7.1 Introduction
Plants have the capability to synthesize diverse secondary metabolites, which are made up of organic molecules with unique carbon arrangements. Secondary metabolites are not required for cells to survive, but it plays a vital character in an interface of the cells with its ambiances, confirming the constant existence of the organism to its biomes (Ncube and Staden 2015). Normally the secondary metabolites are of low molecular weight, and its production is specific to cell, tissue, and organ. These compounds frequently change among germplasm from a similar population of plants in reverence to their quantity and forms (Matsuura et al. 2018). Secondary metabolites look after plants against biotic and abiotic stresses, viz., microorganisms, nematodes, insects, and animals and temperature, moisture, shading, injury, and heavy metals, respectively. Due to the excessive commercial importance, they are extensively used as chemical such as for medications, flavors, essence, insect repellent, and dyes. Most of the significant therapeutic biomolecules are alkaloids which are biosynthesized mainly from amino acids. However plant secondary metabolites can be chemically categorized into different types, i.e., phenolics (Wuyts et al. 2006; Iqbal et al. 2017), terpenes (Singh and Sharma 2015), compounds containing nitrogen (Ejaz et al. 2017), and compounds containing sulfur (Kang and Kim 2007). Due to the occurrence of varied diversity and multifaceted performance of secondary metabolites, it is assumed to be of enormous significance. Secondary metabolites hold various therapeutic properties, and thus it is of immense importance to mankind for health benefits (Forbey et al. 2009).
Metabolomic biosynthesis of primary and secondary metabolites
As an alternative methodology, biotechnologist could isolate and express the genes responsible for the formation of particular secondary metabolite of therapeutic significance in a particular biosynthetic pathway. If this technique will be successful, then recombinant DNA technology on bacteria or yeasts could flourish, which may produce valuable plant secondary metabolites for therapeutic use (Pandey et al. 2014). The biosynthesis of antibiotics by means of genes coding enzymes approach, at present, is of inordinate attainment. In recent years, microorganisms have been exploited, and techniques have been developed to express the genes responsible for alkaloid biosynthesis for its overproduction (Pickens et al. 2011). Eventually, it is conceivable to yield desired alkaloids from cells of yeast or bacteria recombinant DNA technology. Although if particular secondary metabolite of plant or microorganism causes prevention to the growth of pests or pathogens, thus genetic transformation of vulnerable plants may perhaps be added prospective for its utilization. For almost two decades, researchers in the field of biotechnology have attempted to yield beneficial secondary metabolite in cell or tissue/organ cultures. However cell cultures which are undifferentiated have generally botched to form such beneficial compounds judiciously, as distinguished tissue/organ cultures like root cultures linger on as lively as whole plant (Ochoa-Villarreal et al. 2016).
The basic benefit of present techniques is to make available of uninterrupted, consistent source of plant-originated phytochemicals and possibly will be amplified on large-scale production culture of cells or tissue/organ for its extraction. The technique holds prominent advantage for meticulous fabrication of numerous, useful secondary metabolites upon application. The present output and production of secondary metabolite cannot achieve the commercial goal of phytochemicals bioprocessed for the fabrication of most the secondary metabolites of therapeutic importance. In the direction to expand the boundaries, recent progressions and new prospects in plant organ or cell/tissue culture-based procedures are being critically studied and experimented. In such experimentations, novel approaches to develop the preferred secondary metabolites should be taken into consideration for better future. One of the foremost glitches that come across in this arena of research is the absence of elementary information of biosynthesis pathways and mode of action accountable for the fabrication of secondary metabolites. Moreover, it also discusses the various biotechnological techniques for the production of specific and valued secondary metabolites used in the healthcare practices.
7.2 Recent Advancements in Secondary Metabolite Production
Plant cell or tissue/organ cultures embrace prodigious potential for meticulous propagation of innumerable, beneficial secondary metabolites upon requirement and demand. Outcomes associated to cell cultures achieving the fabrication of specific therapeutic phytochemicals at a level parallel or better than that of whole plant have been enhanced in the last few decades (Vijaya et al. 2010; Ncube and Staden 2015). In the course to accomplish amplified yields, appropriate to utilize commercially, attempts have been made to find the biosynthetic actions of cell cultures, accomplished by proper rectification of conditions for growing of culture, choosing high-producing strains with precursor nourishment, biotransformation, and immobilization procedures (DiCosmo and Misawa 1995; Ncube and Staden 2015). Organ culture technique (transgenic root hair cultures) has transfigured the character of secondary metabolite in plant tissue culture. The method is inimitable in their biosynthetic and genetic permanency, quick in growth, and efficiently sustainable. By improvising the approach, an extensive variety of biochemical compounds has been manufactured (Giri and Narasu 2000). Recent progresses in cell or tissue/organ culture technique, associated with enrichment by genetic engineering, have developed biomolecules of pharmaceutical and nutraceutical significance and other additional valued constituents (Hansen and Wright 1999). Furthermore, improvements in the enzyme technology, molecular biology, microbiology, biochemistry, and fermentation technology enhanced the cell culture techniques that have developed a feasible source of secondary metabolites of therapeutic and agricultural importance (Abdin 2007; Barbulova et al. 2014).
Bio-active compounds of nutraceutical and pharmaceutical significance
Class | Known metabolites numbers | Examples | References |
|---|---|---|---|
21,000 | Quinine, cocaine, psilocin, reserpine, caffeine, nicotine, morphine, atropine, berberine, ephedrine, vincristine, galantamine, vincamine, quinidine | ||
Non-protein amino acids (NPAAs) | 700 | Azatyrosine, canavanine | Wink (2010) |
100 | Methylamine, dimethylamine, trimethylamine, aziridine, piperidine | ||
60 | Amygdalin, dhurrin, linamarin, lotaustralin, prunasin | Wink (2010) | |
100 | Wink (2010) | ||
Alkamides | 150 | N-Isobutyl-2E-decenamide and N-isobutyl-decanamide | Molina-Torres et al. (2004) |
Lectins, peptides and polypeptides | 2000 | Reeke et al. (1975) | |
>15,000 | Azadirachtin, artemisinin, tetrahydrocannabinol | Tiwari and Rana (2015) | |
Steroids and saponins | NA | Babiychuk et al. (2008) | |
Phenylpropanoids, lignins, coumarins and lignans | 2000 | Kennedy and Wightman (2011) | |
Polyacetylenes, fatty acids and waxes | 1500 | Oleic acids | Minto and Blacklock (2008) |
750 | Crawford and Townsend (2010) | ||
200 | Formic acid, lactic acid, citric acid | Nakui et al. (2009) |
7.3 Current Biotechnological Exercises Employed in the Production of Secondary Metabolites from Higher Plants
Production of phytochemical can be more consistent, natural, and more foreseeable.
Extraction of the phytochemical can be quick and effective, when compared with isolation from complete plant.
Phytochemicals generated in vitro can directly be equivalent to the phytochemicals produced by complete plant.
Inquisitive phytochemicals can be evaded in cell cultures, which cannot be done in the plants grown infield.
Cell/tissue cultures can harvest definite and customary phytochemicals in bulk quantities.
Cell/tissue cultures are a prospective exemplary to investigate and testify elicitation.
Radiolabeling in cell/tissue cultures could be done, so that the stored secondary metabolites, when delivered as feedstuff to experimental models (animals), can be outlined during metabolic process.
Various research on enhancement of secondary metabolites production, has increased now a days for producing an extensive variety of valued secondary phytochemicals in callus or suspension cultures, however further research is required to establish technique for organ cultures of well-known plant as well (Davioud et al. 1989). This condition repeatedly arises when the phytochemical of importance is only formed in specific plant tissues/organ of the parental plant. A crucial case in point is in Panax ginseng. As in this plant, in vitro saponin and additional valuable metabolites are particularly formed in its organ (root) culture. Likewise, medicinal plant Hypericum perforatum, which holds the hyperforins and hypericins (in foliar glands), has not established the capability to accrue phytochemicals in undistinguished cells (Smetanska 2008). Therefore approaches need to be developed for fabrication of secondary metabolites on large scale. The rigorous accomplishments have been focused on generating natural remedies or chemo-protecting compounds (secondary metabolites) obtained from plant cell or tissue/organ culture by the following subsequent approaches.
7.3.1 Therapeutically Significant Secondary Metabolite Production by Plant Tissue Cultures Technique
Production of valuable phytochemicals under controlled environment without the impact of conditions of soil or fluctuations in climatic.
Microorganisms and pests free cells cultures.
Reproduction of cells from any plant could simply enrich particular metabolites.
Reduction of labor expenses and increased production, as automated regulation of cell growth and balanced parameter of metabolite progressions, could be done.
Callus cultures could be the source of organic constituents which can be easily extractable.
Some of the secondary metabolite productions in culture medium are cathinone alkaloids by suspension culture (Anderson et al. 1987), allicin by callus culture (Malpathak and David 1986), caffeine by callus culture (Waller et al. 1983), anthraquinones by suspension culture (Dornenburg and Knorr 1999), ginkgolide A by suspension culture (Carrier et al. 1991), L-DOPA by suspension culture (Wichers et al. 1993), etc.
7.3.2 Secondary Metabolite Production by Organ Cultures Technique
Organ culture is a technique where rapid propagation can be done by cutting small sections of the plant organ. Small slicing of Fritillaria unibracteata can swiftly propagate the bulb by organ culture process. The sliced bulbs were grown in MS media, supplemented with 4.44 mole indole-3-butyric acid (IBA) and 5.71 mole indole-3-acetic acid (IAA). The cultivated bulbs were collected after 50 days of culture period. The growth rate was enhanced by 30–50 times, which was higher than that of normal conditions. The magnitude of secondary metabolites was elicited like alkaloids and other valuable phytochemicals in the cultured bulbs than in the normal growing bulb (Gao et al. 2004). Micropropagation of shoot development on the MS medium, supplemented with 1-naphthaleneacetic (0.1 mg/l) and thidiazuron (0.1 mg/l) on Frangula alnus, was attained, and the production of secondary metabolite, anthraquinone, was maximum in the shoots than in the plant grown under normal condition (Namdeo 2007).
7.3.3 Secondary Metabolite Improvement by Addition of Precursors
The treatment of plant cells or tissue/organ with factors like biotic and/or abiotic has an expedient approach to enhance secondary metabolite fabrication grown under media culture (Karuppusamy 2009). The utmost commonly used precursors in prior studies were yeast extract, fungus, polysaccharides, methyl jasmonate, and chitosan. One of the most recognized elicitor methyl jasmonate is an established indicator compound and is the most effectual elicitor for Taxol fabrication in T. chinensis Roxb (Wink et al. 2008). Gonsenoside, a secondary metabolite found in P. ginseng, can be illicitly produced in the supplemented media in Meyer cell/organ culture (Yagi et al. 1983; Xu et al. 2008; Yamanaka et al. 1996).
Biosynthesis of hyperforin and adhyperforin by shoot culture process of H. perforatum when treated with amino acids was reported (Kim et al. 2004). Upon supplementation of shoot cultures by amino acids, valine conformed to side chain of hyperforin and isoleucine conformed to side chain of adhyperforin, separately. Nourishing the shoot cultures with amino acid like isoleucine (2 mM) prompted three- to sevenfolds of elicitation in the production of hyperforin (Kim et al. 2004). It was reported that, when amino acids (leucine) were treated in callus and cell suspension cultures of Centella asiatica, the triterpene production was increased. The method was found to be quite impressive for the elicitation of asiaticoside. In the callus culture, multifarious increase of asiaticoside was observed by this approach (Karppinen et al. 2007).
7.3.4 In Vitro Elicitation Technique
In vitro treatment of cells or tissue/organ by microbial, physical, or chemical elements which causes morphological and physiological changes is called “elicitation,” and the compounds are so-called elicitors. An elicitation is a method of tempting or enhancing the fabrication of secondary metabolites by cells or tissue/organ culture to make sure of their existence, perseverance, and effectiveness (Karuppusamy 2009; Kiong et al. 2005). In a study, abiotic elicitor was applied in the hairy roots of P. ginseng to improve growth and to elicit ginseng saponin biosynthesis. However in the study, elicitor treatments were performed to inhibit the development of the root hairs, but at the same time it was enhancing the content of ginseng saponin biosynthesis (Jeong and Park 2006). Elicitor treatment of benzo (1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester and autoclaved lysate of cell suspension of E. sakazaki was done for the fabrication of secondary metabolites in callus culture, cell suspension culture, and hairy roots of Ammi majus. The study showed noteworthy outcomes (Staniszewska et al. 2003). The investigation based on GC and GC-MS estimation of methanolic and chloroform excerpts exhibited greater accretion of umbelliferone in the treated (elicited) tissues (Staniszewska et al. 2003). In a study on Rubia akane cell culture, chitosan (polysaccharide) was used as a biotic elicitor. The results were prompting the multifarious upsurge of anthraquinone fabrication (Jin et al. 1999).
7.3.5 Enhancement of Secondary Metabolites by Cultures of Hairy Root
Secondary metabolite synthesis in plant roots, based on inoculation by Agrobacterium rhizogenes with hairy root system, has become common in the past few years (Palazon et al. 1997; Karuppusamy 2009). However, in the absence of lateral root branching, physical factor like geotropism and factor like genetic stability could affect the growth of the root hair, ultimately affecting the production of secondary metabolite by hairy root culture technique. Hairy roots ascending for the formation of secondary metabolites by the treatment of plant material by A. rhizogenes are analogous to those normally produced by parent roots of whole plant, with parallel or greater yields (Sevón and Oksman-Caldentey 2002). The unperturbed genetic stability and sudden advancement in normal media that are deficient in hormones mark them particularly appropriate for biochemical analysis, which was not easy to undertake in root hair cultures of plant. The hairy roots of the plant are firstly sterilized and then interact with the parts of the plant by infecting it with A. rhizogenes. For the period of the interaction course, A. rhizogenes transmits the portion of DNA (T-DNA) situated in the root-persuading plasmid to plant cells, and the confined genes in the region are expressed in the identical manner as the normal endogenous genes of the plant cells. Some strain of A. rhizogenes (like A4) possess T-DNA divided into two segments (i.e., TL-DNA and TR-DNA). Thus, both are assimilated independently into the plant’s genome (Jouanin 1984).
7.3.6 Production of Secondary Metabolite by Genetic Transformation in Hairy Root Culture
Genetically transformed roots deliver a favorable substitute for biotechnological utilization of the plant cells (Pandey et al. 2014). A. rhizogenes intervened with the transformation of plant’s genome, which could be utilized in the way similar to well-established technique, engaging A. tumefaciens. The transformation of A. rhizogenes enables the growth of plantlets that have been renewed and also to yield transgenic cultures of hairy root of the plant (Karuppusamy 2009). The exclusion of the limiting sequences, not any of the supplementary T-DNA sequences are obligatory for the transmission. The leftover T-DNA could be switched by the external DNA (sequences are firmly hereditary in a Mendelian fashion) and inserted into the cells by which the regeneration of the whole plants can be achieved (Zambryski et al. 1989). Transformations by A. rhizogenes possess advantage of being capable to relocate any external gene of prominence, situated in transformed clone of binary vector of hairy root. In a study, the gene of interest with respect to enhancement of secondary metabolism was introduced into hairy roots. In the process 6-hydroxylase gene of Hyoscyamus muticus was incorporated in hyoscyamine-rich Atropa belladonna by the help of A. rhizogenes. An amplified quantity of enzymatic activity with five times more concentration of scopolamine was observed in engineered roots (Hashimoto et al. 1993).
7.3.7 In Vitro Secondary Metabolite Production by Endophytes
There are three origins of supports on the formation of secondary metabolites produced by plants. There is disagreement that plants and endophytic microorganism are coevolved with similar pathways to form these naturally occurring products. One more assumption states that primeval horizontal gene transmission is attained among plants and endophytic microorganism. The last suggestion is that moreover plants or endophytic fungus yield particular secondary metabolites, thus relocating them to the other symbiotic organism (Karuppusamy 2009; Jennewein et al. 2001). Studies based on radiolabeling of biosynthetic pathways by means of precursor-like amino acids show that fungal endophyte and plants have analogous but different metabolic paths for the fabrication of secondary metabolites (Zhang et al. 2009). It is still under investigation that whether the phytochemicals produced by the plants are naturally produced or it is the result of a mutualistic association of beneficial organisms with the plant. However studies reveal that the blend of influencing factors of plants and fungal endophyte upsurges the secondary metabolite accumulation in both the organisms (Li et al. 2009; Engels et al. 2008). However, the symbiotic relationship among plants and fungal endophytes and the effects on each other in the course of production of substantial bio-active compounds (therapeutically significant) could be processed. This could deliver the background for upcoming natural product fabricated by the process of genetic engineering and metabolic engineering (Komaraiah et al. 2003).
7.3.8 Secondary Metabolites Scaling-Up by the Use of Bioreactors
In this technique bioreactors are used for large-scale production of secondary metabolites. This can be achieved thru scaling-up, by the use of bioreactors, for large-scale modification of plant cells. The process would lead to the formation of exclusive bio-active phytochemicals in a vigorous process. During the process, the plant cells in liquescent suspension provide a distinctive combination of physicochemical environs that is essential for bioreactor’s large-scale progression (Ruffoni et al. 2010; Gupta et al. 2014). The fabrication of secondary metabolites using bioreactor from cell culture of Sandalwood and Periwinkle was done by Valluri (2009). In the study, the activity of phenylalanine ammonia lyase was inhibited by the use of trans-cinnamic acid; as a result substantial upsurge in the formation of alkaloid from the cell culture of periwinkle was observed. When cells were exposed to mannitol-induced osmotic stress, it yields noticeable enhancement in the production of total alkaloid. Biotic and abiotic stresses induce additive stimulation in alkaloid accumulation. However, no secondary metabolites (essential oils) are identified, in the form of phenolics from sandalwood cell cultures manufactured in the bioreactor (Valluri 2009).
7.3.9 Secondary Metabolite Immobilization and Accumulation
Advancements in immobilization techniques and scaling-up methodologies provide significant upsurge in numerous plant cell/tissue culture applications, for the formation of bio-active compounds with prominent and additional importance. Compounds derived from plants with anticancer, chemotherapeutic, or antioxidative properties use Taxol and rosmarinic acid in place of therapeutic agent. Cell cultures of Plumbago rosea were immobilized in calcium alginate. It was then cultured in MS media containing 10 mM calcium chloride for the formation of plumbagin, an essential therapeutic compound. Investigations were performed to elucidate the influence of immobilization on improved deposition of secondary metabolite (plumbagin). Calcium alginate immobilization improved the formation of plumbagin by one- to threefold increase, as compared to control (Vanisree and Tsay 2004).
7.3.10 Secondary Metabolite Production by Metabolic Engineering
Better output, like by increasing the number of cells employed for producing secondary metabolites.
Overexpression of genes could increase the carbon flux by making use of biosynthetic pathway.
Categorize for rate regulation of enzyme or hindering feedback and competitive inhibition mechanism.
Reduction in catabolism.
7.4 Secondary Metabolites of Pharmacological Significance
Structure of secondary metabolites of therapeutic importance
7.5 Secondary Metabolites Significant in Healthcare
Systematic representation of secondary metabolite production and its biological application
An additional barrier in outlining an appropriate dosage of a particular secondary metabolite in food is microbiota of the human, which interacts in the metabolic action and absorption of compounds. An equivalent quantity of secondary metabolite could affect differently, which may cause diverse levels of reaction in different individuals. This deviation in effect is associated to persons’ genetic character, age, fitness level, and medications. On the other hand, an auxiliary difficulty is that for several secondary metabolites to be taken into account its bioavailability. The particular secondary metabolite can consecutively influence the tissues with its various effects; further, it could be responsible for the metabolic transformation of the tissues as well. Therefore, it requires information of the absorption, metabolic action, position, and, eventually, defecation of respective bio-active compound, based on the trials on human/animal models to investigate these factors.
As we know, fruits and vegetables are the source of innumerable sets of secondary metabolites taken by our body. These phytochemicals are significant to understand how every compound is absorbed, and, therefore, its bioavailability can be elucidated. Although, most of the studies emphasize on bioavailability of plant-derived secondary metabolites in human/animal tissues. However the actual effect might be subsidiary, as it is intermediated by means of modifications by gut’s microbiota which may support to elucidate the inconsistency among the comparatively low grade of apparent bioavailability and its definite nutritional advantages (Duynhoven et al. 2011; Manach et al. 2009). Moreover, the existence of particular enterotypes is more persistently linked with nutrition than ethnicity or topography. The association of microbiota enterotype might affect both the individual’s possibility of chronic ailment and also have an impact on the way bio-active compounds are absorbed. It is probably that particular enterotypes will be affected by continuing specific dietary habit, but also short nutritive mediation of merely for a day could modify microbiome association (Wu et al. 2011). Though the multifarious interrelationship with microbiota are usually mutualistic, it could turn out to be pathological, for instance, in the situation of inflammatory bowel disease (Fava and Danese 2011). Minor population of bifidobacteria and greater occurrence of gastrointestinal pathogenic bacteria like E. coli, Campylobacter, Helicobacter, and Salmonella are frequently related to chronic immune diseases. Food can also affect the peril aspects, such as metabolic syndrome, which are generally related with fast food and junk dietaries, and could cause metabolic syndromes like obesity and diabetes (Fava et al. 2013).
Extensive effect of flavonoids, in human-involved investigations, on cardiac risk disease associated with high and low levels of flavonoid intakes over a period of 18 weeks revealed an upsurge in potentially valuable bacterial groups for the high flavonoid intake, for example, as in Bifidobacterium (Chong et al. 2013). Polyphenolics are another category of secondary metabolites. Polyphenols are mostly well-examined biomolecule, and they are permitted through metabolic action and are abdominally absorbed (McGhie and Walton 2007). Relating to the diet, polyphenols are mainly delivered by fruits, vegetables, grains, and beverages like tea, coffee, wine, and beer (Grosso et al. 2014). Anthocyanin is a class of phytosecondary metabolite. Its constancy of structure is determined by the type of sugar component associated. The anthocyanins are derived compounds like gallic acid, protocatechuic acid, syringic acid, and aglycones, which are revealed to be biotransformed by microflora (Forester and Waterhouse 2010). The bacterial-reliant metabolic action of anthocyanins can then, in turn, modify abdominal bacterial inhabitant like Lactobacillus and Bifidobacterium, recommending an optimistic association among bacterial condition and phytochemical intake (Hidalgo et al. 2012). As earlier reviewed, studies associated to human subjects have established the absorption of secondary metabolites derived from plant, but elucidating the consequence of the method and the metabolic outcome still faces methodological challenges. The approaches like using bioinformatical tools (next-generation sequencing or meta-genomics) and animal/human model studies would improve our knowledge of the secondary metabolite interaction with tissue and its metabolism. It would elucidate the pathways of metabolism of secondary metabolite and the development of future nutritional food with enhanced level of secondary metabolites which could be obtained naturally.
7.6 Conclusions and Future Prospects
Secondary metabolites produced by the plants support them to contest and stay alive in extreme environmental conditions. Various biotechnological methodologies are employed for fabrication and enhancement of secondary metabolites by genetic engineering process and plant tissue culture techniques. Genomic knowledge by metabolic engineering for fabrication of secondary metabolites derived from plants is presently well innovated. Thus, metabolic engineering and biotechnological exercises can be applied as a substitute for the production of naturally active, economically valuable, and pharmaceutically important secondary metabolite. Developments in bio-techniques, mainly the technique of plant cell cultures, could deliver new worth for therapeutically and economically bio-active compounds. The main benefit of the in vitro cell cultures comprises the fabrication of secondary metabolites, cultivated in controlled environmental conditions, thus enabling us to extract important and particular phytochemicals in elicited quantities. The practice of genetic engineering is another emerging tool which can regulate the pathways for the fabrication of therapeutically significant secondary metabolites. Knowledge of biosynthesis of desired phytochemicals obtained from plants and its cultures are still in its preliminary stages; therefore accordingly approaches are required to advance the information based on molecular and cellular level. The advancement in new-fangled methods of molecular biology to yield cultures by transgenic and to understand the effect of the expression and regulation of biosynthetic pathways is possibly to be a noteworthy step in the direction of making cell culture technique more relevant to produce commercially important secondary metabolites. These new techniques will contribute to spread and improve the sustained efficacy of higher plants as non-conventional sources of compounds, specifically therapeutic compounds. It is anticipated in this field that prolonged and escalated efforts will lead to contribute efficacious biotechnological fabrication of secondary metabolites. It is further to be explained the effect of particular secondary metabolite associated with health benefits, which could be useful for neutraceutical and pharmaceutical industries. This in turn could permit the elucidation of innovative bio-active compounds and support to fix objectives for the improvement of nutritionally efficient foodstuffs for health benefits.