Study on the extraction of total saponin from folium trevesiae palmatae using ultrasonic method

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.

STUDY ON THE EXTRACTION OF TOTAL SAPONIN FROM

FOLIUM TREVESIAE PALMATAE USING ULTRASONIC METHOD

Le Thi Thanh Thao 1 ; Nguyen Trong Diep 2 ; Nguyen Hong Van 3

Vo Xuan Minh 4 ; Nguyen Nu Huyen My 2

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

extraction on the SM30-CEP device with 50% ethanol, temperature 70 o C, extract 2 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 - 1 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

1 Hadong College of Medicine

2 Vietnam Military Medical University

3 Hadong Traditional Hospital

4 Phenikaa University

Corresponding author: Le Thi Thanh Thao (lethanhthao2604@gmail.com)

Date received: 10/10/2019

Date eccepted: 28/11/2019

MATERIALS AND METHODS

1 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

2 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,

2 hours each time) to remove chlorophyll

The dried to completely remove n-hexane,

extract 2 times with methanol using

by ultrasound for 2 hours at 500C

(100 mL/time), combined the extracts to

200 mL volumetric flask, supplemented

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 5 mL volumetric flask, rinse the tube with ethyl acetate and add ethylacetate to make exactly 5 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 1 g of raw material, calculated according to the following formula:

C × n × V × 100 Saponin (mg/g) =

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 (%)

* 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 2 times, after extracting for the first time, separate the extract, squeeze out the residue, and add the 2nd solvent to continue extracting

RESULTS AND DISCUSSION

1 Quantitative results of total saponin in wild papaya leaves

Table 1: Total saponin content in wild papaya leaves

Papaya

leaves (g)

Moisture (%)

EtOH 50%

Dilution

Saponin (mg/g)

5.0263

0.577

25

Thus, total saponin was 22.39 ± 0.60 mg/g calculated with oleanolic acid to the dried

medicinal herbs

2 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 - 1 mm (DR1 model), 2 - 5 mm

(DR2 model)

Figure 1: Effect of material particle size on total saponin extract from papaya leaves

(n = 3)

Figure 1 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 - 1 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)

Table 2 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)

Saponin content and extraction efficiency

2 h/time

1 h/ time x 2

Table 3 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

1 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 2 times

Table 4: Influence of temperature on saponin extract from wild papaya leaves (n = 3)

Table 4 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

Table 5: Effect of time on saponin extract from papaya leaves (n = 3)

Table 5 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 - 1 mm;

the total saponin content extracted was

21.44 mg/g, corresponding to the extraction efficiency of 95.74%

REFERENCES

1 Đỗ Tất Lợi Những cây thuốc và vị thuốc Việt Nam Nhà xuất bản Y học 2000

2 De Tommasi N, Pizza C, Bellino A et al

Triterpenoid saponins from Trevesia sundaica Journal of Natural Products 1997, 60 (11),

pp.1070-1074