In Vitro Propagation and Secondary Metabolite Production from Medicinal Plants: Current Trends (Part 2)

Bioactive Components in Senna Alata L. Roxb

Archana Pamulaparthi¹, Vamshi Ramana Prathap², Ramaswamy Nanna¹, *

¹ Department of Biotechnology, Kakatiya University, Warangal, India

² Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University, Hyderabad, India

Abstract

Senna alata is an ethnomedicinal plant. The crude extracts of the plants are said to have a large number of medicinal properties due to their phytochemicals. In the present study, we made an attempt to isolate and screen the phytochemical constituents present in the species. In order to determine the bioactive constituents present in S. alata, and the effect of drying on the loss of bioactive constituents, studies on a set of pharmacognostical parameters were conducted on seeds, shade and sun-dried leaves of S. alata as per US pharmacopeia and WHO guidelines. The results of the present studies showed the presence of various important bioactive molecules that are responsible for the medicinal properties of the species. The phytochemical analysis of seed extracts revealed the presence of alkaloids, flavonoids, tannins, saponins, anthraquinones, resins and glycosides in all the extracts, while coumarins, phenols, terpenoids, phlobatannins and quinines are completely absent in all the seed extracts. Preliminary phytochemical investigations from shade and sun-dried leaf extracts showed alkaloids, flavonoids, anthraquinones, saponins, glycosides and tannins in high amounts in all the extracts, resins and phenols are present in moderate amounts. Terpenoids and phlobatannins are present only in fresh leaf extracts. Studies were also conducted on the physicochemical and organoleptic properties of leaves of S. alata that help in the identification and standardization of the leaf extracts for manufacturing of plant-based drugs of S. alata.

Keywords: Bioactive components, Leaf extracts, Preliminary phytochemical screening, Senna alata, Sun-dried, Shade dried.

* Corresponding author Ramaswamy Nanna: Department of Biotechnology, Kakatiya University, Warangal, India; E-mail: [email protected]

INTRODUCTION

Ever since the origin of the human race, plants have been used as medicine because of their potent therapeutic value. Plants have been a source of therapeutic agents for thousands of years, and the majority of drugs or their derivatives used in the present day have been isolated from plants. Since ancient times, conven-

-tional medical systems have been known to play a key role in the primary healthcare needs of the human race [1]. Almost all known civilizations around the world, including the Chinese, Indus Valley, African, and Egyptian, have their own ancient system of medicine that includes various types of naturally occurring compounds derived from medicinal plants.

Indian Vedic literature such as Rig Veda and Atharvana Veda (4500-1600 BC) also mentioned the use of several plants as a source of medicine. Ancient ayurvedic practitioners such as Charakaand Susrutain, and their respective books Charaka Samhita and Susruta Samhita referred to the use of more than 700 herbs as medicine and ancient medical systems such as Ayurveda, Unani, Homeopathic and Siddha have been surviving over 3000 years, by using plant-based drugs or their preparations and formulations for curing diseases.

World Health Organization [2] defined medicinal plants as follows: medicinal plant is any plant in which one or more of its organs possess compounds that can either be used for the therapeutic purposes or act as precursors for the synthesis of practicable drugs. The term herbal drug is used for the part/parts of a plant, viz. leaves, flowers, fruits, roots, bark, and seeds that are used for the preparation of therapeutic compounds. These definitions distinguish medicinal plants whose bioactive ingredients and therapeutic values have been demonstrated scientifically from plants that are considered medicinal but have not been established scientifically. WHO [3] further defines a medicinal formulation as any medicinal plant preparation obtained by subjecting the crude plant material to physical processes such as extraction, purification, fractionation, concentration, or biological processes that can be used for immediate consumption.

Many people from both developing and developed countries across the world do not have adequate access to basic needs, including food, water, clean environment, and medical and health services. The main concern of public health is still the intense need for basic health care, which is lacking even at the elementary level. According to WHO, more than half of the world’s population do not have access to basic healthcare needs as poor people are unable to access the present healthcare services due to their non-affordability. Therefore, the challenge for governments in both developed and developing countries in the near future lies in food and medical security that should necessarily double the production of food and medicine in the next 50 years to meet the needs of the growing population. Medicinal plants not only offer access to medicine to poor people at an affordable price but also help in generating income, employment, and foreign exchange in developing countries, thus contributing significantly to the national economy. It is estimated that plant-derived drugs account for about Rs. 2,00,000 crores in the world market.

During the past century, the formulation and large-scale production of synthetic drugs have brought a revolutionary change in health care across the world. Nevertheless, more than 70-90% of people in both developing as well as developed countries rely on traditional practitioners and herbal medicine as a source of primary medicine [4], which attracted the attention of researchers towards medicinal plants globally. In modern pharmacopoeia, not less than 25% of drugs are derived from plants and many other drugs are synthetic analogues of standard compounds that are already isolated from medicinal plants. Even today, about 121 such active compounds are in use in the pharmaceutical industry [5] and more than 100 herbal-based drugs are under clinical study [6].

Even though modern medicines are effective, they have several disadvantages, including high cost, reducing immunity, causing severe side effects, and physical dependence. On the other hand, plant-based medicines are natural, cost effective, and have minimum or no side effects, that is, leading to an increase in the number of people turning towards herbal medicine, thus being used in achieving the goal of Health for all in a cost-effective manner [7]. This interest in phytomedicine can lead to the exploration of about 500 different plant species in the last few decades, and many species are still being studied.

Escalating faith in herbal medicine is one of the several reasons for the increasing need for recognition of medicinal plants [8]. Medicinal plants play a vital role in various traditional, complementary, and alternate systems of medicine as they contain a broad range of secondary metabolites, such as alkaloids, flavonoids, tannins and terpenoids [9, 10], which are found to play a key role in the regulation of diseases in human beings. The presence of these phytochemicals is responsible for the antioxidant, antimicrobial, and antipyretic effects of these medicinal plants [11]. WHO states that medicinal plants are the best source for obtaining a variety of herbal formulations. Hence, plants with such medicinal properties should be studied for a better understanding of their therapeutic properties, efficacy, and safety issues [12].

Plant metabolites can be divided into two groups as primary metabolites that are directly involved in the growth and metabolism of the plant and secondary metabolites are organic compounds which are the byproducts of primary metabolism that are not generally used by plants for metabolic activities. These secondary metabolites serve as interspecific defenses when the plant interacts with their counter biotic and abiotic partners in the environment [13]. These secondary metabolites are structurally and functionally diverse in nature and can be classified as alkaloids, flavonoids, glycopeptides, phenolics, peptides, steroids, terpenoids, and volatile oils [14]. These secondary metabolites act as precursor

molecules for pharmaceuticals, agrochemicals, cosmetics, and industrial products and as flavouring and colouring agents in the food processing industry.

Bioactive therapeutic agents produced in plants are the products of natural metabolic processes. Each species has its own genetic makeup that governs the production of these bioactive molecules. In addition to the genetic makeup, other factors such as the effect of environmental factors such as temperature, moisture content, and the difference among cultivars within the species also contribute to the variation in the quality and quantity of the compounds [15].

These bioactive compounds either act on one or different systems of the animal physiologically and/or act by interfering with the metabolism of microbes involved in the infection process, thus regulating the host-microbe interactions in favor of the host. Research is being focused on meeting the challenges of identifying bioactive compounds in plants, and establishing evidences on whether the whole plant or extracted compounds are to be used for therapeutic purposes. Therefore, the identification, isolation, characterization, and purification of the bioactive compound from crude extracts of plants play a key role.

Evaluation of the pharmacological activities of medicinal plants and the subsequent increase in the demand for plant-based drugs is leading to overharvesting, thus creating heavy pressure on high-value medicinal plant populations. Moreover, several of the medicinal plants have low population densities, slow growth rates, and narrow geographic ranges and therefore are more prone to extinction [16]. Furthermore, the knowledge of the use of lesser-known medicinal plants is declining rapidly. Hence, there is a need to spread awareness and conservation of these medicinally important species through various conservation techniques available. Furthermore, the lack of availability of plant material throughout the year and the impracticality of conventional propagation and breeding methods for the production of plants on a large scale act as a barrier to the separation of bioactive molecules. In such cases, alternative and economically feasible approaches for the separation of the desired phytochemicals are to be implied. Biotechnological tools help in solving the problems faced by conventional breeding programmes and act as bioreactors for the production of bioactive metabolites from endangered and medicinally important plants [17]. Thus, these biotechnological tools offer a line of approaches for maintenance, genetic improvement, and efficient use of endangered plant resources and products [18].

In both developing and developed countries, the increase in demand for plant-based crude material in pharma, cosmetic, and herbal industries is leading to the frequent contamination of crude products with extraneous/foreign material or with inferior quality crude drugs that resemble the standard drug. To avoid such problems, systematic approaches towards standardization of crude drugs have been developed in modern pharmacology. These standardization procedures include botanical authentification, microscopic and molecular examination, identification of chemical constituents, and biological activity of the whole plant [19]. Identification of bioactive chemicals and macroscopic microscopic evaluation of plant materials for quality control and standardization have been reported by early researchers [20]. Macroscopic evaluation parameters involve sensory characters such as shape, size, colour, texture, odour, and taste, and microscopic parameters involve comparative microscopic inspection of the powdered herbal drugs. Various modern techniques such as chromatography, spectrophotometry, electrophoresis, polarography, fluorescence analysis alone and in combination are currently employed in the standardization of herbal drugs [21].

Based on the above considerations, plant biotechnology can be regarded as an important tool that enables the conservation of desired species and obtains elite clones of pharmaceutically important medicinal plants, and meets the demands of public healthcare systems and pharmaceutical industries, especially in biodiversity rich areas where there is an urgent need for conservation of germplasm for future generations as important species are becoming extinct due to over-exploitation.

Techniques of plant biotechnology also help in the isolation of therapeutically important compounds from a particular tissue/organ without any loss to the whole plant, thus, helping in the conservation of commercially important medicinal plants. Considering the demand for Senna alata L. Roxb. (Syn. Cassia alata) due to its ethnic medicinal properties, use as an ornamental shrub, and therapeutic applications, the species needs conservation. Hence, in the present investigations, attempts have been made to evaluate the pharmacological properties of aqueous leaf extracts using various animal models, to multiply and conserve this medicinally important woody legume using various in vitro culture techniques. Attempts were also made to study the pharmacognotic properties concentrating mainly on screening for the presence of various phytochemicals from leaf explants dried under different conditions in order to study the effect of drying on the loss of chemical constituents and to study the amount of total anthraquinone and anthraquinone glycosides from various explants and extracts to determine the ideal explants/extracts for commercial production of anthraquinones, and to isolate anthraquinones from five different species of Cassia/Senna.

Results and Discussion

About One-fourth of the world's population, accounting for 1.42 billion people generally depends on traditional medicine that particularly consists of plant-based drugs for curing various diseases [8]. Herbal or traditional medicines are gaining importance due to their safety, efficacy, and lack of side effects and are considered a promising choice over modern synthetic drugs [22].

The phytoconstituents present in the plant parts account for various pharmacological activities of these medicinal plants, which form the basis of herbal medicine and herbal industries, which mostly use these fresh or dried plant parts for the manufacturing of herbal drugs. A detailed knowledge of crude drugs is essential in the identification, preparation, safety, and efficacy of herbal products. In order to meet the increasing demand for plant-based crude drugs, phytochemical and pharmaceutical industries in both developing and developed countries are adulterating crude drugs with foreign organic matter or substituting inferior quality crude drugs resembling the standard drugs. Hence, there is a need for the development of a systematic approach to studying crude drugs in modern pharmacognosy.

The process of standardization is a multi-step process and can be achieved by stepwise pharmacognostic studies [23]. Various methods, such as the determination of ash residues, extractive values, and screening of active phytoconstituents, play a significant role in the standardization of indigenous crude drugs [24].

The species Senna alata possesses many valuable medicinal properties, but the knowledge of these properties is still confined to tribal areas because of the absence of proper scientific standardization. In order to determine the usefulness of the species in modern medicine, standardization of various parameters, viz. morphological, physico-chemical and phytochemical constituents, are essential. Based on these parameters, the plants can be made successfully available for the population and herbal industries across the world. Hence, the present study has been undertaken to evaluate the organoleptic, physicochemical, and phytochemical constituents present in the seed and leaf extracts of S. alata in order to elite medicinal properties attributed to the species.

Physicochemical Parameters

Studies on the physicochemical parameters of crude extracts are essential for the identification of the plant material, analyzing the stability of the crude drug, microbial contamination, heavy metal accumulation, avoiding mishandling, and estimating adulteration. In the present study, in order to determine the purity of the drug, various physicochemical parameters have been studied in S. alata. The details of the studies are presented in Tables 1-3.

Table 1 Physical characters of various leaf extracts of S. alata.

Various physicochemical properties such as total ash, acid soluble and insoluble ash, and water insoluble ash were determined using powdered leaf material of S. alata. The results obtained from the present study are presented in Table 2. The total ash and acid insoluble ash content of the leaf of S. alata is 7.84 (% w/w) and 0.94 (% w/w), respectively. The amount of acid insoluble ash is very less than that of water soluble ash (6.90%). These physicochemical properties related to total ash value and acid insoluble ash values help in determining the evaluation of crude drugs at a large scale.

Table 2 Physicochemical properties of Methanol and Aqueous extracts of S. alata.

The results of the fluorescent studies of powdered leaf material are presented in Table 3. The extracts exhibited various fluorescent characteristics under normal/UV light. Among the various solvents tested, acetone extract did not show any fluorescence activity. Whereas, all other tests showed characteristic colouration. The colour due to fluorescence was found to be specific to each compound (Table 3).

Various aspects regarding the physical constituents of five different extracts (Aqueous, Acetone, Benzene, Chloroform and Ether extracts) of S. alata obtained by maceration are presented in Table 1. All the extracts exhibited a characteristic colour ranging from yellowish green to niger brown. The consistency of the extracts was amorphous for all the extracts and the % yield of the extract ranged from 26.1±0.42 (Acetone extract) to 33.9±0.69 (Aqueous extract) (Table 1).

Table 3 Fluorescent studies of leaf powder of S. alata.

Phytochemical Analysis

Preliminary phytochemical screening of both fresh and shade-dried leaf extracts was carried out to determine the effect of loss of plant material on drying and to determine the appropriate solvent for extraction. Different extracts of seed and fresh/shade-dried leaf material were screened for the presence of phytochemicals. The results of preliminary phytochemical screening of seed fresh/shade-dried leaves of S. alata are are shown in Tables 4 and 5.

The phytochemical analysis of seed extracts revealed the presence of alkaloids, flavonoids, tannins, saponins, anthraquinones, resins, and glycosides in all extracts. Sterols were present only in the chloroform extract and phenols only in the aqueous extracts. Coumarins, phenols, terpenoids, phlobatannins and quinines are completely absent in all seed extracts (Table 4). Phytochemical screening of fresh and dry leaves showed the presence of alkaloids, flavonoids, anthraquinones, saponins, glycosides, and tannins in high amounts in all extracts except for methanolic extract. Resins and phenols are present in moderate amounts. Sterols are present only in chloroform extracts. Terpenoids and phlobatannins are present only in fresh leaf extracts, and quinones are present only in benzene and aqueous extracts of dried leaves (Table 5).

Table 4 Phytochemical analysis of seed extracts of S. alata.

++ = Strongly present, + = Present, - = Absent

Table 5 Phytochemical analysis of fresh/dry leaf extracts of S. alata.

F = Fresh leaf extract; D = Dry leaf extract;

++=Strongly present, + = Feebly present, - = Absent

DISCUSSION

According to the results of our study, the total ash content of leaves of S. alata leaves was found to be 7.84%, of which very low quantity (0.94%) of acid insoluble and high amount (6.90%) of water soluble ash, which indicate that the crude extract of S. alata leaves contains more amounts of physiological ash than the non-physiological content. Low moisture content (3.9%) discourages the growth of bacteria, yeast, or fungi during storage of the crude drug, and the high fiber content of the leaves is an indication of the possible microbial contamination due to unfavorable moisture content and rich dietary fiber source of the leaves.

Studies on phytochemical screening revealed the presence of alkaloids, glycosides, resins, tannins, and anthraquinones in all seed extracts except in the methanol extract [25] and the presence of alkaloids, glycosides, saponins, tannins, flavonoids, terpenoids, anthraquinones, resins and steroids in the fresh leaf extracts and glycosides, alkaloids, saponins, tannins, flavonoids and anthraquinones in dry leaf extracts (Table 5). The presence of same phytochemicals was also reported in S. alata [25, 26]. The presence of these phytoconstituents may be responsible for various pharmacological activities associated with the species as different compounds are associated with different pharmacological activities.

Due to the presence of these phytochemicals with pharmacological activities, the demand for crude drugs is increasing day by day, leading to the adulteration of crude drugs. Hence, there is a need for standardization of crude drugs in order to identify the presence of adulterating substances [27]. WHO stated that morpho-

logical characters like epidermal cell features, stomatal index, vein islets, etc. , are to be studied for the proper identification of crude drugs [28, 29].

The preparation of herbal medicines involves the use of fresh or dried plant parts and their extracts. Such preparations usually require a sound knowledge of the crude drugs in order to obtain drugs with high efficacy and safety. A detailed, stepwise pharmacognostic evaluation is therefore required in order to standardize the purity of crude drugs [30]. Pahrmacognostic and organoleptic properties such as extractive value, acid value, fluorescence analysis, and microscopic studies play a major role in the standardization of the native crude drugs and help in the assessment of any adulterating substances in the crude drugs [24].

CONCLUSION

In the present study, leaf extracts of S. alata were tested for various phytochemical and organoleptic characteristics for their successful standardization according to WHO guidelines. The results of the study revealed the presence of various phytochemicals that are responsible for the pharmacological activities of the species. These studies assist in the identification and standardization of the leaf extracts and in carrying out further research on the pharmacological activities based on the phytochemicals present.

REFERENCES

Plant Tissue Culture: A Potential Tool for the Production of Secondary Metabolites

Madhukar Garg¹, Soumi Datta², Sayeed Ahmad³, *

¹ Chitkara College of Pharmacy, Chitkara University, Rajpura, Patiala, Punjab, India

² Dabur Research and Development Center, Dabur India limited, Sahibabad, Ghaziabad-201010, India

³ Hamdard School of Pharmacy, Jamia Hamdard, Hamdard University, Hamdard Nagar, New Delhi, India

Abstract

Plants are an immense source of phytochemicals with therapeutic effects and are widely used as life-saving drugs, and other products of varied applications. Plant tissue culture is a unique technique employed under aseptic conditions from different plant parts called explants (leaves, stems, roots, meristems, etc.) for in vitro regeneration and multiplication of plants and synthesis of secondary metabolites (SMs). Selection of elite germplasm, high-producing cell lines, strain enhancements, and optimization of media and plant growth regulators may lead to increased in vitro biosynthesis of SMs. Interventions in plant biotechnology, like the synthesis of natural and recombinant bioactive molecules of commercial importance, have attracted attention over the past few decades; and the rate of SMs biosynthesis has increased manifold than the supply of intact plants, leading to a quick acceleration in its production through novel plant cultures. Over the years, the production of SMs in vitro has been enhanced by standardising cultural conditions, selection of high-yielding varieties, application of transformation methods, precursor feeding, and various immobilization techniques; however, most often, SM production is the result of abiotic or biotic stresses, triggered by elicitor molecules like natural polysaccharides (pectin and chitosan) that are used to immobilize and cause permeabilization of plant cells. In vitro synthesis of SMs is especially promising in plant species with poor root systems, difficulty in harvesting, unavailability of elite quality planting material, poor seed set and germination, and difficult to propagate species. Thus, the present article reviews various biotechnological interventions to enhance commercially precious SMs production in vitro.

Keywords: Biotecnology, Callus secondary metabolites, Phytomolecules, Plant tissue culture, Suspension cultures.

* Corresponding author Sayeed Ahmad: Hamdard School of Pharmacy, Jamia Hamdard, Hamdard University, Hamdard Nagar, New Delhi, India; Tel: 09891374647; E-mail: [email protected]

Introduction

Plants are renewable sources and form an important part of our daily diet, and provide essential primary metabolites (e.g., carbohydrates, lipids and amino acids) [1] and phytochemicals (low molecular weight compounds-SMs) for different industrial applications like pharmaceuticals, nutraceutical, textile, construction and cosmetic sectors [2]. The majority of the world population’s health and wellness relies on plant-derived components. Therefore, plants with medicinal properties are considered important to support the transition to a bio-economy that is less dependent on fossil resources. The SMs not only play a pivotal role in plants’ adaptation to their environment but also represent an important source of active pharmaceuticals [3] and are synthesised by plants to defend themselves against exogenous stresses, both biotic and abiotic. A study [4] proposed the concept of SMs that were known as opposed to primary ones and an entire volume of plant biochemistry series named as endproduct [5]. It is known that higher plants are a rich source of phyto-pharmaceuticals and are used in the pharmaceutical industry. Some of the plant-derived products include drugs like morphine, codeine, cocaine, pilocarpine, belladonna alkaloids, colchines, phytostigminine, L-DOPA, berberine, reserpine, capsaicin, podophyllotoxin, shikonin derivatives, ajmalicine, vincristine and vinblastine [6] and steroids like ginsenosides, anti-cancer (taxol), diosgenin, digoxin and digitoxin. Significant synthetic substitutes of these drugs with the same efficacy and pharmacological specificity are yet to be found [7].

Previously, chemical synthesis for the production of SMs was achieved through field cultivation; however, the plants originating from particular biotypes were difficult to grow outside their ecosystems and thus led scientists and biotechnologists to consider plant cell, tissue and organ cultures as an alternative to produce secondary metabolites. The major advantages of in vitro synthesis of bioactive secondary metabolites within controlled conditions include: these are climatic and soil stipulations independent, minimal inferences of negative biological parameters affecting the SM production, possible choice of elite germplasm with respect to the presence of SMs, computerization of cell growth control, metabolic processes regulation, and cost price, which can be decreased with increased production. Plants produce alkaloids, flavonoids, lactones, glycosides, quinines, phenylpropanoids, resins, tannins, terpenoids, saponins, sesquiterpene, and steroids [8]. The first large-scale production of commercial plant cells application was carried out in stirred tank reactors to synthesis shikonin by cell cultures of Lithospermum erythrorhizon [9, 10].

Secondary metabolites

Plants are capable of producing different organic molecules called secondary metabolites, having unique carbon skeletons with basic properties. SMs are not necessarily for a cell (organism) to live but also for interaction with its environment. These are organ, tissue and cell-specific with low molecular weights and often differ amid individuals from the same population with respect to their type. SMs protect plants against stress; and are used as drugs, flavors, fragrances, insecticides and dyes and hence are of great economic value. SMs have evolved as molecules imperative for organisms producing them, the majority of these interfere with the pharmacological targets, and thus make them significant for several biotechnological applications.

Primary vs Secondary Metabolites

Primary metabolites (PMs) are compounds that are universally present in all plants, but are not species-specific and, thus might be identical in some organisms. These are directly involved in metabolic activities like growth, development, nutrition and reproduction of a plant whereas secondary metabolites are produced in other metabolic pathways that, although important, but are not essential to the functioning of the plant. Whereas, SMs are species specific and, therefore, unique for each species. The major differences between PMs and SMs are listed in Table 1.

Table 1 Comparison between primary & secondary metabolites in plants.

The Biosynthesis of Secondary Metabolites

SMs are synthesized by diverting energy-generating directions in metabolic pathways like photosynthesis, glycolysis, and Krebs cycle to biosynthetic intermediates; and are classified in separate categories depending upon their biosynthesis, structures and functions. SMs are mostly biosynthesized from acetyl coenzyme A, mevalonic acid, shikimic acid, deoxyxylulose 5-phosphate or various combined pathways [11]. Accordingly, they are classified into terpenoids, steroids, alkaloids, saponin, terpenes, lipids and enzyme cofactors [12, 13]. There are three major pathways to produce SMs-Shikimate, isoprenoid and