Ultrasound-Assisted Extraction of Pristimerin from Celastrus orbiculatus Using Response Surface Methodology

Abstract

This paper describes the ultrasound-assisted extraction (UAE) of pristimerin from Celastrus orbiculatus. Methanol was the most effective for pristimerin extraction, followed by ethanol, ethyl acetate, n-butanol, and water. To optimize the conditions, the Box–Behnken design, a widely used form of response surface methodology, was used to investigate the effects of parameters on UAE. Several variables, such as extraction time, ultrasonic power, extraction temperature, and solvent-to-solid (S/S) ratio were investigated. The highest extraction yield of 1.843 mg/g was obtained using methanol under optimal conditions with an extraction time of 40 min, ultrasonic power of 105 W, an S/S ratio of 40 mL/g, and an extraction temperature of 52°C. The experimental values under optimal conditions agreed well with the predicted values, suggesting that UAE has good potential as an extraction method for pristimerin from C. orbiculatus.

Celastrus orbiculatus TTHUNB. (Nansheteng), a traditional Chinese medicinal plant,¹⁾ is widely distributed in China. Its stems, roots, leaves and fruits have been used for thousands of years as a traditional herb medicine against cancer, arthritis and other inflammatory diseases.²⁾ Up to now, more than sixty compounds were isolated from Celastrus orbiculatus, such as sugiol, friedelane-3-one, salapermic acid, 28-hydroxyfriedelane-3-one, pristimerin, celastrol, β-sitosterol, β-daucosterol and benzoic acid.³⁾ Among them, pristimerin (20α-3-hydroxy-2-oxo-24-nor-friedela-1,10,3,5,7-tetraen-carboxylic acid-29-methylester) is a natural triterpenoid that has been shown to possess a variety of biological activities including anti-cancer activity.^4–9) So, the recovery of pristimerin from Celastrus orbiculatus has strong potential in pharmaceutical industry. The chemical structure of pristimerin is shown in Fig. 1.

Fig. 1. Chemical Structure of Pristimerin

Traditionally, the extraction of various bioactive compounds from medicinal plants can be carried out in a variety of ways, such as refluxing, boiling, heating and Soxhlet extraction. However, although these methodologies have been employed for decades, it is important to mention that the conventional extraction methods have many disadvantages like large amount of solvent utilization, long extraction time and lower extraction yield. In order to overcome these disadvantages, various novel extraction techniques have been successfully applied in the extraction of many phytochemicals from plant materials, including microwave-assisted extraction (MAE),¹⁰⁾ ultrasound-assisted extraction (UAE),¹¹⁾ accelerated solvent extraction (ASE)¹²⁾ and supercritical fluid extraction (SFE).¹³⁾ Among all these novel techniques, UAE offers many advantages involving high extraction efficiency, reduced solvent usage, decreased extraction temperature, short extraction time, easy-operating, and low power consumption.^14–17) UAE is the most economical and the one with less instrumental requirements among all the new techniques. The mechanism of the UAE can be described as cavitation, mechanical and thermal effects. The sound waves that propagate into the solvent media result in alternating high/low pressure cycles, which produces cavitation bubbles. The collapse of bubbles can produce chemical, thermal and mechanical effects,^18–20) which results in disrupting the cell wall allowing greater penetration of solvent into the sample matrix, increasing the contact surface area between the solid and liquid phase,^21,22) as a result, increasing the release of intracellular components into the solvent.²³⁾

This paper was a study about the optimization of extraction of the pristimerin. The experiment in this paper was designed to get the optimization of conditions of extraction and the highest extraction yield of pristimeirn. Based on that the more pristimerin will be gained with the isolation and purification of pristimerin²⁴⁾ discussed in a previous paper. Based on our best knowledge, although UAE has many advantages, there are no reports available on extraction of pristimerin from Celastrus orbiculatus using this technique with Response Surface Methodology (RSM). Here, this paper aims to study the parameters which affect UAE. Optimization of analytical procedures, when one or more responses are affected by several factors and their interactions, is commonly performed by RSM.²⁵⁾ RSM is a collection of statistical and mathematical techniques used for developing, improving and optimizing processes. Therefore, in the present study, a RSM approach was undertaken to optimize the ultrasound assisted extraction parameters such as extraction time, ultrasonic power, extraction temperature and solvent-to-solid (S/S) ratio to maximize extraction of pristimerin from Celastrus orbiculatus.

MATERIALS AND METHODS

Plant Material

The Celastrus orbiculatus were collected from Yangchun, Guangdong Province China and authenticated by the professor Li Shuyuan of Guangdong Pharmaceutical University. The material was allowed to dry naturally and cut into slices, and then ground to pass through a 60 mesh sieve. The powder was kept in sealed polyethylene bags at 4°C until use.

Chemicals

The pristimerin standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol (HPLC grade) was purchased from Tianjin Kermel Laboratory Equipment Co., Ltd. All other organic solvents used in the study were analytical grade.

Ultrasound-Assisted Extraction Process

UAE process was performed with ultrasonic device (KQ3200DE, 40 kHz, 150 W; Ultrasonic Instrument Co., Gongyi, China) equipped with digital timer, power and temperature controller. The powder (1 g) of Celastrus orbiculatus was accurately weighed, and placed in a capped glass tube, and then mixed with an appropriate amount of extraction solvent. Then the tube with the sample was immersed into the water bath in the ultrasonic device, and irradiated at different extraction conditions. After ultrasonic extraction, the sample was filtrated by 0.22 µm microfiltration membrane prior to HPLC analysis.

Extraction Yields Determination

The extraction yields of pristimerin by UAE were calculated using the following equation (Eq. 1):   

(1)

Screening Study for Determination of Appropriate Ranges of Independent Variables

In order to determine the appropriate range of extraction related variables, a screening study was performed prior to extraction optimization, which included extraction time (10, 20, 30, 40, 50, 60 min), extraction temperature (20, 30, 40, 50, 60°C), S/S ratio (50 : 1, 40 : 1, 30 : 1, 20 : 1, 10 : 1 mL/g) and ultrasonic power (75, 90, 105, 120, 135, 150 W). The “one-factor-at-a-time” method was used to study the influence of each factor on the extraction yields.

Experimental Design

RSM was used for investigating the optimal conditions for pristimerin extraction from Celastrus orbiculatus. A four-factor, three-level Box–Behnken design (BBD)²⁶⁾ was applied to evaluate the effects of ultrasonic power (W, X₁), extraction temperature (°C, X₂), extraction time (min, X₃), and the S/S ratio (mL/g, X₄). Table 1 shows the coded values of the independent variables and their levels based on the results of preliminary single factor experiments. The complete design was consisted of 27 combinations including five replicates at central point (Table 2). Experimental runs were randomized to minimize the effects of unexpected variability in the observed responses. As shown in Table 1, the four independent variables were coded at three levels (−1, 0, +1) according to the following equation (Eq. 2):   

(2)

where x_i is the code value of an independent variable, X_i is the real value of an independent variable, X₀ is the real value of an independent variable at the center point, and ΔX is the step change value. The experimental data from BBD were analyzed by multiple regression to fit the following quadratic polynomial model (Eq. 3):   

(3)

where Y is the predicted response, γ₀ is the constant coefficient, γ_i, γ_ii and γ_ij are the the linear coefficient, the squared coefficient, and the interaction coefficient, respectively. x_i and x_j are the independent variables.

Table 1. Values of Independent Variables of the Extraction and Their Corresponding Levels

Independent variable	Symbol	Coded levels
		−1	0	1
Ultrasonic power (W)	X1	75	90	105
Extraction Temperature (°C)	X2	40	50	60
Extraction time (min)	X3	20	30	40
S/S ratio (mL/g)	X4	20	30	40

Table 2. Box–Behnken Design (Coded) Arrangement for Extraction of Pristimerin

Run	X1 (W)	X2 (°C)	X3 (min)	X4 (mL/g)	Pristimerin (mg/g)
1	105	50	40	30	1.51
2	75	50	30	20	1.59
3	90	60	30	40	1.08
4	75	40	30	30	0.96
5	90	50	20	20	1.14
6	75	60	30	30	1.24
7	90	60	20	30	0.79
8	90	50	20	40	1.53
9	90	40	40	30	0.86
10	105	50	20	30	1.01
11	90	60	30	20	1.25
12	95	50	30	30	1.53
13	75	50	20	30	1.42
14	90	50	40	20	1,24
15	90	60	40	30	1.48
16	105	50	30	20	1.12
17	105	40	30	30	0.69
18	75	50	30	40	1.39
19	75	50	40	30	1.53
20	90	50	30	30	1.61
21	105	50	30	40	1.69
22	105	60	30	30	1.12
23	90	40	30	20	0.61
24	90	50	40	40	1.67
25	90	40	20	30	0.99
26	90	50	30	30	1.34
27	90	40	30	40	1.32

HPLC Analysis of Extracts

Pristimerin analysis was carried out on a Agilent 1200 chromatograph using a UV detector and a C₁₈ reversed-phase column (Discovery, 25 cm×4.6 mm, 5 µm, Supelco). The mobile phase was methanol 0.1% phosphoric acid (88 : 12, v/v) at a flow rate of 1.0 mL/min, the column temperature was 30°C and sample volume injected was 20 µL. The absorbance was measured at a wavelength of 424 nm for the detection of pristimerin. Quantification was performed by the integration of the peak using external standard method.

Statistical Analysis

Statistical analysis and 3D graph were conducted using Design-Expert 8.0.6 Trial software (Trial version; State-Ease Inc., Minneapolis, MN, U.S.A.). All experimental results obtained were expressed as mean values. p-Values less than 0.05 were considered to be statistically significant. All experiments were conducted in triplicate unless otherwise noted in the text.

RESULTS AND DISCUSSION

Selection of Solvents

It is very important to select the appropriate solvent for extracting bioactive compounds from plants. Five commonly used solvents, methanol, ethanol, ethyl acetate, n-butanol and water were selected as the extracting solvents. The experiments were carried out at 30°C for 20 min at 150 W of ultrasonic power with a S/S ratio of 20 mL/g. Figure 2 shows that methanol gave the highest extraction yields, followed by ethanol, n-butanol and water. The different extraction efficiencies of these solvents can be attributed to the differences in polarities and viscosities.²⁷⁾ Thus, methanol was selected as an extracting solvent for further study.

Fig. 2. Effect of Different Solvents on the Extracting Yield

Effect of Solvent Concentration

Water as a nontoxic and inexpensive solvent has widely been used for extraction of active components in Chinese traditional medicines. It has been observed that sometimes the extraction yield could be improved by adding small percentages of water to the extraction solvent.^28–30) So, six different solvents concentration of 50, 60, 70, 80, 90, and 100% methanol have been studied for extracting pristimerin from Celastrus orbiculatus. The experimental parameters were set as follows: at 30°C for 20 min at 150 W of ultrasonic power with a S/S ratio of 20 mL/g. The results shown in Fig. 3 indicated that the highest extraction yield was obtained at 100% methanol. Hence, the subsequent experiments were carried out with absolute methanol.

Fig. 3. Effect of Ethanol Concentration on the Extracting Yield

Effect of Independent Variables on Extraction Yield

There are many factors that can affect the extraction efficiency during ultrasound treatment. In the current study, four factors, extraction time, ultrasonic power, extraction temperature and S/S ratio, were taken into account. And a screen study was conducted to screen out the appropriate range of these factors for maximum extraction yields of pristimerin.

Six electrical power levels were studied to evaluate the effect of ultrasonic sound on extraction yield: 75, 90, 105, 120, 135, and 150 W at 30°C for 20 min with a S/S ratio of 20 mL/g. Data shown in Fig. 4 indicated that ultrasonic power had a significant effect on the extraction yield. When the ultrasound power was increased from 75 to 90 W, the yield of pristimerin increased from 0.81 to 1.32 mg/g, a 0.4 mg/g increase. When the ultrasonic power was increased from 90 to 150 W, the extraction yield of pristimerin had a significant decline. Sonication is widely used for extraction of various substances from plant material and generates microscopic bubbles.^31,32) With increase in ultrasonic power more energy was getting transferred for cavitation phenomenon to occur and the cavities formed imploded energetically.³³⁾ With further increase, more bubbles were formed which hampers the propagation of shock waves.³⁴⁾ Also, the bubbles may coalesce to form bigger ones and implode weakly. Hence, the extraction yield decreased. Ninety watts was chosen for optimization.

Fig. 4. Effect of Ultrasonic Power on the Extracting Yield

Figure 5 shows the effect of extraction temperature on extraction yield. Five different temperatures (20, 30, 40, 50, 60°C) (They were real temperatures monitored during the UAE process.) with methanol, were selected to evaluate the influence of temperature on the extraction efficiency of pristimerin. Other conditions were: at 90 W of ultrasonic power with a S/S ratio of 20 mL/g for 20 min. The results show the yield increased as temperature increased from 20 to 50°C. This phenomenon is reasonable since a high temperature can accelerate the softening and swelling of the raw materials, increasing the solubility of extracted compounds.¹⁶⁾ If the temperature continues increasing, the characteristics of ultrasonic cavitation can be altered. Furthermore, a high temperature can lead to a high consumption of energy input. Therefore, 50°C was selected for subsequent treatment.

Fig. 5. Effect of Extraction Temperature on the Extracting Yield

To investigate the influence of extraction time on yield of pristimerin, the experiment conditions were as follows: Ninety watts at 50°C for different time (10, 20, 30, 40, 50, 60 min) with a S/S ratio of 20 mL/g. Figure 6 shows how the yield increased in the initial 30 min, then decreased until reaching an equilibrium. With the extraction time extended, all the plant cells will be completely cracked because of acoustic cavitation effects, and extraction yield increases. However, when the plant cells rupture, various compounds such as insoluble substances and cytosol suspend in the extraction liquid, thus resulting in the lower permeability of the solvent.³⁵⁾ Furthermore, target components also re-absorb into the ruptured tissue particles due to their relatively large specific surface areas, lowering yields of pristimerin.³⁶⁾ Therefore, it is not necessary to do overtime extraction when the maximum extraction yield has been achieved. Based on these results, 30 min was selected as optimum for this treatment.

Fig. 6. Effect of Extraction Time on the Extracting Yield

Five S/S ratios of the effects of extraction yield were studied, including 10, 20, 30, 40, and 50 mL/g using ultrasound at 100 W at 50°C for 30 min, to assess the effectiveness of the ratio of solvent on pristimerin extraction. Figure 7 shows the yield of pristimerin increased with the ratio of S/S increasing from 10 to 30 mL/g, then decreased until reaching an equilibrium. The maximum yield of pristimerin was obtained at a ratio of liquid to material of 30 : 1. This phenomenon was in line with the mass transfer principle, where the driving force during the mass transfer was the concentration gradient between the solid and the bulk of the liquid, which increases when a higher solvent/sample ratio is used.^37,38) But an excessive increase in the ratio also resulted in the decrease of extraction yield.³⁹⁾ Therefore, 30 mL/g was selected as the S/S ratio for further study.

Fig. 7. Effect of S/S Ratio on the Extracting Yield

Model Fitting

RSM using BBD was applied to determine the optimal levels of the four selected variables, such as ultrasonic power (X₁), extraction temperature (X₂), extraction time (X₃) and S/S ratio (X₄). Multiple regression analysis using the quadratic polynomial model (Eq. 4) was performed based on the results listed in Table 2. The model proposed for response (yield of pristimerin) was:   

(4)

Based on the ANOVA results (Table 3), it was found that the sources of X₁, X₂, X₃, X₄, X₁X₄, X₂X₃, X₂X₄, and X₂² were able to explain the individual and interaction effect of variables on the extraction yield of pristimerin, predicting the response with a high significance level. All the other sources (X₁X₂, X₁X₃, X₃X₄, X₁², X₃², X₄²) were insignificant in Eq. 4 and were not required to explain the extraction yield of pristimerin. The analysis confirmed that the sequence of main factors respect to decreasing of influence on yield was X₄>X₂>X₃>X₁ (Table 3).

Table 3. Results of ANOVA and Regression Coefficients

Factor	Coefficient estimate	p Value
Intercept	1.490
X1	−0.082	0.0131*
X2	0.130	0.0007*
X3	0.120	0.0014*
X4	0.140	0.0003*
X1X2	0.037	0.4598
X1X3	0.097	0.0704
X1X4	0.190	0.0020*
X2X3	0.210	0.0013*
X2X4	−0.220	0.0008*
X3X4	0.001	0.8420
X12	−0.056	0.2137
X22	−0.420	< 0.0001*
X32	−0.068	0.1340
X42	−0.011	0.8032
Lack of fit		0.8674

* Statistically important.

The fitness of the model was evaluated by the lack of fit test (p>0.05). The p value of lack of fit for pristimerin was found to be 0.8674, indicating the good fitness of the model (Table 3).

The predicted values obtained by the quadratic polynomial equations (Eq. 4) showed strong correlation with actual experimental values in Fig. 8.

Fig. 8. Comparison between Predicted and Actual Extracting Yield of Pristimerin

As can be seen, there is a good agreement between the predicted values and actual experimental values from the model. The R² value for Eq. 4 was 0.9519, indicating that 95.19% of the total variation in the yield was attributed to the experimental variables studied, namely ultrasonic power, extraction temperature, extraction time and S/S ratio.

Response Surface Optimization of Ultrasonic Extraction Conditions

Based on the information (Eq. 4) using RSM, the 3D response surface plots were generated to determine the levels of the processing variables to achieve the optimal yield of pristimerin (Fig. 9). In the current study, ultrasonic power was one of the parameters used to evaluate the effect of UAE. Variations of the other main factors (extraction temperature, extraction time and S/S ratio) with ultrasonic power are shown in Figs. 9A–C. According to the ANOVA results (Table 3), Figs. 9A and B are not statistically significant. But the variation of extraction yield can also be seen in these figures. With the ultrasonic power of 75 W, the highest extract yield was achieved using temperature of 50–55°C at a fixed time and S/S ratio (Fig. 9A). The interaction of time and ultrasonic power did not affect the transition during extraction (Fig. 9B). Figure 9C shows the yield of pristimerin increased with the increase of S/S ratio at a fixed ultrasonic power and nearly reached a peak with the ultrasonic power of 105 W and the S/S ratio at 40 mL/g.

Fig. 9. Response Surface and Contour Plots for the Effect of Independent Variables on Pristimerin Extraction Efficiency

The other extraction parameter that was evaluated in terms of extraction efficiency was extraction temperature. Variations of other main factors (S/S ratio, ultrasonic power and extraction time) with extraction temperature are shown in Figs. 9A, D, and E. Among the plots, the extraction temperature and time interaction (Fig. 9D) and the extraction temperature and S/S ratio interaction (Fig. 9E) are significant (Table 3) in terms of pristimerin enrichment in the extract. By setting the ultrasonic power and S/S ratio at a fixed values, the optimal pristimerin yield should be in the extraction temperature range of 50–55°C with extraction time at 40 min (Fig. 9D). Obviously, when the ultrasonic power and extraction time were set at fixed values, the highest yield of pristimerin could be achieved at the extraction temperature of 50–55°C and S/S ratio at 40 mL/g (Fig. 9E).

Another extraction parameter investigated in this study was extraction time. According to the results (Table 3), the effect of time on enhancing the extraction efficiency of pristimerin was significant (p=0.0019). Also, the pristimerin increment depending on increasing extraction time can be seen in Figs. 9B, D, and F.

The S/S ratio was the last parameter chosen for the evaluation of UAE conditions in the study. As we can see in the Table 3, the effect of S/S ratio for increasing the extraction yield of pristimerin was significant (p=0.0003). Figures 9C, E, and F show that the pristimerin enhancement depends upon increasing S/S ratio.

By employing the Design-Expert software, the optimal values of the four variables were calculated to be 105 W, 51.66°C, 40 min, and 40 mL/g for ultrasonic power, extraction temperature, extraction time, and the S/S ratio, respectively. The maximum predicted yield of pristimerin was 1.885 mg/g under optimal conditions. For operational convenience, the optimal parameters are 105 W, 52°C, 40 min, and 40 mL/g for ultrasonic power, extraction temperature, extraction time, and the S/S ratio, respectively. To compare the predicted result with the practical value, a control experiment was performed, three times, in the previously defined optimal conditions extraction. A mean value of 1.843±0.06 mg/g (n=3), slightly lower than the one predicted by the model was obtained. This result suggests that the optimized model appropriately explains the actual extraction process of pristimerin.

CONCLUSION

An efficient UAE process was employed to extract pristimerin from Celastrus orbiculatus. RSM was used to determine the optimum extraction parameters that gave high extraction efficiency. The optimum conditions were an extraction temperature of 52°C, ultrasonic power of 105 W, S/S ratio of 40 mL/g, and extraction time of 40 min. The p value indicated that the variable with the largest effect was the S/S ratio (X₄). This was followed by the extraction temperature (X₂), the interaction effect of S/S ratio and extraction temperature (X₂X₄), the extraction time (X₃), the interaction effect of ultrasonic power and S/S ratio (X₁X₄), the interaction effect of extraction temperature and extraction time (X₂X₃) and the ultrasonic power (X₁). The maximum yield of pristimerin (1.843 mg/g) was obtained under optimal conditions. Consequently, the present study was carried out to contribute to the simulation and optimization of pristimerin extraction.

Acknowledgment

We are grateful to the professor Shuyuan Li of Guangdong Pharmaceutical University to help with the identification of the plant.

Conflict of Interest

The authors declare no conflict of interest.

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