Optimization of extraction of flavonoids and triterpenoids from loquat leaves using response surface methodology

Wang S.; Shi Y.; Zhang G.; Meng Y.; Zhang N.; Madhujith T.

УДК 615.322

OPTIMIZATION OF EXTRACTION OF FLAVONOIDS AND TRITERPENOIDS FROM LOQUAT LEAVES USING RESPONSE SURFACE METHODOLOGY

Оптимизация экстракции флавоноидов и тритерпеноидов из листьев японской мушмулы с использованием методологии поверхностного отклика

Wang S., Shi Y., Zhang G., Meng Y, Zhang N., Madhujith T.

Ван Ш, Ши Я., Чжан Г., Менг Я., Чжан Н., Мадхуджис Т.

Abstract

Scientists used the ultrasonic method of boosting extraction speed by response surface method to improve the extraction yield of flavonoids and triterpenoids from the loquat leaves. To determine the extraction yield of flavonoids and triterpenoids authors employed the colorimetry method where rutin and betulin acted as standard solutions. The results showed that the optimal conditions of extraction are ethanol concentration in extraction agent was 60 %, while the ratio of loquat leaves weight to the volume of ethanol concentration was 1:60, the extraction time was 40 min, the extraction temperature was 70 °C. The use of ultrasonic power of 200 W rouse the total flavonoid yield by 4.78 % and triterpene yield by 6.70 %. The study approved the ultrasonic efficiency while extracting plant raw materials aimed at separating flavonoids and triterpenoids.

Реферат

С целью увеличения выхода флавоноидов и тритерпеноидов из листьев японской мушмулы использован ультразвуковой метод повышения скорости экстракции с применением методологии поверхностного отклика. Для определения выхода экстрактов флавоноидов и тритерпеноидов использовали колориметрический метод, согласно которому в качестве стандартных применяли растворы рутина и бе-тулина. Исследования показали, что оптимальными условиями для экстрагирования являются: содержание этилового спирта в экстрагенте - 60 % при соотношении массы листьев японской мушмулы и объема этилового спирта 1:60; время экстрагирования - 40 мин; температура экстрагирования - 70 °C. Использование ультразвука мощностью 200 Вт в процессе экстрагирования позволило увеличить выход флаваноидов на 4,78 %, тритерпеноидов - на 6,7 %. Исследования подтвердили эффективность применения ультразвука при экстрагировании растительного сырья с целью выделения флаваноидов и тритерпеноидов.

Keywords:

methodology;

ultrasound;

extraction;

loquat;

flavonoids;

triterpenoids

Ключевые слова:

методология;

ультразвук;

экстрагирование;

мушмула японская;

флавоноиды;

тритерпеноиды

Wang S., Shi Y. , Zhang G., Meng Y., Zhang N., Madhujith T. Optimization of Extraction of Flavonoids and Triterpenoids from Loquat Leaves Using Response Surface Methodology // Индустрия питания|Food Industry. 2018. Т. 3. № 3. С. 33-45. DOI: 10.29141/25001922-2018-3-3-6.

Introduction

Loquat (Eriobotrya japonica) is a plant belong to the family Rosacea commonly grow in many parts of China [5; 13]. The leaves are oblong shaped with a slight batter taste, a greying appearance. Lo-quat leaves contain a myriad of phenolic substances including flavonoid, triterpenoid thus they are believed to impart many health benefits such as antinflammatory, antioxidant, antimicrobial hypoglycemic and hypolipidemic effects. Loquat leaves have long been used by Chinese to cure cough, fall blood sugar and antivirus, etc [3; 17]. Most of the bioactivity arises from flavonoid and triterpenoid present in loquat leaves hence many researchers have attempted to extract them from loquat leaves in the recent past [4; 8]. Novel meroterpenoid saponin has recently been extracted from loquat leaves.

The objective of this study was to optimize the extraction parameters: temperature, time, etha-nol concentration, material: solvent ratio and extraction power to obtain the maximum amount of flavonoid and triterpenoid from loquat leaves. Materials and Methods

Materials

Rutin and Betulin standard were purchased from Hefei Bomei Biotechnology Co., Ltd. China. Anhydrous Ethanol, Sodium nitrite, Aluminum nitrate, Sodium hydroxide, Sodium nitrite, Aluminum nitrate, Sodium Hydroxide, Vanillin were purchased from Tianjin Guanghua Fine Chemicals Research Institute. China Glacial acetic acid, Perchloric acid were purchased from Tianjin Kemon Chemical Reagent Co., Ltd. China.

Methods

Extraction of flavonoid and triterpenoid from Loquat Leaves

The loquat leaves were dried at 65 °C in a hot air-drying oven for 4 hours, and ground using a grinder (Traditional Chinese medicine grinder, a kind of pulverizing machine) [14].

Computing method of flavonoids and triter-penoids from loquat leaves

Determination of flavonoids

Take 2 g of dried leaf powder with ultrasonic extraction with an ethanol solution. After filtering, take 1 mL of filtrate and add 0.5 mL of 5 % sodium nitrite solution to the colorimetric tube. Let stand for 6 minutes, then add 0.5 mL of 10 % aluminum nitrate solution and let stand 6 times. Minutes, finally add 4 mL of 4 % sodium hydroxide solution and add 60% ethanol to 10mL. The absorbance was measured at 510 nm after standing for 15 minutes, Determine the absorbance value [2]. The calculation formula was obtained:

M sample = {A - 0.0051) / 5.1114 x 10 x N x V (1)

M sample - sample solution total flavonoid content (mg/g)

A - absorbance value N - dilution factor V- sample volume (mL) Determination of triterpenes Take 2g of dried leaf powder with ultrasonic extraction with an ethanol solution, and then take 1 mL of the filtrate after suction filtration, place it in a colorimetric tube, and heat it in a boiling water bath to volatilize all the solvents and take out the solution, then naturally cool to room temperature. Add 0.3 mL of a freshly prepared 5 % vanillin-glacial acetic acid solution and 1 mL of perchloric acid to a 60 °C temperature water bath for 20 minutes, and cool with water to room temperature. After adding 5 mL of glacial acetic acid, the mixture was allowed to stand for 15 minutes, Determine the absorbance value [6; 19]. The formula for calculating triter-penoids can be obtained: M sample = (A - 0.0056) / 6.6086 x 10 x N x V (2) M sample - total triterpene concentration in sample solution (mg/mL) A - Absorbance value N - dilution factor V - sample volume (mL)

Experimental data and solutions

The temperature of the ultrasonic extraction was set to 40, 50, 60, 70 and 80 °C what keeping the lo-quat leaves power: solver ratio at 1:60, the power at 100w, the ultrasonic extraction time at 30 min and the ethanol concentration is 80 %.

The time of the ultrasonic extraction was set to 30, 60, 90, 120 and 150 min what keeping the loquat leaves power: the ultrasonic extraction temperature at 60 °C, the ethanol concentration at 80 %, the solver ratio at 1:60, the power at 100 W.

The ethanol concentration of the ultrasonic extraction was set to 40 %, 50 %, 60 %, 70 % and 80 % min what keeping the loquat leaves power: the ultrasonic extraction temperature at 60 °C, the ultrasonic extraction time at 30 minutes, the solver ratio at 1:60, and the power at 100 W.

The solver ratio of the ultrasonic extraction was set to 1:30, 1:40, 1:50, 1:60 and 1:70 what keeping the loquat leaves power: ultrasonic extraction temperature at 60 °C, ultrasonic extraction time at 30 min, ethanol concentration at 80 %, the power at 100 w.

The ultrasonic power of the ultrasonic extraction was set to 100 W, 125 W, 150 W, 175 W and 200 W what keeping the loquat leaves power: the ratio of material to liquid at 1:60, the ultrasonic extraction temperature at 60 °C, the ultrasonic extraction time at 30 min, the ethanol concentration at 80 %. Response surface optimization experiment Four factors and three levels were selected. The total flavonoids and total triterpenoid extraction

rates were used as response values. The Ben-Benkenken design protocol was used to optimize the central composite experiment set up by Design Expert [12]. The four factors were extraction time, extraction temperature, ethanol content, and feed-liquid ratio. The three levels were low (-1), medium (0), and high (1).

Confirmatory experiment

The optimum extraction conditions of total flavonoids and total triterpenes in loquat leaf were ob-

tained by response surface method [7; 16]. It is used to verify the data obtained from the response surface, so as to know whether the response surface data is available. Results and Discussion

Effect of extraction on total flavonoids and total triterpenoid extraction content.

As can be seen from Fig. 1, the extraction rate of flavonoids and triterpenoids from loquat leaves was highest at 40 min. When the flavonoids were at

от E j-t с

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40 % 50 % 60 % 70 % 80 % Ethanol Content (%) b)

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40 50 60 70 80 Extraction Temperature (°С) c)

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100 125 150 175 200 Ultrasonic Power (W) e)

Fig.1. Effect of Extraction on Total Flavonoids and Total Triterpenoid Extraction Content:

a) Effect of Extraction Time on Total Flavonoids and Total Triterpene Extraction

b) Effect of Ethanol Content on Total Flavonoids and Total Triterpene Content

c) Effect of Extraction Temperature on Total Flavonoids and Total Triterpene Extraction

d) Effect of Solver Ratio on Total Flavonoids and Total Triterpene Extraction

e) Effect of Ultrasonic Power on Total Flavonoids and Total Triterpenoid Extraction

40 min, the extraction rate reached the maximum, while the triterpenoids reached the highest, but it was not obvious. This may be due to the slower rate of ethanol and water entering the cells, which affects the incorporation of flavonoids and triter-penoids, and the triterpenoids enter faster than fla-vonoids and are less abundant [9].

From Fig. 2, we can see that when the ethanol content is 60 %, the extraction rate of total flavo-noids and total triterpenoids from the loquat leaves is the highest. May be due to flavonoids and triter-penoids can be dissolved in a certain concentration of ethanol solution, when the concentration of eth-anol reaches a certain amount, no longer dissolve into ethanol is more likely to precipitate resulting in reduced extraction rate, but the extraction rate is so obvious.

As can be seen from Fig. 3 when the extraction temperature is 70 °C, the extraction amounts of flavonoids and triterpenoids in loquat leaves is the highest. It can be seen that the temperature has a greater influence on flavonoids and triterpenoids,

increasing gradually from 40°C to 70 °C. When the temperature exceeds 70 °C, the flavonoids begin to decrease significantly, while the triterpenoids also decrease, but it is not very obvious. This may be due to the fact that at temperatures between 70 °C and 80 °C, the temperature rise leads to structural fragmentation, and the flavonoids gradually decrease, but this temperature has little effect on the triter-penoids [10; 18].

From Fig. 4, it can be seen that the extraction rate of flavonoids and triterpenoids in loquat leaves is highest when the ratio of material to liquid is 1:60. It can be seen that the fluctuation of the extraction rate of flavonoids and triterpenoids is not very obvious when the ratio of feed to liquid is from 1:30 to 1:70, but the optimal conditions can still be seen. Therefore, the subsequent response surface optimization does not consider the influence of the material-liquid ratio on it.

As can be seen from Fig. 5, when the ultrasonic power is 150 W, the extraction amounts of flavonoids and triterpenoids in loquat leaves is the high-

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Fig. 2. 3D and Contour Map of Interaction Between Time and Temperature

A: Time

Total Flavone

A: Time C: Power A: Time

Fig. 3. 3D and Contour Map of Power and Time Interaction

Total Flavone

A: Time D: Ethanol Content A: Time

Fig. 4.3D Graph and Contour Map of Ethanol Concentration and Time Interaction

Total Flavone

50 55 60 65 70 125

B Temperature C: Power B Temperature

Fig. 5. 3D and Contour Map of Power and Temperature Interaction

est. It can be seen that the ultrasonic power has a greater influence on flavonoids and triterpenoids, increasing from 100 W to 150 W; when the ultrasonic power exceeds 150 W, triterpenoids begin to decrease significantly, while flavonoids also decrease. Fluctuations are not as severe as flavonoids. This may be due to the fact that after 150 W, the increase in ultrasonic power causes the structure to break, the chemical bonds break, and the triter-penoids gradually decrease, but this temperature does not have much effect on the flavonoids.

Response surface method to optimize experimental data The design scheme is shown in Table 1. Response Surface Methodology Analysis The response surface analysis by design-expert 8 and the determination of flavonoids and triter-penoids are shown in Table 2.

Through the data of design-expert 7.2, the results are shown in Table 3. Table 3 shows: time (P < 0.01) and temperature. (P < 0.50) had a significant effect

on the extraction of total flavonoids. With the total flavonoid yield as the response value in the mint leaves, the regression equation was obtained after regression fitting: Analysis of Variance of Flavonoids The above data analysis shows the variance analysis of the extraction conditions of flavonoids after the analysis of variance as shown in Table 3.

Based on the data from Design-Expert 8, the results are shown in Table 3. Table 3 shows: time (P < 0.01) and temperature (P < 0.50) has a significant effect on the extraction of flavonoids. With the extraction rate of flavonoids as the response value of the loquat leaf, the regression equation is obtained by the regression equation Regression equation: Flavonoids = 18.29 - 0.41 A + 0.31 B - 0.40C- 0.098D

- 0.37AB + 0.18AC + 0.028AD - 0.51 BC + 0.40BD -

- 0.53 CD - 0.70A2 - 2.28B2 - 1.41 C2 - 0.98D2. According to the variance analysis above, the relationship between the description of the regression equation and the response surface value of the fla-

№ 3

Table 1

Response Surface Optimization Table

Level I Temperature (°С) Time (min) Power (w) I Ethanol content

-1 60 30 125 60

0 70 40 150 70

1 80 50 175 80

Table 2

Response Surface Experiment Results Table

Number (A) Time (min) (B) Temperature (°С) (C) Power (w) (D) Ethanol content Flavonoids (mg/g) Triterpenes (mg/g)

1 0 -1 0 1 14.18 6.79

2 0 1 1 0 13.99 7.56

3 0 0 0 0 18.51 9.34

4 0 -1 0 -1 15.32 7.43

5 0 0 -1 1 16.93 5.14

6 0 -1 1 0 14.31 7.57

7 0 1 0 1 15.31 7.95

8 0 0 1 1 14.89 6.95

9 -1 0 1 0 15.95 5.91

10 -1 1 0 0 16.67 6.19

11 0 0 0 0 18.29 8.92

12 0 0 0 0 18.27 9.43

13 1 1 0 0 15.08 7.95

14 0 0 1 -1 16.28 6.73

15 1 0 0 -1 16.07 6.70

16 1 -1 0 0 15.03 6.62

17 -1 0 -1 0 16.93 5.38

18 1 0 -1 0 15.82 5.37

19 0 1 -1 0 15.82 6.59

20 0 1 0 -1 14.85 7.53

21 -1 0 0 1 16.98 5.18

22 0 0 -1 -1 16.21 6.10

23 0 -1 -1 0 14.08 6.24

24 1 0 0 1 16.22 8.15

25 -1 0 0 -1 16.95 6.88

26 0 0 0 0 17.99 8.90

27 0 0 0 0 18.39 9.49

28 1 0 1 0 15.55 7.41

29 -1 -1 0 0 14.18 7.21

Explanation: A - Time; B - Temperature; C- Power; D - Ethanol content in Response surface methodology.

Table 3

Variance Analysis of Total Flavonoids

Source Sum of Squares DOF Mean Square F-Value P-value Prob > F Significance

Model 49.83 14 3.56 59.52 < 0.0001 significant

A-Time 1.98 1.98 33.07 < 0.0001 ***

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B-Temperature 1.12 1.12 18.76 0.0007

C-Power 1.93 1.93 32.24 < 0.0001 ***

D- Ethanol content 0.12 0.12 1.93 0.1864

AB 0.55 0.55 9.16 0.0091

AC 0.12 0.12 2.08 0.1709

AD 3.204 3.204 0.054 0.8203

BC 1.04 1.04 17.47 0.0009

BD 0.64 0.64 10.63 0.0057

CD 1.11 1.11 18.60 0.0007

A2 3.19 3.19 53.40 < 0.0001 ***

B2 33.74 33.74 564.13 < 0.0001 ***

C2 12.84 12.84 214.75 < 0.0001 ***

D2 6.20 6.20 103.75 < 0.0001 ***

Residual 0.84 14 0.060

Lack of Fit 0.69 10 0.069 1.85 0.2905 not significant

Pure Error 0.15 4 0.037

Cor Total 50.67 28

vonoid extraction condition is because the P value of the model is less than 0.05 (significant), and the P-test of the differential item is P = 0.2905 (not significant), R2 = 0.9387. This model fully fits the experimental data [11; 20]. This equation is an appropriate mathematical model for the extraction rate of flavonoids from the coriander leaves and various factors and parameters. Therefore, it is possible to determine the total amount of flavonoids in loquat leaves based on this regression equation. This is the best extraction process of flavonoids.

According to the regression equation, the response surface analysis of different factors and the corresponding contour plot results are shown in Figure 5. According to Fig. 5, the interaction between each factor and the effect on the yield of flavonoids in loquat leaves can be seen more intuitively. If the curve is steeper, it indicates that the influence of this factor on the yield of total flavonoids is greater. Correspondingly, the size of the response value changes [1; 15]. From the contour plot, it can be seen that the extreme conditions exist at the center of the circle. According to the figure below, the

extraction time and the ultrasonic power have the greatest influence on the yield of total flavonoids, followed by the extraction temperature, and finally the ethanol concentration. This result was consistent with the regression analysis results. The P value corresponding to the extraction time, extraction temperature and ultrasonic power was less than 0.05, and reached a significant level. Variance analysis of triterpenoids After analysis of the above data, the analysis of variance of the extraction conditions of triter-penoids was obtained after analysis of variance, as shown in Table 4.

Based on the data from Design-Expert 8, the results are shown in Table 3. Table 3 shows: time (P < 0.01) and temperature (P < 0.50) has a significant effect on the extraction of triterpenoids. With the extraction rate of triterpenoids as the response value of the loquat leaves, the regression equation gives the regression equation of the regression equation: Triterpenes = 9.22+0.45A + 0.16B + 0.61 C - 0.10D+ + 0.59AB + 0.38AC + 0.79AD - 0.090BC + 0.26BD + + 0.30 CD - 1.47A2 - 0.63B 2 - 1.72C2 - 1.15D 2.

Table 4

Total Three-WayAnalysis of Variance

Source Sum of Squares DOF Mean Square F-Value P-value Prob > F Significance

Model 43.34 14 3.10 38.07 < 0.0001 significant

A-Time 2.47 2.47 30.38 < 0.0001 ***

B-Temperature 0.30 0.30 3.74 0.0735

C-Power 4.45 4.45 54.77 < 0.0001 ***

D- Ethanol content 0.12 0.12 1.51 0.2398

AB 1.39 1.39 17.13 0.0010

AC 0.57 0.57 7.03 0.0190

AD 2.50 2.50 30.68 < 0.0001 ***

BC 0.033 0.033 0.40 0.5370

BD 0.28 0.28 3.45 0.0844

CD 0.35 0.35 4.35 0.0558

A2 14.02 14.02 172.33 < 0.0001 ***

B2 2.61 2.61 32.13 < 0.0001 ***

C2 19.22 19.22 236.35 < 0.0001 ***

D2 8.54 8.54 104.95 < 0.0001 ***

Residual 1.14 14 0.081

Lack of Fit 0.81 10 0.081 1.01 0.5459 not significant

Pure Error 0.32 4 0.081

Cor Total 44.48 28 significant

According to the analysis of the above variance results, the relationship between the regression equation description and the response surface value of the triterpenoid extraction condition is because the P value of the model is less than 0.05 (significant), and P < 0.0001(not significant) of the differential item tests, R 2 = 0.9785.

This model fully fits the experimental data. The equation is a suitable mathematical model for the extraction rate of triterpenoids in loquat leaves and various factors and parameters. Therefore, the regression equation can be used to determine the total triterpene in the middle loquat leaves.

According to the regression equation, the response surface analysis of different factors and the corresponding contour plot results are shown in Figure 5. According to Fig. 5, the interaction between various factors and the influence on the yield of triterpenoids in loquat leaves can be seen more intuitively. If the curve is steeper, it shows the influence of this factor on the total triterpene yield. The larger, the corresponding performance of the response value changes in size. From the contour plot, it can be seen that the extreme conditions exist at

the center of the circle. From the Figure 6, it can be seen that the extraction time and the ultrasonic power have the greatest impact on the yield of the total tritium, followed by the extraction temperature. The ethanol concentration was not significant, indicating that the ethanol concentration had lesser effect on the triterpenoids (Fig. 7). This result was consistent with the regression analysis results. The P value corresponding to the extraction time, extraction temperature and ultrasonic power was less than 0.05, and reached a significant level (Fig. 8, 9).

Verification experiment

After optimizing the extraction conditions of total flavonoids and total triterpenoids in loquat leaves by response surface methodology, the optimal extraction conditions were obtained, namely, ethanol volume fraction 50 %, liquid to material ratio 1:60, extraction time 40 min, and extraction temperature 70 °C (Fig. 10, 11). Selecting 2 g of loquat leaves and selecting three parallels, the content of total flavonoids in loquat leaves was determined to be 17.10 %. according to the optimal conditions, which was very close to the predicted value of response surface method (19.14 %). The total triterpene content was

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C: Power

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Fig. 7. 3D Graph and Contour Map of Ethanol Concentration and Time Interaction Total Triterpenes

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Fig. 8. 3D and Contour Maps of The Interaction between Time and Temperature

6.69 %, respectively. The response surface method predicted value (9.66 %) was very close, and the repeatability is better. After verification, the content of total flavonoids and total triterpenoids in loquat leaves can be well predicted by the extraction conditions optimized by the response surface.

Conclusion

Ultrasonic assisted extraction of flavonoids in loquat leaf and triterpene compound has the advantages of simple operation, low energy consumption, using the single factor experiment to select optimal condition, to the response surface method and the optimum conditions for extraction of flavonoids, af-

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Fig. 10. 3D graph and contour map of ethanol concentration and time interaction

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Total Triterpenes

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Fig. 11. 3D and contour map of power and temperature interaction

ter verification, the results are available. Ultrasonic assisted extraction conditions of flavonoids in lo-quat leaf: ethanol 50 % volume fraction, liquid 40 min than 1:60, extraction time, extraction temperature is 70 °C, flavonoids yield is 19.30 %; Triterpene

compound: the best condition for ultrasonic power is 200 W, the volume fraction of 50 % ethanol, liquid 40 min than 1:60, extraction time, extraction temperature is 70 °C, triterpene yield was 9.70 % (Fig. 12-13)

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Fig. 12. 3D Graph and Contour Map of Ethanol Concentration and Temperature Interaction

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Total Triterpenes

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C: Power D: Ethanol Content C: Power

Fig. 13. 3D Graph and Contour Map of Ethanol Concentration and Time Interaction

Acknowledgments

This work was financially supported by the Nantong tianlai village agricultural science and technology development co. LTD, the National Natural Science Foundation of China (No.31871747, 31301602), Research and Development of Applied Technology Projects of Heilongjiang Province (No. GC13B215).

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4. Huang Y., Li J., Meng X.M., et al. Effect of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf and MAPK signal trans-duction pathway on inducible nitric oxide synthase expression in alveolar macrophage of chronic bronchitis rats // American Journal of Chinese Medicine. 2009, 37(06):1099-1111. DOI: 10.1142/ S0192415X09007521.