Journal of Pharmacognosy and Phytochemistry 2014; 3 (3): 184-187 ISSN 2278-4136 JPP 2014; 3 (3): 184-187 Received: 07-08-2014 Accepted: 10-09-2014 T.P. Ajeesh Krishna Department of Biology, The Gandhigram Rural I nstitute - Deemed University, Gandigram, Dindigul-624 302, Tamil Nadu, India. T.P. Adarsh Krishna Department of Organic Chemistry, School of Herbal Studies and Naturo Sciences, Dravidian University, K uppam-517 426, A ndhra Pradesh, I ndia. R. Kumuthakallavalli Department of Biology, The Gandhigram Rural I nstitute - Deemed University, Gandigram, Dindigul-624 302, Tamil Nadu, India. V.N. Sanyo Raj Department of Botany, ST. M ary’s College, Sulthan Bathery - 673 592, W ayanad, K erala, India. Sanis Juliet Department of Veterinary Pharmacology and Toxicology, College of V eterinary and A nimal Sciences, K erala V eterinary and A nimal Science University, Pookode-673 625, W ayanad, Kerala, India. T. Shobha Rani Department of Organic Chemistry, School of Herbal Studies and Naturo Sciences, Dravidian University, K uppam-517 426, A ndhra Pradesh, I ndia. U. Darsana Department of Veterinary Pharmacology and Toxicology, College of V eterinary and A nimal Sciences, K erala V eterinary and A nimal Science University, Pookode-673 625, W ayanad, Kerala, India Suresh N Nair Department of Veterinary Pharmacology and Toxicology, College of V eterinary and A nimal Sciences, K erala V eterinary and A nimal Science University, Pookode-673 625, W ayanad, Kerala, India Reghu Ravindran Department of Veterinary Parasitology, College of V eterinary and A nimal Sciences, K erala Veterinary and Animal Science University, Pookode-673 625, Wayanad, K erala, India. Correspondence: Sanis Juliet Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Science University, Pookode-673 625, Wayanad, Kerala, India. Physico-chemical evaluation and biochemical quantification of crude drug powder (stem) of Chassalia curviflora (Wall. ex Kurz.) Thwaites; A folk medicinal plant T.P. Ajeesh Krishna, T.P. Adarsh Krishna, R. Kumuthakallavalli, V.N. Sanyo Raj , Sanis Juliet, T. Shobha Rani, U. Darsana, Suresh N Nair, Reghu Ravindran. Abstract The medicinal plants are sources of important therapeutic aids for alleviating human health and improving the quality of life. The present study was undertaken with an objective to investigate the physico-chemical and biochemical analysis of the crude drug powder of the stem of Chassalia curviflora (Wall. ex Kurz.) Thwaites, an ethno-medicinally important plant belonging to coffee family (Rubiaceae). The physicochemical studies showed foreign content 0.313%, moisture content 11.333 %, total ash content 11.416%, acid soluble ash 56.833%, water soluble ash 15.054 % and alcohol soluble ash 10.595. The result of biochemical contents showed that, the highest value of dry matter was 88.666 ± 0.166%, followed by carbohydrate 63.027 ± 0.023%, crude fibre 14.693 ± 0.170 %, crude protein 13.125 ± 0.004%, total ash 11.416 ± 0.289%, moisture 11.333 ± 0.166% and crude fat 1.099 ± 0.062%.The extractive values of the stem were determined by using different solvents. This information’s are useful for the pharmacognostical evaluation of this plant material. Keywords: Chassalia curviflora, Physico-chemical evaluation, Biochemical quantification. 1. Introduction Ethno-botanical and ethno-pharmacological studies have been defined in many ways. It assumes great importance in enhancing our existing knowledge about the plants used by tribal folklore, the rich diversity assembled by them for their sustenance and the different means adopted by them for its preservation and conservation. The tribes have developed their own traditional ways of diagnosis and treatment of diseases. The medicinal plants constituted the main source of new pharmaceutical drugs. The evaluation of crude drug means confirmation of its identity and determination of its quality and purity. The plant Chassalia curviflora (Wall. ex Kurz.) Thwaites are commonly known as ‘neelakurinji’ in Malayalam. It belongs to the family Rubiaceae which constitutes one of the largest flowering plant. It is an evergreen, erect shrub, up to 2 m tall, leaves elliptic ovate, flower pinkish-white with yellow throat, fruit drupe, purplish-black [1]. It is used as a traditional medicinal plant in Western Ghats of Kerala, India. The Kani tribal folklore in Aarukani Hills, used the root and root bark of the C. curviflora to treat jaundice and wounds [2]. In addition, the different parts of the plant are reportedly used for various medicinal properties such as headache, ulcers, sore throat, phlegm, rheumatism pneumonia eye and ear diseases [3, 4]. At present, studies on the physicochemical and biochemical quantification of this plant are scarce in literature. Therefore, in the present study an attempt has been made to assess the quality control parameters for further pharmacognostical standardization of this plant material. 2. Materials and Methods 2.1 Collection of plant material and identification The taxonomically identified Chassalia curviflora (Wall. ex Kurz.) Thwaites plant stem was collected from “Paingottupuram” near Kuttikkattoor, Kozhikode, Kerala, India. A herbarium for morphological studies was prepared, identified and authenticated by a botanist. ~ 184 ~ Journal of Pharmacognosy and Phytochemistry A voucher specimen No: CALI- 6806 was deposited in the Department of Botany, Calicut University Herbarium, Kozhikode, Kerala. 2.4.2 Determination of moisture content A small amount of powder was placed in a crucible with lid and put in a hot air oven for removal of moisture content at 100 ± 1 0 C, overnight. The dried samples were weighed in crucible with lid after cooling to room temperature. The moisture content was calculated by the following formula. 2.2 Reagents and chemicals All the reagents and chemicals used in present study were procured from M/s Merck India, Ltd. Bombay. Percentage of moisture content = 2.3 Preparation of crude drug powder The collected plant stem of C. curviflora were cleaned and washed with running water and dried at room temperature for 2 to 3 weeks. The dried stem (Figure-1) was then powdered in a plant sample grinder at controlled temperature and stored in plastic container. Weight of sample – Weight of dried sample X 100 Weight of sample 2.4.3 Determination of total ash contents Ash represents the inorganic matter content of the sample which was determined by the method of AOAC [5]. Approximately one gram dried sample in a crucible was charred over a low flame and kept in a muffle furnace set at 550–600 oC for 2-3 hours. It was then cooled in a desiccator and weighed to ensure completion of ashing. It was once again heated in the furnace for half an hour, cooled and weighed. The procedure was repeated consequently till the weight became constant. Total ash content was calculated by the following formula. Percentage of ash = Weight of ashed sample ×100 Weight of sample taken 2.4.4 Determination of acid/alcohol/water soluble ash The soluble ash was determined by using different solvents such as 5N HCl, alcohol and distilled water. The ash obtained was digested with 25 ml of solvent for 20- 30 minutes in a boiling water bath. The content in the crucible was filtered by using ash less filter paper (Whatman filter paper No: 42). The filter paper with residue was removed carefully without any loss, folded, put in the same crucible, dried in hot air oven and ignited in muffle furnace at 600 oC for 1 hour. Then it was cooled in a desiccator and weighed. The soluble ash value was calculated by the following formula. Fig 1: Dried stem 2.4 Physio-chemical analysis 2.4.1 Determination of foreign content Approximately 100 gm of stem powder (Figure-2) of C. curviflora was taken and spread into a thin layer. The foreign matter was being detected by inspection with the unaided eye separated and weighed. The percent aged of foreign content was determined using the formula. Percentage of soluble ash = Weight of soluble ash×100 Total weight of ash 2.4.5 Determination of extracting values Coarsely chopped (5 gm) stem powder of C. curviflora was subjected to macerate for 24 hours in a closed flask using 100 ml of different solvent viz. alcohol, chloroform, ethyl acetate, hexane, petroleum ether and distilled water. The flask was frequently shaken during the first 6 hours and then allowed to stand for 18 hrs. After 24 hrs, the contents in the flask were filtered using Whatman No: 42 filter paper. In a flat bottomed shallow dish, 25 ml of filtrate was evaporated to dryness, dried at 105 oC and weighed. Percentage of soluble extractive was calculated with reference to the air dried powder. Percentage of foreign content = Weight of sample – Weight of foreign matter X 100 Weight of sample Percentage of extracting value = Weight of flask with extract- Weight of empty flask ×100 Weight of sample 2.5 Biochemical quantification The biochemical quantification of ash, crude fibre, crude protein, carbohydrate, crude fat, dry matter and moisture content of the stem powder of C. curviflora were done using standard proximate analysis techniques. [6] Fig 2: Crude drug powder ~ 185 ~ Journal of Pharmacognosy and Phytochemistry 2.5.4.3 Titration The boric acid with trapped ammonia was titrated with 0.1N hydrochloric acid. The colour of boric acid changed again to pink. The percent of protein was calculated by the formula. 2.5.1 Determination of dry matter The dry matter determined using the weight difference method was estimated by deducting percent moisture from hundred as described by James [7]. Dry matter (%) = 100 - % of moisture Protein (%) = V×1.4 x 6.25 x 0.1N HCl x Vol (used) ×100 W×A×1000 Where, 2.5.2 Determination of crude fat One gram of crushed dried sample was taken in the paper thimble kept in a pre-weighed flask of soxhlet extractor. 80 ml of petroleum ether was poured on the flask and refluxed for 8 hours. The flask was cooled in a desiccator and the weight of crude fat extracted was taken. The percent crude fat was determined by using a formula. V - Titer value (Volume of HCl used). 1.4 - Weight of nitrogen expressed in gram in the formula. 6.25 - Protein factor. W - Weight of powdered sample. A - Aliquot digested sample used for distillation. Crude fat (%) = Weight of flask with fat - weight of empty flask x 100 Weight of original sample 2.5.5 Determination of Carbohydrate The percentage of carbohydrate in the sample was calculated by using this formula. 2.5.3 Determination of crude fibre One gram of the defattened plant material was taken in a spout free beaker and boiled in 200 mL of 1.25% sulphuric acid for 30 minutes. The content was then filtered and washed with hot distilled water to neutralize and transferred again to the beaker and boiled in 200 mL of 1.25% sodium hydroxide for 30 minutes. It was again filtered and washed with hot distilled water for neutralization. The crucible was dried in an oven at 100 ± 5 oC in a hot air oven overnight (10-12 hours), cooled in a desiccator and weighed to a constant weight. Latter, the crucible with its content was put in a muffle furnace at 550-600 0C for 23 hour for complete burning of organic matter. It was then cooled in a desiccator and weighed to a constant weight. The percent fibre was determined from the formula. Crude fibre (%) = Carbohydrate (%) = 100 - (ash+moisture + crude protein+crude fat) % 3. Result and Discussion The physicochemical parameters are mainly used in judging the purity and quality of the drug. The parameters of crude stem powder of C. curviflora were estimated based on the standard procedures. The results showed that the stem powder contained foreign content of 0.313% and an ash value of 11.416%. The percentages of acid soluble ash, water soluble ash and alcohol soluble ashes are depicted Fig-3. The highest plant extractive yield was obtained from water. The extractive values of the powdered stem using water, ethyl acetate, ethanol, chloroform, hexane, and petroleum ether are presented in Table-1. Thenmozhi et al (2013) had reported an extractive yield 0.4, 0.3 and 0.6 (w/w) from the fruits of C. curviflora using acetone, ethanol and water respectively as solvents. The ash value and foreign contents gave an idea of the inorganic content or other impurities present along with the crude drug. Besides the extractive values provided the additional information which might be useful in the determination of adulterated drug powder. W1 - W2 x 100 Weight of sample Where, W1- The crucible with crude fibre was cooled and weighed. W2- The content of the crucible was ignited over a low flame until charred and then kept in a muffle furnace and weighed. 2.5.4 Determination of crude protein Total nitrogen (N) content was determined with the help of Kjeldahl method described by Pearson [8]. The protein determination was divided into three steps. Table 1: Data showing the extractive values of C. curviflora stem powder. SL. No. 1 2 3 4 5 6 2.5.4.1 Digestion 0.5 g of dried plant material was taken in the digestion flask. To this 1 g of digestion mixture (copper sulphate and sodium sulphate) and 15 ml of concentrated sulphuric acid was added. The solution was heated until it became clear and frothing ceased. It was then boiled gently for another 2 hours, cooled and digested with 30 ml of water with constant mixing. The digest was transferred to 250 ml standard flask and necessary amount of distilled water up to the mark of the flask was added. Extract Ethanol Chloroform Ethyl acetate Hexane Petroleum ether Water Colour of extract Dark green Green Pale green Yellowish green Yellowish green Brown Yield* (%) 04.294 ± 0.079 03.373 ± 0.109 04.410 ± 0.036 01.700 ± 0.238 01.099 ± 0.062 06.863 ± 0.332 Note:* Mean of 3 readings ± SEM The percentages of dry matter, moisture, crude fibre, crude protein, crude fat, ash and carbohydrate contents of the leaves of C. curviflora are shown in table-2. The plant stem has a crude fibre content of 14.693 ± 0.004%, ash 11.416 ± 0.289%, crude protein 13.125 ± 0.004% and crude fat 0.1099 ± 0.062%. Further, biochemical quantification (proximate content) helps to set up certain standards for dried drugs and the determination of nutritive values. There are no further reports to support the findings. During last few decades, there has been an increasing urge in 2.5.4.2 Distillation The distillation step was carried out in a Kjeldahl apparatus, 20 ml of 4% boric acid was taken and one drop of methyl red indicator was added. 10 ml of aliquot of digested material was transferred to the distillation assembly and 20 ml of sodium hydroxide (40%) solution was added to it. The completion of distillation was indicated by a change of colour of boric acid from pink to blue. ~ 186 ~ Journal of Pharmacognosy and Phytochemistry the study of medicinal plants and their traditional uses in different parts of the world. Herbal remedies are considered as the oldest form of health care known to mankind on this earth. The traditional system of medicine that have evolved over the centuries within various communities, are still maintained as a great traditional knowledge base in herbal medicine. Physicochemical study taken up with C. curviflora suggested that the drug powder has high water soluble extractive values which might be due to its high content of water soluble bioactive components. 4. 5. 6. 7. Table 2: Data showing the biochemical content in C. curviflora stem powder. 8. S. No. 1 2 3 4 5 6 7 Extract Crude fat Crude protein Total ash Moisture contents Dry matter Crude fiber Carbohydrate Yield* (%) 1.099 ± 0.062 13.125 ± 0.004 11.416 ± 0.289 11.333 ± 0.166 88.666 ± 0.166 14.693 ± 0.004 63.027± 0.023 Note:* Mean of 3 readings ± SEM Fig 3: Showing the physicochemical values of C. curviflora stem powder. 4. Conclusion Chassalia curviflora (Wall. ex Kurz.) Thwaites is an ethnomedicinal plant belongs to coffee family. The physiochemical and biochemical analysis of the crude drug powder of stem reported in this research work could be further useful to evaluate pharmacognostical properties of this plant. 5. Acknowledgement Financial support from Indian Council of Agricultural Research through the World Bank funded National Agricultural Innovation Project (NAIP/Com-4/C2066/2007-2008) and National fund project (NFBSFARA/BSA-4004/2013-14) is thankfully acknowledged. 6. References 1. Pullaiah T. Encyclopaedia of World Medicinal Plants, Vol 1, Daya books, 2006, 522. 2. Rajendran DAR, Solomon J, Justsella WP, Johnson MA, Varaprasadham I. Antibacterial activity of selected ethnomedical plants from south India. Asian Paci J of Trop Medi 2011; 375-378. 3. Gustaffson KR, Walton LK, Sowder RC, Johnson DG, Pannell LK, Cardellina JH Jr et al. New circulin ~ 187 ~ macrocyclic polypeptides from Chassalia parvifolia. J Nat Prod 2000; 63:176-178. 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