Saponin Extraction from Gymnema inodorum Decne. Using Ultrasound Extraction Technique

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1 Paper Code: sp003 TIChE International Conference 2011 Saponin Extraction from Gymnema inodorum Decne. Using Ultrasound Extraction Technique Soponrat Rattanasombat *, Chayanoot Sangwichien Department of Chemical Engineering, Faculty of Engineering, Prince of Songkla University, Songkhla 90112, Thailand * Abstract This paper is a study of the ultrasound-assisted ion of saponin from Gymnema inodorum. Ultrasonic ion is an effective technique for the isolation of bioactive compounds from vegetable materials. The s were analyzed by high performance liquid chromatography (HPLC). The results showed that the percentage ion of crude saponin mixture. The effects of main ultrasonic-assisted ion conditions on ion yields of saponin from Gymnema inodorum leaves were evaluated including pre-leaching time (in the range of min). Ultrasonic ion was operated at 45 min of pre-leaching time to give the highest yield of triterpenoid saponins mixture. Compared with the conventional ion method (soxhlet ion), the ultrasonic-assisted ion provided higher percentage ion of triterpenoid saponins mixture at the same period of time. Ultrasonic was found to be the most efficient method for the ion of triterpenoid saponins from Gymnema inodorum leaves which was verified by the experimental results presented in this study. Keywords: Saponin, Gymnema inodorum, Gymnemic acid, Extraction, Ultrasound 1

2 1. Introduction Gymnema inodorum Decne. (GI) is a plant in the Asclepiadacecae Family that grows in the north of Thailand. Gymnemic acid ioned from the leaves of Gymnema sylvestre (GS) is one example of triterpene saponins. On the pharmacological action, crude components ed from the leaves of GS inhibit glucose absorption from the intestinal tract and suppress the increase in glucose level in oral glucose tolerance tests in rat. GI, belonging to the same group as GS, is Gymnemic acids or triterpenoid saponins ioned from the leaves of Gymnema inodorum (GI) and well known to have therapeutic effects similar to those of GS in treating certain diseases such as diabetes mellitus, rheumatic arthritis and gout [1, 2]. Gymnema inodorum has hypoglycemic effect in healthy human. Consumption of GI tea with meal or 15 min after meal can significantly decrease peak plasma glucose. Double dose of GI reduce peak plasma glucose in a dose-responsive manner. Long term consumption of GI for 28 days does not cause fasting hypoglycemia or hepatotoxicity. The mechanism of this hypoglycemic effect does not relate to increase insulin secretion or inhibition of α-glucosidase enzyme [3]. The research report has studied pre-leaching time of ion of vanillin from vanilla pods: a comparison study of conventional soxhlet and ultrasound assisted ion. Ultrasound-assisted ion based on a pre-leaching stage. The aim of pre-leaching was to enhance the permeability of the cell wall to beneficiate increased recovery in the actual ultrasound-assisted ion, which follows the pre-leaching stage. To determine the effect of pre-leaching time before ultrasound-assisted ion, experiments were conducted where powdered cured vanilla beans were pre-leached, in same solvent as used for ultrasonic ion, for various time intervals (30-90 min) in the absence of ultrasonic irradiations. After the pre-leaching stage, ultrasound assisted ion operation was performed for a period of 1 h. It has been observed that an pre-leaching time of 30 min enhances the quantity of vanillin ed after 1 h of ultrasound-assisted ion by about 20% but further increase in the preleaching time (60 and 90 min) results only in marginal beneficial effects. Thus, it can be said that 30 min duration is sufficient to achieve possible soaking and swelling of the cells and increase the permeability of cell walls [4]. In this study, the use of ultrasonic irradiation to assist ion of triterpenoid saponins from the leaves of Gymnema inodorum was presented. The effects of various experimental conditions of the ultrasonicassisted ion were also studied pre-leaching time. 2.1 Materials 2. Materials and methods Fresh Gymnema inodorum leaves were purchased from Lumpang, Thailand. Ethanol, methanol, acetic acid, petroleum ether and n-butanol used in the experimental work were analytical reagent grade chemicals. Acetonitrile, water and acetic acid used for HPLC analysis are liquid chromatography (LC) grade chemicals. 2.2 Ultrasound-assisted ion (UAE) Gymnema inodorum leaves were firstly washed and dried with oven at 60 0 C for 16 h and subsequently crushed into powder. 40 g of the powder was then treated with acetic acid solution (200 ml, ph 2.5) to study pre-leaching time (in the range of min). The treated leaves were ed by ultrasound-assisted ion with 75% ethanol (300 ml) in 60 min of ion time under ambient temperature. Fig. 1. Experimental setup for indirect ion using a cleaning bath [5] Triterpenoid saponins were ed by the indirect sonication in ultrasonic bath at fixed frequency 40 khz. Due to pulsed operation, there was not much heating of the solvent. Use of water bath around the ion flask also ensured that temperature of the solvent reservoir did not increase drastically. The concentrated under vacuum was further treated with n-butanol and water (1:1, 200 ml) and then the layer of n-butanol was concentrated under vacuum. The residue was washed with petroleum ether (30 ml) to remove fatty components and was ed with methanol (100 ml) [1, 2]. After filtering on Whatman No. 1 filter paper, the methanol solution was concentrated under vacuum to give a crude saponin mixture. 2.3 Conventional ion (soxhlet ion) The powdered Gymnema inodorum leaves (40 g) were then treated with acetic acid solution (200 ml, ph 2.5). The treated leaves were ed by soxhlet sp003-2

3 ion with 75% ethanol (300 ml) in 60 min of ion time at C. The powder were packed in filter paper and placed in thimble holder. The arrangement is such that vapors of the solvent are generated from the reservoir, pass through the thimble and get condensed in the condenser. The condensed fresh solvent comes in contact with the powder in the thimble where ion occurs. When the liquid reaches the overflow level in the thimble, the liquid moves through the siphon back into the reservoir, carrying ed solutes into the bulk liquid. In solvent flask, solute is separated from the solvent using distillation. Solute is left in the flask, and solvent vapors pass back into the solid bed material via the condenser for next cycle of ion [4]. The concentrated under vacuum was further treated with n-butanol and water (1:1, 200 ml) and then the layer of n-butanol was concentrated under vacuum. The residue was washed with petroleum ether (30 ml) to remove fatty components and was ed with methanol (100 ml) [1, 2]. After filtering on Whatman No. 1 filtering paper, the methanol solution was concentrated under vacuum to give a crude saponin mixture. 2.4 Total yield of Gymnema inodorum leaves The amount of ed yield was determined gravimetrically after collection. The ion yield is expressed as the percentage ratio of the mass of ed material to the mass of dried Gymnema inodorum leaves as follows [6]: Extracted material( g) Extraction yield 100 Dried leaves( g) 2.5 Analysis of saponin Analytical specifications for the Gymnema inodorum. Identification and estimation of gymnemic acids or saponins in Gymnema inodorum by LC-MS are to be studied. Fig. 2 shows the HPLC chromatogram of crude saponin mixture. 3. Results and discussion 3.1 Ultrasound-assisted ion (UAE): Effect of preleaching time on the ion of saponin The pre-leaching procedure usually consists of the soaking and swelling of the GI leaves in a suitable solvent or mixture of solvents in the absence of ultrasound and is followed by the ion procedure in the presence of ultrasound. The aim of pre-leaching stage is to enhance the permeability of the cell wall to beneficiate the increase in release of triterpenoid saponins in the actual ultrasound-assisted ion, which follows the pre-leaching stage. The pre-leaching stage is also expected to enhance the maximum quantum of crude saponin mixture shown in Table 1. Table 1. Effect of pre-leaching time on the ultrasound-assisted ion (60 min) of GI leaves GI leaves Preleaching time (min) ed material to dried leaves Total yield The effect of ultrasonic and pre-leaching time on the ion yield of triterpenoid saponins are shown in Fig. 3 and Fig. 4. The duration of pre-leaching was 15, 30, 45 and 60 min, respectively. The ion yield of triterpenoid saponins mixture increased very fast during the first 45 min of pre-leaching time. After that, the yield of triterpenoid saponins mixture decreased with ion time. The percentage yield of ion reached the highest point of 9.95% at 45 min of preleaching time. Since swelling and hydration could be accelerated by ultrasonic, thus resulting in a probable enlargement in the pores of cell walls, leading to a better mass transfer of intracellular products into solvent. The probable rupture of cell walls by microjet may also cause an increase of penetration rate of solvent into tissue. Therefore, ultrasound-assisted ion technique allowed target components to dissolve in the solvent in a longer speed, thereby boosting yield with a relevant shorter time. If the pre-leaching time was more than 45 min, the ion percentage of triterpenoid saponins mixture was decreased. Further increase in the pre-leaching time resulted only in marginal beneficial effects. Thus, it can be said that 45 min duration is sufficient to achieve possible soaking and swelling of the cells and increase the permeability of cell walls [4, 7] sp003-3

4 Total yield of ion Total yield of ion TIChE International Conference 2011 capacity for complete condensation [4]. The percentage yield of ion of 8.30% at 60 min of soxhlet ion is shown in Table 2. Table 2. The soxhlet ion (60 min) of GI leaves GI leaves ed material to dried leaves Total yield Fig. 2. HPLC behaviors of crude saponin mixture. Column, Alltech Prevail C18 (4.6 mm IDX 2.5 cm, 5 µm); Eluent, CH 3 CN/H 2 O/AcOH (40/60/0.1); Flow rate, 0.5 ml /min; Detection, UV (210 nm); Column temperature, room temperature Comparison of UAE and soxhlet ion Pre-leaching time (min) Fig. 3. Effect of pre-leaching time (min) on the total yield triterpenoid saponins mixture Pre-leaching time (min) Ultrasonic energy definitely has utility in the ion of triterpenoid saponins from Gymnema inodorum leaves as it results in a substantial enhancement of ion efficiency by increasing the diffusion rates by virtue of eliminating mass transfer resistances. Generally, in soxhlet ion the residue was repeatedly exposed to the solvent. During ultrasonic ion, this physical separation is further enhanced by the localized stirring occurring as a consequence of cavitation. The combination of this stirring effect and the repeated washing of the triterpenoid saponins with solvent were far superior to the simple washing procedure in soxhlet ion [4]. In all the cases investigated in this work, the rate of ion was more rapid with ultrasound than with soxhlet ion. Table 3 shows the extent of triterpenoid saponins mixture recovered using ultrasound assisted and soxhlet ion techniques. The total yield of ion was 9.95% at 45 min of pre-leaching time by 60 min of ultrasound assisted ion. Whereas soxhlet ion, the total yield of ion was 8.30% at 60 min of soxhlet ion. Table 3. Comparison of Ultrasound-assisted ion (60 min) and Soxhlet ion (60 min) Methods ed material to dried leaves Total yield Fig. 4. Effect of pre-leaching time (min) on the total yield triterpenoid saponins mixture 3.2 Conventional ion (soxhlet ion) In the case of conventional soxhlet ion the rate of recycle of the condensed solvent should also play a role in deciding the extent of ion as it is essentially mass transfer-controlled operation. The rate of recycle can be indirectly controlled by modifying the rate of vapor generation and ensuring sufficient cooling Ultrasoundassisted ion (45 min of preleaching time) Soxhlet ion sp003-4

5 4. Conclusions Therefore, ultrasonic is an alternative ion technique for fast ion of triterpenoid saponins mixture from Gymnema inodorum leaves. By studying the effects of pre-leaching time on ion (in the range of min), the percentage yield of triterpenoid saponins mixture reached the highest point of 9.95% at 45 min of pre-leaching time by 60 min of ultrasoundassisted ion. The efficiency of triterpenoid saponins mixture transferring into solvent from the dried leaves of Gymnema inodorum by ultrasonic ion and soxhlet ion were compared. Ultrasonic was found to be the most efficient method for the ion of triterpenoid saponins mixture from Gymnema inodorum leaves which was verified by the experimental results presented in this study. The ultrasonic method employed provided high ion efficiency at the same period of time. Effect of various ion parameters such as ion solvents, solvent volume and temperature are to be studied. 5. Acknowledgements The author gratefully acknowledges the financial support from the Graduate School of Prince of Songkla University, Songkhla, Thailand. References [1] Shimizu, K., Ozeki, M., Iino, A., Nakajyo, S., Urakawa, N., Atsuchi, M Structure-activity relationships of triterpenoid derivatives ed from Gymnema inodorum leaves on glucose absorption. Japanese Journal of Pharmacology, 86, [2] Shimizu, K., Ozeki, M., Tanaka, K., Itoh, K., Nakajyo, S., Urakawa, N., Atsuchi, M Suppression of glucose absorption by s from the leaves of Gymnema inodorum. Journal of Veterinary Medical Science, 59(9), [3] Chiabchalard, A., Tencomnao, T., Santiyanont, R Effect of Gymnema inodorum on postprandial peak plasma glucose levels in healthy human. African Journal of Biotechnology, 9(7), [4] Jadhav, D., Rekha, B.N., Gogate, P.R., Rathod, V.K Extraction of vanillin from vanilla pods : A comparison study of conventional soxhlet and ultrasound assisted ion. Journal of Food Engineering, 93, [5] Vinatoru, M An overview of the ultrasonically assisted ion of bioactive principles from herbs. Ultrasonics Sonochemistry, 8, [6] Yahya, F., Lu, T., Santos, R.C.D., Fryer, P.J., Bakalis, S Supercritical carbon dioxide and solvent ion of 2-acetyl-1-pyrroline from Pandan leaf: The effect of pretreatmen. Journal of Supercritical Fluids, 55, [7] Firdaus, M.T., Izam, A., Prasad, R., Rosli. Ultrasoundassisted ion of triterpenoid saponins from mangrove leaves. The 13th Asia Pacific Confederation of Chemical Engineering Congress, APCChE 2010, October 5-8, 2010, Taipei. sp003-5

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