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Extraction of mangiferin from Mahkota Dewa (Phaleria macrocarpa) using subcritical water
Journal of Industrial and Engineering Chemistry 16 (2010) 425–430
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Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Extraction of mangiferin from Mahkota Dewa (Phaleria macrocarpa) using subcritical water Wan-Joo Kim a, Bambang Veriansyah a, Youn-Woo Lee b, Jaehoon Kim a,**, Jae-Duck Kim a,* a
Supercritical Fluid Research Laboratory, Clean Energy Center, Energy Division, Korea Institute of Science and Technology (KIST), 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea b School of Chemical and Biological Engineering, Seoul National University, Gwanangro 599, Gwanak-gu, Seoul 151-744, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 April 2009 Accepted 18 August 2009
A pharmacological active component, mangiferin, was extracted from Mahkota Dewa using subcritical water extraction. The subcritical water extractions were carried out at temperatures ranging 323–423 K, pressures ranging 0.7–4.0 MPa, and extraction times ranging 1–7 h. Extraction yield of mangiferin was measured using high-performance liquid chromatography (HPLC). The extraction yield was strongly dependent on the temperature while weakly dependent on the extraction pressure. As the extraction temperature increased, the extraction mangiferin yield increased, possibly resulting from the decrease in polarity of subcritical water at higher temperature. At an optimal extraction condition of 373 K, 4.0 MPa and extraction time of 5 h, the extraction yield of mangiferin was 21.7 mg/g. This value was close to the extraction yield with methanol (25.0 mg/g) and higher than those with water (18.6 mg/g) or ethanol (13.2 mg/g) at their boiling points. ß 2010 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.
Keywords: Subcritical water Mahkota Dewa Mangiferin Extraction
1. Introduction Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae) or Phaleria papuana Warb var. Wichanii (Val) Back, typically known as Mahkota Dewa, is a popular herbal plant in the South Asian countries. The stems, leaves and fruits of Mahkota Dewa have been widely used for medicinal treatments. Empirically, Mahkota Dewa is known to be capable of healing cancer, impotency, haemorrhoids, diabetes mellitus, allergies, liver and heart diseases, kidney disorders, blood diseases, rheumatism, high blood pressure, stroke, migraine, various skin diseases, acne and so forth. These beneficial bioactivities result from chemical compounds in Mahkota Dewa that retain antihistamine, antioxidant and anticancer effects [1]. Mahkota Dewa fruit is rich in alkaloid, saponin and flavoid, and Mahkota Dewa leaves contain mangiferin, saponin, and polyphenol [2]. Among these compounds, mangiferin has a wide range of pharmacological effects including antidiabetic, anti-HIV, anticancer, immunomodulatory and antioxidant activity [3,4]. The chemical structure of mangiferin is shown in Fig. 1.
* Corresponding author. Tel.: +82 2 958 5874; fax: +82 2 958 5205. ** Corresponding author. Tel.: +82 2 958 5873; fax: +82 2 958 5205. E-mail addresses: [email protected] (W.-J. Kim), [email protected] (B. Veriansyah), [email protected] (Y.-W. Lee), [email protected] (J. Kim), [email protected] (J.-D. Kim).
Despite of the well-known bioactive effects and qualitative compositions, there are few works on chemical compound extraction from Mahkota Dewa [5–7]. The previous works utilized toxic organic liquid solvents such as methanol or ethanol as the extraction solvents. The organic solvent-based extraction often suffers from low extraction yields, long extraction time, and residual toxic, organic solvents in final extracts. The residual solvents are most problematic because the toxicity can deteriorate the quality of the extracts and can cause serious health problem when the extracts are taken in human body. Therefore, it is highly desired to develop non-organic solvent-based extraction methods with higher extraction efficiency. Supercritical fluid extraction (SFE), especially supercritical carbon dioxide (scCO2) extraction is a potential alternative to conventional organic solvent extractions because of the non-toxic, environmental-friendly, and nonflammable characteristics associated with CO2 [8–10]. Thus no toxic residual solvent is remained in the final extracts. The major disadvantage of scCO2 extraction, however, is that extraction of polar components is highly limited due to the poor solvent power of scCO2 for the polar components. Subcritical water extraction, using water under external pressurization above its boiling point as an extraction solvent, offers an efficient, non-toxic, and environmental-friendly alternative to extract polar or slightly polar compounds [11]. The polarity of subcritical water is much less than that of water at ambient condition (e = 79 at 298 K). The dielectric constant of subcritical water is in the range of 20–40 depending on temperature and pressure. This value is very similar to the dielectric constant of
1226-086X/$ – see front matter ß 2010 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry. doi:10.1016/j.jiec.2009.08.008
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methanol. The flask was then wrapped with aluminum foil to prevent possible light degradation during the extraction. The extractions were carried out at 298 K for 24 h with stirring at 1200 rpm using a magnetic stirrer. The mixture was then filtered through the 0.4 mm Nylon membrane filter to obtain crude extracts. Each solvent extraction was carried out in triplicate.
Fig. 1. Chemical structure of mangiferin.
methanol (e = 33 at 298 K) or ethanol (e = 25 at 298 K) [12]. In addition, the densities of the subcritical water are high (0.98– 0.82 g/cm3 at 4.0 MPa). Thus, desired polar or slightly polar components, that are not soluble well in water at ambient condition or scCO2, can be much more soluble in subcritical water. Other important advantages of subcritical water extraction over the organic liquid solvent extraction include shorter extraction time, higher quality of extracts, and lower solvent extraction costs [13]. Subcritical water as an extraction solvent has been explored to extract polar, bioactive components from herbs and foods. It has been shown that 80% of oxygenates from savory and peppermint can be extracted with subcritical water at 6 MPa and 373 K [14], 54% of nutraceuticals from oregano can be extracted at 10 MPa and 473 K [15], and 90–95% of lignans from whole flaxseed can be extracted at 5.2 MPa and 413 K [16]. Recently, we demonstrated that subcritical water was very efficient to extract bioactive components, asiatic acid and asiaticoside, from Centella asiatica [17]. High yield of asiatic acid (7.8 mg/g) and asiaticoside (10 mg/g) can be extracted at 40 MPa and 523 K. Herein, we demonstrate that mangiferin from Mahkota Dewa can be extracted using subcritical water with high extraction yield. The effects of extraction time, temperature, and pressure on extraction yields of mangiferin as determined by high-performance liquid chromatography (HPLC) will be discussed. The subcritical water extraction yields are compared with those of conventional extraction techniques including liquid solvent extraction, heat of reflux extraction, Soxhlet extraction and scCO2 extraction. Methanol, ethanol or water was used as an extraction solvent for the conventional extraction techniques.
2.2.2. Heat of reflux extraction In the heat reflux extraction, mangiferin was extracted by charging 3 g of Mahkota Dewa in a 250 ml round bottom flask with 100 ml of either water, ethanol, or methanol. The flask was then wrapped with aluminum foil to prevent possible light degradation during the extraction. The extractions were carried out at the solvent boiling point. The solvent was refluxed for 5 h, cooled and filtered through the 0.4 mm Nylon membrane filter to obtain crude extracts. Each heat of reflux extraction was carried out in triplicate. 2.2.3. Soxhlet extraction The Soxhlet extraction was carried out according to standard Soxhlet method (EPA SW 846 method) by charging 3 g of Mahkota Dewa in timble filter (Advantec, Japan) and was loaded into the main chamber of the Soxhlet extractor. The extraction was performed using 100 ml of either water, ethanol, or methanol. The heating power was set to 4 cycles/h so that 20 cycles of extraction were achieved within 5 h of extraction time. Each Soxhlet extraction was carried out in triplicate. 2.3. Supercritical carbon dioxide extraction apparatus and procedure The experiments were carried out using a custom-built highpressure extractor system. Details on the scCO2 extraction apparatus and procedure are given in the previous paper [18]. The extraction was conducted at 40 MPa, 353 K, and a scCO2 flow rate of 41 g/min. 2.4. Subcritical water extraction The subcritical water extraction experiments were conducted using a custom-built, subcritical water extraction apparatus. Fig. 2 shows a schematic diagram of the extraction apparatus. Details on
2. Material and methods 2.1. Materials Dried and ground Mahkota Dewa fruit peal were kindly provided by Dexa Medica Pharmaceutical Company (Jakarta, Indonesia). The original moisture content was 0%. The Mahkota Dewa were ground to an average particle size of 520 mm. Mangiferin standard was purchased from Sigma–Aldrich Co. (St Louis, MO). Acetonitrile (HPLC grade), acetic acid (HPLC grade) and tetrahydrofuran (HPLC grade) were purchased from J.T. Baker (Phillipsburg, NJ). Ethanol and methanol (purity of 99.5%) was obtained from Junsei Chemical Co. (Tokyo, Japan). All chemicals were used as received. Deionized water was prepared using a MilliQ, Ultrapure water purification system with a 0.22-mm filter (MA, USA). Nylon membrane filters with a pore size of 0.4 mm were purchased from Whatman (Maidstone, UK). 2.2. Solvent extraction 2.2.1. Room temperature solvent extraction Three different liquid solvents, water, ethanol or methanol, were used in the room temperature solvent extraction. Mangiferin was extracted by charging 3 g of Mahkota Dewa in a 250 ml round bottom flask (Pyrex, USA) with 100 ml of either water, ethanol, or
Fig. 2. Schematic diagram of subcritical water extraction apparatus. Components: (DW) deionized water reservoir, (HP) high-pressure pump, (PH) preheater, (EV) high-pressure extraction vessel, (HF) heat furnace, (HE) heat exchanger, (BP) backpressure regulator, (V) isolation valves, and (CB) extract collecting bottle.
W.-J. Kim et al. / Journal of Industrial and Engineering Chemistry 16 (2010) 425–430
the subcritical water extraction apparatus and procedure are given in the previous paper [17]. Only a short description of the apparatus will be given here. The extraction vessel (EV) was made of SUS 316 with an internal diameter of 28 mm and a height of 377 mm, giving an internal volume of 232 ml. The preheater (PH) was a 80 cm long SUS 316 coil with an internal diameter of 0.635 cm. The temperature of the extraction vessel and the preheaters were controlled using a furnace (HF, Daepoong Industries, Korea). The temperatures of the extraction vessel were monitored by inserting type-K thermocouples (model TJ36CAXL, Omega Engineering, Inc., USA) with a probe diameter of 0.16 cm inside the extraction vessel. The thermocouples were connected to a multichannel recorder (model DR 240, Yokogawa, Japan). The high-pressure pump (HP) was a model Pulsa 608 diaphragm metering pump, manufactured by Pulsa Feeder. Co. (NY, USA). The pressure of the extraction vessel was controlled using a backpressure regulator (BP), manufactured by Tescom (Model 44-2300 Series, MN, USA). The extraction procedure of mangiferin from Mahkota Dewa consisted of several steps. Mahkota Dewa (50 g) was charged into the extraction vessel. Deionized water was then introduced into the extractor using the high-pressure pump at an experimentally desired pressure. The pressure of the extractor was controlled by the back-pressure regulator. The temperature of the extraction vessel was then increased to an experimentally desired temperature using the furnace. Static extraction was then carried out for an intended period of extraction time. Various extraction conditions were tested at pressures ranging 0.7– 4.0 MPa, temperatures ranging 323–423 K, and extraction times ranging 1–7 h. 2.5. Analytical method Liquid chromatography–mass spectroscopy (LC–MS) was used to identify chemical compounds in Mahkota Dewa. Mass spectrometric analysis was performed using LCQ Deca XP ion trap mass spectrometer (Thermo Electron Corp., San Jose, CA) with atmospheric pressure electrospray source using positive and negativeion mode at 4.5 kV of spray voltage. The heated capillary was maintained at 250 8C, and nitrogen was used as a sheath gas at a flow rate of 20 arbitrary units. The ion entrance capillary voltage and tube lens offset were +15 and +30 V for the positive-ion mode and 15 and 30 V for the negative-ion mode, respectively. The maximum ion collection time was set to 50 ms and three microscans were averaged per scan. Extraction yields of mangiferin from Mahkota Dewa were quantitatively determined with high-performance liquid chromatography (HPLC) analysis. Extracts were sonicated for 10 min to obtain well-dispersed particles in water. During the sonication, 10 ml amount of extracts was collected using a 20 ml syringe. The collected samples were dissolved in 20 ml of methanol and analyzed with HPLC. The HPLC system was comprised of a solvent delivery pump (Young-Lin M930, Seoul, Korea), a column (Optimapak C18 5 mm, 25 cm 0.46 mm, Daejeon, Korea), an absorbance ultra-violet (UV) detector (Young-Lin M720, Seoul, Korea) and built-in software (Autochro 2000, Seoul, Korea). Chromatographic separations were performed at a wavelength of 280 nm. The mobile phase was acetonitrile (88%), water (10%), acetic acid (1%), tertrafurane (1%) [19] at a flow rate of 1.0 mL/min. The amount of sample injection was set at 20 mL. HPLC was calibrated with standard solutions of mangiferin of known solution concentrations. Standard solutions were prepared by first dissolving 0.5 mg of mangiferin in 1 ml methanol. These mixtures were diluted to obtain solutions of desired, known concentrations in the range of 0–500 ppm, and used to construct calibration curves of mangiferin. The relationship between
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concentration of mangiferin y (ppm) and peak area x can be represented by a linear equation as follows: y ¼ 0:0587x
ðR2 ¼ 0:9978Þ
(1)
3. Result and discussion 3.1. LC–MS and HPLC analysis Fig. 3 shows LC–MS chromatograms of the mangiferin standard and Mahkota Dewa extract. The Mahkota Dewa extract was obtained using the room temperature extraction method with methanol as the extraction solvent. The extracts were identified by comparing m/z and retention time values in the total ion current chromatogram to those of the mangiferin standard. The major component of the mangiferin standard and the Mahkota Dewa extract was analyzed to be a peak at 422.83 g/mol and a peak at 422.77 g/mol. These peaks agree very well with the calculated chemical molecular weight of mangiferin (422.34 g/mol). Thus it can be conclude that a considerable amount of mangiferin was present in the Mahkota Dewa extract. Fig. 4 shows the HPLC chromatographic profile of the mangiferin standard and the Mahkota Dewa extract. The retention time at 10.1 min in the Mahkota Dewa extract agrees well with the retention time of the mangiferin standard. Thus we use the peak at 10.1 min to construct a calibration curve and to estimate extraction yields of mangiferin. 3.2. Conventional liquid solvent extraction and scCO2 extraction Table 1 lists the extraction yields of mangiferin using the conventional liquid solvent extractions and scCO2 extraction. Three different extraction methods, room temperature solvent extraction, heat of reflux extraction and Soxhlet extraction, were used in the liquid solvent extraction. When scCO2 at 353 K and 40 MPa was used as the extraction medium, mangiferin apparently was not extracted from Mahkota Dewa. Fig. 5 shows that solubility parameter estimation of mangiferin and scCO2. The solubility parameter difference between mangiferin and scCO2 at any temperature and pressure was higher than 2.0, suggesting that mangiferin solubility in scCO2 is extremely low [20]. This may be because the polar nature of mangiferin makes it difficult to be soluble in the non-polar scCO2 medium. When the room temperature solvent extraction was carried out, the extraction yield of mangiferin with methanol (23.4 mg/g) was higher than those with water (15.1 mg/g) or with ethanol (7.4 mg/g). This suggests that mangiferin is more soluble in the medium polarity solvent (such as methanol, e = 33 at 298 K) than the highly polar solvent (water, e = 79 at 298 K) or the less polar solvent (ethanol, e = 25 at 298 K). The extraction yield of
Table 1 The comparison of mangiferin extraction yield from Mahkota Dewa using various extraction solvents and extraction methods. Extraction methods
Solvents
scCO2 extraction
CO2
Extraction yield (mg/g)
Room temperature extraction
Methanol Ethanol Water
23.4 0.17 7.4 0.19 15.1 0.21
Heat of reflux extraction
Methanol Ethanol Water
25.0 0.11 13.2 0.15 18.6 0.26
Soxhlet extraction
Methanol Ethanol Water
24.6 0.25 12.1 0.26 18.7 0.20
0
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Fig. 3. LC–MS chromatogram of (a) mangiferin standard, and (b) Mahkota Dewa extract. The extraction conditions were 298 K, 0.1 MPa and 24 h using methanol as the extraction solvent.
mangiferin with methanol at room temperature was very close to those of the heat of reflux or of the Soxhlet extraction that used methanol at boiling point (338 K). In contrast, when water or ethanol at its boiling points was used, the extraction yields of mangiferin increased. Typically, extraction yield of desired materials from plants increased at elevated temperature by increasing solubility of desired materials and increasing rate of mass transfer [21]. In addition, heat of reflux extraction works at solvent boiling points, where the surface tension and the viscosity of the solvents are greatly reduced compared to those at ambient condition. Thus the extraction solvents can reach active sites inside the plant matrix far more easily at higher temperature,
resulting in enhancing the extraction yield [22]. Thus the comparable mangiferin extraction yields with methanol at room temperature and at high temperature may indicate that the maximum possible amount of mangiferin in Mahkota Dewa was extracted at room temperature. 3.3. Subcritical water extraction Fig. 6 shows the effect of extraction time on the mangiferin extraction yield using subcritical water as the solvent. The extractions were carried out at 373 K and 4.0 MPa. The extraction yield increased significantly from 12.1 to 21.7 mg/g during the first
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Fig. 6. The effect of extraction time on the mangiferin extract yield at a fixed extraction temperature of 373 K and a fixed pressure of 4.0 MPa.
Fig. 4. HPLC chromatogram of mangiferin standard and Mahkota Dewa extracts. The extraction conditions were 298 K, 0.1 MPa and 24 h using methanol as the extraction solvent.
5 h and appeared to decrease slightly. The extraction yield at 5 h is very close to the extraction yield with methanol as the extraction solvent (23.4–25.0 mg/g). This may indicate that maximum possible amount of mangiferin was extracted during the first 5 h of the subcritical water extraction. A slight decrease of the extraction yield at above 5 h may be caused by degradation of the mangiferin when it was exposed to the high temperature for an extended period of time. Thus further subcritical water extraction time was fixed to 5 h. Fig. 7 shows the effect of subcritical water temperature and pressure on the mangiferin extraction yield. The extractions were
Fig. 5. Dependence of solubility parameters of mangiferin and scCO2 on temperature and pressure.
carried out at temperatures ranging 323–423 K and pressures ranging 0.7–4.0 MPa at a fixed extraction time of 5 h. It can be seen that temperature plays a major role in the extraction yield. The extraction yield of mangiferin increased from 15.5 to 21.7 mg/g when temperature increased from 323 to 373 K at 4.0 MPa. Further increase in temperature, from 373 to 423 K, resulted in slightly increase in the extraction yield to 22.1 mg/g. Note that the extraction yield at 423 K is close to that with methanol (23.4– 25 mg/g). Methanol is highly toxic and not be concerned as generally recognized as safe (GRAS) solvent. Thus, efficient, nontoxic and environmentally benign extraction of mangiferin can be obtained using subcritical water at 423 K. The increase in the extraction yield with an increase in temperature can be because mangiferin solubility increased at higher temperature by decreasing the polarity of subcritical water. The dielectric constant (e) of subcritical water decreases from 82 to 45 at 4.0 MPa with increasing temperature from 298 to 423 K [23], which approaches dielectric constant of methanol (e = 33 at 298 K) [12]. In addition, the viscosity of water decreased significantly from 0.89 to 0.18 cp and the surface tension decreased from 71.97 to 48.74 mN/m when water at ambient condition is pressurized and heated to 423 K at 4.0 MPa [23]. This can lead to facile penetration of subcritical water to the plant matrix and to improved contact between subcritical water and mangiferin. Therefore, higher diffusion rates and lower viscosity, together
Fig. 7. The effect of temperature and pressure on the mangiferin extract yield at a fixed extraction time of 5 h.
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with the enhanced solubility of mangiferin in subcritical water may result in higher extraction yield at elevated temperatures. The extraction yields are not strongly dependent on the extraction pressure, as shown in Fig. 7. As the pressure increased from 0.7 to 4.0 MPa, extraction yield of mangiferin slightly increased from 18.9 to 22.1 mg/g at 423 K. This can be because dielectric constants of subcritical water do not strongly depend on pressure. As pressure increases from 1.0 to 4.0 MPa at 423 K, the dielectric constant of subcritical water stays almost the same (45). Hence the polarity of the subcritical water does not change much with pressure. This may result in the weak dependence of mangiferin extraction yield on pressure. Similar results were observed when asiatic acid and asiaticoside were extracted from C. asiatica using subcritical water [17]. As pressure increased from 10 to 40 MPa at 573 K, the extraction yield of asiaticoside slightly increased from 4.6 to 8.1 mg/g, and the extraction yield of asiatic acid slightly increased from 2.4 to 3.4 mg/g. Thus, in the subcritical water extraction, pressure can be simply selected to maintain the water as a liquid-like state at the given extraction temperature. 4. Conclusion
Table 2 Calculation of the solubility parameter for mangiferin. Group
Quantity
DEi (cal/mol)
DVi (cal/mol)
DTi (K)
CH CH5 5 CH2 C5 5 CO OH (aromatic) OH O 6-Membered ring Conjugated double bond
5 3 1 9 1 7 1 2 4 6
4100 3090 1180 9270 4150 49,840 5200 1600 1000 2400 P DEi = 81830
5 40.5 16.1 49.5 10.8 70 13 7.6 64 13.2 P DVi = 154.3
2.25 4.2 1.34 8.01 5.36 67.55 5.63 5.36 10.72 0.78 P DTi = 111.2
method was found to be useful for estimating the extraction potential of complex molecules using scCO2 [26]. The solubility of solute as a function of temperature can be described by [27]
d2 ðcal=cm3 Þ
In the extraction of Mahkota Dewa using subcritical water, the extraction yield of mangiferin increased with an increase in temperature and in extraction time whereas the yield not markedly increased with pressure. The optimum extraction conditions of mangiferin from Mahkota Dewa were at 373 K, 4.0 MPa, and 5 h. Under these conditions, 21.7 mg/g of mangiferin was extracted. The subcritical water extraction yield at the optimum condition was very close to the extraction yield using methanol as the extraction solvent. Thus mangiferin can be efficiently extracted from Mahkota Dewa with environmentally benign, non-toxic subcritical water. Acknowledgement The authors gratefully appreciate the financial support from PT Dexa Medica. Partial Support by Nano R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (2008-02344) is appreciated.
Appendix A. Solubility parameter estimation The solubility parameter of scCO2 can be estimated by the equation of Giddings et al. [24]: pffiffiffiffiffi r pffiffiffiffiffi 1=2 (2) d ðcal=cm3 Þ ¼ 1:25 Pc r:SF ¼ 0:47rr:SF Pc
rr:L
where Pc is the critical pressure (bar), rr.sF the reduced density (g/ cm3) of the supercritical fluid and rr.L the reduced density of liquid state. This equation reflects the variation of the solvent power of the supercritical fluid as a function of density. Solubility parameter of a given solute can be estimated by using Fedor’s group contribution method when the solute molecular structure is known [25]. Table 2 illustrates the procedure to estimate the solubility parameter of mangiferin by using the Fedor’s method. The solubility parameter of mangiferin was calculated by sP ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðDEv Þi 3 1=2 (3) d2 ðcal=cm Þ ¼ Pi i ðDvÞi where DEv is the summation of all cohesive energy (cal/mol) and Dv is the summation of all molar volume (cm3/mol). The Fedor’s
1=2
¼ d1
1:13 1:13 V1 r T c T 2 0:33 ¼ d1 2 ¼ d1 V2 r1 Tc T1
(4)
where d2 is the solubility parameter of solute at temperature T2, d1 the solubility parameter of solute at temperature T1, and Tc is the critical temperature estimated by a group contribution method [28] as follows: T c ¼ 535 log
X
DT i
(5)
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Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Extraction of mangiferin from Mahkota Dewa (Phaleria macrocarpa) using subcritical water Wan-Joo Kim a, Bambang Veriansyah a, Youn-Woo Lee b, Jaehoon Kim a,**, Jae-Duck Kim a,* a
Supercritical Fluid Research Laboratory, Clean Energy Center, Energy Division, Korea Institute of Science and Technology (KIST), 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea b School of Chemical and Biological Engineering, Seoul National University, Gwanangro 599, Gwanak-gu, Seoul 151-744, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 April 2009 Accepted 18 August 2009
A pharmacological active component, mangiferin, was extracted from Mahkota Dewa using subcritical water extraction. The subcritical water extractions were carried out at temperatures ranging 323–423 K, pressures ranging 0.7–4.0 MPa, and extraction times ranging 1–7 h. Extraction yield of mangiferin was measured using high-performance liquid chromatography (HPLC). The extraction yield was strongly dependent on the temperature while weakly dependent on the extraction pressure. As the extraction temperature increased, the extraction mangiferin yield increased, possibly resulting from the decrease in polarity of subcritical water at higher temperature. At an optimal extraction condition of 373 K, 4.0 MPa and extraction time of 5 h, the extraction yield of mangiferin was 21.7 mg/g. This value was close to the extraction yield with methanol (25.0 mg/g) and higher than those with water (18.6 mg/g) or ethanol (13.2 mg/g) at their boiling points. ß 2010 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.
Keywords: Subcritical water Mahkota Dewa Mangiferin Extraction
1. Introduction Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae) or Phaleria papuana Warb var. Wichanii (Val) Back, typically known as Mahkota Dewa, is a popular herbal plant in the South Asian countries. The stems, leaves and fruits of Mahkota Dewa have been widely used for medicinal treatments. Empirically, Mahkota Dewa is known to be capable of healing cancer, impotency, haemorrhoids, diabetes mellitus, allergies, liver and heart diseases, kidney disorders, blood diseases, rheumatism, high blood pressure, stroke, migraine, various skin diseases, acne and so forth. These beneficial bioactivities result from chemical compounds in Mahkota Dewa that retain antihistamine, antioxidant and anticancer effects [1]. Mahkota Dewa fruit is rich in alkaloid, saponin and flavoid, and Mahkota Dewa leaves contain mangiferin, saponin, and polyphenol [2]. Among these compounds, mangiferin has a wide range of pharmacological effects including antidiabetic, anti-HIV, anticancer, immunomodulatory and antioxidant activity [3,4]. The chemical structure of mangiferin is shown in Fig. 1.
* Corresponding author. Tel.: +82 2 958 5874; fax: +82 2 958 5205. ** Corresponding author. Tel.: +82 2 958 5873; fax: +82 2 958 5205. E-mail addresses: [email protected] (W.-J. Kim), [email protected] (B. Veriansyah), [email protected] (Y.-W. Lee), [email protected] (J. Kim), [email protected] (J.-D. Kim).
Despite of the well-known bioactive effects and qualitative compositions, there are few works on chemical compound extraction from Mahkota Dewa [5–7]. The previous works utilized toxic organic liquid solvents such as methanol or ethanol as the extraction solvents. The organic solvent-based extraction often suffers from low extraction yields, long extraction time, and residual toxic, organic solvents in final extracts. The residual solvents are most problematic because the toxicity can deteriorate the quality of the extracts and can cause serious health problem when the extracts are taken in human body. Therefore, it is highly desired to develop non-organic solvent-based extraction methods with higher extraction efficiency. Supercritical fluid extraction (SFE), especially supercritical carbon dioxide (scCO2) extraction is a potential alternative to conventional organic solvent extractions because of the non-toxic, environmental-friendly, and nonflammable characteristics associated with CO2 [8–10]. Thus no toxic residual solvent is remained in the final extracts. The major disadvantage of scCO2 extraction, however, is that extraction of polar components is highly limited due to the poor solvent power of scCO2 for the polar components. Subcritical water extraction, using water under external pressurization above its boiling point as an extraction solvent, offers an efficient, non-toxic, and environmental-friendly alternative to extract polar or slightly polar compounds [11]. The polarity of subcritical water is much less than that of water at ambient condition (e = 79 at 298 K). The dielectric constant of subcritical water is in the range of 20–40 depending on temperature and pressure. This value is very similar to the dielectric constant of
1226-086X/$ – see front matter ß 2010 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry. doi:10.1016/j.jiec.2009.08.008
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methanol. The flask was then wrapped with aluminum foil to prevent possible light degradation during the extraction. The extractions were carried out at 298 K for 24 h with stirring at 1200 rpm using a magnetic stirrer. The mixture was then filtered through the 0.4 mm Nylon membrane filter to obtain crude extracts. Each solvent extraction was carried out in triplicate.
Fig. 1. Chemical structure of mangiferin.
methanol (e = 33 at 298 K) or ethanol (e = 25 at 298 K) [12]. In addition, the densities of the subcritical water are high (0.98– 0.82 g/cm3 at 4.0 MPa). Thus, desired polar or slightly polar components, that are not soluble well in water at ambient condition or scCO2, can be much more soluble in subcritical water. Other important advantages of subcritical water extraction over the organic liquid solvent extraction include shorter extraction time, higher quality of extracts, and lower solvent extraction costs [13]. Subcritical water as an extraction solvent has been explored to extract polar, bioactive components from herbs and foods. It has been shown that 80% of oxygenates from savory and peppermint can be extracted with subcritical water at 6 MPa and 373 K [14], 54% of nutraceuticals from oregano can be extracted at 10 MPa and 473 K [15], and 90–95% of lignans from whole flaxseed can be extracted at 5.2 MPa and 413 K [16]. Recently, we demonstrated that subcritical water was very efficient to extract bioactive components, asiatic acid and asiaticoside, from Centella asiatica [17]. High yield of asiatic acid (7.8 mg/g) and asiaticoside (10 mg/g) can be extracted at 40 MPa and 523 K. Herein, we demonstrate that mangiferin from Mahkota Dewa can be extracted using subcritical water with high extraction yield. The effects of extraction time, temperature, and pressure on extraction yields of mangiferin as determined by high-performance liquid chromatography (HPLC) will be discussed. The subcritical water extraction yields are compared with those of conventional extraction techniques including liquid solvent extraction, heat of reflux extraction, Soxhlet extraction and scCO2 extraction. Methanol, ethanol or water was used as an extraction solvent for the conventional extraction techniques.
2.2.2. Heat of reflux extraction In the heat reflux extraction, mangiferin was extracted by charging 3 g of Mahkota Dewa in a 250 ml round bottom flask with 100 ml of either water, ethanol, or methanol. The flask was then wrapped with aluminum foil to prevent possible light degradation during the extraction. The extractions were carried out at the solvent boiling point. The solvent was refluxed for 5 h, cooled and filtered through the 0.4 mm Nylon membrane filter to obtain crude extracts. Each heat of reflux extraction was carried out in triplicate. 2.2.3. Soxhlet extraction The Soxhlet extraction was carried out according to standard Soxhlet method (EPA SW 846 method) by charging 3 g of Mahkota Dewa in timble filter (Advantec, Japan) and was loaded into the main chamber of the Soxhlet extractor. The extraction was performed using 100 ml of either water, ethanol, or methanol. The heating power was set to 4 cycles/h so that 20 cycles of extraction were achieved within 5 h of extraction time. Each Soxhlet extraction was carried out in triplicate. 2.3. Supercritical carbon dioxide extraction apparatus and procedure The experiments were carried out using a custom-built highpressure extractor system. Details on the scCO2 extraction apparatus and procedure are given in the previous paper [18]. The extraction was conducted at 40 MPa, 353 K, and a scCO2 flow rate of 41 g/min. 2.4. Subcritical water extraction The subcritical water extraction experiments were conducted using a custom-built, subcritical water extraction apparatus. Fig. 2 shows a schematic diagram of the extraction apparatus. Details on
2. Material and methods 2.1. Materials Dried and ground Mahkota Dewa fruit peal were kindly provided by Dexa Medica Pharmaceutical Company (Jakarta, Indonesia). The original moisture content was 0%. The Mahkota Dewa were ground to an average particle size of 520 mm. Mangiferin standard was purchased from Sigma–Aldrich Co. (St Louis, MO). Acetonitrile (HPLC grade), acetic acid (HPLC grade) and tetrahydrofuran (HPLC grade) were purchased from J.T. Baker (Phillipsburg, NJ). Ethanol and methanol (purity of 99.5%) was obtained from Junsei Chemical Co. (Tokyo, Japan). All chemicals were used as received. Deionized water was prepared using a MilliQ, Ultrapure water purification system with a 0.22-mm filter (MA, USA). Nylon membrane filters with a pore size of 0.4 mm were purchased from Whatman (Maidstone, UK). 2.2. Solvent extraction 2.2.1. Room temperature solvent extraction Three different liquid solvents, water, ethanol or methanol, were used in the room temperature solvent extraction. Mangiferin was extracted by charging 3 g of Mahkota Dewa in a 250 ml round bottom flask (Pyrex, USA) with 100 ml of either water, ethanol, or
Fig. 2. Schematic diagram of subcritical water extraction apparatus. Components: (DW) deionized water reservoir, (HP) high-pressure pump, (PH) preheater, (EV) high-pressure extraction vessel, (HF) heat furnace, (HE) heat exchanger, (BP) backpressure regulator, (V) isolation valves, and (CB) extract collecting bottle.
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the subcritical water extraction apparatus and procedure are given in the previous paper [17]. Only a short description of the apparatus will be given here. The extraction vessel (EV) was made of SUS 316 with an internal diameter of 28 mm and a height of 377 mm, giving an internal volume of 232 ml. The preheater (PH) was a 80 cm long SUS 316 coil with an internal diameter of 0.635 cm. The temperature of the extraction vessel and the preheaters were controlled using a furnace (HF, Daepoong Industries, Korea). The temperatures of the extraction vessel were monitored by inserting type-K thermocouples (model TJ36CAXL, Omega Engineering, Inc., USA) with a probe diameter of 0.16 cm inside the extraction vessel. The thermocouples were connected to a multichannel recorder (model DR 240, Yokogawa, Japan). The high-pressure pump (HP) was a model Pulsa 608 diaphragm metering pump, manufactured by Pulsa Feeder. Co. (NY, USA). The pressure of the extraction vessel was controlled using a backpressure regulator (BP), manufactured by Tescom (Model 44-2300 Series, MN, USA). The extraction procedure of mangiferin from Mahkota Dewa consisted of several steps. Mahkota Dewa (50 g) was charged into the extraction vessel. Deionized water was then introduced into the extractor using the high-pressure pump at an experimentally desired pressure. The pressure of the extractor was controlled by the back-pressure regulator. The temperature of the extraction vessel was then increased to an experimentally desired temperature using the furnace. Static extraction was then carried out for an intended period of extraction time. Various extraction conditions were tested at pressures ranging 0.7– 4.0 MPa, temperatures ranging 323–423 K, and extraction times ranging 1–7 h. 2.5. Analytical method Liquid chromatography–mass spectroscopy (LC–MS) was used to identify chemical compounds in Mahkota Dewa. Mass spectrometric analysis was performed using LCQ Deca XP ion trap mass spectrometer (Thermo Electron Corp., San Jose, CA) with atmospheric pressure electrospray source using positive and negativeion mode at 4.5 kV of spray voltage. The heated capillary was maintained at 250 8C, and nitrogen was used as a sheath gas at a flow rate of 20 arbitrary units. The ion entrance capillary voltage and tube lens offset were +15 and +30 V for the positive-ion mode and 15 and 30 V for the negative-ion mode, respectively. The maximum ion collection time was set to 50 ms and three microscans were averaged per scan. Extraction yields of mangiferin from Mahkota Dewa were quantitatively determined with high-performance liquid chromatography (HPLC) analysis. Extracts were sonicated for 10 min to obtain well-dispersed particles in water. During the sonication, 10 ml amount of extracts was collected using a 20 ml syringe. The collected samples were dissolved in 20 ml of methanol and analyzed with HPLC. The HPLC system was comprised of a solvent delivery pump (Young-Lin M930, Seoul, Korea), a column (Optimapak C18 5 mm, 25 cm 0.46 mm, Daejeon, Korea), an absorbance ultra-violet (UV) detector (Young-Lin M720, Seoul, Korea) and built-in software (Autochro 2000, Seoul, Korea). Chromatographic separations were performed at a wavelength of 280 nm. The mobile phase was acetonitrile (88%), water (10%), acetic acid (1%), tertrafurane (1%) [19] at a flow rate of 1.0 mL/min. The amount of sample injection was set at 20 mL. HPLC was calibrated with standard solutions of mangiferin of known solution concentrations. Standard solutions were prepared by first dissolving 0.5 mg of mangiferin in 1 ml methanol. These mixtures were diluted to obtain solutions of desired, known concentrations in the range of 0–500 ppm, and used to construct calibration curves of mangiferin. The relationship between
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concentration of mangiferin y (ppm) and peak area x can be represented by a linear equation as follows: y ¼ 0:0587x
ðR2 ¼ 0:9978Þ
(1)
3. Result and discussion 3.1. LC–MS and HPLC analysis Fig. 3 shows LC–MS chromatograms of the mangiferin standard and Mahkota Dewa extract. The Mahkota Dewa extract was obtained using the room temperature extraction method with methanol as the extraction solvent. The extracts were identified by comparing m/z and retention time values in the total ion current chromatogram to those of the mangiferin standard. The major component of the mangiferin standard and the Mahkota Dewa extract was analyzed to be a peak at 422.83 g/mol and a peak at 422.77 g/mol. These peaks agree very well with the calculated chemical molecular weight of mangiferin (422.34 g/mol). Thus it can be conclude that a considerable amount of mangiferin was present in the Mahkota Dewa extract. Fig. 4 shows the HPLC chromatographic profile of the mangiferin standard and the Mahkota Dewa extract. The retention time at 10.1 min in the Mahkota Dewa extract agrees well with the retention time of the mangiferin standard. Thus we use the peak at 10.1 min to construct a calibration curve and to estimate extraction yields of mangiferin. 3.2. Conventional liquid solvent extraction and scCO2 extraction Table 1 lists the extraction yields of mangiferin using the conventional liquid solvent extractions and scCO2 extraction. Three different extraction methods, room temperature solvent extraction, heat of reflux extraction and Soxhlet extraction, were used in the liquid solvent extraction. When scCO2 at 353 K and 40 MPa was used as the extraction medium, mangiferin apparently was not extracted from Mahkota Dewa. Fig. 5 shows that solubility parameter estimation of mangiferin and scCO2. The solubility parameter difference between mangiferin and scCO2 at any temperature and pressure was higher than 2.0, suggesting that mangiferin solubility in scCO2 is extremely low [20]. This may be because the polar nature of mangiferin makes it difficult to be soluble in the non-polar scCO2 medium. When the room temperature solvent extraction was carried out, the extraction yield of mangiferin with methanol (23.4 mg/g) was higher than those with water (15.1 mg/g) or with ethanol (7.4 mg/g). This suggests that mangiferin is more soluble in the medium polarity solvent (such as methanol, e = 33 at 298 K) than the highly polar solvent (water, e = 79 at 298 K) or the less polar solvent (ethanol, e = 25 at 298 K). The extraction yield of
Table 1 The comparison of mangiferin extraction yield from Mahkota Dewa using various extraction solvents and extraction methods. Extraction methods
Solvents
scCO2 extraction
CO2
Extraction yield (mg/g)
Room temperature extraction
Methanol Ethanol Water
23.4 0.17 7.4 0.19 15.1 0.21
Heat of reflux extraction
Methanol Ethanol Water
25.0 0.11 13.2 0.15 18.6 0.26
Soxhlet extraction
Methanol Ethanol Water
24.6 0.25 12.1 0.26 18.7 0.20
0
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Fig. 3. LC–MS chromatogram of (a) mangiferin standard, and (b) Mahkota Dewa extract. The extraction conditions were 298 K, 0.1 MPa and 24 h using methanol as the extraction solvent.
mangiferin with methanol at room temperature was very close to those of the heat of reflux or of the Soxhlet extraction that used methanol at boiling point (338 K). In contrast, when water or ethanol at its boiling points was used, the extraction yields of mangiferin increased. Typically, extraction yield of desired materials from plants increased at elevated temperature by increasing solubility of desired materials and increasing rate of mass transfer [21]. In addition, heat of reflux extraction works at solvent boiling points, where the surface tension and the viscosity of the solvents are greatly reduced compared to those at ambient condition. Thus the extraction solvents can reach active sites inside the plant matrix far more easily at higher temperature,
resulting in enhancing the extraction yield [22]. Thus the comparable mangiferin extraction yields with methanol at room temperature and at high temperature may indicate that the maximum possible amount of mangiferin in Mahkota Dewa was extracted at room temperature. 3.3. Subcritical water extraction Fig. 6 shows the effect of extraction time on the mangiferin extraction yield using subcritical water as the solvent. The extractions were carried out at 373 K and 4.0 MPa. The extraction yield increased significantly from 12.1 to 21.7 mg/g during the first
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Fig. 6. The effect of extraction time on the mangiferin extract yield at a fixed extraction temperature of 373 K and a fixed pressure of 4.0 MPa.
Fig. 4. HPLC chromatogram of mangiferin standard and Mahkota Dewa extracts. The extraction conditions were 298 K, 0.1 MPa and 24 h using methanol as the extraction solvent.
5 h and appeared to decrease slightly. The extraction yield at 5 h is very close to the extraction yield with methanol as the extraction solvent (23.4–25.0 mg/g). This may indicate that maximum possible amount of mangiferin was extracted during the first 5 h of the subcritical water extraction. A slight decrease of the extraction yield at above 5 h may be caused by degradation of the mangiferin when it was exposed to the high temperature for an extended period of time. Thus further subcritical water extraction time was fixed to 5 h. Fig. 7 shows the effect of subcritical water temperature and pressure on the mangiferin extraction yield. The extractions were
Fig. 5. Dependence of solubility parameters of mangiferin and scCO2 on temperature and pressure.
carried out at temperatures ranging 323–423 K and pressures ranging 0.7–4.0 MPa at a fixed extraction time of 5 h. It can be seen that temperature plays a major role in the extraction yield. The extraction yield of mangiferin increased from 15.5 to 21.7 mg/g when temperature increased from 323 to 373 K at 4.0 MPa. Further increase in temperature, from 373 to 423 K, resulted in slightly increase in the extraction yield to 22.1 mg/g. Note that the extraction yield at 423 K is close to that with methanol (23.4– 25 mg/g). Methanol is highly toxic and not be concerned as generally recognized as safe (GRAS) solvent. Thus, efficient, nontoxic and environmentally benign extraction of mangiferin can be obtained using subcritical water at 423 K. The increase in the extraction yield with an increase in temperature can be because mangiferin solubility increased at higher temperature by decreasing the polarity of subcritical water. The dielectric constant (e) of subcritical water decreases from 82 to 45 at 4.0 MPa with increasing temperature from 298 to 423 K [23], which approaches dielectric constant of methanol (e = 33 at 298 K) [12]. In addition, the viscosity of water decreased significantly from 0.89 to 0.18 cp and the surface tension decreased from 71.97 to 48.74 mN/m when water at ambient condition is pressurized and heated to 423 K at 4.0 MPa [23]. This can lead to facile penetration of subcritical water to the plant matrix and to improved contact between subcritical water and mangiferin. Therefore, higher diffusion rates and lower viscosity, together
Fig. 7. The effect of temperature and pressure on the mangiferin extract yield at a fixed extraction time of 5 h.
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with the enhanced solubility of mangiferin in subcritical water may result in higher extraction yield at elevated temperatures. The extraction yields are not strongly dependent on the extraction pressure, as shown in Fig. 7. As the pressure increased from 0.7 to 4.0 MPa, extraction yield of mangiferin slightly increased from 18.9 to 22.1 mg/g at 423 K. This can be because dielectric constants of subcritical water do not strongly depend on pressure. As pressure increases from 1.0 to 4.0 MPa at 423 K, the dielectric constant of subcritical water stays almost the same (45). Hence the polarity of the subcritical water does not change much with pressure. This may result in the weak dependence of mangiferin extraction yield on pressure. Similar results were observed when asiatic acid and asiaticoside were extracted from C. asiatica using subcritical water [17]. As pressure increased from 10 to 40 MPa at 573 K, the extraction yield of asiaticoside slightly increased from 4.6 to 8.1 mg/g, and the extraction yield of asiatic acid slightly increased from 2.4 to 3.4 mg/g. Thus, in the subcritical water extraction, pressure can be simply selected to maintain the water as a liquid-like state at the given extraction temperature. 4. Conclusion
Table 2 Calculation of the solubility parameter for mangiferin. Group
Quantity
DEi (cal/mol)
DVi (cal/mol)
DTi (K)
CH CH5 5 CH2 C5 5 CO OH (aromatic) OH O 6-Membered ring Conjugated double bond
5 3 1 9 1 7 1 2 4 6
4100 3090 1180 9270 4150 49,840 5200 1600 1000 2400 P DEi = 81830
5 40.5 16.1 49.5 10.8 70 13 7.6 64 13.2 P DVi = 154.3
2.25 4.2 1.34 8.01 5.36 67.55 5.63 5.36 10.72 0.78 P DTi = 111.2
method was found to be useful for estimating the extraction potential of complex molecules using scCO2 [26]. The solubility of solute as a function of temperature can be described by [27]
d2 ðcal=cm3 Þ
In the extraction of Mahkota Dewa using subcritical water, the extraction yield of mangiferin increased with an increase in temperature and in extraction time whereas the yield not markedly increased with pressure. The optimum extraction conditions of mangiferin from Mahkota Dewa were at 373 K, 4.0 MPa, and 5 h. Under these conditions, 21.7 mg/g of mangiferin was extracted. The subcritical water extraction yield at the optimum condition was very close to the extraction yield using methanol as the extraction solvent. Thus mangiferin can be efficiently extracted from Mahkota Dewa with environmentally benign, non-toxic subcritical water. Acknowledgement The authors gratefully appreciate the financial support from PT Dexa Medica. Partial Support by Nano R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (2008-02344) is appreciated.
Appendix A. Solubility parameter estimation The solubility parameter of scCO2 can be estimated by the equation of Giddings et al. [24]: pffiffiffiffiffi r pffiffiffiffiffi 1=2 (2) d ðcal=cm3 Þ ¼ 1:25 Pc r:SF ¼ 0:47rr:SF Pc
rr:L
where Pc is the critical pressure (bar), rr.sF the reduced density (g/ cm3) of the supercritical fluid and rr.L the reduced density of liquid state. This equation reflects the variation of the solvent power of the supercritical fluid as a function of density. Solubility parameter of a given solute can be estimated by using Fedor’s group contribution method when the solute molecular structure is known [25]. Table 2 illustrates the procedure to estimate the solubility parameter of mangiferin by using the Fedor’s method. The solubility parameter of mangiferin was calculated by sP ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðDEv Þi 3 1=2 (3) d2 ðcal=cm Þ ¼ Pi i ðDvÞi where DEv is the summation of all cohesive energy (cal/mol) and Dv is the summation of all molar volume (cm3/mol). The Fedor’s
1=2
¼ d1
1:13 1:13 V1 r T c T 2 0:33 ¼ d1 2 ¼ d1 V2 r1 Tc T1
(4)
where d2 is the solubility parameter of solute at temperature T2, d1 the solubility parameter of solute at temperature T1, and Tc is the critical temperature estimated by a group contribution method [28] as follows: T c ¼ 535 log
X
DT i
(5)
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