Apigenin (Table 2) is a flavonoid present in various fruits, vegetables, and spices like onions, parsley, oranges, thyme, celery, and sweet red pepper. It possesses several chemopreventive properties, including induction of apoptosis, inhibition of cancer cells proliferation, and

Luteolin (Table 2) is most often found in leaves, but it is also seen in celery, parsley, broccoli, onion, carrots, peppers, cabbages, apples, thyme, dandelion, chamomile tea, carrots, green pepper, olive oil, peppermint, rosemary, oranges, and oregano. The chemopreventive mech‐ anism of action of luteolin includes induction of apoptosis, inhibition of proliferation of cancer cells, and suppression of angiogenesis and metastasis. Luteolin has been reported to inhibit growth of tumors in human skin, hepatoma, and ovarian cancer cells; in lung and breast cancer, luteolin has been shown to inhibit the invasion of cancer cells [2]. Among 68 plant polyphenols examined for their inhibitory ability against hepatocellular carcinoma cells, luteolin was one

Wogonin, baicalein (Table 2), and baicalin (baicalein 7-*O*-glucuronide), all naturally occurring flavonoid extracted from *Scutellaria baicalensis Georgi* (*Lamiaceae*), were reported to have potential antitumor effects in several studies. These phytochemicals are not only cytostatic but also cytotoxic to various human tumor cell lines *in vitro* and inhibit tumor growth *in vivo*. Numerous studies demonstrated that wogonin had a cytostatic effect due to the apoptotic cell death on various tumor cell lines (such as sarcoma, hepatoma cells, breast cancer, and

Quercetin (Table 2) is the most abundant flavonoid in foods. It is present in different fruits (apples, berries, and grapes), vegetables (onions, broccoli), as well as in tea and red wine, mostly in glycosylated forms. The daily intake of quercetin has been estimated at 30 mg. Quercetin is probably the most studied flavonoids, and antioxidant properties are known as outstanding. This flavonol was found to have antiproliferative activity in many situations, such

Myricetin (Table 2) is the next flavonol occurring in fruits, vegetables, or red wine exerting anticancer activity. In lung cancer, this compound has been observed to block invasion and migration of human lung adenocarcinoma cells. According to the study with human leukemia

Kaempferol (Table 2) is another flavonol present in tea, broccoli, grapefruit, brussels sprouts, apples, and other plants. This compound exerts antioxidant, anti-inflammatory, and anticancer effects on several types of cancer. Kaempferol has been reported to inhibit the invasion of the

nasopharyngeal carcinoma cells) and antimetastatic ability as well [48, 49].

as adenomatous polyposis [36] and breast cancer [2].

cell line, myricetin induced apoptosis in these cells [2].

carcinoma and basal cell carcinoma) [47].

60 Phytochemicals - Isolation, Characterisation and Role in Human Health

**• Flavones as anticancer agents**

involvement in angiogenesis [2].

of the most potent [21].

**• Flavonols**

Isoflavone (3-phenyl-4*H*-1-benzopyr-4-one) differs from flavone in location of the phenyl group. Most members of the *Fabaceae* family contain significant quantities of isoflavones. Genistein and daidzein (Table 2), the most prevalent compounds of isoflavonoid class, were found in various legumes including soybean, green bean, alfalfa sprout, mung bean sprout, cowpea, kudzu root, and red clover sprout. Due to the estrogen-like structure of isoflavones found in soybeans and the known role of estrogens in breast carcinogenesis, most soy research has focused on the hormonal activity of these compounds [50]. Some studies showed that daidzein can inhibit hepatoma cell growth and induce apoptosis [21]. Regarding genistein, the major isoflavone in soy, laboratory studies have demonstrated different mechanisms of action, including antiproliferative activity and induction of apoptosis in both animal and human cell lines. In addition, genistein possesses antioxidant activity and the ability to scavenge free radicals, protecting the body against oxidative DNA damage. The chemopreventive activity of genistein has been investigated mainly against breast and prostate cancer.

#### **• Influence of glycosilation in flavonoid family**

The dietary flavonoids in nature almost all exist as their O/C-glycosides, such as glucoside, galactoside, rhamnoside, arabinoside, and rutinoside. The most abundant flavonoid glyco‐ sides in plants are flavone and flavonol O-glycosides. The glycosidation are found mainly as their 3 or 7 O-glycosides, although the 5, 8, and 4′ O-glycosides were also reported in some cases. Regarding the antioxidant and antitumoral properties, it was illustrated that the flavonoid aglycones showed higher anticancer potential than their glycosides in cell level; e.g., rutin did not inhibit cell proliferation of any of the cancer cell lines tested; C/O-glycosylation of apigenin significantly weakened the inhibition of cancer cells; genistein and daidzein glycosides exhibited no noticeable activity on human breast carcinoma cell. It was demon‐ strated that cellulase can remarkably transform baicalin and wogonoside to their aglycones (baicalein and wogonin) with enhance of antiproliferative effects [51]. Generally, glycosylation reduced the antiproliferative activity in flavonoid classes, and a C2-C3 double bond seems important for the antiproliferative activity of flavonoids, and indeed flavones are typically more potent that flavanones [19].

#### *5.2.2. Nonflavonoids as anticancer agents*

#### **• Stilbenoids**

Stilbenoids are hydroxylated derivatives of stilbene with a C6-C2-C6 structure. They are secondary metabolites occurring naturally in various families of plants; grapes and related products are considered the most important dietary sources of these substances. Transresveratrol (3,4′,5-trihydroxystilbene, Table 2), a hydroxylated stilbene, is today a well-known chemopreventive substance for cardiovascular diseases, antiaging, antiviral, and several malignant neoplasms [36]. High amounts of trans-resveratrol can be found in grapes and grape skins used for wine production, raspberries, mulberries, blueberries cranberries, peanuts, and certain types of pine [21]. All anticancer data obtained on preclinical animal studies showed that resveratrol affects all three discrete stages of carcinogenesis (initiation, promotion, and progression) by modulating signal transduction pathways that control cell division and growth, apoptosis, inflammation, angiogenesis, and metastasis [36]. A synthetic stilbene derivative, tamoxifen, is currently used for the treatment of several types of breast cancer in women, and as a hormone treatment for male breast cancer [52].

#### **• Caffeic acid**

Caffeic acid (Table 2) is a hydroxycinnamic acid found in most plants, including coffee beans, nuts, berries, and grains. Several studies have investigated the antitumor potential and hepatoprotective activity of the caffeic acid derivative, which have been identified in particular in honey and propolis [21]. Data showed that caffeic acid phenethyl ester (CAPE), a component of propolis, induces cell cycle arrest and has antiproliferation effect on glioma cells *in vitro* and *in vivo*. In addition, CAPE inhibited the metastasis of glioma cells [53].

Caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE), a natural polyphenol from *Sarcandra glabra*, has potent *in vitro* anticancer activity through multiple targets. This compound significantly decreased tumor growth in hepatoma and sarcoma tumor-bearing mice, and also significantly inhibited ascites development. CADPE did not show any toxicity *in vivo* and anticancer efficacies were equivalent to those of 5-fluorouracil and cyclophosphamide (drugs in cancer treatment).

#### **• Protocatechuic acid**

Protocatechuic acid (Table 2), a dihydroxybenzoic acid widely distributed in the plant kingdom, is the major metabolite of antioxidant polyphenols found in green tea. Protocate‐ chuic acid has antioxidant and anti-inflammatory activity and has been shown to inhibit HepG2 (human liver carcinoma cell line) cell growth and induce apoptosis [21].

#### **• Curcuminoids**

A curcuminoid is a linear diarylheptanoid, with molecules such as curcumin (Table 2) or derivatives of curcumin with different chemical groups that confers increase solubility and make them suitable for drug formulation. Curcumin is the principal curcuminoid of the spice turmeric, which is a member of the ginger family. It has been traditionally used for centuries for treating numerous diseases. Over the past few years, a number of studies uncovered several pharmacological properties of curcumin. It has been shown that curcumin inhibited HepG2 cell growth by inducing apoptosis [21]. Recent studies showed substantial evidence that curcumin inhibited proliferation, migration, invasion and metastasis, and induced apoptosis via modulating multiple signaling pathways in head and neck cancer [36].

#### **• Gallic acid**

malignant neoplasms [36]. High amounts of trans-resveratrol can be found in grapes and grape skins used for wine production, raspberries, mulberries, blueberries cranberries, peanuts, and certain types of pine [21]. All anticancer data obtained on preclinical animal studies showed that resveratrol affects all three discrete stages of carcinogenesis (initiation, promotion, and progression) by modulating signal transduction pathways that control cell division and growth, apoptosis, inflammation, angiogenesis, and metastasis [36]. A synthetic stilbene derivative, tamoxifen, is currently used for the treatment of several types of breast cancer in

Caffeic acid (Table 2) is a hydroxycinnamic acid found in most plants, including coffee beans, nuts, berries, and grains. Several studies have investigated the antitumor potential and hepatoprotective activity of the caffeic acid derivative, which have been identified in particular in honey and propolis [21]. Data showed that caffeic acid phenethyl ester (CAPE), a component of propolis, induces cell cycle arrest and has antiproliferation effect on glioma cells *in vitro* and

Caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE), a natural polyphenol from *Sarcandra glabra*, has potent *in vitro* anticancer activity through multiple targets. This compound significantly decreased tumor growth in hepatoma and sarcoma tumor-bearing mice, and also significantly inhibited ascites development. CADPE did not show any toxicity *in vivo* and anticancer efficacies were equivalent to those of 5-fluorouracil and cyclophosphamide (drugs in cancer treatment).  Protocate‐ chuic acid has antioxidant and anti-inflammatory activity and has been shown to inhibit

A curcuminoid is a linear diarylheptanoid, with molecules such as curcumin (Table 2) or derivatives of curcumin with different chemical groups that confers increase solubility and make them suitable for drug formulation. Curcumin is the principal curcuminoid of the spice turmeric, which is a member of the ginger family. It has been traditionally used for centuries for treating numerous diseases. Over the past few years, a number of studies uncovered several pharmacological properties of curcumin. It has been shown that curcumin inhibited HepG2 cell growth by inducing apoptosis [21]. Recent studies showed substantial evidence that curcumin inhibited proliferation, migration, invasion and metastasis, and induced apoptosis

HepG2 (human liver carcinoma cell line) cell growth and induce apoptosis [21].

via modulating multiple signaling pathways in head and neck cancer [36].

women, and as a hormone treatment for male breast cancer [52].

62 Phytochemicals - Isolation, Characterisation and Role in Human Health

*in vivo*. In addition, CAPE inhibited the metastasis of glioma cells [53].

suppresses tumor growth and angiogenesis [54].

 Gallic acid has been shown to exhibit biological activity, including anticancer and chemopreventive potential, capacity to induce apoptosis in human leukemia. Because gallic acid exhibits anti-invasive and antime‐ tastatic activities in various cancer cells, it might be a potential preventive and therapeutic agent against gastric cancer metastasis [2].

#### **• Ellagic acid**

Ellagic acid (Table 2) is a dimeric derivative of gallic acid occurring mostly in fruits and berries (such as raspberries, strawberries, blackberries, and pomegranates) and a primary constituent of ellagitannins. The data demonstrate that ellagic acid inhibits carcinogenesis, induces apoptosis in pancreatic and leukemia cancer cells, and also can delay the tumor latency and significantly reduce the estrogen-induced mammary tumors in rats. As a consequence, ellagic acid as well as gallic acid may be considered in a promising new class of cancer chemopre‐ ventive and therapeutic agents [2, 55].

#### **• Tannins**

Tannin (also known as tannoid) is an astringent, bitter plant polyphenolic compound that binds to and precipitates proteins and various other organic compounds, including amino acids and alkaloids. The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides, and in plant growth regulation. They showed antimutagenic activity without any obvious toxicity. Pentagalloylglucose and geraniin were among the most active tannins. Geraniin was isolated from the dry leaf of *Geranium thunbergii*. The plant is an official medicine in the Japanese Pharmacopoeia and is used for the treatment of diarrhea and for controlling intestinal function [45]. The grape (*Vitis vinifera*) skin mostly contains the highest amount of condensed tannins, alongside monomeric flavanols and flavonols, phenolic acids, and resveratrol. The grape skin extract induced prostate tumor cell line apoptosis, and the extract from pomace has a signifi‐ cant antiproliferative effect on human colon adenocarcinoma cells [56].

#### **5.3. Polysaccharides**

Polysaccharides are biopolymers with linear or branched side chains, composed of monosac‐ charides linked together through glycosidic bonds. Considering that the repeating units in the polymer backbone are often six-carbon monosaccharides, the general formula can be repre‐ sented as (C6H10O5)*n*, where 40 ≤ *n* ≤ 3000. Natural polysaccharides can be obtained from various organisms, such as plants, algae, microorganisms, and animals, and exist in a variety of chemical compositions, molecular weights, and structures [1]. Most polysaccharides derived from higher plants are relatively nontoxic and do not cause significant side effects; thus, plant polysaccharides are ideal candidates for therapeutics with immunomodulatory and antitumor effects and low toxicity [20]. Natural polysaccharides isolated from herbal plants have been shown to possess bioactivities. For example, the immune-stimulatory properties of *Aloe vera* glucomannan have been confirmed and used for treatment of immune-related diseases [23].

The type of linkage between saccharide units—the glycosidic linkage—seems to be important in immunomodulatory and anticancer activities. The literature also demonstrates that most of antitumor polysaccharides contain B-1,3-glucans, β-1,6-glucans, and α-1,3-glucans [23]. Although today is investigating especially polysaccharides of bacterial, algae, and fungi origins, plants have an important as sources of bioactive polymers especially because they are nontoxic. Here are some examples in the following lines:

**•** *Astragalus membranaceus* is commonly used in Chinese herbalism, where it is considered to be one of the 50 fundamental herbs. The plant, used especially for treatment of the kidneys and also to avoid senility, has been shown to be effective in immune enhancement and in the treatment of diabetes, viral infections, and cancers as well. The polysaccharides isolated from the radix of *A. membranaceus* are an active anticancer component. Structural analysis indicated that the polysaccharides were mainly composed of α-(1 → 4)-glucan with α-(1 → 6)-linked branches or α-(1 → 3)-glucan with side-chains containing arabinose and xylose [1].

shown to possess bioactivities. For example, the immune-stimulatory properties of *Aloe vera* glucomannan have been confirmed and used for treatment of immune-related diseases [23].

The type of linkage between saccharide units—the glycosidic linkage—seems to be important in immunomodulatory and anticancer activities. The literature also demonstrates that most of antitumor polysaccharides contain B-1,3-glucans, β-1,6-glucans, and α-1,3-glucans [23]. Although today is investigating especially polysaccharides of bacterial, algae, and fungi origins, plants have an important as sources of bioactive polymers especially because they are

**•** The genus *Actinidia* (*Actinidiaceae*) consists of over 58 species widely distributed in the Asian continent (China, Taiwan, Korea, Japan, southeast Siberia, and south of Indochina). Some *Actinidia* species, such as *A. macrosperma*, are the important in traditional medicine being used as health foods and medical products. In temperate climate zones, roots of *Actinidia eriantha Benth* (a commonly liana plant) have been used for gastric carcinoma, nasophar‐ yngeal carcinoma, breast carcinoma, and hepatitis in traditional Chinese medicine. The water extracts of this drug possess antitumor and immunopotentiating activities. Four polysaccharides were isolated and purified from the roots of *A. eriantha*, and the chemical composition of these polysaccharides could affect their antitumor and immunomodulatory

**•** *Rhizoma arisaematis* comes from the rhizome of *Pinellia pedatisecta Schott*, which has bitter, warm, pungent, and toxic properties. It has be recorded in Chinese Pharmacopoeia as a traditional Chinese medicine, displaying sedative, stomachic, analgesic, anticoagulant, anti-inflammatory, antiemetic, and antitumor activities. The pharmacognostical study identifies the active components of *R. arisaematis* to be beta-sitosterol, total alkaloids, guanosine, gama-aminobutyric acid, dipeptides, and recently a water soluble polysacchar‐ ide composed of rhamnose, fucose, arabinose, mannose, galactose and glucose, with molar ratios of 0.4:0.5:0.3:0.6:0.9:5.3. This polysaccharide significantly inhibits the growth of tumor in animal experiments; immunomodulation might be the mechanism of the

**•** *Rhodiola rosea L.* belongs to the family Crassullaceae that grows in the Arctic and in the mountainous regions of Europe, Asia, and North America. The rhizome and roots has been widely used for a long time in Russian and Chinese folk medicine to increase human physical and mental performance, longevity, and resistance to high-altitude sickness and to treat fatigue, anemia, cancer, bacterial infection, impotence, nervous system disorders, and cardiovascular diseases. Phytochemical studies of *R. rosea* have revealed the presence of glycosides, flavonoids, essential oils, fats, waxes, sterols, organic acids, tannins, proteins, and polysaccharides. A homogeneous polysaccharide (composed of glucose, galactose, manose, and rhamnose with a relative molar ratio of 4.2:2.4:1.6:1.0) from *R. rosea* was tested for its immunomodulation and anticancer activity *in vitro* and *in vivo* experiments on sarcoma cells. The results showed that this polysaccharide could be used as a novel

promising immunotherapeutic agent in cancer treatment [58].

nontoxic. Here are some examples in the following lines:

64 Phytochemicals - Isolation, Characterisation and Role in Human Health

activity [20].

antitumor activity [57].

polysaccharides, one of the main components of tea extracts, have been demonstrated to have immunological, antiradiation, antioxidant, anticancer, and hypoglycemic effects [63].

Although they are not derived from plants, we must remember some polysaccharides that have been demonstrated to have powerful antitumor activity: chitin, the most abundant renewable natural resource after cellulose, a homopolymer of *N*-acetyl-d-glucosamine, is active on bladder human cancer cells and colon carcinoma [64]; a water-soluble polysaccharide from *Inonotus obliquus* has immunomodulatory and antitumor activity [65]; the polysacchar‐ ides extracted from *Pleurotus eryngii* have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gamacarrageenan sulfated polysaccharides extracted from marine red alga, with different molecular weight, has antitumor and immunostimulating activities [67].

#### **5.4. Alkaloids**

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gama-carrageenan sulfated polysaccharides extracted from

**•** Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant Camptotheca acuminata and Mappia foetida. Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including colorectal, breast, lung, and ovarian cancers. Derivatives of camp‐ tothecin such as 18-OH-camptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have been found to possess a strong antileukemic activity [3, 68]. marine red alga, with different molecular weight, has antitumor and immunostimulating activities [67]. **5.4. Alkaloids**  Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant *Camptotheca acuminata* and *Mappia foetida.* Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including

The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of campto‐ thecin, several new approaches have been investigated such as using drug delivery technolo‐ gies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin nanocrystals were prepared with a sonication-precipitation method with promising results [68]. colorectal, breast, lung, and ovarian cancers. Derivatives of camptothecin such as 18-OHcamptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have been found to possess a strong antileukemic activity [3, 68]. The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of camptothecin, several new approaches have been investigated such as using drug delivery technologies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin

nanocrystals were prepared with a sonication–precipitation method with promising results [68].

Figure 4. Camptothecin (left) and harmine (right). Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was **Figure 4.** Camptothecin (left) and harmine (right).

synthesized by modification of β-carboline nucleus [70].

**5.5. Peptides, proteins, and lectins**

gland cancer and ovarian neoplasm [71].

originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of *P. harmala* have been used for hundreds of years to treat the gastrointestinal cancers and malaria. Harmine was reported to exhibit a diverse range of pharmacological properties such as hallucinogenic, antitumor, antiviral, and antiparasitic activities. In order to search for novel leading compounds with better **•** Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of

antitumor activities and less neurotoxicities, a series of harmine derivatives were designed and

 Peptides are short chains of amino acids linked by peptide bonds. They are naturally, abundantly occurring biological molecules. Many plants are an enormously rich source of peptides with potential antitumor effect, e.g., *Violaceae*, *Rubiaceae*, and *Cucurbitaceae* families. The mechanism of anticancer peptides action consists of inhibition of tumor angiogenesis, induction of tumor apoptosis, induction of tumor necrosis, immunomodulation. Studies have shown that peptides from plants exhibit marked inhibitory effects on the proliferation of various tumor cell lines, such as murine leukemia, rat osteoblastlike sarcoma, human nasopharyngeal carcinoma, lung, liver, and mammary

17

Studies have shown that most peptides isolated from plants are cyclic peptides, so-called cyclopeptides (which usually consist of less than 14 amino acid residues with no disulfide bond). More than 450 cyclopeptides have been discovered in higher plants. They exhibit more potent biological activities, possibly due to the stable configuration provided by their cyclic structure. Some *P. harmala* have been used for hundreds of years to treat the gastrointestinal cancers and malaria. Harmine was reported to exhibit a diverse range of pharmacological properties such as hallucinogenic, antitumor, antiviral, and antiparasitic activities. In order to search for novel leading compounds with better antitumor activities and less neurotoxicities, a series of harmine derivatives were designed and synthesized by modification of β-carboline nucleus [70].

#### **5.5. Peptides, proteins, and lectins**

polysaccharides, one of the main components of tea extracts, have been demonstrated to have immunological, antiradiation, antioxidant, anticancer, and hypoglycemic effects [63].

Although they are not derived from plants, we must remember some polysaccharides that have been demonstrated to have powerful antitumor activity: chitin, the most abundant renewable natural resource after cellulose, a homopolymer of *N*-acetyl-d-glucosamine, is active on bladder human cancer cells and colon carcinoma [64]; a water-soluble polysaccharide from *Inonotus obliquus* has immunomodulatory and antitumor activity [65]; the polysacchar‐ ides extracted from *Pleurotus eryngii* have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gamacarrageenan sulfated polysaccharides extracted from marine red alga, with different molecular

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic

have been demonstrated to have multiple functions, such as antitumor, antioxidant, hepatoprotective activity, and enhance immunity [66]; gama-carrageenan sulfated polysaccharides extracted from marine red alga, with different molecular weight, has antitumor and immunostimulating activities

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic

The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of camptothecin, several new approaches have been investigated such as using drug delivery technologies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin nanocrystals were prepared with a sonication–precipitation method with promising results [68].

Figure 4. Camptothecin (left) and harmine (right).

 Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of *P. harmala* have been used for hundreds of years to treat the gastrointestinal cancers and malaria. Harmine was reported to exhibit a diverse range of pharmacological properties such as hallucinogenic, antitumor, antiviral, and antiparasitic activities. In order to search for novel leading compounds with better antitumor activities and less neurotoxicities, a series of harmine derivatives were designed and

**•** Harmine (Figure 4), the most representative naturally occurring beta-carboline alkaloid, was originally isolated from *Peganum harmala*, which is being widely used as a traditional herbal drug in the Middle East and North Africa. In Northwest China, the extracts of the seeds of

 Peptides are short chains of amino acids linked by peptide bonds. They are naturally, abundantly occurring biological molecules. Many plants are an enormously rich source of peptides with potential antitumor effect, e.g., *Violaceae*, *Rubiaceae*, and *Cucurbitaceae* families. The mechanism of anticancer peptides action consists of inhibition of tumor angiogenesis, induction of tumor apoptosis, induction of tumor necrosis, immunomodulation. Studies have shown that peptides from plants exhibit marked inhibitory effects on the proliferation of various tumor cell lines, such as murine leukemia, rat osteoblastlike sarcoma, human nasopharyngeal carcinoma, lung, liver, and mammary

**•** Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant Camptotheca acuminata and Mappia foetida. Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including colorectal, breast, lung, and ovarian cancers. Derivatives of camp‐ tothecin such as 18-OH-camptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have

The primary limitations of camptothecin are its extremely low water solubility, and the hydrolysis of the active lactone ring to the inactive carboxylate, which reduces the drug efficacy and can lead to side effects. To overcome these stability and solubility problems of campto‐ thecin, several new approaches have been investigated such as using drug delivery technolo‐ gies, e.g., incorporation in liposomes, polymer micelles, microemulsions, and microspheres [69]. Specifically, camptothecin nanocrystals were prepared with a sonication-precipitation

 Camptothecin (Figure 4) is a naturally occurring pyridoindole (quinoline) alkaloid isolated from the seeds of Chinese plant *Camptotheca acuminata* and *Mappia foetida.* Camptothecin and some of its analogs have shown a broad spectrum of antitumor activity against many solid tumors including colorectal, breast, lung, and ovarian cancers. Derivatives of camptothecin such as 18-OHcamptothecin, 11-OH-camptothecin, and 10-OH-camptothecin have been found to possess a strong

17

Studies have shown that most peptides isolated from plants are cyclic peptides, so-called cyclopeptides (which usually consist of less than 14 amino acid residues with no disulfide bond). More than 450 cyclopeptides have been discovered in higher plants. They exhibit more potent biological activities, possibly due to the stable configuration provided by their cyclic structure. Some

weight, has antitumor and immunostimulating activities [67].

66 Phytochemicals - Isolation, Characterisation and Role in Human Health

been found to possess a strong antileukemic activity [3, 68].

synthesized by modification of β-carboline nucleus [70].

**5.5. Peptides, proteins, and lectins**

gland cancer and ovarian neoplasm [71].

**5.4. Alkaloids**

nitrogen atoms.

[67].

method with promising results [68].

**Figure 4.** Camptothecin (left) and harmine (right).

antileukemic activity [3, 68].

**5.4. Alkaloids**

nitrogen atoms.

However, plant lectins attracted increasing attention from cancer biologists due to their possible antitumor properties. A large-scale study in colorectal cancer patients and a control group showed some beneficial effects of consuming plant lectins, but the pathways remain unclear [73].

*Astragalus membranaceus* lectin displays antiproliferative properties toward human leukemia cells *in vitro*. Mistletoe lectin, which is used as an adjuvant in cancer therapy, is known to activate caspases, enzymes involved in the self destruction of cells [74].

*Concanavalin A*, a member of the legume lectin family with a mannose/glucose-binding specificity, was reported to induce apoptosis in human melanoma cells and demonstrated potent therapeutic effect in liver cancer [75].

Recently, some antitumoral lectins have been discovered and researched, such as mistletoe (*V. album*) lectins, rice (*Oryza sativa)* bran agglutinin with remarkable antitumour activities, wheat (*Triticum* spp.) germ agglutinin, a typical chitin-binding lectin with strong inhibitory effects on the growth of the pancreatic tumour cells, and garlic (*Allium sativum L*) lectin, isolated from garlic which induced apoptosis at a low concentration [76]. *Phaseolus vulgaris* lectin can also induce apoptotic cell death toward various types of cancers; more interestingly, induces autophagic cell death in hepatocarcinoma [77].

#### **5.6. Quinones**

Quinones are secondary metabolites generally having a hexacyclic di-ketone system derived from the oxidation of hydroquinones, and isolated principally from plants. Naturally occur‐ ring quinones are widely distributed and include benzoquinones, naphthoquinones, anthra‐ quinones, and polyquinones. Quinones exhibit numerous biological activities, such as neurological, antibacterial, antioxidant, antitumor, and anti-HIV activities that have been proven to be related to the redox properties of their carbonyl functions. Quinones, in general, and naphthoquinones, in particular, are well known for antibacterial, antifungal, and antitu‐ moral activities.

Plumbagin has been shown to have activity against breast, prostate, ovarian, pancreatic, lung, liver, renal, cervical, and skin cancer, in addition to having activity against myeloma and leukemia. It has been shown to inhibit cell proliferation and cell invasion and effectively induces apoptosis and causes cell cycle arrest [80].

**•** Emodin (Figure 5), isolated from *Rheum emodi*, a Himalayan rhubarb, is also produced by many species of fungi, including *Aspergillus* fungi. The pharmacological studies have demonstrated that emodin when isolated from rhubarb exhibits anticancer effects on several human cancers, including human pancreatic cancer [81].

**Figure 5.** Chemical structures of quinones with antitumoral activity.

#### **5.7. Isothiocyanates**

*Astragalus membranaceus* lectin displays antiproliferative properties toward human leukemia cells *in vitro*. Mistletoe lectin, which is used as an adjuvant in cancer therapy, is known to

*Concanavalin A*, a member of the legume lectin family with a mannose/glucose-binding specificity, was reported to induce apoptosis in human melanoma cells and demonstrated

Recently, some antitumoral lectins have been discovered and researched, such as mistletoe (*V. album*) lectins, rice (*Oryza sativa)* bran agglutinin with remarkable antitumour activities, wheat (*Triticum* spp.) germ agglutinin, a typical chitin-binding lectin with strong inhibitory effects on the growth of the pancreatic tumour cells, and garlic (*Allium sativum L*) lectin, isolated from garlic which induced apoptosis at a low concentration [76]. *Phaseolus vulgaris* lectin can also induce apoptotic cell death toward various types of cancers; more interestingly, induces

Quinones are secondary metabolites generally having a hexacyclic di-ketone system derived from the oxidation of hydroquinones, and isolated principally from plants. Naturally occur‐ ring quinones are widely distributed and include benzoquinones, naphthoquinones, anthra‐ quinones, and polyquinones. Quinones exhibit numerous biological activities, such as neurological, antibacterial, antioxidant, antitumor, and anti-HIV activities that have been proven to be related to the redox properties of their carbonyl functions. Quinones, in general, and naphthoquinones, in particular, are well known for antibacterial, antifungal, and antitu‐

**•** Lapachol (Figure 5), the most studied substance of natural quinone class, is the main constituent in Pau D'Arco (a natural remedy) and has been extracted from the bark of South American tree, *Tabebuia impetiginosa*. Lapachol possesses strong biological activity against liver, kidney, breast, prostate, cervix cancer, and leukemia. Unfortunately the toxic effect

**•** Plumbagin (Figure 5) is a naphthoquinone compound that displayed antiproliferative activity on a panel of 60 cancer cell lines. However, plumbagin, identified in the Cameroo‐ nian plants *Diospyros crassiflora* and *Diospyros canaliculata,* was suggested as a promising

Plumbagin has been shown to have activity against breast, prostate, ovarian, pancreatic, lung, liver, renal, cervical, and skin cancer, in addition to having activity against myeloma and leukemia. It has been shown to inhibit cell proliferation and cell invasion and effectively

**•** Emodin (Figure 5), isolated from *Rheum emodi*, a Himalayan rhubarb, is also produced by many species of fungi, including *Aspergillus* fungi. The pharmacological studies have demonstrated that emodin when isolated from rhubarb exhibits anticancer effects on several

activate caspases, enzymes involved in the self destruction of cells [74].

potent therapeutic effect in liver cancer [75].

68 Phytochemicals - Isolation, Characterisation and Role in Human Health

autophagic cell death in hepatocarcinoma [77].

**5.6. Quinones**

moral activities.

limits the use of lapachol [78].

anticancer lead drug [79].

induces apoptosis and causes cell cycle arrest [80].

human cancers, including human pancreatic cancer [81].

Cruciferous vegetables are a rich source of glucosinolates (natural chemicals most likely contribute to plant defense against pests and diseases), which are converted to isothiocyanates. Evidence supports that consumption of cruciferous vegetables has substantial chemopreven‐ tive activity against various human malignancies, including pancreatic cancer. Benzyl isothiocyanate (Figure 6), an agent that is present in cruciferous vegetables such as watercress, cabbage, cauliflower, mustard, and horseradish, is widely consumed as part of a routine diet. Benzyl isothiocyanate is quite effective in suppressing pancreatic tumor growth by inhibiting various key signaling pathways [82].

**Figure 6.** Benzyl isothiocyanate (R=benzyl).

#### **6. Herbs and herbal formulas with antitumor activity**

#### **6.1. Aloe vera**

Aloe vera is one of the most impressive herbs, called by many people "a miracle." The plant works on multiple plans purifying, strengthening, and healing the body. In a single plant, aloe vera offers potent, natural medicine that nourishes the body with minerals, vitamins, enzymes, and saccharides; alkalizes the body, helping to balance overly acidic dietary habits; prevents and treats Candida infections; protects the body from oxidative stress; boosts the oxygenation of your blood; reduces inflammation; purifies the blood, repairs "sludge blood"; halts the growth of cancer tumors; stops colon cancer, heals the intestines and lubricates the digestive tract [83].

Scientific research shows strong immunomodulatory and antitumor properties for aloe vera polysaccharides. Acemannan (Figure 7) is the name given to the major carbohydrate fraction obtained from the gel of the aloe vera leaf. This compound has several important therapeutic properties including acceleration of wound healing, immune stimulation, and antiviral and anticancer effects. The research results suggest that acemannan accelerates the destruction of cancer tumors, improves survival time and results in far better recovery from toxic cancer treatments [83, 84]. Acemannan is currently being used for treatment and clinical management of fibrosarcoma in dogs and cats. Administration of acemannan has been shown to increase tumor necrosis and prolonged host survival [85].

**Figure 7.** Acemannan structure

Also, *Aloe vera* amplifies the antioxidant effects of vitamins C and vitamin E probably due to its effect on enhancing blood quality and allowing the blood to more effectively transport oxygen and nutrients to the body's cells [83].

#### **6.2. Catharanthus roseus or Vinca rosea**

This contains vinca alkaloids, which were the first phytoconstituents ever used to treat cancer. Intense work on *C. roseus*, a folklore hypoglycemic drug, led to isolation of more than 70 dimeric indole alkaloids, which include vinblastine, vincristine, alstonine, ajmalicine, and reserpine. Vinca alkaloids execute anticancer effect by arresting division of the cancerous cells. Vinblastine is used in the treatment of Hodgkin's disease, non-Hodgkin's lymphoma, and cancers of the kidney and the testis. Vincristine is usually given in combination with other anticancer agents to treat acute lymphocytic leukemia, Wilm's tumour, neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, lymphoma, and cancers of the breast, lung, bladder, and the cervix [15].

#### **6.3. Garlic (***Allium sativum***)**

and saccharides; alkalizes the body, helping to balance overly acidic dietary habits; prevents and treats Candida infections; protects the body from oxidative stress; boosts the oxygenation of your blood; reduces inflammation; purifies the blood, repairs "sludge blood"; halts the growth of cancer tumors; stops colon cancer, heals the intestines and lubricates the digestive

Scientific research shows strong immunomodulatory and antitumor properties for aloe vera polysaccharides. Acemannan (Figure 7) is the name given to the major carbohydrate fraction obtained from the gel of the aloe vera leaf. This compound has several important therapeutic properties including acceleration of wound healing, immune stimulation, and antiviral and anticancer effects. The research results suggest that acemannan accelerates the destruction of cancer tumors, improves survival time and results in far better recovery from toxic cancer treatments [83, 84]. Acemannan is currently being used for treatment and clinical management of fibrosarcoma in dogs and cats. Administration of acemannan has been shown to increase

Also, *Aloe vera* amplifies the antioxidant effects of vitamins C and vitamin E probably due to its effect on enhancing blood quality and allowing the blood to more effectively transport

This contains vinca alkaloids, which were the first phytoconstituents ever used to treat cancer. Intense work on *C. roseus*, a folklore hypoglycemic drug, led to isolation of more than 70 dimeric indole alkaloids, which include vinblastine, vincristine, alstonine, ajmalicine, and reserpine. Vinca alkaloids execute anticancer effect by arresting division of the cancerous cells. Vinblastine is used in the treatment of Hodgkin's disease, non-Hodgkin's lymphoma, and cancers of the kidney and the testis. Vincristine is usually given in combination with other anticancer agents to treat acute lymphocytic leukemia, Wilm's tumour, neuroblastoma,

tract [83].

tumor necrosis and prolonged host survival [85].

70 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Figure 7.** Acemannan structure

oxygen and nutrients to the body's cells [83].

**6.2. Catharanthus roseus or Vinca rosea**

Garlic has been used for thousands of years to treat various diseases. Garlic contains approx‐ imately 33 sulfur compounds (aliin, allicin, ajoene, allylpropyl disulfide, diallyl trisulfide, sallylcysteine, vinyldithiines, S-allylmercaptocystein, and others), several enzymes (allinase, peroxidases, myrosinase, and others), 17 amino acids (arginine and others), and minerals (selenium, germanium, tellurium, and other trace minerals). Garlic has shown significant therapeutic effect in stomach, colorectal, and breast cancer in humans. Biological effects of garlic are attributed to its characteristic organ sulfur compounds [86]. The constitutive compounds of garlic can selectively inhibit tumor proliferation by a number of factors, e.g., controlling DNA repair mechanisms, chromosomal stability, and cell cycle regulation. Garlic constituents can suppress carcinogen formation, carcinogen bioactivation, and tumor prolif‐ eration [87, 88].

#### **6.4. Parsley (***Petroselinum crispum***)**

Parsley has a variety of nutrients that protect against developing cancer. It is an excellent source of vitamin C and a good source of beta-carotene, folic acid, vitamin K, flavonoids, and volatile oil components—including myristicin, limonene, eugenol, and alpha-thujene. Parsley's volatile oils—particularly myristicin—have been shown to inhibit tumor formation in animal studies, and particularly tumor formation in the lungs. The flavonoids include apiin, apigenin, crisoeriol, and luteolin [89]. Luteolin have been shown to function as antioxidants that combine with ROS and help prevent oxygen-based damage to cells. Apigenin research studies have associated it with a decreased risk of pancreatic cancer, leukemia, cervical, and ovarian cancer. Apigenin has also been shown to interfere with cancer cell proliferation, exhibiting strong antitumor properties [90].

#### **6.5. Chaparal (***Larrea divaricata Cav***.—Zygophyllaceae)**

This contains five species of evergreen shrubs that are native to the Southwestern United States, and it is a herb derived from the common desert shrubs *Larrea tridentata* and *Larrea divaricata*. It is a plant widely used in popular medicine to treat tumors, infections, and inflammatory diseases. Many studies have shown that chaparral possesses antioxidant properties and exhibits cytotoxic properties in a variety of cell types. The principal ingredient in chaparral is nordihydroguaiaretic acid that is an anticancer agent and a potent antioxidant [91]. This herb also has anti-inflammatory, analgesic, expectorant, emetic, and diuretic properties. Chaparral chelates remove heavy metals in the body and offer protection against the harmful effects of radiation, sun exposure, and the formation of tumors and cancer cells. The lignans found in chaparral that are very similar to estrogen; they are isolated from the flowering tops of *Larrea tridentata* and are effective against human breast cancer, human colon cancer, and human melanoma cell lines [92].

#### **6.6. Essiac**

Essiac is an herbal formula that was originally known to an American tribe and was renamed Essiac in 1920 by a Canadian nurse, Rene Caisse. Essiac is a tea prepared from a mixture of herbs: burdock root (*Arctium lappa*), sheep sorrel (*Rumex acetosella*), slippery elm bark (*Ulmus rubra*), and turkey rhubarb (*Rheum officinale*), and it has been used in alternative medicine for over 50 years. The phytochemicals found in these ingredients are antioxidants protecting cells against oxidative damage manifest interference with DNA replication and have antibacterial effects. Essiac enhances activity of the immune cells and reduces toxic side effects of chemo‐ therapy and radiotherapy. Essiac has been used in the treatment of malignant melanoma, lymphoma, and cancers of the pancreas, breast, ovary, esophagus, bladder, bile duct, and bone. Despite a lack of clinical studies reporting efficacy, 72% of the patients taking Essiac reported a positive opinion of the product [93].

#### **6.7. Hoxsey herbal formula**

Hoxsey herbal formula was discovered incidentally in by Elder Hoxsey, when one of his horses suffering from cancer on the leg, got cured after grazing certain herbs. Later, his grandson Harry Hoxsey tried these herbs on the human cancer. The cancer treatment practiced by Harry M. Hoxsey is one of the longest-lived unconventional therapies of this century. Hoxsey herbal formula contains *Arctium lappa*, *Berberis vulgaris*, *Glycyrrhiza glabra*, *Larrea tridentata*, *Picramnia antidesma*, *Rhamnus purshianus*, *Stillingia sylvatica*, *Trifolium pratense*, and *Xanthoxylum ameri‐ canum*. Hoxsey herbal formula has been used to treat malignant melanoma, lymphoma and cancer of the skin [94].

#### **6.8. Iscaddor**

Iscaddor is a fermented extract of *Viscum album* (mistletoe) that was discovered by the Austrian scientist, Rudolf Steiner, and has been used in the treatment of cancer by European physicians since 1920s. Mistletoe extracts are complex multicomponent mixtures, containing various biologically active substances such as glycoproteins, in particular the mistletoe lectins, polypeptides, amino acids, and oligo- and polysaccharides. More than 20 prospective clinical trials using mistletoe extracts in patients with various malignancies have been reported, and in most of these studies, it was concluded that mistletoe extracts had therapeutic benefit in terms of response rate, overall survival, quality of life, and reduce side effects [95].

#### **7. Conclusion**

Conventional chemotherapy, radiotherapy, and surgical treatments of cancer mainly focus on mass cell killing without high specificity and often cause severe side effects and toxicities. Because of these reasons and high mortality rate associated with cancer, many cancer patients seek alternative methods of treatment and herbal medicines have a vital role to play in the prevention and treatment of cancer.

Over the past decade, there has been an increasing demand of drug development against cancer; at the same time there is considerable scientific and commercial interest in the con‐ tinuing discovery of new anticancer agents from natural product sources, especially herbs. Already a great number of modern drugs in clinical use, having the ability to control cancer cells, are of natural product origins; with advanced knowledge and modern device in isolation and structure elucidation techniques and in molecular science, a more efficient research for finding new plant remedies is possible. There are an impressive number of higher plants, and it is estimated that only 10% of all plants have been examined for their chemical composition and only about 100,000 types of chemical structures have been identified. Thus, there is still a lot to be done in the search for novel natural chemopreventive compounds.

Chemically speaking, it can notice a large structural diversity in herbs: alkaloids, polyphenols, tannins, fatty acids, terpenes, polysaccharides, etc. We can say that nature has cures for serious diseases, in every place all around the globe; most of them are waiting to be discovered and used for the good of people.

Until now, the scientific researchers have investigated mainly particular herbs well known in worldwide alternative medicine ; far less have been investigated the mixtures of plants, e.g., some recognized herbs formulations (e.g., Essiac, Hoxsey, Iscador) that proved to be effective on hundreds of patients. The synergisms arising among the biologically active compounds are waiting to be demonstrated.

In scientific terms, no magic overall formula has been found so far to be applicable to all types of cancer. However, there are enough people who had a longer life or even healed with plants; therefore, medical and herbal research must continue.

#### **Author details**

**6.6. Essiac**

a positive opinion of the product [93].

72 Phytochemicals - Isolation, Characterisation and Role in Human Health

**6.7. Hoxsey herbal formula**

cancer of the skin [94].

**6.8. Iscaddor**

**7. Conclusion**

prevention and treatment of cancer.

Essiac is an herbal formula that was originally known to an American tribe and was renamed Essiac in 1920 by a Canadian nurse, Rene Caisse. Essiac is a tea prepared from a mixture of herbs: burdock root (*Arctium lappa*), sheep sorrel (*Rumex acetosella*), slippery elm bark (*Ulmus rubra*), and turkey rhubarb (*Rheum officinale*), and it has been used in alternative medicine for over 50 years. The phytochemicals found in these ingredients are antioxidants protecting cells against oxidative damage manifest interference with DNA replication and have antibacterial effects. Essiac enhances activity of the immune cells and reduces toxic side effects of chemo‐ therapy and radiotherapy. Essiac has been used in the treatment of malignant melanoma, lymphoma, and cancers of the pancreas, breast, ovary, esophagus, bladder, bile duct, and bone. Despite a lack of clinical studies reporting efficacy, 72% of the patients taking Essiac reported

Hoxsey herbal formula was discovered incidentally in by Elder Hoxsey, when one of his horses suffering from cancer on the leg, got cured after grazing certain herbs. Later, his grandson Harry Hoxsey tried these herbs on the human cancer. The cancer treatment practiced by Harry M. Hoxsey is one of the longest-lived unconventional therapies of this century. Hoxsey herbal formula contains *Arctium lappa*, *Berberis vulgaris*, *Glycyrrhiza glabra*, *Larrea tridentata*, *Picramnia antidesma*, *Rhamnus purshianus*, *Stillingia sylvatica*, *Trifolium pratense*, and *Xanthoxylum ameri‐ canum*. Hoxsey herbal formula has been used to treat malignant melanoma, lymphoma and

Iscaddor is a fermented extract of *Viscum album* (mistletoe) that was discovered by the Austrian scientist, Rudolf Steiner, and has been used in the treatment of cancer by European physicians since 1920s. Mistletoe extracts are complex multicomponent mixtures, containing various biologically active substances such as glycoproteins, in particular the mistletoe lectins, polypeptides, amino acids, and oligo- and polysaccharides. More than 20 prospective clinical trials using mistletoe extracts in patients with various malignancies have been reported, and in most of these studies, it was concluded that mistletoe extracts had therapeutic benefit in

Conventional chemotherapy, radiotherapy, and surgical treatments of cancer mainly focus on mass cell killing without high specificity and often cause severe side effects and toxicities. Because of these reasons and high mortality rate associated with cancer, many cancer patients seek alternative methods of treatment and herbal medicines have a vital role to play in the

terms of response rate, overall survival, quality of life, and reduce side effects [95].

Mariana Albulescu1,2

Address all correspondence to<EMAIL_ADDRESS>
1 Faculty of Chemistry, Biology, Geography, Department of Biology-Chemistry, West Uni‐ versity of Timişoara, Timişoara, Romania

2 Advanced Environmental Research Laboratories, West University of Timişoara, Timişoara, Romania

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an Journal of Integrative Medicine 2012; 3: 55-62.

Asteracea family (Asteraceae) includes more than 23,000 species and is the group of Angio‐ sperms with the richest biodiversity existing in the world. *Vernonia* (*Vernonanthura*) is the most representative gene among 1000 species [1 - 4].

Various chemical components have been reported for the genus: sesquiterpene lactones of glaucolide type, hirsutinolide, vernomargolide, eudesmanolide, cardinanolide, and elephan‐ topus, reported for most species, with some exceptions [5-11]. Moreover, diterpenes derived from ent-kaurano and kaurano, pentacyclic triterpenoids from oleanane and ursane, phytos‐ terols, and flavonoids have been isolated and identified [12-15], while the antimicrobial and molluscicidal pharmacological activities have been the most investigated [15-17].

Within the genus *Vernonia*, *Vernonanthura patens* has been one of the less studied species. It is a shrub that grows wild in Ecuador and is widely used by people in rural areas, who attribute various medicinal properties among them: analgesic, anti-inflammatory, anticancer, and antileishmanial. However, medicinal use, studies on the chemical composition, and pharma‐ cological activities until 2010 were very few. The first information collected on the chemical composition of the species reported the absence of sesquiterpene lactones [18], which consti‐ tuted a chemical marker of gender. Nevertheless, in 1986, the aerial parts were studied, isolated and 10 lactones of a species were identified, existing contradictions on the composition [19]. Jakupovic and Schmedia-Hirschmann, With respect to biological activity, less information exists. The first, was the effect demonstrated by a methanol extract of the leaves against *Artemia salina*, the inertness to the potato disc tumor produced by the introduction of *Agrobacterium tumefaciens*, and the lack of cytotoxicity against cell lines V79 [20]. The antimalarial activity of

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aqueous extract of leaves has been described and a mean inhibitory concentration (IC50) of 38.7 ± 10.1 μg/ml against *Plasmodium falciparum* has been reported [21], *whereas* others authors have reported that an aqueous extract of leaves, got antileishmanial activity against strains of *Leishmania amazonensis* with an IC50 >100 μg/ml, being these the only reports found for the species [22].

#### **1.1. Studies in the species** *V. patens* **in the Ecuadorian coast**

In an earlier chapter of this book, has been published the results for the fractionation of a methanol extract of the species, the study of fractions as antifungal against strains of *Fusarium oxysporum* and *Penicilium notatum*, and the chemical characterization by gas chromatography/ mass spectrometry of the fraction that showed that activity. The hexane fraction presented antifungal activity against these strains, and 33 compounds were possible to identify. Three pentacyclic triterpenoids (lupeol, epilupeol, and lupeol acetate), presented as major compo‐ nents, were also isolated and characterized for fractions[23]

Unlike other species of *Vernonanthura* and the own *V. patens* living in other regions of South America, the one that grows in Ecuador presents as major components pentacyclic triterpe‐ noids and no sesquiterpene lactones. These results forced to perform a genetic characterization of the genes from the plastid rbcL and matK of leaf chloroplast DNA fraction, and it confirmed that the species was *V. patens* [24, 25]

This research group has continued with studies to verify the identity of the species, obtaining the identification of 53 compounds from leaves of a methanolic extract within the terpene compounds are highlighted (oxygenated sesquiterpenoids and triterpenoids), aliphatic hydrocarbons, fatty acids, and their methyl and ethyl esters and sugars [26, 27].

In others studies, two types of waxes of leaves and fat fractions of stems and flowers were isolated and were identified 29 fatty acids and 8 triterpenoids as components of the lipid fractions of these organs, which had not been previously reported (Tables 1 and 2) [28]. Figure 1 presents some of the chemical structures identified in this species.

Regarding biological studies, the antileishmanial evaluation of ethanolic extract of the leaves and stems was performed, proving the traditional use of the leaves of the species as antileish‐ manial. The ethanol extract of stems was highly toxic. Leaves extract showed a higher selectivity index than pentamidine, used as reference drug [29].

aqueous extract of leaves has been described and a mean inhibitory concentration (IC50) of 38.7 ± 10.1 μg/ml against *Plasmodium falciparum* has been reported [21], *whereas* others authors have reported that an aqueous extract of leaves, got antileishmanial activity against strains of *Leishmania amazonensis* with an IC50 >100 μg/ml, being these the only reports found for the

In an earlier chapter of this book, has been published the results for the fractionation of a methanol extract of the species, the study of fractions as antifungal against strains of *Fusarium oxysporum* and *Penicilium notatum*, and the chemical characterization by gas chromatography/ mass spectrometry of the fraction that showed that activity. The hexane fraction presented antifungal activity against these strains, and 33 compounds were possible to identify. Three pentacyclic triterpenoids (lupeol, epilupeol, and lupeol acetate), presented as major compo‐

Unlike other species of *Vernonanthura* and the own *V. patens* living in other regions of South America, the one that grows in Ecuador presents as major components pentacyclic triterpe‐ noids and no sesquiterpene lactones. These results forced to perform a genetic characterization of the genes from the plastid rbcL and matK of leaf chloroplast DNA fraction, and it confirmed

This research group has continued with studies to verify the identity of the species, obtaining the identification of 53 compounds from leaves of a methanolic extract within the terpene compounds are highlighted (oxygenated sesquiterpenoids and triterpenoids), aliphatic

In others studies, two types of waxes of leaves and fat fractions of stems and flowers were isolated and were identified 29 fatty acids and 8 triterpenoids as components of the lipid fractions of these organs, which had not been previously reported (Tables 1 and 2) [28]. Figure

Regarding biological studies, the antileishmanial evaluation of ethanolic extract of the leaves and stems was performed, proving the traditional use of the leaves of the species as antileish‐ manial. The ethanol extract of stems was highly toxic. Leaves extract showed a higher

 Nonanedioic acid dimethyl ester 0.37 - 0.01 - Tetradecanoic acid 0.31 0.26 0.03 0.23 Pentadecanoic acid 0.31 0.24 0.06 0.15 Acid, 9-hexadecenoic (ISOM) 0.33 0.45 0.04 -

**Percent relative abundance HSCB HSCN TSC FSC**

hydrocarbons, fatty acids, and their methyl and ethyl esters and sugars [26, 27].

1 presents some of the chemical structures identified in this species.

selectivity index than pentamidine, used as reference drug [29].

**Compounds**

**1.1. Studies in the species** *V. patens* **in the Ecuadorian coast**

82 Phytochemicals - Isolation, Characterisation and Role in Human Health

nents, were also isolated and characterized for fractions[23]

that the species was *V. patens* [24, 25]

species [22].

HSCB = saponifiable white wax fraction leaves; HSCN = saponifiable orange wax fraction leaves; TSC = saponifiable wax fraction stems, FSC = saponifiable wax fraction flowers.

**Table 1.** Compounds identified in the fatty acid fractions of leaves, stems and flowers of *Vernonanthura patens*

All these studies have contributed to knowledge of the species growing in the Ecuadorian coast and serve as reference for other studies.

HICB and HICN, unsaponifiable leaves fractions; TIC, unsaponifiable stems fractions; FIC, unsaponifiable flowers fractions

+

**Table 2.** Compounds identified in the unsaponifiable fractions *Vernonanthura patens*

**Figure 1.** Some compounds isolated from *Vernonanthura patens.*

#### **2. Materials and methods**

Leaves, flowers, and stems of the species in the phenological stage of flowering were used and collected around the Biotechnology Research Center of Ecuador located at Km. 30.5 via Perimeter province of Guayas, Ecuador. A sample of the plant material was taken for botanical identification, which was botanized at the National Herbarium of Ecuador (QCNE), Quito, with the key CIBE37a.

#### **2.1. Extraction**

**Pico Compound**

84 Phytochemicals - Isolation, Characterisation and Role in Human Health

HO

**PALMITATO DE LUPEOL**

**2.  A sample of the plant material was taken for botanical

+

**Table 2.** Compounds identified in the unsaponifiable fractions *Vernonanthura patens*

**ACETATO DE LUPEOL**

N

O

**ACETATO DE TARAXASTEROL**

N

**LUPANINA**

CH3COO

CH2CH3

> H3CCOO

**ESTIGMASTEROL**

**Figure 1.** Some compounds isolated from *Vernonanthura patens.*

The extraction from the aerial parts of the species (leaves, flowers, and stems) was performed in water and ethanol by triplicate in an ultrasonic bath VWR of 35 KHz power [30]. In all cases, 10 g of sample was extracted in the solvent (water or ethanol), in the following time intervals: 5, 15, 30, 45, and 60 min.

#### **2.2. Determination of antioxidant activity**

The determination of antioxidant activity was performed based on the stability of 1,1 diphenyl-2-picrylhydrazyl (DPPH) radical to alcoholic and aqueous extracts obtained from the aerial parts. A total of 800 μl of 0.1 N DPPH and 200 μl of the extracts were taken, and the absorbance was measured at 517 nm after 30 min using 200 μl of ethanol and 800 μl of DPPH as control. Determinations were performed in triplicate.

#### **2.3. Determination of total polyphenols**

The total polyphenol content was measured by the Folin-Ciocalteu method in a spectropho‐ tometer (BioTek), at 760 nm using gallic acid monohydrate (CAS 149-91-7) as patron of the calibration curve. Results were expressed as milligrams of gallic acid per gram of sample (mg GA/g sample). A total of 250 μl of sample and 350 μl of Folin-Ciocalteu 1 N were mixed; 5 min after, 350 μl of 20% of sodium carbonate was added. After the 90-min incubation at room temperature, the absorbance was measured at 760 nm. Determinations were performed in triplicate.

#### **2.4. Chromatographic profile of the extracts by HPLC**

Chromatographic profiles by high-resolution liquid chromatography (HPLC) were performed in all extracts obtained at different extraction times to determine whether the extraction time produces chemical changes in the extracts. HPLC analysis was performed with Perkin Elmer Series 2000 HPLC with TotalCrom Software operating system. The phenolic compounds were detected at 280 nm with a flow rate of 1 ml/min. C18 column was used at a temperature of 30°C. Separations were carried out in a pumping system by varying the proportion of 2.5% (v/ v) acetic acid in water (mobile phase A) and 70% methanol in water (mobile phase B). The solvent gradient elution program was as follows: 10% to 26% B (v/v) in 10 min, to 70% B at 20 min, and finally to 90% B at 25 to 31 min. The injection volume for all samples was 10μL.

#### **2.5. Statistical analysis**

A factorial design 2 × 3 × 5 was used involving the categorical factors: extraction solvent (water and alcohol), plant organ (leaves, flowers, and stems), and extraction time (5, 15, 30, 45, and 60 min), with antioxidant activity and total polyphenol content as response variables.

#### **2.6. Antileishmanial activity**

This test was performed with aqueous extracts obtained as described.

#### *2.6.1. Aqueous extract*

Extraction was carried out by decoction of plant materials in proportion of 10% in water during 20 min. The aqueous extract was evaporated to 87ºC and 400 mmHg. Finally, the concentrated extract was lyophilized to 120 × 10-3 mbar and 47ºC below zero.

#### *2.6.2. Reference drug*

Pentamidine (Richet, Buenos Aires, Argentina) diluted in sterile distilled water was used for *L. amazonensis*. *L. amazonensis* (MHOM/77BR/LTB0016) was kindly provided by the Depart‐ ment of Immunology, Oswaldo Cruz Foundation (FIOCRUZ), Brazil. Parasites were routinely isolated from mouse lesions and maintained as promastigotes at 26ºC in Schneider's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (HFBS) (Sigma, St. Louis, MO, USA), 100 mg of streptomycin/ml, and 100 U penicillin/ml. The parasites were not used after the tenth passage.

#### *2.6.3. Antileishmanial activity*

The activity of the extracts against intracellular amastigotes was evaluated as described previously. The peritoneal macrophages were harvested and plated at 106/ml in 24-well Lab-Tek (Costarâ, USA) and incubated at 37ºC under an atmosphere of 5% CO2 for 2 h. Nonadherent cells were removed by washing with prewarmed phosphate-buffered saline (PBS). Stationaryphase *L. amazonensis* promastigotes were added at a 4:1 parasite/macrophage ratio, and the cultures were incubated for further 4 h. The cell monolayers were washed three times with prewarmed PBS to remove free parasites. Then 999 ml of RPMI completed medium and 1 ml of the different products dissolved in DMSO were added in duplicate for further 48 h. The cultures were then fixed in absolute methanol, stained with Giemsa, and examined under a light microscope. The number of intracellular amastigotes was determined by counting the amastigotes in 100 macrophages per each sample. In addition, the percentage of infected macrophages was calculated. The results were expressed as percent of reduction of the infection rate in comparison to that of the controls, where the infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages [31]. The IC50 value was determined from the linear regression of concentration– response curves.

The IC50 of the extracts for the viability of mouse peritoneal macrophages was determined. Twenty-two macrophages were collected from peritoneal cavities of normal BALB/c mice in ice-cold RPMI 1640 medium (Sigma, St. Louis, Mo, USA) supplemented with antibiotics and seeded at 30,000 cells/well. The cells were incubated for 2 h at 37ºC in 5% CO2. Nonadherent cells were removed by washing with PBS, and then 1 μl of product solution was added to 200 μl medium containing 10% HFBS and antibiotics. Macrophages treated with 1 μl DMSO were included as controls. The cytotoxicity was determined using the colorimetric assay with 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). MTT solutions were prepared at 5 mg/ml in saline solution, filtered, and sterilized at the moment of use, and 15 μl was added to each well. After incubation for an additional 3 h, the formazan crystals were dissolved by addition of 100 μl DMSO. The optical density was determined using an EMS Reader MF Version 2.4-0, at a test wavelength of 560 nm and a reference wavelength of 630 nm [32]. The IC50 was obtained by fitting a sigmoidal Emax model to dose–response curves. Selectivity indexes were calculated by dividing the IC50 for perito‐ neal macrophage of BALB/c mice by the IC50 for *L. amazonensis* amastigotes. The IC50 of the extracts for the viability of mouse peritoneal macrophages was determined [31].

#### **3. Results and discussions**

**2.6. Antileishmanial activity**

*2.6.1. Aqueous extract*

*2.6.2. Reference drug*

were not used after the tenth passage.

*2.6.3. Antileishmanial activity*

response curves.

This test was performed with aqueous extracts obtained as described.

86 Phytochemicals - Isolation, Characterisation and Role in Human Health

extract was lyophilized to 120 × 10-3 mbar and 47ºC below zero.

Extraction was carried out by decoction of plant materials in proportion of 10% in water during 20 min. The aqueous extract was evaporated to 87ºC and 400 mmHg. Finally, the concentrated

Pentamidine (Richet, Buenos Aires, Argentina) diluted in sterile distilled water was used for *L. amazonensis*. *L. amazonensis* (MHOM/77BR/LTB0016) was kindly provided by the Depart‐ ment of Immunology, Oswaldo Cruz Foundation (FIOCRUZ), Brazil. Parasites were routinely isolated from mouse lesions and maintained as promastigotes at 26ºC in Schneider's medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (HFBS) (Sigma, St. Louis, MO, USA), 100 mg of streptomycin/ml, and 100 U penicillin/ml. The parasites

The activity of the extracts against intracellular amastigotes was evaluated as described previously. The peritoneal macrophages were harvested and plated at 106/ml in 24-well Lab-Tek (Costarâ, USA) and incubated at 37ºC under an atmosphere of 5% CO2 for 2 h. Nonadherent cells were removed by washing with prewarmed phosphate-buffered saline (PBS). Stationaryphase *L. amazonensis* promastigotes were added at a 4:1 parasite/macrophage ratio, and the cultures were incubated for further 4 h. The cell monolayers were washed three times with prewarmed PBS to remove free parasites. Then 999 ml of RPMI completed medium and 1 ml of the different products dissolved in DMSO were added in duplicate for further 48 h. The cultures were then fixed in absolute methanol, stained with Giemsa, and examined under a light microscope. The number of intracellular amastigotes was determined by counting the amastigotes in 100 macrophages per each sample. In addition, the percentage of infected macrophages was calculated. The results were expressed as percent of reduction of the infection rate in comparison to that of the controls, where the infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages [31]. The IC50 value was determined from the linear regression of concentration–

The IC50 of the extracts for the viability of mouse peritoneal macrophages was determined. Twenty-two macrophages were collected from peritoneal cavities of normal BALB/c mice in ice-cold RPMI 1640 medium (Sigma, St. Louis, Mo, USA) supplemented with antibiotics and seeded at 30,000 cells/well. The cells were incubated for 2 h at 37ºC in 5% CO2. Nonadherent cells were removed by washing with PBS, and then 1 μl of product solution was added to 200 μl medium containing 10% HFBS and antibiotics. Macrophages treated with 1 μl DMSO were included as controls. The cytotoxicity was determined using the colorimetric assay with 3-[4,5-

#### **3.1. Determination of antioxidant activity**

The percentage of antioxidant activity by the method of DPPH of the aqueous and the alcoholic extracts of different plant organs, which were obtained at different times of ultrasonic extraction, were determined and differences in time of extraction were observed.

The highest percentage of antioxidant activity was obtained by employing an extraction time of 5 min for the aqueous extracts of leaves and flowers. However, the highest percentage of activity was obtained at 15 min of extraction for the aqueous extract of stems.

Alcoholic extracts of leaves and stems showed no activity at any time of extraction; in contrast, a greater activity at 45 and 60 min of extraction was observed in flowers of *V. patens* with no differences of significance between these times (*p* > 0.05) (Table 3, Figure 2).

Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not significantly different (*p* > 0.05).

**Table 3.** Antioxidant activity (%) of aerials parts of *Vernonanthura patens*

There is only one antecedent of antioxidant activity for the *Vernonia* genre, for the species *Vernonia tweedieana*, with no previous studies for *V. patens* [33].

**Aerial part of the plant**

**Flowers**

Table 3. Antioxidant activity (%) of aerials parts of *Vernonanthura patens*

**Leaves** 80.98 ± 1.7 Aa 48.36 ± 6.69 Ba 51.68 ± 3.14 Ba 54.16 ± 4.95 Ba 44.72 ± 4.33 Ba

**Flowers** 82.85 ± 3.79 Aa 73.85 ± 5.83 ABb 62.63 ± 2.69 BCb 68.05 ± 8.77 BCa 56.26 ± 5.38 Cb

**Stems** 35.10 ± 9.18 Ab 78.76 ± 6.09 Bb 59.14 ± 5.65 Cab 61.35 ± 2.84 Ca 54.60 ± 2.97 Cb

**(ethanol)** 34.24 ± 6.36 Ab 28.07 ± 3.29 Ac 69.34 ± 3.09 Bb 79.42 ± 3.29 BCb 80.82 ± 2.60 Cc

**Extraction time 5 min 15 min 30 min 45 min 60 min**

**Figure 2.** Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

#### **3.2. Determination of total polyphenols**

The part of the plant, the extraction time, and the solvent employed have influence in the content of polyphenols. There is only one antecedent of antioxidant activity for the *Vernonia* genre, for the species *Vernonia*

Figure 2. Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the spectrophotometric determination of polyphenols in the extract. *tweedieana*, with no previous studies for *V. patens* [33]. **3.2 Determination of total polyphenols**  The part of the plant, the extraction time, and the solvent employed have influence in the content of

When water is used as solvent, there is a direct relationship between the extraction time and the concentration of polyphenols; that is, the longer the extraction, the higher the concentration independent of the plant organ used (Figure 3). polyphenols. No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the

In all cases, the highest percentage of total polyphenols was obtained at 45 min of aqueous extraction without significant differences (*p* > 0.05) with 60 min of extraction. At this time of extraction (45 min), the leaves of the species exhibited the highest percentage of polyphenols, followed by flowers. Stems exhibited the lowest percentage of polyphenol (Table 4). spectrophotometric determination of polyphenols in the extract.

> There are several reports regarding the presence of phenolic compounds in the different plant organs for the *Vernonanthura* (*Vernonia*) genre. Flavonoids have been designated as constituents of gender [34]. Flavonoids, tannins, and phenolics are reported for leaves of *V. cinerea* [35], whereas others authors have reported the presence of flavonoids [14]. For flowers of the same species, flavonoids, tannins, and phenolic compounds have been identifiedas well [35]. Flavonoids and tannins has been detected for leaves of *Vernonia amigdalina*, [36, 37].

The part of the plant, the extraction time, and the solvent employed have influence in the content of polyphenols.

Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not

There is only one antecedent of antioxidant activity for the Vernonia genre, for the species Vernonia tweedieana, with no

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the

When water is used as solvent, there is a direct relationship between the extraction time and the concentration of

Figure 2. Antioxidant activity of aerials parts of Vernonanthura patens according to extraction time.

Table 3. Antioxidant activity (%) of aerials parts of Vernonanthura patens

significantly different (p > 0.05).

previous studies for V. patens [33].

3).

3.2. Determination of total polyphenols

spectrophotometric determination of polyphenols in the extract.

**Figure 3.** Polyphenol concentration *vs* time for different plant organs.

Figure 3. Polyphenol concentration vs time for different plant organs.

**3.2. Determination of total polyphenols**

88 Phytochemicals - Isolation, Characterisation and Role in Human Health

independent of the plant organ used (Figure 3).

spectrophotometric determination of polyphenols in the extract.

content of polyphenols.

**3.2 Determination of total polyphenols**

**Antioxidant**

**Activity**

**%**

*tweedieana*, with no previous studies for *V. patens* [33].

extract.

polyphenols.

not significantly different (*p* > 0.05).

**Aerial part of the plant**

**Flowers**

The part of the plant, the extraction time, and the solvent employed have influence in the

ethanol

5 min 15 min 30 min 45min 60 min

**Extraction time**

There is only one antecedent of antioxidant activity for the *Vernonia* genre, for the species *Vernonia*

Figure 2. Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

Leaves Flowers Stems Flowers

**Aerial Part of** *V. patens*

**Figure 2.** Antioxidant activity of aerials parts of *Vernonanthura patens* according to extraction time.

Table 3. Antioxidant activity (%) of aerials parts of *Vernonanthura patens*

**Leaves** 80.98 ± 1.7 Aa 48.36 ± 6.69 Ba 51.68 ± 3.14 Ba 54.16 ± 4.95 Ba 44.72 ± 4.33 Ba

**Flowers** 82.85 ± 3.79 Aa 73.85 ± 5.83 ABb 62.63 ± 2.69 BCb 68.05 ± 8.77 BCa 56.26 ± 5.38 Cb

**Stems** 35.10 ± 9.18 Ab 78.76 ± 6.09 Bb 59.14 ± 5.65 Cab 61.35 ± 2.84 Ca 54.60 ± 2.97 Cb

**(ethanol)** 34.24 ± 6.36 Ab 28.07 ± 3.29 Ac 69.34 ± 3.09 Bb 79.42 ± 3.29 BCb 80.82 ± 2.60 Cc

Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are

**Extraction time 5 min 15 min 30 min 45 min 60 min**

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the spectrophotometric determination of polyphenols in the

When water is used as solvent, there is a direct relationship between the extraction time and the concentration of polyphenols; that is, the longer the extraction, the higher the concentration

The part of the plant, the extraction time, and the solvent employed have influence in the content of

No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the

> In all cases, the highest percentage of total polyphenols was obtained at 45 min of aqueous extraction without significant differences (*p* > 0.05) with 60 min of extraction. At this time of extraction (45 min), the leaves of the species exhibited the highest percentage of polyphenols,

> There are several reports regarding the presence of phenolic compounds in the different plant organs for the *Vernonanthura* (*Vernonia*) genre. Flavonoids have been designated as constituents of gender [34]. Flavonoids, tannins, and phenolics are reported for leaves of *V. cinerea* [35], whereas others authors have reported the presence of flavonoids [14]. For flowers of the same species, flavonoids, tannins, and phenolic compounds have been identifiedas well [35].

followed by flowers. Stems exhibited the lowest percentage of polyphenol (Table 4).

Flavonoids and tannins has been detected for leaves of *Vernonia amigdalina*, [36, 37].

the plant 5 min 15 min 30 min 45 min 60 min Leaves 6.07 ± 1.7 Aa 5.57 ± 0.22 Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not significantly different (*p* > 0.05)

Aa 8.53 ± 1.2 Aa 10.95 ± 0.91 Ba 11.46 ± 1.94 Ba 1.41 ± 0.17 **Table 4.** Total polyphenol content (mg GA/g) of aerial parts of *Vernonanthura patens* using water as solvent.

Flowers 1.12 ± 0.12 Ab Ab 2.55 ± 0.67 Ab 5.74 ± 0.06 Bb 6.85 ± 0.96 Bb Stems 0.98 ± 0.06 Ac 1.09 ± 0.14 Ac 1.47 ± 0.22 ABc 2.06 ± 0.48 BCc 2.99 ± 0.49 Cc Phenolic compounds, tannins, and flavonoids among other compounds in the leaves, stems, and flowers of the species *V. patens* of Ecuadorian coast have been reported [38]. This may explain the total polyphenol content found in different plant organs studied, although in not a very high proportion.

#### **3.3. Chromatographic profile of the extracts by HPLC**

Considering the fact that the extraction method using ultrasound sometimes causes changes in the chemical composition of the extracts, a chromatographic profile was performed to the aqueous extracts obtained at 5 and 45 min of extraction.

Some changes in the chromatographic profile of the aqueous extract of leaves at 45 min are observed (Figure 4), especially in the baseline, although the highest chromatographic peak intensity observed at about 56.9 min has no variation.

Chromatographic profile changes are minor in the aqueous extract of the flowers; a major chromatographic peak around 56.6 min is also seen (Figure 5).

Nevertheless, the aqueous extract of the stems suffered no change in the chromatographic profile at different extraction times analyzed (5, 15, and 45 min). The retention time of the major peak was found at 56.9 min (Figure 6).

Only the study for alcoholic extracts of flowers was done because it was the only one that showed antioxidant activity. Minimal changes were observed in the chromatographic profile of 5 and 45 min extraction, which may be due to the transmission of the ultrasonic waves in the ethanol solvent, which are lower than when water is used [38, 39]. A chromatographic peak around 57 min was also observed in this extract (Figure 7).

The results lead us to believe that in all plant organs of the species obtained with water and the alcoholic extract of the flowers, there is a major component or a mixture thereof, which eluted at a similar retention time by HPLC, in the studied conditions, which may or may not be responsible for the antioxidant activity found for these compounds. However, it is impor‐ tant to note that no correlation between the polyphenol content of the extracts and the antioxidant activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the percentage of total polyphenolics (Figure 8).

These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or more compounds are influential in this activity.

**Figure 4.** HPLC chromatographic profile of the aqueous extract of the leaves of *Vernonanthura patens* obtained with 5 and 45 min of ultrasound extraction

Chromatographic profile changes are minor in the aqueous extract of the flowers; a major

Nevertheless, the aqueous extract of the stems suffered no change in the chromatographic profile at different extraction times analyzed (5, 15, and 45 min). The retention time of the major

Only the study for alcoholic extracts of flowers was done because it was the only one that showed antioxidant activity. Minimal changes were observed in the chromatographic profile of 5 and 45 min extraction, which may be due to the transmission of the ultrasonic waves in the ethanol solvent, which are lower than when water is used [38, 39]. A chromatographic peak

The results lead us to believe that in all plant organs of the species obtained with water and the alcoholic extract of the flowers, there is a major component or a mixture thereof, which eluted at a similar retention time by HPLC, in the studied conditions, which may or may not be responsible for the antioxidant activity found for these compounds. However, it is impor‐ tant to note that no correlation between the polyphenol content of the extracts and the antioxidant activity was found. In all cases, the higher the percentage of antioxidant activity,

These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or more compounds are influential in

**Figure 4.** HPLC chromatographic profile of the aqueous extract of the leaves of *Vernonanthura patens* obtained with 5

chromatographic peak around 56.6 min is also seen (Figure 5).

90 Phytochemicals - Isolation, Characterisation and Role in Human Health

around 57 min was also observed in this extract (Figure 7).

the lower the percentage of total polyphenolics (Figure 8).

peak was found at 56.9 min (Figure 6).

this activity.

and 45 min of ultrasound extraction

**Figure 5.** HPLC chromatographic profile of the aqueous extract of the flowers of *Vernonanthura patens* obtained with 5 and 45 min of ultrasound extraction

**Figure 6.** HPLC chromatographic profile of the aqueous extract of the stems of *Vernonanthura patens* obtained with 5, 15, and 45 min of ultrasound extraction

**Figure 7.** HPLC chromatographic profile of the ethanolic extract of the flowers of *Vernonanthura patens* obtained with 5 and 45 min of ultrasound extraction However, it is important to note that no correlation between the polyphenol content of the extracts and the antioxidant activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the percentage of total

#### These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of **3.4. Evaluation of antileishmanial activity**

3.4. Evaluation of antileishmanial activity

polyphenolics (Figure 8).

polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or more compounds are influential in this activity. Aqueous extracts of leaves and stems showed activity and selectivity against *L. amazonensis* (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a

Figure 8. Total polyphenol content vs antioxidant activity of the leaves, flowers, and stems of Vernonanthura patens

Aqueous extracts of leaves and stems showed activity and selectivity against L. amazonensis (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a different behavior to that reported for the ethanol extract different behavior to that reported for the ethanol extract of this plant organ [28]. The evalu‐ ation was reported as not detectable because it caused a minimal toxicity in the concentrations tested, destroying the cell monolayer.

The results obtained for the aqueous extract support the traditional use of the species and are highly relevant as these extracts would enhance the usefulness of a possible low-cost source for the development of an effective herbal drug for the treatment against Leishmania.

However, it is important to note that no correlation between the polyphenol content of the extracts and the antioxidant **Table 5.** Antileishmanial activity and cytotoxicity of aqueous extracts of *Vernonanthura patens* against *L. amazonensis*

> IC50, half maximal inhibitory concentration, is expressed as the concentration of extract (mg/ml) that inhibits 50% of the parasite growth. CC50, median cytotoxic concentration, is expressed as the concentration of extract (mg/ml) causing 50% of parasite mortality. *X*¯(*S*), mean (standard deviation). SI, selectivity index: CC50 macrophages / IC50 Leishmania. SI ≤ 5 more selective for the cell, SI ≥5 more selective for parasite.

#### **4. Conclusion**

**Figure 7.** HPLC chromatographic profile of the ethanolic extract of the flowers of *Vernonanthura patens* obtained with 5

R² = 0.239

R² = 0.588

Figure 8. Total polyphenol content vs antioxidant activity of the leaves, flowers, and stems of Vernonanthura patens

Aqueous extracts of leaves and stems showed activity and selectivity against L. amazonensis (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a different behavior to that reported for the ethanol extract

activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the percentage of total

Leaves Flowers Stems

Linear (Leaves) Linear (Flowers) Linear (Stems)

and 45 min of ultrasound extraction

0

2

4

6

Total polyphenols content (mgEGA/g)

8

10

12

polyphenolics (Figure 8).

more compounds are influential in this activity.

**3.4. Evaluation of antileishmanial activity**

3.4. Evaluation of antileishmanial activity

R² = 6E-05

20 40 60 80 100

Antioxidant Activity %

**Figure 8.** Total polyphenol content *vs* antioxidant activity of the leaves, flowers, and stems of *Vernonanthura patens*

Aqueous extracts of leaves and stems showed activity and selectivity against *L. amazonensis* (Table 5). The activity and selectivity present in the aqueous extract of the stems showed a

92 Phytochemicals - Isolation, Characterisation and Role in Human Health

The extraction time ultrasound influences the concentration of polyphenolic compounds in all organs tested, when water is used as solvent. Ethanol extracts are unable to determine the concentration of phenolic compounds due to the possible interference of colored compounds extracted with this solvent. Aqueous extracts of the leaves and flowers and the alcoholic extract of flowers showed higher antioxidant activity than the aqueous extracts of the stems. Antiox‐ idant activity in the alcoholic extracts of leaves and stems was not observed.

The HPLC chromatographic profiles of all extracts tested showed a majority chromatographic peak between 56 and 57 min, which could correspond to a mixture of compounds responsible for the antioxidant activity found.

No correlation between antioxidant activity and polyphenol content was found, so presumably they are not solely responsible for the antioxidant activity found.

These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or Aqueous extracts of the leaves and stems of the species *V. patens* showed higher activity and selectivity than the alcoholic extracts, against *L. amazonensis*, corroborating the traditional use of the species.

#### **5. Future directions**

The results obtained in the chemical and biological study of the species *V. patens* show its potential as antimicrobial, antioxidant, and particularly antileishmanial, antiparasitic diseases of high incidence in Ecuador and other countries. However, inclusion in the therapeutic still requires further studies among which may be mentioned toxicology to demonstrate the safety of their preparations and as a development of a suitable dosage form to the pathology to be treated, aspects that should motivate future research of our work group.

### **Acknowledgements**

This study was supported by grants from SENESCYT, Prometheus program, and ESPOL (Ecuador).

#### **Author details**

P.I. Manzano1\*, M. Miranda2 , M.F. Quijano2 and L. Monzote3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Escuela Superior Politécnica del Litoral, Facultad de Ciencias Naturales y Matemáticas (ESPOL-FCNM), Guayaquil, Ecuador

2 Escuela Superior Politécnica del Litoral, Centro de Investigaciones Biotecnológicas del Ecuador (ESPOL-CIBE), Guayaquil, Ecuador

3 Institute of Tropical Medicine Pedro Kouri, Havana, Cuba

#### **References**

[4] Bremer K, Anderber A. (1994). *Asteraceae:* Cladistics & Classification*.* Universidad de Michigan, Timber Press. ISBN-10:<PHONE_NUMBER>. ISBN-13:<PHONE_NUMBER>752.

**5. Future directions**

94 Phytochemicals - Isolation, Characterisation and Role in Human Health

**Acknowledgements**

(Ecuador).

**Author details**

**References**

P.I. Manzano1\*, M. Miranda2

(ESPOL-FCNM), Guayaquil, Ecuador

Ecuador (ESPOL-CIBE), Guayaquil, Ecuador

The results obtained in the chemical and biological study of the species *V. patens* show its potential as antimicrobial, antioxidant, and particularly antileishmanial, antiparasitic diseases of high incidence in Ecuador and other countries. However, inclusion in the therapeutic still requires further studies among which may be mentioned toxicology to demonstrate the safety of their preparations and as a development of a suitable dosage form to the pathology to be

This study was supported by grants from SENESCYT, Prometheus program, and ESPOL

1 Escuela Superior Politécnica del Litoral, Facultad de Ciencias Naturales y Matemáticas

2 Escuela Superior Politécnica del Litoral, Centro de Investigaciones Biotecnológicas del

[1] Rodríguez A. (2005). *Asteraceae*. In: Hammel BE, Zamora N, Grayum MH (eds.). Plant Manual of Costa Rica. Missouri Bot. Gard. Press, St. Louis & Inst. Nac. de Biodiversi‐

[2] Stevens PF. (2006). «Asteraceae». Angiosperm Phylogeny Website. Version 7, Mayo.

[3] Jeffrey C. (2007). Compositae: Introduction with Key to Tribes. 7 in Families and Genera of Vascular Plants. Flowering Plants, Eudicots, *Asterales*. Springer-Verlag,

and L. Monzote3

treated, aspects that should motivate future research of our work group.

, M.F. Quijano2

\*Address all correspondence to<EMAIL_ADDRESS>
3 Institute of Tropical Medicine Pedro Kouri, Havana, Cuba

dad, Santo Domingo de Heredia, Costa Rica.

Berlin: J. W. Kadereit and C. Jeffrey. Vol. VIII: 61–68.

[28] Manzano Santana PI, Miranda Martínez M, de Oca Porto RM, Orellana León T, Abreu Payrol J, Peralta García EL. (2013c). Chemical study of lipid compounds of the leaves, stems and flowers *Vernonanthura patens* (Kunth) H. Rob. (Asteraceae). Cuban Journal of Medicinal Plants, 18(4).

[17] Borkosky S, Ponce S, Gabriela Juárez G, González M, Bardón A. (2009). Molluscicidal sesquiterpene lactones from species of the tribe Vernonieae (Compositae). Chemistry

[18] Mabry TJ, Abdel-Baset Z, Padolina WG. (1975). Systematic implications of flavo‐ noides and sesquiterpene lactones in species of *Vernonia*. Biochemical Systematics

[19] Jakupovic J, Schmedia-Hirschmann G. (1986). Hirsutinolides, glaucolides and sesqui‐

[20] Gupta M, Monge A. (1986). Screening of Panamanian medicinal plants for brine shrimp toxicity, crown gall tumor inhibition, cytotoxicity and DNA intercalation. In‐

[21] Fournet A, Barrios A. (1994). Leishmanicidal and trypanocidal activities of Bolivian

[22] Valadeau C, Pabon A, Deharo E, Albán-Castillo J, Estévez Y, Lores F, Rojas R, Gam‐ boa D, Sauvain M, Castillo D, Bourdy G. (2009). Medicinal plants from the Yanesha (Peru): evaluation of the leishmanicidal and antimalarial activity of selected extracts.

[23] Manzano Santana PI, Silva Osorio M, Sterner O, Peralta Garcìa EL. (2012). Phyto‐ chemical Studies of Fractions and Compounds Present in *Vernonanthura patens* with Antifungal Bioactivity and Potential as Antineoplastic, Phytochemicals—A Global Perspective of Their Role in Nutrition and Health, Dr Venketeshwer Rao (Ed.). ISBN:

[24] Santos E, Manzano P, Navarrete O, Hidalgo L, Miranda M, Peralta E. (2012a). *Verno‐ nanthura patens* maturase K (matK) gene, partial cds; chloroplast. National Center for Biotechnology Information. GenBank: JX276535.1. http://www.ncbi.nlm.nih.gov/

[25] Santos E, Manzano P, Navarrete O, Hidalgo L, Miranda M, Peralta E. (2012b). *Verno‐ nanthura patens* ribulosa-1,5-bifosfato carboxilasa/oxigenasa subunidad grande (rbcL) de genes, cds parcial; cloroplasto National Center for Biotechnology Information. GenBank: JX276536.1. http://www.ncbi.nlm.nih.gov/nuccore/JX276535.1; http://

[26] Manzano Santana P, Miranda Martínez M, Abreu Payrol J, Silva M, Olov S, Peralta E. (2013a). Pentacyclic triterpenoids with antimicrobial activity from the leaves of *Ver‐ nonanthura patens* (Asteraceae). Emirates Journal of Food and Agriculture, 25(7). doi:

[27] Manzano Santana PI, Orellana León T, Miranda Martínez M, Abreu Payrol J, Ruíz O, Peralta García EL. (2013b). Some pharmacognostic parameters of *Vernonanthura pat‐ ens* (Kunth) H. Rob. (Asteraceae) endemic to Ecuador. Cuban Journal of Medicinal

terpene lactones in species of *Vernonia*. Phytochemistry, 25(2): 145–158.

ternational Journal of Pharmacognosy, 34: 19–27.

Journal of Ethnopharmacology, 123(3): 413–422

www.ncbi.nlm.nih.gov/nuccore/JX276536.1

medicinal plants. Journal of Ethnopharmacology, 42: 19.

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96 Phytochemicals - Isolation, Characterisation and Role in Human Health

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Plants, 18(1).

### **Phytochemical Profile of Honey**

Aldina Kesić, Nadira Ibrišimović-Mehmedinović and Almir Šestan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60087

#### **1. Introduction**

During the process of cellular respiration in living organisms some reactive oxygen species (ROS) are constantly forming, sometimes as a result of exogenous sources such as pollution, radiation and ionizing radiation and drugs [1, 2, 3, 4]. They can harm vitally important structures, such as cell membranes, destroying deoxyribonucleic acid (DNA) that is an essential core component of every cell and damaging the respiratory enzymes and genetic material, thus creating the preconditions for the emergence of degenerative and malignant diseases [5, 6, 7, 8].

Production of prooxidant species, especially ROS, is in equilibrium with the antioxidant protection of the organism under normal conditions.

Increased production of pro-oxidants and / or reduced antioxidant protection of the organism can lead to tissue damage and disease. This situation is called oxidative stress, which is the cause or contributing factor in the pathology of many diseases [9].

So, if there is a genetic predisposition or exposure to external factors such as cigarette smoke, sun light, pollution, etc., the balance of prooxidants / antioxidants may be impaired (Figure 1).

Oxidative stress, induced mainly by ROS is now recognized as a major cause of several human health disorders (Figure1) [10, 11]. Antioxidants of both endogenous and exogenous sources have the ability to donate electrons and are effective in mitigating the damaging effects of ROS on cellular components [12]. Honey is a complex mixture of more than seventy different compounds present in varying proportions [13,14]. Many of these phytochemicals such as polyphenols and flavonoids present in honey have antioxidant properties [15,16, 17,18,19]. In view of the importance of the antioxidant phytochemicals present in honey, we undertook a

**Figure 1.** ROS: Occurrence and major consequences on the human body.

study to characterize the photochemical profiles of several honey samples from different botanical and geographical origins.

#### **2. Methods and materials**

The study included 60 samples of honey from different botanical and geographical origin. Samples of honey were collected in cooperation with the Union of Beekeepers of Tuzla Canton, Bosnia and Herzegovina. Information about the botanical and geographical origin of the samples was collected from the producers themselves. The analysis of total antioxidants in the samples analyzed, and an analysis of the content of polyphenols were made [20, 21, 22]. Then, statistical analysis of the data was performed and examined the correlation between the total antioxidant capacity of honey and polyphenol content. The total content of antioxidants in the samples was determined by Ferric Reducing Antioxidant Power (FRAP) method [23, 24, 25]. Total polyphenols in the samples were determined using the Folin-Ciocalteu (FC) method [26].

#### **2.1. Determination of total antioxidant capacity of honey using the FRAP method**

In order to determine the antioxidant capacity, a lot of methods based on different mechanisms of antioxidant defensce systems were developed, such as the removal or inhibition of free radicals or chelating metal ions, which would otherwise lead to the formation of free radicals.

The method used in our study is an indirect FRAP method. The FRAP is a method that was described in 1996 by Benzie and Strain, but in 1999 was further modified [25, 27]. This is a simple, quick test method based on the reduction of iron from ferric Fe3+ into the ferrous Fe2+ form in the presence of antioxidants, where at low pH level, a deep- blue colored complex of ferro tripyridyltriazine is developed, which has an absorption maximum at 593 nm (Figure 2.). The reaction is not specific. The results are expressed as the μmol Fe2+ equivalent of (Fe) / mL sample.

**Figure 2.** Ferrum reduction reaction - 2,4,6-tripyridyl-s-triazine (TPTZ).

Analysis results were determined by measuring absorbance of standard solutions, which have been incorporated in the calibration line.

The concentration of antioxidants in the measured sample on the basis of the obtained value is calculated according to the formula:

$$X = \frac{Y - 0.0243}{0.0011}$$

where:

study to characterize the photochemical profiles of several honey samples from different

The study included 60 samples of honey from different botanical and geographical origin. Samples of honey were collected in cooperation with the Union of Beekeepers of Tuzla Canton, Bosnia and Herzegovina. Information about the botanical and geographical origin of the samples was collected from the producers themselves. The analysis of total antioxidants in the samples analyzed, and an analysis of the content of polyphenols were made [20, 21, 22]. Then, statistical analysis of the data was performed and examined the correlation between the total antioxidant capacity of honey and polyphenol content. The total content of antioxidants in the samples was determined by Ferric Reducing Antioxidant Power (FRAP) method [23, 24, 25]. Total polyphenols in the samples were determined using the Folin-Ciocalteu (FC) method [26].

botanical and geographical origins.

**Figure 1.** ROS: Occurrence and major consequences on the human body.

100 Phytochemicals - Isolation, Characterisation and Role in Human Health

**2. Methods and materials**

X – antioxidant concentration

Y – sample average absorbance

The results are expressed in μmol FeII/L 10 % of honey solution.

#### **3. Determination of total polyphenols using the FC method**

The content of total polyphenols in the honey samples was determined using the FC method. The FC method is one of the oldest indirect methods that is sensitive to phenolic and poly‐ phenolic compounds. This method has been standardized and widely used in the determina‐ tion of polyphenols.

### **4. Results and discussion**

The components of honey, with antioxidant properties, are phenolic acids and flavonoids, enzymes, ascorbic acid, organic acids, amino acids, proteins and some micro biogenic elements [28, 29]. Honey characterization helps us to understand its antioxidant characteristics, thereby, its use as natural foodstuff, i.e., as sources of antioxidant human nutrition.

Our results have shown that antioxidant activity of honey from Bosnia and Herzegovina is in the range of 4.7 μMFeII/L to 1606.54 μMFeII/L.

According to Jerkovic and co-workers´ research, antioxidant activity of honey from the Croatian territory was from 101.5 μMFe to 955.9 μMFe [30].

With the analysis of total antioxidant activity along with the concentration of polyphenols, we came to the conclusion that there has been a significant correlation between these two parameters.

This conclusion is confirmed by research scientist Al-Mamary et-al. [31], who showed that antioxidant activity of several types of honey originating from different countries depends on the concentration of phenolic groups.

In addition, researches and other antioxidant activities of honey that support this assertion were also included [32, 33, 34, 35].

Our results are also in correlation with the results of the Slovenian scientists [26] who, with their studies, confirmed the polyphenol content of 4.48 mg/100 g in locust honey and 24.14 mg/ 100g in forest honey. Italian scientists [35] confirmed the contents of polyphenols in honey in the value of 3 mg/100g to 17.5 mg/100g. These results show slightly lower values of polyphe‐ nols in Italian honey samples compared to the honey from the territory of Bosnia and Herze‐ govina.

A high correlation coefficient between the antioxidant activity of honey and total polyphenol content has also been confirmed in our studies (Figure 3):

With statistical analysis of the given results, we got Pearson's correlation coefficient that is equal to 0.957. This means that 95.7% of honey samples with a stronger antioxidant activity have higher polyphenol content.

The chemical composition of honey largely depends on botanical origin as an important factor. According to the botanical origin, analyzed honey samples are divided into several groups:

**Figure 3.** Correlation between total antioxidant activity of honey and polyphenol content

**3. Determination of total polyphenols using the FC method**

its use as natural foodstuff, i.e., as sources of antioxidant human nutrition.

tion of polyphenols.

parameters.

govina.

**4. Results and discussion**

the range of 4.7 μMFeII/L to 1606.54 μMFeII/L.

102 Phytochemicals - Isolation, Characterisation and Role in Human Health

the concentration of phenolic groups.

were also included [32, 33, 34, 35].

have higher polyphenol content.

Croatian territory was from 101.5 μMFe to 955.9 μMFe [30].

content has also been confirmed in our studies (Figure 3):

The content of total polyphenols in the honey samples was determined using the FC method. The FC method is one of the oldest indirect methods that is sensitive to phenolic and poly‐ phenolic compounds. This method has been standardized and widely used in the determina‐

The components of honey, with antioxidant properties, are phenolic acids and flavonoids, enzymes, ascorbic acid, organic acids, amino acids, proteins and some micro biogenic elements [28, 29]. Honey characterization helps us to understand its antioxidant characteristics, thereby,

Our results have shown that antioxidant activity of honey from Bosnia and Herzegovina is in

According to Jerkovic and co-workers´ research, antioxidant activity of honey from the

With the analysis of total antioxidant activity along with the concentration of polyphenols, we came to the conclusion that there has been a significant correlation between these two

This conclusion is confirmed by research scientist Al-Mamary et-al. [31], who showed that antioxidant activity of several types of honey originating from different countries depends on

In addition, researches and other antioxidant activities of honey that support this assertion

Our results are also in correlation with the results of the Slovenian scientists [26] who, with their studies, confirmed the polyphenol content of 4.48 mg/100 g in locust honey and 24.14 mg/ 100g in forest honey. Italian scientists [35] confirmed the contents of polyphenols in honey in the value of 3 mg/100g to 17.5 mg/100g. These results show slightly lower values of polyphe‐ nols in Italian honey samples compared to the honey from the territory of Bosnia and Herze‐

A high correlation coefficient between the antioxidant activity of honey and total polyphenol

With statistical analysis of the given results, we got Pearson's correlation coefficient that is equal to 0.957. This means that 95.7% of honey samples with a stronger antioxidant activity

The chemical composition of honey largely depends on botanical origin as an important factor. According to the botanical origin, analyzed honey samples are divided into several groups: meadow, acacia, forest, mixed, lime, chestnut honey, mountain honey, hawthorn, heather, sage and rosemary. Different antioxidant activity is shown in honey samples of different botanical origin. Antioxidant activity of honey samples divided according to botanical origin grows according to the following sequence: hawthorn <acacia <lime <meadow <chestnut <mixed <sage <ling <forest <mountain <rosemary. This relationship is shown in Figure 4:

**Figure 4.** Total antioxidant activity of honey samples of different botanical origin

Rosemary honey has got the highest antioxidant activity. Because of its relatively high content of antioxidants and low content of essential oil and chlorophyll, rosemary is the best source of natural antioxidants. Plants such as rosemary, sage, oregano, thyme, pepper and green tea, contain active substances with strong antioxidant activities. Rosemary extracts have a high concentration of active substances carnosol acid, carnosol, methyl carnosol and rosemary acid. Besides carnosol acid and its derivatives which are the most powerful natural antioxidants, rosemary extract also contains ursolic and oleanolic acid (in its chemical composition triter‐ penoids).

Rosemary honey showed the highest antioxidant activity since rosemary is a significant source of natural antioxidants. Although the data showed [36] that hawthorn is a significant source of antioxidants, in our analysis, hawthorn honey showed a lower antioxidant activity com‐ pared to other analyzed honey samples. Since our study determined the total antioxidant activity of aqueous solutions of honey, there is a possibility that hawthorn contains more antioxidants, that are not soluble in water.

If we divide the analyzed honey samples according to their geographical origin, we can say that the samples from the middle Bosnia are of the richest sources of antioxidants, followed by specimens from North-eastern Bosnia, and slightly weaker source of antioxidants are samples of honey from the north and west of Bosnia and Herzegovina (Figure 5).

**Figure 5.** Antioxidant activity of honey samples from different geographic origin

The content of polyphenols in honey is affected by the botanical origin of honey besides having a significant impact on the overall antioxidant activity (Figure 6).

**Figure 6.** Polyphenol content in honey of different botanical origin.

Rosemary honey has got the highest antioxidant activity. Because of its relatively high content of antioxidants and low content of essential oil and chlorophyll, rosemary is the best source of natural antioxidants. Plants such as rosemary, sage, oregano, thyme, pepper and green tea, contain active substances with strong antioxidant activities. Rosemary extracts have a high concentration of active substances carnosol acid, carnosol, methyl carnosol and rosemary acid. Besides carnosol acid and its derivatives which are the most powerful natural antioxidants, rosemary extract also contains ursolic and oleanolic acid (in its chemical composition triter‐

Rosemary honey showed the highest antioxidant activity since rosemary is a significant source of natural antioxidants. Although the data showed [36] that hawthorn is a significant source of antioxidants, in our analysis, hawthorn honey showed a lower antioxidant activity com‐ pared to other analyzed honey samples. Since our study determined the total antioxidant activity of aqueous solutions of honey, there is a possibility that hawthorn contains more

If we divide the analyzed honey samples according to their geographical origin, we can say that the samples from the middle Bosnia are of the richest sources of antioxidants, followed by specimens from North-eastern Bosnia, and slightly weaker source of antioxidants are

samples of honey from the north and west of Bosnia and Herzegovina (Figure 5).

**Figure 5.** Antioxidant activity of honey samples from different geographic origin

a significant impact on the overall antioxidant activity (Figure 6).

The content of polyphenols in honey is affected by the botanical origin of honey besides having

penoids).

antioxidants, that are not soluble in water.

104 Phytochemicals - Isolation, Characterisation and Role in Human Health

According to the results of the study, the polyphenol content is increased according to the following order: acacia <lime <chestnut <meadow <mixed <winter savory <sage <forest <rosemary <hawthorn <mountain.

By analyzing these results we can say that the botanical origin significantly affects the polyphenol content and the total antioxidant activity of honey samples. Influence of polyphe‐ nol content to total antioxidant activity is shown in Figure 7:

**Figure 7.** Polyphenol influence on antioxidant activity of honey samples of different botanical origin.

Most samples showed a correlation between polyphenol content and total antioxidant activity.

Exceptions are the samples of honey from the hawthorn where this correlation does not exist. Polyphenols also exhibit antioxidant activity in the presence of copper ions and prooxidant action. So, it can be assumed that the honey samples contain higher amounts of this micro biogenic element, as it affects the overall antioxidant activity.

#### **5. Conclusion**