Phytochemical investigation of natural and in vitro raised Vṛddhadāruka plants.

BACKGROUND: Argyreia nervosa commonly known as elephant creeper (English) and Vṛddhadāruka (Sanskrit) is a woody climber that belongs to the family Convolvulaceae. Seeds of this plant contain hallucinogens including ergot alkaloids and a naturally occurring lysergic acid amide. Traditionally the plant is used in the treatment of gonorrhea, strangury, chronic ulcers, diabetes, anemia and cerebral disorders. The plant is also used as appetitiser, brain tonic, cardiotonic, aphrodisiac. It possesses anti-inflammatory, immunomodulatory, antibacterial, antiviral and antifungal activities.
OBJECTIVE: To give an account of information on in vitro regeneration and phytochemical analysis of the plant.
MATERIALS AND METHODS: Nodal explants were selected for in vitro regeneration. Different aerial parts viz., seeds, natural and in vitro leaf, stem and callus were dried and extracted with different solvents and were subjected to various phytochemical analyses.
RESULTS: Different concentrations of 6-benzylaminopurine showed shoot and root initiation. The study of phytochemical screening of different extracts showed the presence of bioactive substances like flavonoids, alkaloids, terpenoids, etc.
CONCLUSION: The study will provide an efficient in vitro protocol for micropropagation as an alternative method to conserve the plant and shows the presence of some important secondary metabolites in the nature grown and in vitro raised plants which can be useful for treatment of various diseases.

INTRODUCTION

Medicinal plants are the richest bio-resource of drugs for traditional systems of medicine.[1] About 80% population of Asian and African countries relies on traditional medicines for primary health care.[2] Herbal medicines are the most lucrative form of traditional medicine, generating billions of dollars in revenue.[3] Nowadays, the crude extracts and dry powder samples from medicinal plants and their species have been the object of interest for the development and preparation of alternative traditional medicines.[4567] Therefore, there is an urgent need to search for herbal medicinal plants with potential medicinal properties on the basis of their ethno-medicinal uses and also for their isolation, characterization along with conservation.

Argyreia nervosa (Convolvulaceae) is a perennial climbing shrub with woody tomentose stem, native to Indian subcontinent. The hallucinogenic properties of A. nervosa are because of the seeds that contain the highest concentration of psychoactive compounds in the entire family.[8] Śūla, śopha, apasmāra, arṣa, aruci, āmavāta, anāha, graharbādhā, gulma, hrdrujā, kāsa, kṛmi, mūtrakrchra, meha, pāṇḍu, taktapitta, udara, unmāda, vātarakta are some of the therapeutic uses of A. nervosa in Ayurvedic system of medicine.[9] Leaves of the plant have nematicidal,[10] antibacterial,[1112] and antimicrobial activity,[13] root of this plant is used in rheumatism, gonorrhea, chronic ulcer and diseases of nervous system. It is also used us a tonic, diuretic and aphrodisiac.[1415] It also has hypoglycemic,[16] immunomodulatory,[17] hepatoprotective,[18] anti-inflammatory,[19] and analgesic activities.[20] The plant is listed as critically endangered in Vindhya region.[21]

The main aim of the present investigation is to develop an in vitro regeneration system from nodal explants as there are very few previous reports on in vitro studies and preliminary phytochemical screening of in vitro and natural grown A. nervosa leaf, stem, callus and seed extract in different solvents like petroleum ether, methanol, diethyl ether, water and ethyl acetate and subsequent qualitative analysis of some primary and secondary metabolites.

MATERIALS AND METHODS

Plant material

Nature grown plant of A. nervosa was procured from the nursery of Jawaharlal Nehru Krishi Vishwavidyalaya, Jabalpur. Plants were identified by Institutional Botanist, and voucher specimen (No. 19617) was deposited. In vitro, plant was obtained by inoculating the nodal explants on Murashige and Skoog media fortified with different concentrations of plant growth regulators. The pH of the medium was adjusted to 5.7 before autoclaving at 121°C for 20 min. Nodal segments were aseptically transferred onto MS medium supplemented with different concentrations (0.44, 4.44, 17.76 and 22.22 μM) of 6-benzylaminopurine (BAP). All cultures were maintained at 16 h photoperiod with 3000 Lux light intensity at 25 ± 2°C. Seeds, callus, natural and in vitro leaves and stem were shade dried. Each sample of the material was ground separately into a fine powder and stored in airtight containers at ambient temperature.

Preparation of crude extracts

Approximately, 5 g of sample was weighed and dissolved in solvents viz., petroleum ether, methanol, diethyl ether, ethyl acetate and water separately and was allowed to stay overnight for 24 h. After overnight incubation the sample was filtered using Whatman filter paper, the filtrate was used for phytochemical screening.

Phytochemical tests

The phytochemical analysis was carried out to determine the presence of following bioactive compounds using the standard qualitative procedures [Table 1].[2223]

Table 1

Procedure for phytochemical analysis

RESULTS

Morphogenetic responses of nodal explants were observed on MS media alone and MS supplemented with different concentrations of BAP is presented in [Table 2]. Callus induction was also observed on MS media supplemented with different (0.44–22.2 μM) concentration of BAP. Profuse callusing was obtained in higher concentration (17.76 and 22.2 μM) of BAP. Hard green callus were formed after 30 days of inoculation. Maximum frequency of shoot initiation (58.33) was obtained on BAP (0.44 μM) whereas maximum mean shoot length (3.23 cm) was obtained on BAP (4.44 μM) the same concentration also gives the maximum frequency of root initiation (43.2). Maximum mean root length (3.03 cm) was obtained on BAP (0.44 μM). Anthocyanine and anthocyanidine are present in all parts of the plant except in in vitro leaf, coumarin is mostly present in methanolic extracts, whereas anthracene is present in methanolic extract of natural leaf. Nature grown leaf and callus doesn’t contain tannins, saponin is present only in seeds, steroids are present in all parts except in in vitro stem and in vitro leaf. Alkaloids and flavonoids are almost present in all parts of A. nervosa. Starch is present in seeds, callus and in vitro leaves. Terpenoids are present in all parts except in vitro leaves. Methanolic extracts and water extracts show maximum presence of secondary metabolites.

Table 2

Effects of different levels of BAP on shoot and root growth from nodal explants of Argyreia nervosa

DISCUSSION

In micropropagation, the nodal explants, responded most frequently than other explants as shown in Figure 1. Similar results were reported in Ceropegia intermedia,[24] Leptadenia reticulate[25] and Clitoria ternatea.[26] The type and concentration of cytokinin has an immense effect on shoot bud proliferation and in some cases for root organogenesis. BAP was observed to be best in shoot and root organogenesis in A. nervosa. The reason for the effectiveness of the BAP may lie in its ability to stimulate the plant tissues to metabolize the natural endogenous hormones or could induce the production of natural hormone system for the induction of organogenesis.[2728] Plants are of great medicinal value as they are rich in secondary metabolites like alkaloids, glycosides, flavonoids, steroids, related active metabolites and have been extensively used in the drug and pharmaceutical industry. Alkaloids possess many medicinal properties, like anti-inflammatory, anti-asthmatic, and may alter immunological status in vivo, it has further effect on human health.[29] In the present study the presence of alkaloids in various fractions of different parts of the nature grown and in vitro plant of A. nervosa has shown that the plant is useful for treating different ailments and has a potential of providing useful drugs for human use. The presence of flavonoids in nature grown and in vitro plant in different fractions is also been shown in this study. Flavonoids are potent antioxidants with free radical scavenging activity that prevent cell damage and have strong anti-cancer activity.[3031]

Figure 1

(a) Natural plant of Argyreia Nervosa. (b) Fruits of Argyreia nervosa. (c) Seeds of Argyreia nervosa. (d) In vitro regenerated plant of Argyreia nervosa

Therefore from the present investigation a promising method has been developed for an efficient regeneration of A. nervosa from nodal explants using BAP. Phytochemical analysis in different parts of the plant was also performed and the results were comparatively assessed. Phytochemical analysis in different parts of the plant was also performed and the results were comparatively assessed [Table 3a and 3b]. Exploitation of these pharmacological properties involves further investigation and identification of these active ingredients by implementing techniques like extraction, purification, separation and crystallization.

Table 3a

Phytochemical screening

Table 3b

Phytochemical screening