Preparation of Phytopharmaceuticals for the Management of Disorders: The Development of Nutraceuticals and Traditional Medicine

Egypt

Part I

Phytopharmaceuticals and technology

Chapter 1: General principle of primary and secondary plant metabolites: Biogenesis, metabolism, and extraction

Charles Oluwaseun Adetunjia; Santwana Palaib; Chika Precious Ekwuabuc; Chukwuebuka Egbunad,e; Juliana Bunmi Adetunjif; Chioma Bertha Ehis-Eriakhag; Shyam Sundar Keshh; Andrew G. Mtewai,j,k    a Department of Microbiology, Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Edo University Iyamho, Iyamho, Edo State, Nigeria

b Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, OUAT, Bhubaneswar, Odisha, India

c Department of Clinical Pharmacy and Pharmacy Practice, University of Benin, Benin City, Edo State, Nigeria

d Department of Biochemistry, Faculty of Natural Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria

e Nutritional Biochemistry and Toxicology Unit, World Bank Africa Centre of Excellence, Centre for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, Port Harcourt, Rivers State, Nigeria

f Department of Biochemistry, Nutrition and Toxicology Research Laboratory, Osun State University, Osogbo, Osun, Nigeria

g Environmental and Molecular Biology Laboratory, Edo University Iyamho, Iyamho, Edo State, Nigeria

h Department of Veterinary Clinical Complex (Veterinary Biochemistry), Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal, India

i Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom

j Chemistry Department, Malawi Institute of Technology, Malawi University of Science and Technology, Thyolo, Malawi

k Pharmbiotechnology and Traditional Medicine Center of Excellence, Mbarara University of Science and Technology, Mbarara, Uganda

Abstract

Plants are repository of novel products of medicinal value that are necessary for production of new drugs with application in the medical and pharmaceutical industries. The recent advances in the application of metabolomics techniques have enhanced the application of analytical techniques for effective quantification useful metabolites. This chapter gives an insight on the principles and classification of primary and secondary plant metabolites and provides detailed information on the biogenesis of carbohydrates, lipids, volatile oils, and resins. Also, this chapter provides information on the medicinal application of these metabolites.

Keywords

Medicinal plants; Alkaloids; Saponins; Flavonoids; Carbohydrates; Lipids; Volatile oils and resins; Antimicrobial effect; Health benefits

1.1: Introduction

Since the beginning of life, man has utilized plants for different purposes. The early man intensified effort in search of food to cope, which led him to distinguish between plants that serve as food from those that have medicinal value [1]. Ever since, man has depended on plants for their daily needs for food, medicine, and other things including shelter, clothing, and fertilizers.

Plants synthesize hundreds of thousands of metabolites. Metabolites are products and intermediates of metabolism with numerous functions including signaling, inhibitory effects on enzymes, defense, cofactor to enzyme, and protection of the plant against several pathogens and diseases. Various plants of different species and genera have been recognized to produce both primary and secondary metabolites of different types. The advances in the application of metabolomics have helped in the profiling of numerous types of metabolites. Metabolomics in effect entails the comprehensive analysis of various metabolites present in any biological system. Also, it helps give a better knowledge of the organization and principle of cellular purposes at several levels including an insight of biological processes in an integral system [2–8].

1.2: Plant metabolites

Biochemical reactions that occur in organisms are collectively referred to as metabolism. The products of these reactions, as well as their intermediates, which are usually small molecules, are called metabolites. As earlier mentioned, they are classified as either primary or secondary, based on their intrinsic functions within the organism.

1.2.1: Primary metabolites

Primary metabolites are different types of organic compounds secreted by plants that are involved in the various development and growth, synthesis of hormone and protein, photosynthesis, and respiration. Primary metabolites are present in almost every part of the plant species within extensive phylogenetic clusters, which are synthesized using the same or almost biochemical pathways. Examples of primary metabolites include carbohydrates, that is, glucose, fructose, sucrose, raffinose, stachyose, mannitol, xylitol, and sorbitol; the nonstarch includes lignin; amino acids, that is, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, and valine; vitamin B complex, that is, thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, and folic acid; organic acids; ascorbic acid; oxalic acid; aconitic acid; and unsaturated fatty acids, that is, monounsaturated and polyunsaturated [9].

1.2.2: Secondary metabolites

Plants secrete some useful biologically active chemical substances that are not involved in the development and growth of plants. These substances are called secondary metabolites and are often differentially circulated among taxonomic sets within the plant kingdom. Although the function of many of the plant secondary metabolites still remains unknown, there are four major groups of secondary metabolites that include the sulfur-containing compounds and alkaloids, which are almost about 12,000 compounds; phenolic and polyphenolic compounds, which are about 8000 compounds; and the terpenoids, which are about 25,000 compounds. Moreover the function of these secondary metabolites mainly is to prevent the body from diseases. Examples of secondary metabolites includes flavonoids, that is, quercetin, apigenin, luteolin, myricetin, and cyanidin; phenolic acids, that is, chlorogenic acid, caffeic acid, ferulic acid, p-coumaric acid, vanillic acid, sinapic acid, and protocatechuic acid; lignans, that is, lariciresinol, pinoresinol, secoisolariciresinol, and matairesinol; carotenoids, α-carotene, β-carotene, β-cryptoxanthin, zeaxanthin, and lycopene; tocopherols and tocotrienols, that is, α-tocopherol, β-tocopherol, γ-tocopherol, α-T3, and γ-T3; quinones, that is, phylloquinone; sterols, that is, campesterol, campestanol, stigmasterol, β-sitosterol, β-sitostanol; alkaloids, that is, α-tomatine, dehydrotomatine, α-solanine, α-chaconine, lactucin, and lactucopicrin; glucosinolates, that is, glucoiberin; and glucosinolates, that is, glucoraphanin, glucoalyssin, sinigrin, gluconapin, progoitrin, and gluconasturtiin [10].

These bioactives are capable of eliciting pharmacological/toxicological response in humans (Table 1.1) while increasing healthy nutrition as the major component of nutraceuticals and dietary supplements. They also help increase agricultural yields as growth enhancers of plants and animals as well as being used as dyes, fragrances, flavors, and cosmetics [23].

Table 1.1

Traditionally, secondary metabolites are obtained from plant parts by extraction methods such as steam, solvent (organic/inorganic), and super critical extraction. Other modern methods exist [24]. However, this has encountered a lot of challenges, which the scientific community has constantly been working to overcome. Plant tissue and cell culture is a method in metabolic engineering that has been found as a worthy alternative to mitigating the limitations associated with the conventional methods of extraction. This involves the isolation of advantageous cells and culturing them under special organized environments. The metabolites obtained are free from contaminants; the metabolic process can be rationally controlled, labor cost is decreased, increased yield of high-value metabolites is achieved, and cells are cultured under controlled condition free from climatic variations/soil types [23]. Improved methods of biotechnology have been applied in plant cell and tissue culture to further increase yield of metabolites for commercial and industrial purposes. As seen in Table 1.1, the most prominent and bioactive principles of medicinal and toxicological significance are as follows:

1.Alkaloids: These are group of nitrogenous heterocyclic bases obtained from various parts of the plant. Alkaloids are bitter in taste. They are waste product of plant metabolism, and their names usually end with ine. Alkaloids are alkaline secondary metabolites with pharmacological activity.

2.Flavonoids: These are pigments found in plant more specifically in the colored areas of the plant, like petals of flower or leaf itself.

3.Anthocyanins: These are antioxidants made up of aromatic rings of the cyanidin with substituents such as hydroxyl, methyl, or glycosyl groups. They exhibit antioxidant and antiviral properties.

4.Saponins: These are group of compounds with sugar moieties bound to a sapogenin-steroid alkaloids, steroids, or triterpene resulting to structural varieties of saponins. They reduce the digestion of protein due to formation of less soluble saponin-protein complexes and stop the absorption of micronutrients. They exhibit antioxidant properties.

5.Tannins: These are bitter astringents, condensed, or hydrolyzable, water-soluble polyphenols. They bind to protein and precipitate it as well as to other various organic compounds including amino acids and alkaloids. Due to its astringent action, it produces a tangy sensation in mouth. Condensed tannins of polyphenolic compounds exhibit powerful antioxidant activity.

6.Phenols: Phenols are associated with lignin as ester groups. They are extracted as alcohol-soluble fraction bound to simple glycosides and insoluble fractions.

7.Glycosides: Glycosides are found in all plants with large range of therapeutic value. They have glycosylated principles of medicinal and toxicological value. Cardiac glycosides derivatives of digitalis and strophanthus such as ouabain are notable inhibitor of Na +/K + -ATPase activity of biomembranes.

8.Terpenoids: Terpenoids combine in many combinations to get a wide range of isoprenoids. These are present as glycosides.

9.Coumarins: The coumarins exist in different forms, for example, as benzopyrene derivatives in many plants. They are found as aroma present in essential oil [25].

1.3: Biogenesis of primary and secondary plant metabolites

Biogenesis is a term that refers to the processes involved in the synthesis of organic substances from intermediate compounds. Cell metabolism includes two distinct processes: anabolism, which involves production of complex compounds using energy and reducing power, and catabolism, which involves the degradation of complex molecules to produce energy and reducing power. These processes enable the plant cell to utilize absorbed nutrients and stay alive. Plant cell metabolism involves a complex cascade of chemical reactions called metabolic pathways. As earlier stated, same biochemical pathway may produce both primary and secondary metabolites with common intermediates, which further complicates the process.

Plants undergo photosynthesis and respiration, which uses carbon dioxide (as the sole source of carbon), water, sunlight energy, and ammonia as starting materials to produce primary metabolites such as glucose, amino acids, lipids, and nucleic acids. Major metabolic pathways involved in plant metabolism include photosynthesis, Calvin's cycle, glycolysis, citric acid (Kreb’s) cycle, pentose phosphate pathway, oxidative phosphorylation, fatty acid oxidation, and respiration (anaerobic).

1.3.1: Biogenesis of carbohydrates

Plants are capable of synthesizing carbohydrate from carbon dioxide (CO2) and water (H2O) in the presence of sunlight (energy) via photosynthesis [26]. The assimilation of carbon from CO2 to produce carbohydrate is a cyclic process that involves various intermediates and enzymatic catalysts and is referred to as the Calvin's cycle. The light energy from sunlight is captured and converted to chemical energy, adenosine triphosphate (ATP) and nicotinamide dehydrogenase (NADH) via ATP synthase, to initiate subsequent reactions. Another enzyme, ribulose-1,5-diphosphate carboxylase oxygenase (RuBisCO), then catalyzes the reduction of CO2 and its subsequent covalent bonding to a 5-carbon compound, ribulose 1,5 bisphosphate to form an unstable 6-carbon compound, which splits into two molecules of 3-phosphoglycerate. Then, 3-phosphoglycerate kinase catalyzes the introduction of a phosphoryl group to yield 1,3-bisphosphoglycerate, which is acted upon by glyceraldehyde 3-phosphate dehydrogenase to form glyceraldehyde 3-phosphate. Next, triosephosphate isomerase catalyzes the conversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, most of which is used to reproduce ribulose 1,5 bisphosphate (as starting material to continue the cycle), and the rest carbohydrate. Two molecules of dihydroxyacetone phosphate combine to produce fructose 1,6-bisphosphate, which is further converted by fructose 1,6-bisphosphatase to fructose 6-phosphate and complex carbohydrates like starch.

1.3.2: Biogenesis of lipids

Fatty acids are biosynthesized in seed plastids from acetyl CoA, produced from pyruvate, the end product of glycolysis [27], and then transferred to the ER for lipid storage. Enzymes act on glucose in glycolysis pathway to produce little amount of ATP and NADH, as well as intermediates (glycerates and pyruvates), with the most important being pyruvate. Pyruvate is infused into Kreb's cycle (also called citric acid or tricarboxylic cycle) and undergoes oxidation to produce greater amount of energy (ATP) and several biosynthetic intermediates, one of which being acetyl CoA, the precursor of fatty acids. Plant studies have shown that plant lipids are made up of a triacylglycerol (TAG) core, surrounded by proteins [28], which eventually bud off as lipid bodies. This is formed at specific sites in the endoplasmic reticulum [29, 30], where substantial amounts of lipids are stored in plant seeds as TAG, although not exclusively, as lipids have also been found in other types of cells within the plant [28]. It has been proven that lipid biogenesis in plants is one of the various functions that occurs within the endoplasmic reticulum [31–33]. Certain regions in the ER stores diacylglycerol acyltransferase (DGAT), which is the precursor enzyme for TAG biosynthesis, and proteins such as oleosin, which is the major structural protein of lipid bodies formed in seeds [28, 34–38], others being caleosin and steroleosin. There is a possibility that the mechanism of lipid formation differs in other cell types (nonseed) or varying species and may be specific to them [39, 40].

1.3.3: Biogenesis of volatile oils

Volatile oils (also known as essential oils) are odoriferous liquids produced by diverse (several hundreds) odor-active chemicals in plants. Characteristically, they evaporate quickly when exposed and are produced in various plant parts. They are products of metabolism, with primary metabolites as the building blocks, and as such are yields of the basic processes such as glycolysis, Kreb's cycle, fatty acid oxidation, and pentose phosphate pathway. Precursors include fatty acids, amino acids, terpenoids, phenolics, glucosinolates [41], and carbohydrates [27]. They may be classified as primary or secondary metabolites depending on the point of formation in the metabolic pathway [42].

1.3.3.1: Volatile oil derivation by carbohydrate pathway

Carbohydrate-derived essential oils are uncommon, and two types have been reported: terpenoid compounds and fluranones [42]. Terpenes represent a group of organic compounds synthesized from active isoprene C5 units dimethylallyl diphosphate (DMPP) and isopentenyl diphosphate (IPP). They are classified as hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, and triterpenes having 1, 2, 3, 4, and 6 isoprene units respectively; however, monoterpenes and sesquiterpenes make up most terpenoid volatile oils [42]. Terpenes are generated from mevalonic acid (MVA) or an alternative methylerythritol phosphate (MEP) pathway. They exist majorly as hydrocarbons and their oxidized forms as aldehydes, ketones, phenols, alcohols, oxides, peroxides, and ester. Both MVA and MEP pathways have acetyl-CoA and pyruvate, respectively, as the starting material to produce isoprene units, which combines in head-to-tail fashion to produce terpenes. Multiple reactions including hydrolysis, ionization, isomerization, cyclisation, and oxidoreduction occur, and large group of enzymes, generally referred to as terpene synthases, act on the pathways, to yield the different classes of terpenes. According to Reineccius [43], oxygenated monoterpenes account for the volatile oils in many citrus fruit species. The most prominent examples of furanone group are 4-hydroxy2,5-dimethyl-3(2H)-furanone, HDMF known as furaneol, and 2,5-dimethyl-4-methoxy-3(2H)-furanone, DMMF known as methoxyfuraneol. They are considered key odorants in the plants where they occur such as pineapple and strawberry fruits. The furanones are generated via multiple pathways. d-fructose 6-diphosphate has been identified as the biosynthetic precursor, which undergoes a sequence of reactions and enzymatic action to yield the intermediate 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF), and then the product, furaneol and methoxyfuraneol. Furaneol is also generated in heated foods during Millard reaction as is observed to give an attractive look [27, 42].

1.3.3.2: Volatile oil derivation by fatty acid pathway

Fatty acids that are derived from acetyl CoA, produced from pyruvate, the end product of glycolysis, act as nonvolatile precursors in the biosynthesis of some volatile oils. Fatty acids are stored in plant ER as TAG and released by lipases to produce volatiles such as alcohols, lactones, esters, aldehydes, ketones, and acids through three major pathways, namely, alpha-oxidation, beta-oxidation, and lipoxygenase (LOX) pathway. The LOX pathway occurs majorly in disrupted plant cells associated with wounding or damage as oxygen is introduced. Alpha- and beta-oxidation occurs in intact plants. They cause the degradation of fatty acids to produce short- to medium-chain fatty acids, which undergo series of enzymatic reactions to yield diverse odoriferous compounds [27, 42].

1.3.3.3: Volatile oil derivation by amino acid pathway

Volatile oils can also be formed directly from the amino acid pathway [27], and this depends on the availability of free amino acids, which in turn depends on degree of ripeness and environmental conditions during growth [43, 44]. Several amino acids such as methionine, cysteine, alanine, phenylalanine, leucine, isoleucine, and aspartic acid are involved as precursors. Importantly, branched-chain amino acids produce branched-chain volatile oils such as methyl 2-methylbutanoate in prickly pear, isoamyl acetate in banana, and 2-methyl butyl acetate in apples [27]. The biosynthesis of these volatiles proceeds from amino acid pathway through diverse reactions and enzyme action. Initially, amino acid undergoes deamination by aminotransferase to yield alpha-ketoacid, which loses one carbon atom by decarboxylase enzyme to form an aldehyde. Series of subsequent reactions follows to produce corresponding volatile oils [42].

1.3.3.4: Glucosinolate-derived flavors

Glucosinolates are naturally occurring compounds found majorly among plants of the Brassicaceae family like cabbage, broccoli, mustard, and horseradish, majorly for the purpose of repelling insects, and it is also responsible for the sulfury or sharp flavor characteristic to these plants [27, 42]. In humans the hydrolyzed products of glucosinolate have been reported to possess anticancer properties [45, 46]. In intact plants, glucosinolate and thioglucosinolate (also known as myrosinase) exist in the vacuoles and cytoplasm, respectively, and are thus separated physically. Once cells disrupt (damage), myrosinase acts to hydrolyze glucosinolate to yield the corresponding nitriles, thiocyanates, and isothiocyanates and by-products of glucose and sulfates [42].

1.3.4: Biogenesis of resins

Plant resins are secondary metabolites of complex mixtures, which include volatile and nonvolatile terpenoid and/or phenolic compounds. They often exist in combination with essential oils (oleoresins), gums (gum resins), oil and gum (oleo-gum resins) sugars (as glycosides), and benzoic/cinnamic acid (balsams). They not only are formed and stored in specified structures internally or externally on the tree surface but also may be found in other plant cells and can be produced spontaneously at the site of injury to the plant. They occur mainly as exudates from the tree trunk and are generally insoluble in water but soluble in alcohol, oil, chloroform, and ether. Terpenoids and phenolic resins have been identified with the former consisting majorly of the internally formed resins while the latter largely constitute resins formed on the surface. Terpenes as mentioned in the metabolism of volatile oils are made up of isoprene C5 unit building blocks and are classified according to the number of isoprene units they contain. Unlike volatile oils, most resins consist of di- and triterpenes, rarely both; however, they occur in coexistence with other classes of terpenes such as mono- and sesquiterpenes (volatile oils) and tetra- and polyterpenes (gums, rubber, and gutta-percha). Phenolic resin metabolism involves multiple pathways. The shikimic acid pathway yields the aromatic amino acid, phenylalanine, which is converted to cinnamic acid (phenylpropane) by phenylalanine ammonia-lyase (PAL). Cinnamic acid is a C9 compound occurring as a benzene ring with C3 side chain. It serves as the substrate from which other phenolic compounds are derived via cleavage of the side chain or substitution. Malonyl acid pathway from acetyl CoA also produces aromatic benzene ring that combines with the benzene ring and C3 side chain from the shikimic acid pathway, to yield flavonoid, components of phenolic resins. Many plant resins have been observed to elicit pharmacological effects; hence, they find application in medicine and pharmacy.

1.4: Medicinal potentials of plant secondary metabolites

1.4.1: Anticancer effects

Many plant metabolites including isothiocyanate [45–47], luteolin [48], resveratrol [49], genistein [50], vitamin A derivatives [51], curcumin [52], green tea extract [53], soybean extract [54], and lycopene [55] have been investigated and found to possess antitumor properties. Nutraceuticals have gained increasing interest due to minimal side effects and general acceptance and hence have been applied as preferred alternative in the prevention of cancer. Most plant extracts have been studied for prevention rather than treatment of cancer resulting in minimal efficacy and adoption in practice. An investigation carried out showed that combination of anticancer plant extracts, produced a more effective result when compared with the efficacy of individual compounds.

1.4.2: Antiinflammatory activity

Inflammation is a natural physiological response to cell injury, disease, or other forms of disturbance in animals. While it may be protective in certain cases, it can trigger a cascade of events resulting in a continuum of disorders, acute and chronic pain/diseases such as rheumatoid arthritis, allergies, hyperventricular dystrophy, atherosclerosis, osteoarthritis, inflammatory bowel disease, cancer, and coronary heart failure. Inflammation is associated with redness, heat, swelling, pain, and loss of function. During the process the vessels are dilated, which allows increased blood flow and mobilization of plasma proteins, leucocytes, and fluid to the affected site. Inflammation is the result of a number of biological pathways including the synthesis of plasma proteins, which act as chemical mediators for inflammation such as prostaglandins, cytokines, leukotrienes, vasoactive amines, and nitric oxide [56]. At the onset, cyclooxygenase and lipoxygenase together with their by-products, prostaglandin (PGE2 and PGH2), leukotrienes (LTC4 and LTB4), and cytokines (IL-1 and TNF-a) are produced by enzymatic action in response to a trigger (cell injury, disease, allergen, and so on). These by-products are referred to as proinflammatory mediators and make up the principal mediators of inflammation. Currently, antiinflammatory chemical agents such as aspirin and nonsteroidal antiinflammatory drugs (NSAIDs) exist but are however known to cause side effects such as suppression of the immune system and gastrointestinal bleeding [57]. For this reason, alternative therapy is being sought. Several plants have been investigated and discovered to have capacity to interrupt the inflammatory process at various points and hence possess substantial antiinflammatory properties (Table 1.2), some of which have been applied in therapy.

Table 1.2

1.4.3: Antimicrobial effect of compound present in plant metabolites

Chang et al. [107] investigated the antimicrobial activity of essential oils obtained from Cinnamomum osmophloeum in an in vitro trial. The result obtained shows that the tested essential oil exhibited an antimicrobial effect against methicillin resistant Staphylococcus aureus, Enterococcus faecalis, Vibrio parahaemolyticus, and Escherichia coli. The high antibacterial activity might be linked to the presence of cinnamaldehyde as the active agent in the essential oil of Cinnamomum osmophloeum. Moreover, some other essential oil with high antimicrobial activity includes oregano oil, which inhibit Escherichia coli O157:H7 [108], peppermint and spearmint oil against pathogenic bacterial [109], essential oil from herbs most especially from Lamiaceae and Compositae family [110], essential oil against Helicobacter pylori [109]; and essential oil against oral pathogens [111].

The composition and the rich nature of plant extract have influenced and enhanced their antimicrobial activity against pathogenic microorganisms. Some of the factor responsible for their antimicrobial activity might be linked to the presence of various compounds with low molecular weight [112]. The nature of chemical structure, the position, and number of substitutions, for example, in the benzene ring of phenolic acids and saturated side chain length are factors to consider. It has been observed that some of these compounds have the capability to destroy the proton motive force, cytoplasmic membrane, and the active transport available in these pathogenic microorganisms [113, 114].

Also, some other compounds present in plant metabolites are organic acid, which form part of plant extract. It has been observed that in the nondissociated nature, they have the potential to migrate through the bacterial cell wall and move into the cell where they dissociate and bring down the cytoplasmic pH and destabilize the normal physiology of pH-sensitive bacteria. Moreover, because the organic acids are present in anionic form, they cannot penetrate the cell membrane, and therefore they accumulate within the cell surface, which later increase the level of osmotic pressure and interrupt some of the metabolic process.

Volatile oil has been observed to possess antimicrobial action [115, 116]. It has been reported that for plant extract to be effective against their target microorganism, they have to be in contact with the cell, but essential oil are more volatile. It should be noted that hydrophilic compounds are less volatile, but there is a need for the equilibrium achieved between volatile compounds available in the headspace [117]. Moreover the presence of lipophilic molecules in the essential oil present in the lipid bilayer can affect the lipid-protein interaction [118] and enhance the increase in the rate of membrane permeability, which later leads to functional and structural impairment of the targeted microbial cells. This might also lead to seepage of intracellular components and damage of microbial enzyme systems, which could result to autolysis of the affected microbial cell [112, 119]. On the whole, lipophilic compounds have the potential to interreact with the hydrophobic parts of cell proteins [120]. This might enhanced the hydrophobicity nature of the essential or volatile oil to overcome the compound accumulation, which might led to destruction of cell membrane [117, 121].

1.5: Processing, extraction, and isolation of plant metabolites

The processes involved in the extraction, isolation, and identification of plant secondary metabolites were summarized.

1.5.1: Selection of specific species of plant

For the investigation of plants for medicinal effects, a common methodology is undertaken. Whole plant or its parts are randomly collected and taken for investigation in which a common chemical test is carried out to identify the phytochemicals present. The selection of plant is done on the basis of being traditionally used, and the approach is termed ethnobotanical bioprospecting approach. The plants used traditionally have generally got biologically active compounds with medicinal properties. The selection of plants can also be done on the basis of chemotaxonomical approach where taxonomically related plant is established with structurally similar biomolecules. Plants are also selected on the basis of local field observation with immunity against bacteria, fungi, or virus as they synthesize defensive natural products with bactericidal, fungicidal, or virucidal effects. Plant selection also utilizes a combination of these three, that is, ethnopharmacological, taxonomic, and random approaches with a database of relevant information of a particular plant. The database prioritizes the plant species for extraction and screening of medicinal activity. This is the accepted approach by pharmacological companies.

1.5.2: Collection of plant material and its identification

Either whole or parts of a plant are collected. The portions where metabolites are stored in the plant are selected. It is better to select aboveground parts like leaf, stem, flower, fruit, seed, and bark and underground parts like tuber, bulb, and root separately. Various factors like age of the plant, environmental condition of light, rainfall, and humidity should also be taken into consideration for perfect selection of active plant material. Accurate identification of the plant for isolation of active principles is a must. The plant must be authenticated by a botanist or a plant taxonomist. The plant name, portion of the plant collected, place, and date of collection should be noted and be deposited in the herbarium for future reference.

1.5.3: Drying and grinding of plant material

After collecting the plant, it is usually dried on trays at room temperature with proper ventilation. Ideally, dry conditions prevent the growth of microbial organisms, which lead to fermentation or deterioration of metabolites of the plant. The plant materials should be ground into fine powder to give a uniform sample with increased surface area. The plant material can be cut into more small pieces for easy grinding. Mechanical grinding can be done with the help of grinders, mixture, hammer, or cutting mills. However, sometimes the process results in clogging of sieve due to fat and volatile oils present in plant. Plants can be cut into small pieces to facilitate uniform and faster drying. Plants should not be dried in direct sunlight to minimize formation of artifacts by unwanted reactions. Simple air drying, microwave drying, oven drying, and freeze-drying of plants samples are the preferred methods of drying.

Air drying usually takes long say 3–7 days to months depending on the types of sample. As high temperature is not involved, heat-sensitive compound is not affected. The only drawback is the longer time and contamination. Microwave drying utilizes electromagnetic radiation with electrical and magnetic fields. The method will decrease the drying time but will also lead to deterioration of the phytochemicals. Oven drying is the easiest method using rapid heat energy to decrease the moisture content along with preserving phytochemicals. This method nowadays is common because it is faster and minimizes the chemical reactions in the plant material. After drying the samples, they are to be kept in cool and dry place. Longtime storage of samples is not advisable as it leads to degradation of plant metabolites. In freeze-drying, the sample is frozen overnight at − 80°C to − 20°C before lyophilization to avoid the frozen liquid in the sample from melting. It is a complex and expensive method [122].

1.5.4: Extraction protocol

Extraction is the process by which we separate the bioactive principle from the plant using solvents like diethyl acetate, ethanol, chloroform, acetone, and so on through standardized procedures to get the active ingredient, which can provide medicinal effect. The solvent is chosen based on the solubility and polarity of the bioactive principle to be extracted. The most common solvents of use are water, organic and inorganic solvents. The extraction procedure is chosen based on the nature of the source of plant material and the bioactive compound to be isolated. The soluble extract is separated from insoluble residue. The most common extraction techniques used by researchers in the field of phytopharmacology are as follows:

Maceration: By this technique, the plant material is soaked in closed container containing a solvent for 4–5 days at room temperature with intermittent agitations. This process softens and breaks the plant cell wall leading to release of the phytochemicals. After the intended days, the mixture is filtered. The type of compound extracted is determined by the solvent used.

Infusion and decoction: Infusion and decoction have the same principle as maceration. The sample is boiled for short time to extract volatile ingredients in infusion and to get mineral salts, bitter samples from hard materials like root, bark, wood, and seed in decoction.

Percolation: Percolation is slowly passing a liquid through filter. The equipment percolator is used for percolation where boiling water and acid are added with shaking till extraction is done.

Soxhlet extraction/hot continuous extraction: The sample is finely grounded and placed in thimble or a porous bag made up of filter paper in the Soxhlet apparatus. The solvent is heated by heating mantle in the solvent chamber, which vaporizes in thimble and then condenses in the condenser and drips back to the solvent chamber. It has the drawbacks of exposure to flammable solvents, toxicity, costly, and nonenvironmental friendly.

Microwave-assisted extraction: Here, microwave energy is utilized for the separation of bioactive principles from the sample to the solvent. This method favors polar solvents with high dielectric constant more than nonpolar solvents. This method needs less time and volume of solvent. The drawback is thermal degradation.

Ultrasound-assisted extraction or sonication extraction: Ultrasound-assisted extraction (UAE) uses ultrasound of 20–2000 kHz. The ultrasound enhances the contact surface of solvents and samples. It increases the permeability and disrupts plant cell wall, thus facilitating extraction. The procedure has advantages of being simple and of low costly.

Accelerated solvent extraction: Accelerated solvent extraction (ASE) is an efficient method that uses minimal amount of solvent. The plant sample is packed with an inert material in the extraction cell. This helps to prevent sample aggregation leading to further blocking of system. The method has advantages of temperature and pressure control and requires less time.

Supercritical fluid extraction: Supercritical fluid or dense gas is a substance that has characteristics of both gas and liquid at its critical point, for example, CO2 that becomes supercritical fluid at above 31.1°C and 7380 kPa. It is a better solvent for nonpolar analytes. The addition of ethanol or methanol makes it a solvent for polar compounds too. The advantages are low cost and have low toxicity of CO2. Also the strength is easily alterable by change of temperature and pressure.

The methods employing solvent for extraction are highly influenced by the type of solvent used. However, the amount of solvent used for extraction causes no difference in effect. All the processes done for extraction or preextractions are very much important in studying the medicinal effect of those plants. The steps like sample preparation by grinding and drying also affect the level of phytochemicals in final extractions. All the methods have their own benefits and drawbacks [123].

1.5.5: Identification and characterization of bioactive principles

Plant samples usually contain various bioactive principles whose isolation, identification, and characterization need recent analytical protocol and instruments. Mostly, thin-layer chromatography (TLC), column chromatography, flash chromatography, Sephadex chromatography, and high-performance liquid chromatography (HPLC) are used for isolation. Also, nonchromatographic techniques like immunoassay using monoclonal antibodies (MAbs) and phytochemical screening assay are also remarkable. The bioactive fractions separated can be further identified or characterized by Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS).

Hyphenated techniques like LC/UV, LC/MS, and LC/NMR can be used to differentiate between new and already established bioactive principles from medicinal plants. Also, spectroscopic techniques like UV/VIS and IR spectrophotometry, carbon and proton NMR, MS, and X-ray diffraction are helpful for structural elucidation of bioactive principles. Furthermore, chemical reactions involving hydrolysis and hydration reaction are done to confirm structural or functional relationships.

1.6: Quality and safety

Pure compounds isolated from medicinal plants are usually vetted in the laboratories through standard protocols. To establish the effects of medicinal plants, the ethnobotanical claims and pharmacological, observational, and clinical studies are taken into account. The dose of bioactive compound varies. The changes coming to these compounds over time may also be a drawback of its efficacy. So based on this, the United States and Europe saw the need to regulate the market and license these products. A minimum level of quality phytoingredients is needed in the product for appropriate response. Standards for active ingredients of medicinal products may be found in monographs and/or pharmacopeias. Standardized products provide a better sense of security for using herbal drugs. At the international level, WHO developed a strategy to review traditional medicines by developing monographs for herbal ingredients. Chemical compounds in herbs have properties that add to the pharmacological activity of the extract [124–130]. However, adulteration of herbal products can be intentional and sometimes accidental. In some cases, herbal remedies have been found to contain heavy metal residues, which can lead to harmful effects when consumed. Also, microbial contamination is of great concern. So it is needful that all herbal products go through some quality control processes before being allowed to be released into the market.

1.7: Conclusion

This chapter provides detailed information on the different types of primary and secondary metabolites and their application for the treatment of diseases. The advances in plant metabolomics have facilitated the detection of some other new metabolites that might be useful in the production of useful drugs for the management of diseases. However, there is need to intensify efforts on the application of plant metabolomics in synergy with other functional genomics, proteomics, and bioinformatics for better results.

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