JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES

Gupta P K et.al. / JPBMS, 2011, 6 (16) Available online at www.jpbms.info ISSN NO- 2230-7885 Research Article JPBMS JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES PHARMACOGNOSTICAL AND PHYTO-CHEMICAL EVALUATION OF OLEO GUM- RESIN OF SHALLAKI (BOSWELLIA SERRATA ROXB.) * Gupta P.K.1, Chandola H.M 2, Harisha C.R.3, Shukla V.J.4, Varun B. Gupta5, Pankaj Nariya6 Institute for Post graduate Teaching and Research in Ayurveda,Gujarat Ayurveda University, Jamnagar -361008 1 M.D. Scholar, Dept of Kayachikitsa, (Senior Medical Officer), Gujarat Ayurveda University, Jamnagar -361008, India. 2 Professor & Head-Kayachikitsa, Gujarat Ayurveda University, Jamnagar -361008, India. 3 Head- Pharmacognosy Laboratory, Gujarat Ayurveda University, Jamnagar -361008, India. 4 Head, Pharmaceutical Chemistry Laboratory, Gujarat Ayurveda University, Jamnagar -361008, India. 5 PhD Scholar, Pharmacology lab, Gujarat Ayurveda University, Jamnagar -361008, India. 6 PhD Scholar, Pharmaceutical chemistry lab, Gujarat Ayurveda University, Jamnagar -361008, India. Abstract Oleo-gum-resin of Shallaki (Boswellia serrata Roxb.) is a drug used by all systems of medicines, traditional as well as modern and prescribed in the management of inflammatory diseases viz bronchitis, osteo-arthritis etc. Pharmacognostical study counting both macroscopic and powder microscopy of raw drug exposed the quality and genuineness of all the constituents of Shallaki. Organoleptic features along with microscopically and chemical tests of coarse powder made out of the crude drugs were within the standard range. Loss on drying 20.74%, Total ash 0.949% w/v, water soluble and alcohol soluble extract were recorded 24.20% and 68.80% respectively. Volatile oil was present 2.4%. TLC and HPTLC were carried out after organizing appropriate solvent system in which maximum 5 were distinguished in TLC and up to 8 in HPTLC and most of the values were identical when done with different sample extractive methods. This shows the presence of certain definite constituents in the drug sample and is helpful for the easy separation of these particles. Keywords:- Boswellia serrata Roxb., oleogum-resin, Pharmacognosy, Phytochemistry Introduction: Since ancient time the production of herbal medicine was chiefly on individual patient basis where the standardization techniques were mainly based on judgment of the physician. But in modern times the scenario has changed utterly and currently most of the times herbal medicines are prepared in huge scale. So to prevent the adulteration of drugs and also to ensure the quality control it is essential to pursue certain protocols [1] starting from the identification of single drugs to the formation of the end product. Accurate identification and guarantee of purity through pharmacognosy and pharmaceutical chemistry measures is inescapable ladder needed for the quality assurance and standardization of any of the herbal medicine whether it is single drug or formulation Shallaki (Boswellia serrata Roxb.) Family Burseraceae, commonly known as Indian olibanum and frankincense is a middle sized tree that widely grows in dry hilly forests of India especially Rajasthan, Madhya Pradesh, Gujarat, Bihar, Assam, Orissa as well as central peninsular regions of Andhra Pradesh, Assam etc. [Plate 1.1, and 1.2] Plate - 1: Photographs of Boswellia serrata Roxb. Oleo-gumresin 1.1 A branch with leaves 1.2 A trunk with exfoliating bark 1.3 Exudation of resin after tapping 1.4 Crude Gum-resin of B. serrata 1.5 Trichomes in powder microscopy 1.6 Sclereides in powder microscopy Corresponding Author Dr P. K. Gupta, Contact: +919624452001 Chaman Vihar, Niranjanpur, Dehradun. Uttarakhand. - 248001 1 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 06, Issue 06

1.7 Resin-yellowish brown colored 1.8 Aloerone grain with oil globules The word frankincense derived from the Old French word frauk-encens and it means the true, authentic, pure, or free lighting incense. The word olibanum derived from the Arabic al-luban and it means the milk or authentic incense (SEPASAL database). [2] The oleo-gum-resin and the bark are used. Oleo-gum-resin is a term used to describe ole (looks fat or oily in nature), gum (parts are soluble in water), and resin (whole or partly soluble in alcohol). (Felter & Lloyd, 1898). [3] The gum is tapped from the incision made on the trunk [Plate 1.3] of the tree which is then stored in specially made bamboo basket and converted into different grades of material according to flavor, color, shape and size. The fresh gum obtained from the tree is hot dry with a Materials and Methods: Drug Collection The sample of the Shallaki [Plate 1.4] which have been used in the present study were collected from Pharmacy I.P.G.T & R.A; GAU Jamnagar. Later the organoleptic and Pharmacognostical Evaluation: The initial purpose of the study was to confirm the authenticity of the test drug. For that coarse powder was subjected for organoleptic feature estimation and its solubility was also assessed in water, alcohol as well as in Table 1: OrganolepticCharacters: Part used Consistensy Nature of powder Shallaki Oleo-gum-resin Coarse powder Hygroscopic White yellow * Felter & Lloyd, 1898, Evans, 1996 and Dabur Research Foundation & Dabur Ayurvet Limited, 2002 ** Felter & Lloyd, 1898, Holmes, 1999 pleasant flavor and slightly bitter in taste. It is the frankincense of ancient Egyptians, Greeks and Romans who used it as prized incense, fumigant as well as a multipurpose aromatic. It is generally used in making incense powder and sticks. [4] The oleo gum resin of Boswellia serrata is used in various Unani and Ayurvedic preparation. It is reported to be useful in the treatment of bronchitis, asthma, cough, bad throat and various intestinal problems. It is a diaphoretic and astringent prescribe in various syphilitic and pulmonary diseases [5-8]. Historically Boswellia serrata is recommended for osteoarthritis, juvenile rheumatoid arthritis (JRA), soft tissue fibrosis and spondylitis without any side effect. [1] powder microscopy of the drugs was carried out in Pharmacognosy Lab and physicochemical analysis was carried out in Pharmaceutical Chemistry Lab of I P G T & R A Jamnagar. The drug was confirmed to be authentic and good-quality. acid. Powder microscopy was made with the coarse powder of the dried samples before and after staining. Phloroglucinol and HCl were used for the staining purpose. Results are reported in the Table 1, 2 and 3. Color Taste Odor Sub-acrid, Bitter, pungent, sweet * Sweet balsamic aromatic** Table 2: Results of Powder Microscopy S. No Parameters Description 1 Irregular shape, translucent, Brittle irregular droplets or tear shaped Appearance lumps or globes, dusty. 2 Trichome Present 3 Sclereides Present 4 Oil Globules Present-yellow colored Table 3: Results of Chemical Tests S. No Parameters Description 1 Water solubility Soluble 2 Alcohol solubility Soluble 3 Acid solubility Insoluble 4 With Glycerin & Concentrated HCl soluble & give Reddish or Brown Colour Physico chemical analysis: 2 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 06, Issue 06

Oleo-gum-resin of Shallaki was subjected to physico chemical parameters like Loss of drying% [9], Total Ash Value% [10] and ph value. The Water soluble Extract% [11], Alcohol (Methanol) soluble extract% [12] and Volatile Oil% Gupta P K et.al. / JPBMS, 2011, 6 (16) [13] was assessed by methods described in API (Ayurvedic Pharmacopeia of India). Results are reported in the Table 4. Table 4: Physico chemical parameters Sr No Test Result 1 Loss on drying 20.74 % w/w 2 Total Ash Value 06.00 % w/w 3 Water soluble Extract 24.20 % w/w 4 Volatile Oil 02.40 %w/w 5 ph value 06.09 Chromatographic Evaluation: TLC and HPTLC were carried out after making appropriate solvent system with Ethyl acetate extract and Methanolic extract of Shallaki resin. TLC [14] : Thin layer chromatography and High performance thin layer chromatography were performed for the normal phase separation of components of ethyl acetate and methanol extracts of shallaki resin. Solvent system was prepared by taking Toluene: Ethyl acetate: Table 5: Results of TLC S.No Track Hexane: Formic acid in the proportion of 8:2:0.5:0.3 respectively. The obtained from both the extracts were examined under ultra violet light of wavelength 254nm and 366nm.The resolution factor (R f) was calculated by using the formula R f = Distance travelled by solute/distance travelled by solvent. The final results are reported in the Table 5. 254nm 366nm After Spray 0.21,0.34,0.47, 1 E.A 5 1 0.35 3 0.21,0.34,0.47 0.61,0.80 2 Me- OH 3 0.34,0.46, 0.78 1 0.34 1 0.34 Sample preparation: Track - 1: Ethyl acetate extract of Shallaki (EA) Track - 2: Methanol extract of Shallaki (MeOH) Solvent system - Toluene: Ethyl acetate: Hexane: Formic acid: (8:2:0.5:0.3) Stationary phase- Silica gel G Visualization-Under long U.V (366nm) and short U.V (254nm) Spray Reagent : Methanolic sulphuric acid HPTLC [15] : HPTLC study of the Ethyl acetate extract (Track 1) and Methanol extract (Track 2) was also carried out by using the same solvent system of Toluene: Ethyl acetate: Hexane: Formic acid (8:2:0.5:0.3).. Table 6: Results of HPTLC S.No Track 1 E.A 8 2 Me- OH 7 After completion of HPTLC, post chromatographic deprivation was done with Methanolic sulphuric acid. All the results are shown in the Table 6. 254nm 366nm After Spray 0.21,0.34,0.47, 0.58,0.61,0.70, 1 0.35 6 0.21,0.34,0.47,0.58,0.70 0.80,0.93 0.34,0.46,0.56, 0.68, 0.78, 0.82, 0.93 Track 1: Ethyl acetate extract of Shallaki Tack 2: Me-OH extract of Shallaki Solvent system: Toluene:Ethyl acetate:hexane:formic acid: (8:2:0.5:0.3) Spray Reagent: Methanolic sulphuric acid 1 0.34 6 0.34, 0.46,0.56, 0.68, 0.78, 0.82, Results and discussion: Pharmacognostical Study: Detailed pharmacognostical evaluation was carried out for test drug. Organoleptic characters were represented in Table 1, shows hygroscopic nature of resin powder of Shallaki. The colour of the powder observed white yellow, which was some different from brownish yellow colored resin crystals after tapping the plant. Taste and odor were specific as per quoted in API and other sources. The powder microscopy revealed presence of simple trichomes [Plate 1.5] and fragments of sclereids [Plate 1.6] are from the outer portion of the bark. The yellowish brown coloured resin was observed microscopically [Plate 1.7]. Some yellow colored oil globules along with aleorone granules [Plate 1.8] were also observed in the microscopical examination, which determined the presence of certain fat soluble chemical constituents in the resin powder of Shallaki. The details of results 3 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 06, Issue 06

obtained in pharmacognostical study are enlisted in the Table 2. The various solubility and chemical tests were Analytical study Physico chemical parameters: The results observed after experiments are mentioned in the Table 4. Loss on drying test is a method to measure the loss in mass of the sample, when dried under the specific conditions. This method is applied to determine the amount of water, all or a part of water for crystallization, or volatile matter in the sample [16]. The loss on drying 20.74w/w in sample was observed within normal limits as in standardized by API. [17] The less value of moisture content could prevent bacterial, fungal or yeast growth [18].(Table 4). The ash remaining after the ignition of the prescribed amount of substance is calculated as Total Ash value. Total ash is designed to measure the total amount of material remaining after ignition. It includes both physiological (which is derived from the plant tissue itself) and nonphysiological ash (residue of the extraneous matter like sand etc adhering to the plant substance). [19] For Shallaki oleo-gum resin the total ash determined was 6.0%w/v. (Table 4). Water soluble and Alcohol (methanol) soluble extractive values were found to be 24.20% w/w and 68.80% w/w respectively. The percent yields of different extracts are given in Table 4. Volatile oils are characterized by their odour, oil-like appearance and ability to volatilize at room temperature. Chemically, they are usually composed of mixtures of, for example, monoterpenes, sesquiterpenes and their oxygenated derivatives. Aromatic compounds predominate in certain volatile oils. [20] The percent yields of volatile oil is given in (Table 4) which prove the presence of certain chemical constituents in the form of mono-terpenoides and terpenoides.ph is the measure of acidity or alkalinity of a solution. In the present sample ph was detected by using digital ph meter-335 (Systronics) and it was 6.09 showing the mild acidic nature of the solution. (Table 4). Thin layer chromatography is the most common form of chromatographic method used by ayurvedic research workers to detect the number of compounds present in a product. It also helps to determine the purity of the sample. Identity of a compound is also possible by comparing it with the R f value of a known (standard) compound. Here for the purpose of conducting TLC two tracks were made; in track 1 the sample was ethyl acetate extract of Shallaki resin and in track 2 with methanol extract used. After careful analysis and discussion with experts the mobile phase was fixed to be Toluene: Ethyl acetate: Hexane: Formic acid in the proportion of References: 1. Dermarderosian. The review of natural products. JB Lippincott: 2002 2. SEPASAL Database.http://www.rbgkew.org.uk/ceb/ sepasal/bsacra.htm. (accessed December 2009.) 3. Felter H, Lloyd J. King s American Dispensatory, vol I and II. Ohio Valley Company. 1898. 4. Aman Upaganlawar, Balu Ghule. Pharmacological carried out for the test drug. The tests and the results are depleted in the Table 3. 8:2:0.5:0.3 respectively. The sample tracks and mobile phase remained the same for all the experiments related to TLC and HPTLC. The produced by TLC were observed in day light, short UV and long UV and R f value was calculated. Track 1 showed five with R f 0.21, 0.34, 0.47, 0.61 and 0.80 whereas three were available in track 2 with R f 0.34, 0.46 and 0.78.The matter of significance is that in both the tracks the R f value of two compounds are same. So this implies the definite presence of certain constituents in the sample. HPTLC is a more convenient and simple procedure in which finger printing profile is available in the form of graph and densitogram. [21] In present HPTLC study maximum eight obtained in track 1 and seven obtained in track 2 in which once again most of the R f values were matching when compared between both the tracks. After completion of chromatographic procedure spraying of the plate was done with methanolic sulphuric acid and the obtained were observed in day light. [Plate 2 Densitograms of Boswellia serrata oleo-gumresin]. Conclusion: The preliminary step to reach the goal of standardization in case of traditional medicine is the strict and vigilant following of the parameters of pharmacognosy and phyto chemistry. Pharmacognosy study helps in authentication of the commonly used drugs through morphological, histological and physico-chemical parameters. This can prevent the accidental misuse of drugs and adulteration to a greater extent. [22] With this aim pharmacognosy and phyto chemical evaluation of Shallaki [Boswellia serrata Roxb.] was performed which is a gifted medicine in the management of Inflammatory diseases especially in arthritis. Preliminary Organoleptic features and results of powder microscopy as well as chemical tests were compared with the parameters mentioned in API and all the ingredients were proved to be authentic. In phytochemical analysis, Loss on drying, Total Ash value, water soluble extract value, presence of volatile oil and ph were assessed. In the present study the drug was proved to be genuine by assessing the organoleptic characters and powder microscopy features. Though the groundwork requisites for the authentication of Shallaki are covered in the current study, additional cavernous analysis and investigations are required for the identification of all the active chemical constituents of the test drug to substantiate the clinical efficacy. 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