To establish a process of extracting total saponin from wild papaya leaves using ultrasonic method. Research method: Ultrasonic extraction; investigation of process parameters: herbal particles size, solvent type, solvent/material ratio, number of extraction, extraction temperature and extraction time; quantify saponin by UV-Vis method.
Journal of military pharmaco-medicine no9-2019 STUDY ON THE EXTRACTION OF TOTAL SAPONIN FROM FOLIUM TREVESIAE PALMATAE USING ULTRASONIC METHOD Le Thi Thanh Thao1; Nguyen Trong Diep2; Nguyen Hong Van3 Vo Xuan Minh4; Nguyen Nu Huyen My2 SUMMARY Objectives: To establish a process of extracting total saponin from wild papaya leaves using ultrasonic method Research method: Ultrasonic extraction; investigation of process parameters: herbal particles size, solvent type, solvent/material ratio, number of extraction, extraction temperature and extraction time; quantify saponin by UV-Vis method; Results: The influence of process parameters on the content and efficiency of saponin extraction were investigated, from which the appropriate extraction parameters were selected: Ultrasonic o extraction on the SM30-CEP device with 50% ethanol, temperature 70 C, extract times with the ratio of solvent/material was 15/1, extraction time was 90 minutes each time, particle size of raw herbal materials were 0.5 - mm Conclusion: A process of extracting total saponin from papaya leaves has been established using ultrasonic extraction with an efficiency of 95.74% * Keywords: Folium Trevesiae palmatae; Saponin; Ultrasonic extraction INTRODUCTION The wild papaya (Trevesia palmata) is popular tree, which grow easily at the edge of the forest or regenerating forest Their leaves are abundant and available in Ba Vi, Cuc Phuong, Phu Tho, Ha Giang area, thus the collection does not affect the ecological environment or biodiversity [1] Up to now, the papaya leaves in Vietnam have only been used based on traditional experience which used the core of the trunk instead of the pine herb [2] The studies in the world have shown that saponin is the main chemical component, related to some important biological effects of papaya leaves However, at present in Vietnam, there have not been any researches on saponin extract from papaya leaves for application in drug preparation Although this is a natural source of raw material, with high potential for exploitation Therefore, the study of saponin extract from papaya leaves is very necessary Research results are an important prerequisite for preparing extracts, contributing to the development of medicinal products from papaya leaves Hadong College of Medicine Vietnam Military Medical University Hadong Traditional Hospital Phenikaa University Corresponding author: Le Thi Thanh Thao (lethanhthao2604@gmail.com) Date received: 10/10/2019 Date eccepted: 28/11/2019 175 Journal of military pharmaco-medicine no9-2019 MATERIALS AND METHODS Materials and equipment - Materials: Papaya leaves collected in Ha Giang were washed, dried (40°C), minced and stored in sealed plastic bags, kept at laboratory temperature - Chemicals: FeCl3, NaOH, ammonia, glacial acetic acid, vanillin, HCl, HClO4 All chemicals meet the analytical standards; solvents: ethanol, n-hexane, dichloromethane, ethyl acetate, acetone, methanol; standard compound: oleanolic acid (97.0% purity) - Equipment: Ultrasonic extraction equipment SONY MEDI SM30-CEP (Korea); spectrophotometer Hitachi U-1900, Japan; analytical balance Mettler Tolendo ML204 (Switzerland) has an accuracy of 0.1 mg Methods * Quantification of total saponin in wild papaya leaves: Proceed by UV-Vis spectroscopy method: - Standard solution: The oleanolic acid standard stock solution was accurately prepared at a concentration of approximately 92.50 µg/mL From the standard stock solution, diluted to get the standard working solution ranging from 3.70 to 11.10 µg/mL - Sample preparation: Accurately weigh about 5.0 g of medicinal plant powder, extracted with n-hexane (2 times x 125 mL, hours each time) to remove chlorophyll The dried to completely remove n-hexane, extract times with methanol using by ultrasound for hours at 500C (100 mL/time), combined the extracts to 200 mL volumetric flask, supplemented 176 methanol to get the marked volume, shook thoroughtly, accurately aspirate 1.0 mL for clear solution at the top and poured into a 100 mL volumetric flask, added methanol to the marked volume, shook thoroughtly to get the solution for quantifitation Blank solution was methanol - Proceed by Rosenthaler coloring reaction: Accurately draw 0.2 mL of test solution (or standard solution) into a test tube, then add 0.2 mL of vanillin 5% solution/glacial acetic acid and 1.2 mL of perchloric acid Closed the tubes and incubated in liquid paraffin at 70°C for 40 minutes Immerse the test tube in ice and transfer to a mL volumetric flask, rinse the tube with ethyl acetate and add ethylacetate to make exactly mL, shook thoroughly Determine the absorbances at the wavelength of 550 nm Blank solution ethanol 50% was proceeded at the same procedure - Examining process parameters: Size of material particles, solvent type, solvent/material ratio, number of extracts, extraction temperature and extraction time; The output parameter was the total saponin content (mg) extracted per g of raw material, calculated according to the following formula: Saponin (mg/g) = C × n × V × 100 M × (100-h) C: Total saponin concentration in the extract calculated according to oleanoic acid (mg/mL); V: Volume of extract (mL), m: Weight of medicinal material (g); n: Dilution factor; h: Medicinal moisture (%) Journal of military pharmaco-medicine no9-2019 * Extraction total saponin from papaya leaves: - Method of extraction: Proceed on SM30 ultrasonic extraction device Set parameters for temperature, extraction time, working mode When the device reached the temperature specification, place the material in an intermediate extraction flask in the ultrasonic bath, add solvent and extract according to the conditions determined for each test After extraction, collect the extract, filter and determine the total saponin content In case of extraction times, after extracting for the first time, separate the extract, squeeze out the residue, and add the nd solvent to continue extracting RESULTS AND DISCUSSION Quantitative results of total saponin in wild papaya leaves Table 1: Total saponin content in wild papaya leaves Papaya leaves (g) Moisture (%) EtOH 50% (mL) Absorbance 5.0217 5.0704 Dilution factor 0.606 6.27 500 5.0263 0.582 0.577 Mean ± SD 25 C (mcg/mL) Saponin (mg/g) 8.69 23.08 8.38 22.04 8.31 22.06 22.39 ± 0.60 Thus, total saponin was 22.39 ± 0.60 mg/g calculated with oleanolic acid to the dried medicinal herbs The results of the survey on the influence of extraction process parameters * Effect of material particles size: Conduct ultrasonic extraction under the same conditions: The solvent was 50% ethanol, the solvent/material ratio was 20/1, the extraction temperature 60oC in 60 minutes, but with two different types of particles: 0.5 - mm (DR1 model), - mm (DR2 model) Figure 1: Effect of material particle size on total saponin extract from papaya leaves (n = 3) 177 Journal of military pharmaco-medicine no9-2019 Figure showed that the reduction of material particle size significantly increased saponin content and efficiency Although papaya leaves were a relatively thin and soft material, the reduction of material particle size created favorable conditions for the solvent to penetrate, dissolved the pharmaceutical substance and diffuse out, so the extraction efficiency increased by nearly 10% Therefore, the material size from 0.5 - mm was selected for further investigation * Effect of extraction solvent: Carry out ultrasonic extraction of papaya leaves under the same conditions: 1-time extraction with solvent/material ratio of 10/1, 60ºC extraction temperature in 120 minutes, but with extracting solvents such as water, ethanol of different concentrations (30, 50, 70, 90%) Table 2: Effects of solvents on saponin extraction from papaya leaves (n = 3) Sample Extraction solvents Saponin (mg/g) Extraction efficiency (%) DR3 H2O 9.72 ± 0.30 43.4 ± 1.32 DR4 EtOH 30% 11.43 ± 0.46 51.03 ± 2.04 DR5 EtOH 50% 13.89 ± 0.22 62.03 ± 0.98 DR6 EtOH 70% 13.15 ± 0.58 58.75 ± 2.59 DR7 EtOH 90% 12.26 ± 0.44 54.74 ± 1.97 Table showed that when extracting once under the same conditions, different concentration of ethanol solvent, the content and efficiency of saponin extraction reached the highest level when using 50% ethanol Therefore, 50% ethanol was selected for further investigation * Effect of solvent/material ratio: Conducted ultrasonic extraction of wild papaya leaves under the same conditions: The solvent was 50% ethanol, 60°C extraction temperature for 120 minutes, but with different solvent/material ratios Table 3: Effect of solvent/material ratio to saponin extract from wild papaya leaves (n = 3) Sample Solvent/material (ratio x times) DR5 10/1 x time DR8 20/1 x time DR9 30/1 x time DR10 40/1 x time DR11 10/1 x times DR12 15/1 x times 178 Times h/time h/ time x times Saponin content and extraction efficiency Saponin (mg/g) Extraction efficiency (%) 13.89 ± 0.22 62.03 ± 0.98 16.53 ± 0.30 73.81 ± 1.34 18.17 ± 0.58 81.15 ± 2.60 20.51 ± 0.85 91.62 ± 3.81 17.46 ± 0.16 77.99 ± 0.71 19.02 ± 0.88 84.95 ± 3.94 Journal of military pharmaco-medicine no9-2019 Table showed that with single extraction, saponin content and efficiency increased significantly when increasing solvents/material ratio At the solvent/ material ratio of 10/1, the efficiency was 62.03 ± 0.98%, but it increased to 73.81 ± 1.34% at the solvents/material ratio of 20/1 and 81.15 ± 2.60% at the solvent/material ratio of 30/1 For double extraction (samples DR11 and DR12), increasing solvents/material ratio at each extraction also increased content and efficiency of saponin extraction Comparing between one-time and two-fold extraction (samples DR8 with DR11, samples DR9 with DR12), the results showed that: with the same solvent/material ratio, but if divided into two extracts and each time for hour, the 2-fold extraction generated higher content and efficiency than two-fold extraction (up about 4%) To increase the efficiency of saponin extraction from papaya leaves, the relative amount of solvent was required and at solvent/ material ratio of 30/1 or higher, the extraction efficiency was over 80% In particular, double extraction was higher extraction efficiency, so the condition of the DR15 sample was more suitable because of the high efficiency, whereas increasing the solvents/material ratio or the number of extraction times will take more solvent, time and expense * Effect of extraction temperature: An ultrasonic extraction was conducted in the same condition: 50% ethanol solvent, extracted twice, each time with solvent/material ratio of 15/1, 60 minutes/ time x times Table 4: Influence of temperature on saponin extract from wild papaya leaves (n = 3) Sample Extraction temperature Saponin (mg/g) Extraction efficiency (%) DR13 50⁰C 17.70 ± 0.46 79.05 ± 2.07 DR12 60⁰C 19.02 ± 0.88 84.95 ± 3.94 DR14 70⁰C 19.94 ± 0.38 89.06 ± 1.68 DR15 80⁰C 19.66 ± 0.47 87.79 ± 2.10 Table showed as the extraction temperature gradually increased, the content and efficiency of saponin extraction gradually increased and reached the highest level at 70°C Continuing to increase the extraction temperature to above 70°C did not increase the saponin extraction efficiency Thus, the most suitable temperature for saponin extraction from papaya leaves was 70ºC, because increasing the temperature did not increase the extraction efficiency but required more energy * Effect of extraction times: Ultrasonic extract was carried out in the same condition: 50% ethanol solvent, extracted twice, each time with solvent/ material ratio of 15/1, 70ºC extraction temperature, but at different extraction times 179 Journal of military pharmaco-medicine no9-2019 Table 5: Effect of time on saponin extract from papaya leaves (n = 3) Sample Time Saponin (mg/g) Extraction efficiency (%) DR16 30 mins/time x times 12.63 ± 0.58 56.39 ± 2.59 DR17 45 mins/time x times 14.37 ± 0.48 64.16 ± 2.13 DR14 60 mins/time x times 19.94 ± 0.38 89.06 ± 1.68 DR18 75 mins/time x times 20.55 ± 0.55 91.76 ± 2.44 DR19 90 mins/time x times 21.44 ± 0.63 95.74 ± 2.81 DR20 120 mins/time x times 21.58 ± 0.40 96.38 ± 1.80 Table showed as the time of extraction increased, the content and efficiency of saponin extraction from wild leaves gradually increased The increase was the highest when extraction time was about 60 minutes/time, then increased slightly With the extraction conditions as surveyed, the extraction time was 90 minutes/time for the highest content and efficiency of saponin extraction, when extending the extraction time, it did not increase Therefore, the choice of time for each extraction was 90 minutes CONCLUSION The process of extracting total saponin from papaya leaves has been established by ultrasonic extraction method with the following parameters: SM30-CEP device, 50% ethanol solvent, 2-fold extraction, each time with solvent/material ratio was 15/1 at 700C in 90 minutes/time, the herbal particles size was 0.5 - mm; the total saponin content extracted was 180 21.44 mg/g, corresponding to the extraction efficiency of 95.74% REFERENCES Đỗ Tất Lợi Những thuốc vị thuốc Việt Nam Nhà xuất Y học 2000 De Tommasi N, Pizza C, Bellino A et al Triterpenoid saponins from Trevesia sundaica Journal of Natural Products 1997, 60 (11), pp.1070-1074 ... minutes/time, then increased slightly With the extraction conditions as surveyed, the extraction time was 90 minutes/time for the highest content and efficiency of saponin extraction, when extending the extraction. .. extracting once under the same conditions, different concentration of ethanol solvent, the content and efficiency of saponin extraction reached the highest level when using 50% ethanol Therefore,... to the dried medicinal herbs The results of the survey on the influence of extraction process parameters * Effect of material particles size: Conduct ultrasonic extraction under the same conditions: