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Simple and effective large-scale preparation of geniposide from fruit of Gardenia jasminoides Ellis using a liquid–liquid two-phase extraction
Fitoterapia 83 (2012) 1558–1561
Contents lists available at SciVerse ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Simple and effective large-scale preparation of geniposide from fruit of Gardenia jasminoides Ellis using a liquid–liquid two-phase extraction Min Zhou a, Jiaxiong Zhuo a, Wanxing Wei a,⁎, Jianwen Zhu b, Xiurong Ling c a b c
Department of Chemistry, Guangxi University, Nanning, 530004, PR China Shan Yun Biochemical Science and Technology Corporation, Liuzhou, 545600, PR China School of Environment, Guangxi University, Nanning, 530004, PR China
a r t i c l e
i n f o
Article history: Received 27 April 2012 Accepted in revised form 30 August 2012 Available online 11 September 2012 Keywords: Geniposide Extraction Preparative isolation Gardenia jasminoides Ellis
a b s t r a c t Geniposide was prepared on a large-scale using a selective two-phase liquid–liquid extraction. The aqueous residue from the fruit of Gardenia jasminoides Ellis was treated with sodium carbonate and extracted with n-butanol several times. The n-butanol extracts were treated with activated granular charcoal to remove pigments and were then concentrated to produce a residue with a high solid content. The residue was crystallized to obtain geniposide with 98% purity. For large-scale synthesis, the residue (solid content 45%, geniposide 5.5%) was extracted to generate 70 g of geniposide with 98% purity and 84.8% recovery using 1500 g residue. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Geniposide [C17H24O10, Fig. 1] is the main bioactive iridoid glycoside in ripe Gardenia fruit (Gardenia jasminoides Ellis). Studies have shown that geniposide exhibits a multitude of bioactivities. Geniposide can inhibit liver fibrosis in rats [1] and promote the adhesion of monocytes to HUVECs and the expression of human umbilical vein endothelial cells (CAMs) induced by high glucose concentrations [2]. This compound also upregulates the expression of heme oxygenase-1 (HO-1) and enhances the antioxidant capacity in primary hippocampal neurons [3]. In addition, it decreases the blood glucose, insulin and total cholesterol levels in diabetic mice in a dose-dependent manner [4]. Geniposide also inhibits CoCl2induced PC-12 cell death [5], increases the expression of Bcl-2 preventing oxidative damage induced by free radical in PC-12 cells [6], and inhibits IL-2 secretion by phorbol myristate acetate and the anti-CD28 monoclonal antibody co-stimulated activation of human peripheral blood T-cells
⁎ Corresponding author. Tel.: +86 7713272601. E-mail address: [email protected] (W. Wei). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.09.001
[7]. This target compound showed significant anti-HCV entry, anti-infectious [8] and anti-inflammatory activities [9], and can be used as an anti-lipopolysaccharide agent [10]. A derivative of geniposide, penta-acetyl geniposide, exhibited anti-tumor activity [11]. A hydrolyzate of geniposide, genipin, was used to synthesize genipin blue pigment [12], which acts as a cross-linking agent in bone substitutes [13] and biologic tissues [14]. Recently, the preparation of geniposide was primarily performed using column chromatography methods, such as HPLC [15] and silica gel column chromatography [12]. Counter current chromatography was also used to separate geniposide on a 390 mg scale [16]. A similar method, centrifugal partition chromatography (CPC), was performed for the preparation of geniposide [17]. The disadvantages of these chromatography methods include a limited production scale and time-consuming methods. Thus, a more effective method for the preparation of geniposide is necessary. Herein, we report a simple and effective large-scale preparation of geniposide by extraction that does not require any chromatography procedures. In a 5 L container, 1500 g residue (45% solid content, geniposide 5.5%) of ripe Gardenia fruit was extracted with water to obtain 70 g geniposide with 98% purity and 84.8% recovery in a matter of hours.
M. Zhou et al. / Fitoterapia 83 (2012) 1558–1561
2. Materials and methods 2.1. Instrumentation An Agilent 1100 high performance liquid chromatography system (Agilent Technologies, USA) equipped with a vacuum degasser, four single solvent delivery pumps, a thermostat column compartment, a 0.1–100 μL sample loop manual injector and a diode-array detector was used in this study. The chromatography of geniposide was performed on a Reliasil C18 column (4.6×250 mm, 50 μm) at 25 °C. The mobile phase used for analysis was methanol:water (18:82, v/v) at a flow rate of 1.0 mL/min, monitored at 239 nm. Ultraviolet absorption analysis was performed using a spectrophotometer (UV2501, Shimadzu, Japan) with distilled water as the reference. The melting point was measured using a WRX-4 micro melting point apparatus (Shanghai Yice Apparatus & Equipments Co. Ltd, China). The nuclear magnetic resonance (NMR) spectrometer used in this study was a Bruker 500 Avance NMR system (Bruker, Germany). 2.2. Reagents and materials All solvents used for HPLC were of chromatographic grade and purchased from WuLian Chemical Factory, Shanghai, China. The G. jasminoides Eillis material was provided and characterized by Shan Yun Biochemical Science and Technology Corporation. Sodium carbonate, ethyl acetate, granular activated charcoal (CAS 64365-11-3) and n-butanol are of analytic grade from Xilong Chemical Engineering Co. Ltd. The geniposide (98.5%) standard was provided by the National Institute for Food and Drug Control (China). 2.3. Preparation of extraction residue from Gardenia fruit material Ten kilograms of dried Gardenia fruit was ground up and extracted with 40 L of water using a circulation pump for
1559
24 h at room temperature. This procedure was repeated three times. All extracts were combined, filtered and evaporated under vacuum to obtain a residue (2400 g, 45% total content, geniposide 5.5%). 2.4. Extraction with n-butanol At room temperature, 50 g residue was extracted directly using 50 mL n-butanol for 20 min under constant stirring at different pH values. The organic phase was removed when the phase separation was complete. Water was added to the heavier phase to maintain the same volume as in the original solution. The extraction procedure was repeated four times to produce 240 mL butanol solution, which was subsequently extracted with water (240 mL × 3). 2.5. Pigment removal using an activated granular charcoal column The n-butanol extract (mentioned in Section 2.4) was treated with an activated granular charcoal column (20 × 2.5 cm, 30 g) to obtain a colorless solution. This colorless solution was evaporated under vacuum to produce residues with various concentrations. 2.6. Crystallization and purification n-Butanol residues obtained from the method in Section 2.5 mentioned above were added to 500 mL ethyl acetate under constant stirring for 10 min to obtain a precipitate. The precipitate was filtered to generate a filtrate cake and then washed with 10 mL ethyl acetate three times. The solid was dissolved in hot ethanol to form a saturated solution, cooled and recrystallized to produce compound 1. 2.7. Structural determination of compound 1 Compound 1 was analyzed by HPLC, UV, NMR and melting point determination to elucidate the structure. 2.8. Large-scale preparation of geniposide
Fig. 1. Structural configuration of geniposide.
One thousand five hundred grams (45% solid content, geniposide 5.5%) of residue from the water extract of Gardenia fruit was treated with sodium carbonate (pH = 11), and extracted with 1500 mL n-butanol under constant stirring for 20 min. The two phases were formed in 10 min after the stirring had stopped, and the lighter phase (the extract) was removed. Water was added to the heavier phase to maintain the same volume as in the original solution. The heavier phase was then extracted with n-butanol three times. All butanol extracts were combined (5000 mL) and extracted with water (5000 mL× 3). The lighter phase then treated using the activated granular charcoal column (120 g, 5 × 20 cm). Once all of the butanol passed through the carbon column, a colorless solution was obtained, and 500 mL butanol was used to wash the carbon column. All the butanol solutions were combined and evaporated to obtain a residue (60% solid, 150 mL). The 150 mL butanol residue was added to 1500 mL ethyl acetate under constant stirring at room temperature. The resulting mixture was then filtered to obtain a cake. This cake (85 g) was washed with 200 mL ethyl acetate and then
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dissolved in hot ethanol to produce a saturated solution. The saturated solution was cooled and recrystallized at room temperature. The crystal was separated, and then dried under vacuum at 45 °C to yield 70 g of compound 1 with an 84.8% recovery from the 1500 g residue. This preparation was accomplished using a simple extraction method without any required chromatography. Thus, it is possible to manufacture this compound on a large scale.
3. Results and discussion The extraction of the residue with n-butanol was monitored by TLC. Different concentrations of water extraction residues were assayed. Low concentrations of residue correspond to low viscosity solutions. On the one hand, a very high concentration of residue makes it difficult to form phases during the n-butanol extraction. On the other hand, low concentration of residue is better for extractions but results in a low yield. Adequate solution fluidity and extraction operation and a good yield were observed at 45% concentration. During the purification process, the pH value and concentration of the decoloring n-butanol residue significantly affected the separation.
3.1. Effect of pH value on extraction Performing the extraction under different pH values significantly affected results (Figs. 2 and 3). For pH values of 5, 7, 9 and 11 geniposide of the extraction yields were 34%, 36%, 70%, 82%, respectively. Above a pH of 13, geniposide was not detected in the extraction medium. The results demonstrated that the geniposide content in the butanol phase was low when the extraction occurred at low and high pH values. In acidic and neutral solutions, many acidic components were extracted unselectively by n-butanol. In the basic medium, the acidic components became salts and could not be extracted by n-butanol. When the pH value was above 13, nearly all of geniposide was hydrolyzed.
Fig. 3. Purification yields.
3.2. Effect of condensation of butanol solution on purification of products The butanol extracts decolored by activated carbon were evaporated to obtain different solution concentrations. These solutions were added to ethyl acetate under constant stirring to precipitate the solute. Different concentrations of solutions produced different results [Fig. 2]. When the solution concentrations were 30%, 45% and 60%, the purities of the product were almost identical. Above the concentration of 70%, the purity of the products decreased. The results also demonstrated that higher concentrations of butanol solution resulted in higher yields but lower purities of the products. When considering the purities and yields of products, the 60% solution was used in the large-scale preparation. 3.3. Structural determination of compound 1 The structure of compound 1 was determined using spectroscopy. The melting point of 1 was 163.5–164.3 °C, λmax = 239 nm. 1H NMR (600 MHz, H2O) δ 7.42 (s, 1H, H-3), 5.73 (s, 1H, H-7), 5.12 (m, 1H, H-1), 4.68 (dd, J = 8.0, 2.2 Hz, 1H, H-1′), 4.17 (d, J = 14.2 Hz, 1H, H-10), 4.10 (d, J = 14.2 Hz, 1H, H-10), 3.75 (d, J = 12.5 Hz, 1H, H-6′), 3.63 (s, 3H, OCH3), 3.58 (m, 1H,H-5′), 3.39 (dd, J = 12.5, 4.9 Hz, 1H, H-6′), 3.29 (m, J = 6, 1 H, H-2′), 3.27 (m, 1H, H-3′), 3.22 (m, J = 6, 1H, H-4′), 3.07 (m, J = 6.0 Hz, H-5), 2.70 (m, 1H, J = 12 Hz, H-6), 2.67 (m, J = 6 Hz, 1H, H-9), 1.98 (m, 1H,H-6). 13C NMR (150 MHz, H2O) δ 170.00 (\CO2\), 152.51 (C-3), 141.35 (C-8), 128.92 (C-7), 111.51 (C-4), 98.83 (C-1′), 97.14 (C-1), 76.18 (C-5′), 75.61 (C-3′), 72.70 (C-2′), 69.42 (C-4′), 60.54 (C-6′), 59.71 (C-10), 51.77 (\OCH3), 45.66 (C-9), 38.00 (C-6), 34.23 (C-5). The carbon signals at δ 128.9 and δ 141.3 were assigned to the vinyl carbons (C-7 and C-8, respectively). All physical properties and spectroscopic data confirm the structure of geniposide [12,16]. HPLC also confirmed that the product was geniposide when compared with the geniposide standard. 4. Conclusions
Fig. 2. HPLC chromatogram of the extracted liquor obtained using different pH media.
The results of our investigation demonstrate that a high concentration of the Gardenia fruit residue could be extracted with butanol in a basic medium (pH=11) at room temperature
M. Zhou et al. / Fitoterapia 83 (2012) 1558–1561
to produce an enriched geniposide fraction. This fraction was concentrated, precipitated and recrystallized to obtain a high purity product after removing the pigment with a short activated carbon column. This process is simple and could easily be applied to a large-scale manufacturing process. The separation step was performed in less than approximately 30 min, which is an improvement over the traditional chromatography methods that require several hours and result in very low yields. Acknowledgments This work was supported by the Natural Science Foundation of the Guangxi province (grant no. 2011GXNSFD018016), the National Natural Science Foundation (grant no. 81060261) and the science and technology development project, Liuzhou city (grant no. 2009051101). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2012.09.001. References [1] Ma T, Huang C, Zong G, Zha D, Meng X, Li J, et al. Hepatoprotective effects of geniposide in a rat model of nonalcoholic steatohepatitis. J Pharm Pharmacol 2011;4:587-93. [2] Wang GF, WU SY, Xu W, Jin H, Zhu ZG, Li ZH, et al. Geniposide inhibits high glucose-induced cell adhesion through the NF-κB signaling pathway in human umbilical vein endothelial cells. Acta Pharmacol Sin 2010;31:953-62. [3] Yin F, Liu J, Zheng X, Guo L, Xiao H. Geniposide induces the expression of heme oxygenase-1 via PI3K/Nrf2-signaling to enhance the antioxidant capacity in primary hippocampal neurons. Biol Pharm Bull 2010;11: 1841-6.
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[4] Wu SY, Wang GF, Liu ZQ, Ro JJ, LÜ L, Xu W, et al. Effect of geniposide, a hypoglycemic glucoside, on hepatic regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin. Acta Pharmacol Sin 2009;12:202-8. [5] Guo LX, Liu JH, Xia ZN. Geniposide inhibits CoCl2-induced PC-12 cell death via mitochondrial pathway. Chin Med J 2009;23:2886-92. [6] Yin F, Liu JH, Xiao H, Kong SZ. geniposide prevents PC12 cells from peroxynitrite via the mitogen-activated protein kinase signaling pathway. J Health Sci 2010;2:195-9. [7] Chang WL, Wang HY, Shi LS, Lai JH, Lin HC. Immunosuppressive iridoids from the fruits of Gardenia jasminoides. J Nat Prod 2005;68:1683-5. [8] Zhang HJ, Rothwangl K, Mesecar AD, Sabahi A, Rong LJ, Fong Harry HS. Lamiridosins, hepatitis C virus entry inhibitors from Lamium album. J Nat Prod 2009;12:2158-62. [9] Koo HJ, Lim KH, Jung HJ, Park EH. Anti-inflammatory evaluation of Gardenia extract, geniposide and genipin. J Ethnopharmacol 2006;103: 496-500. [10] Zheng XC, Yang D, Liu X, Wang N, Li B, Cao HW, et al. Identification of a new anti-LPS agent, geniposide, from Gardenia jasminoides Ellis, and its ability of direct binding and neutralization of lipopolysaccharide in vitro and in vivo. Int Immunopharmacol 2010;10:1209-19. [11] Peng CH, Huang CN, Wang CJ. The anti-tumor effect and mechanisms of action of penta-acetyl geniposide. Curr Cancer Drug Targets 2005;5: 299-305. [12] Park YS, Lee CM, Cho MH, Hahn TR. Physical stability of the blue pigments formed from geniposide of Gardenia fruits: effects of pH, temperature, and light. J Agric Food Chem 2001;49:430-2. [13] Liu BS, Yao CH, Chen YS, Hsu SH. In vitro evaluation of degradation and cytotoxicity of a novel composite as a bone substitute. J Biomed Mater Res A 2003;4:1163-9. [14] Tsai CC, Huang RN, Sung HW, Liang HC. In vitro evaluation of the genotoxicity of a naturally occurring crosslinking agent (genipin) for biologic tissue fixation. J Biomed Mater Res 2000;1:58-65. [15] Sheu SJ, Hsin WC. HPLC separation of the major constituents of gardeniae fructus. J High Resolut Chromatogr 1998;9:523-6. [16] Zhou TT, Fan GR, Hong ZY, Chai YF, Wu YT. Large-scale isolation and purification of geniposide from the fruit of Gardenia jasminoides Ellis by high-speed counter-current chromatography. J Chromatogr A 2005;1100: 76-80. [17] Kim CY, Kim JW. Preparative isolation and purification of geniposide from gardenia fruits by centrifugal partition chromatography. Phytochem Anal 2007;18:115-7.
Contents lists available at SciVerse ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Simple and effective large-scale preparation of geniposide from fruit of Gardenia jasminoides Ellis using a liquid–liquid two-phase extraction Min Zhou a, Jiaxiong Zhuo a, Wanxing Wei a,⁎, Jianwen Zhu b, Xiurong Ling c a b c
Department of Chemistry, Guangxi University, Nanning, 530004, PR China Shan Yun Biochemical Science and Technology Corporation, Liuzhou, 545600, PR China School of Environment, Guangxi University, Nanning, 530004, PR China
a r t i c l e
i n f o
Article history: Received 27 April 2012 Accepted in revised form 30 August 2012 Available online 11 September 2012 Keywords: Geniposide Extraction Preparative isolation Gardenia jasminoides Ellis
a b s t r a c t Geniposide was prepared on a large-scale using a selective two-phase liquid–liquid extraction. The aqueous residue from the fruit of Gardenia jasminoides Ellis was treated with sodium carbonate and extracted with n-butanol several times. The n-butanol extracts were treated with activated granular charcoal to remove pigments and were then concentrated to produce a residue with a high solid content. The residue was crystallized to obtain geniposide with 98% purity. For large-scale synthesis, the residue (solid content 45%, geniposide 5.5%) was extracted to generate 70 g of geniposide with 98% purity and 84.8% recovery using 1500 g residue. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Geniposide [C17H24O10, Fig. 1] is the main bioactive iridoid glycoside in ripe Gardenia fruit (Gardenia jasminoides Ellis). Studies have shown that geniposide exhibits a multitude of bioactivities. Geniposide can inhibit liver fibrosis in rats [1] and promote the adhesion of monocytes to HUVECs and the expression of human umbilical vein endothelial cells (CAMs) induced by high glucose concentrations [2]. This compound also upregulates the expression of heme oxygenase-1 (HO-1) and enhances the antioxidant capacity in primary hippocampal neurons [3]. In addition, it decreases the blood glucose, insulin and total cholesterol levels in diabetic mice in a dose-dependent manner [4]. Geniposide also inhibits CoCl2induced PC-12 cell death [5], increases the expression of Bcl-2 preventing oxidative damage induced by free radical in PC-12 cells [6], and inhibits IL-2 secretion by phorbol myristate acetate and the anti-CD28 monoclonal antibody co-stimulated activation of human peripheral blood T-cells
⁎ Corresponding author. Tel.: +86 7713272601. E-mail address: [email protected] (W. Wei). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.09.001
[7]. This target compound showed significant anti-HCV entry, anti-infectious [8] and anti-inflammatory activities [9], and can be used as an anti-lipopolysaccharide agent [10]. A derivative of geniposide, penta-acetyl geniposide, exhibited anti-tumor activity [11]. A hydrolyzate of geniposide, genipin, was used to synthesize genipin blue pigment [12], which acts as a cross-linking agent in bone substitutes [13] and biologic tissues [14]. Recently, the preparation of geniposide was primarily performed using column chromatography methods, such as HPLC [15] and silica gel column chromatography [12]. Counter current chromatography was also used to separate geniposide on a 390 mg scale [16]. A similar method, centrifugal partition chromatography (CPC), was performed for the preparation of geniposide [17]. The disadvantages of these chromatography methods include a limited production scale and time-consuming methods. Thus, a more effective method for the preparation of geniposide is necessary. Herein, we report a simple and effective large-scale preparation of geniposide by extraction that does not require any chromatography procedures. In a 5 L container, 1500 g residue (45% solid content, geniposide 5.5%) of ripe Gardenia fruit was extracted with water to obtain 70 g geniposide with 98% purity and 84.8% recovery in a matter of hours.
M. Zhou et al. / Fitoterapia 83 (2012) 1558–1561
2. Materials and methods 2.1. Instrumentation An Agilent 1100 high performance liquid chromatography system (Agilent Technologies, USA) equipped with a vacuum degasser, four single solvent delivery pumps, a thermostat column compartment, a 0.1–100 μL sample loop manual injector and a diode-array detector was used in this study. The chromatography of geniposide was performed on a Reliasil C18 column (4.6×250 mm, 50 μm) at 25 °C. The mobile phase used for analysis was methanol:water (18:82, v/v) at a flow rate of 1.0 mL/min, monitored at 239 nm. Ultraviolet absorption analysis was performed using a spectrophotometer (UV2501, Shimadzu, Japan) with distilled water as the reference. The melting point was measured using a WRX-4 micro melting point apparatus (Shanghai Yice Apparatus & Equipments Co. Ltd, China). The nuclear magnetic resonance (NMR) spectrometer used in this study was a Bruker 500 Avance NMR system (Bruker, Germany). 2.2. Reagents and materials All solvents used for HPLC were of chromatographic grade and purchased from WuLian Chemical Factory, Shanghai, China. The G. jasminoides Eillis material was provided and characterized by Shan Yun Biochemical Science and Technology Corporation. Sodium carbonate, ethyl acetate, granular activated charcoal (CAS 64365-11-3) and n-butanol are of analytic grade from Xilong Chemical Engineering Co. Ltd. The geniposide (98.5%) standard was provided by the National Institute for Food and Drug Control (China). 2.3. Preparation of extraction residue from Gardenia fruit material Ten kilograms of dried Gardenia fruit was ground up and extracted with 40 L of water using a circulation pump for
1559
24 h at room temperature. This procedure was repeated three times. All extracts were combined, filtered and evaporated under vacuum to obtain a residue (2400 g, 45% total content, geniposide 5.5%). 2.4. Extraction with n-butanol At room temperature, 50 g residue was extracted directly using 50 mL n-butanol for 20 min under constant stirring at different pH values. The organic phase was removed when the phase separation was complete. Water was added to the heavier phase to maintain the same volume as in the original solution. The extraction procedure was repeated four times to produce 240 mL butanol solution, which was subsequently extracted with water (240 mL × 3). 2.5. Pigment removal using an activated granular charcoal column The n-butanol extract (mentioned in Section 2.4) was treated with an activated granular charcoal column (20 × 2.5 cm, 30 g) to obtain a colorless solution. This colorless solution was evaporated under vacuum to produce residues with various concentrations. 2.6. Crystallization and purification n-Butanol residues obtained from the method in Section 2.5 mentioned above were added to 500 mL ethyl acetate under constant stirring for 10 min to obtain a precipitate. The precipitate was filtered to generate a filtrate cake and then washed with 10 mL ethyl acetate three times. The solid was dissolved in hot ethanol to form a saturated solution, cooled and recrystallized to produce compound 1. 2.7. Structural determination of compound 1 Compound 1 was analyzed by HPLC, UV, NMR and melting point determination to elucidate the structure. 2.8. Large-scale preparation of geniposide
Fig. 1. Structural configuration of geniposide.
One thousand five hundred grams (45% solid content, geniposide 5.5%) of residue from the water extract of Gardenia fruit was treated with sodium carbonate (pH = 11), and extracted with 1500 mL n-butanol under constant stirring for 20 min. The two phases were formed in 10 min after the stirring had stopped, and the lighter phase (the extract) was removed. Water was added to the heavier phase to maintain the same volume as in the original solution. The heavier phase was then extracted with n-butanol three times. All butanol extracts were combined (5000 mL) and extracted with water (5000 mL× 3). The lighter phase then treated using the activated granular charcoal column (120 g, 5 × 20 cm). Once all of the butanol passed through the carbon column, a colorless solution was obtained, and 500 mL butanol was used to wash the carbon column. All the butanol solutions were combined and evaporated to obtain a residue (60% solid, 150 mL). The 150 mL butanol residue was added to 1500 mL ethyl acetate under constant stirring at room temperature. The resulting mixture was then filtered to obtain a cake. This cake (85 g) was washed with 200 mL ethyl acetate and then
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M. Zhou et al. / Fitoterapia 83 (2012) 1558–1561
dissolved in hot ethanol to produce a saturated solution. The saturated solution was cooled and recrystallized at room temperature. The crystal was separated, and then dried under vacuum at 45 °C to yield 70 g of compound 1 with an 84.8% recovery from the 1500 g residue. This preparation was accomplished using a simple extraction method without any required chromatography. Thus, it is possible to manufacture this compound on a large scale.
3. Results and discussion The extraction of the residue with n-butanol was monitored by TLC. Different concentrations of water extraction residues were assayed. Low concentrations of residue correspond to low viscosity solutions. On the one hand, a very high concentration of residue makes it difficult to form phases during the n-butanol extraction. On the other hand, low concentration of residue is better for extractions but results in a low yield. Adequate solution fluidity and extraction operation and a good yield were observed at 45% concentration. During the purification process, the pH value and concentration of the decoloring n-butanol residue significantly affected the separation.
3.1. Effect of pH value on extraction Performing the extraction under different pH values significantly affected results (Figs. 2 and 3). For pH values of 5, 7, 9 and 11 geniposide of the extraction yields were 34%, 36%, 70%, 82%, respectively. Above a pH of 13, geniposide was not detected in the extraction medium. The results demonstrated that the geniposide content in the butanol phase was low when the extraction occurred at low and high pH values. In acidic and neutral solutions, many acidic components were extracted unselectively by n-butanol. In the basic medium, the acidic components became salts and could not be extracted by n-butanol. When the pH value was above 13, nearly all of geniposide was hydrolyzed.
Fig. 3. Purification yields.
3.2. Effect of condensation of butanol solution on purification of products The butanol extracts decolored by activated carbon were evaporated to obtain different solution concentrations. These solutions were added to ethyl acetate under constant stirring to precipitate the solute. Different concentrations of solutions produced different results [Fig. 2]. When the solution concentrations were 30%, 45% and 60%, the purities of the product were almost identical. Above the concentration of 70%, the purity of the products decreased. The results also demonstrated that higher concentrations of butanol solution resulted in higher yields but lower purities of the products. When considering the purities and yields of products, the 60% solution was used in the large-scale preparation. 3.3. Structural determination of compound 1 The structure of compound 1 was determined using spectroscopy. The melting point of 1 was 163.5–164.3 °C, λmax = 239 nm. 1H NMR (600 MHz, H2O) δ 7.42 (s, 1H, H-3), 5.73 (s, 1H, H-7), 5.12 (m, 1H, H-1), 4.68 (dd, J = 8.0, 2.2 Hz, 1H, H-1′), 4.17 (d, J = 14.2 Hz, 1H, H-10), 4.10 (d, J = 14.2 Hz, 1H, H-10), 3.75 (d, J = 12.5 Hz, 1H, H-6′), 3.63 (s, 3H, OCH3), 3.58 (m, 1H,H-5′), 3.39 (dd, J = 12.5, 4.9 Hz, 1H, H-6′), 3.29 (m, J = 6, 1 H, H-2′), 3.27 (m, 1H, H-3′), 3.22 (m, J = 6, 1H, H-4′), 3.07 (m, J = 6.0 Hz, H-5), 2.70 (m, 1H, J = 12 Hz, H-6), 2.67 (m, J = 6 Hz, 1H, H-9), 1.98 (m, 1H,H-6). 13C NMR (150 MHz, H2O) δ 170.00 (\CO2\), 152.51 (C-3), 141.35 (C-8), 128.92 (C-7), 111.51 (C-4), 98.83 (C-1′), 97.14 (C-1), 76.18 (C-5′), 75.61 (C-3′), 72.70 (C-2′), 69.42 (C-4′), 60.54 (C-6′), 59.71 (C-10), 51.77 (\OCH3), 45.66 (C-9), 38.00 (C-6), 34.23 (C-5). The carbon signals at δ 128.9 and δ 141.3 were assigned to the vinyl carbons (C-7 and C-8, respectively). All physical properties and spectroscopic data confirm the structure of geniposide [12,16]. HPLC also confirmed that the product was geniposide when compared with the geniposide standard. 4. Conclusions
Fig. 2. HPLC chromatogram of the extracted liquor obtained using different pH media.
The results of our investigation demonstrate that a high concentration of the Gardenia fruit residue could be extracted with butanol in a basic medium (pH=11) at room temperature
M. Zhou et al. / Fitoterapia 83 (2012) 1558–1561
to produce an enriched geniposide fraction. This fraction was concentrated, precipitated and recrystallized to obtain a high purity product after removing the pigment with a short activated carbon column. This process is simple and could easily be applied to a large-scale manufacturing process. The separation step was performed in less than approximately 30 min, which is an improvement over the traditional chromatography methods that require several hours and result in very low yields. Acknowledgments This work was supported by the Natural Science Foundation of the Guangxi province (grant no. 2011GXNSFD018016), the National Natural Science Foundation (grant no. 81060261) and the science and technology development project, Liuzhou city (grant no. 2009051101). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2012.09.001. References [1] Ma T, Huang C, Zong G, Zha D, Meng X, Li J, et al. Hepatoprotective effects of geniposide in a rat model of nonalcoholic steatohepatitis. J Pharm Pharmacol 2011;4:587-93. [2] Wang GF, WU SY, Xu W, Jin H, Zhu ZG, Li ZH, et al. Geniposide inhibits high glucose-induced cell adhesion through the NF-κB signaling pathway in human umbilical vein endothelial cells. Acta Pharmacol Sin 2010;31:953-62. [3] Yin F, Liu J, Zheng X, Guo L, Xiao H. Geniposide induces the expression of heme oxygenase-1 via PI3K/Nrf2-signaling to enhance the antioxidant capacity in primary hippocampal neurons. Biol Pharm Bull 2010;11: 1841-6.
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