In vitro Study of Tinospora cordifolia (Wild.) Miers

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In vitro Study of Tinospora cordifolia (Wild.) Miers (Menispermaceae)– a Multipurpose Plant, by Using Different Plant Bark Extracts for Secondary Metabolite Production a

Meghna P. Patel & Kalpesh B. Ishnava

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Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences, Sardar Patel University, New Vallabh, Gujarat, India Published online: 02 May 2014.

To cite this article: Meghna P. Patel & Kalpesh B. Ishnava (2014) In vitro Study of Tinospora cordifolia (Wild.) Miers (Menispermaceae)– a Multipurpose Plant, by Using Different Plant Bark Extracts for Secondary Metabolite Production, Journal of Herbs, Spices & Medicinal Plants, 20:4, 341-349, DOI: 10.1080/10496475.2013.876482 To link to this article: http://dx.doi.org/10.1080/10496475.2013.876482

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Journal of Herbs, Spices & Medicinal Plants, 20:341–349, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1049-6475 print/1540-3580 online DOI: 10.1080/10496475.2013.876482

In vitro Study of Tinospora cordifolia (Wild.) Miers (Menispermaceae)– a Multipurpose Plant, by Using Different Plant Bark Extracts for Secondary Metabolite Production


MEGHNA P. PATEL and KALPESH B. ISHNAVA Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences, Sardar Patel University, New Vallabh, Gujarat, India

Vegetative parts (stem, leaf, and nodal explants) of Tinospora cordifolia were excised from an in vivo grown mature plant and thereafter cultured on MS medium supplemented with 2, 4-D and bark extracts of Azadirachta indica and Acacia nilotica prepared in hexane, ethyl acetate, chloroform and methanol. The best callus growth was observed on MS medium supplemented with 2, 4-D (1 mg.L−1 ) and A. indica bark methanolic extract (1 mg.L−1 ). The methanol extracts of the dried callus showed the presence of alkaloids and sterols. HPLC, HPTLC, and GC-MS analysis of callus [MS+2, 4-D (1 mg.L−1 )] and callus [MS+2, 4-D (1 mg.L−1 ) + A. indica methanolic extract (1 mg.L−1 )] were carried out for comparative study. The new-found peaks revealed the presence of methyl hexadecanoic acid with molecular weight of 270. KEYWORDS Barks extracts, callus, Secondary metabolites, 2, 4 –D

INTRODUCTION Natural products play an important role in the drug development and programs in the pharmaceutical industry (2). In rural areas of the developing Received May 12, 2013. Address correspondence to Kalpesh B. Ishnava, Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences (ARIBAS), Sardar Patel University, New Vallabh Vidyanagar 388121, Gujarat, India. E-mail: ishnavakb203@ yahoomail.com Color versions of one or more of the figures in the article can be found online at www. tandfonline.com/whsm. 341


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M. P. Patel and K. B. Ishnava

countries, they continue to be used as the primary source of medicine (7). About 80% of individuals from developing countries use traditional medicine that has compounds derived form medicinal plants (9). Depending on the plant species, traditional agricultural methods often require months to years to obtain a crop (18). The prospective cultivator of medicinal plants must make the difficult decision of which particular species to grow in what is a rapidly shifting and fashion-prone market (5). The target compounds are almost invariably secondary metabolites, which, for the plants, frequently served as adaptation to fluctuating temperature and light conditions (29). Tinospora cordifolia (Menispermaceae) is distributed throughout the tropical region of India. Studies on T. cordifolia report anti-inflammatory, antiarthritic, antiosteoporotic (16), antiallergic (30), antioxidant (30), antineoplastic (13), anti-malarial, radio-protective, anti-hyperglycemic (25), antidiabetic (28), anti-pyretic (32,33), anti-infective (14), immunomodulatory (8), hepatoprotective (18), antileprotic (3), gastrointestinal-anti ulcer (4), diuretic (22), antifertility (4), osteoprotective (17) and cardioprotective activities (27). The present study was undertaken to increase secondary metabolite production by adding plants extracts in plant tissue culture medium.

MATERIAL AND METHOD Mature plants of T. cordifolia were collected June to October, 2012, and bark of Azadirachta indica (Neem) and Acacia nilotica (Baval) trees were collected in June, 2012 from the campus of New Vallabh Vidyanagar. The plants were identified at the Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences, New Vallabh Vidyanagar, Gujarat, India. Murashige and Skoog medium (20) with sucrose 3%, 0.8% agar, and growth hormones (2, 4-D) were used. The medium was prepared with combination (1.0 mg.L−1 , 1.5 mg.L−1 , and 2.0 mg.L−1 of Neem and Baval bark extracts. The concentrated stock solutions of ingredients were prepared and refrigerated. The surface sterilization of explants (leaves and nodal segments) was carried out as per as stranded protocol (20) with slight modifications to include immersion of explants in 0.1% mercuric chloride (HgCl2 ) solution for 4 min. Explants were inoculated and experimental manipulations carried out under strictly aseptic conditions in a laminar air flow bench (20). The tubes and bottles were shifted to a culture room with controlled facility of diffused light (2,000 lux) for 10 h daily at 28 ± 2◦ C and 50 to 60% relative humidity. The calluses were collected after 30 to 40 days, washed with distilled water to remove all adhering particles, and dried at room temperature. Callus extracts were prepared as described earlier (15), evaporated, and dissolved in methanol for further analyses.

In vitro Study of Tinospora cordifolia for Secondary Metabolite Production

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Phytochemical Analysis PREPARATIVE THIN-LAYER CHROMATOGRAPHY The analytical separation of extracts was carried out on silica gel 60 F254 plates 10 cm × 10 cm developed with methanol: acetic acid: water (8:1:1) solvent system. The chromatograms were observed under both visible light and UV radiation (at 254 nm and 346 nm) and photographed.


HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY ANALYSIS For chemical profile analysis, methanol: acetic acid: water (8:1:1) were used as the mobile phase. The plates were run up to 8 cm height. The bands were observed and scanned at 366 nm, and photographs were taken. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS One gram dried callus (experimental (2, 4-D+Neem methanolic extract) and control (Standard Berberin)) powder was extracted thrice in 5 mL methanol. Separations were achieved using column C-18, RP-18 and PIGel, 5 µm particle size and mobile phase of acetonitrile, and 1% acetic acid in water in the ratio of 60:40 at a flow rate of 1 mL.min−1 . Both sample control and experimental (methanolic extract) were analyzed in duplicates. GAS CHROMATOGRAPHY–MASS SPECTROSCOPY ANALYSIS GC–MS analysis was by electron impact ionization (EI) on Auto system XL GC at Sophisticated Instrumentation Centre for Applied Research and Training, Vallabh Vidyanagar, Gujarat. The column was fused with silica capillary column, 30 · 0.25 mm ID; coated with D-I, 0.25 l m film thickness. The temperature of the column was programmed at 70◦ to 250◦ C at the rate of 10◦ C.min−1 ; injection port was at 250◦ C. Helium was used as carrier gas at constant pressure of 100 kPa and flow rate of 20 mL.min−1 . Samples dissolved in chloroform were run fully at of 60 to 550 amu, and the results were compared using NIST 107 Spectral library search program.

RESULTS Callus formation occurred from nodal, inter-nodal, and leaf explants when planted on the medium containing the combination of 2, 4 – D (1 mg.L−1 ) and A. indica (1 mg.L−1 , 1.5 mg.L−1 , and 2 mg.L−1 ) (Figure 1 A–E) and


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FIGURE 1 Morphological features of Tinospora cordifolia and callus induction using plant extracts. (Row I) M.S medium + 2, 4-D (1 mg.L−1 ): (A) Callus induction in Tinospora cordifolia (nodal explants); (B) Explants inoculation- Callus induction after 10 days; (C) Callus after 20 days. (Row II) M.S Medium +2, 4-D (1 mg.L−1 ) + Azadirachta indica methanol extracts (1 mg.L−1 ): (D) Explants inoculation; (E) Callus induction after 10 days; (F) Callus after 20 days. (Row III) M.S medium + 2, 4 –D (1 mg.L−1 ) + Acacia nilotica methanol extract (1 mg.L−1 ): (G) Explants inoculation; (H) Callus induction after 10 days; (I) Callus after 20 days.

A. nilotica (1 mg.L−1 , 1.5 mg.L− 1, and 2 mg.L−1 ) bark extract, respectively (Figure 1 G–I; Table 1). In hormone-free basal medium and hormone 2, 4-D (1 mg.L−1 ) callus production occurred after 36, 15, and 10 days, respectively, in leaf explants was 98% (see Table 1). In the hormone 2, 4-D (1 mg.L−1 ) containing basal medium, callus production was observed after 10 days in nodal explants response was 98 % (see Table 1). When compared to leaf


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TABLE 1 Production of Callus from Tinospora cordifolia Explants Induction of callus from different explants


Nutrient medium Leaf M.S medium without growth hormone M.S. medium + 2, 4 –D (1 mg.L−1 ) Node M.S. medium + 2, 4 –D (1 mg.L−1 ) M.S. medium + 2, 4 –D (1 mg.L−1 ) +NME (1 mg.L−1 ) M.S. medium + 2, 4 –D (1 mg.L−1 ) +NME (1.5 mg.L−1 ) M.S. medium + 2, 4 –D (1 mg.L−1 ) +NME (2 mg.L−1 ) M.S. medium + 2, 4 –D (1 mg.L−1 ) +AME (1 mg.L−1 ) M.S. medium + 2, 4 –D (1 mg.L−1 ) +AME (1.5 mg.L−1 ) M.S. medium + 2, 4 –D (1 mg.L−1 ) +AME (2 mg.L−1 )

% of explants Duration of callus showing callus production (days)

Response

95

36

Light brown callus

98

15

Whitish friable callus

98

10

100

4–6

95

7

Nodulated dark brown callus Light brownish nodulated callus Nodulated dark brown callus

90

15

Brownish callus

100

10

95

14

Brownish nodulated callus Brownish nodulated callus

98

18

Brownish nodulated callus

explants, the nodal explants required less time for callus induction and produced nodulated brownish mass of callus. In hormone (2, 4-D 1 mg.L−1 ) containing medium callus was occurred within 10 days in stem explants, and the response was 80%. The control callus was collected after 42 days from nodal culture [M.S. + 2, 4-D (1 mg.L−1 )]. The sample callus was collected after 34 days from nodal culture [M.S. + 2, 4-D (1 mg.L−1 ) + Neem methanol extract (1 mg.L−1 )]. Maximum % yield of extract was 8.4855 % in sample H1- M.S + 2, 4-D (1 mg.L−1 ) + Acacia nilotica (1.5 mg.L−1 ), and minimum % yield of extract was 2.7016% in the sample H- M.S + 2, 4-D (1 mg.L−1 ) + Acacia nilotica (1 mg.L−1 ) (see Figure 1). The phytochemical test was carried out for all callus extracts (nodal extracts), sample (M.S + 2, 4-D (1 1 mg.L−1 ) – callus collection after 42 days), sample G (M.S + 2, 4-D (1 mg.L−1 ) +A. indica (1 mg.L−1 ) – callus collection after 34 days), sample H(M.S + 2, 4-D (1 mg.L−1 ) + A. nilotica (1 mg.L−1 )), sample G1(M.S + 2, 4-D (1 mg.L−1 ) + A. indica (1.5 mg.L−1 )), and sample H1 (M.S + 2, 4-D (1 mg.L−1 ) + A. nilotica (1.5 mg.L−1 )) gave positive test in alkaloid and sterols in all the samples. The other compounds were not present in the samples.


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The present HPLC method was conducted to identify and quantify the berberine from T. cordifolia callus extracts. Berberine peaks (18901580.92) from solutions of various extract like pet-ether; methanol, aqueous, and chloroform were identified by comparing their Rf values with standard under the same conditions. Methanolic extracts of 42- and 34–dayold callus were used for HPLC analysis to compare production of secondary metabolites. The sample concentration used was 2 mg.L−1 . Control sample of callus was grown in basal M.S. medium with 2, 4 – D (1 mg.L−1 ), and sample G of callus was grown in basal M.S. medium with 2, 4-D (1 mg.L−1 ) and neem methanol extract (1 mg.L−1 ) of peak area 57187.32 and 43096.48, respectively. The control callus was collected after 42 days, and sample was after 34 days. Methanolic extract of 42- and 34-day-old callus extract were used for GC-MS analysis to compare production of secondary metabolites. Analyses of the extract confirmed with the help of mass spectrometric characteristics and compared with the already reported compounds from the same species and other. The results on the present study indicate that some of the peaks were newly found in sample (18.67 peak) compared to control. The peak showing maximum percentage area at RT 18.67 in GC–MS analysis and scan 2.88e5 through mass spectrophotometer revealed the presence of methyl hexadecanoic acid (molecular weight −270).

DISCUSSION Leaves of T. cordifolia are rich in protein (11.2%) and are fairly rich in calcium and phosphorus and containing anti-oxidant activity in vitro models (24,31,33). In modern medicine, the use of T. cordifolia for the treatment of general weakness and fever, the immunomodulatory properties, hypoglycemic activities have been reported (28). Three major groups of compounds— protoberberine alkaloids, terpenoids, and polysaccharides—are considered as putative active constituents of T. cordifolia (6). Protoberberine alkaloids such as berberine and palmatine are reported to have anti-cancer (11), anti-infective (12), anti-diabetic (19) and immunomodulatory activities (23). Callus formation occurred from nodal and inter-nodal explants when planted on the medium containing the combination of 2, 4 – D (1 mg.L−1 ) and A. indica (1 mg.L−1 , 1.5 mg.L−1 , and 2 mg.L−1 ) and A. nilotica (1 mg.L−1 , 1.5 mg.L−1 , and 2 mg.L−1 ; see Table 1). In basal MS medium containing hormone 2, 4 – D (1 mg.L−1 ) and A. indica (1 mg.L−1 ) extract and A. nilotica (1 mg.L−1 ) combination, the callus induction occurred within 10 days, and the response was 100 %. In this combination [2, 4-D (1 mg.L−1 ) and Neem extract (1 mg.L−1 ) and A. nilotica (1 mg.L−1 )] nodulated brownish mass of callus was produced. In very short time, a large quantity of callus



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was produced. Callusing from the nodal, inter-nodal, and leaf explants on the medium supplemented with different concentrations of BAP and NAA have been reported (1). However, late shoot growth was seen from the explants on the medium supplemented with higher concentration of NAA. NAA and 2, 4-D alone could initiate callusing from stem, leaf, and nodal segments, but callus may grow slowly, and Kn along with auxins considerably enhanced callus growth (21). Shoot was induced from nodal explants on MS medium supplemented with higher concentrations of Kn, whereas nodal and intermodal explants cultured on MS medium supplemented with NAA and BAP produced roots. Others also reported that higher concentrations of Kn induced shoots in nodal explants and induction of a single shoot by higher concentrations of BAP in Tinospora sp. (10), while some others reported benzyl adenine to be more effective than Kn for auxiliary shoot proliferation while Kn was better for shoot elongation (26). Callus formation occurred from nodal, inter-nodal, and leaf explants when planted on the medium containing the combination of BAP and NAA. No response was shown by nodal and inter-nodal explants. In such conditions, callusing was seen only on leaf explants. Shoot growth was observed from callus after 40 days of culture. However, the shoot was induced only from nodal explants and root from nodal and inter-nodal explants. The shoot and root induction from cultured explants were influenced by the type and concentration of hormone.

ACKNOWLEDGEMENTS The authors thank the director of the Sophisticated Instrument Centre for Applied Research and Testing, Vallabh Vidyanagar, Gujarat, India for HPLC and GC-MS analysis. Authors are also thankful to Charutar Vidya Mandal, Vallabh Vidyanagar, Gujarat, and the director of Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences, New Vallabh, Gujarat, India, for providing necessary support for research and laboratory facility.

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