Accelerated extraction of Xanthone from Mangosteen pericarp using ultrasonic technique

Mangosteen pericarp has been used for long time as traditional medicine. One of main active ingredient in M. pericarp is xanthone. Xanthone has remarkable effects on cardiovascular health, antiviral and antiinflammatory. Ultrasonic Assisted Extraction (UAE) was employed to extract xanthone from dried M. pericarp and compared with soxhlet extraction and maceration. The UAE was applied by various temperature (33, 45 and 55°C), amplitude (25, 50 and 75%) and solvent (0, 50 and 95% ethanol) for 1 h extraction time. The conditions for the highest xanthone recovery were determined at temperature 33°C, and 50% ethanol at 50% amplitude, resulting 0.16 mg/g of dried M. pericarp. The Box-Behnken design was applied to investigate the optimum condition of UAE. The optimum conditions from Box-Behnken design to obtain the highest xanthone recovery were determined to be temperature 33°C, amplitude 75 and 80% ethanol. The results presented that for UAE in 0.5 h, soxhlet extraction in 2 h and maceration in 2 h, the extracted xanthones were 0.1760, 0.1221 and 0.0565 mg/g of dried M. pericarp respectively.


INTRODUCTION
Mangosteen (Garcinia mangostana L.) is known as "the queen of fruits" is in Guttiferae family.Mangosteen is grown in Thailand and Southeast Asian countries.The fruit pericarp of this plant has been used for long time as a traditional medicine for treatment of abdominal pain, diarrhea, dysentery, infected wound, suppuration and chronic ulcer.The major active substances in Mangosteen pericarp is xanthone.The xanthone has a remarkable effect on cardiovascular health, antioxidant, antibiotic, antiviral and anti-inflammatory (Suksamran et al., 2002;Sabphon et al., 2012).Due to its pharmacological activities, it is popularly applied to herbal cosmetics and pharmaceutical products.There are many ways to extract active substances such as xanthone from M. pericarp.The conventional extraction methods such as soxhlent or maceration are time-consuming methods or operate at elevated temperature that could thermally damage the active substances.In contrast, novel extraction technique such as ultrasonic operates at moderate or room temperature and promise much more yield compare to conventional extraction methods.Ultrasonic extraction is proved to be economical and effective method.It is used as alternative extraction technique at the laboratory or industry scale due to shorter extraction time and higher extraction yield (Toma et al., 2001;Nasri et al., 2012).Ultrasonic has ability to penetrate the cellular wall, reduce the particle size, and increases the mass transfer between the cell walls and the outside because of the cavitation effect (Entezari et al., 2004;Asgarpanah and Ramezanloo, 2012).
In this study, the effect of ultrasonic on extraction of xanthone from M. pricarp was investigated and compared to conventional soxhlet and maceration methods.For design of experimental was response surface methodology using Box-Behnken method applied that could reduce the number of total experiments during ultrasonic extraction for three level of investigation (temperature, ultrasonic power and solvent).The Box-Behnken design has advantages that reduce experiments due to its requirement of only three levels and it is efficiency to prepare and explain when compare to the full factorial design and other methods (Ferreira et al., 2007).

MATERIALS AND METHODS
Dried mangosteen (G.mangostana L.) pericarps were obtained from Government Pharmaceutical Organization (GPO, Thailand).For lab study, the dried sample was crushed by hammer and grounded with grinder (5657 HAAN, Retsch, Germany) to obtain 3 mm particle size.The dried sample powder was packed in plastic bags and stored in darkness.

Chemicals and reagents
95% ethanol was purchased from Alcoh-A (Thailand).99.9% methanol was purchased from Burdick & Jackson (Korea).Xanthone standard was purchased from Sigma Chemical Company (St. Louis, MO, USA).

Maceration
The experiment was performed in a 250 ml beaker filled with 5 g dried sample powder in 100 ml of 95% v/v ethanol.The extraction was carried out at room temperature for 0.5, 1 and 2 h without shaking.

Soxhlet extraction
A classical Soxhlet apparatus was employed in which 5 g of grounded sample was placed into cartridge with 250 ml of 95% v/v ethanol in round bottom flask.Extraction was carried out at a boiling point (78.1°C) of ethanol for 0.5, 1 and 2 h.

Ultrasonic assisted extraction
5 g of dried sample powder mixed with 100 ml solvent (0, 50 and 95% etanol content) was applied for each ultrasonic extraction.The extraction was performed in a 200 ml beaker with heating jacket.The temperature in heating jacket was adjusted using a water bath with accuracy of about ±1°C (ISOTEMP 2150, Fisher Scientific, US).The extraction process was performed using an ultrasonic probe (200 W max. power, HD 3200, 20 kHz, SONOPULS, Ultrasonic Homogenizers, Germany) at different ultrasonic conditions (temperature of 33 to 55°C, ultrasonic amplitude of 25 to 75% and ethanol concentration of 0 to 95%) for constant extraction time of 1 h.The extracts of soxhlet, maceration and ultrasonic was filtrated through filter paper no. 1 (Whatman, Germany) and removed solvent by using rotary evaporator (R-215, BUCHI Rotavapor, Switzerland) at 60°C under vacuum.99% v/v methanol was added for adjust volume to 25 ml.After that, the extract was subjected to analysis of xanthone concentration.The Box-Behnken design was applied to determine the response pattern of temperature (X1), ultrasonic amplitude (X2) and solvent (X3), respectively, with three levels for each variable, while the dependent variable was the xanthone recovery.The symbols and levels are shown in Table 1.The whole design consisted of 15 experimental points, which were carried out in a randomized order, to maximize the effect of unexplained variability in the observed response due to extraneous factors.

Analytical method for xanthone recovery
The concentration of xanthones was determined by UVspectrophotometer (1001 Plus, Milton Roy Spectronic, USA).The absorbance of the solutions was measured at 517 nm.99% v/v methanol was used as the blank.

Statistical analysis
All of the experiments were carried out in triplicate, and the average of the xanthone recovery was taken as a responsive value.ANOVA statistic analysis of variance was applied using Microsoft EXCEL 2010 to evaluate the data.Analyses of the variance were used to Extraction time (h) determine the significant difference between results.Differences were considered significant when the probability of a type I error was less than 5% (P0.05).

Soxhlet extraction
The effect of extraction time on xanthone yield is demonstrated in Figure 1.With increasing the extraction time increased the yield.Soxhlet extraction after 2 h resulted to a xanthone recovery up to 0.12 mg/g dried sample (Figure 1).

Maceration
In general was the xanthone yield during maceration lower than soxhlet extraction.Increasing the maceration time has slight effect on xanthone recovery.The amount of extracted xanthone during 2 h maceration was about 0.06 mg/g dried sample (Figure 1).

Effect of ultrasonic temperature
The effect of temperature on the release of xanthones extracted in 95% ethanol as a solvent at 50% ultrasonic amplitude is shown in Figure 2. It was found that the increase in temperature from 33 to 45°C enhanced the xanthone recovery and the increasing was leveled up when the temperature raised from 45 to 55°C.Temperature affected many physical properties such as viscosity, diffusivity, solubility, vapor pressure and surface tension.The main effect of ultrasounds is cavitation.At high vapor pressure of the liquid, more bubbles will be created that enhance cavitation effect (Hemwimol et al., 2006).

Effect of ultrasonic amplitude
The effect of amplitude on the release of xanthones extracted in 95% ethanol as solvent at 33°C ultrasonic temperature is shown in Figure 3.It was found that the increase in ultrasonic amplitude enhanced the xanthone recovery significantly.Amplitude is the objective measurement of the degree of change in atmospheric pressure caused by sound waves.In general, an increase in intensity will provide an increase in the cavitation effects (Mason, 1999).

Effect of solvent (% ethanol)
The ratio of water in ethanol solution had a significant effect on the extraction of xanthones (Figure 4).It was found that water, as solvent (without ethanol) was not effective for xanthone extraction.This is maybe due to the differences in polarity between the water and xanthone.The polarity of xanthones is much lower than water, so that it is not well soluble in water.50% ethanol was found to be the more effective solvent.Water content in the applied solvent played an important role in the extraction.Ultrasound enlarged the pores of the cell walls so that the diffusion process and mass transfer were improved (Soares et al., 2006).The intensity of ultrasonic cavitation in the ethanol mixture in the presence of water  was also increased because of the increase in surface tension and the decrease in viscosity.
The results of xanthone extraction using UAE at process conditions according to Box-Behnken design are shown in Figures 5a to c.The effects of ultrasonic amplitude and solvent on the recovery of xanthone as well as their interactions are shown in Figure 5a.The xanthone recovery decreased with increasing ultrasonic amplitude when water was applied as solvent.However, a reverse interaction between xanthone recovery and ultrasonic amplitude was observed when the solvent was close to 95% ethanol.Similar results were observed in the case of the effect of temperature and solvent compo-sition on the xanthone extraction during ultrasonic extraction (Figure 5b).For 50% ethanol as solvent, increasing the process temperature resulted to decreasing the xanthone extractability.This was at higher ultrasound power obviously (Figure 5c).
In this research, the value of R 2 (0.9330) indicated a good agreement between the experimental and predicted values of xanthone recovery.The value of adjusted R 2 (0.8828) suggests that the total variation of 88% for xanthone recovery was attributed to the independent variables, and about 12% of the total variation could not be explained by the model.However, the value of the model was not significant (P> 0.05) that indicated the model did not exhibited a good fitness to the true behavior.

Validation of the model
In order to validate the adequacy accuracy of the model equation (Equation 1), a verification experiment was carried out under the optimum condition (temperature 33°C, 75% amplitude and 80% ethanol) within the experimental range.Under the optimum condition, the model predicted a maximum response of 0.1273 mg/g dried sample.The experimental result showed xanthone extraction of 0.1681 mg/g dried sample.It is not significantly different from the predicted value within the 95% confidence interval.

Comparison of ultrasonic assisted extraction and conventional technique
The xanthone recovery by ultrasonic assisted extraction was compared to maceration and Soxhlet extraction by applied extraction time 0.5, 1 and 2 h as shown in Figure 6.UAE was extracted by optimum condition from Box-Behnken design (33°C, 75% amplitude and 80% ethanol).This recovery by UAE applied with Box-Behnken design, Soxhlet extraction and maceration increased with increasing extraction time.The optimum conditions of UAE applied with Box-Behnken, Soxhlet extraction and maceration presented that extracted xanthones in 2 h were 0.1760, 0.1221 and 0.0565 mg/g of dried sample respectively.The UAE applied with Box-Behnken design showed the highest xanthone recovery (Figure 6).The response surface methodology was proved to be useful for investigating the optimum conditions of xanthone extraction.The statistical analysis showed that the optimum conditions for ultrasonic assisted extraction were ultrasonic temperature 33°C, ultrasonic amplitude 75 and 80% ethanol as solvent.

Figure 2 .
Figure 2. Effect of ultrasonic temperature on the ultrasonic assisted extraction of xanthone at 50% ultrasonic amplitude in 95% (v/v) ethanol.

Figure 3 .
Figure 3.Effect of ultrasonic amplitude on the ultrasonic assisted extraction of xanthones at 33°C ultrasonic temperature in 95% (v/v) ethanol.

Figure 5b .Figure 5c .Figure 6 .
Figure 5b.Response surface plot showing the effect of ultrasonic temperature and solvent on xanthone recovery.The ultrasonic amplitude was constant at 50%.
a The values in parentheses mean practical levels.