° 231/2012.

(vitamins, phenolic compounds, etc.) are degraded by heat.

pharmaceutical and cosmetic industries [115].

variations to the method.

*4.1.2 Heat assisted extraction*

*4.1.3 Cold pressure extraction*

unpopular among consumers [117].

*4.1.4 Organic solvent extraction*

**140**

ethanol [116].

Capsicum

This group includes several techniques with the aim of not only preventing pollution but also reducing sample preparation costs by, for example, lowering solvents consumption.

#### *4.2.1 Microwave-assisted extraction*

Microwave-assisted extraction (MAE) is based on the heating of solvents that are in contact with solid samples with the use of microwaves. This allows the partition of compounds of interest from the sample into the solvent. Microwave energy accelerates a great variety of chemical reactions as well as the extraction of organic compounds from different matrices [98]. During the process, electromagnetic energy is transformed into calorific energy by two mechanisms: ionic conduction and dipole rotation [119]. It is a method that could be an alternative for avoiding thermal degradation and oxidation, with no influence on cell integrity and shape. However, due to thermal stress and localized high pressures, cell rupture is more rapid than with another techniques, which is an inconvenience, for example, in the extraction of volatile oils [91].

Some of the advantages of this technique are simplicity, effectiveness, low processing time, low solvent consumption and energy, no generation of secondary waste and can be used for larger volumes [120]. Another advantage of this method is that it produces a uniform heating, so extraction is simultaneous regardless of the area where the compound is [121]. By using this method, lipids, pigments, carbohydrates, vitamins and proteins can be extracted [122]. Moreover, it is characterized by having a superior extraction rate of volatile compounds in *Capsicum* spp.

The selectivity and efficiency of MAE largely depends on the dielectric constant of the extraction solvent mixture, which defines its chemical polarity and thus what compounds will be extracted [91]. It can be expected that by using water as a single solvent, the quantity of capsaicinoids extracted decrease due to its negligible polarity [109]. Therefore, the parameters to take into account in this type of extraction are time, power, temperature and type of solvent and its ratios.

Several experiments can be seen in a summarized form in **Table 7**, which shows the variation between the values of the parameters among different experiments with solvents of different polarity. It is worth highlighting the values obtained for capsaicinoids using ethanol [105] as solvents since it is the one with the highest yield.

#### *4.2.2 Ultrasound assisted extraction*

In every extraction process there is a critical step which is the degradation of cell walls and membranes. Ultrasound assisted extraction (UAE) makes this possible by applying pressure waves that are transmitted across a medium as compression and expansion (rarefaction) cycles, finding an area with maximum pressure in the compression phase and one with minimum pressure in the rarefaction phase. This pressure difference makes the cavitation phenomenon possible, which breaks the


#### **Table 7.**

*Different experimental conditions carried out in peppers by microwave-assisted extraction.*

structure to make the compounds accessible and therefore available to extract them [123].

This technique has been applied to extract different bioactive compounds from herbs or algae due to its ease to disrupt their cell walls despite its resistance which differentiates this method from the previous ones, since strong disruption of the cell wall is achieved and as a result the extraction of intracellular materials is increased with the increment of energy input [106, 120, 124].

The ultrasonic technique, as well as the other green techniques, has been proven to have several advantages such as reduction of solvents consumption, temperature and time, very important parameters in the extraction of thermolabile and unstable compounds as it has no effect on the chemical structure and biological properties. Furthermore, UAE has low equipment investment and easy implementation, so it can be basically industrially employed in local companies [106]. Moreover, there are studies that showed that this method also achieves a greater supercritical extraction of pungent compounds from ginger owing to physical effects on the surface of particles [106].

*4.2.3 Enzyme-assisted extraction*

Antioxidants *Capsicum*

Oleoresin *Capsicum*

*—, not described.*

**Table 8.**

**143**

Capsaicinoids *Capsicum*

*chinense*

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

*Capsicum chinense*

*Capsicum annuum*

*Capsicum annuum*

*Capsicum annuum*

*Capsicum frutescens*

*Capsicum frutescens*

*Capsicum frutescens*

*Capsicum baccatum*

*baccatum*

*baccatum*

tain the quality and yield of multiple biomolecules [125].

Phytochemicals *Capsicum* 35 — 20 50 Methanol

*Different experimental conditions carried out in peppers by ultrasound assisted extraction.*

Enzyme-assisted extraction (EAE) is an extraction system that allows the avoidance of processing conditions like temperature or drastic pH changes and so, main-

This type of extraction supports isolation for recovering bioingredients from different plant materials. An enzymatic pre-treatment before applying traditional methods will help to isolate high yields of bioingredients due to enzyme assisted extraction facilities. This is due to the fact that degradation of cell walls and membranes is the critical step of extraction which [101]. Among its advantages over traditional methods are high selectivity, overall efficacy, eco-friendly procedures, low-energy consumption, minimal usage of harsh chemicals, maximum yield, low to no wasteful protection or deprotection steps, easy recovery, and process recyclability [126]. However, it also presents some drawbacks such as the cost of enzymes, requirement of holding tanks that may require long term incubation, lack of knowledge about optimal or compatible enzyme formulations for cell disruption

**Target Species Conditions Yield Ref.**

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

**Freq. Intensity Time Temp. Solvent kHz W/cm<sup>3</sup> min °C (%)**

20 — 10 40 Acetone

40 — 10 24 Methanol

31.5 — 20 76 Methanol

— — 20 50 n-hexane

20 360 10 50 Methanol

20 360 10 50 Methanol

— — 40 25 Acetone

20 150 20 40 Methanol

20 150 20 40 Methanol

20 450 20 60 Methanol

(100)

(100)

(60)

(100)

(100)

(100)

(100)

(100)

(100)

(100)

(80)

35 600 180 45 Ethanol (95) 87.4% [106]

0.31 mg/g [109]

2.88 mg/g [110]

200 ppm [97]

— [99]

100% [100]

100% [100]

3.92 mg/g [107]

50% [112]

27% [112]

26% [112]

— [114]

and inability to completely hydrolyse the bonds in plant cell wall [127].

There are several parameters to take into account to optimize the method which include ultrasound power intensities, frequency, wavelength and time. There are several studies about it (**Table 8**). One of them studied the influence of several of these parameters on the extraction of capsaicinoids, observing that their release was very fast in the first 5 min of the process and then decreased. Temperature influence was also observed since an increase of 15°C (up to 45°C) improved the extraction using 95% ethanol (v/v) as a solvent. Higher temperatures did not lead to significant improvements [106]. The effect of the solvent was also studied, being the most common solvents used for extracting capsaicinoids methanol, ethanol, acetone, acetonitrile and water. It was proved that the yield of extraction is worse with the addition of water. Methanol and ethanol have similar recoveries but ethanol was better than acetone, the best concentration of ethanol being 95% [100, 106].


Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties… DOI: http://dx.doi.org/10.5772/intechopen.91785*

#### **Table 8.**

structure to make the compounds accessible and therefore available to extract

*Different experimental conditions carried out in peppers by microwave-assisted extraction.*

**Target Species Extracting conditions Yield Ref.**

Capsaicinoids *Capsicum* spp. 21 15 300 Acetone (30%) 0.48 mg/g [98]

**°C min W**

**Temp. Time Power Solvent ratios**

60 2 50 Acetone-water

55 10 250 n-hexane (pure) 42

(50%)

50 5 800 Methanol (100%) 0.32 mg/g [109]

— 20 320 Ethanol (99.5%) 5.3 mg/g [105]

120 15 150 Acetone (pure) 0.673% [104]

76 1 500 Methanol (60%) 230 ppm [97]

compounds

[59]

— [91]

with the increment of energy input [106, 120, 124].

This technique has been applied to extract different bioactive compounds from herbs or algae due to its ease to disrupt their cell walls despite its resistance which differentiates this method from the previous ones, since strong disruption of the cell wall is achieved and as a result the extraction of intracellular materials is increased

The ultrasonic technique, as well as the other green techniques, has been proven to have several advantages such as reduction of solvents consumption, temperature and time, very important parameters in the extraction of thermolabile and unstable compounds as it has no effect on the chemical structure and biological properties. Furthermore, UAE has low equipment investment and easy implementation, so it can be basically industrially employed in local companies [106]. Moreover, there are studies that showed that this method also achieves a greater supercritical extraction of pungent compounds from ginger owing to physical effects on the

There are several parameters to take into account to optimize the method which include ultrasound power intensities, frequency, wavelength and time. There are several studies about it (**Table 8**). One of them studied the influence of several of these parameters on the extraction of capsaicinoids, observing that their release was very fast in the first 5 min of the process and then decreased. Temperature influence was also observed since an increase of 15°C (up to 45°C) improved the extraction using 95% ethanol (v/v) as a solvent. Higher temperatures did not lead to significant improvements [106]. The effect of the solvent was also studied, being the most common solvents used for extracting capsaicinoids methanol, ethanol, acetone, acetonitrile and water. It was proved that the yield of extraction is worse with the addition of water. Methanol and ethanol have similar recoveries but ethanol was better than acetone, the best concentration of ethanol

them [123].

**Table 7.**

Capsicum

*—, not described.*

Volatiles *Capsicum*

Pigments *Capsicum*

*annuum*

*annuum*

*Capsicum chinense*

*Capsicum frutescens*

*Capsicum frutescens*

*Capsicum annuum*

surface of particles [106].

being 95% [100, 106].

**142**

*Different experimental conditions carried out in peppers by ultrasound assisted extraction.*

#### *4.2.3 Enzyme-assisted extraction*

Enzyme-assisted extraction (EAE) is an extraction system that allows the avoidance of processing conditions like temperature or drastic pH changes and so, maintain the quality and yield of multiple biomolecules [125].

This type of extraction supports isolation for recovering bioingredients from different plant materials. An enzymatic pre-treatment before applying traditional methods will help to isolate high yields of bioingredients due to enzyme assisted extraction facilities. This is due to the fact that degradation of cell walls and membranes is the critical step of extraction which [101]. Among its advantages over traditional methods are high selectivity, overall efficacy, eco-friendly procedures, low-energy consumption, minimal usage of harsh chemicals, maximum yield, low to no wasteful protection or deprotection steps, easy recovery, and process recyclability [126]. However, it also presents some drawbacks such as the cost of enzymes, requirement of holding tanks that may require long term incubation, lack of knowledge about optimal or compatible enzyme formulations for cell disruption and inability to completely hydrolyse the bonds in plant cell wall [127].

The factors to take into account in this technique are fundamentally temperature, pH, and type of enzyme. A range of enzymes (lipases, carbohydrase, celluloses, proteases, pectinases) have been widely used as specific catalysts. Each enzyme has different substrates. For example, cellulase and hemi-cellulase have their greater hydrolysing activity on the cellulose found in plant cell walls, hence their name. This enzymatic processing increases the permeability of the cell wall, resulting in a better recovery of some compounds like volatile oil and resin, which are prone to degradation when extracted with more disruptive methods [101].

gives higher extraction yields and fewer impurities [131] giving, unlike other preservation technologies as thermal treatment, uniform and nearly instantaneous effects throughout the foodstuff and thus independent of foodstuff geometry and equipment size which makes an easy scale-up from laboratory findings to full-scale

This method can be applied to multiple matrices for the extraction of natural compounds [133] like fruit and vegetables for different targets as for example

The main parameters to be taken into account are pressure, time, temperature and type and quantity of solvent, studying each parameter for each variety in

HHP can change physiological and biochemical properties of pepper based on a study carried out with *C. annuum* [136]. In this research, different pressures (50, 100, 200 and 300 MPa) where applied for 5 min at 25°C observing that it produces remarkable changes in seedlings, but no or very little physiological and biochemical variations, creating an antioxidant system that is positive for the plant itself in the process. Therefore, HHP (500 MPa) can be used as a preservation treatment similar to pasteurization which was the ttraditional thermal treatment for sweet pepper

In supercritical-fluid extraction (SFE), the fluid must reach temperature and pressure above the critical point, so as the fluid behaves like liquid and gas simultaneously which makes extraction easier. This technology has been used in a wide diversity of fields (food, pharmaceutical, chemical and fuel industries) due to its advantages, there is an absence of toxic residue in the final product which allows not only the extraction valuable active compounds (fatty acids, pigments, polyphenols and vitamins) free of solvents, but also to remove undesirable compounds (pollutants, toxins and pesticides) [138]. Additional advantages are great extraction selectivity, short processing times, requirement of minimal solvents, low degradability of the extracted product and the fact that the remaining biomass can be

The most important conditions are temperature, pressure and co-solvent. The selection of each parameter will depend on the specific compound searched [138]. The most used solvent is CO2 due to its thermodynamics and heat transfer properties. Moreover, it has a low critical point (31°C, 73 bar). Furthermore, the polarity of CO2 can be modified by the use of co-solvents such as ethanol, and in this way also

Studies demonstrate that the extracts obtained with this technique are better than natural spice for flavoring purposes as SFE could reduce aflatoxin in the final products [139]. In addition, numerous bibliographic references show that caroten-

Furthermore, when analyzing the extraction of β-carotene and capsaicin at the same time, capsaicin shows lower solubility, yet the solubility of β-carotene did not change in the presence of capsaicin, a factor to bear in mind when designing the separation process of coloring and hot components from paprika [140]. Several pepper species have been used to obtain natural compounds of interest using supercritical fluid extraction including capsaicinoids, oleoresins, pigments, tocopherols and even aflatoxins (**Table 10**) observing large variations between the pressures applied in each of the methods referred to in the bibliography. The greatest amount of studies is related to the specie *C. annuum* since it is the best known,

carotenoids [134], antioxidants [131] and pigments [135].

Capsicum *Seeds as a Source of Bioactive Compounds: Biological Properties…*

treated with other techniques in order to continue extraction.

oid extraction is better at higher pressures [113].

widespread and cultivated specie of the genus *Capsicum*.

production possible [132].

*DOI: http://dx.doi.org/10.5772/intechopen.91785*

particular.

preservation [137].

*4.3.2 Supercritical-fluid extraction*

extract polar components.

**145**

Several studies (**Table 9**) show that viscozyme L is an enzyme with a superior recovery rate of bioactive compounds fractions like total carotenoid content, total phenolic content, total flavonoids and total antioxidant activity with high total suspended solids (TSS). It also has at this moment the better extract yield [95, 96]. Among its applications is the extraction of pigments or capsaicinoids. In almost all cases the pH of the medium is 4.5 as it is the pH of the sample and it is in the range of optimal activity of the different enzymes [95].
