International Journal of Botany and Research (IJBR) ISSN 2277-4815 Vol. 3, Issue 4, Oct 2013, 1-6 TJPRC Pvt.

. Ltd.

IN VITRO CALLUS PRODUCTION AND ANTI-BACTERIAL ACTIVITY OF BARLERIA PRIONITIS LINN. AGAINST DENTAL CARIES PATHOGENS
PURNIMA KUMARI1, POONAM YADAV2, ARVIND ARYA3 & SANDEEP KUMAR4
1,2,4 3

Department of Biotechnology, NIET, NIMS University, Jaipur, Rajasthan, India

Department of Biotechnology, Meerut Institute of Engineering and Technology, Meerut, Uttar Pradesh, India

ABSTRACT
Callus culture of Barleria prionitis Linn were established from leaf explants on MS medium supplemented with IAA and BAP in various combinations. The maximum production of calli was observed in the combination of IAA (2.0 mg/l) and BAP (1.5 mg/l). The methanolic extract from fresh callus cultures of Barleria prionitis which was cultured on MS medium fortified with IAA (2.0 mg/l) and BAP (1.5 mg/l) for 2 - 12 weeks was observed and examined for the potency of their phytochemicals against dental caries pathogens. Phytochemical screening revealed the presence of alkaloids, tannin and phenolic compounds, terpenoids, steroids, saponins, flavonoids, carbohydrates, glycoside and cardiac glycoside. The antibacterial activity of the methanolic extract of the calli showed the activity against the Streptococcus mutans, Staphylococcus aureus, Lactobacillus acidophilus and Pseudomonas sp. Barleria prionitis leaf derived callus extract showed antibacterial activity against all but the strongest anti-bacterial activity against Lactobacillus acidophilus with MIC value of 0.250 mg/ml. The anti-bacterial potency of callus of leaf explants of Barleria prionitis against Lactobacillus acidophilus was compared with the standard antibiotic drug Ciprofloxacin.

KEYWORDS: Callus Culture, Bacterial Strains, Streptococcus mutans, Staphylococcus aureus, Lactobacillus
acidophilus, Pseudomonas sp., Barleria prionitis Linn., In vitro, Minimum Inhibitory Concentration

ABBREVIATION: IAA (Indole-3- acetic acid), MS (Murashige and Skoog), BAP (6-Benzylaminopurine), MIC
(Minimum inhibitory concentration), DMSO (Dimethyl sulphoxide).

INTRODUCTION
Barleria prionitis Linn. (Acanthaceae) is a threatened perennial medicinal shrub. It is commonly known as Porcupine flower, Barleria, Kundan, Mullu goranti, Pilikantashelio etc. The plant is especially well known for treating bleeding gums and toothache. Because of its antidontalgic property it is also known as Vajradanti (Bhogaonkar and Lande, 2012). In India it is mainly found in Andeman and Nicobar Island, Andhra Pradesh, Assam, Bihar, Delhi, Chhatisgarh, Uttarakhand, Uttar Pradesh and West Bangal, Kerala and Rajasthan etc. (Shendage and Yadav, 2010) and also found in many parts of the world, like USA, Australia, Indonesia, Malaysia, Philippines, Asia, Africa and Yemen. The whole plant parts (leaves, flowers and roots) are used in traditional Indian medicines for treatment of catarrhal affections, whooping cough, inflammations, urinary infection, jaundice, fever, gastrointestinal disorders, bronchial asthma, cough, boils and glandular swellings (Khare, 2008). The plant is used in many formulations and preparations like Rasnadi kvatha, Rasnadi churna, Sahachara ghritha, Sahachara taila and Dantaroganashani churna (Bhogaonkar and Lande, 2012; Sharma et al., 2002). The leaves are used to promote healing of wounds and to relieve joint pains (Parrotta, 2001). Extracts and isolated phytochemicals from this plant have been found to possess wide range of pharmacological include antimicrobial (Shukla et al., 2011), antifertility (Gupta et al., 2000), antihelminthic, antioxidant

Purnima Kumari, Poonam Yadav, Arvind Arya & Sandeep Kumar

(Chavan et al., 2010), antidiabetic (Dheer and Bhatnagar, 2010), anti-inflammatory (Singh et al., 2013), anti-arthritic, cytoprotective, hepatoprotective, diuretic, antidiarrhoeal, and enzyme inhibitory activities without any toxic effects (Amoo et al., 2009). The extracts of the plant are incorporated into herbal cosmetics and hair products to promote skin and scalp health (Yadav et al., 2011). Whole-plant extracts of Barleria contain iridoid glycosides, barlerin and verbascoside which have potent activity against respiratory syncytial virus in vitro and may account for the plants use in treating fever and several respiratory diseases in herbal medicine (Chen et al., 1998). Due to its wide area of medicinal and theraupatic value large scale production of this plant is required. Barleria prionitis L have low percentage of seed viability (Menges and Gordon, 1996). So it is not possible to narrow down the gap between the requirement and its production. Plant tissue culture techniques can provide an alternate answer to its cultivation related problems in short span of time with large number of plantlets. Keeping all the above mentioned points in view the present study was undertaken to save the endangered plant and to determine the anti-bacterial activity of plant leaf and its callus against dental caries causing pathogens.

MATERIAL AND METHODS

Plant Materials Young leaves of Barleria prionitis Linn. were used as explant to grow callus culture. The leaves were washed with distilled water and sterilized by 0.1% solution of mercuric chloride for 2 minutes and 70% ethyl alcohol for 30 seconds. The explants was followed by washing with sterile double distilled water and after that small piece of explants were inoculated in sterilized MS basal media with concentration of IAA and BAP. Drugs and Chemicals The drugs and chemicals used were ciprofloxacin (Indocap, Jagsonpal Pharmaceuticals Ltd., Faridabad) and methanol (Himedia Laboratories Pvt. Ltd., Mumbai). MS basal medium (Murashige and Skoog, 1962) (Himedia Laboratories Pvt. Ltd., Mumbai), Muller Hinton agar media (Bacteriological Himedia, India), IAA, (Central Drug House Pvt. Ltd., New Delhi) and BAP (Sigma, China) was used in this study. Callus Culture Conditions The sterilized explants were cultured in sterile polystyrene flasks containing 50 ml of MS media fortified with IAA (0.5-3.0 mg/l) and BAP (0.1-2.0 mg/l) prior to autoclaving at 121C (15 psi or 1.5 kg/cm2) for 30 min, the pH of all media was adjusted to 5.8 with 0.1N NaOH and 0.1N HCl. All cultures were incubated at 25C with photoperiod for 16 hrs and darkness for 8 hrs in a culture room. The initiated calli were routinely subculture onto a fresh (multiplication) medium fortified with same concentration of growth regulators as in initiation medium to proliferate the calli at faster rate. Different 2-12 weeks of the callus were taken for the extract preparation. Preparation of Extracts Different weeks of callus of Barleria prionitis were shade dried at room temperature. A powdered callus was successively extracted with methanol using soxhlet apparatus. The extracts were filtered and concentrated under vacuum below 40C for complete removal of the solvent. These dried extracts were exposed to UV rays for 24 hrs and checked for sterility on nutrient agar plates and stored at 4C in glass vials for further use. Phytochemical Screening The methanolic extract was subjected to preliminary qualitative phytochemical screening for the presence of various primary and secondary metabolites like alkaloids, Steroids, flavonoids, saponins, tannins, carbohydrate,

In Vitro Callus Production and Anti-Bacterial Activity of Barleria prionitis Linn. against Dental Caries Pathogens

glycosides/cardiac glycoside and phenolics compounds by employing standard phytochemical tests (Parekh and Chanda, 2006). Media Preparation for Microbes Dehydrated nutrient agar medium (28 g) was accurately weighed and suspended in 1000 ml of distilled water in a conical flask. It was heated on a water bath to dissolve the medium completely and followed by autoclaving. Test Organisms Used Streptococcus mutans, Staphylococcus aureus, Lactobacillus acidophilus and Pseudomonas sp. were isolated from dental caries for the study of antibacterial activity of different stages of callus of Barleria prionitis. Screening of Bacterial Activity Different weeks of callus extracts were tested for antibacterial activity using agar well diffusion assay method (Okeke et al., 2001). The isolated bacteria strains from dental caries were inoculated in different conical flask containing 100 ml of nutrient broth. These conical flasks were incubated at 37C for 24 hrs and were called seeded broth. Prepared media was poured on petri dishes and inoculated with the test organisms from the seeded broth using cotton swabs. With the help of sterile borer 8mm diameter of the well were made for different weeks of callus extracts. Extracts were dissolved in 20% DMSO (Dimethyl sulphoxide) and then 100L of each extract were poured in the wells with the help of sterilized micropipette (Rajasekaran, 2008). The solvent DMSO was used as a negative control and Ciprofloxacin (10 g/disc) was used as positive control. Plates were then incubated overnight at 37C. Antibacterial activity was observed by calculating the zone of inhibition. The experiment was done three times and the mean values were presented. The experiment was performed in triplicates and average diameter of zone of inhibition was obtained. Determination of MIC MIC is the minimum concentration of extract/drug that inhibits the growth of microorganism within 24 hrs (Aneja et al., 2010; Thongson et al., 2004). The MIC for methanolic callus extracts was determined by following the modified agar well diffusion method (Cappuccino and Sherman, 1995). A twofold serial dilution of each extract was prepared by first reconstituting the different weeks of callus extract (1-5 mg/ml) in 20% DMSO followed by dilution in sterile distilled water to achieve a decreasing concentration range of 50 mg/ml to 0.39 mg/ml. A 100 l volume of each dilution was introduced into wells (in triplicate) in the seeded nutrient media agar plates with 100 l of standardized inoculum (106 cfu/ml) of the test microbial strain. All test plates were incubated aerobically at 37C for 24 hrs and observed for the inhibition zones. The lowest concentration of each extract showing a clear zone of inhibition (>8 mm) (in triplicates), considered as the MIC, was recorded for each test organism (Aneja and Joshi, 2009; Nkere and Iroegbu, 2005).

RESULTS AND DISCUSSIONS

Callus growth from leaf explants was observed for twelve weeks on the MS media supplemented with IAA (2.0 mg/l) and BAP (1.5 mg/l) and data was recorded at the interval of two weeks. The callus cultures were maintained in dark conditions in culture room. For further multiplication the callus were transferred on to the multiplication medium. The physical appearance of all calli was friable and ranging from light brown and blackish brown. Callus cultures (2, 4, 6, 8, 10 and 12 weeks) expressed as the increase in fresh and increase or decrease in dry weights from second week to twelve weeks (Table.1). The weights of fresh callus extracts significantly increased with the duration of cultivation. The dried callus weight was maximum of eight week of callus culture as compare to others. (Yadav et al., 2011) was reported best growth on MS media supplemented with NAA (0.5 mg/l) & BAP (2.0 mg/l) and also observed the effect of TDZ

Purnima Kumari, Poonam Yadav, Arvind Arya & Sandeep Kumar

(thidiazuron) on shoot regeneration was with concentration of (0.4 mg/l) combined with BAP (1.5 mg/l) in the same basic media. Barleria lupulina Lindl showed maximum callus production from leaf explants on MS medium supplemented with the combination of IAA (2.0 mg/l) and BAP (2.0 mg/l) and anti-bacterial screening of methanolic extracts of this callus showed activity against Staphylococcus aureus and Bacillus pumilus (Moin et al., 2012). Methanolic extract of callus was subjected to qualitative phytochemical tests having alkaloids, terpenoids, steroids, flavonoids, carbohydrates, glycosides/cardiac glycosides, tannin and phenolic compounds (Table 2). Chavan et al. (2010) reported that the medicinal value of plants lies in some chemical substances like alkaloids, flavonoids, tannins and phenolic compounds which serve as defend against many microorganisms, insects and herbivores. Kumari et al. (2012) also reported that the natural phenolic, alkaloids, tannins, glycosides and flavonoids compounds function as antioxidants. Amoo et al. (2009) assayed COX-1 and COX-2 cyclooxygenase from leaf root and stem of Barleria prionitis having anti-inflammatory activity against acute inflammation. Comparison of MIC value was made between the best recorded 8 th week callus and the positive control (Ciprofloxacin 10 g/ml). The MIC of the methanolic extract was shown in Table 3. Callus showed the strongest antibacterial activity with MIC value of 0.25 mg/ml against Lactobacillus acidophilus and 0.375 mg/ml against Streptococcus mutans, Staphylococcus aureus and Pseudomonas sp. whereas the positive control (Ciprofloxacin) produced MIC values of 10 g for Lactobacillus acidophilus and 20 g for Streptococcus mutans, Staphylococcus aureus and Pseudomonas sp. First time antibacterial activity of Barleria prionitis was reported by Chavan et al. (2010). In present study, the well diffusion method applied for anti-bacterial activity against dental caries causing pathogens showed significant reduction in bacterial growth in terms of zone inhibition around the well. The obtained results showed the gradual increase of zone inhibition from second to eight week of callus extract and after eight week inhibition zones were decreased gradually. But there was maximum zone inhibition of eight week of callus extract of mean value 26.44 0.23 mm, 19.23 0.23 mm, 16.26 0.23 mm and 14.85 0.23 mm against Lactobacillus acidophilus, Pseudomonas sp., Staphylococcus aureus and Streptococcus mutans respectively (Table.4). The negative control of 20% DMSO showed no any zone inhibition against any strains had been taken. Positive control (Ciprofloxacin) produced significant zone inhibition against the test bacteria. The results detected in present study were comparable with Aneja et al. (2010) who advocated that Sapindus mukorosis and Emblica officinalis possesses good antifungal and antibacterial activities respectively to cure dental caries. Chavan et al. (2010) also explained the antibacterial activity by stem and leaf extracts of Barleria prionitis. As compare to present study on anti-bacterial activity of callus extract was much greater than the stem and leaf extracts of Barleria prionitis.

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10. Khare, C. P. (2008). Indian medicinal plants: an illustrated dictionary. Springer. 11. Kumari, P., Sachan, R., Yadav, P., Tomer, P., Arya, A., Tripathi, S. and Kumar, S. (2012). Antiinflammatory activity of in vitro and in vivo Kigelia pinnata (jacq.) Dc from Indian origin. Indian J. Fundam. Appl. Life Sci. 2, 261268. 12. Menges, E. S. and Gordon, D. R. (1996). Three levels of monitoring intensity for rare plant species. Nat. Areas J. 16,. 13. Moin, S., Babu, S. S. and Mahalakshmipriya, A . (2012). In vitro callus production and antibacterial activity of Barleria lupulina Lindl. Asia-Pac. J. Mol. Biol. Biotechnol. 20, 5964. 14. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and biassays with tobacco tissue cultures. Physiol. Plant. 15, 473497. 15. Nkere, C. K. and Iroegbu, C. U. (2005). Antibacterial screening of the root, seed and stem bark extracts of Picralima nitida. Afr J Biotechnol 4, 5226. 16. Okeke, M. I., Iroegbu, C. U., Eze, E. N., Okoli, A. S. and Esimone, C. O. (2001). Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity. J. Ethnopharmacol. 78, 119 127. 17. Parekh, J. and Chanda, S. (2006). In vitro antimicrobial activities of extracts of Launaea procumbens Roxb.(Labiateae), Vitis vinifera l.(Vitaceae) and Cyperus rotundus l.(Cyperaceae. Afr. J. Biomed. Res. 9,. 18. Parrotta, J. A. (2001). Healing plants of peninsular India. CABI publishing. 19. Rajasekaran, C. (2008). Investigations on Antibacterial Activity of Leaf Extracts of Azadirachta indica A. Juss (Meliaceae): A Traditional Medicinal Plant of India. Ethnobot. Leafl. 2008, 161. 20. Sharma, P. C., Yelne, M. B., Dennis, T. J. and Joshi, A. (2002). Database on Medicinal Plants Used in Ayurveda & Siddha. Central Council for Research in Ayurveda & Siddha, Deptt. of ISM & H, Min. of Health & Family Welfare, Government of India. 21. Shendage, S. M. and Yadav, S. R. (2010). Revision of the genus Barleria (Acanthaceae) in India. Rheedea 20, 81130.

Purnima Kumari, Poonam Yadav, Arvind Arya & Sandeep Kumar

22. Shukla, P., Singh, A., Gawri, S., Alexander, A. and Sonwane, S. (2011). In vitro propagation of Barleria prionitis Linn and its antibacterial activity. Int. J. Pharma Prof. Res. 2, 198 200. 23. Singh, K., Kaur, R., Singh, S., Bajwa, B. S. and Prasad, D. N . (2013). Anti-inflammatory Activity of Barleria prionitis Linn. J. Nat. Remedies 13, 13. 24. Thongson, C., Davidson, P. M., Mahakarnchanakul, W. and Weiss, J. (2004). Antimicrobial activity of ultrasound-assisted solvent-extracted spices. Lett. Appl. Microbiol. 39, 401406. 25. Yadav, A., Badkhane, Y., Sharma, A. K., Bakshi, S. H., Raghuvanshi, D. and Lone, S. A. (2011). Effect of different plant growth regulators on in vitro propagation of Barleria prionitis l. A threatened medicinal plant. 2, 438444.

APPENDICES
Table 1: Weights (gms) of Callus Cultures after 2, 4, 6, 8, 10 and 12 Weeks in MS Medium Fortified with IAA (2.0 mg/l) and BAP (1.5 mg/l)
Callus Texture (Friable) Light brown Brown Light brown Light green Light brown Dark brown Blackish brown Fresh Callus Weight (gm SD) 1.70 0.22 1.75 0.19 3.66 0.10 4.70 0.52 6.54 0.48 7.12 0.92 8.40 0.77 Dried Callus Weight (gm SD) 0.11 0.01 0.11 0.01 0.24 0.03 0.30 0.02 0.36 0.01 0.31 0.03 0.36 0.02 Loss on Drying (gm SD) 93.52% 89.14% 93.44% 93.61% 94.49% 95.65% 95.71%

Control 2 week 4 week 6 week 8 week 10 week 12 week

Table 2: Qualitative Screening of Phytochemicals of Methanolic Extract of Barleria prionitis Linn Leaf Derived Callus
Phytochemical Constituents Alkaloids Terpenoids Steroids Tannin and phenolic compounds Flavonoids Carbohydrates Glycoside/Cardiac glycoside Test Used Dragendorff test Salkowski test Libermann-Burchard test Ferric chloride test Sodium hydroxide test Fehlings test Keller-Killani test Methanolic Extract of Callus + + + + + + +

Table 3: MIC of Methanolic Extract of 8 Weeks (Best Callusing Stages) Callus of Barleria prionitis against Dental Caries Causing Pathogens
Organisms Streptococcus mutans Staphylococcus aureus Lactobacillus acidophilus Pseudomonas sp. MIC of Callus ( mg/ml) 0.375 0.375 0.250 0.375 Positive Control (g/ml) 20 20 10 20

Table 4: Antibacterial Activity of Methanolic Extract of Leaf Derived Callus of Barleria prionitis against Dental Caries Pathogens by Agar Well Diffusion Method
Dental Pathogens from Dental Caries Streptococcus mutans Staphylococcus aureus Lactobacillus acidophilus Pseudomonas sp. Zone of Inhibition (mm) Methanolic Callus Extract of Different Weeks 2 Weeks 10.87 0.01 12.12 0.05 21.65 0.21 12.56 0.04 4 Weeks 12.91 0.11 14.96 0.21 23.78 0.12 15.78 0.01 6 Weeks 14.21 0.03 15.67 0.31 25.54 0.06 17.98 0.11 8 Weeks 14.85 0.23 16.26 0.11 26.44 0.22 19.23 0.01 10 Weeks 13.93 0.32 14.95 0.20 25.12 0.04 17.89 0.31 12 Weeks 11.96 0.42 14.12 0.11 23.98 0.22 15.87 0.01 Negative Control (DMSO) Positive Control (Ciprofloxacin) 27.34 0.03 34.98 0.23 28.65 0.22 33.12 0.13

(-) = no activity, value including diameter of well (8mm), are means of three replicates, standard deviation