Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 4(9), 1-6, September (2014) Res. J. Chem. Sci. International Science Congress Association 1 Evaluation of Phytochemical constituents, Proximate and Fluorescence analysis of ethanolic extract and its fractions of Clerodendrumphilippinum Schauer found in Wayanad region of Kerala, IndiaDarsana Udayan, Suresh Narayanan Nair*, Sreelekha Kana Padinchareveetil, Ajeesh Krishna Thumadath Palayullaparambil, Adarsh Krishna Thumadath Palayullaparambil, Sanis Juliet, Reghu Ravindran and Ayilliath Kuttyeri PradeepDepartment of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Pookode, Wayanad, Kerala, INDIA Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Pookode, Wayanad, Kerala, INDIA Department of Botany, University of Calicut, Kerala, INDIAAvailable online at: www.isca.in, www.isca.me Received 16th July 2014, revised 20th August 2014, accepted 6th September 2014Abstract Clerodendrum philippinum Schauer, a member of the family Verbenaceae, is a semi-woody shrub widely distributed in the tropics and subtropics of India. The present study was conducted forphytochemical evaluation, proximate analysis and fluorescent characteristicsof leaves of C. philippinumcollected from Western Ghats of Wayanad. The ethanolic extract and its different fractions subjected to preliminary phytochemical analysis by standard procedures. The extract revealed alkaloids, saponins, glycosides, steroids, phenolic compounds, tannins, flavonoids,fixed oils and fats . The proximateanalysis of the plant leaves revealed dry matter (94.54%), moisture (5.47%), carbohydrates(56.86%), crude fibre (11.36%), ash (8.80%), crude protein (26.25%) and crude fat (2.62%).Fluorescence analysis showed difference in characteristic colour when examined with specificchemical reagents in powdered plant leaves, crude extract and fractions of C. philippinum under visible light and UV light. Keywords: Clerodendrum philippinum, ethanolic extract, fractions, phytochemical analysis, proximate analysis, fluorescence analysis. Introduction Traditional medicine continue to play a pivotal roles in the healthcare system of large proportion of the world’s population because of their cost-effective, eco-friendly attributes, and true relief from disease condition1,2. Scarcity and high costs of orthodox medicine are also attributed to this. The medicinal plants constitute a rich source of pharmacologically active secondary metabolites such as alkaloids, steroids, tannins, and phenol compounds, which are available abundantly at low cost and easily biodegradable. The plants of the genus Clerodendrum have more than five hundred member species and are widely distributed in the tropics and subtropics especially in the southern Asia. Morphologically, the plant is characterized by opposite and estipulate leaves, terminal or axillary cymose inflorescence or panicles, hypogynous bisexual flowers and persistent calyx(figure1). Clerodendrum philippinum Schauer, commonly called as Chinese Glory Bower and locally as Kattumulla in Wayanad is an ornamental plant. It has a semi-woody shrub of approximately10 feet height and morphologically characterised by broad leaves up to 1 feet long and wide, toothed margins, white with pink or red tinged fragrant flowers in tight clusters. Traditionally the extracts of root and leaves of . philippinum were used for the treatment of rheumatism, asthma and inflammatory diseases. It is also used for the treatment of jaundice, syphilis and typhoidDaily external application of C. philippinum leaf paste, after bath in the morning on the affected area of the stem was reported to relieve from all types of skin diseases. Srivastava et al8 reported that this plant is used externally as a poultice for rheumatism and decoction of leaves for scabies. The plant also has many scientifically validated pharmacological activities like antibacterial as well as anti-inflammatory activity. The ethanolic extract of plant showed anti bacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiell sp., and Bacillus substillus. The aqueous extract exhibited significant anti inflammatory activity in acute and chronic inflammatory animal models10. In the present study, an attempt is made to explore the phytochemical constituents, proximate analysis and fluorescence characteristics of the plant . Philippinum found in Wayanad regions of Kerala. The fluorescence and proximate analysis and information on phytochemical constituents of C. Philippinum revealed its identification, standardization and diverse medicinal and pharmacological properties respectively. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 1-6, September (2014) Res. J. Chem. Sci. International Science Congress Association 2 Figure-1 Photograph of Clerodendrum philippinum Schauer.in wild Material and MethodsPlant materials: The leaves of Clerodendrum philippinum Schauer (figure 1) were collected from the campus of College of Veterinary and Animal Sciences, Pookode, and were authenticated and a voucher specimen was deposited at Department of Botany, University of Calicut (Accession No: CALI 6770). The plant leaves were cleaned, dried in shade and ground in a temperature controlled plant sample pulveriser. The powdered plant material was used for extraction using ethanol in soxhlet extraction apparatus (M/s Buchi, Switzerland) under reduced vapour temperature. Solvents were evaporated off by rotary vacuum evaporator (M/s Buchi, Switzerland) at 175 mbar at a temperature in the range of 40C to 50C. The weight of the dried extract was recorded and the extractive yield was calculated as, Extractive yield = Weight of the extract × 100 Weight of the sample taken Fractionation of crude ethanolic extract: The ethanolic extract was fractionated using four solvents of ascending polarity index namely hexane, chloroform, n-butanol and water. The crude extract (200 g) was transferred to a separating funnel and extracted with hexane to obtain a hexane soluble fraction and a hexane insoluble fraction. After collecting the hexane soluble fraction, the hexane insoluble fraction was extracted with chloroform to yield chloroform soluble fraction and insoluble fraction. After collecting the chloroform soluble fraction, the chloroform insoluble fraction was further extracted with n-butanol and subsequently with water to yield n-butanol soluble and aqueous fractions. Solvents were initially removed using a rotary vacuum evaporator (M/s Buchi, Switzerland) and completely dried by keeping at room temperature. Phytochemical analysis: The crude ethanolic extract and its four fractionswere subjected to phytochemical analysis for secondary metabolites like steroids, tannins, flavonoids, glycosides, phenolic compounds, diterpines, triterpines, saponins and alkaloids11-13Proximate analysis: The analysis of total ash, crude fibre, crude protein, total carbohydrate, crude fat, dry matter and moisture content of the leaves of C. philippinum were carried out using standard techniques of proximate analysis14 as briefly mentioned below. Determination of dry matter: The dry matter was determined using the weight difference method and estimated by deducting per cent moisture from hundred as described by James15. Dry matter (%) = 100 - % of moisture The moisture content was determined by using the formula, Moisture Percentage = Weight of the sample – Weight of the dried sample X 100 Weight of the sample Determination of total ash: Ash represents the inorganic content of the sample which is determined by the method as described by Association of Official Analytical Chemists.16For this, dried sample approximately one gram was taken in a crucible, charred over low flame and was kept in a muffle furnace set at 550-600C for 2-3 hours. It was cooled in a desiccator to prevent re-absorption of moisture and the procedure repeated till constant weight of ash was obtained after three repeated weighing when the ash becomes white or greyish. The total ash content was calculated using the formula, Ash (%) = Weight of the ashed sample ×100 Weight of the sample taken Determination of crude fat: One gram of dried crushed sample was taken in the filter paper thimble and kept in a pre-weighed flask of the soxhlet extractor. 80 ml of petroleum ether was poured into the flask and the system was refluxed for 8 hours. The flask was cooled in a desiccator and the weight of crude fat extracted was taken. The per cent crude fat was determined by using formula, Percentage of Crude fat = Weight of the flask with fat - Weight of empty flask x 100 weight of the original sample Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 1-6, September (2014) Res. J. Chem. Sci. International Science Congress Association 3 Determination of Crude fibre: One gram of the de-fattened plant material was taken in a spout-less beaker and boiled with 200 ml of 1.25% sulphuric acid for 30 minutes. The boiled content was then filtered and washed with hot distilled water to neutralize and transferred again into the beaker and boiled with 200 ml of 1.25% sodium hydroxide for 30 minutes followed by filtration and the filtrate was washed with hot distilled water for neutralization. The crucible was dried in a hot air oven at 100 ± C overnight (10-12 hr) and cooled in a desiccator to a constant weight. Subsequently, the desiccator along with its content was put in a muffle furnace at 550-600C for 2-3 hour for complete burning of organic matter and cooled in a desiccator and weighed to a constant weight. The per cent of crude fibre was determined from the formula,Crude fibre (%) = (w 1 – w 2 ) x 100 weight of the sample where, w- The crucible having crude fibre was cooled and weighed, w- The content of the crucible was ignited over a low flame until charred and then kept in a muffle furnace and weighed. Determination of crude protein: Total nitrogen (N) content was determined with the help of Kjeldahl method described by Pearson17 which consisted of digestion, distillation and titration steps. Digestion: Approximately 5g of dried plant material was taken in a 50 ml digestion flask and digested with 1 g of digestion mixture (copper sulphate and sodium sulphate) and 15 ml of concentrated sulphuric acid. The solution was heated until the frothing was ceased and the solution became clear. The solution was gently boiled for another 2 hours, cooled and digested with 30 ml of water with constant stirring. The final digest was transferred to 250 ml standard flask and required amount of distilled water (up to the mark of the flask) was added. Distillation: The distillation was done in a Kjeldahl distillation apparatus. Four per cent boric acid (20 ml) was taken with one drop of methyl red as indicator. Digested material (10 ml) was transferred to the distillation assembly and mixed with 20 ml of 40 per cent sodium hydroxide solution. The distillation was stopped in 6 minutes as indicated by the change of colour of boric acid from pink to blue. Titration: The boric acid having trapped the ammonia from the nitrogen of protein was titrated with 0.1N hydrochloric acid, changing the colour back to pink. The per cent of protein was calculated by the formula.Protein (%) = V×1.4 x 6.25 x 0.1N HCl x Vol (used) ×100 W×A×1000 Where; V= Titre value, 1.4 = Weight of nitrogen expressed in gram in formula, 6.25 = Protein factor, W= Weight of sample, A= Aliquot digested sample used for distillation. Determination of carbohydrate: Determination of available carbohydrate in the sample was calculated from the above parameters by using the formula. Carbohydrate (%) = 100 - (moisture + crude fat + ash + protein) % Fluorescence analysis: The fluorescence analysis of the plant was done by placing dry powdered leaves on a slide and observing the colour changes under visible and UV lights after treating with several drops of specific reagents (acetic acid, 10% copper sulphate, 1N hydrochloric acid, 1N nitric acid, 1N sulphuric acid, 10% ferric chloride and 1N sodium hydroxide). Similarly, the crude ethanolic extract and its fractions along with the respective solvent were observed without adding any reagents under visible light and UV light. The development of colour was noted within 1-2 minutes in order to avoid drying and resultant colour change18,19Results and Discussion Extractive yield: The extractive value of the ethanolic crude extract of leaves of C. Philippinum was 6.13 per cent whereas for n-hexane, chloroform, n-butanol and aqueous fractions, the extractive yield varied from 11.24, 27.96, 25.58, 25.02 per cent respectively. Phytochemical Analysis: The results of qualitative phytochemical analysis are summarized in the table 1. The crude ethanolic extract of C. philippinum revealed the presence of flavonoids, steroid, glycosides, phenolic compounds, tannins, saponins, carbohydrates, alkaloids, fixed oils and fats. The hexane fraction of C. philippinum was positive for the presence of alkaloids, carbohydrates, flavonoids, fixed oils and fats. The chloroform fraction was positive for alkaloids, carbohydrates, flavonoids, saponins, fixed oils and fats. The n-butanol fraction revealed the presence of steroids, glycosides, flavonoids, saponins and carbohydrates. Steroids, glycosides, saponins and carbohydrates were detected in the water fraction. Previous reports on C. Philippinum revealed that the leaves of the plant was quite rich in alkaloids, flavonoids, terpenoids, phenols, tannins, saponins, anthraquinones, lignin, glycosides, phytosterols, quinines, amino acids and proteins6,20,21Terpenoids, proteins and amino acids were not found in the present study. Proximate analysis: The dry matter showed the highest percentage of 94.54%, the moisture content 5.47%, carbohydrate has 56.86% while crude fibre has a percentage of 11.36%, ash 8.80%, crude protein 26.25% and crude fat 2.62% (table 2). In another study the proximate analysis of C. volubile revealed energy value of 3.98± 0.12%, dry matter 93.3± 0.28%, Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 1-6, September (2014) Res. J. Chem. Sci. International Science Congress Association 4 crude fibre 5.31± 0.3%, ash 12.14± 0.23%, crude protein 11.2± 0.36%, crude fat 4.87± 0.2% and NFE 59.78± 0.2%22. Fluorescence analysis: The different colours shown by the powdered leaf in presence or absence of chemical reagents are presented in table 3. On exposure to UV light, the crude ethanolic extract of C. philippinum and its fractions elicited characteristic colours as given in table 4. Table-1 Qualitative phytochemical analysis of ethanolic extract of leaves of C. philippinum and its four fractionsPhytochemicals Inferences Ethanol Fractions of ethanolic extract of Clerodendrum-philippinumHexane Chloroform Butanol Water Alkaloids ++ ++ + - - Carbohydrate + + + + + Saponin ++ - + ++ ++ Steroids + - - + ++ Phenolic compounds and Tannins ++ - - - - Terpenoids - - - - - Flavanoids + + + ++ - Protein and amino acids - - - - - Fixed oils and fats + ++ + - - Glycosides + - - + + Gums and mucilages - - - - - Note: (+) Present and (-) Absence Table-2 Proximate composition of leaves of C. philippinumPlant Proximate contents (%) Dry Matter Moisture Crude fibre Crude protein Crude fat Ash Carbohydrate C. philippinum 94.54 5.47 11.36 26.25 2.62 8.80 56.86 Table-3 Fluorescence characteristics of powdered leaves of C. philippinum treated with different chemical reagents No. Reagents Visible light Long UV (356 nm) Short UV (254 nm) 1 Powder Green Black Green 2 Powder + Water Light green Black Yellowish green 3 Powder + CH 3 COOH Yellowish green Black Yellowish green 4 Powder +10% CuSO 4 Green Black Green 5 Powder +1NHCl Grey Black Pale green 6 Powder +1N HNO 3 Grey Black Pale green 7 Powder +1N H 2 SO 4 Yellowish grey Black Yellowish light green 8 Powder +10% FeCl 3 Dark green Black Dark green 9 Powder + 1N NaOH Yellowish dark green Black Yellowish dark green Table-4 Fluorescence characteristics of crude ethanolic extract of leaves of C. philippinum and its four fractions No. Fractions Visible light Long UV (356 nm) Short UV (254 nm) 1 Crude extract Dark brown Black Dark brown 2 Hexane fraction Lightbrown Dark brown Dark brown 3 Chloroform fraction Dark green Black Black 4 n-butanol fraction Light green Brownish black Light green 5 Water fraction Yellowish green Black Light green Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 1-6, September (2014) Res. J. Chem. Sci. International Science Congress Association 5 The information on proximate and nutrient contents of herbal products are required for their standardization and oral use23. Results of proximate analysis indicated high content of mineral and crude protein in leaves of C. philippinum. Fluorescence is the phenomenon exhibited by various chemical constituents present in the plant material. Some constituents show fluorescence in the range of visible light or when exposed to ultra violet radiation19. The organic molecules absorb light over a specific range of wave length and reemit radiations and hence it can be used for the identification of the powdered drug, extract or fractions of herbs24. Taking in to consideration of existing medical importance, the plant can be explored for its additional pharmacological properties and explanations for existing activities. Conclusion The present study revealed the phytochemical constituents, proximate and fluorescence characteristics of ethanolic extract and its fractions of Clerodendrum philippinum Schauer. found in Wayanad region of Kerala, India. Further studies are required to characterize the bioactive compounds by suitable techniques. Acknowledgement Financial assistance from Indian Council of Agricultural Research through the National Agricultural Innovation Project (NAIP/Com-4/C2066/2007-2008) funded by World Bank and National Fund project (NFBSFARA/BSA-4004/2013-14) is thankfully acknowledged. References 1.Fredrick C. A., Onyekaba T.U., Charity C. E., Chibueze I. and Valentine C. E., Preliminary phytochemical investigations and evaluation of antimicrobial activity of n-hexane extract of the leaves of Synclisia scabrida family menispermaceae, Res. J. Pharmaceutical Sci., 2(3),1-5 (2013)2.Tamilarasi T. and Ananthi T., Phytochemical analysis and anti microbial activity of Mimosa pudica Linn. Res. 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