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Phytochemicals are chemical compounds that occur naturally in the plant kingdom. Some are responsible for the organoleptic properties of the natural sources in which they are present. The term is generally used to refer to those chemicals that may have biological significance, for example carotenoids, flavonoids, coumarins, or chromones, but not all are established as essential nutrients. There may be as many as 4,000 different phytochemicals having potential activity against several diseases such as cancer and metabolic or degenerative diseases.
Among them, coumarins are a family of benzopyrones (1,2-benzopyrones or 2H-1-benzopyran-2-ones) widely distributed in the nature. They represent an important family of naturally occurring and/or synthetic oxygen-containing heterocycles, bearing a typical benzopyrone framework (Figure 1) .
The name coumarin comes from a French term for the Tonka bean, coumarou, seeds of Dipteryx odorata (Coumarouna odorata) (Fabaceae/Leguminosae), one of the sources from which coumarin was first isolated as a natural product in 1820. It has a sweet odor, easy to be recognized as the scent of new-mown hay; because of that, coumarin has been used in perfumes since 1882. It is presumed to be produced by plants as a chemical defense to discourage predation [2, 3].
Coumarinic compounds are a class of lactones structurally constructed by a benzene ring fused to α-pyrone ring, and essentially possess - conjugated system with rich electron and good charge-transport properties [4, 5]. The simplicity and versatility of the coumarin scaffold make it an interesting starting-point for a wide range of applications [6-8]. There are coumarins as perfumes, cosmetics, and industrial additives. Some of its derivatives have been used as aroma enhancers in tobaccos and certain alcoholic drinks [9, 10]. But their most relevant role is described in natural products, organic chemistry, and medicinal chemistry [11, 12]. The extraction, synthesis, and evaluation of coumarins have become an extremely attractive and rapidly developing topic [13, 14]. Moreover, a lot of coumarin compounds as medicinal candidates for drugs with strong pharmacological activity, low toxicity and side effects, fewer drug resistance, high bioavailability, broad spectrum, better curative effects, etc., to treat various types of diseases are being actively studied . Several efforts have been made mainly in developing coumarin-based anticoagulant, antioxidant , antimicrobial (anti-viral, antifungal, and anti-parasitic) [10, 17], anticancer [18-20], anti-diabetic, analgesic, anti-neurodegenerative, and anti-inflammatory agents [10, 21]. Moreover, the unique and versatile oxygen-containing heterocyclic structure makes coumarin compounds occupy an important place in medicinal chemistry [22, 23]. In addition, studies have been done regarding coumarins as bioactive agents , as well as supramolecular medicinal drugs, diagnostic agents and pathologic probes, and biological stains . Particularly, the large - conjugated system in the coumarinic ring, with electron-rich and charge-transport properties, is important in the interaction of this scaffold with molecules and ions. Coumarin-based ion receptors, fluorescent probes, and biological stains are growing quickly and have extensive applications to monitor timely enzyme activity, complex biological events, as well as accurate pharmacological and pharmacokinetic properties in living cells [26, 27].
Coumarin was first synthesized in 1868, and it was used in the pharmaceutical industry as a precursor in the synthesis of a number of synthetic anticoagulant pharmaceuticals, starting with dicoumarol (removed from the current therapy) . So far, some interesting coumarin-based anticoagulant drugs have extensively been used in clinics . Coumarins are a type of vitamin K antagonists . The most notable ones are warfarin, acenocumarol, and phenprocoumon, currently in use in several countries [31, 32]. Warfarin is employed more frequently than acenocoumarol because of its longer half-life (36 h), theoretically providing more stable anticoagulation and avoiding factor VII fluctuations that potentially occur during acenocoumarol treatment (half-life 10 h) . Nowadays, some coumarins proved to be enzymatic inhibitory agents [monoamine oxidase (MAO) inhibitors, acetylcholinesterase (AChE) inhibitors, and butyrylcholinesterase (BuChE) inhibitors] with great potential in neurodegenerative diseases (ND) [34-38]. These studies represent an important tendency in the coumarin’s chemistry and biological evaluation [39-41].
Therefore, the coumarin ring is prevalently applied to construct several functional molecules in the medicinal field. A great deal of work has been done directed towards the separation and purification of naturally occurring biological coumarins from a variety of plants, animals, and microorganisms, as well as towards the artificial synthesis of coumarin compounds with novel structures and properties . Coumarin compounds as medicinal drugs have been increasingly attracting special interest due to their underlying outstanding contributions in the prevention and treatment of diseases, and the related researches and developments have become an extremely attractive highlighted area.
In this context, an overview of the role of coumarins as important phytochemicals and their interesting applications will be presented and discussed. The origin, natural sources, biosynthesis, and applications are going to be presented in this chapter.
2. Natural occurring coumarins
Coumarin (Figure 1) and its derivatives are an important group of natural compounds widely distributed in the natural kingdom . They can be found in the integument of seeds, fruits, flowers, roots, leaves, and stems, although the largest concentration is generally in fruits and flowers . Originally, coumarin was isolated from the seed of D. odorata. Coumarins are secondary metabolites of higher plants, few microorganisms (bacteria and fungi), and sponges . The function of this type of end product of secondary metabolism is related to defense mechanisms against herbivores and attacks by microorganisms. These compounds are biosynthesized from phenylalanine via the shikimic acid . Natural coumarins are generally unsaturated lactones and comprise another class of compounds C6C3. Almost all the natural coumarins have an oxygenated substituent at position 7 , either free as in hydroxylated umbelliferone, or combined (methyl, sugars, etc.) in other derivatives. Structurally, they are considered derivatives of the ortho-hydroxy-cinnamic acid.
There are different classifications for the coumarin derivatives. Generally, they can be chemically classified according to the most common cores: simple coumarins, complex coumarins, and various coumarins. More complex coumarins are generally fused with other heterocycles . Therefore, they can be classified as: simple coumarins, furocoumarins, dihydrofurocoumarins, pyranocoumarins (linear and angular), phenylcoumarins, and biscoumarins . As said before, hundreds of coumarins have been identified in natural sources, especially plants [48, 49]. Major coumarin constituents isolated from plants include: simple hydroxycoumarins, furocoumarins and isofurocoumarins, pyranocoumarins, biscoumarins, and dihydroisocoumarins (Figure 2) .
Coumarins have been isolated from hundreds of plants species distributed in more than 40 different families. There were isolated more than different 1300 coumarins, well distributed in Angiospermae, Monocotyledoneae and Dicotyledoneae families. Orders with occurrence numbers > 100 are Araliales, Rutales, Asterales, Fabales, Oleales, Urticales, and Thymelaeales. Families with occurrence numbers > 100 are Apiaceae (Umbelliferae), Rutaceae, Asteraceae (Compositae), Fabaceae (Leguminosae), Oleaceae, Moraceae, and Thymelaeaceae, respectively (Figure 3) . The best known and researched coumarins in the field of phytochemistry, pharmacology, medicinal chemistry, and the food science can be found in these families. Therefore, these are the coumarins that are going to be further addressed in the next sections of this chapter.
Coumarins usually are in the free state in plants as they are polar structures, and many of them can sublimate. They might also be found in the form of glycosides, including psoralen core-related structures . They are characterized by UV light absorption, resulting in a very characteristic blue fluorescence; they are also very photosensitive as they can be altered by natural light . These features are used in the isolation and analysis, as well as in unusual therapies such as photochemotherapy and the industry of chemical sensors [51, 52].
3. Biosynthesis of coumarins
Simple coumarins are biogenetically derived from shikimic acid, via cinnamic acid. The specificity of the process is the C-2 hydroxylation, producing a break (β-oxidation) of the side chain (i.e. Salix spp.), or chain isomerization and subsequent lactonization, generating the umbelliferone. Figure 4 explains the entire process [46, 53].
Pyrano and furocoumarins (Figure 2) are also biogenetically derived from shikimic acid. These coumarins could be divided in two groups—lineal and angular—depending on the position where the isopentenyl pyrophosphate is condensed to further cyclize and form the heterocycle. The biosynthesis of these complex coumarins could also be the result of the cyclization of a simple coumarin previously prenylated .
Among the coumarins classified as “various” is the dicoumarol, which is formed by bacterial fermentation of Yellow Sweet Clover, and was isolated for the first time from decomposed leaves of Melilotus albus (Fabaceae/Leguminosae).
An approximation for the dicumarol biogenesis is the hydroxylation of the 4-position of the coumarin, that then captures a molecule of formaldehyde and is condensed with another molecule of 4-hydroxycoumarin, and finally enolize the keto group forming the dicumarol .
From a chemotaxonomic approach, Ribeiro & Kaplan (2002) evidenced that the diversity and structural complexity of the coumarins constitute an example of higher plant evolution. Simple coumarins are the most common in all angiosperms, especially in Oleaceae and Asteraceae, and their occurrence is of 100% and 98, 68%, respectively . The second most prevalent coumarins are furocoumarins and pyranocoumarins. Coumarin in some families are high (Thymelaeaceae, Rutaceae, Apiaceae, Fabaceae, and Moraceae). In the case of well-diversified structural types in Apiaceae and Rutaceae, coumarins are considered as chemotaxonomic markers .
Apiaceae is the major source of coumarins (Figure 3) and one of the more diverse, containing five different types of coumarin derivatives (simple coumarins, lineal furocoumarins, angular furocoumarins, lineal pyranocoumarins, and angular pyranocoumarins) [50, 54]. Rutaceae is also highlighted in both occurrence and diversification. Generally, the division Angiospermae is preferably rich in simple coumarins, followed by the furo and pyranocoumarins .
4. Coumarins in medicinal plants
A large number of valuable species used commonly as medicinal plants, aromatic plants, and edible plants for human and animal feeding belongs to coumarin-rich plant families. Among them are species with well-documented biological activity, in which coumarins are part of the active principles. Table 1 shows a selection of plants of these families (first listed seven families with number of occurrence > 100) and some other families with species of particular pharmacological interest on chronic diseases. Coumarins presenting great pharmacological interest have been isolated in different geographical regions from other botanical families. Also shown are the coumarin compounds having species and their yield (if available).
Most of these plants are well known by people and scientists as part of herbal medicine repertories in Europe, Asia, or the Americas [55-58]. From the list, several coumarin-containing species or genera have also ethnomedical records in Cuba and the Caribbean Basin [59, 60]. Among of plant included are species with a great historical record of ethnomedicinal uses, and are present in traditional medicine systems: Ayurveda Medicine, Traditional Chinese Medicine and Unani Medicine, or in other recent cultures. Also, renowned species used on conventional therapeutics and modern herbal medicine are included, ie. Aesculus hippocastanum (Horse-chestnut), Passiflora incarnata (Passion Flower), Lawsonia inermis (Henna), Hypericum perforatum (Saint John Wort), Tilia cordata (Lime Tree) and Uncaria tomentosa (Cat’s Claw).
Coumarins are also present in several species belonging to different botanical families, which are widespread in the northeastern region of Brazil . Some of them are reported in folk medicine as traditional remedies drugs for the treatment of respiratory diseases . Many pharmacological activities have been ascribed to coumarins such as anticlotting, hypotensive, antimicrobial, anti-inflammatory, and antitumor activities .
Recent studies and review manuscripts regarding the coumarin scaffold describe the huge variety of biological activities that may be present in the natural coumarins [8, 18, 62-64]. Venugopala et al. (2013) presented several coumarins displaying activities such as anti-inflammatory, anticoagulant, antibacterial, antifungal, antiviral, anticancer, anti-hypertensive, antitubercular, anticonvulsant, anti-adipogenic, Cytochrome P450 inhibiting, anti-hyperglycemic, antioxidant, and neuroprotective. Several recent reviews summarize and highlight advances in the application of coumarins, especially concerning their antioxidant and anticancer properties [62-70].From Calophyllum spp., it is remarkable the antiviral activity of calanolides and other related pyranocoumarins on Epstein-Barr virus and HIV . As active compound of molluscicidal effects on Biomphalaria glabrata of C. brasiliense extracts were determined (-) mammea A/BB, also found in C. Verticillatum .
It is the great structural diversity of coumarinic compounds that allows for their several applications, and also allows for the high interest of these derivatives as phytochemicals. The pharmacological and biochemical properties and therapeutic applications of simple coumarins depend upon the pattern of substitution .
Family-specie (vernacular name)
Ammi majus (Bishop's flower)
Imperatorin, bergapten, oxypeucedanin, pabulenol, marmesin, xanthotoxin, isopimpinellin and heraclenin.
Medicinal and food plant uses of some species from major coumarin-containing families.
* M: Medicine, F: Food.
5. Natural coumarins, non-nutrients presented in the food
Phytochemicals are defined as bioactive non-nutrient plant compounds presented in fruits, vegetables, grains, and other food plants that have been linked to reducing the risk of major chronic diseases. It is estimated that > 5,000 individual phytochemicals have been identified in fruits, vegetables, and grains, but a large percentage still remain unknown and need to be identified before we can fully understand the health benefits of phytochemicals in whole foods .
Phenolics are compounds possessing one or more aromatic rings with one or more hydroxy groups, and generally are categorized as phenolic acids, flavonoids, stilbenes, coumarins, and tannins . Phenolics are the products of secondary metabolism in plants, providing essential functions in the reproduction and the growth of the plants, acting as defense mechanisms against pathogens, parasites, and predators, as well as contributing to the color of plants . In addition to their roles in plants, phenolic compounds in our diet may provide health benefits associated with reduced risk of chronic diseases. Among the 11 common fruits consumed in the United States, cranberry has the highest total phenolic content, followed by apple, red grape, strawberry, pineapple, banana, peach, lemon, orange, pear, and grapefruit . Some of these fruits as important antioxidant and antiproliferative activities . Among the 10 common vegetables consumed in the United States, broccoli possesses the highest total phenolic content, followed by spinach, yellow onion, red pepper, carrot, cabbage, potato, lettuce, celery, and cucumber . Some of these vegetables proved to display interesting antioxidant and antiproliferative activities . It is estimated that flavonoids account for approximately two thirds of the phenolics in our diet and the remaining one third are from phenolic acids .
Epidemiological studies have consistently shown that a high dietary intake of fruits and vegetables as well as whole grains is strongly associated with reduced risk of developing chronic diseases, such as cancer and cardiovascular disease [107-109]. Even if it is not so described in the bibliographic sources, most of the food plants, spice plants, and culinary herbs used regionally or worldwide are coumarin-containing plants, thus its effect on health cannot be ignored. For example, the potentially health-promoting role of popular vegetables and spices proved to be derived from Apiaceae . Additionally, the above vegetables and spices also contain several bioactive phytochemicals such as flavonoids (quercetin, rutin) and coumarins (bergapten, isopimpinellin, xanthotoxin), which are reported to have curative, preventive, or nutritive value . The above coumarins have also been found to inhibit multiplication of bacteria, fungi, and viruses  and demonstrated anti-allergy , anti-inflammation , and immunosuppression activities .
Table 1 also shows the importance of a number of families containing coumarins in human nutrition. Among other species of interest, the Apiaceae family is a prominent food source of coumarins: carrots, celery, parsley, coriander, cumin, fennel, and aniseed are present in the culinary practice around the world and in the food industry (fixative) . Rutaceae also proved to contain a great number of coumarins with nutritional and economic interest, particularly the citrus and some other fruits such as Bael . Besides fruits and vegetables, olive oil and beverages such as coffee, wine, and black and green tea are also important dietary sources of coumarins .
It is also known that essential oils derived from some plants also contain coumarin derivatives and are used as flavoring in foods. Some essential oils such as Chinese cinnamon oil , cinnamon bark oil , and lavender oil  have important amounts of coumarins. Coumarin’s aroma has been described as sweet, aromatic, creamy vanilla bean odor with nut-like tones that are heavy, but not sharp or brilliant . A major source in alcoholic beverages is Hierochloe odorata, which is used to flavor a special kind of vodka, produced mainly in Eastern Europe .
According to Lake (1999), the main source of coumarin in human diet is the cinnamon. Cinnamon comes from the dried bark of Cinnamomum verum and C. cassia/C. aromaticum, and is considered a spice. Cinnamon is widely used in various cultures in preparation of desserts, cakes, candy, etc., to decorate and some flavoring dishes. It is also used in some places as a beverage or tea. It is also an ingredient in many curries and other dishes Eastern. Intake levels [tolerable daily intake (TDI)] of coumarin derivatives are 0.1 mg/kg bw . For food and beverages in general, the maximum permissible level is 2 mg/kg [122.
It has been estimated that human exposure to coumarins diet is approximately 0.02 mg/kg/day (Lake, 1999). The theoretical maximum daily intake (TAMDI) of coumarin via food was estimated to be 4.085 mg/day (0.07 mg/kg bw/day) .
Evidence has suggested that coumarin is not a genotoxic agent [121, 124]. The International Agency for Research on Cancer  has classified coumarin as belonging to group 3 (‘‘not classifiable as to its carcinogenicity in humans”). No epidemiological data relevant to the carcinogenicity of coumarin were available and there was only limited evidence in experimental animals for the carcinogenicity of coumarin .
The field of food science is of great interest to develop research related to consumer safety and also those designed to elucidate the potentially health-promoting capacity and biological activity of bioactive components that are part of so-called functional foods. However, the beneficial role of these phytocompounds when in synergy on the original food matrix or when isolated (nutraceutics or food supplement) is currently a hot topic .
Due to the structural diversity and versatility of applications of coumarins, not only in food sciences (including diet supplements), it is necessary to continue research related to the safety, and also its bioavailability, interactions with other dietary compounds, and therapeutic and environmental components. It is also important to amplify omics techniques, including epigenetic studies.
Different environmental insults can influence epigenetics and nutrition is one of the major factors that contribute to epigenetic regulation of diseases. Particularly, non-communicable diseases phenotypes can be determined by the role of prenatal or early-age nutrition and epi-mutations can have transgenerational effects, while some “epi-nutrients” and food products can also stabilize the genome [127, 128]. Therefore, the role of food-based “epi-bioactive” compounds has become an emerging field. Nutri-epigenomics is part of this new era too, since this approach on research has been carried out on chronic or degenerative diseases [129-133].
Besides micronutrients (folate, selenium, retinoic acid, and vitamins D and E) effects on epigenome, investigations on dietary phytochemicals has been carried out to determine their ability to reverse adverse epigenetic marks, mainly in cancer, for instance: polyphenols (resveratrol, curcumin, catechin, ellagitannin); genistein and soy isoflavones; sulfur-containing compounds (sulforaphane, phenylethyl isothiocyanat, phenylhexyl isothiocyanate, diallyldisulfide, allyl mercaptan) from Allium spp. (Alliaceae); and cruciferous (Brassicaceae) vegetables [129, 133].
Histone modifications are one of the major epigenetic mechanisms and its acetylation is mediated by the interplay of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The class III HDACs, called sirtuins (SIRT), has been shown to deacetylate the transcription factor p53. Given the regulatory functions of p53 in cell metabolism, inhibition of SIRT1 might contribute to inhibition of glycolysis and inhibit cell proliferation and the apoptosis [129, 133, 134].
Dihydrocoumarin (DHC), which is found in Melilotus officinalis (Fabaceae) (sweet clover) or synthetized, is commonly added as flavoring agent to food and cosmetics. This compound was studied by Olaharski et al. (2005), on different assays to evaluate it as an “epi-bioactive” agent and resulted that DHC inhibited the deacetylase activities of yeast SIRT2p and human SIRT1. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation, cytotoxicity, and flow cytometric analysis, demonstrating that DHC increased apoptosis more than 3-fold over controls .
Authors also stated that these findings on DHC could be potentially worrisome, since SIRT1 inhibition may lead to epigenetic alterations, as well as possible stem cell depletion and early tissue senescence, a phenotype associated with senescence and aging. However, on the possibility of deleterious human exposition to epigenetic toxicants that inhibit SIRT deacetylases, this effect can be desirable in cancer treatment mediated chemopreventive potential epigenetic mechanism [36, 129].
6. Extraction techniques and identification of coumarins
There have been a variety of methods described for extraction of coumarins. Generally, coumarins extraction can be performed either on dry or fresh material, with solvents of different polarities, depending on the type of structure. Some coumarins are sparingly soluble in apolar solvents and often they can be crystallized directly by cooling or concentrating the solvent.
Miranda and Cuéllar (2001), in their book entitled "Farmacognosia y Productos Naturales ", raised the possibility of following four variants using the Soxhlet method:
Soxhlet extracts the dry powdered material with petroleum ether continuously for 3 days. The ether extract is concentrated until 1/5 of the original volume and it is cooled to obtain the crystallization of the extract. Coumarins are presented in the obtained solid.
The dry material is removed and sprayed with ethanol, continuously using the Soxhlet for 2–3 days, at least. The extract is concentrated under vacuum to an oily residue. This residue is repeatedly washed with portions of hot water. The aqueous washings are combined and concentrated to the minimum volume, acidifying with hydrochloric acid solution 10%. The mixture is refluxed for 30 minutes. If some precipitate appears, it is filtered (hot filtration) and the solution is allowed to cool. The crystals are collected by filtration and found therein the coumarins.
Available materials are extracted with ethyl ether or by successive macerations with the Soxhlet apparatus. The extract is concentrated to dryness and the residue contains the coumarins.
The dried and ground plant material is extracted with acetone continuously in a Soxhlet apparatus, for at least 3 days, and the extract is concentrated to dryness giving a residue that contains the coumarins.
Purification and separation of coumarins contained in various extracts could be performed by using chromatographic columns, using as a carrier aluminum oxide and as solvent the eluotropic series: benzene-hexane (1:2.5); benzene; chloroform; chloroform-acetone, in proportions of a linear gradient to pure acetone . For recognition of the described structures some trials were described, within which there are:
Those that recognize coumarin’s phenolic substitutions where Emerson's Reagent is used, developing color.
The presence of lactone groups can be observed leading to changes of pH in the medium. When coumarins are dissolved in ethanol, solutions change the color when acidified (yellow color disappears).
The furan ring can be recognized by using the Erlich test. The extract is treated with a solution of dimethylamino-benzaldehyde (5% ethanol), and then acidified by bubbling gaseous hydrochloric acid. The orange color indicates a positive test.
The last test is commonly used for phytochemical screening that is initially performed in plant research. Carrying on this approach, Payo et al., (1996), from 39 Cuban species screened, detected that 51.2% were positive for coumarin test.
Other extraction methods used in coumarins are: microwave, sonication, and supercritical fluid extraction (SFE) , these tests also propose capillary electrophoresis for natural products isolation.
Therefore, on the isolation and analysis of coumarins diverse methods have been used: chromatography (paper chromatography, thin layer chromatography, gas chromatography, and high-performance liquid chromatography), titrimetric, and spectrophotometric (colorimetric and polarographic) methods .
Due to coumarin-characteristic chromophore groups and its strong UV absorption at around 300 nm, it is routinely possible to be detected by feasible methods such as ultraviolet–visible spectroscopy (UV–vis) . UV–vis detector is used in high performance liquid chromatography (HPLC) and also other hyphenated techniques are employed to characterize and quantify natural products such as Liquid Chromatography (LC)–Photo Diode Array Detector (PDA), coupling of Mass Spectrometry to LC–, or Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS) .
A simple spectroscopic technique  and HPLC  were employed to determine coumarins in the Brazilian medicinal plant "guaco", Mikania glomerata (Asteraceae). Coumarin HPLC detection is also used in Cuba to standardize a sedative herbal medicine based on Justicia pectoralis (Acathanceae) [97, 139].
Coumarins have been increasingly attracting special interest as phytochemicals due to their underlying outstanding contributions in the prevention and treatment of diseases. Coumarins represent a diverse class of phytochemicals that are ubiquitous in the human diet. Some of the medicinal usages of extracts of plants containing coumarins have been proven in experimental models, which suggested that the extracts possess various pharmacological actions. Several related researches and developments make coumarins an extremely attractive scaffold. The role of coumarins as important phytochemicals and their interesting applications were presented and discussed in this book chapter. The origin, natural sources, biosynthesis, and applications were also described.
The authors thank the partial financial support of University of Santiago de Compostela, University of Camagüey Ignacio Agramonte Loynaz and Galician Plan of research, innovation and growth 2011-2015 (Plan I2C, ED481B 2014/086-0)
Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A. Abreu, Enrique Molina and Estela Guardado Yordi (September 30th 2015). Coumarins — An Important Class of Phytochemicals, Phytochemicals - Isolation, Characterisation and Role in Human Health, A. Venket Rao and Leticia G. Rao, IntechOpen, DOI: 10.5772/59982. Available from:
Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents
By James Hamuel Doughari
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