Pankaj Pradhan et al. / Journal of Pharmacy Research 2010, 3(4),

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1 Research Article ISSN: Pankaj Pradhan et al. / Journal of Pharmacy Research 2010, 3(4), Available online through Pharmacognostic, Phytochemical & Quantitative Investigation of Saraca asoca leaves Pankaj Pradhan *1, Lincy Joseph 1, Mathew George 1, Neha Kaushik 1, Rahul Chulet 1. * 1 School of Pharmaceutical Sciences, Jaipur National University,Jagatpura, Jaipur (Rajasthan), India Received on: ; Revised on: ; Accepted on: ABSTRACT Saraca asoca family (Caesalpiniaceae) commonly known as Ashoka briksh. According to our traditional system of medicine, bark of Saraca asoca has tremendous therapeutic effects in menstrual problems. Establishment of Pharmacognostic profile of the leaves will assist in standardization which can guarantee quality, purity and identification of samples. In the present study we describe Pharmacognostical feature, including macroscopy, microscopy and analytical profile and to investigate the phytochemical and quantitative determination of phytoconstituents from the leaves of Saraca asoca. These observations would be of immense value in the botanical identification and standardization of the drug in crude form. This study would help distinguish the drug from its adultered species. Keywords: Microscopy, Physico-Chemical Parameter, Phytochemical Analysis, Quantitative Determination INTRODUCTION Plants are the essential and integral part in Complementary and Alternative medicine and due to this they develop the ability for the formation of secondary metabolite substances which are in turn used to restore health and heal many diseases. 1 After decades of serious obsession with the modern medicinal system, people have started looking at the ancient healing systems like Ayurveda, Siddha and Unani. This is because of the adverse effects associated with synthetic drugs. Herbal drugs play an important role in health care programs especially in developing countries. Ancient Indian literature incorporates a remarkably broad definition of medicinal plants and considers all plant parts to be potential sources of medicinal substances. 2 Saraca asoca is an indigenous herbal drug belonging to the family Caesalpiniaceae. Saraca asoca is distributed in evergreen forests of India up to an elevation of about 750 meters. It is cultivated in many gardens because of its decorative orange red flowers and evergreen beautiful foliage. The leaves are parpinnate with 6 to 12 leaflets, oblong and rigidly sum-coriaceous. The fruit is a pod flat, leathery with 4 to 8 ellipsoid- oblong and compressed seeds. The whole plant is claimed to possess medicinal properties. For example, the bark of Saraca asoca is used by traditional healers for the treatment of menorrhagia, leucorrhoea, visceromegaly, dipsia, ulcers and fevers. 3, 4 The flowers also regarded medicinal property and used in diabetes, cancer and haemorrhagic dysentery. Seeds of S. asoca used for treating bone fracture, strangury and vesical calculi. Despite these, the antimicrobial property of different parts of plant including bark, 5, 6, 7, 8 flower and flower buds are reported. MATERIALS AND METHODS: Collection and Authentication of Plant Material: *Corresponding author. Pankaj Pradhan School of Pharmaceutical Sciences, Jaipur National University Jagatpura, Jaipur (Rajasthan), India Tel.: pnkj.pradhan@gmail.com The leaves of S. asoca were collected in February, 2009, from Mahaveer Nagar, Jaipur, Rajasthan, India. The species was authenticated by Mr. Vinod Sharma, Herbarium incharge, Department of botany, Rajasthan University, Jaipur. A voucher specimen (Voucher No. RUBL ) was deposited in the herbarium of the Department of Botany, Rajasthan University, Rajasthan, India. Macroscopy: The following macroscopic characters for the fresh leaves 9, 10 were noted: colour, odour, taste, size, shape, texture and fracture. Microscopy: The pieces of fresh leaves were boiled in a test tube with chloral hydrate for several minutes until completely clarified and then examine them in chloral hydrate solution. After clarification, leaf pieces are divided into two parts with the help of a scalpel or needle, and carefully turn one part. The leaf can be examined from both the dorsal and ventral surfaces. Transverse section is obtained by cutting the leaf portion including mid rib with the help of sharp blade and staining is done by safranin to impart a red colour to the lignified tissue. 11 Physico-Chemical Parameter: Foreign Matter: The 50 gm sample was spread in a thin layer, and the pieces of foreign matter were sorted out by visual inspection. The powder of foreign matter was sifted through a 250 micron sieve. All portions of the foreign matter were pooled and weighed. Loss on Drying: 10 gm of the drug was weighted in a tarred evaporating dish. It was dried at 105 C for 5 hours and weighed. The drying and weighing was continue at 1 hour interval until difference two successive weighing correspond not more than 0.25%.

2 Ash Values: Total ash value: Pankaj Pradhan et al. / Journal of Pharmacy Research 2010, 3(4), Table 1. Leaf Characters Characters Observation About 3 gm accurately weighed powdered drug was incinerated in a silica dish at a temperature not exceeding C until free from carbon. It was then cooled and weighed. The % w/w of ash with reference to the air-dried drug was calculated. Acid insoluble ash value: To the crucible containing the total ash was added 25 ml of hydrochloric acid. The crucible was then covered with a watch-glass and the mixture was boiled gently for 5 minutes. The watch-glass was rinsed with 5 ml of hot water and this liquid was added in to the crucible. The insoluble matter was collected on an ashless filter-paper and washed with hot water until the filtrate was neutral. The filter-paper contain the insoluble matter was transferred to the original crucible, dried on a hotplate and ignite to constant weight. The residue was allowed to cool in a desiccator for 30 minutes and then weighed. Water soluble ash value: Total ash obtained was boiled for 5 minute with 25 ml of water. Insoluble matter was collected in a crucible or an ashless filter paper. Washed with hot water and ignite for 15 minute at temperature not exceeding 450 C. Weight of insoluble matter was substracted from the weight of the ash, the difference in weight was representing the water soluble ash. Percentage of water soluble was calculated with reference to the air dried drug. Sulphated ash value: 1 gm of substances was taken in an accurately weighed crucible, ignited gently at first until a substance is thoroughly charred. Cooled and the residue was moistened with 1 ml of sulphuric acid, heat gently until white fumes was no longer evolved and ignite at C until black particle were disappeared. Allowed the crucible to cool, few drops of sulphuric acid were added and heat. Ignite as before, allowed to cool and weigh. Operation was repeated until two successive weighing was not differing by more than 0.5 mg. Extractive Values: Alcohol soluble extractive value: 5 gm of the air dried coarsely powdered drug was macerate Figure 1: Transverse Section of Midrib. Colour Odour Taste Size Texture Fracture with 100 ml of alcohol of the specified strength in a closed flask for 24 hour. Shaking frequently during 6 hour and allowed to stand for 18 hour. Filter rapidly and evaporate 25 ml of the filtrate to dryness in tarred flat bottomed shallow dish and dry at 105 C to constant weight and weigh. Water soluble extractive value: 5 gm of the air dried coarsely powdered drug was macerate with 100 ml of distilled water in a closed flask for 24 hour. Shaking frequently during 6 hour and allowed to stand for 18 hour. Filter rapidly and evaporate 25 ml of the filtrate to dryness in tarred flat bottomed shallow dish and dry at 105 C to constant weight and weigh. Foaming Index: 1 gm coarse powder was weighted and transferred to a 500 ml conical flask containing 100 ml of water. It was maintained at moderate boiling for 30 minute. It was cool and filtered in to a 100 ml volumetric flask. Volume was diluted by adding sufficient amount of water. The decoction was poured in to 10 stoppered test tubes in successive portion of 1 ml, 2 ml, 3 ml-etc. upto 10 ml and the volume of liquid in each test tubes was adjusted to 10 ml with water. The tubes were stoppered and they were shaken in a lengthwise motion for 15 seconds, two shake per second. They were allowed stand for 15 minutes and the height of foam was measured. Crude Fibre: 2 gm of powder drug was taken in a beaker, add 50 ml of 10% v/ v nitric acid. Heated to boil with constant stirring (till about 30 second after boiling start). Strain through fine cotton cloth on buchner funnel and residue was washed with boiling water. Transfer the residue from cloth to a beaker and 50 ml of 2.5% v/v Sodium hydroxide solution was added and heat to boil, maintain at boiling point for 30 second with constant stirring. Strain and washed the residue with hot water, transfer in cleaned & dried crucible for quantitative determination. Weigh the 12, 13, 14 residue and percentage of crude fibre was determined. Preparation of Extracts: Green to Yellow Characteristic Bitter cm in length, 2 to 3 cm in width Rough Soft Plant materials were washed with water and shade dried. The dried leaves were crushed to coarsely powdered by wood-grinder. The powdered material was defatted with petroleum ether (60-80 ºC) and then successively extracted in Soxhlet apparatus with different solvents according to increasing polarity. Mark is dried in oven at 40 ºC during solvent changing. The extract was concentrated for further studies on water bath at 40ºC. 15 Phytochemical Analysis: All the extracts obtained by the powdered leaves of Saraca

3 Pankaj Pradhan et al. / Journal of Pharmacy Research 2010, 3(4), asoca were subjected to various qualitative tests for the identification of phytoconstituents present. Table 3. Phytochemical Analysis Test for Alkaloids: Dragendorff s test: To the 1 ml of extract, add 1 ml of Dragendorff s reagent (potassium bismuth iodide solution). An orange-red precipitate indicates the presence of alkaloids. Mayer s test: To the 1 ml of extract, add 1 ml of Mayer s reagent (Potassium mercuric iodide solution). Whitish yellow or cream coloured precipitate indicates the presence of alkaloids. Hager s test: To 1 ml of extract add 3ml of Hager s reagent (saturated aqueous solution of picric acid) yellow colored precipitate indicates the presence of alkaloids. Wagner s test: To the 1 ml of extract add 2 ml of Wagner s reagent (iodine in potassium iodide) formation of reddish brown precipitate indicates the presence of alkaloids. Test for Saponins:Take small quantity of alcoholic and aqueous extract separately and add 20 ml of distilled water and shake in a graduated cylinder for 15 minutes lengthwise. Layer of foam indicates the presence of Saponins. Test for Glycosides: Legal s test: Dissolve the extract in pyridine and add sodium nitroprusside solution to make it alkaline. No formation of pink to red colour shows absence of glycosides. Baljet s test: To 1ml of the test extract, add 1ml of sodium picrate solution and the yellow to orange color reveals the presence of glycosides. Keller-Killani test: 1gm of powdered drug is extracted with 10ml of 70% alcohol for 2 minutes, filtered, add to the filtrate, 10ml of water and 0.5ml of strong solution of lead acetate and filtered and the filtrate is shaken with 5ml of chloroform. The chloroform layer was separated in a porcelain dish and remove the solvent by gentle evaporation. Dissolve the cooled residue in 3ml of glacial acetic acid containing 2 drops of 5% ferric chloride solution. Carefully transfer this solution to the surface of 2ml of concentrated sulphuric acid. A reddish brown layer forms at the junction of the two liquids and the upper layer slowly becomes bluish green, darkening with standing. Borntrager s test: Add a few ml of dilute Sulphuric acid to 1ml of the extract solution. Boil, filter and extract the filtrate with chloroform. The chloroform layer is treated with 1ml of ammonia. The formation of red color of the ammonical layer shows the presence of anthraquinone glycosides. Test for Carbohydrates: Molisch s test: To 2ml of the extract, add 1ml of α-napthol solution, add concentrated sulphuric acid through the side of the test tube. Purple or reddish violet color at the junction of the two liquids reveals the presence. Table 2. Physiochemical Parameters S.No. Parameter Values Obtained 1. Foreign Matter 0.7% 2. Moisture Content 0.06% 3. Total Ash Value 6.33% 4. Acid Insoluble Ash Value 1.4% 5. Water Soluble Ash Value 1.03% 6. Sulphated Ash Value 3.9% 7. Alcohol Soluble Extractive Value 15.6% 8. Water Soluble Extractive Value 12.4% 9. Foaming Index Less than Crude Fibre Content 14.5% Phytoconstituents Pet. Ether Chloroform Ethyl Acetate Methanol Water Carbohydrates Positive Positive Positive Positive Positive Proteins Negative Positive Positive Positive Positive Amino acids Negative Positive Positive Positive Positive Glycosides Negative Negative Positive Negative Negative Alkaloids Negative Negative Negative Negative Negative Flavonoids Negative Positive Positive Positive Positive Tannins Negative Negative Positive Positive Positive Saponins Negative Positive Positive Positive Positive Steroids Negative Negative Positive Positive Negative Resins Negative Negative Negative Negative Negative Fehling s test: To 1ml of the extract, add equal quantities of Fehling solution A and B, upon heating formation of a brick red precipitate indicates the presence of sugars. Benedict s test: To 5ml of Benedict s reagent, add 1ml of extract solution and boil for 2 minutes and cool. Formation of red precipitate shows the presence of sugars. Test for Tannins: i)take the little quantity of test solution and mixed with basic lead acetate solution. Formation of white precipitates indicates the presence of tannins. ii) To 1ml of the extract, add ferric chloride solution, formation of a dark blue or greenish black color product shows the presence of tannins. The little quantity of the extract is treated with potassium ferric cyanide and ammonia solution. A deep red color indicates the presence of tannins. iii) To the test extract, add strong potassium dichromate solution, a yellow color precipitate indicates the presence of tannins and Phenolic compounds. Test for Flavonoids: Shinoda s Test: i)the alcoholic extract is treated with magnesium foil and concentrated HCl give intense cherry red color indicates the presence of flavonones or orange red color indicates the presence of flavonols. ii)the extract is treated with sodium hydroxide; formation of yellow color indicates the presence of flavones. iii)the extract is treated with concentrated H 2 SO 4, formation of yellow or orange color indicates flavones. Test for Steroids: Salkowski test: Dissolve the extract in chloroform and add equal volume of conc. H 2 SO 4. Formation of bluish red to cherry color in chloroform layer and green fluorescence in the acid layer represents the steroidal components in the tested extract. Test for Proteins: Biuret test: Add 1ml of 40% sodium hydroxide solution and 2 drops of 1% CuSO4 solution till a blue color is produced, and then add to the 1ml of the extract. Formation of pinkish or purple violet color indicates the presence of proteins. Ninhydrin test: Add two drops of freshly prepared 0.2% Ninhydrin reagent (0.1% solution in n-butanol) to the small quantity of extract solution and heat. Development of blue color reveals the presence of proteins, peptides or amino acids. Xanthoproteic test: To 1ml of the extract, add 1ml of concentrated nitric acid. A white precipitate is formed, it is boiled and cooled. Then 20% of sodium hydroxide or ammonia is added. Orange color indicates the presence of aromatic amino acids.

4 Table 4. Quantitative Phytochemical Analysis S. No Phytoconstituents % Yield Pankaj Pradhan et al. / Journal of Pharmacy Research 2010, 3(4), like at the base and with a shot pestistipules are intra-petiolar and completely united. The leaf characters showing in table no Flavonoids 1.82 % 2. Saponins 2.18 % 3. Tannins 0.76 % Millon s test: 1ml of test solution is made acidify with sulphuric acid and add Millon s reagent and boil this solution. A yellow precipitate is formed indicates the presence of protein. 16 Quantitative Determaination: Any estimation method applied to plant tissue should allow determination of a particular compound or of a group of compounds in weight percent. Quantitative Determination of Flavonoids: 10 g of the powdered crude sample was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through whatman filter paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated into dryness over a water bath and weighed to a constant weight. 17 Quantitative Determination of Tannins: Drug sample was analysed by adding 1 ml sample (1 gm extract in 1 ml ethyl acetate) and some drop of methylene blue to the 500 ml flask and adding 200 ml double distilled water. Titration was carried out against the 0.005M potassium permanganate solution, until the royal blue fades to a light green. Then titration was continuing drop wise until the lime green changes to yellow. This value was recorded as X ml. Then blank (only indicator and water) titration also carried out and this value recorded as Y ml. According to these reading total tannins was calculated. 18 Quantitative Determination of Saponins: The leaves were ground and 20 g powdered leaves put into a conical flask and 100 ml of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4h with continuous stirring at about 55 C. The mixture was filtered and the residue re-extracted with another 200 ml 20% ethanol. The combined extracts were reduced to 40 ml over water bath at about 90 C. The concentrate was transferred into a 250 ml separatory funnel and 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 60 ml of n- butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the samples were dried in the oven to a constant weight; the saponin content was calculated as percentage. 19 RESULTS AND DISCUSSIONS: Macroscopy: Leaves are parpinnate cm long and the leaflets 6-12, oblong and rigidly sub-coriaceous. Leaves are narrowly lanceolate, cork Microscopy: Transverse section of leaf shows common dicotyledonous characters. Epidermis is single layered. Mesophyll consists of single layered palisade and multilayered spongy tissue with intercellular spaces. In the midrib region, vascular bundle is encircled by a schlerenchymatous ring. Bundle sheath, Xylem and Phloem are clearly visible. The upper and lower epidermal cells are polygonal in shape and their walls are slightly wavy. Physico-Chemical Parameter: The Physicochemical parameters were investigated and reported in table no. 2. The above studies enable the identification of the plant material for future investigation and form an important aspect of drug studies. Phytochemical Analysis: The extracts obtained were subjected to various phytochemical tests, to identify the active constituents, which shows in table no. 3From this analysis, ethyl acetate extract found to have more active constituents compare to other extracts. In ethyl acetate extract presence of Carbohydrate, Proteins, Glycosides, Flavonoids, Tannins and Saponins are determined. Quantitative Determination: From the Qualitative test we determine that, in the Saraca asoca leaves presence of tannins, flavonoids and Saponins in excess and glycoside and steroids in minute amount. Therefore the quantitative amount of tannins, saponins and flavonoids estimated. The percentage amounts of these are shown in table no. 4. From the quantitative investigation we found presence of Flavonoids, Tannins and Saponins in the Saraca asoca leaves. These Phytoconstituents are responsible for various therapeutic effects of Saraca asoca leaves. CONCLUSION: As there is no Pharmacognostic / anatomical work on Saraca asoca leaves record of this much valued traditional drug, the present work was taken up with a view to lay down standards which could be useful to detect the authenticity of this medicinally useful plant. In other words, the Pharmacognostic features examined in the present study may serve as tool for identification of the plant for validation of the raw material and for standardization of its formulations. 20 ACKNOWLEDGEMENT: Authors are thankful to the Mr. Vinod Sharma, Department of Botany, Rajasthan University, Jaipur for identification and authentication of plant

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