Kanakasabapathi Devaki et al. IRJP 2012, 3 (7) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407 Research Article BARLERIA CRISTATA LINN.: PHYTOCHEMICAL SCREENING AND HPTLC ANALYSIS Rajasekaran Narmadha and Kanakasabapathi Devaki* Department of Biochemistry, Karpagam University, Coimbatore-641 021 India Article Received on: 15/04/12 Revised on: 21/05/12 Approved for publication: 11/06/12 *Dr.K.Devaki, Assistant Professor in Biochemistry, Karpagam University, Coimbatore-641 021 Tamilnadu, India Email: [email protected] ABSTRACT Phytochemical examination (qualitative and quantitative) and HPTLC analysis of phytochemicals of the crude extract Barleria cristata Linn. leaves were investigated. Preliminary phytochemical screening of various extracts of the leaves revealed the presence of compounds such as amino acids, carbohydrates, flavonoids, proteins, phenolic groups, saponins, steroids, tannins and terpenoids. HPTLC finger printing analysis support the presence of alkaloids and phenolic compounds (Quercetin) in this plant extract. The present study provides information with respect to phytochemicals of Barleria cristata L. Keywords: HPTLC, secondary metabolites, Barleria cristata Linn., sequential extraction INTRODUCTION MATERIALS AND METHODS Plants are rice source of large amount of drugs comprising to Collection of Plant Material: different groups such as antispasmodics, emetics, anti-cancer, The leaves of Barleria cristata used for the investigation antimicrobials etc. They have been an important source of were obtained from Coimbatore district, Tamilnadu, India. medicine for thousands of years. Even today, the World The plant was authenticated by Botanical Survey of India, Health Organization estimates that up to 80 percent of people TNAU Campus, Coimbatore. The voucher number is still rely mainly on traditional remedies such as herbs for BSI/SRC/5/23/2011-12/Tech.-n62. their medicines. Its civilization is very ancient and the Preparation of Plant Extract: country as a whole has long been known for its rich resources The leaves of Barleria cristata L. were air dried in the of medical plants1-3. The use of traditional medicines holds a absence of sunlight and powdered well using a mixer and great promise as a easily available source as effective stored in an air tight container. The powdered plant material medicinal agents to cure a wide range of ailments among the (50 g) was taken and subjected to successive solvent people particularly in tropical developing countries like India. extraction (250ml) with increasing order of polarity like Use of several plants or plant derived formulations to cure petroleum ether, chloroform, ethyl acetate, ethanol and water. helmintic infections and treatment of wounds were also The plant extracts were concentrated and stored in an airtight recorded 4, 5. vial for further studies. Herbal medicines are known to be oldest health care products Phytochemical screening that have been used by mankind all over the world in the Qualitative estimation of phytoconstituents form of folklore medicines or traditional medicines or ethnic Phytochemical screening was carried out to assess the medicines. The therapeutic use of herbal medicines is gaining qualitative chemical composition of crude extracts using considerable momentum in the world during the past decade. commonly employed precipitation and coloration to identify The World Health Organization (WHO) estimates that herbal the major natural chemical groups such as steroids, reducing medicine is still the mainstay of about 75-80% of the world sugars, alkaloids, phenolic compounds, saponins, tannins, population, mainly in the developing countries, for primary flavonoids, amino acids, fixed oils and fats and cardio health care because of better cultural acceptability, better glycosides. General reactions in these analysis revealed the compatibility with the human body and lesser side-effects6. presence or absence of these compounds in the crude extracts Barleria cristata Linn (family acanthaceae) is a shrub found tested 8,9. widely in subtropical Himalaya, Sikkim, Khasi Hills, central, Quantitative estimation of phytoconstituents and southern India at a height of 1,350 m. The chemical Estimation of Protein: constituents of the plant have been identified as flavonoid The amount of protein present in the ethanolic extract of type phenolic compounds, especially apigenin, quercetin, Barleria cristata was determined by the standard method quercetin-3-O-β-D-glucoside, naringenin, luteolin, and given by Lowry et al.,10 apigenin glucuronide. Barleria cristata L. have been used Estimation of Total Phenolics: traditionally for the treatment of variety of diseases including Total phenolic content of ethanolic extract of Barleria anemia, toothache, and cough. Leaves were used to reduce cristata was measured based on Folin-Ciocalteu assay11. swellings in inflammation7. Briefly, 0.5 ml of ethanolic extract was mixed with distilled The main objective of the present study was to investigate the water (2.5 ml) and 0.5 ml of Folin-ciocalteu reagent was phytochemical screening and HPTLC finger printing analysis added. After 3 minutes 2 ml of 20% sodium carbonate was of the leaves of Barleria cristata. added and mixed thoroughly. The tubes were incubated in a boiling water bath for exactly one minute. It was then cooled and the absorbance was measured at 650nm using spectrophotometer against the reagent blank. Total phenolic Page 301 Kanakasabapathi Devaki et al. IRJP 2012, 3 (7) content was expressed as mg gallic acid equivalents (GAE)/g RESULT AND DISCUSSION of extract. The phytoconstituents are the major important compounds Estimation of Total Flavonoids: which are responsible for the medicinal properties of the The flavonoid content was examined by adopting the herbs. Hence medicinal plants could be a potential source for methodology of Ordon et al., 200612. Briefly, a volume of 0.5 nutraceuticals. The phytochemical substances namely, ml of 2% AlCl3 in ethanol solution was added to 0.5 ml of phenols and flavonoids are the major important substances sample solution. After one hour incubation at room responsible for the medicinal values of the plants including temperature, yellow colour was developed. This was antioxidant, anticancer, antimicrobial activities, etc13. In the measured at 420 nm with UV-Visible spectrophotometer. A present study the phytochemical screening results revealed standard graph was prepared using the quercetin and the total the presence of phytoconstituents such as alkaloids flavonoid content was expressed as quercetin equivalent carbohydrates, glycosides, proteins and amino acids, phenols, (mg/g) of extract. flavonoids, phytosterols, steroids, tannins and triterpenes HPTLC analysis (Table 1). Among extracts, most of the phytochemicals were 2µl of the above test solution and 2µl of standard solution found to be present in ethanol and aqueous extract of were loaded as 5mm band length in the 3 x 10 Silica gel Barleria cristata L. 60F254 TLC plate using Hamilton syringe and CAMAG Table. 2 shows the percentage yield of different extracts of LINOMAT 5 instrument. The samples loaded plate was kept Barleria cristata L. In that, the maximum yield was found in in TLC twin trough developing chamber (after saturated with both ethanolic and water extract. So the quantitative analyses Solvent vapor) with its respective mobile phases (alkaloid, of secondary metabolites were carried out by using these two flavonoids and phenols) and the plate was developed up to extracts. In quantitative assay the ethanolic extract showed 90mm. The developed plate was dried by hot air to evaporate the high concentration of alkaloids (1.43±0.02), phenols solvents from the plate. The plate was kept in photo- (0.725±0.01), flavonoids (0.47±0.05), and proteins documentation chamber (CAMAG REPROSTAR 3) and (42.5±5.13) when compared with aqueous extract (Table 3). captured the images at white light, UV 254nm and Therefore, ethanolic extract of Barleria cristata was used for UV366nm. The developed plate was sprayed with respective the subsequent studies. spray reagent and dried at 100º C in hot air oven. The plate HPTLC profile of ethanolic extract of Barleria cristata L. was photo-documented at daylight and UV 366nm mode was recorded in Table 3, 4 and 5 for alkaloids, flavonoids and using Photo-documentation (CAMAG REPROSTAR 3) phenols respectively. Yellow-brown coloured zones were chamber. After derivatization, the plate was fixed in scanner detected in day light and UV after derivetaization for stage (CAMAG TLC SCANNER 3) and scanning was done alkaloid, yellow coloured fluorescent zone at UV 366nm for at 500nm. The peak table, peak display and peak flavonoid and blue coloured zones for phenols in the densitogram were noted. chromatogram. The extracts were run along with the standard alkaloid, flavonoid and phenolic compounds. The leaf extract that shows the presence of these compounds in the chromatogram and densitogram are depicted in Figure 1-6. Table. 1. Qualitative analysis of selected phytochemicals in Barleria cristata Solvents Constituents Petroleum ether Chloroform Ethyl acetate Ethanol Water Alkaloids + + + + + Carbohydrates + + - + + Glycosides - - - + + Proteins and amino acids + + + + + Phenols - - - + + Flavonoids - - - + + Phytosterols - - + + + Steroids - - - + - Tannins + - - + + Triterpenes - + - + + Table. 2. Percentage yield of different extracts of Barleria cristata Extracts % of yield (g) Petroleum ether 3 Chloroform