ISSN: 2277-8713 Tushar Brahmbhatt,, IIJJPPRRBBSS,, 22001122:: VVoolluummee11 ((3): 317-327 IJPRBS RREESSEEAARRCCHH AARRTTIICCLLEE IIINNNTTTEEERRRNNNAAATTTIIIOOONNNAAALLL JJJOOOUUURRRNNNAAALLL OOOFFF PPPHHHAAARRRMMMAAACCCEEEUUUTTTIIICCCAAALLL RRREEESSSEEEAAARRRCCCHHH AND BIO-SCIENCE AA PPaatthh ffoorr HHoorriizziinngg YY oouurr IInnnnoovvaattiivvee WWoorrkk DDDEEEVVVEEELLLOOOPPPMMMEEENNNTTT AAANNNDDD EEEVVVAAALLLUUUAAATTTIIIOOONNN OOOFFF VVVAAARRRIIIOOOUUUSSS OOORRRAAALLL HERBAL FFOORRMMUULLAATTIIOONNSS FFOORR AANNTTII-AASSTTHHMMAATTIICC PPLLAANNTT EEXXTTRRAACCTT *TTUUSSHHAARR BBRRAAHHMMBBHHAATTTT,, BBIIRREENN SSHHAAHH,, DDr.. UUPPEENNDDRRAA PPAATTEELL,, HHIIRREENN KKAADDIIKKAARR DDDeeepppaaarrrtttmmmeeennnttt ooofff PPPhhhaaarrrmmmaaacccyyy,,, AAArrriiihhhaaannnttt SSSccchhhoooooolll ooofff PPPhhhaaarrrmmmaaacccyyy aaannnddd BBBiiiooo RRReeessseeeaaarrrccchhh IIInnnssstttiiitttuuuttteee,,, AAAdddaaalllaaajjj,,, Gandhinagar CCoorrrreessppoonnddiinngg AAuutthhoorr EEmmaaiill:: ttuusshh77552288@@yyaahhoooo..ccoomm AAvvaaiillaabbllee OOnnlliinnee AAtt wwwwww..iijjpprrbbss..ccoomm ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS Accepted Date: 31/05/2012 Publish Date: 27/06/2012 Abstract: To develop and evaluate herbal oral dosage forms (Conventional Tablet and Syrup) for ease of dispensing and consumption of dry powder of ethanolic extract of leaves of Hiptage benghalensis (L) Kurzz used to treat asthma and to check pharmacological effect of formulated dosage form by suitable experimental animal model. Tablet form was developed by wet granulation and direct compression methods to determine the more suitable method using various excipients like MCC, Lactose etc. By considering difficulty of solubility of herbal drugs in a vehicle, in one of the liquid class, decoction form of drugs in specific vehicle was used. Formulated dosage forms then subjected to evaluation of production quality by different methods stated as per official compendia. the evaluated formulation were than subjected to check for its efficacy by using experimental animal model like histamine and Ach induced bronchoconstriction in guinea pigs. Such evaluation has unique position in development of new herbal formulations. The prepared tablets were spherical puffy green colour with smooth surface having acceptable elegance. W form of 5 tablets was of good quality with regard to hardness, friability & weight variation. W form of 5 the tablets formulated with starch paste (6% w/v) as disintegrating agent & binder show disintegration within 549±0.57 second. The liquid oral herbal dosage forms like liquid oral prepared showed good elegance. The liquid oral evaluated for measurement of pH, specific gravity & stability. The final formulation found to have pH 6.94±0.023 and specific gravity 1.34±0.045 gm/ml. The results of stability study of final liquid oral form of drugs indicate the homogeneity of syrup without turbidity at storage temperature. Formulated dosage forms significant protection from histamine and Ach induced brocnchospasm when compared to control group and is comparable to crude extract of Hiptage benghalensis and marketed anti- asthmatic product. Keywords: Hiptage benghalensis, Anti-asthmatic activity, Herbal formulation Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS INTRODUCTION According to WHO, asthma is a disease Madhavi lata (Hiptage benghalensis), native characterized by recurrent attacks of from India to the Philippines, is a vine like breathlessness and wheezing, which vary in plant that is often cultivated in the tropics severity and frequency from person to for its attractive and fragrant flowers. It is person. It attacks all age groups but often used medicinally in India. The bark, leaves starts in childhood. This condition is due to and flowers are aromatic, bitter, acrid, inflammation of the air passages in the lungs astringent, refrigerant, vulnerary, and affects the sensitivity of the nerve expectorant, cardiotonic, anti-inflammatory endings in the airways so they become and insecticidal. They are useful in burning easily irritated. sensation, wounds, ulcers, cough, and asthma. The currently used drugs for the treatment of asthma are so far from satisfactory as they MATERIALS AND METHODS provide only symptomatic relief; produce several adverse effects, like Muscle tremor Materials and hypokalemia are major adverse effects Hiptage Benghalensis fresh leaves collected of β agonists. Theophyline has narrow 2 from Ayurvedic udhyan, Gandhinagar. therapeutic index and requires monitoring of Micro crystalline cellulose was obtained drug levels. from FMC biopolymers, USA. Ethanol, Hence, ayurveda has recommended a Acetone, Magnesium stearate and talc were number of drugs from indigenous plant obtained from S.D. fine chemicals, Mumbai. sources for the treatment of asthma and allergic disorder, and have been successful Methods in controlling these diseases as well. Some Collection & authentication of plant herbal drugs which are mainly used in Fresh plant of Hiptage benghalensis was treatment of asthma are, Albezzia Lebbeck, collected from ayurvedic udhyan, Euphorbia Hirta, Adhatoda Vasica, and Gandhinagar. The plant identified and Allium Capa. Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS authentified by Mr. Divyakant Patel, These studies were carried out as per the Assistant professor, Department of standard procedures. In the present study, Pharmacognosy, Arihant School of the leaf powder was treated with 1.0 N Pharmacy & BRI, Adalaj. The plant was aqueous sodium hydroxide and 1.0 N dried in shade and ground to get a coarse alcoholic sodium hydroxide, acids like 1.0 N powder. hydrochloric acid and 50% sulphuric acid. These extracts were subjected to Preparation of plant extract fluorescence analysis in visible/daylight and The coarse powder (500 g) of the dried plant UV light (254nm & 365nm). Various ash was exhaustively extracted using 95% types and extractive values were determined ethanol (2,000 ml) in a soxhlet extractor at a by following standard method.1 temperature of 60–70 0C (yield 3%, w/w). Alcoholic and aqueous extract of Hiptage The extract was concentrated under reduced benghalensis was subjected to standard pressure to yield a syrupy mass and stored in chemical test for detection of different air tight container in cool place and used phytochemicals.2-5 throughout the project study. FORMULATION OF HERBAL Phytochemical analysis of plant extracts TABLETS CONTAINING PLANT of Hiptage benghalensis EXTRACT Preliminary phytochemical analysis Preparation of herbal tablets by direct Shaded dried and powdered leaf samples compression method were successively extracted with petroleum, benzene, chloroform and alcohol. The Herbal tablets containing plant extract were extracts were filtered and concentrated using prepared by direct compression method. The vaccum distillation. The different extracts composition of various formulations is given were subjected to qualitative tests for the in Table 1. All ingredients were weighed identification of various phytochemical accurately and mixed well in dry mortar. constituents as per standard procedure.1 Microcrystalline cellulose was used as a disintegrating agent. Lactose was use as Physico-chemical constant and diluents. Talc and magnesium stearate were fluorescence analysis used as a lubricants. Tablets were Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS compressed each of 500 mg weight on a 10 density, tapped density, hausner’s ratio, station rotary tablet compression machine carr’s index and angle of repose. using 9 mm round flat punch. Tablet Post compression parameters8-9 formulations prepared by direct compression method were coded as D1 to D3. Tablets were evaluated for hardness by using a Monsanto type hardness tester. Preparation of herbal tablets by wet Friability of the tablets was evaluated by a granulation method Roche Friabilator (Mumbai, India). Herbal tablets of plant extract were prepared Thickness of the tablets was measured by by wet granulation method by using PVP using Vernier calipers. K30 and starch mucilage with varying concentration (4% w/v, 6% w/v and 8% Weight variation w/v) as binder. Composition is given in Randomly selected twenty tablets were Table 2. All ingredients were weighed weighed individually and together in a accurately and mix well in dry mortar. Talc single pan balance. The average weight was and magnesium stearate were used as a noted and standard deviation calculated. The lubricants. Granules were prepared by tablet passes the test if not more than two passing wet mass from 12 mesh standard tablets fall outside the percentage limit and sieve. Granules were dried and passed none of the tablet differs by more than through 20 mesh standard sieve. Granules double the percentage limit. were mixed with lubricants. Tablets were Disintegration test prepared by compressing granules in rotary Disintegration test was performed on 6 core tablet compression machine using 9 mm flat tablets at 37 ± 0.5° C in 900 ml of distilled punch. Tablet formulations prepared by wet water using Electro lab disintegration tester granulation were coded as W1 to W6. (USP, Model ED2L). EVALUATION OF HERBAL TABLETS CONTAINING PLANT EXTRACT FORMULATION OF ORAL HERBAL LIQUID FORMULATION Pre compression parameters6-7 CONTAINING PLANT EXTRACT10 The flow property of powder ready to Preparation of liquid oral compress was evaluated by measuring bulk Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS To prepare liquid oral form of plant extract EVALUATION OF ORAL HERBAL of Hiptage benghalensis, following steps LIQUID FORMULATION were carried out. It was prepared by CONTAINING PLANT EXTRACT11-12 decoction method. The herbal syrup was evaluated for various parameters such as physical appearance Method of preparation of decoction (colour, odour and taste), pH, specific 500 gm each of powder Hiptage gravity and viscosity. benghalensis was taken. Powder was mixed with 4000 ml (4 litres) of water. The Determination of pH powdered material was boiled until total The pH of herbal oral liquids was obtained volume become one fourth of previous. by potentiometer. The pH method was After boiling liquid was cooled and filtered. calibrated using distilled water, buffer (at Filtrate was taken to prepare final liquid oral pH 4 and 9) pH till constant reading. form. Determination of viscosity Method of preparation of simple syrup Ostwald viscometer was used to determine 850 gm of sucrose was dissolved in the viscosity of all samples of oral liquid. sufficient water to get 1000 ml of The method was followed as per the concentrated simple syrup. Then the solution standard procedure. was filtered. This simple syrup was used as vehicle. Determination of specific gravity Pcynometer was used to determine the Method of preparation of final liquid oral specific gravity at 25 0C. It was determined form dividing the weight of sample (expressed in To prepare final liquid oral of Hiptage gm) by the weight of water (in ml). benghalensis, one part of decoction was mixed with five parts of simple syrup (1: 5). Stability testing of oral herbal liquid dosage Solubility was checked by observing the form clarity of solution visually. The final liquid Stability study of prepared syrup was carried oral form of Hiptage benghalensis was then out for 3 days. The syrup was kept at subjected to evaluation of production quality different temperature and relative humidity as per official standards. for short term stability study at 40C, 470C and at room temperature. Humidity was kept Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS at 75% RH. The parameters checked were Guinea pigs of either sex weighing 350 - turbidity, colour and taste. Syrup was stored 500 gm were selected and randomly divided in ambered colour glass bottle. into six groups each containing six animals. The drugs were administered orally in 0.5% PHARMACOLOGICAL EVALUATION sodium carboxy methyl cellulose (CMC). OF PREPARED TABLETS AND SYRUP The single dose treatments were given one BY USING HISTAMINE AND ACH and half hour before the study. INDUCED BRONCHOSPASM IN GUINEA PIGS. (IN VIVO) Later the animals were exposed to an aerosol of 0.25% histamine and time for pre- Selection of animals convulsion state was noted for each animal. Male albino rats of wistar strain, weighing After about 15 days of wash out period, the 225-250 g and guinea pigs, 6-7 weeks old same animals were given the above were used for study. All animals were treatments and time for pre-convulsion state housed at ambient temperature (22 ± 1°C), was noted for 0.5% acetylcholine bromide relative humidity (55 ± 5%) and 12/12 h aerosol spray. light/dark cycle. Rats have access to standard pellet diet and water given ad STATISTICAL ANALYSIS libitum. Guinea pigs have access to standard Results were analyzed by one way analysis pellet diet, tomatoes, grass and soaked black of variance (Dunnet’s test) (n=6), were grains. The protocol of the experiment has expressed as mean ± S.E.M. at the been submitted to the IAEC as per the probability level of 95% and P< 0.05 was guidelines of the Committee for the Purpose considered as significant. of Control and Supervision of Experiments on Animals (Protocol no. = RESULTS AND DISCUSSION ASP&BRI/AH/12/02), Ministry of Social Justice and Empowerment, Government of India for approval. Qualitative phytochemical analysis of plant extracts of Hiptage benghalensis Studies on Acetylcholine and Histamine Shaded dried and powdered leaf samples induced bronchospasm in guinea pigs were successively extracted with petroleum, Procedure benzene, chloroform and alcohol. The Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS extracts were filtered and concentrated using aqueous sodium hydroxide and 1.0 N vaccum distillation. The different extracts alcoholic sodium hydroxide, acids like 1.0 N were subjected to qualitative tests for the hydrochloric acid and 50% sulphuric acid. identification of various phytochemical These extracts were subjected to constituents as per standard procedure. fluorescence analysis in visible/daylight and UV light (254nm & 365nm). Various ash The detailed and systematic types and extractive values were determined pharmacognostical evaluation would give by following standard method.59 valuable information for the future studies. The physico-chemical constant like ash and The results of various types of ash provided extractive values were determined (Table 4). a basis to identify the quality and purity of the drug. In fluorescence analysis revealed The result of Table 5 shows that that the powdered leaves of Hiptage phytochemical constituent of crude leaves benghalensis was treated various chemical extract and prepared formulation. reagents to give different colours (Table 6). Fluorescence is the phenomenon exhibited The result shows presence of Sterols, by various chemical constituents present in Saponins, Coumarins whereas absence of the plant material. Many phytocompounds alkaloids in both crude plant extract and fluoresce when suitably illuminated. The prepared formulations. This result confirms fluorescence colour is specific for each the preliminary phyto constituent in compound. A non-fluorescent compound formulation as found in crude extract. may fluoresce if mixed with impurities that This similarity in preliminary phyto- are fluorescent. constituents in both crude plant extract and The fluorescent method is adequately prepared formulations supports comparable sensitive and enables the precise and pharmacological activity of prepared accurate determination of the analyze over a pharmacological activity with crude satisfactory concentration range without ethanolic extract of Hiptage benghalensis. several time consuming dilution steps prior These studies were carried out as per the to analysis of pharmaceutical samples. standard procedures59. In the present study, the leaf powder was treated with 1.0 N Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS EVALUATION OF HERBAL TABLETS EVALUATION OF HERBAL TABLETS CONTAINING PLANT EXTRACT CONTAINING PLANT EXTRACT PREPARED BY DIRECT PREPARED BY WET GRANULATION COMPRESSION Pre compression parameters Pre compression parameters Results of pre compression parameters of The results of powder blend for direct granules are shown in Table 9. From result it compression is given in Table 7. From result was found that granules prepared for the it was found that the powder blend prepared tablets have Angle of repose (27.45 ± 0.45 for tablets have Angle of repose (31.25 ± to 32.36 ± 0.73), Hausner’s ratio (1.15 ± 0.082 to 33.67 ± 1.70), Hauser’s ratio (1.28 0.033 to 1.26 ± 0.012) and Carr’s index ± 0.23 to 1.32 ± 0.36) and Carr’s index (13.20 ± 0.034 to 20.92 ± 0.31), which (15.53 ± 0.786 to 16.79 ± 0.654), which shows good flow property and shows good flow property and compressibility of granules when compared compressibility of powder when compared with standard data of pre-compression with standard data of pre compression parameters. parameters . Post compression parameters Post compression parameters Results of tablets prepared by wet The results of post compression parameters granulation method are given in Table 10. are given in Table 8. Tablets prepared using Tablets prepared using direct compression direct compression techniques were found techniques were found within range of within range of uniform thickness and uniform thickness and acceptable weight acceptable weight variations limit as per variations limit as per Pharmacopoieal Pharmacopoeial specifications. specifications. Hardness was found in the range of 3-5 kg/cm2 for all the formulation Hardness was found in the range of 2-3 of the tablet and the friability for the same kg/cm2 for all the batches of the tablets. The was found to be less than 1 indicating friability was found to be more than 1 so, sufficient mechanical integrity and strength direct compression method was not of the prepared tablets. acceptable for the compression of the tablets. Available Online At www.ijprbs.com ISSN: 2277-8713 Tushar Brahmbhatt, IJPRBS, 2012: Volume1 (3): 317-327 IJPRBS EVALUATION OF ORAL HERBAL clear solution was obtained and passes LIQUID FORMULATION through an appropriate filter. CONTAINING PLANT EXTRACT The pH value of herbal oral liquids was Results of prepared liquid oral are given in obtained by potentiometer. The pH of all Table 11. Syrup has sweet test and dark samples was in the range of 6.0 – 7.9. The brown in colour. specific gravity of all the samples was in the range of 1.33 to 1.55. The viscosity of STABILITY TESTING OF ORAL herbal oral liquids when tested by Ostwald HERBAL LIQUID DOSAGE FORM viscometer was in the range of 190 to 195 cps throughout the period of stability. The result of stability study is shown in Table 12. The results of stability study of PHARMACOLOGICAL EVALUATION syrup indicate no change in colour, taste of OF PREPARED TABLETS AND SYRUP herbal liquid dosage form. No turbidity was BY USING HISTAMINE AND ACH found INDUCED BRONCHOSPASM IN The physical parameters like colour, odour, GUINEA PIGS. (IN VIVO) specific gravity, hydrogen ion concentration Pretreatment with Marketed tablet, marketed and viscosity for the formulated herbal syrup, and crude ethanolic extract of cough syrup are mention in Table 11. Five Hiptage benghalensis increased Pre samples of 500 ml of herbal oral liquids Convulsion Time in guinea pigs in both developed with various concentrations of histamine and ach induced Hiptage benghalensis studied throughout the bronchoconstriction. (Table 13, Figure 1, 2) period by its applicable parameters and it reveals that there was no any major change This increased pre-convulsion time was in values as shown in Table 12 that proves a significant when compare to control group good stability. The yellowish brown colored and was comparable to marketed tablet and oral liquids with pleasant odor having marketed syrup group. appropriate sweet taste were obtained. A Available Online At www.ijprbs.com