Invitro Studies on Callus Culture and Enhanced Andrographolide Production Using Sodium Azide in Andrographis Paniculata

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Invitro Studies On Callus Culture And Enhanced Andrographolide Production Using Sodium Azide In Andrographis Paniculata.

Anitha Chandran N1, Susila Kuruvilla2 and P R Unnikrishna Pillai 3

1,3 Department of Botany, S.D.College, Alappuzha, Kerala-688 003 2Department of Botany, St.Joseph's College for Women, Alappuzha, Kerala-688 001

Abstract

Mutagenic effect of different concentrations of sodium azide on callus production in Andrographis paniculata was examined. Proliferated calli were obtained from Andrographis paniculata leaf explants on Murashige and Skoog’s (MS)medium supplemented with 2mg/l NAA,1mg/l Kinetin , and 50mg/l Phenyl alanine. Various concentration of Sodium Azide was used for this study. In 0.01gm/l concentration of sodium azide treatment for 5 hours showed significant result in fresh weight (1.67+.18gm) and dry weight (0.13+0.01 gm) of callus induction frequency (83.3+5.7) when compared to the control and other samples. Treatment of Sodium Azide at 0.01gm/l concentration for 3 hour resulted with 7 fold increase in andrographolide production compared to control. This study also proved that treatment concentration and treatment time of sodium azide influenced the fresh weight, dry weight of callus, callus induction frequency and andrographolide production .Most effective and significant results were obtained from 0.01gm/l concentration of sodium azide treatments compared to the control

Key words : Andrographis paniculata, sodium azide, Andrographolide, callus proliferation, callus induction frequency

I. INTRODUCTION

Andrographis paniculata (wall.ex. nees) of family Acanthaceae is most popular medicinal plant for the pharmaceutical values. The plant is widely seen in India, China, Srilanka, Taiwan, and other south East Asian countries. It is commonly used for the treatment of common cold, diarrhea, fever, respiratory tract infection,[1],[2],[3]. This herb has many common names such as Kalmegh, Nelavepu, etc. This herb is commonly known as bhui-neem, because of its bitter taste as that of neem (Azadiracta indica). This plant contains pharmaceutically important molecules such as diterpenoids, flavanoids, and polyphenols [4].Andrographis paniculata has diverse therapeutic potential including anti malarial, [5], antioxidant [6], antibacterial [7] and for anticancer activity [8]. In clinical study, andrographolide was reported to inhibit human immunodeficiency virus (HIV) induced cell cycle dyqsregulation and to increase CD4+ lymphocytes in HIV-I infected patients [9]. Andrographolide, neoandrographolide and 14 deoxy 11 12 didehydroandrographolide have been studied for their anti allergic, anti inflammatory and cardiovascular effects. [10],[11]. Genetic variability is fundamental for successful breeding programs in vegetatively and sexually propagated plants. The variation can be occurred naturally or can be induced through mutations, using physical and chemical mutagens. Mutations are used to produce many cultivars with improved economic value. [12]. Genetic variation were created by physical and chemical induced mutation and it resulting in improved varieties with better characteristics [13].

Sodium azide is one of the most powerful chemical mutagen used to induce mutations in plants .Sodium azide is used to generate genetic variability in plant breeding has been reported in Helianthus annuus [14] and other crops. The present study was aimed to analyze the effect of various concentrations of sodium azide on callus induction, fresh weight, dry weight of callus and production of andrographolide in Andrographis paniculata

II. MATERIALS AND METHODS

One month old healthy seedlings and the chemical mutagen sodium azide were used in the study. Plants were maintained in the green house of S.D. College. Plants were divided into 4groups. The first one was maintained as control and the other 3 groups were treated with 0.01gm/l concentrations of sodium azide, for1 hour, 3 hour and 5 hour respectively. The leaves explants were washed using mild detergent (labolene solution) followed by washing under tap water for 30 minutes . Leaf explants were surface sterrilized with 0.1% (w/v) mercuric chloride solution for 5 minutes and washed 4-5 times with sterile double distilled water .

A. Culture medium and culture condition.

Full MS medium [15] was the basal medium used for this study. Full ms medium and half ms medium with different concentration and combinations of growth hormones like kinetin, NAA and 2 -4 D were used in trial and error methods. Maximum response of callus induction and proliferation were gained from full ms medium . Hence the remaining parts of this work were conducted in full ms medium .Culture was incubated in the culture room at 24+ 10 C under white light condition. Different parameters such as fresh weight, dry weight, callus induction frequency and days for callus initiation were analyzed in the study.

III. RESULTS AND DISCUSSION

Leaf explants inoculated into ms medium containing 2mg/l NAA, 1mg/l kinetin and 50mg/l phenyl alanine showed faster and better response. Control calli were green in colour after 50 days and then turned into brown. All explants were treated with 0.01gm/lconcentrations of sodium azide produced calli with creamish white and later became brownish in colour. Experimental parameters were studied after 70 days of inoculation. Callus initiation was observed after 14-15 days of explants inoculation in the case of control. Fresh weight and dry weight of control calli were 0.61+0.02 gm/l and .04+.009gm/l respectively. Callus induction frequency of control calli was 56.6+5.7%. Amount of andrographolide present in the control calli was 0.01%w/w.

A. Effect of sodium azide in callus.

The influence of sodium azide treatment on the production of andrographolide from callus culture in Andrographis paniculata reveled irregular response . Explants treated with 0.01gm/l sodium azide for 1 hour inoculated into the medium, the callus induction started within 11 -12 days. Callus induction frequency was 76.6+_5.7% in this concentration. Fresh weight (1.23+.19gm) and dry weight (.07+0.009 gm) were more than control. High amount of andrographolide ( 0.06%w/w) were obtained from this concentration and time of the sodium azide treatment compared to control. 0.0 1gm/l concentration of sodium azide for 3 hour showed better results in all the parameters. Callus proliferated from treated explants after 11-12 days of inoculation. Callus induction frequency was 73.3+_5.7% and

@IJAPSA-2016, All rights Reserved Page 255 International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 02, Issue 06, [June- 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X its fresh weight was 1.35+-0.07gm and dryweight was 0.12+-.01gm respectively. An increase in andrographolide content (0.07%w/w) was observed with 0.03 gm/l sodium azide treatment compared to control (0.01%w/w). However, a significantly large increase in andrographolide content (0.07%w/w) was obtained from this concentration . In the 0.01 gm/l concentration of sodium azide for 5 hour explants showed rapid callus initiation after 10-11 days and the Callus induction frequency was 83.3+5.7 %.(table 1) After the treatment of sodium azide in this concentration, a marginal increase in callus induction frequency was observed. Fresh weight and dry weight of the calli produced from the explants treated with 0.01gm/l sodium azide for 5hour were 1.67+.18 gm and 0.13+0.01gm respectively. Samples treated with 0.01gm/l concentration of the sodium azide for 5 hour showed very effective results(Table 1). Fresh weight and dry weight of the callus of these treated samples were higher than the control and other treated samples. Further a marginal decline in andrographolide content (0.06%w/w) was observed in this case .

Table 1: Effect of 0.01 concentration of Sodium azide at different time intervals in callus culture and andrographolide production. Sample Concentration of Time Day for callus % of Fresh Dry Sodium Azide (Hr) initiation (%) response weight(gm weight(gm) s) 1 Control -- 14 -15 56.6 - 5.7 b 0.61-0.02 c .04-.009 c 2 0.01 1 11-12 76.6-5.7 a 1.23-.19 b .07-0.009 b 3 0.01 3 11-12 73.3-5.7 a 1.35-0.07 b 0.12-.01 a

4 0.01 5 10-11 83.3-5.7 a 1.67-.18 a 0.13-0.01 a F value=df(n-1)=3 11.58*** 29.5*** 29.13***

Means with in column followed by same letters are not significantly (p<0.05) different as determined by Duncan’s Multiple Range Test.* significant(p<0.05)F value, ** significant(p<0.01) F value ,*** significant (p<0.001) F value.

Figure 1: Control Figure 2: .01 concentration of sodium azide for 1 hour.

Figure 3: Figure 2: .01 concentration of sodium Figure 4: .01 concentration of sodium azide for 3 hours. azide for 5 hours.

In all concentration of sodium azide treatments showed significant response compared to control. Significant amount of andrographolide (0.07%w/w) were obtained from explants which treated with 0.01mg/l concentration of sodium azide for 3 hour . This result reveled that most efficient concentration for production of andrographolide in Andrographis paniculata using sodium azide is 0.01mg/l for 3 hour . Treatment of sodium azide at 0.01gm/l for 3 hour promoted 7 fold (0.07%w/w) enhancement in andrographolide production compared to control (0.01%w/w) .This result indicated , lower amount of sodium azide were positively influences the production of andrographolide in callus and callus growth.

HPLC chromatograms of a Andrographolide standard; b Control ; c 0.01 Concentration of Sodium azide for 1 hour; d 0.01 Concentration of Sodium azide for 3 hour: e 0.01 Concentration of Sodium azide for 5 hour

Table 2: Amount of andrographolide present in different samples

samples Concentration Time Amount of andrographolide(%w/w) 1 control - 0.01 2 0.01 1 0.06 3 0.01 3 0.07 4 0.01 5 0.06

Sodium azide is more effective in 0.05 for callus induction in Stevia rebaudiana.[16] In Andrographis paniculata the treated leaf explants showed higher callus induction response when compared to the control. In Daucus carota L , multiplication of callus was only achieved on ms medium containing 200µM sodium azide from all the different concentration of sodium azide.[17] Callus growth was decreased by the higher concentration of sodium azide, was supported by the result in different plants like Mirabilis jalapa .[18]

IV. CONCLUSION

The effect of Sodium azide treatment on fresh weight, dry weight, callus induction frequency , days for callus induction and production of andrographolide on invitro callus were recorded in the present study. Amount of all the growth parameters were increased in all treated concentrations of sodium azide when compared to the control. Maximum response of andrographolide production(0.01%w/w) was noted in the explants treated with 0.01gm/l sodium azide for 3 hours . Concentration and treatment time of chemical mutagen played a major role in this experiment.

BIBLIOGRAPHY

[1] Negi, AS; Kumar, JK; Luqman, S; Shanker ,K; Gupta ,MM and Kbanuja SP (2008) recent advances in plant hepatoprotectives: a chemical and biological profile of some important leads. Med Res Rev 28:746–772 [2] Sareer ,O; Ahmad, S and Umar ,S (2014) Andrographis paniculata: a critical appraisal of extraction, isolation and quantification of andrographolide and other active constituents. Nat Prod Res 9:1–21[11]. [3] Wang, J; Yang ,W; Wang ,G; Tang ,P and Sai Y (2014) Determination of six components of Andrographis paniculata extract and one major metabolite of andrographolide in rat plasma by liquid chromatography–tandem mass spectrometry. J Chromatogr B 951–952:78–88 [4] Chao, WW and Lin BF (2010) Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian). Chin Med 17:1–15 [5] Mishra, K; Dash, AP and Dey N (2011) Andrographolide: a novel antimalarial diterpene lactone compound from Andrographis paniculata and its interaction with curcumin and artesunate. J Trop Med 2011 Article ID 579518:1–6 [6] Lin, FL; Wu, SJ; Lee ,SC and Ng, LT (2009) Antioxidant, antioedema and analgesic activities of Andrographis paniculata extracts and their active constituent andrographolide. Phyto Res 23:958–964 [7] Burm ,F; Kumar, OA; Naidu, LM and Rao KG (2010) In vitro antibacterial activity in the extracts of Andrographis paniculata. Int J Pharm Tech Res 2:1383–1385 [8] Subramanian, R; Asmawi ,MZ and Sadiku A (2012) A bitter plant with a sweet future. A Comprehensive review of an oriental medicinal plant: Andrographis paniculata. Phytochem Rev 11:39–75 [9] Calabrese, C; Berman, SH and Babish, JG (2000) A phase I trial of andographolide in HIV positive patients and normal volunters. Phytother Res 14:333–338 [10] Yoopan, N; Thisoda ,P; Rangkadilok, N; Sahasitiwat, S; Pholphana ,N; Ruchirawat, S and Satayavivad J (2007) Cardiovascular effects of 14deoxy11, 12didehydroandrographolide and Andrographis paniculata extracts. Planta Med 73:503–511

[11] Wang ,J; Yang, W; Wang, G; Tang, P and Sai ,Y (2014) Determination of six components of Andrographis paniculata extract and one major metabolite of andrographolide in rat plasma by liquid chromatography–tandem mass spectrometry. J Chromatogr B :951–952 [12] Broertjes, C. and Van Harten,A M 1988. Applied Mutation breeding vegetatively propogated Crops. Elsevier, New York, USA., ISBN-13: 9780444427861, pages:345. [13] Wongpiyasatid, A.; Chotechuen, s; Hormchan ,p; Ngampongsai ,S and Promcham,W. 2000. Induced mutations in mungbean breeding: regional yield trial of mungbean mutant lines. Kasetsart J.(Nat.Sci.), 34:443-449 [14] Mostafa, G.G, 2011. Effect of Sodium Azide on the growth and variability induction in Helianthus Annus L. Int. J.Plant Breed. Genet, 5:76-85 [15] Murashige, T.and F.Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant.,15:473-497 [16] Snehal pande,and Madhukar khetmalas (2012) Biological effect of sodium azide and colchicines on seed germination and callus induction in Stevia Rebaudiana .Asian j.exp.biol.sci , vol.3 (1):93-98. [17] Nagananda, G.S; Rajath, s; Disha Agarwal; Ruby Mathew, k. and Sundara Rajan ,s (2013) Induced mutation in callus cell lines of Daucus carota L. Asian journal of plant science 12(1): 46-50 [18] Al-Gawwad, A. and Makka ,A H 2009. Effect of chemical mutagens on Mirabilis jalapa. M.Sc. Thesis, Floriculture, Faculty of Agriculture, AlexandriaUniverity .