Continuous Pressurized Fluid Extraction of Gynostemma

Taiwan 402, Republic of China, Department of Biochemistry, China Medical UniVersity, ... Taiwan, Republic of China, and Education and Research Departm...
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SEPARATIONS Continuous Pressurized Fluid Extraction of Gynostemma pentaphyllum and Purification of Gypenosides Chun-Hao Chen,† Tai-Yuan Huang,† Miau-Rong Lee,‡ Shih-Lan Hsu,§ and Chieh-Ming J. Chang*,† Department of Chemical Engineering, National Chung Hsing UniVersity, 250, Kuo-Kuang Road, Taichung, Taiwan 402, Republic of China, Department of Biochemistry, China Medical UniVersity, Taichung 404, Taiwan, Republic of China, and Education and Research Department, Veterans General Hospital, Taichung, Taiwan, Republic of China

This work studied extractions of gypenosides from Gynostemma pentaphyllum using batch and continuous pressurized fluids. Temperature was found to be a dominant factor in extracting gypenosides from this plant material. Using low solid to solvent ratio (50 g/L) was evidenced to avoid a rate limitation on batch pressurized water extraction of gypenosides. Within 3 h of continuous pressurized water extraction at 1.48 MPa, 373 K, 10 mL/min, a high solid to solvent ratio (200 g/L) resulted in 106.7 mg/g gypenosides. Our experimental results showed that 100% recovery of gypenosides, i.e., 164 mg/g, was obtained following pressurized 80% ethanol extraction at the same operation condition. Purification of the extract using Amberlite XAD7-HP column chromatography indicated that the purity of gypenosides increased from 24% to 83% by weight. In the bioassay aspect, the effect of concentrations of gypenosides in the extracts on the growth inhibition of two cancer cells was examined. 1. Introduction Gynostemma pentaphyllum (GP) is a kind of perennial liana of Cucurbitaceae, growing wild throughout China, Japan, Korea, India, and Taiwan. This liana has long been used in Chinese traditional medicine to treat maladies such as bronchitis. Previous studies have found and isolated more than 90 dammarane-type saponins (called gypenosides) from several GP species, structurally related to ginseng saponins.1 Figure 1 shows a few gypenosides that have structures identical with those from Panax ginseng, P. quinquefolium, and P. notoginseng.1-6 Pharmacological studies have demonstrated that gypenosides possess a few biochemical functionalities including anticancer, antiaging, antifatigue, antiulcer, hypolipidemic, and immunomodulatory activation.7,8 Owing to its lower retail price and easier availability than ginseng, GP has been widely commercialized in such products as herbal teas and extracts recently. A few studies in the literatures reported that the extraction of ginsenosides from P. ginseng requires an enormous amount of organic solvents, resulting in environmental pollution and the remaining residue of solvents.9 Compared to organic solvents, hot water extracts a small amount of fat-affinitive ginsenosides. Hawthorne et al. and Clifford et al. reported applications of pressurized water extraction for natural materials.10,11 Studies have shown that elevated pressure could modulate the dielectric constant of water, even lower it to that of ethanol at atmospheric conditions, and enabled easy extraction of organic-affinity compounds.12-19 There is no literature found on exploring the * To whom correspondence should be addressed. Tel.: +011-8864-2285-2592. Fax: +011-886-4-2286-0231. E-mail: cmchang@ dragon.nchu.edu.tw. † National Chung Hsing University. ‡ China Medical University. § Veterans General Hospital.

Figure 1. Chemical structure of gypenosides Rb1, Rb2, Rb3, and Rd.1

effect of pressurized fluids on the extraction of gypenosides from GP plant material. Hence, this study examined hot fluid extraction of gypenosides from the leaves and stems of G. pentaphyllum using batch and continuous processes under accelerated pressures. 2. Experimental Section 2.1. Plant materials. The aerial part of G. pentaphyllum, collected from Guilin, Guangxi province of China, was purchased from a Taiwan herbal supplier. Dried GP materials were ground by a mixer and passed through an international standard screen of 60 mesh (i.e., 250 µm) to obtain powder having small particles. It was then stored in a vacuum container at room temperature before use.

10.1021/ie0705709 CCC: $37.00 © 2007 American Chemical Society Published on Web 10/12/2007

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Figure 2. Schematic flow diagram of pressurized liquid extraction of gypenosides from G. pentaphyllum.

2.2. Chemicals and Reagents. Methanol (99%), ethanol (99.8%), 1-butanol (99%), ethyl acetate (99.5%), perchloric acid (70-72%), vanillic acid (99%), and glacial acetic acid (99.8%) were of analytical reagent grade (Merck, Germany) and purchased from a local supplier. Seven gypenoside standards (Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1) were purchased from a local agent (Genay, France). The 80% pure gypenosides mainly containing Rb1, Rb2, Rb3, and Rd, donated by Luster Co. (Taichung, Taiwan), were used as standards in the UV quantification without further purification. A few standardized media were used for the cell assay including L-glutamine (Sigma, U.S.A.), sodium dodecyl sulfate (SDS; Sigma, U.S.A.), Dulbecco’s modified Eagle’s medium (DMEM; Gibco, U.S.A.), fetal calf serum (FCS; Gibco, U.S.A.), nonessential amino acids solution (NEAA; Gibco, U.S.A.), penicillin, streptomycin (VGH Pharmaceutical, Taiwan), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma, U.S.A). Human lung cancer cells (CH-27) and rat leukemia cells (WEHI-3) were kindly donated by the Department of Biochemistry, School of Medicine, China Medical University (Taichung, Taiwan). 2.3. Extraction and Analysis. The GP powder was extracted by Soxhlet solvent extraction, batch pressurized water extraction, and continuous pressurized fluid extraction, respectively. A 20 g sample of the powder was loaded into a cellulose thimble inside a 25 g reflux Soxhlet cell and extracted using 250 mL of water, methanol, and ethanol for 2-24 h, individually. All of the extracts were collected, filtered, dried, and weighed. The total extracted amount and the extraction efficiencies of the gypenosides in the extracts were then determined. The maximal amount of gypenosides in the extract was 164 mg/g (grams of gypenosides/gram of powder), which was reached in 24 h Soxhlet extraction with ethanol at 351 K. This result represents 100% recovery of gypenosides from the GP powder. Figure 2 indicates a schematic flow diagram of the pressurized fluid extraction, as described in detail in previous work.15,17-19 Briefly, pressurized fluid extractions were conducted in a 1 L autoclave (Parr Instrument Company, Moline, IL) equipped with an agitator, a PIT temperature controller, and a pressure indicator and operating at a 200 rpm stirring speed. From 50 to 200 g of the GP powder was used for these extractions. For continuous

Figure 3. Effect of pressure on amount of gypenosides extracted in batch pressurized water at 2 h period (duplicated data).

Figure 4. Effect of temperature on amount of gypenosides extracted in batch pressurized water at 1.48 MPa. Table 1. Experimental Data on 3 h Continuous Pressurized Fluid Extractions of Gypenosides from G. pentaphyllum at 1.48 MPa and 373 Ka expt

solvent

SSR (g/L)

Q (mL/min)

WGyp (mg/g)

R (%)

C1 C2 C3 C4 C5 C6 C7 C8 C9

H2O H2O H2O H2O 20% EtOH(aq) 40% EtOH(aq) 60% EtOH(aq) 80% EtOH(aq) 95% EtOH(aq)

50 50 83 200 200 200 200 200 200

5 10 10 10 10 10 10 10 10

52.9 72.3 77.0 106.7 83.9 109.5 161.6 164.0 163.5

32.3 44.1 47.0 65.1 51.2 66.8 98.5 100 99.7

a SSR, solid to solvent ratio ) (W /V); V, volume of 1 L autoclave; Q, GP flow rate of pressurized fluid (mL/min); WGyp, weight of gypenosides in the extract (mg/g); R, recovery of gypenosides from the GP powder) (WGyp/ WGyp,80%EtOH).

fluid extraction, the GP powder suspended with 1000 mL of water was loaded into the autoclave before each experiment started. After the desired pressure, temperature, and retention time were reached, samples were taken every 10 min at the outlet and a small amount of water at room temperature was

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Figure 5. (A) Effect of SSR value on outlet gypenoside concentration, following batch pressurized water extraction at 1.48 MPa and 373 K. (B) Effect of SSR value on outlet gypenoside concentration, following continuous pressurized water extraction at 1.48 MPa and 373 K.

continuously added into the autoclave by use of an LC pump to maintain the extraction pressure. The colorimetric content of the gypenosides in the extract was determined using a UV-3000 spectrophotometer (Hitachi, Japan), similar to that reported recently by Chang et al.20 The standard calibration curve of gypenosides having a 0.9995 correlation coefficient (R2) was constructed and was linear from 250 to 2000 µg/g in a certain substrate solution. HPLC was employed to quantify the content of Rb1, Rb2, Rb3, and Rd in the extracts. This analytical HPLC system consists of one Inertsil ODS-3V-5 µm (3.0 mm × 150 mm) column, which is equipped with a Perkin-Elmer 785A UV detector, a 410 pump, and a NCI-900 control interface (PerkinElmer, USA). The column temperature was stabilized at 30 °C (303 K). The UV absorption of the samples was detected at a wavelength of 203 nm, and the injection volume was 20 µL. A two-solvent (water and acetonitrile) gradient mobile phase controlled at 0.6 mL/min was used for this analysis. Substrate

Figure 6. (A) Effect of SSR value on amount of gypenosides extracted, following batch pressurized water extraction at 1.48 MPa and 373 K. (B) Effect of SSR value on amount of gypenosides extracted, following continuous pressurized water extraction at 1.48 MPa and 373 K.

solutions were formed by infinitely diluting the gypenoside extracts, and calibration curves were built up for quantifying the content of seven gypenosides in the extracts. These curves were linear between 20 and 250 mg/L, and correlation coefficients (R2) were at least 0.99. 2.4. Purification by Column Chromatography. The extract was manually loaded and fractionated on the 50 mL Amberlite XAD7-HP packed column and eluted gravimetrically by deionized water, followed by 80% methanol and 95% ethanol solutions. Every 10 mL of eluate was collected at flow rate of one time of bed volume per hour. A total eight times of bed volume were collected. Every collected solution was dried and dissolved in a few milliliters of 99.8% absolute ethanol to form a sample for quantitatively analyzing the purity of gypenosides by a UV method at 550 nm and by an HPLC method at 203 nm. 2.5. Bioassay. 2.5.1. Preparation for Cell Growth Medium. A 10 g powder sample of Dulbecco’s modified Eagle’s medium

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Figure 7. HPLC chromatography of (A) seven standards and (B) purified gypenosides.

(DMEM) was dissolved in 900 mL of deionized water and formed a pH 7.0-7.2 solution by adding NaHCO3 and HCl. This prepared solution was finally set to 1 L, and it was passed through 0.45 and 0.2 µm nylon filters and stored in a 4 °C refrigerator. A 1 mL DMEM solution finally contained 100 IU (IU/mL) penicillin, 100 µg (µg/mL) streptomycin, 2 mM L-glutamine, 100 µg of NEAA, and 5% fetal calf serum. 2.5.2. Cell Proliferation Assay. After the DMEM solution containing cancer cells lysed with trypsin reagent, 100 µL of this solution with cell density of 2.5 × 103 was cultivated in a plate of 96 wells. These adherent cells were stuck on the surface of wells and incubated at 37 °C from 48 to 72 h in the absence or presence of various specific concentrations (20, 30, 40, 60,

80, 90, 120 µg/mL) of the pressurized fluid extracts. The calculation of the cell viability was done by an MTT method. 3. Results and Discussion Compared with the 24 h Soxhlet ethanol extraction, using pressurized fluid extraction (or so-called accelerated fluid extraction) might result in the same amount of gypenosides in the extract within a short extraction time. Therefore, batch and continuous pressurized fluid extractions of gypenosides from the GP powder was investigated herein. 3.1. Batch and Continuous Pressurized Water Extractions. In order to maintain pressurized water in a liquid state, the

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Ind. Eng. Chem. Res., Vol. 46, No. 24, 2007 Table 2. Effect of Gypenoside Concentration on Viability of CH-27 and WEHI-3 Cellsa viability (%) concn (mg/L)

CH-27

WEHI-3

0 15.63 31.25 62.5 125 250 500 1000

100.0 ( 0.0 98.4 ( 5.0 87.1 ( 5.5 81.9 ( 5.2 79.8 ( 7.4 75.0 ( 1.7 72.1 ( 0.7 56.6 ( 3.2

100.0 ( 0.0 95.2 ( 3.8 97.8 ( 2.2 98.9 ( 5.0 100.5 ( 7.1 99.9 ( 5.8 88.5 ( 7.4 50.9 ( 4.8

a Viability was measured by the MTT assay. Cells were exposed to the dosed amount of the extracts for 24 h, and viable cells were expressed as a percentage of the control cells. Data points represent mean values of four independent experiments.

Figure 8. Purity and recovery of gypenosides in fractionated samples of the continuous water extract. Each fraction contained 10 mL of elute.

pressure range of 0.79-6.99 MPa was investigated for extractions at the temperature range of 333-393 K within the 2 h period. The 433 K extraction was carried out only at 1.48 MPa from 0.5 to 3 h. Figures 3 and 4 describe effects of pressure and temperature respectively on the contents of gypenosides in the extracts following batch pressurized water experiments. Pressure ranging from 0.79 to 6.99 MPa did not significantly affect the amount of gypenosides extracted, shown by two-repeat data in Figure 3. However, the temperature was an important factor in extracting the amount of gypenosides, shown by timerelated one-repeat data in Figure 4. Extractions at high temperature (393 or 433 K) and long retention time might destroy gypenosides in the extract. The 373 K batch pressurized water extraction at 1.48 MPa and the 1 h period was found to be suitable in obtaining high recovery of gypenosides. 3.2. Effect of the Solid to Solvent Ratio (SSR) on Extracted Gypenosides. Several experiments with varied SSR values were carried out to determine the concentration of gypenosides in the outlet for batch and continuous pressurized water extractions. The flow rate of continuous experiments was set at 10 mL/min. Table 1 lists these experimental data at 1.48 MPa and 373 K. Pressurized fluid extractions of gypenosides from the powder using hot water added with ethanol concentration varied from 20% to 95% were also conducted at the same pressure and temperature with the SSR value of 200; these data are also shown in Table 1. Figure 5 shows that the concentration of gypenosides in the outlet for both batch and continuous extractions increased with the increasing SSR value from 50 to 200 g/L. The low concentration of gypenosides at t ) 0 indicates the amount of gypenosides extracted before the desired temperature and pressure were attained. During batch extraction, the higher the SSR value, the higher the outlet concentration of gypenosides (Figure 5A). This high concentration of gypenosides was gradually diminished as fresh water was continuously pumped into the extractor, shown in Figure 5B. When plotted as the extracted gypenosides with respect to the extraction time, Figure 6 presents a different effect of the SSR value on the batch (Figure 5A) and continuous (Figure 5B) water extractions of gypenosides. In view of the low extracted gypenosides following the batch process at the 200 g/L SSR value (Figure 6A), it was observed that the extraction becomes slower when

one uses a high SSR value; this may simply be a rate limitation resulting from a lower driving force. In other words, the extracted amount of gypenosides (milligrams per gram of the GP), shown by Figure 6A, decreased with increasing SSR values. Experimental results indicated that 50 g/L was a suitable SSR value in obtaining high recovery of gypenosides following the batch pressurized water extraction. Figure 6B shows that continuous water extraction at the 200 g/L SSR value could obtain the largest amount of gypenosides in water (i.e., 106.7 mg/g), which was not a rate limitation process and was different from that of the batch extraction (Figure 6A). Fresh water continuously pumped into the autoclave as the extraction time progressed provided a high driving force to extract the gypenosides in this continuous water extraction. By doing this, the gypenosides in the outlet were continuously collected in the sample and the amounts of gypenosides increased in the extract (Figure 6B). The amount of gypenosides finally attained 106.7 mg/g of the solid. From this point of view, continuous pressurized water extraction belongs to a low rate limitation process, better than batch water extraction for recovering gypenosides from G. pentaphyllum. A continuous pressurized 80% ethanol extraction was further demonstrated on completely recovering gypenosides from the GP powder at the same operation condition; the experimental data are represented by row C8 in Table 1. 3.3. Purification of Gypenosides. Figure 7A shows the HPLC spectrum of seven ginsenoside standards. Figure 7B shows the spectrum of a purified fraction containing 83% gypenosides. Experimental data following column purifications of the extracts indicate that the purity of the purified fraction containing mainly Rb1, Rb3, and Rd was above 80 wt %. The purest fraction exhibited a dark yellowish color and was collected between the preliminary deionized water fractions and the final methanol fractions. Figure 8 shows that the purity and the recovery of gypenosides in the fractions changed with the number of fractions collected. Experimental data demonstrated that an average of 83% purity of gypenosides was continuously obtained from the third to the sixth fractions, while the accumulated recovery of gypenosides was also shown in this column chromatography purification. 3.4. Effect on Cell Viability. Table 2 presents growth suppression of human lung cancer cells (CH-27) and rat leukemia cells (WEHI-3) individually cultivated by the pressurized fluid extracts at gypenoside concentrations up to 1000 µg/mL. The results reveal that there are some compounds in the extracts that might have a significant inhibition effect on these cancer cells at high concentrations. Cell viabilities were found to be concentration dependent for both cancer cells treated

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by the extracts. Further studies should be conducted for the evaluation of results obtained herein.

(7) Tanaka, O. Recent studies on glycosides from plant drugs of Himalaya and south-western China: chemogeographical correlation of Panax species. Pure Appl. Chem. 1990, 62 (7), 1281-1284.

4. Conclusions

(8) Li, L.; Lau, J. H. S. Protection of vascular endothelial cells from hydrogen peroxide-induced oxidant injury by gypenosides, saponins of Gynostemma pentaphyllum. Phytother. Res. 1993, 7 (4), 299-304.

This study demonstrated that continuous pressurized fluid extraction could be a viable alternative for extracting gypenosides from G. pentaphyllum. Temperature and the solid to solvent ratio were two important parameters affecting the amount of gypenosides in the extracts for both batch and continuous pressurized water extractions. Continuous water extraction presented little rate limitation and was a better process for recovering gypenosides from the plant material. Experimental results indicated that continuous pressurized 80% ethanol at 1.48 MPa, 373 K, and 10 mL/min completely recovered gypenosides within 3 h extraction, producing 160 mg/g gypenosides of the plant material. Purification of gypenosides from these extracts using column chromatography indicated that the purity of gypenosides in collected fractions increased up to 83% by weight. The growth inhibition on two treated cancer cells was also positively related to the gypenoside concentration in the samples. Acknowledgment The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research (NSC94-2214-E005-001). They would also like to thank the Taichung Veterans General Hospital and National Chung Hsing University (TCVGH-NCHU-947605). The green chemistry project (NCHU) funded by the Taiwan ministry of education is also gratefully acknowledged. Literature Cited (1) Hu, L. H.; Chen, Z. L.; Xie, Y. Y. New triterpenoid saponins from Gynostemma pentaphyllum. J. Nat. Prod. 1996, 59 (12), 1143-1145. (2) Hu, L. H.; Chen, Z. L.; Xie, Y. Y. Dammarane-type glycosides from Gynostemma pentaphyllum. Phytochemistry 1997, 44 (4), 667-670. (3) Cui, J. F.; Eneroth, P.; Bruhn, J. G.; Arihara, S.; Yoshikawa, K. Alkaline cleavage gypenosides and characterization of dammarane-type aglycones by gas chromatography mass spectrometry. Phytochem. Anal. 1998, 9 (3), 128-133. (4) Cui, J. F.; Eneroth, P.; Bruhn, J. G. Gynostemma pentaphyllum: identification of major sapogenins and differentiation from Panax species. Eur. J. Pharm. Sci. 1999, 8 (3), 187-191. (5) Kuwahara, M.; Kawanishi, F.; Komiya, T.; Oshio, H. Dammarane saponins of Gynostemma pentaphyllum Makino and isolation of malonylginsenosides Rb1, Rd, and malonylgyoenoside V. Chem. Pharm. Bull. 1989, 37, 135-139. (6) Piacente, S.; Pizza, C.; Tommasi, N.; Simone, F. New dammaranetype glycosides from Gynostemma pentaphyllum. J. Nat. Prod. 1995, 58, 512-519.

(9) Wang, H. C.; Chen, C. R.; Chang, C. J. Carbon dioxide extraction of ginseng root hair oil and ginsenosides. Food Chem. 2001, 72, 505509. (10) Hawthorne, S. B.; Yang, Y.; Miller, D. J. Extraction of organic pollutants from environmental solids with sub- and supercritical water. Anal. Chem. 1994, 66, 2912-2921. (11) Clifford, A. A.; Basile, A.; Al-Saidi, S. H. R. A comparison of the extraction of clove buds with supercritical carbon dioxide and superheated water. Fresenius’ J. Anal. Chem. 1999, 364, 635-637. (12) Miller, D. J.; Hawthorne, S. B. Solubility of liquid organic flavor and fragrance compounds in subcritical (hot/liquid) water from 298 K to 473 K. J. Chem. Eng. Data 2000, 45, 315-318. (13) Curren, M. S. S.; King, J. W. Ethanol-modified subcritical water extraction combined with solid-phase microextraction for determining atrazine in beef kidney. J. Agric. Food Chem. 2001, 49 (5), 2175-2180. (14) Chang, L. H.; Cheng, Y. C.; Chang, C. J. Extracting and purifying isoflavones from defatted soybean flakes using superheated water at elevated pressures. Food Chem. 2004, 84 (2), 279-285. (15) Chen, P. Y.; Tu, Y. X.; Wu, C. T.; Jong, T. T.; Chang, C. J. Continuous hot pressurized solvent extraction of 1,1-diphenyl-2-picrylhydrazyl free radical scavenging compounds from Taiwan yams (Dioscorea alata). J. Agric. Food Chem. 2004, 52, 1945-1949. (16) Shotipruk, A.; Kiatsongserm, J.; Pavasant, P.; Goto, M.; Sasaki, M. Pressurized hot water extraction of anthraquinones from the roots of Morinda citrifolia. Biotechnol. Prog. 2004, 20 (6), 1872-1875. (17) Chen, C. R.; Lee, Y. N.; Chang, C. J.; Lee, M. R.; Wei, I. C. Hot pressurized fluid extraction of flavonoids and phenolic acids from Brazilian propolis and their cytotoxic assay in-Vitro. J. Chin. Inst. Chem. Eng. 2007. Available online; http://dx.doi.org/10.1616/j.jcice.2007.04.004. (18) Chang, L. H.; Chang, C. J. Continuous hot pressurized fluids extraction of isoflavones and soyasaponins from defatted soybean flakes. J. Chin. Inst. Chem. Eng. 2007. Available online; http://dx.doi.org/10.1016/ j.jcice.2007.03.003. (19) Lin, C. C.; Hsieh, S. J.; Hsu, S. L.; Chang, C. J. Hot-pressurized water extraction of syringin from Acanthopanax senticosus and in Vitro activation on rat blood macrophages. Biochem. Eng. J. 2007. Online available; http://dx.doi.org/10.1016/j.bej.2007.05.003. (20) Chang, C. K; Chang, K. S.; Lin, Y. C.; Liu, S. Y.; Chen, C. Y. Hairy root cultures of Gynostemma pentaphyllum (Thunb.) Makino: A promising approach for the production of gypenosides as an alternative of ginseng saponins. Biotechnol. Lett. 2005, 27 (16), 1165-1169.

ReceiVed for reView April 23, 2007 ReVised manuscript receiVed August 29, 2007 Accepted August 29, 2007 IE0705709