The present study was aimed to evaluate and compare the phytochemical and anti-bacterial potential of mother plants (aerial parts), in vitro leaves and stems derived callus and in vitro callus mediated shootlets of Rhinacanthus nasutus (L.) Kurz. Preliminary phytochemical analysis and extraction was performed on stem and leaves segments derived calli (4 weeks old), aerial portions of mother plants and calli mediated shootlets of R. naustus. Anti-bacterial activity of different extracts (25µg) (ethanol, chloroform, aqueous and ethyl acetate) of stem and leaves segments derived calli, aerial portions of mother plants and calli mediated shootlets were investigated by well-diffusion method against Staphylococcus aureus, Pseudomonas aeruginosa,Klebsiella pneumoniae, Bacillus subtilis and Escherichia coli. The preliminary phytochemical studies confirmed that the leaves and stem derived calli mediated shootlets showed the higher degree of metabolite constituents and extraction value compared to the in vitro derived calli and in vivo leaves and stem (aerial portions). The ethyl acetate extracted solvents showed the maximum bio-efficacy compared with other solvents due to the presence of more compounds such as saponins, steroids, tannins, phenolics, triterpenoids, alkaloids, coumarins, anthraquinones, glycosides and flavonoids. The present study observation suggested that a possibility to establish high yielding genotypes by in vitro culture for production of medicinally important bioactive compounds. The methods developed in this work make possible for the low volume and high potential production of active principles under in vitro condition in short duration with less amount of explants utilization.

Keywords: Rhinacanthus nasutus, bio-efficacy, phytochemical, anti-bacterial

Due to the less side effects and safety, the importance of medicinal and aromatic plants has drawn attention from time to time.¹ Rhinacanthus nasutus (L.) Kurz is the best known member of the family Acanthaceae. In the ayurvedic system of medicine, R. nasutus is practiced as a traditional medicinal plant in the Indian sub continent, China and Southeast Asia including Thailand.² The literatures reported that the traditional medicine preparation from roots, stems and leaves of this shrub has long been used in Thai traditional medicine for treatment of various diseases such as eczema, pulmonary tuberculosis, herpes, hepatitis, diabetes, cancer, obesity, leprosy, eczema, scurvy, dhobi’s itch hypertension and various skin diseases.^3,4 Several reports of investigation states that leaves and stems of R. nasutus contains various types of flavonoids, benzenoids, coumarin, anthraquinone, quinone, glycosides, carbohydrate, triterpenes, sterols, and chlorophyll.⁵ The naphthoquinone; rhinacanthins (A-D, G-Q), rhinacanthone and lignan groups are the main bioactive compounds.⁶ The leaf petroleum ether fraction shows significant mosquitocidal activity.⁷ It has been reported that rhinacanthin-C, rhinacanthin-D and rhinacanthin-N isolated from R. nasutus possessed various biological activities.^8-10

Naphthoquinone compounds have been reported to possess anti-proliferative activity against a panel of cancer cells. Rhinacanthone (3, 4-dihydro-3, 3-dimethyl-2H-naphtha [1,2-b]pyran-5, 6-dione) was isolated from the shrub, R. nasutus, which is essential for the tumor activity.¹¹ Due to its high demand resulted over exploitation from the wild and leads to depletion of this important medicinal plant demands to develop an alternative protocol for the propagation two of R. nasutus. Johnson et al.¹² reported the micro propagation and calli mediated organogenesis of R. nasutus. To fulfill the commercial requirements and growing demand of this herb, an alternative source is needed to meet the demand. In vitro cell culture or cell line culture is an effective alternative pathway as it has already proved successful in many other cases.¹³ To date, only a few plant metabolites have been produced via cell culture production in large scale production. With reference to quantity and quality of secondary metabolites, few cell line cultures proved to possess more amounts of secondary metabolites than the differentiated mother plant.¹⁴ The main intention of this study is to evaluate and compare the phytochemical and anti-bacterial potential of mother plants (in vivo), in vitro leaves and stem derived callus and calli mediated plantlets of R. nasutus.

Phytochemical analysis

The mother plants (leaves and stem aerial portions) and calli mediated shootlets of R. nasutus were air and shade dried for 15 d at room temperature and pulverized to powder using the electric homogenizer. In vitro derived callus (leaves and inter-nodal) were dried in the hot air oven and powdered using the electric homogenizer. The powdered (2 g) samples were extracted with 100ml of solvent (chloroform, ethanol, ethyl acetate and aqueous) for 8h by using the soxhlet apparatus.¹⁵ The preliminary phytochemical screening was performed by Harborne method.¹⁶

Antibacterial activity

The crude extracts of R. nasutus leaves, stem and calli mediated shootlets derived extracts were concentrated and subjected for their antibacterial activity against the selected pathogenic bacteria. Pure bacterial cultures viz., Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis (ATCC 10707)and E. coli (ATCC 35218) were maintained on nutrient broth at 37°C for 24 h. Antibacterial efficacy was performed by well diffusion method with 25µg/ml of extract and incubated for 24 h at 37°C.¹⁷ The inhibition zone and antibacterial activity against the pathogenic bacteria were recorded. Amoxicillin and tetracycline discs were used as a positive control. The solvents (chloroform, ethanol, ethyl acetate and aqueous) alone are used as negative control. The experiments were repeated in triplicate and the results were documented.

The preliminary phytochemical studies confirmed that the occurrence of more quantity of secondary meabolites in the calli mediated plantlets than mother plants in vivo leaves and stem (aerial portions) and in vitro derived calli. The phytochemical study documented the high quantity of steroids, triterpenoids, glycosides, coumarins, anthroquinnones, saponins, alkaloids, flavonoids, phenolic compounds, tannins, sugar etc in calli mediated shootlets compared to in vitro derived calli and in vivo aerial portions (mother plants). Different kinds of solvents were used for extraction, of which ethyl acetate extracts of R. nasutus showed maximum metabolites occurrence (13/13) compared with other extracts (Table 1). Similarly the ethyl acetate extracts of R. nasutus showed the maximum bio-efficacy (3/5) due to the presence of more metabolites (Table 2).

Various compounds known as pharmaceuticals, antimicrobials, secondary metabolites such as coumarins, flavonoids, anthocyanins, tannins, anthraquinones, napthoquinones, sterols, triterpenes, carotenoids and nicortine have been produced using tissue culture technique. In the present study the secondary metabolites have been produced using tissue culture techniques; and the metabolites were compared for the degree of presence and efficacy of the extracts of aerial portions, stem and leave derived calli and calli mediated shootlets of R. nasutus. The result of the present study revealed that the antibacterial efficacies of ethanol, chloroform, ethyl acetate and aqueous extracts of leaves and leaves segment derived calli, stem and stem derived calli and calli mediated shootlets extracts of R. nasutus was diverse in effectiveness which may be attributed due to the presence of the secondary metabolites. Results of the present study are directly correlated with the previous observations.^18-23 The various extracts of aerial portions and calli mediated shootlets showed the inhibition against the selected pathogenic bacteria. The earlier observations on Balisospermum montanum and Alternanthera sessilis aerial portions and in vitro derived calli and calli mediated shootlets extract demonstrated significant antibacterial activity.^14,15

The result of the present study revealed that the antibacterial efficacies of acetone, ethanol, chloroform and ethyl acetate extracts of mother plants, in vitro derived calli and calli mediated shootlets of R. nasutus with varied frequency which may be attributed to the presence of the secondary metabolites (Table 1). The ethyl acetate extracts of R. nasutus calli mediated shootlets showed the maximum bio-efficacy compared with other solvents due to the presence of more compounds such as saponins, steroids, tannins, phenolics, triterpenoids, alkaloids and flavonoids (Table 1 & 2). Results of the present study are directly correlated with the previous observations.^12-14,17-25 The ethyl acetate extracts R. nasutus in vitro calli derived shootlets showed the inhibition against the selected pathogenic bacteria. This study observations were supplemented by earlier observations on Baliospermum montanum, Hypericum perforatum and Mimosa hamata leaf and callus extract demonstrated significant antibacterial and antimicrobial activity.^13,17,25 The methods developed in this work enable high potential production of active principles with low volume input under in vitro condition in short duration with less amount of explants without any seasonal influence. The results of the present study demonstrated that in vitro cultivation of calli mediated shootlets significantly enhanced production of secondary metabolites compared to micro propagated plants. The results of the present study observation indicated the possibility to establish high yielding genotypes by in vitro culture for production of medicinally important bioactive compounds.

The authors declare no conflict of interest.

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1. Manish D, Arun N, Ansari SH. Pterocarpus marsupium Roxb. – A Comprehensive Review. Pharmacognosy Review. 2009;3(6): 359–363.
2. Tunsaringkarn T, Palanuvej C, Issaravanich S, et al. Quality assessment of Rhinacanthusnasutus. J Health Res. 2009;3(3):111–115.
3. Supinya T, Tansakul P, Panichayupakaranant P. Anti–allergic principles of Rhinacanthus nasutus leaves. Phytomedicine. 2009;16(10):29–34.
4. Siripong P, Wongseri V, Piyaviriyakul S. Antiproliferative naphthoquinone esters from Rhinacanthus nasutus Kurz. roots on various cancer cells. J Trad Med. 2006;23(5):166–172.
5. Gotoh A, Sakaeda T, Kimura T. Anti–proliferative activity of Rhinacanthus nasutus (L.) Kurz extracts and the active moiety, rhinacanthin C. Biol Pharm Bull. 2004;27:1070–1074.
6. Siripong P, Yahuafai J, Shimizu K. Induction of apoptosis in tumor cells by three naphthoquinone esters isolated from Thai medicinal plants: Rhinacanthus nasutus Kurz. Biol Pharm Bull. 2006;29:2070–2076.
7. Shaalan EAS, Canyon D, Younes MWF. A review of botanical phytochemicals with mosquitocidal potential. Environment International. 2005;31(8):1149–1166.
8. Puttarak P, Charoonratana T, Panichayupakaranant P. Antimicrobial activity and stability of rhinacanthins–rich Rhinacanthus nasutus extract. Phytomedicine. 2010;17(5):323–327.
9. Panichayupakaranant P, Charoonratana T, Sirikatitham A. RP–HPLC analysis of rhinacanthins in Rhinacanthus nasutus: validation and application for the preparation of rhinacanthin high–yielding extract. J Chromatogr Sci. 2009;47(8):705–708.
10. Kongkathip N, Kongkathip B, Siripong P. Potent Antitumor Activity of Synthetic 1,2–Naphthoquinones and 1,4–Naphthoquinones. Bioorganic & Medicinal Chemistry. 2003;11(14):3179 –3191.
11. Johnson M, Berhanu A, Mulugeta K. Regeneration from callus cultures of Rhinacanthus nasutus L. Kurtz. Eth J Sci & Technol. 2005;3(1):17–24.
12. Johnson M, Wesely EG, Selvan N, et al. In vivo and In vitro Anti–Bacterial Efficacy of Alternanthera sessilis (Linn.). IJPRD. 2010;2(10):72–79.
13. Johnson M, Wesely EG, Zahir Hussain MI, Selvan N (2010) In vivo and in vitro phytochemical and antibacterial efficacy of Baliospermum montanum(Willd.) Muell Arg Asian Pacific Journal of Tropical Medicine. 3(11): 894–897.  
14. Johnson M, Maridass M, Irudayaraj V. Preliminary Phytochemical and Anti–Bacterial Studies on Passiflora edulis. Ethnobotanical Leaflets. 2008;12:425–432.
15. Harborne JB. “Phytochemical Methods”. Chapman and Hall, London, UK. 1973.
16. Victor B, Maridass M, Ramesh U. Antibacterial activity of flower and fruit extracts in Citrullus lanatus. Journal of Eco–Physiology. 2002;5:1–3.
17. Jain SC, Jain R, Vlietinck AJ. In vivo and in vitro antimicrobial efficacy of Mimosa hamata. Ind J Biotech. 2004;3:271–273.
18. Arafeh RM, Shibli RA, Al Mahmoud M, et al. Callusing, Cell Suspension Culture and Secondary Metabolites Production in Persian Oregano (Origanum vulgare L.) and Arabian Oregano (O. syriacum L.). Jordan Journal of Agricultural Sciences. 2006;2(3):274–281.
19. Ana Ciric, Branka Vinterhalter, Katarina Savikin Fodulovic, et al. Chemical analysis and antimicrobial activity of methanol extracts of Celandine (Chelidonium majusL.) plants growing in nature and cultured in vitro. Arch Biol Sci. 2008;60(1):7–8.
20. Johnson M. Somoclonal variation studies on Phyllanthus amarus Schum and Thonn. Iranian Journal of Biotechnology. 2007;5(4): 240–245.
21. Johnson M, Babu A, Irudayaraj V. Antibacterial studies on in vitro derived Calli of Ocimum basilicum L. J Chem Pharm Res. 2011;3(1): 715–720.
22. Johnson M, Babu A. Somoclonal Variation Studies on Passiflora mollussima (H.B.K.) Bailey using Phytochemical methods. Natural Products: An Indian Journal. 2008;6(1):5–10.
23. Karmarkar SH, Keshavachandran R, Augustin A, et al. Biochemical evaluation of root tubers and in vitro induced callus of adapathiyan (Holostemma ada–kodien k.schum.). J Trop Agric. 2009;39: 108–110.
24. Shinde AN, Malpathak N, Fulzele DP. Induced high frequency shoot regeneration and enhanced isoflavones production in Psoralea corylifolia. Rec Nat Prod. 2009;3(1):38–45.
25. Pasqua G, Pinarosa A, Monacelli B, et al. Metabolites in cell suspension cultures, calli and in vitro regenerated organs of Hypericum perforatum cv. Topas Plant Sci. 2003;165:977–982.