Optimization of ultrasound-assisted extraction of charantin from Momordica charantia fruits using response surface methodology.

BACKGROUND: Momordica charantia Linn. (Cucurbitaceae) fruits are well known for their beneficial effects in diabetes that are often attributed to its bioactive component charantin.
OBJECTIVE: The aim of the present study is to develop and optimize an efficient protocol for the extraction of charantin from M. charantia fruits.
MATERIALS AND METHODS: Response surface methodology (RSM) was used for the optimization of ultrasound-assisted extraction (UAE) conditions. RSM was based on a three-level, three-variable Box-Behnken design (BBD), and the studied variables included solid to solvent ratio, extraction temperature, and extraction time.
RESULTS: The optimal conditions predicted by the BBD were: UAE with methanol: Water (80:20, v/v) at 46°C for 120 min with solid to solvent ratio of 1:26 w/v, under which the yield of charantin was 3.18 mg/g. Confirmation trials under slightly adjusted conditions yielded 3.12 ± 0.14 mg/g of charantin on dry weight basis of fruits. The result of UAE was also compared with Soxhlet extraction method and UAE was found 2.74-fold more efficient than the Soxhlet extraction for extracting charantin.
CONCLUSIONS: A facile UAE protocol for a high extraction yield of charantin was developed and validated.

Momordica charantia Linn. fruits are vegetable cucurbits, commonly known as bitter gourd. Traditionally, these are considered as useful against diabetes and its related complications.[1] The fruits reduce elevated blood glucose level,[2] improve glucose tolerance,[3] decrease insulin resistance,[4] and increase the mass of β-cells in pancreas.[5] Charantin, the main bioactive component, has been found to be more potent than oral hypoglycemic agent tolbutamide in alloxan-induced diabetic rabbits.[6] Charantin is also reported to have antihyperglycemic and antifertility effects in rabbits.[7]

Ultrasound-assisted extraction (UAE) utilizes sonic energy to assist solvent extraction of phytoconstituents from plants.[89] TheUAE has been applied for the extraction of phytosterols,[1011] flavonoids,[1213] polysaccharides,[1415] and polyphenols[16] from plants. Response surface methodology (RSM) is an efficient technique for the optimization of different parameters used in any process. It has been successfully applied to different processes for achieving their optimization using experimental designs.[1718] It is widely used to optimize liquid-liquid and liquid-solid extraction processes.[192021] Earlier polyethylene glycol/salt aqueous two-phase systems have been optimized for the extraction of charantin using RSM.[22] Pressurized liquid extraction utilizing ethanol or ethanol: water (1:1) at pressure of 10 MPa has been also reported.[23] As UAE combines the advantages of short time, less solvent requirement, and low-temperature operation, the main aim of this study is to develop a facile UAE protocol for extraction of charantin from M. charantia fruits and to optimize its conditions by RSM.

Materials and Methods

Materials

Fresh unripe fruits of M. charantia were collected from local vegetable market. A voucher specimen (PRL/JH/11/03) was deposited in Phyto-pharmaceuticals Research Laboratory, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India.

Extraction method

Fruits were cleaned and cut into small pieces and dried in an oven at 45°C. Dried sample was pulverized to a powder using a grinder. About 10 g of powder was placed in a stoppered conical flask and extracted under designed conditions in an ultra-sonicator (Toshniwal, India) at 200 W ultrasonic power. The extract was filtered and evaporated under reduced pressure at 50°C in a rotary evaporator (Buchi, Switzerland). For comparative studies, about 10 g fruit powder was extracted with 500 mL methanol: water (80:20, v/v) for 120 min at sub-boiling temperature in a Soxhlet apparatus. The extract was filtered and evaporated in-vacuo to obtain viscous crude residue. The residue was analyzed for charantin content by high-performance thin layer chromatography (HPTLC).

Quantification of charantin by high-performance thin layer chromatography

The charantin was analyzed in the extracts of M. charantia fruits by HPTLC densitometric method as reported earlier.[24] Briefly, ascending development with toluene:ethyl acetate:methanol:formic acid (68:20:10:2, v/v/v/v) as mobile phase was carried out in a twin trough chamber. After development, the plates were dried in air and sprayed with anisaldehyde-sulfuric acid reagent, heated at 105°C for five min and scanned at 525 nm.

Selection and optimization of extraction conditions

Given the UAE, factors such as extraction solvent composition, solid to solvent ratio, extraction temperature, and time significantly affect the extraction yield. The independent variables affecting the extraction efficiency were screened by single factorial design. The response to change in each parameter was assessed by keeping the other factors constant at their median level. The effect of methanol content in extraction solvent (70–100% v/v), temperature (30–60°C), time (30–90 min), and solid to solvent ratio (1:10–1:25, w/v) were screened accordingly. Total extractive of dried fruits (% w/w) was selected as the response for the selection of variables.

Further experiments were designed with the higher ranges of the selected significant variables for optimization by Box-Behnken design (BBD). The selected factors viz., solid to solvent ratio (X1), extraction temperature (X2), and extraction time (X3) at three levels were used. The range and center point values of three independent variables are presented in Table 1. The experimental design consisted of seventeen runs as presented in Table 2. Charantin content (mg/g) on dry weight basis of fruits was selected as the response for the combination of the independent variables.

Table 1

Independent variables and their levels used for BBD

Table 2

BBD experimental results and predicted values of charantin yield Y (mg/g)

The model proposed for predicting the values of response variable was a quadratic one and was expressed according to the equation.

where Y is the yield of charantin (mg/g); b0 is the model constant; bi, bii, and bim represent the linear, quadratic, and interaction coefficient, respectively.

Extractions at predicted levels were carried to verify the validity of the statistical experimental design. The analysis of the BBD and calculation of predicted responses were carried out using Design Expert software (Version 8.0.7.1, Stat-Ease, Inc., Minneapolis, USA). P < 0.05 was considered as statistically significant.

Results and Discussion

Selection of extraction variables

In order to develop an efficient method for charantin extraction, UAE has been proposed. Charantin being a steroidal glycoside, is slightly soluble in more acceptable organic solvents such as ethanol, methanol, and their mixture with water; however, the extraction with ethanol alone requires longer extraction time.[23] Thus, methanol: water mixture was used as solvent for extraction. The efficiency of extraction process was ascertained by the estimation of charantin by HPTLC. It appeared as a sharp, symmetrical, and well-resolved band at Rf value of 0.71 ± 0.12 [Figure 1].

Figure 1

High-performance thin layer chromatography chromatogram of extract from Momordica charantia fruits

Solvent compositions with 100, 90, 80, and 70% v/v methanol were tested, and the results are shown in Figure 2a. UAE efficiency increased with increasing percentage of water in the solvent mixture until up to 20%. Water enhances the solubility of glycosides such as charantin and thus its extraction. The effect of solid to solvent ratio was studied over the range of 1:10 to 1:25 w/v. It was found that greater the amount of solvent used, higher the extraction efficiency [Figure 2b]. The effect of extraction time on extraction was examined over the range of 30–90 min and the results revealed that UAE efficiency was greatly influenced by extraction time [Figure 2c]. The yield increased with increasing extraction time up to 70 min. The influence of extraction temperature on extraction efficiency was assessed at 30, 40, 50, and 60°C [Figure 2d]. When the temperature was increased from 30°C to 40°C, the extraction efficiency increased. Higher temperature resulted in increased solvent transfer into the plant matrix. However, when the temperature increased beyond 40°C, the extraction efficiency did not change.

Figure 2

Effect of solvent composition (a); solid to solvent ratio (b); time of extraction (c) and temperature (d) on extraction efficiency

On the basis of these observations, the extraction conditions: Solvent methanol: water (80:20, v/v), solid to solvent ratio (1:25, w/v), extraction time (90 min), and temperature (40°C) were found appropriate for optimization by BBD.

Optimization of extraction variables by Box-Behnken design

The results of regression analysis (F-test) of the predicted model for optimization of charantin yield are presented in Table 3. The regression coefficient of X1, X2, X3,X1 X2, X1 X3, X12, and X22 were significant at 5% level for charantin yield. The coefficient of correlation (R2) of the predicted of the predicted model was found as 0.9826. The high F-value (44.02) and low P- value (<0.0001) indicated that the model was highly significant. The value of 1.49 for lack-of-fit implied that it was insignificant relative to the pure error. The predicted R2 (0.8406) is in reasonable agreement with the adjusted R2 (0.9603). Thus, the model was adequate to predict the extraction yield of charantin within the range of experimental variables.

Table 3

Results of regression analysis of the predicted model for optimization of charantin yield

Optimization of predicted models

The optimal conditions for UAE giving maximum charantin yield with methanol: water (80:20 v/v) as calculated from the equation were: Extraction at 46°C for 120 min with a solid to solvent ratio of 1:26, w/v, under which the predicted response value of charantin yield was 3.18 mg/g. In order to validate the adequacy of the model, verification experiments were carried out with methanol: water (80:20, v/v) with a solid to solvent ratio of 1:25, w/v at 50°C for 120 min. A charantin yield of 3.12 ± 0.14 mg/g was obtained that was in good agreement with the predicted value with a validity of 98.11%. The charantin content in the fruit extracts prepared by exhaustive Soxhlet extraction using methanol: water (80:20, v/v) with a solid to solvent ratio of 1:50, w/v at sub-boiling temperature for 120 min was 1.17 ± 0.13 mg/g on dry weight basis. UAE was 2.74 times more efficient than the conventional method with regard to charantin extraction.

Conclusion

UAE technique was developed for the fast and efficient extraction of charantin from M. charantia fruits. UAE with methanol-water (80:20, v/v) at 46°C for 120 min with a solid to solvent ratio of 1:26, w/v was found to maximize the charantin yield (more than 3 mg/g). The UAE protocol was 2.74 times more efficient than the soxhlet method in extracting charantin.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.