Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered Herbs/Medicinal Plants

*Habeeb O. Yusuf and Ruth T.S. Ofongo*

## **Abstract**

The sole aim of raising pullet hens in the poultry industry is to produce eggs for human consumption in a large scale when they commence laying. Eggs are important dietary components to humans both adult and children and is classified as complete protein. However, certain quality of eggs produced by laying hens is further influenced by the diet consumed which in turn is determined by the quality of the feed ingredients making up the diet. Antibiotic residue in eggs and antimicrobial resistance are few concerns to consumers of poultry products. The current era of limiting antimicrobial utilization for livestock production has increased research into medicinal plants and herbs as suitable alternative. Antioxidant and anti-inflammatory activities reported in literature indicate the invaluable benefits of these plants both for humans and livestock. This book chapter attempts to present the 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant scavenging activity of eggs from laying hens fed medicinal plants – *Vernonia amygdalina* and *Ocimum gratissimum* as component of feed or administered orally as an aqueous extract. The DPPH antioxidant scavenging activity was present in eggs sampled but was better (p < 0.05) in eggs of laying hens administered aqueous *O. gratissimum* extract.

**Keywords:** antioxidants, antioxidant scavenging activity, medicinal plants, eggs, nutrition, laying hens

## **1. Introduction**

Healthy food and healthy diet are a major concern to health-conscious individuals; since health lost is wealth lost. The concern of such individuals cannot be over emphasized. The genetic modification of plants and animals is an evolving development which is yet to be generally accepted by consumers of agricultural products. The current shift towards natural sources of antioxidants reported [1] can be attributed to the unfavorable effects of synthetic sources of antioxidants resulting from prolonged usage.

Reactive oxygen species (ROS) otherwise referred to as free radicals are byproducts unavoidably produced in biological systems in the course of normal cellular energy function [1, 2]. They are also produced through exogenous sources such as environmental pollutants, radiation, pesticides [1, 3, 4] etc. just to mention a few. Free radicals are important for some biological functions physiologically by acting as cell signaling molecules which function against cellular responses as reported in literature [5, 6]. Oxidative stress elicits adverse effects on lipids, proteins and nucleic acids thereby resulting into a number of degenerative conditions [7–10]. Free radical scavengers or antioxidants delay/inhibit damage made to cells by converting ROS to non-reactive radical species [1]. Common antioxidants include vitamin E (α-tocopherol), vitamin C (ascorbic acid), and β-carotene [11, 12].

Generally speaking; consumption of antioxidant rich food by health-conscious individuals has more to do with preventing damage in tissues and privation of cellular functions resulting from free radicals' intermediates generated by cells during normal metabolism [2] or avoiding degenerative diseases. Antioxidant activities of plants are regarded as safe. This activity is attributed mainly to phenolic compounds [5, 13] which act as hydrogen donors, reducing agents, oxygen quenchers besides their metal chelating potential [3]. These properties play a significant role in neutralizing free radicals [3].

## **1.1 Eggs as quality food**

Eggs are considered high quality protein which is readily digestible, however, with recent developments in human health, animal nutrition, it is gradually developing into a functional food for health and better wellbeing. The amino acid profile of eggs is adequate to meet both essential and non-essential amino acid needs of both adults and young children making it a complete protein. Eggs are also considered cheap, extremely nutritious, palatable and readily accessible across the globe [14, 15].

Egg yolk, egg white/albumen and egg shell with membrane respectively; accounts for approximately 27.5%, 63% and 9.5% of the whole egg [16]. The various constituent of egg nutrients are proteins and lipids 12% respectively; while the edible portion is made up of 74% water [17]. Eggs contain less than 1% carbohydrate along with vitamins and minerals [17–20]. Both high density and low-density lipoproteins in addition to livetin's is located in egg yolk. Ovalbumin, ovotransferrin, ovomucoid, ovomucin just to mention a few are protein fractions present in egg white [21]. The quality of egg protein is such that it is used as a golden standard for measuring the quality of other food proteins [18].

Greater part of lipids in egg yolk are present as triglycerides—almost 65%, however, carotenoids constitute less than 1% while phospholipids and cholesterol are 30% and 4% respectively [22]. Lipids from egg yolk have been used to supply long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA) and phospholipids incorporated into infant formula [23, 24]. They are also regarded as an excellent source of micronutrients—vitamins and minerals. According to published work [18] eggs contain approximately 16% required daily intake (RDI) 0f phosphorus. The RDI of eggs was reported for selenium (29%); iron (9%) and zinc (9%) respectively. In addition, eggs also provide 10% of the RDI for fat soluble vitamins, vitamin B2, B12, biotin and pantothenic acid [18]. As it is in spite of the nutritive quality of eggs, it appears the need to limit its cholesterol concentration, especially high-density lipoproteins are focused on meeting healthy outcomes for consumers of eggs. This has led to manipulating poultry diets to influence the nutritional constituents in eggs [25]. Several studies have been carried out in this regard and reported in literature [14, 15, 26–35]. Besides the fatty acid constituents of eggs, some minerals like selenium and iodine have been enriched in eggs via enrichment of feed [35, 36] and feed formulation [37].

Intake of antioxidants through diet is known to be important in reducing oxidative damage in cells and improving human health. Although eggs are known for their exceptional, nutritional quality, they are not generally considered as antioxidant foods [21].

#### **1.2 Antioxidant content of eggs**

Many of the compounds present in eggs—vitamin E, and A, selenium, phospholipids and carotenoids—show evidence of antioxidant properties; eggs are not normally considered as antioxidant foods [21]. Even though certain polyunsaturated fatty acids (PUFA) exhibit antioxidants properties; eggs are mostly consumed for their protein constituents and minerals which are components of egg.

Furthermore, it is important to note that with recent developments in enriching eggs via feed thereby making eggs as functional foods then consumption of eggs might just be a source of antioxidants for healthy living.

A recent report [38] testing the antioxidant property of a product containing pectic oligosaccharides, with prebiotic, chlorogenic as well as antioxidant effect on the possibility of enhancing egg laying performance and egg quality of laying hens. The results showed that the tested product enhances laying hens egg quality and performance, particularly by means of its antioxidant properties that play a part to sustain oxido-redox balance, consequently reducing the negative effects triggered by oxidation like degradation of egg quality [38]. By this, may the various nutrient components of egg which make up the quality of eggs can be improved upon by feed manipulation. The antioxidant properties of eggs are exhibited by the different components' present either in the egg yolk or egg white.

Ovalbumin, ovatransferrin, ovomucin, lysozyme, cystatin, in egg white reportedly have antioxidant properties. Phosvitin, phospholipids, carotenoids and vitamin E which are components of egg yolk.

Ovalbumin in egg white has free thiol groups that regulate redox status and bind metal ions thereby exerting antioxidant properties. In conjugation with saccharides increased antioxidant activity takes place [39, 40]. Ovomucin in egg white inhibit hydrogen peroxide H2O2-induced oxidative stress inhuman embryonic kidney [41]. Furthermore, lysozyme in egg white Suppress reactive oxygen species (ROS) and oxidative stress genes [42]. In the case of nutrient components with antioxidant properties in egg yolk; phospholipids are 10% of egg yolk dry matter. Hydrolyl amines in the side chains of egg yolk phospholipids play a role in radical scavenging with antioxidant properties [43]. The unsaturated backbone and aromatic rings of carotenoids present in egg yolk help in neutralizing singlet oxygen, free radicals what is more is protective against oxidative damage [44–46].

## **2. Antioxidant properties of medicinal plants**

Current research thrust has exposed the numerous benefits of herbs and medicinal plants in the nutrition of farm animals. Needless to say, several herbs and medicinal plants formerly taken as traditional medicine have been used for animal feeding trials. In an earlier report [47, 48] a wide range of extracts from herbal plants have antioxidant properties.

In particular is extracts of herbs from the Labiatae family—oregano, thyme, basil, mint, rosemary, sage, savory, marjoram, etc. The antioxidant properties of hyssop and lavender were attributed to phenolic terpenoid compounds such as carvacrol, thymol, menthol and eugenol just to mention a few [2]. Evidence in literature regarding antioxidant properties of herbal plants [49] further revealed that both phenolic

and nonphenolic compounds exhibit antioxidant properties in them. Example of nonphenolic compounds with antioxidant properties include glycosides [50]. A recent report [49] showed the antioxidant properties of phenols from *Vernonia amygdalina* Delile*.* (Asteraceae). High levels of phenolic content 54.61 ± 0.94 mg GAE/g in *V. amygdalina* [49] has also been reported from phytochemical analysis of *Vernonia amygdalina.* The methanolic extract indicated that phenolic compounds were highly detected (+++) while flavonoids were detected (++) [51]. The medicinal plant *V. amygdalina* is rich in flavonoids, tannins and saponins, which may possibly play a part in anti-oxidative effect [49, 51]. From literature *V. amygdalina* has a flavonoid content of 22.53 ± 0.91 mg QE/g [49]. Phenolics hydroxy groups present in the molecular structure of flavonoids earlier stated as exhibiting antioxidant properties are involved in antioxidant properties of flavonoids. The powerful antioxidant property exhibited by phenolic compounds [52] present in plants constituents can be attributed to the hydroxyl groups [53] present in them. The medicinal plant *Ocimum gratissimum* also has both antioxidant anti-inflammatory properties already attributed to its therapeutic benefits from literature [54, 55]. The presence of other phytochemicals - saponins, terpenoids, glycosides and alkaloids—in aqueous *O. gratissimum* besides flavonoids and phenols (Ofongo, 2023, unpublished data). These phytochemicals may further play a part in its anti-inflammatory and anti-oxidative activities [54, 55].

## **2.1 Medicinal plants as component of feed and their benefits to nutrition of laying hens**

Medicinal as component of feeds is becoming a common practice in the livestock industry either as suitable alternative to antibiotics or for their growth performance and health promoting benefits to livestock and ultimately to consumers of livestock consumers. Other benefits of medicinal plants as components of feed include; antimicrobial; anti-inflammatory; immune modulatory effect and antioxidant effect.

As earlier stated, the efficacy of herbal extracts as antioxidant feed additives needs to be evaluated and correlated with their phenolic content [2]. The use of medicinal to feed laying hens is more or less an evolving practice in the poultry industry.

Firstly, from improving productive performance, modulation of cholesterol contents in eggs and egg quality have been reported [14, 15, 34, 38, 56, 57]. However, gradually, inclusion of medicinal plants in diets of laying hens is focusing at making eggs functional food by improving or fortifying the content of nutrients present in eggs with benefits to consumers of eggs such as antioxidant properties [34, 58–60].

This book chapter tries to illustrate possibility for antioxidant scavenging activity enhancement in eggs from laying hens by administering medicinal plants via feed or orally.

## **3. Materials and methods**

### **3.1 Collection and identification of plant materials**

Two medicinal plants (*Vernonia amygdalina* Ochile (Compositae) NDUP/21/14— **Figure 1** and *O. gratissimum* L. Lamiaceae) NDUP/12/13—**Figure 2**) were used in this study. Authentication of the plants were done at Herbarium Unit of the Department of Pharmacognosy, Faculty of Pharmacy, Niger Delta University. Fresh leaves of *V. amygdalina* and *O. gratissimum* were collected from Niger Delta University Teaching and Research Farm, Wilberforce Island, Bayelsa State, Nigeria.

*Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered... DOI: http://dx.doi.org/10.5772/intechopen.111658*

**Figure 1.** V. amygdalina*.*

**Figure 2.** Ocimum gratissimum*.*

## **3.2 Preparation of extracts and chaff**

A large amount of each leave was collected either first thing in the morning or late in the evening. The leaves were separated from the stalk and placed in a large container filled with clean drinkable water into which little salt to brine the water. This was done to remove durst and debris from the leaves. Thereafter, the leaves were placed in a sieve to remove the brine and rinsed in clean drinkable water without salt again to remove any brine water on the leaves. The leaves were afterwards placed in

a sieve to drain out excess water. Each leave was chopped separately into fine particle size then one thousand gram (1000 g) of each leave was weighed separately for milling to obtain the aqueous extract. Seven hundred and fifty mills (750 ml) of clean drinkable water were used to mill 1000 g of each leave sample separately by means of an electric milling machine. The aqueous filtrate was obtained by passing the milled product through a cheese cloth. The obtained chaff was set aside, air dried then packaged in zip lock bag (**Figure 3A**) for use as component of feed (**Figure 3B**). The aqueous extract was administered to 22 weeks old bovan brown layer at an inclusion rate of 1 ml/bird administered twice a week. The obtained dried chaff was further milled to obtain fine particles and added to standard layers mash at an inclusion rate of 50 g/kg of complete feed. Feed mixed with chaff not for immediate use were stored (**Figure 4**).

## **3.3 Animal experiment**

A feeding trial was carried out to access and determine improved DPPH concentration in eggs from laying hens administered medicinal plants either as component of feed of as aqueous extract. The experiment was arranged as a complete randomized

**Figure 3.** *Dried chaff of leaves in zip lock bag (A); fine milled chaff mixing into feed (B).*

**Figure 4.** *Feed incorporated with chaff packaged in zip lock bag.*

*Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered... DOI: http://dx.doi.org/10.5772/intechopen.111658*

design having five (5) treatment groups of five (5) replicates and four (4) birds per replicate. A total of 100 bovan brown laying hens were purchased at 20 weeks of age randomly distributed to the above stated design then allowed to acclimatize for 2 weeks under the experimental treatments. Experimental collection of eggs for sampling commenced at week 22. The experiment was terminated after eight (8) weeks on day 56. Birds allocated to treatment 1 (T1) served as the control group. They were fed a standard layer's mash but where not administered aqueous plant extract neither was their feed supplemented with the chaff of *V. amygdalina* or *O. gratissimum*. Birds allocated to treatment 2 (T2) were fed diet supplemented with *V. amygdalina* chaff while birds in treatment 3 (T3) were administered 1 ml/bird of aqueous *V. amygdalina* twice a week. Birds assigned to treatment four (T4) had their diet supplemented with *O. gratissimum* chaff while birds in treatment five (T5) were administered 1 ml/bird of aqueous *O. gratissimum* extract twice a week.

#### **3.4 Sample collection**

On day 56, one egg per replicate was collected to determine antioxidant scavenging activity in inhibiting DPPH according to procedure illustrated below. The collected eggs were first cracked, homogenized then lyophilized to obtain a powder version of each egg before carrying out antioxidant scavenging activity in inhibiting DPPH using ascorbic acid as standard.

## **3.5 Proximate composition of feed and antioxidant scavenging activity in inhibiting DPPH**

Samples of feed already supplemented with the respective chaff of *V. amygdalina* and *O. gratissimum* was collected into sample containers (100 g) for proximate analysis of feed as well as antioxidant activity in scavenging DPPH. The aqueous extract (30 ml) of either plant was also collected into sample bottles for antioxidant activity analysis in scavenging DPPH. Proximate composition of the experimental diets with and without each leave chaff as well as each respective chaff alone was carried out according to AOAC method.

Antioxidant activity of the plant extracts and feed supplemented with plant chaff in scavenging DPPH were evaluated on the basis of free radical scavenging effect of stable 2,2-diphenyl-1-picrylhydrazyl (DPPH). This was evaluated in comparison with Ascorbic acid standard, using a slightly modified method [61]. The compound DPPH is a synthetic compound not occurring in nature but it is utilized to evaluate the antioxidant activity of organic compounds such as vitamins, polyphenols and other phytochemicals.

Standard concentrations of Ascorbic acid standard were prepared at concentrations of 20, 40, 60, 80, and 100 ᶙ g/ml; respectively from a stock solution in triplicates using Methanol. Thereafter, a solution of DPPH was prepared using 0.1 mM of DPPH in methanol of which 2 ml of this solution was mixed with 3 ml of the test and standard solutions in test tubes. The solutions were shaken, then allowed to stand for 30 min in the dark before absorbance was measured at 517 nm using UV-VIS Spectrophotometer (Biomate 3, USA).

A standard control was prepared by mixing Methanol (3 ml) with 2 ml DPPH solution (0.1 mM, 1 ml). Methanol was used as a blank. The same procedure carried out with the Ascorbic acid standard was repeated with the test samples (leave chaff, aqueous extract and egg samples).

Percentage inhibition of DPPH was carried out using the formula below:

Inhibition of DPPH Ac Aa / Ac % =− × ( ) 100

Ac: absorbance of control sample. Aa: absorbance of test samples or standard.

### **3.6 Statistical analysis**

Collected data on DPPH scavenging activity in each sample (leaf chaff, aqueous extract, feed supplemented with leaf chaff and eggs) were subjected to analysis of variance (ANOVA). Statistically significant means were separated with Duncans Multiple Range test [62].

## **4. Results and discussions**

#### **4.1 Proximate composition of experimental diets**

The proximate composition of layers mash fed to experimental birds as well as the proximate composition of *V. amygdalina* and *O. gratissimum* chaff is presented below in **Table 1**.

Crude protein concentration in *O. gratissimum* and *V. amygdalina* chaff was within the same range 12.00–13.00 g/kg DM. Ash concentration of *V. amygdalina* chaff was numerically higher (12.48 g/kg DM) than in *O. gratissimum* (9.17 g/kg DM). Dry matter concentration (943.15 g) was higher in *V. amygdalina* than in *O. gratissimum* (929.05 g). Proximate composition of feed showed an adequate crude protein concentration in the feed. Nitrogen free extract (carbohydrate) concentration was 61.61 g/kg DM in *O. gratissimum* which was numerically higher than value recorded in *V. amygdalina* and layers mash used in this study.

The synthetic compound 2,2-diphenyl-1-picrylhydrazyl (DPPH) is used as a reagent in antioxidant assays. It is used to evaluate the antioxidant activity of organic compounds such as vitamins, polyphenols and other phytochemicals. The addition of an organic compound to a solution of DPPH is used to measure the antioxidant activity of the compound. This activity is dependent on the ability of the compound to scavenge DPPH radical thereby limiting the ability of DPPH to absorb light at 517 nm. The lesser the absorbance of the solution, the higher the antioxidant scavenging


#### **Table 1.**

*Proximate composition of layers mash and leaf chaff (g/kg DM except otherwise stated).*

*Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered... DOI: http://dx.doi.org/10.5772/intechopen.111658*

activity of the test material. The DPPH scavenging activity is reported as percentage DPPH inhibition by the test compound. The lower the absorbance, the higher the percentage inhibition or free radical scavenging activity.

The antioxidant scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) by medicinal plants administered to layers or supplemented into complete feed is presented in **Table 2** below. Based on the DPPH scavenging activity (UV-absorbance at 517 nm); absorbance of DPPH was significantly (*p* < 0.05) low at 10 ug/ml concentration in *O. gratissimum* aqueous extract. The higher concentration resulted in significantly lower (*p* < 0.05) DPPH scavenging activity in *V. amygdalina* and *O. gratissimum* either in chaff or aqueous extract.

**Table 3** shows the DPPH scavenging activity of *V. amygdalina* was high compared to *O. gratissimum* either as an aqueous extract or chaff. Values recorded was comparable to value obtained in Ascorbic acid standard. As indicated in **Table 3**: the least absorbance closest to value obtained using ascorbic acid was at a concentration of 200 ug/ml. The herb, *V. amygdalina* has antioxidant properties [63, 64].

The DPPH scavenging activity (% inhibition) is presented in **Figure 5**. *Vernonia amygdalina* chaff earlier reported in **Table 3** above elicited DPPH scavenging activity which was close to Ascorbic acid was significantly higher than values recorded for *O. gratissimum* leave chaff or aqueous extract. This value was also higher than that recorded for *V. amygdalina* aqueous extract in a dose dependent manner.

Expectedly, one will think the same trend will follow in DPPH scavenging activity for eggs collected from laying hens fed medicinal leaf chaff as component of feed or administered aqueous extract of either medicinal plant (**Table 4**). Rather; it was eggs from laying hens administered aqueous *O. gratissimum* that had numerically higher


*abcd: means along the same column with different superscripts are significantly different (*p < 0.05*); DPPH: 2,2-diphenyl-1-picrylhydrazyl; AE: aqueous extract; SEM: standard error of mean.*

#### **Table 2.**

*DPPH scavenging activity (UV – Absorbance at 517 nm) in medicinal plants – Aqueous extract and chaff.*


#### **Table 3.**

*DPPH scavenging activity (UV-absorbance at 517 nm) in* V. amygdalina *chaff compared to ascorbic acid standard.*

#### **Figure 5.**

*DPPH scavenging activity (% inhibition). VA: Vernonia amygdalina; VA extract: Vernonia amygdalina aqueous extract; Og extract:* Ocimum gratissimum *Aqueous extract; Og chaff:* O. gratissimum *Chaff.*


#### **Table 4.**

*DPPH scavenging activity (% inhibition) in eggs from laying hens fed or administered medicinal plants.*

DPPH scavenging activity which was better than that recorded for birds feed the layers mash control diet. Although values obtained in the study were lower than that recorded for Ascorbic acid; it can be said that may be the antioxidant components in *O. gratissimum* were better absorbed and incorporated into eggs of layers administered *O. gratissimum* aqueous extract than the chaff and *V. amygdalina* either as component of feed or aqueous extract for laying hens. The compound DPPH does not occur naturally and is not present in any organic compound; however, it is readily used to evaluate the antioxidant activity of organic compounds such as vitamins, polyphenols and other phytochemicals which can be present in medicinal plants and their extracts.

Natural products such as sesquiterpenoids and flavonoids were reported to be potential antioxidants [65–67]. These products are naturally obtained from food and use of medicinal plants. This fact further strengthens the antioxidative potential of *V. amygdalina*. Furthermore, results of earlier study added that not only flavonoids are responsible for any antioxidant effects of this plant species [68] but the presence of sesquiterpene lactones [69] might also play a part. Phytochemical screening of bitter leaf extract revealed a high concentration of flavonoids reported to be as the most abundant phytochemical present [63]. Antioxidant assay also indicated high levels of antioxidant activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity which was concentration dependent.

*Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered... DOI: http://dx.doi.org/10.5772/intechopen.111658*

The possibility of enhancing egg quality of laying hens by means of utilizing products exhibiting antioxidant properties can contribute to maintain oxido-redox balance thereby reducing the effect of oxidation on egg quality—degradation of eggs [38].

Possibly consumption of medicinal plants by laying hens either as feed component or aqueous extract can fortify the antioxidant properties of eggs. Poultry feed is composed mostly of plant ingredients which can be attributed to the antioxidant scavenging activity recorded in eggs from laying hens fed the control diet. Besides spoilage of eggs which is a concern to both consumers and producers of table eggs, the possibility of improving the not only the shelf life [38] but also improvement in good egg quality parameters [14] as well as designing eggs as functional foods.

Increased content of optimal ω-3 fatty acids, better ω-6/ω-3 fatty acids ratio, as well as good sensory profile of eggs optimal yolk color [14]. Furthermore, it has been shown in previous studies that fatty acid composition of eggs is dependent on fatty acid composition of feed given to laying hens which is subsequently transferred to the eggs [31].

A significant dose dependent DPPH scavenging activity (UV-absorbance at 517 nm) was observed in either medicinal plant consumed by laying hens and in eggs collected from the laying hens irrespective of treatment as presented in **Table 5**.

**Table 6** further corroborated the possibility of plant to influence antioxidant scavenging activity of eggs from laying hens. The UV-absorbance at 517 nm for DPPH scavenging activity from eggs sampled showed significant improvement in antioxidant scavenging activity treatment 5 as earlier indicated in **Table 4**. Eggs from birds administered aqueous *O. gratissimum* significantly improved % inhibition of DPPH.

The possibility of producing functional eggs from laying hens for human consumption is wide. Apart from improving egg quality [38, 58], shelf life [38]; cholesterol content [57, 58], fatty acids profile and sensory profile [14, 15]. opportunities exist to improve antioxidant properties of eggs using medicinal plants. With increasing demand for enriched and functional foods which will provides various benefits to human health, eggs can be enriched with desirable nutrients [15] by means of dietary manipulation to achieve this goal [26, 27].

The all-important role of certain fatty acids such as linoleic and α-linolenic besides their long-chain (LC) *n*-6 and *n*-3 polyunsaturated fatty acids (PUFA) for humans have been reported some of the benefits ascribed to consumption of n-3 PUFA enriched eggs [15, 29]. Evidently this is made possible by feeding laying hens with


*abcde: means along the same column with different superscripts are significantly different (*p < 0.05*); DPPH: 2,2-diphenyl-1-picrylhydrazyl; SEM: standard error of mean.*

#### **Table 5.**

*DPPH scavenging activity (UV – Absorbance at 517 nm in plants and eggs sampled.*


*abcd: means along the same column with different superscripts are significantly different (*p < 0.05*); DPPH: 2,2-diphenyl-1-picrylhydrazyl; SEM: standard error of mean; T1: control group; T2: treatment 2 –* V. amygdalina *chaff; T3: treatment 3 – 1 ml aqueous* V. amygdalina*; T4: Treatment 4 –* Ocimum gratissimum *chaff; T5: Treatment 5 – 1 ml* O. gratissimum *aqueous extract.*

#### **Table 6.**

*Effect of treatment on DPPH scavenging activity (UV – Absorbance at 517 nm) in eggs from layers administered medicinal plant extract.*

different by-products rich in PUFA [15, 27, 28, 31] such as flaxseed, rapeseed, microalgae, canola, chia (seed, meals or oils) etc.

Eggs for human consumption can have their fatty acid components enhanced by manipulating their feed by means of medicinal plants having antioxidant properties which may be transferred into the eggs. Furthermore, such eggs can serve as functional food for human consumption due to health benefits ascribe to eggs from laying hens fed with certain plants cum medicinal plants. In addition, the two medicinal plants reported here have antioxidant properties which can be of health benefit to consumers.

## **5. Conclusion**

Although there are several variables for measuring antioxidant properties of medicinal plants; however, this study only looked at DPPH scavenging activity in eggs from laying hens fed *V. amygdalina* and *O. gratissimum* leaf chaff as components of the feed. It also evaluated DPPH scavenging activity when the aqueous extract of both plants is administered to laying hens. The DPPH scavenging activity of *V. amygdalina* chaff was high and comparable to ascorbic acid; but it was aqueous *O. gratissimum* administration to laying hens that yielded improved DPPH scavenging activity.

## **Acknowledgements**

Miss Dressman, Mary Paul is gratefully acknowledged for collecting the eggs and participating in the study as part of her BSc project.

## **Conflict of interest**

The authors declare no conflict of interest.

*Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered... DOI: http://dx.doi.org/10.5772/intechopen.111658*

## **Author details**

Habeeb O. Yusuf1 and Ruth T.S. Ofongo2 \*

1 Faculty of Pharmacy, Department of Pharmacognosy, University of Lagos, Lagos State, Nigeria

2 Faculty of Agriculture, Department of Animal Science, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria

\*Address all correspondence to: ruthofongo@ndu.edu.ng; ruthofongo@gmail.com

© 2023 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.

## **References**

[1] Lourenço SC, Moldão-Martins M, Alves VD. Antioxidants of natural plant origins: From sources to food industry applications. Molecules (Basel, Switzerland). 2019;**24**(22):4132. DOI: 10.3390/molecules24224132

[2] Gholami-Ahangaran M, Ahmadi-Dastgerdi A, Azizi S, Basiratpour A, Zokaei M, Derakhshan M. Thymol and carvacrol supplementation in poultry health and performance. Veterinary Medicine and Science. 2022;**8**:267-288. DOI: 10.1002/vms3.663

[3] Fadzai B, Elaine C, Stanley M. Evaluation of nitrite radical scavenging properties of selected Zimbabwean plant extracts and their phytoconstituents. Journal of Food Processing. 2014. Article ID 918018:7. DOI: 10.1155/2014/918018

[4] Masoko P, Eloff JN. Screening of twenty-four south African *Combretum* and six *Terminalia* species (Combretaceae) for antioxidant activities. African Journal of Traditional, Complementary and Alternative Medicines. 2007;**4**:231-239. DOI: 10.4314/ajtcam.v4i2.31213

[5] Huyut Z, Beydemir S, Gülçin I. Antioxidant and antiradical properties of selected flavonoids and phenolic compounds. Biochemistry Research International. 2017;**2017**, Article ID 7616791:10. DOI: 10.1155/2017/7616791

[6] Comunian TA, Ravanfar R, de Castro IA, Dando R, Favaro-Trindade CS, Abbaspourrad A. Improving oxidative stability of echium oil emulsions fabricated by microfluidics: Effect of ionic gelation and phenolic compounds. Food Chemistry. 2017;**233**:125-134. DOI: 10.1016/j.foodchem.2017.04.085

[7] Sagar BK, Singh RP. Genesis and development of DPPH method of

antioxidant assay. Journal of Food Science and Technology. 2011;**48**(4):412- 422. DOI: 10.1007/s13197-011-0251-1

[8] Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy Reviews. 2010;**4**(8):118- 126. DOI: 10.4103/0973-7847.70902

[9] Phaniendra A, Jestadi DB, Periyasamy L. Free radicals: Properties, sources, targets, and their implication in various diseases. Indian Journal of Clinical Biochemistry. 2015;**30**(1):11-26. DOI: 10.1007/s12291-014-0446-0

[10] Haseeb A, Ghulam H and Imtiaz M. Antioxidants from Natural Sources. In Antioxidants in Foods and Its Application. Emad Shalaby and Ghada Mostafa Azzam Eds. Intech. 2018. DOI: 10.5772/intechopen.75961

[11] Traber MG, Stevens JF. Vitamins C and E: Beneficial effects from a mechanistic perspective. Free Radical Biology & Medicine. 2011;**51**(5):1000- 1013. DOI: 10.1016/j.freeradbiomed

[12] Idamokoro EM, Falowo AB, Oyeagu CE, Afolayan AJ. Multifunctional activity of vitamin E in animal and animal products: A review. Animal Science Journal. 2020;**91**:e13352. DOI: 10.1111/asj.13352

[13] Loi M, Paciolla C, Logrieco AF, Mulè G. Plant bioactive compounds in pre- and postharvest management for aflatoxins reduction. Frontiers in Microbiology. 2020;**11**:243. DOI: 10.3389/ fmicb.2020.00243. PMID: 32226415; PMCID: PMC7080658

[14] Spasevski N, Peulić T, Banjac V, Rakita S, Puvača N, Kokić B, et al.

*Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered... DOI: http://dx.doi.org/10.5772/intechopen.111658*

Effects of adding the functional co-extrudates and natural pigments in the diet of laying hens on egg quality. In: 26th World's Poultry Congress, book of abstracts. 2020. p. 445

[15] Vlaicu PA, Panaite TD, Turcu RP. Enriching laying hens' eggs by feeding diets with different fatty acid composition and antioxidants. Scientific Reports. 2021;**11**:20707. DOI: 10.1038/ s41598-021-00343-1

[16] Cotterill OJ, Geiger GS. Egg product yield trends from shell eggs. Poultry Science. 1977;**56**:1027-1031

[17] Li-Chan ECY, Kim HO. Structure and chemical composition of eggs. In: Mine Y, editor. Egg Bioscience and Biotechnology. Hoboken, NJ, USA: John Wiley & Sons, Ltd; 2008. pp. 1-95

[18] Seuss-baum I. Nutritional evaluation of egg compounds. In: Huopalahti R, López-Fandiño R, Anton M, Schade R, editors. Bioactive Egg Compounds. Berlin, Heidelberg. Germany: Springer; 2007. pp. 117-144. DOI: 10.1007/978-3-540-37885-3\_18

[19] Kovacs-Nolan J, Phillips M, Mine Y. Advances in the value of eggs and egg components for human health. Journal of Agricultural and Food Chemistry. 2005;**53**:8421-8431. DOI: 10.1021/ jf050964f

[20] United States Department of Agriculture. United States Department of Agriculture: National Nutrient Database for standard reference Release 27. Available online: http://ndb.nal.usda.gov/ ndb/ [Accessed: February 25, 2023]

[21] Nimalaratne C, Bandara N, Wu J. Purification and characterization of antioxidant peptides from enzymatically hydrolysed chicken egg white. Food Chemistry. 2015;**188**:467-472. DOI: 10.1016/j.foodchem.2015.05.014

[22] Hatta H, Kapoor M, Juneja L. Bioactive components in egg yolk. In: Mine Y, editor. Egg Bioscience and Biotechnology. Hoboken, NJ, USA: John Wiley & Sons, Ltd.; 2008. pp. 185-237

[23] Carlson S, Montalto M, Ponder D. Lower incidence of necrotizing enterocolitis in infants fed a preterm formula with egg phospholipids. Pediatric Research. 1998;**44**:491-498. DOI: 10.1203/00006450-199810000- 00005

[24] Hoffman DR, Theuer RC, Castaneda YS, Wheaton DH, Bosworth RG, O'Connor AR, et al. Maturation of visual acuity is accelerated in breast-fed term infants fed baby food containing DHA-enriched egg yolk. Journal of Nutrition. 2004;**134**:2307- 2313. DOI: 10.1093/jn/134.9.2307

[25] Surai PF. Effect of selenium and vitamin E content of the maternal diet on the antioxidant system of the yolk and the developing chick. British Poultry Science. 2000;**41**:235-243. DOI: 10.1080/713654909

[26] Ao T et al. Effects of supplementing microalgae in laying hen diets on productive performance fatty-acid profile and oxidative stability of eggs. Journal of Applied Poultry Research. 2015;**24**(3):394-400. DOI: 10.3382/japr/ pfv042

[27] Gonzalez-Esquerra R, Leeson S. Alternatives for enrichment of eggs and chicken meat with omega-3 fatty acids. Canadian Journal of Animal Science. 2001;**81**(3):295-305. DOI: 10.4141/ A00-092

[28] Carrillo S et al. Potential use of seaweeds in the laying hen ration to improve the quality of n-3 fatty acid enriched eggs. In: Nineteenth International Seaweed Symposium. Dordrecht: Springer; 2008. pp. 271-278 [29] FAO/WHO (Food and Agricultural Organization of the United Nations and World Health Organization). 2010 Fats and fatty acids in human nutrition. Report of an Extract Consultation Vol. 91 (FAO Food Nutrition Papers). 2010;**91**: 1-166. PMID: 21812367

[30] Hayat Z, Cherian G, Pasha TN, Khattak FM, Jabbar MA. Oxidative stability and lipid components of eggs from flax-fed hens: Effect of dietary antioxidants and storage. Poultry Science. 2010;**89**(6):1285-1292. DOI: 10.3382/ps.2009-00256

[31] Fraeye I, Bruneel C, Lemahieu C, Buyse J, Muylaert K, Foubert I. Dietary enrichment of eggs with omega-3 fatty acids: A review. Food Research International. 2012;**48**(2):961-969. DOI: 10.1016/j.foodres.2012.03.014

[32] Shahidi F, Ambigaipalan P. Omega-3 polyunsaturated fatty acids and their health benefits. Annual Review of Food Science and Technology. 2018;**9**:345-381

[33] Shinn S, Proctor A, Baum J. Egg yolk as means for providing essential and beneficial fatty acids. Journal of the American Oil Chemists' Society. 2018;**95**:5-11. DOI: 10.1146/ annurev-food-111317-095850

[34] Kralik G, Kralik Z, Galovic O, Hanžek D. Cholesterol content and fatty acids profile in enriched n-3 PUFA and conventional eggs. In: World Poultry Congress 2022; Abstracts presented as webinar. Book of abstract page 57

[35] Charoensiriwatana W, Srijantr P, Teeyapant P, Wongvilairattana J. Consuming iodine enriched eggs to solve the iodine deficiency endemic for remote areas in Thailand. Nutrition Journal. 2010;**9**:68. DOI: 10.1186/1475-2891-9-68

[36] Bourre JM, Galea F. An important source of omega-3 fatty acids, vitamins D and E, carotenoids, iodine and selenium: A new natural multi-enriched egg. Journal of Nutrition, Health & Aging. 2006;**10**:371-376

[37] Naber EC. Modifying vitamin composition of eggs: A review. Journal of Applied Poultry Research. 1993;**2**:385-393

[38] Oueslati K, Ribeiro B, Chavatte D, Alleno C, Bouvet R. Positive impact of prebiotics and antioxidants on egg quality at the end of the laying hen production cycle. In: 26th World's Poultry Congress, Abstracts selected in 2020. p. 145

[39] Nakamura S, Kato A, Kobayashi K. Enhanced antioxidative effect of ovalbumin due to covalent binding of polysaccharides. Journal of Agricultural and Food Chemistry. 1992;**40**:2033-2037. DOI: 10.1021/jf00023a001

[40] Huang X, Tu Z, Xiao H, Wang H, Zhang L. Characteristics and antioxidant activities of ovalbumin glycated with different saccharides under heat moisture treatment. Food Research International. 2012;**48**:866-872. DOI: 10.1016/j. foodres.2012.06.036

[41] Chang O, Ha G, Han G. Novel antioxidant peptide derived from the ultrafiltrate of ovomucin hydrolysate. Journal of Agricultural and Food Chemistry. 2013;**61**:7294-7300. DOI: 10.1021/jf4013778

[42] Liu H, Zheng F, Cao Q, Ren B, Zhu L, Striker G, et al. Amelioration of oxidant stress by the defensin lysozyme. American Journal of Physiology. Endocrinology and Metabolism. 2006;**290**:E824-E832. DOI: 10.1152/ ajpendo.00349.2005

[43] Sugino H, Ishikawa M, Nitoda T, Koketsu M, Juneja LR, Kim M, et al. Antioxidative activity of egg yolk phospholipids. Journal of Agricultural *Antioxidant Fortification of Eggs through Nutrition of Laying Hens Administered... DOI: http://dx.doi.org/10.5772/intechopen.111658*

and Food Chemistry. 1997;**45**:551-554. DOI: 10.1021/jf960416p

[44] Stahl W, Sies H. Antioxidant activity of carotenoids. Molecular Aspects of Medicine. 2003;**24**:345-351. DOI: 10.1016/ s0098-2997(03)00030-x

[45] Ma L, Lin XM. Effects of lutein and zeaxanthin on aspects of eye health. Journal of the Science of Food and Agriculture. 2010;**90**:2-12. DOI: 10.1002/ jsfa.3785

[46] Zhang LX, Cooney RV, Bertram JS. Carotenoids enhance gap junctional communication and inhibit lipid peroxidation in C3H/10T1/2 cells: Relationship to their cancer chemopreventive action. Carcinogenesis. 1991;**12**:2109-2114. DOI: 10.1093/ carcin/12.11.2109

[47] Cuppett SL, Hall CA. Antioxidant activity of the Labiatae. In: Taylor S, editor. Advances in Food and Nutrition Research. Vol. 42. Academic Press Inc.; 1998. pp. 245-271. DOI: 10.1016/ S1043-4526(08)60097-2. Available from: https://www.sciencedirect.com/science/ article/pii/S1043452608600972

[48] Brenes A, Roura E. Essential oils in poultry nutrition: Main effects and modes of action. Animal Feed Science and Technology. 2010;**158**(1-2):1-14. DOI: 10.1016/j.anifeedsci.2010.03.007

[49] Harahap U, Dalimunthe A, Hertiani T, Mahatir MN, Satria D. Antioxidant and antibacterial activities of ethanol extract of *Vernonia amygdalina* Delile. Leaves. 2021. The International Conference on Chemical Science and Technology (ICCST – 2020) AIP Conf. Proc. 2342, 080011-1-080011-4; DOI: 10.1063/5.0045447

[50] Milos M, Mastelic J, Jerkovic I. Chemical composition and antioxidant effect of glycosidically bound volatile

compounds from oregano (*Origanum vulgare* L. ssp. *hirtum*). Food Chemistry. 2000;*71*(1):79-83. DOI: 10.1016/ S0308-8146(00)00144-8

[51] Ofongo RTS, Ohimain EI, Iyayi EA. Qualitative and quantitative phytochemical screening of bitter leaf and neem leaves and their potential as antimicrobial growth promoter in poultry feed. European Journal of Medicinal Plants. 2021;**32**(4):38-49. DOI: 10.9734/ejmp/2021/v32i430383

[52] Shahidi F, Janitha PK, Wanasundara PD. Phenolic antioxidants. Critical Reviews in Food Science and Nutrition. 1993;**32**:67-103. DOI: 10.1080/10408399209527581

[53] Hatano T, Edamatsu R, Mori A. Effects of the interaction of tannins with Co-existing substances. VI.: Effects of tannins and related polyphenols on superoxide anion radical, and on 1, 1-Diphenyl-2-picrylhydrazyl radical. Chemical & Pharmaceutical Bulletin. 1989;**37**:2016-2021. DOI: 10.1248/ cpb.37.2016

[54] Olamilosoye KP, Akomolafe RO, Akinsomisoye OS, Adefisayo MA, Alabi QK. The aqueous extract of *Ocimum gratissimum* leaves ameliorates acetic acid induced colitis via improving antioxidant status and haematological parameters in male Wistar rats. Egyptian Journal of Basic and Applied Sciences. 2018;**5**(3):220-227. DOI: 10.1016/j. ejbas.2018.05.006

[55] Oyem JC, Chris-Ozoko LE, Enaohwo MT, Otabor FO, Okudayo VA, Udi OA. Antioxidative properties of *Ocimum gratissimum* alters Lead acetate induced oxidative damage in lymphoid tissues and haematological parameters of adult Wistar rats. Toxicology Reports. 2021;**8**:215-222. DOI: 10.1016/j. toxrep.2021.01.003

[56] Dauksiene A, Klementaviciute J, Gruzauskas R, Klupsaite D, Bartkiene E. Laying hens' production effectiveness increasing and quality improving by including to their diet sustainable plants (*Helianthus tuberosus* l.). In: 26th World's Poultry Congress, abstracts selected in 2020. p. 443

[57] Abdel-Wareth AAA, Lohakare JD. Productive performance, egg quality, nutrients digestibility, and physiological response of bovans brown hens fed various dietary inclusion levels of peppermint oil. Animal Feed Science and Technology. 2020;**267**:114554. DOI: 10.1016/j.anifeedsci.2020.114554

[58] Vlaicu PA, Panaite TD. Effect of dietary pumpkin (*Cucurbita moschata*) seed meal on layer performance and egg quality characteristics. Animal Bioscience. 2022;**35**(2):236-246. DOI: 10.5713/ab.21.0044

[59] Vakili R, Toroghian M, Torshizi ME. Saffron extract feed improves the antioxidant status of laying hens and the inhibitory effect on cancer cells (PC3 and MCF7) growth. Veterinary Medicine and Science. 2022;**8**:2494-2503. DOI: 10.1002/ vms3.910

[60] Omri B, Alloui N, Durazzo A, Lucarini M, Aiello A, Romano R, et al. Egg yolk antioxidants profiles: Effect of diet supplementation with linseeds and tomato-red pepper mixture before and after storage. Foods. 2019;**8**:320. DOI: 10.3390/foods8080320

[61] Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958;**181**:1199-1200. DOI: 10.1038/1811199a0

[62] Steel RGD, Torrie JH. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. New York, USA: McGraw-Hill; 1980. pp. 20-90

[63] Oriakhi K, Oikeh EI, Ezeugwu NO, Anoliefo O, Aguebor OES. Comparative antioxidant activities of extracts of *Vernonia amygdalina* and *Ocimum gratissimum* leaves. Journal of Agricultural Science. 2014;**6**:13-20. DOI: 10.5539/jas.v6n1p13

[64] Ekaluo UB, Ikpeme EV, Ekerette EE, Chukwu CI. *In vitro* antioxidant and free radical activity of some Nigerian medicinal plants: Bitter leaf (*Vernonia amygdalina* L.) and Guava (*Psidium guajava* Del.). Research Journal of Medicinal Plant. 2015;**9**(5):215-226. DOI: 10.3923/rjmp.2015.215.226

[65] Bork PM, Schimitz ML, Kuhnt M, Escher C, Heinrich M. Sesquiterpene lactone containing Mexican Indian medicinal plants and pure sesquiterpene lactones as potent inhibitors of transcription factor NF-*j*B. FEBS Letters. 1997;**402**:85-90. DOI: 10.1016/ s0014-5793(96)01502-5

[66] Haraguchi H, Ishikawa H, Sanchez Y, Ogura T, Kubo Y, Kubo I. Antioxidative constituents of *Heterotheca inuloides*. Bio-organic & Medicinal Chemistry. 1997;**5**:865-871. DOI: 10.1016/ S0968-0896(97)00029-1

[67] Jodynis-Liebert J, Murias M, Bloszyk E. Effect of sesquiterpene lactones on antioxidant enzymes and some drug-metabolizing enzymes in rat liver and kidney. Planta Medica. 2000;**66**:199-205. DOI: 10.1055/s-2000-8566

[68] Igile GO, Oleszek W, Jurzysta M, Burda S, Fanfunso M, Fasanmade AA. Flavonoids from Vernonia amygdalina and their antioxidant activities. Journal of Agricultural and Food Chemistry. 1994;**42**:2445-2448. DOI: 10.1021/ jf00047a015

[69] Erasto P, Grierson DS, Afolayan AJ. Antioxidant constituents in *Vernonia amygdalina* leaves. Pharmaceutical Biology. 2007;**45**(3):195-199. DOI: 10.1080/13880200701213070

## **Chapter 3** The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds

*Mostafa Fazeli*

## **Abstract**

Plant-associated microorganisms that live symbiotically in the plant body without causing disease symptoms are called endophytic microorganisms. Endophytes, including bacteria and fungi, can enhance the growth of the host plant and increase its resistance to pests, phytopathogens, and environmental stresses. In addition, endophytes can regulate the synthesis of plant secondary metabolites. Endophytes are a new reservoir for the discovery and production of valuable active substances. Some endophytic secondary metabolites are the same as host plants, such as paclitaxel. This finding has increased the importance of endophytes because the production of effective substances on an industrial scale in microorganisms is easier than in plants and has lower environmental costs. Therefore, endophytes need more attention in the pharmaceutical industry.

**Keywords:** endophyte, symbiosis, secondary metabolites, Taxol, endophytic fungi

## **1. Introduction**

The rapid growth of human societies has increased the need to improve health standards and intensify food production. On the other hand, the emergence of drug resistance in pathogens and pests has become an increasing need to promote the search for new pharmaceutical and agricultural sources. Medicinal plants have been a valuable source of bioactive substances for a long time; however, environmental considerations, labor-intensive, high cost, and time-consuming have limited the use of these plant resources. On the other hand, the production of plant material in cell cultures faces technical challenges. The production of effective plant substances entered a new age with the discovery of the endophytic fungus *Taxomyces andreanea* in the yew, which could produce bioactive such as its host. Microorganisms are an attractive source of new biomaterials; also, they have the potential to increase the production of existing valuable materials. Plant-associated microorganisms called endophytes live in symbiosis with the tissues of their host plants. Many microorganisms, such as fungi, bacteria, and actinomycetes, have been discovered in endophytic relationships with plants [1].

The endophytes live asymptomatically in mutual association with plants. The endophytic lifestyle of microbes plays an important role in maintaining the health of plants by providing nutrients and defending plants against abiotic and abiotic stresses [2]. In addition, endophytes can produce many bioactive. Some of these substances are similar to the profile of the host plant's bioactive, which has increased the hope for cost-effective and environmentally friendly production. In the pharmaceutical and agricultural industries, bioactive compounds are known for their many applications. During the last two decades, endophytes have been recognized as important sources of bioactive compounds. Also, the proportion of new structures produced by endophyte isolates (51%) is significantly higher than that of soil isolates (38%), which has made endophytes one of the main natural product screening programs [3].

## **2. Endophytes**

Microorganisms colonize many living plants in nature, and the degree of this microbial colonization varies by plant species. If the host plant tissue remains stable during this colonization, the relationship may vary from latent pathogenesis to mutual symbiosis. These microorganisms may be epiphytes, endophytes, or latent pathogens. Endophyte refers to microorganisms that are found under normal conditions in the tissues of living plants, without causing apparent diseases or visible symptoms of disease [4]. Endophytes are ubiquitous and spend a significant part of their life cycle without causing negative or obvious symptoms in the living tissues of the host plant. The word endophyte was first coined in 1866, where "endo" means "inside" and "phyte" means plant. They are mostly located in internal tissues such as roots, stems, leaves, flowers, and seeds. Endophytes may be transmitted horizontally or vertically [2], and some may even be seed-borne and passed on to the next generation [4]. A large community of endophytes lives inside the tissues of any plant. The diversity of endophytes is influenced by the host plant and its characteristics, including genotype, tissue, growth stage (age), and health status [5].

Endophytes have been isolated from all different parts of the plant. More than 200 genera from 16 bacterial phyla have been documented to be associated with endophytes [6]. It is also estimated that out of about 1.5 million species of fungi, one million of them are endophytic [7].

#### **2.1 Endophytes: Plant interaction**

Endophytes can provide benefits to their host plants. They mediate abiotic and biotic stress tolerance, reduce water consumption, and defend against pests and phytopathogens [8]. This interaction is controlled by endophyte and plant genes. The endophytic relationship is a novel and cost-effective plant-microbe evolutionary relationship that is driven by location and not defined by function [9]. Endophytic microbes are chemical synthesizers inside plants [10]. The imperceptible association of endophytes with the plant enables them to evolve [9]. It is the coevolution between endophytes and their host plant that determines the production of bioactive compounds. These compounds often play a role in the plant-microbe interaction in different ways and can bring different fitness benefits to the host plant [11, 12].

Plant compounds can be of plant origin or derived from endophytes or even can be produced by both. In the latter case, the endophyte may be involved in the entire pathway, but another scenario may be that only parts of the biosynthesis originate

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

from the endophyte. In plant-endophyte interactions, significant changes appear in the secondary metabolism of symbionts, and these changes can be as a result of (i) induction of host metabolism by endophyte, (ii) induction of endophyte metabolism by the host, (iii) host and endophyte share part of a specific pathway, (iv) the host metabolizes endophyte products, and (v) the endophyte can metabolize host secondary compounds. [13]. Endophytes isolated from medicinal plants can produce bioactive metabolites and play a vital role in inducing secondary metabolite production by host plants [5, 14].

## **3. Secondary metabolites**

Endophytes play a critical role in enhancing plant growth and are also known for their ability to produce bioactive with biotechnological applications. The use of herbal medicines is common in developing countries and up to 80% of people use this medicine. This traditional medicine has a long history. Medicinal plants are known for their rich sources of natural products. They are very valuable for disease prevention and treatment [15]. Endophytes communicate with their host plant through metabolic interactions [1, 16], which enable them to produce signaling molecules with interesting biological activities. In addition, the coevolution of endophytes with the host plant enables them to mimic the biological properties of the host and produce similar bioactive compounds [16].

Endophytes synthesize various bioactive compounds. However, compounds that have shown anticancer properties have attracted more attention, and in the meantime, the discovery of paclitaxel production by endophytic fungi has been a turning point in endophyte research.

#### **3.1 Paclitaxel (Taxol)**

Paclitaxel, with the brand name Taxol, is a terpenoid that was mainly obtained from the tissues of the yew plant; due to its amazing properties in binding to microtubules and inhibiting the division spindle, it is used in the treatment of various types of cancer, especially breast and ovarian cancer. It has been used a lot. However, extraction from plant sources due to the slow growth of the plant, the difficulty of purifying paclitaxel, and also its low amount in the plant tissues did not meet the needs of the market. Therefore, several methods, such as chemical synthesis, were also developed and commercialized. The scientists were also looking for alternative sources until the ability to synthesize it in the endophytic fungi of the host plant was discovered.

The discovery of *Taxomyces andreanea* from the Pacific yew,*Taxus brevifolia*, was undoubtedly a turning point in the field of bioprospecting for endophytes. This endophytic fungus demonstrated the ability to synthesize paclitaxel in the culture broth same as its host plant [17]. Microbial production of paclitaxel is very important for the development of the first billion-dollar anticancer drug business [17, 18]. Since this important discovery, several other endophytic fungi and bacteria showing paclitaxel production from yew and other plant species have been discovered (**Table 1**), including *Alternaria*, *Bartalinia*, *Fusarium*, *Lasiodiplodia*, *Metarhizium*, *Monochaetia*, *Pestalotiopsis*, *Penicillium*, *Phoma*, and *Spomatoichoanthermium* [13, 81–83]. The efficiency of paclitaxel among these fungal species varies [from nanograms to milligrams per liter], and their productivity is often lost during several generations of cultivation in laboratory conditions [84]. Microbial production of paclitaxel by endophytes has

#### *Medicinal Plants – Chemical, Biochemical, and Pharmacological Approaches*



*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*


*Some strains that are only capable of producing precursors, such as baccatin III and 10-DAB, are listed separately. \* Reveals bacterial producers.*

#### **Table 1.**

*Production of paclitaxel and some of its precursors by endophytic microorganisms; due to the multiplicity of different isolates from the same species, the name of the strain is also mentioned, as well as the amount of production in the strains noticed without subsequent manipulations and optimizations.*

been observed mostly in fungal isolates. However, there are limited reports of the production of paclitaxel and some of its precursors by several strains of endophytic bacteria, such as *Erwinia taxi*, *Micromonospora* sp., *Streptomyces* sp., *Kitasatospora* sp., *Bacillus cereus*, *B. megaterium*, *Sphingomonas* ssp. *taxi*, *B. subtilis*, *Pantoea* sp., and *Curtobacterium* sp. [85]. Also, the discovery of paclitaxel-producing bacteria symbiotic with marine macroalgae *Sargassum polycystum* and *Acanthaphora specifera* showed that the search for endophytic sources of paclitaxel should not be limited to plants and terrestrials [68].

In addition to paclitaxel production, some endophytes can increase paclitaxel production in plants. Endophytic *Pseudodidymocyrtis lobariellae* fermentation broth can effectively increase paclitaxel accumulation in *T. chinensis* by regulating phytohormone metabolism and signal transduction and further regulating the expression of several key genes involved in paclitaxel biosynthesis [86]. The fermentation broth of *Kocuria* sp., *Micromonospora* sp., and *Sphingomonas* sp. also significantly increased the accumulation of taxanes in the stem cells of *T. yunnanensis* [87].

#### **3.2 Vinca alkaloids**

Vinblastine and vincristine are vinca alkaloids from *Catharanthus roseus* plant [88]. These compounds were the first herbal anticancer agents that were introduced to the

## *The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

clinical market. In the 1960s, vinblastine was used to treat breast cancer, testicular cancer, and Hodgkin's disease. Three years later, its oxidized derivative, vincristine, was introduced, which was widely used in the treatment of leukemia. These compounds inhibit the division spindle by irreversibly binding to microtubules and finally induce apoptosis. Vinblastine production from endophytic *Alternaria* was first described in 1998, followed by Lingqi et al. discovered an endophytic *Fusarium oxysporum* from *C. roseus* that successfully produced vincristine [89, 90]. These discoveries sparked a global hunt for new alternative sources of vinblastine and vincristine. Vincristine is most valuable as an anticancer agent. Endophytic *F. oxysporum* successfully biotransformed vinblastine to vincristine [91].

Palem et al. isolated an endophytic *Thalaromyces radicus* from *C. roseus* that could produce vinblastine and vincristine [92]. Ayob et al. isolated an endophyte *Nigrospora sphaerica* from *C. roseus* that can produce vinblastine. This fungus produced vinblastine with 10-fold better cytotoxicity to a breast cancer cell line compared to vinblastine extracted from *C. roseus* [93]. Endophytic fungal and bacterial species were found to have the ability to synthesize Vindoline —the precursor of vinca alkaloids— and have a high potential to be used as a biological elicitor in the production of vincristine [94, 95]. Also, a species of *Streptomyces* spp. was isolated from the rhizosphere soil of *C. roseus*, which can produce vinblastine and vincristine, (**Table 2**) [104].


*Some of these isolates can biotransform vinblastine into vincristine. Also, some isolates only can synthesize vindoline as a valuable precursor of anticancer drugs.*

#### **Table 2.**

*Microbial production of vinca alkaloids by endophytic microorganisms; endophytic producers of vinca alkaloids have so far only been isolated from* C. roseus *or its rhizosphere soil.*

## **3.3 Camptothecin**

Camptothecin (CPT) is a pentacyclic quinoline alkaloid isolated from the wood of *Camptotheca acuminata* and the root of *Nothapodytes foetida*. Several reports show the therapeutic potential of CPT and its derivatives for the treatment of colon, cervical, uterine, lung, and ovarian cancer. Most of the two promising anticancer activities [107] are related to its main derivatives, 9-methoxycamptothecin and 10-hydroxycamptothecin, because CPT is not directly used as an anticancer drug due to its low solubility, short half-life, and toxicity [108–110]. These cytotoxic agents act by selectively inhibiting topoisomerase 1. and thereby disrupting the DNA replication process.

In 2005, the CPT-producing endophytic fungus *Entrophospora infrequens* was isolated from *N. foetida* [109]. Endophyte *Neurospora crassa* and *Nodulisporium* sp. isolated from *N. foetida* produces CPT in culture medium [111, 112]. There are also examples of endophytes that can produce hydroxylated CPT derivatives, for example, Mycelia sterilia XK001 can produce 10-hydroxycamptothecin, which is the clinically active derivative of CPT [107]. Most CPT-producing endophytes are fungi; however, there are also reports of bacterial producers (**Table 3**) [116, 121, 123, 126]. A CPT-producing endophytic fungus from the marine sponge *Cliona* sp. It has been isolated that unlike other endophytes isolated from soil and plant environments, and it has been isolated and identified from the marine environment and aquatic organisms [131].




*CPT and active derivative 10-hydroxyCPT, podophyllotoxin, deoxypodophyllotoxin act as anticancer, and Huperzine A approved for treatment of Alzheimer's disease.*

#### **Table 3.**

*Production of plant-derived secondary metabolites by endophytic microorganisms.*

### **3.4 Podophyllotoxin**

Podophyllotoxin is an aryltetralin lignin that uses in the synthesis of anticancer drugs. It is originally isolated from the resins of the *Podophyllum emodi*, which is traditionally used to treat genital warts [16]. Podophyllotoxin is a strong inhibitor of microtubules, while its derivatives inhibit topoisomerase 2. These derivatives are used to treat bronchial and testicular cancers. Podophyllotoxin production from endophytic fungi isolated from *Podophyllum* [syn. *Sinopodophyllum*] *hexandrum*, *Diphylleia sinensis*, and *Dysosma veitchii* were reported for the first time [134]. After that, two strains of the endophytic fungus *Phialocephala fortinii* from the rhizome of *P. peltatum*, which could produce podophyllotoxin under axenic culture conditions, were isolated and identified [138]. The fungus *Trametes* isolated from *P. hexandrum* is another endophyte capable of producing podophyllotoxin and podophyllotoxin glycosides [139]. In addition, *F. oxysporum* and *Aspergillus* endophytes isolated from

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

*Juniperus recurva* and *Juniperus communis* produced podophyllotoxin and deoxypadophyllotoxin, respectively [141, 151]. Podophyllotoxin production has also been reported from *Mucor fragilis*, and *Alternaria tenuissima* isolated from *P. emodi* was found to produce podophyllotoxin [143, 144]. Podophyllotoxin-producing endophytic fungi *Penicillium* sp.,*Trametes* sp., *Purpureocillium* sp., *Aspergillus* sp. *Ganoderma* sp., and *Fusarium* spp. were isolated from plants of *Dysosma* spp. [149, 150]. Most podophyllotoxin-producing fungi belong to *Penicillium* sp., *Alternaria* sp., and *Fusarium* spp. genera, respectively, While there is no report of podophyllotoxin production among endophyte bacteria (**Table 3**).

Fungal production of podophyllotoxin is promising for mass production, and it is possible to provide affordable resources for commercial production by optimizing the cultivation methods and genetic changes of the producing microorganisms and reducing the pressure of harvesting from plant resources and giving the chance to producing plants for save from extinction.

#### **3.5 Huperzine A**

The lycopod *Huperzia serrata* is the main source of a natural lycopodium alkaloid called Huperzine A (HupA), which has attracted worldwide attention for its potential in the treatment of Alzheimer's disease. This compound is an acetylcholinesterase inhibitor that increases the availability of acetylcholine in central cholinergic synapses by highly selective and reversible inhibition of this enzyme and blocking its activity. The bulk of HupA is obtained from the *Huperziaceae* family. The *H. serrata* has a narrow geographical distribution, slow growth rate, and very low HupA content, which limits its natural harvest and HupA extraction. In the first report, the endophyte *Acremonium* sp. isolated from *H. serrata* has been capable of production of HupA [152]. Similarly, the endophyte *Shiraia* sp. Slf14 and *Cladosporium cladosporioides* isolated from *H. serrata* leaves also produced HupA [155, 156, 158]. In general, 32 endophytic fungi belonging to 15 genera were recorded to produce Hup A. These fungal endophytes were isolated from members of *Huperziaceae* family, including *H. serrata*, *Phlegmariurus phlegmaria*, and *Phlegmariurus taxifolius* (**Table 3**) [171].

Xia and colleagues isolated endophytic fungi *Mucor racemosus*, *M. fragilis*, *Fusarium verticillioides*, *F. oxysporum*, and *Trichoderma harzianum* from the *H. serrata*, which can inhibit acetylcholinesterase enzyme [168]. The endophyte *Ceriporia lacerate* successfully transformed HupA into five different compounds that showed potential acetylcholinesterase inhibitory activity [172]. Biotransformation, using fungal endophytes, is also a valuable approach to producing HupA derivatives. Microbial production of HupA has positive economic and environmental effects. This will be a practical strategy to meet the global market demand through microbial fermentation and genetic manipulation of the source fungi.

## **4. Industrial aspects**

The role of plant compounds in the production of many clinically effective anticancer drugs is undeniable, but the production of herbal drugs is not always as expected. Because their production from plant resources faces serious challenges, many of these compounds are produced at a certain stage of plant growth or under certain environmental conditions, stress, or availability of nutrients. Also, the growth of plants is slow, and to collect and extract some products, they must reach acceptable growth. On the other hand, production in plant cell culture also faces technical challenges. Also, due to the extent and variety of bioactive in plants, the purification processes of the desired effective substances will be complicated and therefore expensive. Due to the limitations identified with the productivity and vulnerability of plant species as sources of new metabolites, microorganisms act as an available and inexhaustible resource of new pharmaceuticals [173].

Over many years, seasonal and climatic factors have caused failure in traditional methods of extracting bioactive from natural resources. The environmental issues that researchers face during the extraction of bioactive from plants make it necessary to adopt new approaches to obtain these compounds [174]. In the future, with the increase in population, the demand for pharmaceutical and agricultural products will increase day by day, and the future of endophytic fungi for the isolation of various beneficial compounds is bright. There is a great need to discover bioactive compounds from natural resources that can be used to treat various diseases. Recently, more attention has been paid to the production of bioactive from endophytic fungi because they are excellent for exploiting the biosynthetic pathway for the synthesis of bioactive. The main challenge is the low yield of desired active compounds obtained from endophytes. However, to meet the demand of pharmaceutical companies to increase the commercial production of drugs, genetic engineering technologies, drug design techniques, and microbial fermentation technology can be solutions to increase the rate of endophyte production [2]. In addition, the use of cell cocultures of host plants and endophytes has improved the production rate. Some secondary metabolites may be produced by combined endophyte and host activity. Some endophytic bacteria produce secondary metabolites in medicinal plants. For example, *Bacillus altitudinis*, *Burkholderia* sp., and *Flavobacterium* sp. act as effective stimulators that increase ginsenoside concentrations by converting the major ginsenoside Rb1 to the minor ginsenoside Rg3 in the valuable medicinal plant ginseng [175–177]. Such biotransformations using endophytic bacteria have significant potential to intensify the accumulation of rare active substances in medicinal plants. The endophytic *Pseudomonas fluorescens* can increase the production of sesquiterpenoids in *Macrocephala Atractylodes* [178]. The endophyte *Bacillus subtilis* in the plant *Chuanxiong Ligusticum* enhances ligustrazine accumulation [179].

The interaction of endophytes with plant tissues asymptomatically increases the production of secondary metabolites. A double synthesis of podophyllotoxin was obtained from the interaction of endophytic fungi *Phialocephala fortinii* and rhizomes of *P. peltatum* [138]. Endophytic fungi *Stemphylium amaranthi* and *Gliomastix masseei* can be used as fungal stimulants to improve indole alkaloid production from *C. roseus* [180].

## **5. Conclusion**

Throughout history, humans have used plants and plant-derived products to treat various ailments. Plant secondary metabolites or bioactive are known to be synthesized by plants. Microbes living inside host plant tissues are also known for their ability to synthesize substances similar to those synthesized by the host plant. Secondary metabolites, such as alkaloids, flavonoids, terpenoids, steroids, etc. synthesized by microbes, are known for their vital role as antioxidants and anticancer. The discovery of the ability to produce plant secondary metabolites in endophytes has

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

raised many hopes for the production of these compounds on an industrial scale. Microorganisms reduce environmental concerns about the production of biological substances in plants because endophytic microbes have a high reproduction ability, the possibility of their genetic manipulation is easier, and the fermentation conditions for them are simpler, cheaper, and more diverse.

## **Author details**

Mostafa Fazeli Mehrdad Zist Tous Ltd, R&D Centers, Mashhad, Iran

\*Address all correspondence to: fazelim921@mums.ac.ir

© 2023 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.

## **References**

[1] Ezeobiora CE, Igbokwe NH, Amin DH, Mendie UE. Endophytic microbes from Nigerian ethnomedicinal plants: A potential source for bioactive secondary metabolites—A review. Bulletin of the National Research Centre. 2021;**45**(1):1-10

[2] Rana KL, Kour D, Kaur T, Devi R, Negi C, Yadav AN, et al. Endophytic fungi from medicinal plants: Biodiversity and biotechnological applications. In: Microbial Endophytes. Sawston, UK: Elsevier; 2020. pp. 273-305

[3] Segaran G, Sathiavelu M. Fungal endophytes: A potent biocontrol agent and a bioactive metabolites reservoir. Biocatalysis and Agricultural Biotechnology. 2019;**21**:101284

[4] Hassani M, Durán P, Hacquard S. Microbial interactions within the plant holobiont. Microbiome. 2018;**6**(1):1-17

[5] Wu W, Chen W, Liu S, Wu J, Zhu Y, Qin L, et al. Beneficial relationships between endophytic bacteria and medicinal plants. Frontiers in Plant Science. 2021;**12**:646146

[6] Gouda S, Das G, Sen SK, Shin H-S, Patra JK. Endophytes: A treasure house of bioactive compounds of medicinal importance. Frontiers in Microbiology. 2016;**7**:1538

[7] Priyadarshini MS, Panigrahi S, Rath C. Endophytes: Novel Microorganisms for Plant Growth Promotion. Tamil Nadu, India: Darshan publishers; 2022

[8] Hodkinson TR, Doohan FM, Saunders MJ, Murphy BR. Endophytes for a Growing World. Cambridge, UK: Cambridge University Press; 2019

[9] Kusari S, Spiteller M. Metabolomics of endophytic fungi producing

associated plant secondary metabolites: Progress, challenges, and opportunities. In: Metabolomics. Rijeka, Croatia: InTechOpen; 2012. pp. 241-266

[10] Kaul S, Gupta S, Ahmed M, Dhar MK. Endophytic fungi from medicinal plants: A treasure hunt for bioactive metabolites. Phytochemistry Reviews. 2012;**11**:487-505

[11] Kusari S, Spiteller M. The promise of endophytic fungi as sustainable resource of biologically relevant pro-drugs: A focus on Cameroon. In: Fungi. Boca Raton, FL, USA: CRC Press; 2018. pp. 1-13

[12] Saxena S, Meshram V, Kapoor N. Muscodor tigerii sp. nov.-volatile antibiotic producing endophytic fungus from the Northeastern Himalayas. Annals of Microbiology. 2015;**65**(1):47-57

[13] Ludwig-Müller J. Plants and endophytes: Equal partners in secondary metabolite production? Biotechnology Letters. 2015;**37**:1325-1334

[14] Ek-Ramos MJ, Gomez-Flores R, Orozco-Flores AA, Rodríguez-Padilla C, González-Ochoa G, Tamez-Guerra P. Bioactive products from plantendophytic gram-positive bacteria. Frontiers in Microbiology. 2019;**10**:463

[15] Pan S-Y, Zhou S-F, Gao S-H, Yu Z-L, Zhang S-F, Tang M-K, et al. New perspectives on how to discover drugs from herbal medicines: CAM's outstanding contribution to modern therapeutics. Evidence-Based Complementary and Alternative Medicine. 2013;**2013**:627375

[16] Meshram V, Gupta M. Endophytic fungi: A quintessential source of

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

potential bioactive compounds. Endophytes for a Growing World. 2019; **277**:277-309

[17] Stierle A, Strobel G, Stierle D. Taxol and taxane production by taxomyces andreanae, an endophytic fungus of Pacific yew. Science. 1993;**260**(5105): 214-216

[18] Stierle AA, Stierle DB. Bioactive secondary metabolites produced by the fungal endophytes of conifers. Natural Product Communications. 2015;**10**(10): 1671-1682

[19] Strobel G, Hess W, Ford E, Sidhu R, Yang X. Taxol from fungal endophytes and the issue of biodiversity. Journal of Industrial Microbiology. 1996;**17**:417-423

[20] Li J-y, Strobel G, Sidhu R, Hess W, Ford EJ. Endophytic Taxol-producing fungi from bald cypress, taxodium distichum. Microbiology. 1996;**142**(8): 2223-2226

[21] Landry N. Bacterial Mass Production of Taxanes with Erwinia. US5561055A: Google Patents; 1996

[22] Strobel GA, Hess W, Li J-Y, Ford E, Sears J, Sidhu RS, et al. Pestalotiopsis guepinii, a Taxol-producing endophyte of the Wollemi pine, Wollemia nobilis. Australian Journal of Botany. 1997;**45**(6): 1073-1082

[23] Li J, Sidhu R, Ford E, Long D, Hess W, Strobel G. The induction of Taxol production in the endophytic fungus—Periconia sp from Torreya grandifolia. Journal of Industrial Microbiology and Biotechnology. 1998; **20**:259-264

[24] Su K. Screening of Taxol-producing endophytic fungi from Ginkgo biloba and Taxus cuspidate in Korea.

Agricultural Chemistry and Biotechnology. 1999;**42**:97-99

[25] Caruso M, Colombo A, Fedeli L, Pavesi A, Quaroni S, Saracchi M, et al. Isolation of endophytic fungi and actinomycetes taxane producers. Annals of Microbiology. 2000;**50**(1):3-14

[26] Page M, Landry N, Boissinot M, Helie M-C, Harvey M, Gagne M. Bacterial Mass Production of Taxanes and Paclitaxel. WO1999032651A1: Google Patents; 2000

[27] Wang B, Li A, Wang X. An endophytic fungus for producing Taxol. Science in China Series C. 2001;**31**:271-274

[28] Guo B, Wang Y, Zhou X, Hu K, Tan F, Miao Z, et al. An endophytic Taxol-producing fungus BT2 isolated from Taxus chinensis var. mairei. African Journal of Biotechnology. 2006; **5**(10):875-877

[29] Hu K, Tan F, Tang K, Zhu S, Wang W. Isolation and screening of endophytic fungi synthesizing Taxol from Taxus chinensis var. mairei. Journal of Southwest China Normal University (Natural Science Edition). 2006;**31**: 134-137

[30] Renpeng T, Qiao Y, Guoling Z, Jingquan T, Luozhen Z, Chengxiang F. Taxonomic study on a Taxol producing fungus isolated from bark of Taxus chinensis var. mairei. Wuhan zhi wu xue yan jiu= Wuhan Botanical Research. 2006;**24**(6):541-545

[31] Cheng L, Ma Q, Tao G, Tao W, Wang R, Yang J, et al. Systemic identification of a paclitaxel-producing endophytic fungus. Industrial Microbiology. 2007;**37**:23-30

[32] Zhou X, Wang Z, Jiang K, Wei Y, Lin J, Sun X, et al. Screening of Taxolproducing endophytic fungi from Taxus chinensis var. mairei. Applied Biochemistry and Microbiology. 2007; **43**:439-443

[33] Gangadevi V, Muthumary J. Taxol, an anticancer drug produced by an endophytic fungus Bartalinia robillardoides Tassi, isolated from a medicinal plant, Aegle marmelos Correa ex Roxb. World Journal of Microbiology and Biotechnology. 2008;**24**:717-724

[34] Gangadevi V, Murugan M, Muthumary J. Taxol determination from Pestalotiopsis pauciseta, a fungal endophyte of a medicinal plant. Chinese Journal of Biotechnology. 2008;**24**(8): 1433-1438

[35] Dai W, Tao W. Preliminary study on fermentation conditions of Taxolproducing endophytic fungus. Chemical Industry and Engineering Progress. 2008;**27**(6):883-886

[36] Kumaran RS, Muthumary J, Hur B-K. Taxol from Phyllosticta citricarpa, a leaf spot fungus of the angiosperm Citrus medica. Journal of Bioscience and Bioengineering. 2008;**106**(1):103-106

[37] Sun D, Ran X, Wang J. Isolation and identification of a Taxol-producing endophytic fungus from Podocarpus. Wei sheng wu xue bao= Acta Microbiologica Sinica. 2008;**48**(5): 589-595

[38] Venkatachalam R, Subban K, Paul MJ. Taxol from Botryodiplodia theobromae (BT 115)—AN endophytic fungus of Taxus baccata. Journal of Biotechnology. 2008;**136**:S189-SS90

[39] Chang-Tian L, Yu L, Wang Q-J, Sung C-K. Taxol production by Fusarium arthrosporioides isolated from yew, Taxus cuspidata. Journal of Medical Biochemistry. 2008;**27**(4):454-458

[40] Senthil Kumaran R, Muthumary J, Hur B. Production of Taxol from Phyllosticta spinarum, an endophytic fungus of Cupressus sp. Engineering in Life Sciences. 2008;**8**(4):438-446

[41] Kumaran RS, Muthumary J, Hur B-K. Isolation and identification of an anticancer drug, Taxol from Phyllosticta tabernaemontanae, a leaf spot fungus of an angiosperm, wrightia tinctoria. The Journal of Microbiology. 2009;**47**(1): 40-49

[42] Chakravarthi B, Das P, Surendranath K, Karande AA, Jayabaskaran C. Production of paclitaxel by Fusarium solani isolated from Taxus celebica. Journal of Biosciences. 2008;**33**: 259-267

[43] Zhao K, Ping W, Li Q, Hao S, Zhao L, Gao T, et al. Aspergillus Niger var. taxi, a new species variant of Taxolproducing fungus isolated from Taxus cuspidata in China. Journal of Applied Microbiology. 2009;**107**(4):1202-1207

[44] Deng BW, Liu KH, Chen WQ, Ding XW, Xie XC. Fusarium solani, Tax-3, a new endophytic Taxol-producing fungus from Taxus chinensis. World Journal of Microbiology and Biotechnology. 2009;**25**:139-143

[45] Liu K, Ding X, Deng B, Chen W. Isolation and characterization of endophytic Taxol-producing fungi from Taxus chinensis. Journal of Industrial Microbiology and Biotechnology. 2009; **36**(9):1171

[46] Zhang P, Zhou P-P, Yu L-J. An endophytic Taxol-producing fungus from Taxus media, Cladosporium cladosporioides MD2. Current Microbiology. 2009;**59**:227-232

[47] Zhang P, Zhou P-P, Yu L-J. An endophytic Taxol-producing fungus *The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

from Taxus x media, aspergillus candidus MD3. FEMS Microbiology Letters. 2009;**293**(2):155-159

[48] Miao Z, Wang Y, Yu X, Guo B, Tang K. A new endophytic taxane production fungus from Taxus chinensis. Applied Biochemistry and Microbiology. 2009;**45**:81-86

[49] Kumaran RS, Muthumary J, Kim E-K, Hur B-K. Production of Taxol from Phyllosticta dioscoreae, a leaf spot fungus isolated from Hibiscus rosasinensis. Biotechnology and Bioprocess Engineering. 2009;**14**:76-83

[50] Gangadevi V, Muthumary J. A novel endophytic Taxol-producing fungus Chaetomella raphigera isolated from a medicinal plant, Terminalia arjuna. Applied Biochemistry and Biotechnology. 2009;**158**:675-684

[51] Gangadevi V, Muthumary J. Taxol production by Pestalotiopsis terminaliae, an endophytic fungus of Terminalia arjuna (arjun tree). Biotechnology and Applied Biochemistry. 2009;**52**(1):9-15

[52] Zhao K, Sun L, Ma X, Li X, Wang X, Ping W, et al. Improved Taxol production in Nodulisporium sylviforme derived from inactivated protoplast fusion. African Journal of Biotechnology. 2011;**10**(20):4175-4182

[53] Pandi M, Kumaran RS, Choi Y-K, Kim HJ, Muthumary J. Isolation and detection of Taxol, an anticancer drug produced from Lasiodiplodia theobromae, an endophytic fungus of the medicinal plant Morinda citrifolia. African Journal of Biotechnology. 2011; **10**(8):1428-1435

[54] Bi J, Ji Y, Pan J, Yu Y, Chen H, Zhu X. A new Taxol-producing fungus (Pestalotiopsis malicola) and evidence for Taxol as a transient product in the

culture. African Journal of Biotechnology. 2011;**10**(34):6647-6654

[55] Kumaran RS, Choi Y-K, Lee S, Jeon HJ, Jung H, Kim HJ. Isolation of Taxol, an anticancer drug produced by the endophytic fungus, Phoma betae. African Journal of Biotechnology. 2012; **11**(4):950-960

[56] Mirjalili MH, Farzaneh M, Bonfill M, Rezadoost H, Ghassempour A. Isolation and characterization of Stemphylium sedicola SBU-16 as a new endophytic Taxol-producing fungus from Taxus baccata grown in Iran. FEMS Microbiology Letters. 2012;**328**(2): 122-129

[57] Garyali S, Kumar A, Reddy MS. Taxol production by an endophytic fungus, Fusarium redolens, isolated from Himalayan yew. Journal of Microbiology and Biotechnology. 2013; **23**(10):1372-1380

[58] Yang Y, Zhao H, Barrero RA, Zhang B, Sun G, Wilson IW, et al. Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431. BMC Genomics. 2014;**15**(1):1-14

[59] Zaiyou J, Li M, Xiqiao H. An endophytic fungus efficiently producing paclitaxel isolated from Taxus wallichiana var. mairei. Medicine. 2017; **96**(27):e7406

[60] Qiao W, Ling F, Yu L, Huang Y, Wang T. Enhancing Taxol production in a novel endophytic fungus, Aspergillus aculeatinus Tax-6, isolated from Taxus chinensis var. mairei. Fungal Biology. 2017;**121**(12):1037-1044

[61] El-Sayed AS, Safan S, Mohamed NZ, Shaban L, Ali GS, Sitohy MZ. Induction of Taxol biosynthesis by Aspergillus terreus, endophyte of Podocarpus

gracilior Pilger, upon intimate interaction with the plant endogenous microbes. Process Biochemistry. 2018;**71**: 31-40

[62] El-Sayed AS, Ali DM, Yassin MA, Zayed RA, Ali GS. Sterol inhibitor "fluconazole" enhance the Taxol yield and molecular expression of its encoding genes cluster from Aspergillus flavipes. Process Biochemistry. 2019;**76**:55-67

[63] Gill H, Vasundhara M. Isolation of Taxol producing endophytic fungus Alternaria brassicicola from non-taxus medicinal plant Terminalia arjuna. World Journal of Microbiology and Biotechnology. 2019;**35**:1-8

[64] Kumar P, Singh B, Thakur V, Thakur A, Thakur N, Pandey D, et al. Hyper-production of Taxol from Aspergillus fumigatus, an endophytic fungus isolated from Taxus sp. of the Northern Himalayan region. Biotechnology Reports. 2019;**24**:e00395

[65] El-Sabbagh SM, Eissa OAE, Sallam MHE. Taxol production by an endophytic fungus cladosporioides isolated from Catheranthus roseus Cladosporium. Egyptian Journal of Experimental Biology (Botany). 2019; **15**(1):13-28

[66] Suresh G, Kokila D, Suresh TC, Kumaran S, Velmurugan P, Vedhanayakisri KA, et al. Mycosynthesis of anticancer drug Taxol by Aspergillus oryzae, an endophyte of Tarenna asiatica, characterization, and its activity against a human lung cancer cell line. Biocatalysis and Agricultural Biotechnology. 2020;**24**:101525

[67] El-Sayed E-SR, Zaki AG, Ahmed AS, Ismaiel AA. Production of the anticancer drug Taxol by the endophytic fungus Epicoccum nigrum TXB502: Enhanced production by gamma irradiation

mutagenesis and immobilization technique. Applied Microbiology and Biotechnology. 2020;**104**(16):6991-7003

[68] Subramanian M, Marudhamuthu M. Hitherto unknown terpene synthase organization in Taxol-producing endophytic bacteria isolated from marine macroalgae. Current Microbiology. 2020;**77**:918-923

[69] Abdel-Fatah SS, El-Batal AI, El-Sherbiny GM, Khalaf MA, El-Sayed AS. Production, bioprocess optimization and γ-irradiation of Penicillium polonicum, as a new Taxol producing endophyte from Ginko biloba. Biotechnology Reports. 2021;**30**:e00623

[70] Jagan EG, Sharma P, Sureshkumar S, Pandi M. Isolation of Taxol and flavinlike fluorochrome from endophytic fungi of Mangifera indica. Journal of Pure & Applied Microbiology. 2021;**15** (4):2195-2208

[71] Gauchan DP, Vélëz H, Acharya A, Östman JR, Lundén K, Elfstrand M, et al. Annulohypoxylon sp. strain MUS1, an endophytic fungus isolated from Taxus wallichiana Zucc., produces Taxol and other bioactive metabolites. 3 Biotech. 2021;**11**(3):152

[72] Koutb M, Hassan E, El-Sokkary G, Saber S, Hussein N. Paclitaxel production by endophytic fungus, neopestalotiopsis clavispora KY624416 and subsequent extraction of chitosan from fungal biomass wastes. Global Nest Journal. 2021;**23**(3):370-380

[73] Abdel-Fatah SS, El-Sherbiny GM, Khalaf MA, El-Batal AI. Enhancement of Taxol production by endophytic fungi from Hibiscus and moringa plant using gamma irradiation. Egyptian Journal of Medical Microbiology. 2021; **30**(4):9-17

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

[74] Mohammadi Ballakuti N, Ghanati F, Zare-Maivan H, Alipour M, Moghaddam M, Abdolmaleki P. Taxoid profile in endophytic fungi isolated from Corylus avellana, introduces potential source for the production of Taxol in semi-synthetic approaches. Scientific Reports. 2022;**12**(1):9390

[75] Chowdhury DR, Chattopadhyay SK, Roy S. Isolation and partial characterization of bioactive components of Endophytic fungi Penicillium singorense, isolated from two Indian medicinal plants: Calotropis procera and Catharanthus roseus. American Journal of Microbiological Research. 2022;**10**(3):84-93

[76] Pandy R, Kumar SS, Suresh P, Annaraj J, Pandi M, Vellasamy S, et al. Screening and characterization of fungal Taxol-producing endophytic fungi for evaluation of antimicrobial and anticancer activities. Open Chemistry. 2023;**21**:1

[77] Adhikari P, Singh M, Pandey A. Production of Taxol by endophytic fungi isolated from roots of Himalayan yew (Taxus wallichiana Zucc.). Journal of Graphic Era University. 2022;**10**(2): 195-216

[78] Wang Y, Tang K. A new endophytic Taxol-and baccatin III-producing fungus isolated from Taxus chinensis var. mairei. African Journal of Biotechnology. 2011;**10**(72):16379-16386

[79] Zaiyou J, Li M, Guifang X, Xiuren Z. Isolation of an endophytic fungus producing baccatin III from Taxus wallichiana var. mairei. Journal of Industrial Microbiology and Biotechnology. 2013;**40**(11):1297-1302

[80] Li Y, Yang J, Zhou X, Zhao W, Jian Z. Isolation and identification of a 10-deacetyl baccatin-III-producing

endophyte from Taxus wallichiana. Applied Biochemistry and Biotechnology. 2015;**175**:2224-2231

[81] Omeje EO, Ahomafor JE, Onyekaba TU, Monioro PO, Nneka I, Onyeloni S, et al. Endophytic fungi as alternative and reliable sources for potent anticancer agents. In: Natural Products and Cancer Drug Discovery. London, UK, Norderstedt, Germany: IntechOpen; 2017. pp. 52-60

[82] Vasundhara M, Kumar A, Reddy MS. Molecular approaches to screen bioactive compounds from endophytic fungi. Frontiers in Microbiology. 2016;**7**:1774

[83] Zhao J, Zhou L, Wang J, Shan T, Zhong L, Liu X, et al. Endophytic fungi for producing bioactive compounds originally from their host plants. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. 2010;**1**:567-576

[84] Gond S, Kharwar R, White J Jr. Will fungi be the new source of the blockbuster drug Taxol? Fungal Biology Reviews. 2014;**28**(4):77-84

[85] Tejesvi MV, Pirttilä AM. Endophytic fungi, occurrence, and metabolites. In: Anke T, Schüffler A, editors. Physiology and Genetics: Selected Basic and Applied Aspects. Cham: Springer International Publishing; 2018. pp. 213-230

[86] Cao X, Xu L, Wang J, Dong M, Xu C, Kai G, et al. Endophytic fungus Pseudodidymocyrtis lobariellae KL27 promotes Taxol biosynthesis and accumulation in Taxus chinensis. BMC Plant Biology. 2022;**22**(1):1-18

[87] Liu Q, Li L, Chen Y, Wang S, Xue L, Meng W, et al. Diversity of endophytic microbes in Taxus yunnanensis and their potential for plant growth promotion

and taxane accumulation. Microorganisms. 2023;**11**(7):1645

[88] Cragg GM, Pezzuto JM. Natural products as a vital source for the discovery of cancer chemotherapeutic and chemopreventive agents. Medical Principles and Practice. 2016;**25**(Suppl. 2):41-59

[89] Bo G, Haiyan L, Lingqi Z. Isolation of an fungus producting vinbrastine. Journal of Yunnan University (Natural Sciences). 1998;**20**(3):214-215

[90] Lingqi Z, Bo G, Haiyan L, Songrong Z, Hua S, Su G, et al. Preliminary study on the isolation of endophytic fungus of Catharanthus roseus and its fermentation to produce products of therapeutic value. Zhong Cao Yao= Chinese Traditional and Herbal Drugs. 2000;**31**(11):805-807

[91] Kumar A, Ahmad A. Biotransformation of vinblastine to vincristine by the endophytic fungus Fusarium oxysporum isolated from Catharanthus roseus. Biocatalysis and Biotransformation. 2013;**31**(2):89-93

[92] Palem PP, Kuriakose GC, Jayabaskaran C. An endophytic fungus, Talaromyces radicus, isolated from Catharanthus roseus, produces vincristine and vinblastine, which induce apoptotic cell death. PLoS One. 2015;**10**(12):e0144476

[93] Ayob FW, Simarani K, Zainal Abidin N, Mohamad J. First report on a novel Nigrospora sphaerica isolated from Catharanthus roseus plant with anticarcinogenic properties. Microbial Biotechnology. 2017;**10**(4):926-932

[94] Anjum N, Chandra R. Endophytic bacteria of Catharanthus roseus as an alternative source of vindoline and application of response surface

methodology to enhance its production. Archives of Biological Sciences. 2019; **71**(1):27-38

[95] Birat K, Siddiqi TO, Mir SR, Aslan J, Bansal R, Khan W, et al. Enhancement of vincristine under in vitro culture of Catharanthus roseus supplemented with Alternaria sesami endophytic fungal extract as a biotic elicitor. International Microbiology. 2022;**25**(2):275-284

[96] Xianzhi Y, Lingqi Z, Bo G, Shiping G. Preliminary study of a vincristine-proudcing endophytic fungus isolated from leaves of Catharanthus roseus. Zhong Cao Yao= Chinese Traditional and Herbal Drugs. 2004;**35**(1):79-81

[97] Kumar A, Abnave P, Ahmad A. Cultural, morphological and molecular characterization of vinca alkaloids producing endophytic fungus Fusarium solani isolated from Catharanthus roseus. International Journal of Botany and Research. 2013;**3**(2):2277-4815

[98] Kumar A, Patil D, Rajamohanan PR, Ahmad A. Isolation, purification and characterization of vinblastine and vincristine from endophytic fungus Fusarium oxysporum isolated from Catharanthus roseus. PLoS One. 2013; **8**(9):e71805

[99] Kuriakose GC, Palem PP, Jayabaskaran C. Fungal vincristine from Eutypella spp-CrP14 isolated from Catharanthus roseus induces apoptosis in human squamous carcinoma cell line-A431. BMC Complementary and Alternative Medicine. 2016;**16**(1):1-8

[100] Ashoka H, Hegde P, Manasa K, Madihalli C, Pradeep S, Shettihalli A. Isolation and detection of vinca alkaloids from endophytes isolated from Catharanthus roseus. European Journal

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

of Biomedical and Pharmaceutical Sciences. 2017;**10**:675-683

[101] Zafari D, Leylaiee S, Tajick MA. Isolation and identification of vinblastine from the fungus of Chaetomium globosum Cr95 isolated from Catharanthus roseus plant. Biological Journal of Microorganism. 2019;**8**(32):1-14

[102] Parthasarathy R, Shanmuganathan R, Pugazhendhi A. Vinblastine production by the endophytic fungus Curvularia verruculosa from the leaves of Catharanthus roseus and its in vitro cytotoxicity against HeLa cell line. Analytical Biochemistry. 2020;**593**: 113530

[103] Bandara CJ, Siriwardhana A, Karunaratne DN, Ratnayake Bandara BM, Wickramasinghe A, Krishnarajah SA, et al. Production of vincristine and vinblastine by the endophytic fungus Botryosphaeria laricina strain (CRS1) is dependent on stimulating factors present in Catharanthus roseus. The Natural Products Journal. 2021;**11**(2):221-230

[104] Andriambeloson OH, Noah RMA, Rigobert A, Jean-Marc C, Luciano R, Rado R. Isolation of Novel Vincristine and Vinblastine Producing Streptomyces Species from Catharanthus Roseus Rhizospheric Soil. Research Square. 2021. DOI: 10.21203/rs.3.rs-1082130/v1

[105] Ashraf J, Sharma MK, Biswas D. Separation, purification and characterization of vincristine and vinblastine from fusarium oxysporum, an endophytic fungus present in catharanthus roseus leaves. Journal of Advanced Scientific Research. 2021;**12** (01 Suppl 2):128-136

[106] Birat K, Binsuwaidan R, Siddiqi TO, Mir SR, Alshammari N, Adnan M, et al.

Report on vincristine-producing endophytic fungus Nigrospora zimmermanii from leaves of Catharanthus roseus. Metabolites. 2022; **12**(11):1119

[107] Min C, Wang X. Isolation and identification of the 10-hydroxycamptothecin-producing endophytic fungi from Camptotheca acuminata decne. Acta Botanica Boreali-Occidentalia Sinica. 2009;**29**(3):614-617

[108] Kusari S, Zühlke S, Spiteller M. An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. Journal of Natural Products. 2009;**72**(1):2-7

[109] Puri SC, Verma V, Amna T, Qazi GN, Spiteller M. An endophytic fungus from Nothapodytes f oetida that produces Camptothecin. Journal of Natural Products. 2005;**68**(12):1717-1719

[110] Ran X, Zhang G, Li S, Wang J. Characterization and antitumor activity of camptothecin from endophytic fungus Fusarium solani isolated from Camptotheca acuminate. African Health Sciences. 2017;**17**(2):566-574

[111] Rehman S, Shawl A, Kour A, Andrabi R, Sudan P, Sultan P, et al. An endophytic Neurospora sp. from Nothapodytes foetida producing camptothecin. Applied Biochemistry and Microbiology. 2008;**44**:203-209

[112] Rehman S, Shawl A, Kour A, Sultan P, Ahmad K, Khajuria R, et al. Comparative studies and identification of camptothecin produced by an endophyte at shake flask and bioreactor. Natural Product Research. 2009;**23**(11): 1050-1057

[113] Gurudatt P, Priti V, Shweta S, Ramesha B, Ravikanth G, Vasudeva R, et al. Attenuation of camptothecin

production and negative relation between hyphal biomass and camptothecin content in endophytic fungal strains isolated from Nothapodytes nimmoniana Grahm (Icacinaceae). Current Science. 2010; **98**(8):1006-1010

[114] Shweta S, Zuehlke S, Ramesha B, Priti V, Kumar PM, Ravikanth G, et al. Endophytic fungal strains of Fusarium solani, from Apodytes dimidiata E. Mey. ex Arn (Icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin. Phytochemistry. 2010;**71**(1):117-122

[115] Pu X, Qu X, Chen F, Bao J, Zhang G, Luo Y. Camptothecin-producing endophytic fungus Trichoderma atroviride LY357: Isolation, identification, and fermentation conditions optimization for camptothecin production. Applied Microbiology and Biotechnology. 2013; **97**:9365-9375

[116] Shweta S, Bindu JH, Raghu J, Suma H, Manjunatha B, Kumara PM, et al. Isolation of endophytic bacteria producing the anti-cancer alkaloid camptothecine from Miquelia dentata Bedd. (Icacinaceae). Phytomedicine. 2013;**20**(10):913-917

[117] Shweta S, Gurumurthy BR, Ravikanth G, Ramanan US, Shivanna MB. Endophytic fungi from Miquelia dentata Bedd., produce the anti-cancer alkaloid, camptothecine. Phytomedicine. 2013;**20**(3–4):337-342

[118] Su H, Kang J-c, Cao J, Mo L, Hyde KD. Medicinal plant endophytes produce analogous bioactive compounds. Chiang Mai Journal of Science. 2014;**41**(1):1-13

[119] Musavi SF, Dhavale A, Balakrishnan RM. Optimization and kinetic modeling of cell-associated camptothecin production from an endophytic Fusarium oxysporum NFX06. Preparative Biochemistry and Biotechnology. 2015; **45**(2):158-172

[120] Venugopalan A, Srivastava S. Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte, Fusarium solani. Bioresource Technology. 2015; **188**:251-257

[121] Pu X, Chen F, Yang Y, Qu X, Zhang G, Luo Y. Isolation and characterization of Paenibacillus polymyxa LY214, a camptothecinproducing endophytic bacterium from Camptotheca acuminata. Journal of Industrial Microbiology and Biotechnology. 2015;**42**(8):1197-1202

[122] Bhalkar BN, Patil SM, Govindwar SP. Camptothecine production by mixed fermentation of two endophytic fungi from Nothapodytes nimmoniana. Fungal Biology. 2016;**120**(6–7):873-883

[123] Soujanya KN, Siva R, Mohana Kumara P, Srimany A, Ravikanth G, Mulani FA, et al. Camptothecinproducing endophytic bacteria from Pyrenacantha volubilis Hook. (Icacinaceae): A possible role of a plasmid in the production of camptothecin. Phytomedicine. 2017;**36**: 160-167

[124] Aswini A, Soundhari C. Production of camptothecin from endophytic fungi and characterization by high-performance liquid chromatography and anticancer activity against colon cancer cell line. Asian Journal of Pharmaceutical and Clinical Research. 2018;**11**(3):166-170

[125] Clarance P, Lalitha J, Sales J, Khusro A, Agastian P. Anticancer *The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

activity of camptothecin producing endophytes isolated from Chonemorpha fragrans (moon) Alston. (Apocynaceae). Research Journal of Biotechnology. 2019; **14**(5):74-82

[126] Ghiasvand M, Makhdoumi A, Matin MM, Vaezi J. Exploring the bioactive compounds from endophytic bacteria of a medicinal plant: Ephedra foliata (Ephedrales: Ephedraceae). Advances in Traditional Medicine. 2020; **20**:61-70

[127] Aswani R, Jasim B, Arun Vishnu R, Antony L, Remakanthan A, Aravindakumar CT, et al. Nanoelicitor based enhancement of camptothecin production in fungi isolated from Ophiorrhiza mungos. Biotechnology Progress. 2020;**36**(6):e3039

[128] Mohinudeen IAHK, Kanumuri R, Soujanya KN, Shaanker RU, Rayala SK, Srivastava S. Sustainable production of camptothecin from an Alternaria sp. isolated from Nothapodytes nimmoniana. Scientific Reports. 2021; **11**(1):1478

[129] Dhakshinamoorthy M, Ponnusamy SK, Nyayiru Kannaian UP, Srinivasan B, Shankar SN, Kilavan PK. Plant-microbe interactions implicated in the production of camptothecin – An anticancer biometabolite from Phyllosticta elongata MH458897 a novel endophytic strain isolated from medicinal plant of Western Ghats of India. Environmental Research. 2021; **201**:111564

[130] El-Sayed ASA, Khalaf SA, Azez HA, Hussein HA, El-Moslamy SH, Sitohy B, et al. Production, bioprocess optimization and anticancer activity of Camptothecin from aspergillus terreus and aspergillus flavus, endophytes of Ficus elastica. Process Biochemistry. 2021;**107**:59-73

[131] El-Sayed ASA, Hassan WHB, Sweilam SH, Alqarni MH, El Sayed ZI, Abdel-Aal MM, et al. Production, bioprocessing and anti-proliferative activity of Camptothecin from Penicillium chrysogenum, an endozoic of marine sponge, Cliona sp., as a metabolically stable Camptothecin producing isolate. Molecules. 2022;**27**:9

[132] El-Sayed ASA, George NM, Abou-Elnour A, El-Mekkawy RM, El-Demerdash MM. Production and bioprocessing of camptothecin from Aspergillus terreus, an endophyte of Cestrum parqui, restoring their biosynthetic potency by Citrus limonum peel extracts. Microbial Cell Factories. 2023;**22**(1):4

[133] Degambada KD, Kumara PAASP, Salim N, Abeysekera AM, Chandrika UG, Diaporthe sp. F18; a new source of camptothecin-producing endophytic fungus from Nothapodytes nimmoniana growing in Sri Lanka. Natural Product Research. 2023;**37**(1): 113-118

[134] Xianzhi Y, Shiping G, Lingqi Z, Hua S. Select of producing podophyllotoxin endophytic fungi from podophyllin plant. Natural Product Research and Development. 2003;**15**(5): 419-422

[135] Zeng S, Shao H, Zhang L. An endophytic fungus producing a substance analogous to podophyllotoxin isolated from Diphylleia sinensis. Journal of Microbiology. 2004;**24**:1-2

[136] Guo S, Jiang B, Su Y, Liu S, Zhang L. Podophyllotoxin and its analogues from the endophytic fungi derived from Dysosma veitchii. Biotechnology. 2004;**14**:55-57

[137] Lu L, He J, Yu X, Li G, Zhang X. Studies on isolation and identification of endophytic fungi strain SC13 from harmaceutical plant Sabina vulgaris ant. and metabolites. Acta Agriculturae Boreali-occidentalis Sinica. 2006;**15**: 85-89

[138] Eyberger AL, Dondapati R, Porter JR. Endophyte fungal isolates from Podophyllum peltatum produce podophyllotoxin. Journal of Natural Products. 2006;**69**(8):1121-1124

[139] Puri SC, Nazir A, Chawla R, Arora R, Riyaz-ul-Hasan S, Amna T, et al. The endophytic fungus Trametes hirsuta as a novel alternative source of podophyllotoxin and related aryl tetralin lignans. Journal of Biotechnology. 2006; **122**(4):494-510

[140] Li C. Fermentation conditions of Sinopodophyllum hexandrum endophytic fungus on production of podophyllotoxin. Food and Fermentation Industries. 2007;**33**(9):28

[141] Kour A, Shawl AS, Rehman S, Sultan P, Qazi PH, Suden P, et al. Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva. World Journal of Microbiology and Biotechnology. 2008;**24**:1115-1121

[142] Nadeem M, Ram M, Alam P, Ahmad MM, Mohammad A, Al-Qurainy F, et al. Fusarium solani, P1, a new endophytic podophyllotoxin-producing fungus from roots of Podophyllum hexandrum. African Journal of Microbiology Research. 2012;**6**(10): 2493-2499

[143] Huang J-X, Zhang J, Zhang X-R, Zhang K, Zhang X, He X-R. Mucor fragilis as a novel source of the key pharmaceutical agents podophyllotoxin and kaempferol. Pharmaceutical Biology. 2014;**52**(10):1237-1243

[144] Liang Z, Zhang J, Zhang X, Li J, Zhang X, Zhao C. Endophytic fungus from Sinopodophyllum emodi (wall.) ying that produces Podophyllotoxin. Journal of Chromatographic Science. 2016;**54**(2):175-178

[145] Aharwal RP, Kumar S, Sandhu SS. Endophytic mycoflora as a source of biotherapeutic compounds for disease treatment. Journal of Applied Pharmaceutical Science. 2016;**6**(10): 242-254

[146] Wang T, Ma Y, Ye Y, Zheng H, Zhang B, Zhang E. Screening and identification of endophytic fungi producing podophyllotoxin compounds in Sinopodophyllum hexandrum stems. Chinese Journal of Experimental Traditional Medical Formulae. 2017;**39**: 402-408

[147] Tan X-m, Zhou Y-q, Zhou X-l, Xia X-h, Wei Y, He L-l, et al. Diversity and bioactive potential of culturable fungal endophytes of Dysosma versipellis; a rare medicinal plant endemic to China. Scientific Reports. 2018;**8**(1):5929

[148] Gohar UF, Attia Majeed BM, Mukhtar H. Optimum conditions for enhanced production of Podophyllotoxin from Penicillium sp. isolated from Khanspur, Pakistan. Pakistan Journal of Zoology. 2022;**54**(6):2775

[149] Thi Tran H, Thu Nguyen G, Thi Nguyen HH, Thi Tran H, Hong Tran Q, Ho Tran Q, et al. Isolation and cytotoxic potency of endophytic fungi associated with Dysosma difformis, a study for the novel resources of Podophyllotoxin. Mycobiology. 2022;**50**(5):389-398

[150] Nguyen GT, Nguyen HTH, Tran HT, Tran HT, Ho AN, Tran QH, et al. Enhanced podophyllotoxin production of endophyte Fusarium

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

proliferatum TQN5T by host extract and phenylalanine. Applied Microbiology and Biotechnology. 2023;**107**(17): 5367-5378

[151] Kusari S, Lamshöft M, Spiteller M. Aspergillus fumigatus Fresenius, an endophytic fungus from Juniperus communis L. Horstmann as a novel source of the anticancer pro-drug deoxypodophyllotoxin. Journal of Applied Microbiology. 2009;**107**(3): 1019-1030

[152] Li W, Zhou J, Lin Z, Hu Z. Study on fermentation condition for production of huperzine a from endophytic fungus 2F09P03B of Huperzia serrata. Chinese Medicinal Biotechnology. 2007;**2**(4): 254-259

[153] Zan J, Wang J, Pan S. Isolation and preliminary identification of the endophytic fungi which produce Hupzine a from four species in Hupziaceae and determination of Huperzine a by HPLC. Fudan University Journal of Medical Sciences. 2009;**36**(4): 445-449

[154] Zhou S, Yang F, Lan S, Xu N, Hong Y. Huperzine a producing conditions from endophytic fungus in SHB Huperzia serrata. Journal of Microbiology. 2009;**3**:32-36

[155] Zhu D, Wang J, Zeng Q, Zhang Z, Yan R. A novel endophytic Huperzine A–producing fungus, Shiraia sp. Slf14, isolated from Huperzia serrata. Journal of Applied Microbiology. 2010;**109**(4): 1469-1478

[156] Zhang ZB, Zeng QG, Yan RM, Wang Y, Zou ZR, Zhu D. Endophytic fungus Cladosporium cladosporioides LF70 from Huperzia serrata produces Huperzine A. World Journal of Microbiology and Biotechnology. 2011; **27**:479-486

[157] Wang Y, Yan R, Zeng Q, Zhang Z, Wang D, Zhu D. Producing huperzine a by an endophytic fungus from Huperzia serrata. Mycosystema. 2011;**30**(2): 255-262

[158] Wang Y, Zeng QG, Zhang ZB, Yan RM, Wang LY, Zhu D. Isolation and characterization of endophytic huperzine A-producing fungi from Huperzia serrata. Journal of Industrial Microbiology and Biotechnology. 2011; **38**(9):1267-1278

[159] Shu S, Zhao X, Wang W, Zhang G, Cosoveanu A, Ahn Y, et al. Identification of a novel endophytic fungus from Huperzia serrata which produces huperzine a. World Journal of Microbiology and Biotechnology. 2014; **30**:3101-3109

[160] Dong L-H, Fan S-W, Ling Q-Z, Huang B-B, Wei Z-J. Indentification of huperzine A-producing endophytic fungi isolated from Huperzia serrata. World Journal of Microbiology and Biotechnology. 2014;**30**:1011-1017

[161] Su J, Yang M. Huperzine a production by Paecilomyces tenuis YS-13, an endophytic fungus isolated from Huperzia serrata. Natural Product Research. 2015;**29**(11):1035-1041

[162] Han W, Song T, Yang S, Li X, Zhang H, Wu Y, et al. Identification of alkaloids and huperzine A-producing endophytic fungi isolated from wild Huperzia serrata. Journal of International Pharmaceutical Research. 2015;**6**:507-512

[163] Zhang F, Wang M, Zheng Y, Liu H, Zhang X, Wu S. Isolation and characterzation of endophytic Huperzine A-producing fungi from Phlegmariurus phlegmaria. Microbiology. 2015;**84**:701-709

[164] Wang Y, Lai Z, Li X-X, Yan R-M, Zhang Z-B, Yang H-L, et al. Isolation, diversity and acetylcholinesterase inhibitory activity of the culturable endophytic fungi harboured in Huperzia serrata from Jinggang Mountain, China. World Journal of Microbiology and Biotechnology. 2016;**32**:1-23

[165] Thi Minh Le T, Thi Hong Hoang A, Thi Bich Le T, Thi Bich Vo T, Van Quyen D, Hoang CH. Isolation of endophytic fungi and screening of Huperzine A–producing fungus from Huperzia serrata in Vietnam. Scientific Reports. 2019;**9**(1):16152

[166] Zaki AG, El-Shatoury EH, Ahmed AS, Al-Hagar OE. Production and enhancement of the acetylcholinesterase inhibitor, huperzine a, from an endophytic Alternaria brassicae AGF041. Applied Microbiology and Biotechnology. 2019;**103**:5867-5878

[167] Kang X, Liu C, Shen P, Hu L, Lin R, Ling J, et al. Genomic characterization provides new insights into the biosynthesis of the secondary metabolite huperzine a in the endophyte Colletotrichum gloeosporioides Cg01. Frontiers in Microbiology. 2019; **9**:3237

[168] Wen-Xia H, Zhong-Wen H, Min J, Han Z, Wei-Ze L, Li-Bin Y, et al. Five novel and highly efficient endophytic fungi isolated from Huperzia serrata expressing huperzine a for the treatment of Alzheimer's disease. Applied Microbiology and Biotechnology. 2020; **104**:9159-9177

[169] Cruz-Miranda OL, Folch-Mallol J, Martínez-Morales F, Gesto-Borroto R, Villarreal ML, Taketa AC. Identification of a Huperzine A-producing endophytic fungus from Phlegmariurus taxifolius. Molecular Biology Reports. 2020;**47**(1): 489-495

[170] Le TTM, Hoang ATH, Nguyen NP, Le TTB, Trinh HTT, Vo TTB, et al. A novel huperzine A-producing endophytic fungus Fusarium sp. Rsp5.2 isolated from Huperzia serrate. Biotechnology Letters. 2020;**42**(6): 987-995

[171] Putri NWPS, Ariantari NP. Production of huperzine a by fungal endophytes associated with huperziaceae plants. Journal Pharmaceutical Science and Application. 2023;**5**(1):45-52

[172] Ying Y-M, Shan W-G, Zhan Z-J. Biotransformation of Huperzine a by a fungal endophyte of Huperzia serrata furnished sesquiterpenoid–alkaloid hybrids. Journal of Natural Products. 2014;**77**(9):2054-2059

[173] Thirumalanadhuni V, Yerraguravagari LL, Palempalli UMD. Endophytic microflora: The fountainhead of anticancer metabolites —A systematic review. Recent Developments in Applied Microbiology and Biochemistry. 2021;**2**:13-20

[174] Madhusudhan CM, Bharathi RT, Prakash SH. Isolation and purification of bioactive metabolites from fungal endophytes–a review. Current Biochemical Engineering. 2015;**2**(2): 111-117

[175] Song X, Wu H, Yin Z, Lian M, Yin C. Endophytic bacteria isolated from Panax ginseng improves ginsenoside accumulation in adventitious ginseng root culture. Molecules. 2017;**22**(6):837

[176] Fu Y, Yin ZH, Yin CY. Biotransformation of ginsenoside Rb1 to ginsenoside Rg3 by endophytic bacterium Burkholderia sp. GE 17-7 isolated from Panax ginseng. Journal of Applied Microbiology. 2017;**122**(6): 1579-1585

*The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds DOI: http://dx.doi.org/10.5772/intechopen.112931*

[177] Fu Y. Biotransformation of ginsenoside Rb1 to gyp-XVII and minor ginsenoside Rg3 by endophytic bacterium Flavobacterium sp. GE 32 isolated from Panax ginseng. Letters in Applied Microbiology. 2019;**68**(2): 134-141

[178] Yang H-R, Yuan J, Liu L-H, Zhang W, Chen F, Dai C-C. Endophytic Pseudomonas fluorescens induced sesquiterpenoid accumulation mediated by gibberellic acid and jasmonic acid in Atractylodes macrocephala Koidz plantlets. Plant Cell, Tissue and Organ Culture (PCTOC). 2019;**138**:445-457

[179] Yin DD, Wang YL, Yang M, Yin DK, Wang GK, Xu F. Analysis of chuanxiong Rhizoma substrate on production of ligustrazine in endophytic Bacillus subtilis by ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Journal of Separation Science. 2019;**42**(19):3067-3076

[180] Hemmati N, Azizi M, Spina R, Dupire F, Arouei H, Saeedi M, et al. Accumulation of ajmalicine and vinblastine in cell cultures is enhanced by endophytic fungi of Catharanthus roseus cv. icy pink. Industrial Crops and Products. 2020;**158**:112776

## **Chapter 4**
