JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.625, ISSN: 2320-5083, Volume 3, Issue 6, July 2015 1 www.jiarm.com IN VITRO ANTIOXIDANT AND ANTIPROLIFERATIVE ACTIVITIES OF WILD PLANT AND CALLUS EXTRACTS OF INDIGOFERA ASPALATHOIDES VAHL.EX.DC RAAMAN NANJIAN* SRIRAM CHANDRASEKARAN** *Professor, Natural Products and Tissue Culture Laboratory, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India **Research Scholar, Natural Products and Tissue Culture Laboratory, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India ABSTRACT Indigofera aspalathoides Vahl. ex. DC, belongs to the family Fabaceae and it is commonly called as wiry indigo. The present study was carried out to study the comparative account on antioxidant potential and antiproliferative activity between the methanolic extracts of wild plant and in vitro grown callus. Murashige and Skoog (MS) medium supplemented with NAA (0.5 mg/L) showed maximum callus induction from root (92%) and nodal explants (89%). The quantitative phytochemical analysis showed maximum total phenol (382 mg/g) and total flavonoid content (324 mg/g) in methanolic extract of wild plant followed by callus (370 mg/g and 318 mg/g). Antioxidant activities by different methods such as, 2,2’-azinobis (3-ethylbenzo-thiazo-line-6- sulfonate) (ABTS•+) radical scavenging activity, phosphomolybdenum activity, hydroxyl radical scavenging activity and iron chelating activity showed maximum antioxidant potential in methanolic extract of wild plant followed by methanolic extract of callus and standard. Antiproliferative activity of A375 cells showed IC 50 value of 86.29 μg/mL for methanolic extract of callus and 90.76 μg/mL for methanolic extract of wild plant. KEYWORDS: Indigofera Aspalathoides, Murashige and Skoog (MS) Medium, Callus Induction, Antioxidant Activity, Antiproliferative Activity, A375 Cell Line. INTRODUCTION Medicinal plants play a key role in the human healthcare. About eighty percent of the world populations rely on the traditional medicine, which is predominantly based on plant materials. Large number of medicinal plants and their purified isolated metabolites have been shown to have beneficial therapeutic potential (Agbar et al., 2008). The plant Indigofera aspalathoides is commonly called as “Sivanar vembu” in tamil. In the traditional medicinal system, leaves, flowers and tender shoots are said to posses cooling and demulcent effect. They are used in the form of decoction for the treatment of leprosy and cancer (kirtikar and
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RAAMAN NANJIAN* SRIRAM CHANDRASEKARAN** ABSTRACT · SRIRAM CHANDRASEKARAN** *Professor, Natural Products and Tissue Culture Laboratory, Cent re for Advanced Studies in Botany, University
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JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.625, ISSN: 2320-5083, Volume 3, Issue 6, July 2015
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IN VITRO ANTIOXIDANT AND ANTIPROLIFERATIVE ACTIVITIES OF WILD PLANT AND CALLUS EXTRACTS OF INDIGOFERA ASPALATHOIDES VAHL.EX.DC
RAAMAN NANJIAN*
SRIRAM CHANDRASEKARAN**
*Professor, Natural Products and Tissue Culture Laboratory, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India
**Research Scholar, Natural Products and Tissue Culture Laboratory, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India
ABSTRACT
Indigofera aspalathoides Vahl. ex. DC, belongs to the family Fabaceae and it is
commonly called as wiry indigo. The present study was carried out to study the comparative
account on antioxidant potential and antiproliferative activity between the methanolic extracts
of wild plant and in vitro grown callus. Murashige and Skoog (MS) medium supplemented
with NAA (0.5 mg/L) showed maximum callus induction from root (92%) and nodal explants
(89%). The quantitative phytochemical analysis showed maximum total phenol (382 mg/g)
and total flavonoid content (324 mg/g) in methanolic extract of wild plant followed by callus
(370 mg/g and 318 mg/g). Antioxidant activities by different methods such as, 2,2’-azinobis
and 0.97 under 365 nm and 0.32, 0.39 under iodine vapor for methanolic extract of wild
plant. Methanolic extract of callus showed Rf values of 0.10, 0.14, 0.39, 0.43, 0.48, 0.79 and
0.89 under 365 nm and iodine vapor showed Rf values of 0.84 and 0.90.
According to Puratchikody and Swarnalatha (2011), TLC profile of methanolic
extract of stem of I. aspalathoides showed spots under 365 nm and 254 nm in the solvent
system methanol: chloroform: acetic acid: formic acid (90:5.0:2.5:2.5). Pavala rani
et al. (2013) have reported that ethanolic extract of leaf and aerial parts showed different
spots at different Rf values under 365 nm with the solvent system toluene: ethyl acetate:
acetic acid: methanol (2.5:7.0:0.25:0.25).
Antioxidant activity of I. aspalathoides
With regarding to ABTS•+ activity, methanolic extract of wild plant showed
maximum absorption of 98.84 µg/mL followed by methanolic extract of callus (96.23
µg/mL) which is found to be higher when compared with the standard ascorbic acid (79.72
µg/mL) at 100 µg/mL concentration. The IC50 was 19.16 µg/mL for methanolic extract of
wild plant and 20.78 µg/mL for methanolic extract of callus followed by 41.11 µg/mL for
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standard (Fig. 5). Hydroxyl radical scavenging activity showed maximum absorption of 95.71
µg/mL for methanolic extract of wild plant followed by methanolic extract of callus (92.85
µg/mL) and standard ascorbic acid (94.12 µg/mL) at 100 µg/mL concentration. The IC50
value for methanolic extract of wild plant was 38.89 µg/mL and 50 µg/mL for methanolic
extract of callus followed by standard (41.31 µg/mL) (Fig. 6). Philips et al. (2010) stated that
ethanol and chloroform fractions of I. aspalathoides showed IC50 value for ABTS•+ activity
as 12.1 µg/mL, 8.6 µg/mL and standard as 14.6 µg/mL and hydroxyl radical scavenging
activity of ethanol and chloroform fractions of I. aspalathoides showed IC50 value as 32.5
µg/mL, 21.85 µg/mL and standard as 67.8 µg/mL.
The reduction of Mo (VI) to Mo (V) showed maximum in methanolic extract of wild
plant (72%) followed by methanolic extract of callus (65.27%) which was less when
compared with that of standard (95.65%) at 100 µg/mL concentration (Fig. 7). Fe3+
reduction is often used as an indicator of electron donating activity, which is an important
mechanism of phenolic antioxidant action and can be strongly correlated with other
antioxidant properties. The reducing power of both methanolic extracts of wild plant and
callus was observed maximum (98.5 µg/mL and 92.53 µg/mL) followed by standard
Na2EDTA (91.04 µg/mL) at 100 µg/mL concentration. The IC50 value for methanolic extract
of wild plant was 21.61 µg/mL followed by methanolic extract of callus (31.40 µg/mL) and
standard (41.87 µg/mL) (Fig. 8). According to Thangavel et al. (2014), of the various types
of extracts of Indigofera cordifolia, ethanol extract had more reducing power than other
extracts used. Ebrahimzadeh et al. (2008) have reported that methanol extract of Melilotus
arvensis showed chelating activity of 80 µg/mL concentration.
Antiproliferative activity of A375 cells
Methanol extract of wild plant and callus of I. aspalathoides with the varying
concentrations from 10 μg/mL to 100 μg/mL showed anti-proliferative activity on A375 cell
lines. The normal structural morphology was altered on dose dependent when compared with
that of the normal cell line. IC50 value for the methanolic extract of callus was 86.29 μg/mL
followed by methanolic extract of wild plant (90.76 μg/mL) (Fig. 9, 10).
The DNA fragmentation assay was observed on the IC50 concentrations of the extracts
treated on A375 cells after 24 h incubation. The control cells in the lane 4 of A375 cells
showed intact DNA while the fragmentation was observed in other lanes treated with the
extracts. The DNA fragmentation pattern was observed because of the chromatin
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condensation and nuclear fragmentation which occurred during apoptotic cell death. The
fragmentation pattern showed maximum cell death with the methanolic extract of wild plant
followed by methanolic extract of callus treated on A375 cells (Fig. 11).
According to Ranjitha kumari et al. (2013), methanolic extract of root of
I. aspalathoides showed IC50 value of 50.62 μg/mL and 50.63 μg/mL against NCI h460 (lung
cancer) cells after 24 and 48 h incubation. IC50 value of the extract also showed induction of
DNA fragmentation of the cells treated for 24 and 48h and the maximum was observed after
48h incubation. Chanda and Nagani (2013) suggested that stem extract of
I. aspalathoides possess 95% anticancer activity against various skin disorders and cancer
including Ehrlich’s ascites carcinoma cancer.
CONCLUSION
In the present work, an effective study was carried out to evaluate the antioxidant
potential between methanolic extracts of wild plant and in vitro callus of
I. aspalathoides. The study revealed that the callus also showed maximum antioxidant
activity with that of wild plant due to the presence of secondary metabolites accumulation
present in them. Antiproliferative activity of the A375 cells showed that both methanolic
extracts of wild plant and callus balanced activity against the growth of the cells. Methanolic
extract of callus of I. aspalathoides showed potential inhibitory activity against oxidation due
to the presence of various secondary metabolites present in them.
ACKNOWLEDGEMENTS
One of the author (Sriram Chandrasekaran) thank to University Grants Commission,
New Delhi, for the award of UGC-BSR Herbal Science research fellowship.
REFERENCES
1. Agbar, Z.A.E., Shakya, A.K., Khalaf, N.A. and Haroon, M.A. 2008. Comparative antioxidant activity of some edible plants. Turk j Biol., 32: 193-196.
2. Anusuya, N. and Manian, S. 2013. Antioxidant and free radical scavenging potential of different solvent extracts of Indigofera tinctoria leaves. International Journal of Pharmacy and Pharmaceutical Sciences, 5: 142-147.
3. Bakasso, S., Lamien meda, A., Lamien, C.E., Kiendrebeogo, M., Millogo, J., Ouedraogo, A.G. and Nacoulma, O.G. 2008. Polyphenol contents and antioxidant activities of five Indigofera species (Fabaceae) from Burkina Faso. Pak. J. Biol. Sci., 11: 1429-1435.
4. Cenkci, S., Kargioglu, M., Dayan, S. and Konuk, M. 2008. In vitro propagation of an endangered plant species, Thermopsis turcica (Fabaceae). Biologia, 63: 652-657.
5. Chanda, S. and K. Nagani. 2013. In vitro and in vivo methods for anticancer activity evaluation and some Indian medicinal plants possessing anticancer properties: An Overview. Journal of Pharmacognosy and Phytochemistry, 2: 140-152.
JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.625, ISSN: 2320-5083, Volume 3, Issue 6, July 2015
11 www.jiarm.com
6. Deinstrop, E.H. 2000. “Applied Thin-Layer Chromatography”, Wiley-VCH, Weinheim. 320 p. 7. Dinis, T.C.P., Madeita, V.M.C. and Almeida, L.M. 1994. Action of phenolic derivatives
(acetaminophen, salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys., 315: 161-169.
8. Ebrahimzadeh, M.A., Pourmorad, F. and Bekhradnia, A.R. 2008. Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran. African Journal of Biotechnology, 7: 3188-3192.
9. Gurav, S., Vijay, G., Nandkishore, D. and Arun, P. 2007. Pharmacognosy, Phytochemistry, pharmacology and clinical applications of Gymnema sylverstre RcBr., 1: 414-433.
10. Halliwell, B., Gutteridge, J.M.C. and Aruoma, O.I. 1987. The deoxyribose method: A simple test tube assay for determination of rate constants for hydroxyl radicals. Anal. Biochem., 156: 215-219.
11. Karadeniz, F., Burdurulu, H.S., Koca, N. and Soyer, Y. 2005. Antioxidant activity of selected fruits and vegetables grown in Turkey. Journal of Agriculture and Food Chemistry, 29: 297-303.
12. Kirtikar, K.R. and Basu, B,D. (1975). Glossary of Indian Medical Plants. Volume I. M/S Periodical Experts, Second Edition. Jayed Press, New Delhi, 338 p.
13. Pavala rani, N., C. Moorthi, R. Senthamarai and K. Kathiresan. 2013. Identification and phytochemical evaluation of ethanolic extract of Indigofera aspalathoides (Shivanar vembu). International Journal of Pharmacy and Pharmaceutical Sciences, 5: 508-511.
14. Philips, A., Philip, S., Arul, V., Padmakeerthiga, B., Renju, V., Santha, S. and Sethupathy S. 2010. Free Radical Scavenging Activity of Leaf Extracts of Indigofera Aspalathoides – An in vitro Analysis. J. Pharm. Sci. and Res., 2: 322-328.
15. Prieto, P., Pineda, M. and Aguilar, M. 1999. Spectophotometric quantitative of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal. Biochem., 269: 337‐341.
16. Puratchikody, A. and S. Swarnalatha. 2011. Preliminary phytochemical investigation of methanolic stem extract of Indigofera aspalathoides Vahl. International Journal of Pharmaceutical Sciences Review and Research, 11: 116-118.
17. Rajkapoor, B., Murugesh, N., Chodon, D. and Sakthisekaran, D. 2005. Chemoprevention of N-nitrosodiethylamine induced phenobarbitol promoted liver tumors in rat by extract of Indigofera aspalathoides. Biol. Pharm. Bull., 28: 364-366.
18. Ranjitha kumari, B.D., Rajaperumal, S. and M. Nimmi. 2013. In vitro studies on antimicrobial and antioxidant effect of methanolic extract of Indigofera aspalathoides (Vahl ex DC) and its cytotoxic property against human lung cancer cell line NCI H460. European Journal of Experimental Biology, 3: 18-29.
19. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. 1999. Antioxidant activity applying an improved ABTS radical cation decolourisation assay. Free Radical Biology and Medicine, 26: 1231-1237.
20. Sahranavard, S., F. Naghibi and S. Ghaffari. 2012. Cytotoxic activity of extracts and pure compounds of Bryonia aspera. International Journal of Pharmacy and Pharmaceutical Sciences, 4: 541-543.
21. Selvakumar, S., Karrunkaran, C.M., Rao, M.R.K. and Balasubramanian, M.P. 2011. Inhibitory Effect of Indigofera aspalathoides on 20-methylcholanthrene-induced chemical carcinogenesis. Journal of carcinogenesis, 10: 1-7.
22. Sen, C.K. 1995. Oxygen toxicity and antioxidants: state of the art. Indian journal of physiol. Pharmacol., 39: 177-196.
23. Singleton, V.L. and Jr. Rossi, J.A. 1965. Colorunetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Amer. J. Enol. Viticult., 16: 144-158.
24. Solowey, E., M. Lichtenstein, S. Sallon, H. Paavilainen, E. Solowey and H. L. Galski. 2014. Evaluating medicinal plants for anticancer activity. The Scientific World Journal, 2014: 1-12.
25. Thangavel, D., Govindasamy, J. and Senthil Kumar, R. 2014. In vitro antioxidant and anticancer activities of various extracts of Indigofera cordifolia Roth. Journal of Pharmaceutical Biology, 4: 85-93.
26. Vidoz, M.L., Quesenberry, K.H., Real, D. and Gallo, M. 2012. Plant regeneration of Lotononis bainesii Baker (Fabaceae) through cotyledon and leaf culture. African Journal of Biotechnology, 11: 9724-9731.
27. Vipranarayana, S., Prasad, T.N.V.K.V. and Damodharam, T. 2012. In vitro seed germination and induction of enhanced shoot multiplication in Pterocarpus santalinus Linn.f: An endemic medicinal plant of Seshachalam hills, Tirumala. Int. J. Pure Appl. Sci. Technol., 9: 118-126.
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