File 2541 (12).qxd Bangladesh J. Sci. Ind. Res. 41(1-2), 83-88, 2006 Introduction Cassia alata is an indigenous flowering plant which grows almost in all the districts of Bangladesh but it grows abundantly in the districts of Dhaka, Rajshahi and Dinajpur. Cassia alata is found to grow in the lower Bengal, Western Peninsula, Burma and Malacca, very probably introduced into India, as it does not appear to occur far away from human dwellings. At present it is predominantly found in Indo-Pak- Bangladesh subcontinent.1 The Cassia alata is of considerable importance from a medicinal point of view.2 The fresh leaves were often employed to cure ring worm.2 Cassia alata is used in West Africa to treat parasitic skin diseases and pustular skin infections.3 In the search for therapeutic agents from natural sources for the treatment of AIDS patients the antibacterial and antifungal activities of water extracts of Cassia alata were investigated.4 Cassia alata has wide range of medicinal uses in India and the West Indies for the treatment of various aliments.5 Cassia alata plants produce considerable amounts of seeds which are generally left as waste. The physico-chemical properties of oils are believed to be directly related to their glyceride and lipid. So, a first hand knowl- edge of its chemical composition seems to be very much essential for its potential use as a medicinal plant. Studies on the Lipid and Glyceride Compositions of Cassia alata Seed Oil M. S. Rahman,a M. Y. Ali,b M. U. Alib and AJM Moynul Hasana aBCSIR Laboratries, Binodpur Bazar, Rajshahi-6206 and bDepartment of chemistry, University of Rajshahi, Rajshahi, Bangladesh Abstract The oil composition of Cassia alata seed collected from three different districts of Bangladesh was investigated. All the seed samples contain about 3.2 % oil. The triglyceride component of the oil varied from 91.5 to 92.0 % diglyceride from 3.4 to 3.9 % and monoglyceride from 1.5 to 2.1 %. The lipid components were almost the same in all the samples, the neutral lipid accounted for over 93 % of the total lipids present. The analysis of the fatty acid composition showed 25.6 % oleic acid, 45.5 % linoleic acid, 18.7 % palmitic acid, 3.5 % stearic acid, 3.4 % arachidic acid and 3.2 % behenic acid. 84 Studies on the Lipid and Glyceride Compositions 41(1-2) 2006 Despite considerable works were done on the isolation of various compounds from leaves by some workers,2,6 information on the characteristics of the Cassia alata seed oil is lacking. Hence the present investigation was made with a view to finding out the approximate compositions of glyceride and lipid of the oil of the locally available seeds, as the location of Bangladesh is typical in the subcontinent because of seasonal variations of monsoon. Materials and Methods Ripe and matured Cassia alata seeds were colleted from the districts of Dhaka, Rajshahi and Dinajpur in the month of August. The seeds were deshelled and decorticeted manu- ally and dried in the sunlight for four consec- utive days. The sun dried seeds were finally crushed into powder with the help of a grind- ing machine and dried in the oven at a tem- perature with the help of a grinding machine and dried in a Soxhlet extractor with n-hexa- ne for an 8 hour. The solvent was removed by using a rotary vacuum evaporator and the percentage of oil content was calculated. The crude oil thus obtained was purified in a column (Neutral alumina in pet. ether) using pet. ether (70:30) as the eluting solvent. The purity of the oil was checked by normal TLC. The specific gravity of the oil was calculated at 28O C with the help of a Pycnometer. Refractive index of clear oil free from water and air bubbles were determined at 28O C by standard IUPAC method.7 Moisture and volatile matter in the oil were also deter- mined by IUPAC method.7 The Free fatty acid (FFA), saponification value, peroxide value and unsaponifiable matters in the oil were determined by the standard AOCS methods.8 Hanus method9 was followed to determine the iodine value of the oil. Separation of glyceride Each sample of oil of the seeds collected from the above three districts was separated into mono-, di- and triglycerides on silicic acid (E. Merck, Darmstadt, Germany 70-230 mesh) column. The silicic acid was activated at 120O C overnight and again for 1 hour immediately before the column was prepared. Then the silicic acid was hydrated with 5 % (w/w) water. A slurry of silicic acid in chloroform was poured into the column (2.2 cm i.d). One g oil was dissolved in 15 ml of chloroform and quantitatively transferred to the column. The triglyceride components from each of the three samples (0.917, 0.920 and 0.915 g) was eluted with 200 ml of ben- zene, the diglyceride (0.039, 0.034 and 0.036g) with 200 ml of a 1:9 (v/v) mixture of diethy1 ether and benzene and the monoglyc- eride (0.021, 0.018 and 0.015g) with 200ml dieth1 ether.10 The percent compositions of the glyceride are shown in Table II. The elu- tion was controlled at a flow rate of 1.5- 2ml/min. The elution of each fraction was monitored by micro slide thin layer chromatography (TLC) to ensure complete separation of each class of the glycerides during slilicic acid chromatography and the eluted solvents were collected in weighed flasks. The fractions thus obtained were evaporated in a rotary vacuum evaporated in a rotary vacuum evap- orator and were dried under reduced pressure before being weighed. The purity of the different glyceride components was further checked by TLC using silica gel developed with n-hexene : diethy1 ether checked by TLC using silica gel developed with n-hex- ene: diethy1 ether (80/20 v/v) and identified by chromic-sulphuric acid spot tests at 180O C. The glyceride were identified by comparison of Rf values with standard refer- ences. The weight percentage of each glyc- eride class was based on total glyceride recovered, which averaged to 99.3 of the total glyceride present. Fractionation of Cassia alata seed lipid Total lipid extracted by Bligh and Dyer method11 was fractionated into three major lipid groups, neutral lipid, glycolipid and phospholipid by silicic acid chromatography on about 150mg of Cassia alata seed lipids Neutral lipids were eluted with chloroform, glycolipid with acetone and phospholipid. with methanol.12 The elution was controlled at a flow rate of 0.5ml-1.0ml/min. The com- plete elution of each fraction was monitored by micro slide TLC during silicic acid column chromatography and eluted solvents were collected in weighed vials. The percent- ages of these fractions were determined by gravimetric method. Separation of saturated and unsaturated fatty acid present in the seed oil Separation of saturated and unsaturated fatty acids was carried out by lead-salt-ether method.14 50gm of the oil was saponified with alcoholic caustic soda to obtain soap solution. A slight excess of lead acetate solu- tion was added to the soap solution to form lead salt of fatty acid which were then sepa- rated. Ether was added to the mixture of lead salt and the whole mixture was added to the soap solution to form lead salt of fatty acid which were then separated. Ether was added to the mixture of lead salts and the whole mixture was boiled and then cooled at OO C for 24 hours. The upper ether layer was decanted off. The lead of the unsturated fatty acid were obtained by removing the ether from the upper ethereal solution. The precip- itated part contained lead salts of the saturat- ed fatty acids. Each group of lead salts was suspended in water and treated with suffi- cient hydrochloric acid and extracted with ether. On evaporating the ether, the fatty acid were obtained mainly in two groups. Finally masses of saturated and unsaturated fatty acids were obtained by weighing them sepa- rately. Fatty acid composition of Cassia alata seed oil Fatty acid composition of Cassia alata seed oil was analysed as their methy1 ester, which was prepared by the Boron trifluoride Rahman, Ali, Ali and Hasan 85 methanol method.14 A GCD Pye Unicam gas chromatograph equipped with a flame ioniza- tion detector was used to determine the fatty acid methy1 esters. Nitrogen carrier gas was used at a flow rate of 30ml/min. Fatty acid were separated on a 18X 1/8 I. d glass column packed with 6 % BDS (Butanediol Succinate Polyesters) on solid support Anakorm ABS (100/120) mesh. Analysis was carried out at isothermal column temperature 190O C, injector and detertor temperatures for all GLC analyses were 230O C. Gas chromato- graphic peaks were identified by comparison with standard methy1 ester with respect to retertion times against equivalent carbon length (ECL). Peak areas were measured by a Pye Unicam electronic integrator. The per- centage of each peak was calculated as the percentage of total area of all the peaks. Results and Discussion The solvent extraction of Cassia alata seed yielded 3.2 % light yellow coloured oil. Purification of the oil was carried out by column chromatography and the purity was verified by normal TLC. The physico-chemi- cal characteristics of purified oil were deter- mined by the conventional methods and results were given in Table I. No. appreciable change in physical and change in physical and chemical characteristics among the three samples collected from three different dis- tricts was observed. Specific gravity and refractive index were normal in comparison with other vegetable oils.13 The whole oil was fractionated into mono-, di- and triglycerides by means of column chro- matography and the results were presented in Table II. From the results in Table II, it is evi- dent that triglycerides in all the three samples irrespective from where they were collected accounted for over 91.5 % of the total weight of the oil. Total extracted Cassin alata seed lipid were separated into neutral lipid, glycolipid and 86 Studies on the Lipid and Glyceride Compositions 41(1-2) 2006 Table I. Physical and chemical characteristics of Cassia alata seed oil Mean results of three experiments for each sample Physical and chemical characteristics 1. Specific gravity at 28 O C 2. Refractive index at 28 O C 3. Moisture and volatile matter (%) 4. Melting point 5. Free fatty acid as oleic (%) 6. Iodine value (Hanus method) 7. Saponification value 8. Unsaponifiable matter (%) 9 Peroxide value (m.e.v/mg) Name of the districts from where seeds were collected Rajshahi Dinajpur Dhaka 0.9215 0.9226 0.9248 1.4590 1.4628 1.4625 0.1171 0.1173 0.1170 28-29O C 28-29O C 29-30O C 2.1 2.5 2.7 94.3 94.5 95.0 189.0 188.4 188.7 1.5 1.8 2.0 1.80 1.82 1.85 phosphopid by slilicic acid column and presented in Table III. The results indicate that no significant change in the lipid compo- sition among the three samples was noticed. From the results shown in Table III, it is observed that neutral lipids in all the three samples were in Table III, it is observed that neutral lipid in all the three samples were found to be over 93 % of the total weights of the lipid. The saturated and unsaturated fatty acid present in the oil were separated by lead salt- ether method and the results were depicted in Table IV. From the results in Table IV, it is seen that the percentage of sat- urated and unsaturated fatty acid present in the oil collected from Rajshahi and Dinajpur districts were almost similar. But the percentage of fatty acid composition of the oil in the sample collected from Dhaka dis- trict differs from fatty acid composition of the samples from other two districts. The fatty acid composition of the oil was determined by GLC and the results were pre- sented in Table V. Gas chromatographic analysis showed that unsaturated fatty acid present in Cassia alata seed oil were mainly oleic (25.6 %) and linoleic (45.5 %) which altogether accounted for over 70 % of the total fatty acid. Rahman, Ali, Ali and Hasan 87 Table II. Glyceride composition of Cassia alata seed oil (weight %) Mean results of three experiments results for each sample Name of the districts from where seeds were collected Rajshahi Dinajpue Dhaka Monoglyceride 2.1 1.8 1.5 Diglyceride 3.9 3.4 3.6 Triglyceride 91.7 92.0 91.5 FFA 2.1 2.5 2.7 Table III. Lipid composition of Cassia alata seed oil (weight %) Mean results of three experiments results for each sample Name of the districts from where seeds were collected Rajshahi Dinajpur Dhaka Neutral lipid (%) 93.2 94.3 92.8 Glycolipid (%) 4.6 3.3 4.6 Phospholipid (%) 2.6 2.1 2.3 Table IV. Percentage of saturated and unsat- urated fatty acid Name of the districts from where seeds were collected Rajshahi Dinajpur Dhaka Saturated fatty acid 25.5 23.7 29.0 Unsaturated fatty acid 70.7 72.5 67.2 Conclusion The Cassia alata seed has been found to con- tain about 3.2 % oil. The oil is mainly com- posed of unsaturated fatty acid (over 70 % of which 25 % accounts for oleic acid and 45.5 % linoleic acid. Acknowledgement The authors express their gratitude to Professor M. Mosihuzzaman and Professor Nilufar Nahar of Dhaka University for the fatty acid analysis by GLC. References 1. The wealth of India, A Dictionary of Indian Raw Materials and Industrial Products, Vol. III. C.S.I.R. Delhi, India, (1992) 332. 2. R. N. Chopra, S. L. Nayar and I. C. Chopra: Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research, New Delhi, India, (1980). 3. E. O. Ogunti, A. J. Aladesomani, S. A. Adesonya. Fitoterapia 62(6)(1991) 537-539, 4. C. O. Crockett, F. G. Guina, D. Pugh, M. V. Manda, T. J. Robinson. J. O. Olubadewo. R. F. Ochillo. Cellular and Molecular-Biology- Oxford, 38(5) (1992) 505-511. 5. S. Palanichamy, S. Nagarajan. Journal of Ethnopharmcology, 29(3) (1990) 337-340. 6. H. Moriama, T. Iizaka, M. Nagai and Y. Murata. Fitoterapia, 74(5) (2003) 405.11. 7. International Union of Pure and Applied Chemistry: Standard Methods for the Analysis of Oils, Fats and Derivatives, Pargamon press, 6th, Ed. 8. Official and Tentative Methods of the American Oil Chemicsts Society (1 & 11), 3rd Ed. (1980). 9. Association of Official Agricultural Chemists: Official Methods of Analysis 8th Ed. Washington, (1955). 10. M. A. Gafur, M. S. Rahman, G. M. Ahmed, A. Hossain and M.E. Haque. Bangladesh J. Sei Ind. Res. XXVIII 3 (1993) 25. 11. J. Jayarama. Laboratory Manual in Bio- chemistry, Wilely Eastern Ltd. (1985) 96. 12. M. A. Gafur, M. S. Rahman, G. M. Ahmed, A. Hossain and M. E. Haque. Bangladesh J. Sci. Ind. Res. XXVIII 3, 10. 13. T. P. Hilditch. The Industrial Chemistry of the Fats and Waxes, 3rd Ed. Bailliers Tindall and Cox London, (1949) 80. 14. W. R. Morrison and L. M. Smith. J. Lipid. Res. 5 (1964) 600. 88 Studies on the Lipid and Glyceride Compositions 41(1-2) 2006 Table V. The fatty acid composition of Cassia alata seed oil of Rajshahi variety (weight %) Fatty acid C16:0 (Palmilic acid) C18:0 (Stearic acid) C18:1 (Oleic acid) C18:2 (Linoleic acid) C20:0 (Arachidic acid) C22:0 (Behenic acid) Weight percent 18.7 3.5 25.6 45.5 3.4 3.2