Vol-3, Issue-4, Suppl-1, Nov 2012 ISSN: Bidari et al

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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES IN VITRO REGENERATION OF ANDROGRAPHIS PANICULATA NEES AN IMPORTANT MEDICINAL PLANT Sanjeevkumar Bidari 1*, Anitha S 1, Rajshekaran P E 2, Shashidhara S 1, Praveen Ningappa Tatti 1 1 Department of Pharmacognosy, Government College of Pharmacy, Bangalore-27 2 Indian Institution of Horticulture Research, Hesaragatta, Bangalore, Karnataka. ABSTRACT The present study reveals that the in vitro regeneration of nodal explants of Andrographis paniculata were transferred in MS (Murashige & Skoog s) medium supplemented with various auxins and cytokinins at different combinations and concentrations. Nodal explants of Andrographis paniculata showed better response with the combination 2mg/l BAP+0.1mg/l BAP +0.1mg/l Kinetin and 2mg/l BAP + 2mg/l Kinetin. And best rooting was recorded with half strength MS +2mg/l IBA + 1% sucrose.ms media without growth regulators shows better growth. Hence it is concluded that, it is rapid and efficient method for the large scale propagation of a highly valuable medicinal plant Andrographis paniculata Nees through in vitro culture of nodal explants. Keywords: Andrographis paniculata, in vitro regeneration, MS (Murashige & Skoog s) medium, BAP (Benzyl amino purine) Kinetin and IBA (Indole butyric acid). INTRODUCTION The drug consists of dried leaves and tender parts of Andrographis paniculata Nees belonging to the family Acanthacea [1], it is an annul herb found throughout India and tropical Asia. Andrographis paniculata is distributed in tropical Asian countries, often in isolated patches. It can be found in a variety of habitats, e.g. plains, hill slopes, waste lands, farms, dry or wet lands, sea shores.native populations of Andrographis paniculata are spread throughout south India and Sri Lanka which perhaps represent the center of origin and diversity of the species. The herb is also available in northern parts of India, Java, Malaysia, Indonesia, West Indies and elsewhere in the Americas where it is probably an introduced species. The species also occurs in Hong Kong, Thailand, Brunei and Singapore etc. However, precise data are lacking on the introduction and naturalization of the species in these countries. This plant is currently collected from different geographical sources like Bangalore [2].Due to its bitterness property it is also known as King of bitters. It contains diterpene lactones i.e. andrographolide ( %), and a bicyclic diterpenoid lactone and kalmeghin, neoandrographolide, IC Value

2 andrographanines A,B,C,D & E, 14-deoxy-11, 12-deoxyandrographolide and andrographanin [3]. It is known to possess various pharmacological activities like hepatoprotective, anti-inflammatory, anthelmintic, antimalarial, antipyretic, antibiotic, immunostimulant, antiallergic, antihypertensive, anticholastatic, antithrombotic activities [1,3]. In the present communication in vitro regeneration of Andrographis paniculata from the nodal explants is reported. Micropropagation is a unique method for the production of uniform and disease free plants. This technique is to facilitate the large number of uniform plants in irrespective of season and will serve as an alternative source of seed materials. MATERIALS AND METHODS Collection and authentification of plant material: It was collected from Bangalore and authenticated by Dr.P. Santhan, plant taxonomist, Natural Remedies pvt. Ltd. Bangalore. The research work was carried out in the department of Pharmacognosy, Govt. college of Pharmacy and tissue culture room in Indian Institute of Horticulture Research, Hesarghatta, Bangalore. Materials required: Hot air oven, autoclave, laminar air flow, ph meter, culture tubes, paraffin film, scalpels, scissors, forceps and culture stands. Chemicals: Hormones or phytohormones i.e. BAP (benzyl amino purine), kinetin, IBA (indole butyric acid) and MS (Murashige & Skoog s) medium obtained from Hi media chemicals corporation [4,5]. Preparation of media and its sterilization: MS (Murashige & Skoog s) media collected from Hi media was used as basal medium 4.4g of the medium was taken with 100ml of distilled water and solubilized with the aid of slight heat. ph of the medium was adjusted to before sterilization using 0.1N NaOH. Different combinations of the hormones were then added and mixed thoroughly to dissolve the contents. Homogeneous solution was taken into different culture tubes with autoclavable caps and sterilized in autoclave at 121 ºC temperature and 15 lb psi pressure for 15 to 20 minutes. After sterilization the medium was allowed to solidify in test tubes under aseptic conditions [6]. Growth regulators/phytohormones: Different hormones like BAP, Kinetin and IBA were accurately weighed and dissolved in 2-3 drops of 1M sodium hydroxide and the final volume was adjusted with sterile double distilled water to get a concentration of IC Value

3 100μg/ml. This stock solution was added in different ratio as per the requirements. All the stock solutions were freshly prepared and used. Maintenance of aseptic conditions: The aseptic room was sprayed with Acetone: ethanol (2:8) prior to aseptic transfer, the laminar airflow bench was swabbed with 70% alcohol using absorbent cotton. scissors, forceps and scalpels used for aseptic transfer were dipped in 70% alcohol first and sterilized by flaming. The hands were also swabbed with ethanol before transfer. Method: The fresh nodal explants were collected from three months old planted and washed thoroughly under running tap water for 15 min to remove any adhering dirt, followed by distilled water and then washed with savlon soap solution for 10 min, followed by distilled water for 3 mins. Then the washed explants were taken in to aseptic room and they were soaked in 70% alcohol for 30 seconds and rinsed with double distilled water for 3-4 times. Explants were surface disinfected with 0.01% Hgcl 2 (mercuric chloride) for 3 minutes followed by 3 times with distilled water. The surface sterilized explants were inoculated on MS medium with different concentrations of cytokinins for shoots and auxins for root formation. Fifteen explants were used for each hormonal combination and data was recorded after 10 of initial culture, sucrose was used as carbon source into the media and agar (solidifying agent) 8g/l and hormones as per requirement were added. The ph was adjusted to before sterilization of media using 0.1 N NaOH and dispensed in to glass tubes. The medium was sterilized by autoclaving at C temperature, 15 lb pressure for 20 minutes. After inoculation all the culture tubes were kept under photo period of 16 hours light (with white fluorescent tubes) at a room temperature 25±1 0 C. The culture tubes were observed at regular intervals for the initiation of growth. After 30 of culturing, the shooted plantlets were transferred to a MS medium supplemented with auxins at different concentration for rooting. The culture tubes were observed at regular intervals for the initiation of growth of adventitious roots from shooted plantlets. Rooted plantlets were removed from the culture tubes and media was washed thoroughly with sterilized double distilled water. The rooted plantlets were transferred to a polythen bag containing soil sand: cocopeat: compost at the ratio 1:1:1. IC Value

4 Plantlets were watered every two for two weeks in green house. The plantlets were initially established in the field for hardening [7,8,9]. RESULTS Regeneration of shoots from nodal explants of Andrographis paniculata was observed at different hormonal combinations and concentrations (Fig. No 1). The highest percentage of shoot proliferation was recorded at 2mg/l BAP and 0.1mg/l BAP+0.1mg/l Kinetin. MS media without growth regulators shows better growth. Best rooting was recorded with half strength MS medium+2mg/liba+1% sucrose (Fig. No 2). The root induced plantlets were successfully transferred for hardening after 30 (Fig. No. 3). About 90% of the in vitro regenerated plants were healthy and useful for propagation. DISCUSSION The combination of soap solution %w/v mercuric chloride+70%v/v alcohol with the treatment time of 10+30sec+2 respectively was found to be the best surface sterilizing agent. Increase in the concentration of mercuric chloride more than 0.1%, led to the browning followed by subsequent death of the explants, without any growth. Decrease in the concentration of mercuric chloride and increase in treatment time with less concentration of mercuric chloride was not contaminated. The culture media was sterilized at 15lb psi for 15-20minutes after adding the requisite concentrations of phytohormones and allowed to set and solidify at room temperature. The explants were trimmed at the cut edges after surface sterilization and inoculated into the culture tubes. The inoculated culture tubes were incubated at 25±1 0 C under the fluorescent tube light in the culture tube rack and a photoperiod of 16 hours was maintained. The frequency of initiation was observed after 15 of incubation. The growth of explants however started at 7 8. A combinations of 2mg/l BAP and 0.1mg/l BAP+0.1mg/l kin was found to give very good results. Initially multiple shoots were not formed on the nodal explants. Genotype, tissue type and developmental stage may all be determining factors in the comparative ability to respond to auxin or cytokinin. Shoot multiplication was observed only after the subculturing of nodal explants of regenerated shoots on same medium. For root regeneration individual regenerated healthy shoots (4 to 5cm long) were excised and transferred to half strength and quarter strength of MS medium supplemented with different concentrations and combinations of IBA and IAA. Best IC Value

5 rooting was recorded on half strength MS medium supplemented with 2mg/l IBA + 1% sucrose. Root induction was not observed on quarter strength MS + 1mg/L IAA +.05mg/l IBA even after 40 of incubation. The in vitro regenerated plants from nodal explants were washed thoroughly to remove adhered media and then transferred to a small plastic pot containing sand, coco peat and compost in the ratio of 1:1:1. Plants were watered every two for two weeks with the survival rate of 86%. A photoperiod of 16 hours was maintained in indigenously designed green house to acclimatize the plantlets with the new environment. After 15, plants were transferred to normal soil conditions [10, 11]. ACKNOWLEDGEMENT We are greatly thankful to Indian Institution of Horticulture Research, Hesaragatta, Bangalore, for providing facilities for doing this work. We are also grateful to Mr. Havagirai Shrigire for providing plants at the time of our research work. TABLE 1: HORMONAL COMBINATIONS USED FOR INITIATION AND PROLIFERATION OF SHOOTS OF ANDROGRAPHIS PANICULATA BY USING NODAL EXPLANTS. Sl.No. Media composition 1) MS media 2) MS + 1mg/l TDZ 3) MS + 1mg/l IBA 4) MS + 2mg/l BAP 5) MS + 2mg/l Kin 6) MS + 2mg/l BAP + 2mg/l Kin 7) MS + 0.1mg/l BAP + 0.1mg/l Kin 8) MS + 0.4mg/l IBA + 0.8mg/l BAP 9) MS + 4mg/l BAP + 0.5mg/l NAA 10) MS + 2mg/l BAP + 0.5mg/l NAA Based on the above observations, four different hormonal combinations and concentrations were selected for optimization purpose. IC Value

6 TABLE 2: HORMONAL COMBINATIONS SELECTED FOR SHOOT REGENERATION FROM NODAL EXPLANTS Sl. No. Growth regulators Observations after 10 after 20 after MS + 2mg/l BAP Growth Growth Growth 2. MS + 2mg/l Kin Growth Growth Growth 3. MS + 2mg/l BAP + Growth Growth Growth 2mg/l Kin 4. MS + 0.1mg/l BAP + 0.1mg/l Kin Growth Growth Growth TABLE 3: COMPOSITION OF MEDIA FOR IN VITRO ROOT REGENERATION ON ANDROGRAPHIS PANICULATA. Sl. No. Media composition 1. Half strength MS + 2mg/l IAA + 1% sucrose 2. Quarter strength MS + 1mg/l IAA + 0.5mg/l IBA after 10 No response Observations after 20 Small whitish colored root was induced after 30 Main root was elongated & adventitious roots were formed No response No response No response IC Value

7 Figure 1 In vitro shoot regeneration. IC Value

8 Figure 2 In vitro root regeneration IC Value

9 Figure 3 Hardening of the rooted plantlets of Andrographis paniculata REFERENCES 1. Kokate CK, Purohit AP, Gokhale SB: Pharmacognosy. Nirali Prakashan, Pune, 26th edition Coon, JT, Ernst: Andrographis paniculata in the Treatment of Upper Respiratory Tract Infections: A Systematic Review of Safety and Efficacy. Planta Medica Journal 70, Indian Herbal Pharmacopoeia, Indian Drug Manufacturers, Mumbai, Revised edition, Gupta PK: Plant Cell and tissue culture. Elements of biotechnology, Rastogi Publication, New Dehli, 2000: Satyanarayana U. Bitechnology. Plant Tissue Culture Media. Anuradha sen books and Allied Pvt. Ltd., Kolkatta,2009: Chawla HS: Types of culture. Introduction to Plant Biotechnology. Oxford and IBH Publishing, New Dehli, 2 nd edition, 2008: IC Value

10 7. Altaf Ahmed. Shashidhara S, Rajshekharan PE, Harish Kumar V and Honnesh NH: In vitro regeneratiom of Acorus calamus an important medicinal plant. Journal of Current Pharmaceutical Research 2010;2: Praveen N et al:production of andrographolide from adventitious rootcultures of Andrographis Paniculata. Current Science2009; 96: Martin KP: Plant regeneration protocol of medicinally importance of Andrographis paniculata (Burm.F.)Wallach Ex Nees via somatic embryogenesis. In Vitro Cellular Development Biology-Plant2004; 40: Kalimuthu K, Saravanakumar M and Senthilkumar R: In vitro micropropagation of Musa sapientum L. (Cavendish Dwarf). African Journal of Biotechnology 2007; 6: Saroj K Sah et.al: In vitro study of Tinospora cordifolia (Wild) Miers (Menispermaceae). Journal of Plant Science 2009; 6: For Correspondence: Sanjeevkumar Bidari sanjuklespharma@gmail.com IC Value