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IN VITRO CONSERVATION and PROTECTIVE EFFECT of Premna Serratifolia L

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IN VITRO CONSERVATION and PROTECTIVE EFFECT of Premna Serratifolia L International Journal of Pharmaceutical Applications ISSN 0976-2639.Online ISSN 2278 – 6023 Vol 3, Issue 2, 2012, pp 332-343 http://www.bipublication.com IN VITRO CONSERVATION AND PROTECTIVE EFFECT OF Premna Serratifolia L. – AN IMPORTANT MEDICNAL TREE. *1C. Ravinder Singh, 2 R. Nelson, 1 N. Sithranga Boopathy, 1K. Kathiresan and 3Chinta.Sudheer Kumar *1 ,Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai- 608 502, Tamil Nadu, India. 2Department of Botany, Government Arts College, Ariyalur- 621 713, Tamil Nadu, India. 3Department of Bio- Technology, IST, University College of Engineering, JNTU-Kakinada, A.P-533 003. *1 Corresponding Author E- mail: [email protected] [Received 25/03/2012, Accepted27/04/12] ABSTRACT An attempt for conservation of medicinal important shrub Premna serratifolia L. through i n vitro morphogenesis was achieved from callus tissue derived from leaf explants, production of Luteolin through callus culture using different explants and screening of callus derived Luteolin for anti inflammatory effect of Premna serratifolia L. The leaf explants excised from 8 years old Premna serratifolia L. and achieved highest frequency of callus mass from MS medium supplemented with 3% sucrose as carbon source, 0.8% agar and 5.0mg/l IAA. Regeneration of adventitious buds from calli and maximum number of shoots were achieved when calli were cultured on MS medium fortified with 3.0mg/l BAP in combination with 0.5mg/l KN. Regenerated shoots produced prominent roots when they were transferred on MS medium with 1.0mg/l of NAA. Rooted plantlets, thus developed were hardened and successfully established in the soil. In this search 89% of field survivals have been achieved. Among the various callus extracts root and root callus found to be the best biomass source for luteolin production and this root and root callus extracts of Premna serratifolia L. augments that it is having good anti-inflammatory activity against carrageenan induced paw edema. Key Words : IAA: Indole-3- acetic acid, BAP: Benzyl amino purine, KN: Kinetin, paw edema. 1. INTRODUCTION notable interest in medicinal plants for its Medicinal plants are an important source of medicinal properties, approximately 85% of medicines and play a key role in world health traditional medicine preparations involve the use [1,2]. In the past few decades there has been a of plants or plant extracts [3]. Many traditional IN VITRO CONSERVATION AND PROTECTIVE EFFECT OF Premna Serratifolia L. plant based remedies are back in use and find and mass multiplication through controlled increasing application as, (i) source of direct environments. Advances in tissue culture, therapeutic agent (ii) as a raw material base for combined with improvement in genetic the elaboration of more complete semi-synthetic engineering, specifically transformation chemical (iii) as model for new synthetic technology, have opened new avenues for high compounds for the production [4]. Premna volume production of pharmaceuticals, serratifolia L. [syn. Premna obtusifolia R. Br., nutraceuticals, and other beneficial substances Premna taitensis Schauer., Premna. integrifolia [23]. Premna serratifolia L. is dwindling at an Var.] Shrub or small tree about 10 m tall. Leaves alarming rate due to overexploitation by the opposite, petiolate, blades elliptic to oblong, up to pharmaceutical industry and low seed viability 15 cm long and 9 cm wide, with base usually [24]. It is a vulnerable mangrove associates [25] cordate, and the tip pointed. Flowers minute, and also this important species listed in the Red white, 4-5 parted, borne in densely packed Data book of Andhra Pradesh, India [50]. clusters. Fruit is a black globose drupe to 8 mm Conventional breeding in some of the medicinal broad. Flowers and fruits are available throughout plants including Premna serratifolia is hampered the year, belonging to the family Lamiaceae due to poor seed viability and low percentage of (previously: Verbenaceae) is a native of Konkan germination through stem cutting [26]. near the sea from Bombay to Malacca, Immediate measures have to be taken to save widespread throughout the South Pacific, from these species from extinction . Application of India to Malaysia, South-East Asia, and Africa tissue culture methods used for conservation and [5,6]. Root, leaf and stem bark of the plant large scale propagation of plant species [27,28]. produce medicinal substances which are found to This is our second scientific report for possess anti-malarial, febrifuge, galactogenic and conservation of Premna serratifolia L. through in hypoglycemic activity [8-10]. Pepper along with vitro technique. The present search was leaf extract shown significant effects in patients undertaken in order to contribute in developing a of fever and cold, cough, constipation, low cost and efficient protocol for the in vitro Hypertension and cardiac insuffiency [10,11] conservation and screening of anti inflammatory Hepato protective activity [12-15] It is also used effect L. through callus extract of Premna in the ayurvedic preparation of arishtam, serratifolia L.. Kvatham, ghritham [16,17]. Cardio tonic 2. Materials and methods: [18].The decoction of roots is also used to treat Leaf explants were collected from the 8 years old gonorrhea [19], Cancer [20], anti- parasitic agent Premna serratifolia L. growing in [21] and used for infectious disease [22]. Keelathaniyam, Pudukottai District, Tamilnadu, Traditionally it is used to promote menstruation, India. For the present investigation shoot tip, to treat shortness of breath and illness after leaves, node, stem and root of Premna childbirth, to remedy deep pains in bones, to treat serratifolia were selected as the explants. The bone fractures, appendicitis, rheumatic aches, young leaf explants were collected and immersed swellings, headaches, diarrhoea, wounds, in water immediately then they were thoroughly migraine and testicles swollen from hernia and washed under running tap water for 30minutes. also used for the treatment of eye injuries and Teepol (2%) wash for 5minutes, leaf explants inflammations [5]. were sterilized with 80% ethanol for 30 seconds. Medicinal plant products have created a need for Followed by treatment with 0.1% (w/v) mercuric the development of solid scientific protocols for chloride solution (Qualigens, India) for 2-3 the phytochemical investigations and protections, minutes and then thorough washing with C. Ravinder Singh, et al 333 IN VITRO CONSERVATION AND PROTECTIVE EFFECT OF Premna Serratifolia L. sterilized double-distilled water using 5-10 fold 25 ± 1 oC under a relative humidity of 50 to 60% volume rinses to completely remove the mercuric and 12/12 – hour photoperiod, with irradiance of chloride. And inoculations of plant materials 30 µmol m 2 s-1 provided by cool white were carried out in clean laminar air flow fluorescent tubes (Philips India Ltd. Mumbai). chamber under aseptic condition. The callus growth was measured in terms of fresh and dry weight. Fresh weights of callus were 2.1. Methods and culture condition: taken after removing the excess of moisture on MS [29] salts and vitamin medium supplemented the surface using blotting paper. Dry weight of with 30% sucrose (w/v) (Qualigens, India) was callus was determined by drying in a hot air oven H used in all the experiments. P of the medium at 60 0C for 24 h. was adjusted to 5.7-5.8 with 0.1N NaOH or HCL perior to adding agar 0.8% (w/v), (Qualigens, 2.3. Plant materials preparation and India). Culture tubes were plugged with non- Chromatographic separation of Luteolin adsorbent cotton wrapped in double layered gauze cloth. The media was steam sterilized for The in vivo plant parts (leaf, stem and root) were 20 minutes at 121°C. All the culture tubes were collected from 8 years old tree and washed in tap incubated at 16-h photoperiod under illumination water and then chopped into small fragments. of cool-white fluorescent tube with a light Then materials were dried under shade conditions intensity of 3000 lux at 22±1°C. Cultures were for 30 days and the drying operation was carried examined regularly. All the experiments were out under controlled conditions to avoid chemical repeated thrice. Data which showed advantageous changes. The dried samples were powdered effect were induced in the tables and presented in roughly with hands. The powdered samples were mean±SE of 80 explants per treatment. Mean stored in polythene containers at room values with the same superscript were not temperature. The in vitro derived callus materials significantly different (p=0.05%) according to obtained from leaf stem and roots were dried in ο Duncan’s Multiple Range Test (DMRT). hot air oven at 50 C for 48 h. Then the dried 2.2. Luteolin production through callus callus materials were powdered using mortar and culture: pestle and the powdered samples were stored in Various explants (leaf, stem and root) of Premna polythene containers at room temperature. serratifolia L were excised into 0.5 – 1.5 cm long segments and small incision was made on the 2.4. Preparation of TLC plates (Harbone, surface of each explant using a surgical sterile 1998) blade. Stem and root explants were placed The glass plates to be used in TLC were cleaned horizontally on culture medium. In the case of carefully with ethanol in order to remove grease. leaf, the abaxial side of leaflet was in contact Then the slurry of silica gel G in water (20 g of with the medium. All the explants were cultured silica gel per plate) was shaken vigorously for 90 on MS basal medium supplemented with various seconds and coated on the plates to a thickness of concentrations of different auxins ranging from 0.5 mm using a commercial spreader. Then the 1.0 – 7.0 mg/l of 2.4-D, NAA, IAA and IBA for plates were activated at 105° C for 30 minutes callus initiation.
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