Studies on Chemical Constituents, Phytochemical Profile and Pharmacological Action of Datura alba

Middle-East Journal of Medicinal Plants Research 1(1): 14-18, 2012 ISSN 2309-8929 IDOSI Publications, 2012 DOI: 10.5829/idosi.mejmpr.2011.1.1.1104 Studies on Chemical Constituents, Phytochemical Profile and Pharmacological Action of Datura alba Ghias Uddin, Abdur Rauf and Samina Akhtar Institute of Chemical Sciences, University of Peshawar, Peshawar-25120, KPK, Pakistan Abstract: Phytochemical screening of various fractions of Datura alba showed bioactive secondary metabolites based on microbial and antioxidant activities. n-hexane fraction showed a significant activity against three selected bacterial strains; Bacillus subtilis, Klebsiella pneumoniae and Staphylococcus aureus while other isolated fraction showed moderate activity. The ethyl acetate (88.89 %) and methanolic (76.94 %.) fraction exhibited significant antioxidant properties at 100 µg/ml against querceitin. -Sitosterol (1) and stigmasterol (2) were isolated hither unreported from the methanolic extract of plant. Key words: Datura alba Phytochemical profiling -Sitosterol Stigmasterol Antioxidant Antibacterial activity INTRODUCTION discover new compounds with diverse chemical structures and novel mechanisms of action for new and Medicinal plants are an important source of reemerging infectious diseases [3]. producing valuable bioactive compounds which is great Datura alba belong to family Solanaceae which is importance for the health of individuals and communities. commonly known as is also known as angel s devils The medicinal values of the plants are due to the chemical trumpet. D. alba is an annual herb grown up to 3 ft in substances that produce a definite physiological action height [4]. It belongs to genus Datura, which consists of on human body and are called phytochemicals [1]. fifteen species. All parts of the Thorn apple have Infectious diseases are the leading cause of death in all medicinal value, but only the leaves and seeds are over the world. Plants constitute of various naturals officially used. D. alba is a very well-known plant for its products that are important form medicinal point of view. various medicinal activities in various parts of the world. As plants are abundance of these products and the It is widely used for the treatment of asthma, healing expanding areas of health and medicine demands to potential of burn wounds, muscle spasm, whooping evaluate the presence of these products, it is reported cough, hemorrhoids and skin ulcers etc. This plant is that over 50% of all modern clinical drugs are of natural used by the Curve s practitioners for all types of wounds product origin. They therefore, play an important role in [1]. The leaf extracts ofd. alba show very good toxicity development in the pharmaceutical industry. All these against aphids and ants [5]. On the basis of such factors demand for more vast study of natural products interesting activity it was considered to carry out the and biologically active compounds [2]. Many infectious phytochemical screening, antibacterial activity and radical diseases have been known to be treated with herbal scavenging activity of the plant to evaluate more remedies throughout the history of mankind. Natural important side of the plant. The plant appears in late products, either pure compounds or as standardized winter/early spring season and bloom repeatedly.in plant extract, provide unlimited opportunities for new current study, we have made an effort to identify potential drug leads because of unmatched availability of chemical bioactive secondary metabolite and their antioxidant diversity. There is a continuous and urgent need to activities. Corresponding Author: Ghias Uddin, Institute of Chemical Sciences, University of Peshawar, Peshawar-25120, KPK, Pakistan. 14

MATERIALS AND METHODS Plant Materials: The of D. alba were collected from the Ground of Institute of Chemical, Sciences, Peshawar, Peshawar, Khyber Pakhtunkhwa Pakistan. The plants were identified by Prof Dr Abdur Rashid Department, of Botany, University of Peshawar, Peshawar, Pakistan. Middle-East J. Med. Pl. Res., 1(1): 14-18, 2012 Extraction, Fractionation and Isolation: Shade dried of Datura alba were soaked in methanol for 5 days.the extract were concentrated using electric water 0 bath at 50 C The solvent extract was concentrated under 0 reduce pressure at 40C using rotavapor, the extracts obtained were suspended in water and successively partitioned with n-hexane, chloroform, ethyl acetate and methanolic fraction. The chloroform fraction was subjected to column chromatography which led to the isolation of two known compounds -Sitosterol (1) and stigmasterol (2).The structure of these compounds was elucidated by comparing their physical and spectra data with already reported data inliterature [6]. HO -Sitosterol (1) Phytochemical Profiling: The chemical tests were performed on the hexane, chloroform, ethyl acetate and methanolic extracts of Datura alba using standard procedure [7-10] to identify the bioactive secondary metabolite. Test for Alkaloids: 0.2g of each fraction was warmed with 2%H2SO 4for 2 min. The reaction mixture was filtered and added a few drops of Dragendorff reagent to each filtrate. Orange red precipitate indicates presence of alkaloids. Test for Tannins: A small quantity of each extract was mixed with water and heated on water bath and filtered. A few drops of ferric chloride were added. A dark green color indicates tannins. Test for Glycosides: Each extract was hydrolyzed with HCl and neutralized with NaOH solution. A few drops of Fehling solution A and B were added. Red color indicates presence of Glycosides. Test for Reducing Sugars: Each extract were shaken with distilled water and filtered. The filtrate was boiled with fews drops of Fehling solution A and B. An orange red precipitate indicates the presence of sugars. HO Stigmasterol (2) Test for Saponins: 0.2 g of each extract was shaken with 5ml of distilled water and heated to boiling. Frothing (appearance of creamy miss of small bubbles) shows presence of saponins. Test for Flavonoids: 0.2g of each extract was dissolved in diluted NaOH and few drops of HCL were added. Yellow solutions that turn colorless indicate the presence of flavonoids. Test for Phlobatanins: 0.5 g of each extract was dissolved in distilled water and filtered. The filtrate was boiled with 2%HCL solution. Red precipitate indicates the presence of phlobatanins. Test for Steroids: 2ml of acetic anhydride was added to the mixture of 0.5 g of each extract and H2SO 4(2ml). The color from violet to green in some samples indicates the presence of steroids. Test for Terpenoids: 0.2 g of each extract was mixed with 2ml of chloroform and concentrated (3ml) H2SO 4 was carefully added to form a layer. The formation of reddish 15

brown coloration at the interface indicates the presence Antibacterial Assay: The antibacterial activity was done of terpenoids. using modified agar well diffusion method as earlier discuss [12]. The Muller-hinton agar was used as medium. Test for Cardiac Glycoside: To 2ml of plant extract, 1ml of The cultures were taken in triplicates at incubation glacial acetic acid and 5% ferric chloride was added. temperature of 37 Cfor 24 to 72 hours. The broth culture Then few drops of concentrated H2SO4 were added. (0.6 ml) of the test organism was placed in a sterile Presence of greenish blue colour indicates the presence Petri-dish to which 20 ml of the sterile molten MHA was of cardiac glycosides. added. Holes were bored in to the medium using 0.2ml of the extract. Streptomycin was the standard antimicrobial Test for Anthraquinones: 0.5 g of each extract agent at concentration of 2 mg /ml. Inoculation was done was boiled with 10%hcl for few min. The reaction for 1 hour to make possible the diffusion of the mixture were filtered and allowed to cool. Equal antimicrobial agent into the medium. Incubation was volume of chloroform was added to each filtrate. done at 37 Cfor 24 hours and the diameters of the zone of Few drops of 10% ammonia was added to each mixture inhibition of microbial growth were measured in the plate and heated. Rose-pink color indicates the presence of in millimeters. anthraquinones. RESULTS AND DISCUSSION DPPH Radical Assay: The hydrogen atom or electron donation abilities of the corresponding extracts/fractions The results obtained of phytochemical screening of and standards were measured from the bleaching of the Dature alba is presented in Table 1. The n-hexane purple-colored methanol solution of 2, 2-diphenyl-1- fraction exhibited the presence of terpeniods, tanins and picrylhydrazyl (DPPH.) Experiments were carried out saponins while rest of phytochemical was not detected. according to the standard procedure [11]. Briefly, a 1mM The chloroform fraction showed the accumulation of solution of DPPH radical solution in methanol was terpeniods, tanins and saponins, glycosides, reducing prepared and 1ml of this solution was mixed with 3ml of sugar and amino acid. When ethyl acetate was tested for sample solutions in methanol (containing 20-100ug) and various phytochemical tests, it confirmed the presence control (without sample). The solution was stand for of terpeniods, tanins and saponins, reducing sugar, beta 30 min, in dark the absorbance was measured at cyanin and amino acids. 517 nm. Decreasing of the DPPH solution absorbance The antibacterial effect of various solvent fraction indicates an increase of the DPPH radical-scavenging of D. alba is presented in Figure 1. The medicinal value activity. Scavenging of free radicals by DPPH as of the plant can be correlated to thepresence of various percent radical scavenging activities (%RSA) was bioactive secondary metabolites. The n-hexane and ethyl calculated as follows. acetate fractions and methanolic extract of the D. alaba showed the presence of terpenoids which exhibits % DPPH = Control absorbance-extract absorbance X 100/control absorbance antiviral and antibacterial activities. Table 1: Phytochemical profiling of crude methanolic extract and various fractions of of D. alba Chemical components n-hexane chloroform EtOAc MeOH Crude Alkaloids - - - - - Terpenoids + + + + + Flavonoids + - + + + Anthraquinones - - - - - Tannins - - - + + Phlobatanins - - - - - Saponins + + + - + Glycosides - - - - - Reducing sugars - + - + + Steroids + + + + + Cardiac glycosides + + + + + Phenol - + + + + Key words: present: +, absent: - 16

Fig. 1: Anti-bacterial activities of crude extract and various solvent extracted fractions D. alba Key: Well size: 6 mm, B.C =Bacillus subtilus, K.P = Klebsiella pneumoniae, S.A = Staphylococcus aureus Fig. 2: DPPH radical scavenging activity of the crude extract and fractions of D. alba Our findings correlate with the observations of Staphylococcu saureus and Bacillus subtilis with zone previous screened medicinal plants for antimicrobial of inhabation ranging from 10-12. activity, where most of the active plants showed activity The antioxidant effect of various solvent fractions against selected bacterial strains. The pharmacological Datura alba of is presented in Figure 2. The maximum activity of D. alba wasconfirmed by the antimicrobial (88.3%) free radical scavenging effect was observed with assay of various fractions and methanolic extract. Among ethyl acetate fraction at higher concentration (100 µg/ml), the tested bacteria Klebsiella pneumonia and while the effect was weak (22.4%) at 10 µg/ml. the Staphylococcu saureus exhibited complete resistance antioxidant effect of n-hexane was higher (73.40%, at 100 against all the tested solvent fractions except n- hexane. µg/ml) but the effect at lowest concentration (10 µg/ml) The chloroform fraction exhibited 16, 12, 10 and 10 mm was 5.01%. the free radical scavenging effect of zone of inhibition against Klebsiella pneumonia, chloroform and chloroform fraction was moderate (29.89 Staphylococcu saureus, S. Typhimuriun and Bacillus %) at highest concentration. Our results suggest that subtilis respectively. While the ethyl acetate was tested further work is needed to locate the active principles against these bacterial strains, it caused activity only from the various extracts or fractions and such efforts against Staphylococcus aureus 10 mm zone on inhibition. could result in the discovery of new compounds Methanol fraction demonstrated moderate antibacterial possessing a wide range of bioactivity for the treatment effect against Klebsiella pneumonia, S. Typhimuriunand of infectious disease. 17

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