An Application of β-glycosidase to Transformation of Ginsenosides for the Effective Production of Specific Ginsenosides with Biological Efficacy
Biotechnology and Bioprocess Engineering 17: 000-000 (2012) DOI 10.1007/s12257-011-0678-2
Youl Her, Young-Chul Lee, Jin-Hwan Oh, Yoon-E Choi, Chang-Woo Lee, Jin-Suk Kim, Hwan Mook Kim, and Ji-Won Yang Received: 12 December 2011 / Revised: 7 February 21012 / Accepted: 7 February 21012 © The Korean Society for Biotechnology and Bioengineering and Springer 2012
Abstract Over the past several decades, the pharma- cological effects of ginsenosides in Panax ginseng roots have been extensively investigated. Here, we developed a method for producing specific ginsenosides (F1 and F2) with good yields (F1:162 mg/g, F2:305 mg/g) using β- glycosidase purified from Aspergillus niger. In addition, each ginsenoside (at least 25 species) was separated and purified by high performance liquid chromatography (HPLC) using five different types of solvents and different purification steps. In addition, the Rg3:Rh2 mixture (1:1, w/w) was shown to inhibit a specific lung cancer cell line (NCI-H232) in vivo, displaying an anticancer effect at a dose lower than achieved using treatments with single Rg3 or Rh2. This finding suggests that the combination of ginsenosides for targeting anticancer is more effective than the use of a single ginsenoside from ginseng or red ginseng.
Youl Her, Jin-Hwan Oh, Jin-Suk Kim BTGin Co. Ltd., Daejeon 305-500, Korea
Young-Chul Lee, Ji-Won Yang
Department of Chemical and Biomolecular Engineering (BK21 program), KAIST, Daejeon 305-701, Korea
Yoon-E Choi, Ji-Won Yang*
Advanced Biomass R&D Center, KAIST, Daejeon 305-701, Korea Tel: +82-42-350-3924, Fax: +82-42-350-3910
Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Chungcheongbuk-do 363-883, Korea
Hwan Mook Kim*
College of Pharmacy, Korea University, Chungnam 339-700, Korea Tel: +82-41-860-1613, Fax: +82-41-860-1606
Keywords: ginsenosides, ginsenoside combination, lung cancer cell line (NCI-H23), enzymatic process, solvent system, purification
Recent Progress in Research on Anticancer Activities of Ginsenoside-Rg3
Ginsenosides in ginseng and red ginseng not only possess major pharmacological activity but are also comprised of more than 30 species [1,2]. However, the number of ginsenosides is over 40 species when considering the ginsenoside metabolites created in the intestines in humans. All of these species can be classified into three groups, protopanaxadiol, protopanaxatriol, and ocotillol-type, which is based on the structural properties of their aglycone moieties (Fig. 1).
Recently, the pharmacological activities of ginsenosides in a broad spectrum of biological regimes and their mechanisms have been reported [3-12]. Of particular note are the anti-tumor effects produced by ginsenosides. Ingredients of ginsenosides, such as Rb1, Rg1, Rg3, Rh2, PPT, and compound-K, have shown pharmacological effects through a variety of mechanisms [7-12]. In early anticancer- related research (in the early 1980s) regarding ginsenosides, the majority of studies investigated the effects of Rb1 and Rg1 [7,8]. However, from the late 1980s to recently, the majority of studies have been based on metabolites (Rg3, Rh1, Rh2, compound-K, Rg5, and Rk1) of natural ginsenosides (Rb1 and Rg1) that have their sugar moieties partially removed [9-15] and these compounds were shown to produce better pharmacological results. Thesefindings are related to the metabolites (e.g., compound-K) produced by the intestinal microflora of natural ginsenosides in the
course of in vivo experiments.
Due to the complex and unknown pharmacological mechanisms of ginsenosides, researchers are confronted with the challenge of determining the pharmacological mechanism of ginseng extracts, which contain a mixture of ginsenosides. Despite these difficulties, it can be possible to determine whether the use of a ginsenoside mixture or a single ginsenoside is more effective, provided that the pharmacological effects of each ginsenoside depend on different mechanisms [16,17].
In order to produce the metabolites from natural ginsenosides, studies have been performed to examine the physical processes, fermentation, and enzymatic methods involved in producing ginsenosides. The focus of these studies has been on enzymatic approaches for the production of a specific ginsenoside [18-21] because not only are the structural transformation of the ginsenosides effective and reproducible, but also the reaction conditions are insensitive and the production of byproducts is negligible depending on the specific substrate. Nevertheless, an enzyme that can produce ginsenosides with a high yield has not yet been identified and the enzymatic activity after purifying the target has not yet been characterized.
β-glycosidase (β-D-glucoside glucohydrolase, EC 220.127.116.11) catalyzes the hydrolysis of aryl- an alkyl- β-D-glycosides as well as glycosides with only carbohydrate moiety (e.g., cellobiose) . In addition, previous studies have indicated
that this plant enzyme may be involved in the metabolism of plant hormones such as auxin, gibberellins, and cytokinin, which are stored as β-glycosides and are activated upon cleavage by β-glycosidase.
In this study, the purification of a ginseng extract and a new enzymatic reaction were developed with the goal of producing Rg3, Rh2, and Rb1 efficiently. For the production of Rh2 and PPT, which are important agents for anticancer treatment, a novel method of producing the ginsenoside F1 and F2 (intermediates between PPT and Rh2) with robust yields was developed using an enzymatic process (β-glycosidase purified from Aspergillus niger) and five different solvent systems that could be used to effectively separate and purify each of the ginsenosides were developed. In addition, the synergistic anticancer effect of a combined ginsenoside mixture (Rg3:Rh2 = 1:1, w/w) was compared to treatment with single Rg3 or Rh2.
1. Materials and Methods
- Chemicals (Materials)
All of the solvents used in this study were from Sigma- Aldrich (St. Louis, USA) as received. Silica-gel 60 (particle size: 55 ~ 65 µm) for the purification of ginsenosides was purchased from Merck (Darmstadt, Germany). All standard ginsenosides were supplied by BTGin Co., Ltd. (Daejeon,Korea). For convenience, formula and molecular structures of all ginsenosides referred in this study are summarized in the Supplementary data. Dried Panax Notoginseng root, which is widely sold in Chinese markets, was purchased from from HongJiu Gingeng (Fusong, Jilin, China).
- Separation and purification of natural ginsenosides (GM1)
Fig. 1 shows a schematic diagram of all of the steps used for the GM1, GM2, GM3, and GM4 formula. Dried ginseng (1 kg) was extracted and concentrated with ethanol (EtOH, 70%, w/v) for 3 h, and the final solid (100 g) was obtained after being extracted twice from the EtOH solution (90%) at 60oC for 3 h. After dissolving this solid in water, the extract was subsequently adsorbed by being passed through a column packed with synthetic adsorption resin (HP-20, Diaon Co.). The ginsenoside mixture (GM1, 30 g, ≥ 90% purity) was obtained by desorbing with EtOH (95%). The resultant GM1 contained Rb1, Rb2, Rc, Rd, Re, Rg1, and traces of other compounds. Detailed ingredients and pathways for each compound are displayed in Fig. 2. These pathways are composed of GM1 (Rb1, Rd, Re, and Rg1), GM2 (Rg3 and Rh1), GM3 (F1 and F2), and GM4 (PPT and Rh2).
- Preparation of Rg3 and Rh1 (GM2)
GM1 (10 g) was reacted with 1 L of 5% acetic acid (AcOH) at 80oC for 12 h. A mixture (6 g) containing
∆Rg3, Rg3 (structural isomers, Rk1+Rg5), Rh1, and ∆Rh1 (structural isomers, Rk3+Rh4) was obtained after removing major impurities, chemical catalyst, and non-saponin com- pounds from the reaction mixture. The extract was then passed through a column packed with synthetic adsorption resin (HP-20, Diaion Co.), desorbed with EtOH (95%), and subsequently evaporated.
- Preparation of F2 and F1 mixture (GM3)
GM1 (10 g) was reacted in 1 L of sodium acetate-acetic acid buffer solution (50 mM, pH 4.5) with β-glycosidase powder (1 g), which was purified from Aspergillus niger (at 45oC , pH 4.5) for more than 2 days [23,24]. Next, a mixture (6.2 g) containing the ginsenosides F1 and F2 was obtained following the procedures described above.
- Preparation of Rh2 and PPT mixture (GM4) GM3 (5 g) was reacted with 500 mL of AcOH solution (10%) at 80oC for 12 A mixture (3.5 mg/g) containing Rh2, PPT, and ∆PPT were obtained following the procedures described above.
Fig. 1. Chemical structures of ginsenosides, (A) PPD type and (B) PPT type. Note: Glu β–D-glucopyra-Nosyl, Arap α-L-arabinopyrano- Syl, Araf α-L-arabinofuranosyl, Xyl β-D-xylopyranosyl, and Rha A-L-rhamnopyranosyl.
Fig. 2. Transformation steps for the production of Rg3, Rh2, and Rh1. Note: Glu and Rha indicate β–D-glucopyranosyl and α-L- rhamnopyranosyl, respectively.
- Separation of Rg3 from GM2
The supernatant and precipitate were separated by centrifugation (× 6,000 g, 15 min) after GM2 (1 g) was dissolved in MeOH (20 mL). Next, EA (75 mL) and standard Rg3 (10 mg) were added to the supernatant. After being placing in the above precipitate at 4oC for 20 h, the solid to be filtered was mixed with the precipitate and dried in an oven. The resulting white powder weighed approximately 192 mg (75% yield, 83% purity).
- Separation of Rh2 from GM4
After dissolving GM4 (1 g) in MeOH (10 mL), EA (43 mL) was poured into the tube. Subsequently, standard Rh2 (10 mg) was added. After incubation at 4oC for 17 h, the solid to be filtered was dried in a vacuum oven. The resulting white powder was approximately 165 mg (75% yield, 88% purity).
- Mice and tumors
Specific pathogen-free female BALB/c-nu/nu mice (nude mice), which were 6 ~ 8 weeks old, were used for all human tumor xenograft experiments and were obtained from SLC Japan Inc. The mice were housed in a pathogen-free barrier facility where ambient light was automatically controlled to produce 12-h light and dark cycles. The human tumor cell line NCI-H23 (adenocarcinoma; non-small cell lung cancer) was obtained from the Korea Research Institute of Biotechnology (KRIBB). The human cell line NCI-H23 was implanted in nude mice at concentrations of 9 × 106 cells/mouse.
- Evaluation of activity in human tumor models
To evaluate the effects of single Rg3, Rh2, and Rg3:Rh2 on in vivo tumor growth, NCI-H23 (9 × 106 cells/mouse) was injected s.c. (subcutaneously) at day 0 into the right flank of nude mice. Drug treatment was initiated on day 1 after tumor transplantation. Rg3 or Rh2 was administered
p.o. (oral administration, per os) every day at a dose of 3 mg/kg on days 1-19 or 1-33. Tumor volume was measured at days 8, 10, 13, 16, and 19 or 12, 17, 23, 27, and 33. Rg3:Rh2 was administered p.o. every day at a dose of 3 mg/kg on days 1-23. Tumor volume was measured at days 12, 13, 15, 17, 20, 21, and 24. Tumor volume was estimated using a two-dimensional caliper and the following formula for an ellipsoid :
Tumor volume = (L × W × H) / 2
where L is the major axis, W is the width, and H is the height of the tumor. Tumor growth inhibition was analyzed using Student’s t-test for statistical significance.
Analyses of ginsenosides were carried out on an HPLC
system (Waters, USA) using a UV detector set to record at a wavelength of 203 nm (Waters 2487 DAD), a pump (Waters 1525), a C18 column (Chromolith Performance, RP-18e, 100-4.6 mm, Merck, Germany), and two solvents (water:acetonitrile, 10:90 and 80:20, v/v) for the mobile phase. Linear regressions of ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2, Compound K, and PPT etc.) were plotted to quantify each ginsenoside. The 1H NMR (400 MHz) and 13C-NMR (100 MHz) techniques (Varian, model UNITY 300) were used to characterize the molecular structures of ginsenosides in C5D5N using tetramethylsilane (TMS) as an internal standard.
2. Results and Discussions
- Purification condition and characterization for GM1, GM2, and GM4
Fig. 3 shows a high-resolution chromatogram of a mixture of ginsenosides with well-separated peaks for 25 pure species. Detailed results are shown in Table 1. The structures of the single ginsenosides was confirmed using standard substances. For mixtures of ginsenosides, GM1 primarily contained protopanaxadiol group-Rb1 (319 mg/g), but also contained protopanaxatriol group-Rg1 (351 mg/g), a small amount of Rd (86 mg/g), Re (52 mg/g), and a minor amount of Rc (7 mg/g) and Rb2 (15 mg/g) (Fig. 4A). GM2 produced from GM1 contained Rh1 (173 mg/g), which was converted from Rg1 and Re, and Rg3 (241 mg/g), which was converted from Rb1 and Rd. The other peaks corresponded to ∆Rg3 and ∆Rh1, which are structural isomers that have a similar structure as Rh1 and Rg3, respectively (Fig. 4B). Fig. 4C shows GM3, which is another mixture of ginsenosides produced from GM1 by the enzymatic reaction. It was confirmed that F1 (161 mg/g) was converted from Rg1 and Re, and F2 (305 mg/g) was converted from Rb1 and Rd (Fig. 2). GM4 was produced from GM3 by the AcOH reaction and F1 and F2 were converted to PPT (63 mg/g) and Rh2 (218 mg/g), respectively (Figs. 2 and 4D).
- GM3 (F1 and F2) produced by enzymatic reaction The structures of F1 and F2, which are ginsenoside metabolites formed by the β-glycosidase purified from Aspergillus niger and GM1 at step 3 (Fig. 2), were confirmed by 1H-NMR, 13C-NMR, and HPLC data. Although the methods for preparing F2 have been reported previously, it is difficult to fully convert to F2 and prevent the reaction from proceeding past this conversion step . Thus, an enzymatic approach for efficiently producing F2 is an attractive F1 and F2 were initially produced by β-glycosidase with higher yields and their molecular
structures were confirmed (F1: 1H-NMR δ: 5.16(1H, d, J = 7.67Hz, 20-glu.), 5.21(1H, t, J = 6.34Hz, H-24). 13C-NMR: Table 4, and F2: 1H-NMR δ: 4.92(1H, d, J = 7.02Hz, 3-glu.), 5.17(1H, d, J = 7.27Hz, 20-glu.). 13C-NMR: Table 2).
F2 is an important intermediate for producing Rh2 in high yields rather than other substances that display pharmacological effects. Although enzymes could produce Rh2 by a one-step reaction from protopanaxadiol-type ginsenosides, such as Rb1 and Rd, it is more favorable to produce Rh2 from F2 when comparing the final yields of Rh2 by the
two methods. This finding is related to the fact that the pathway for the production of compound-K is more downregulated than the Rh2 pathway when F2 is additionally hydrolyzed [26,27] (Fig. 2). Metabolites of Rb1 and Rd were divided into Rg3 and F2, depending on the location of the hydrolysis of the sugar moieties of protopanaxadiol ginsenosides (GM1). Thus, this enzymatic technology produced Rh2 from F2 with a higher yield compared to the
yield produced from Rg3 . To examine the anticancer effects of these compounds, the chromatograms of Rg3 and Rh2 were confirmed (Figs. 4E and 4F).
3.3 Rg3 and Rh2 from GM2 and GM4
Synthetic Resin (HP-20) is usually used for separating ginsenosides from ginseng extracts. All ginsenosides have notably different properties depending on the distinction
between protopanaxadiol and protopanaxatriol in types and numbers of sugar moieties. Single ginsenosides can be separated based on their solubility in different solvents. Due to the similar properties of ginsenosides and their low content in extracts, it is difficult to confirm the structures of single ginsenosides. Therefore, different solvent systems and HPLC methods to effectively separate and purify the various ginsenosides, including F1, F, Rg3, Rh2, and Rb1,
should be established (Tables 3 and 4). After separating and purifying with the proper solvent systems, the structures of the ginsenosides were confirmed by 1H and 13C-NMR spectroscopic data.
- Antitumor activity of Rg3 and Rg3:Rh2 for human tumor xenograft models
Nineteen days (Rg3), 33 days (Rh2), and 23 days (Rg3:Rh2)
Although the many biological effects of various ginsenosides, such as Rb1, Rg3, Rh2, and compound-K, have been examined, there is no commercial product of single or combined ginsenosides that has been developed for use as a therapeutic agent and its use is limited. This is the case because of the difficulty in preparing a single ingredient, i.e., dried ginseng is comprised of approximately 2% of the ginsenosides, which are divided into various species, and the negative opinions of the efficacy of ginsenosides.
Despite the large number of studies that have been conducted on a variety of ginsenosides, sufficient pharma- cological activity has not been demonstrated with a single ginsenoside. Thus, the approach described here may provide evidence of the efficacy of ginensoside-based medicines. Among the ginsenosides with protopanaxadiol and protopanaxatriol groups, the very low levels of Rg3 and Rh2 in ginseng or red ginseng, which have various pharmacological activities, are desired for efficient mass- production. However, this calibration process for precursors of Rg3 and Rh2 with high yield would require adjusting several variables, such as the acidity or enzyme, reaction temperature, and time.
In regards to Rh2 production, which has been shown to produce anticancer effects, β-glycosidase purified from Aspergillus niger was shown to effectively produce ginse- noside F2 at a good yield (F2: 305 mg/g). This process fully converted the compounds to F2 and prevented the reaction from proceeding further. Ginsenosides F1 and F2 produced by β-glycosidase with high yield and the
Fig. 5. Growth curves of human tumor xenograft treated with Rg3 (A), Rh2 (B), and Rg3:Rh2 (C). Note: Human NCI-H23 lung cells were implanted s.c. on day 0. Rg3 or Rh2 was administered p.o. every day at 3 mg/kg on days 1-19 or 1-33. Tumor volume was measured at days 8, 10, 13, 16, and 19. Rg3:Rh2 was administered
p.o. every day at 3 mg/kg on days 1-23. Tumor volume was measured days 12, 13, 15, 17, 20, 21, and 24. Vehicle control (treated with distilled water) and Rg3, Rh2, and Rg3:Rh2 indicate symbols (□) and (■), respectively.
production of Rh2 from F2 are more favorable because the pathway from F2 to Rh2 was predominant as opposed to the pathway creating compound-K [26,27].
Since the anticancer effect of a single ginsenoside was lower than that obtained using synthetic drugs, treatment with a combination of ginsenosides was examined. Rg3:Rh2 (1:1, w/w) was shown to inhibit specific lung cancer line (NCI-H23) in vivo at a low dose when compared to the pharmacological effect of single treatments. Interestingly,
the inhibitory effect of these compounds against cancer was in the following order: Rg3 < (Rg3: Rh2 = 1:1, w/w) when administrated at an equivalent dose; however, it is possible that other combinations of ginsenosides would be more effective.
This work was supported by the Advanced Biomass R&D Center (ABC) of Korea Grant funded by the Ministry of Education, Science and Technology (ABC-2010-0029728).
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