Minor Ginsenosides Research Articles

Biotransformation of Ginsenosides Re and Rg1 into Rg2 and Rh1 by Thermostable β-Glucosidase from Thermotoga thermarum

The recombinant thermostable β-glucosidase from Thermotoga thermarum DSM 5069T exhibited high selectivity to catalyze the conversion of ginsenoside Re and Rg1 to the more pharmacologically active minor ginsenoside Rg2 and Rh1, respectively. At a concentration of 1.36 U/mL of the enzyme, a temperature of 85°C, and pH 5.5, 10 g/L ginsenoside Re was transformed into 8.02 g/L Rg2 within 60 min, and 2 g/L ginsenoside Rg1 was transformed into 1.56 g/L Rh1 within 60 min. This paper provides the first report on the production of ginsenoside Rg2 and Rh1 by a highly thermostable β-glucosidase.

A literature update elucidating production of Panax ginsenosides with a special focus on strategies enriching the anti-neoplastic minor ginsenosides in ginseng preparations.

Ginseng, an oriental gift to the world of healthcare and preventive medicine, is among the top ten medicinal herbs globally. The constitutive triterpene saponins, ginsenosides, or panaxosides are attributed to ginseng's miraculous efficacy towards anti-aging, rejuvenating, and immune-potentiating benefits. The major ginsenosides such as Rb1, Rb2, Rc, Rd., Re, and Rg1, formed after extensive glycosylations of the aglycone "dammaranediol," dominate the chemical profile of this genus in vivo and in vitro. Elicitations have successfully led to appreciable enhancements in the production of these major ginsenosides. However, current research on ginseng biotechnology has been focusing on the enrichment or production of the minor ginsenosides (the less glycosylated precursors of the major ginsenosides) in ginseng preparations, which are either absent or are produced in very low amounts in nature or via cell cultures. The minor ginsenosides under current scientific scrutiny include diol ginsenosides such as Rg3, Rh2, compound K, and triol ginsenosides Rg2 and Rh1, which are being touted as the next "anti-neoplastic pharmacophores," with better bioavailability and potency as compared to the major ginsenosides. This review aims at describing the strategies for ginsenoside production with special attention towards production of the minor ginsenosides from the major ginsenosides via microbial biotransformation, elicitations, and from heterologous expression systems.

Bioconversion Using Lactic Acid Bacteria: Ginsenosides, GABA, and Phenolic Compounds.

Lactic acid bacteria (LAB) are used as fermentation starters in vegetable and dairy products and influence the pH and flavors of foods. For many centuries, LAB have been used to manufacture fermented foods; therefore, they are generally regarded as safe. LAB produce various substances, such as lactic acid, β-glucosidase, and β-galactosidase, making them useful as fermentation starters. Existing functional substances have been assessed as fermentation substrates for better component bioavailability or other functions. Representative materials that were bioconverted using LAB have been reported and include minor ginsenosides, γ-aminobutyric acid, equol, aglycones, bioactive isoflavones, genistein, and daidzein, among others. Fermentation mainly involves polyphenol and polysaccharide substrates and is conducted using bacterial strains such as Streptococcus thermophilus, Lactobacillus plantarum, and Bifidobacterium sp. In this review, we summarize recent studies of bioconversion using LAB and discuss future directions for this field.

Preparation of ginseng extract with enhanced levels of ginsenosides Rg1 and Rb1 using high hydrostatic pressure and polysaccharide hydrolases

Background:Ginsenosides are the principal components responsible for the pharmacological activities of ginseng. Ginsenosides Rg1 and Rb1 are the major compounds recognized as marker substances for quality control of ginseng-based products. These major compounds can be transformed to several pharmacologically active minor ginsenosides by chemical, microbial, and enzymatic means.Materials and Methods:In the present study, a combination of polysaccharide hydrolases and high hydrostatic pressure (HHP) were used to extract ginseng saponins enriched with ginsenosides Rg1 and Rb1. Temperature, pH, time, ginseng-to-water ratio, and pressure were optimized to obtain the maximum amount of Rg1 and Rb1 in the resulting extract using commercial polysaccharide hydrolases.Results:This study showed that treatment with a combination of cellulase, amylase, and pectinase at 100 MPa pressure, pH 4.8, and 45°C for 12 h resulted in higher Rg1 and Rb1 levels in the extract.Conclusion:This study describes a cheap and ecofriendly method for preparing ginseng extract enriched with Rg1 and Rb1.SUMMARY Ginsenosides are the principal bioactive components present in ginsengGinsenosides Rg1 and Rb1 are the most abundant compounds in ginsengHigh hydrostatic pressure (HHP) and Polysaccharide hydrolases (PH) were combined to extract ginseng saponins enriched with Rg1 and Rb1Extraction conditions were optimized to obtain the maximum amount of Rg1 and Rb1Extraction with a combination of cellulase, amylase, and pectinase at 100 MPa pressure at pH 4.8, and 45°C for 12 h resulted in higher levels of Rg1 and Rb1 in the ginseng extract Abbreviations used: ATCC: American Type Culture Collection, Mpa: Mega Pascal

Complete genome sequencing of Arachidicoccus ginsenosidimutans sp. nov., and its application for production of minor ginsenosides by finding a novel ginsenoside-transforming β-glucosidase

A novel bacterial strain (BS20T), which has ginsenoside-transforming ability, was whole genome sequenced for the identification of a target gene.

산양삼의 품질특성 및 항산화 활성에 미치는 영향

In this study, we investigated the nutritional and functional constituents as well as quality characteristics and antioxidant activity of Korean cultivated wild ginseng (KG). The chemical compositions and amino acid content of KG were 7.56% water, 73.01% carbohydrates, 12.58% protein, 1.99% lipids, and 5.54% ash as well as 16.17 mg/g of amino acids, respectively. The major ginsenoside and minor ginsenoside contents of KG were 15.94 mg/g and 0.04 mg/g, respectively. The total polyphenol and flavonoid contents of KGE (Korean cultivated wild ginseng with 70% ethanol extract) were 8.93 mg GAE/g and 3.96 mg RHE/g, respectively. KGE also showed higher antioxidant activity than the other extracts (KGW, Korean cultivated wild ginseng with water extract) with regard to DPPH and ABTS radical scavenging activities (57.75% and 70.73%, respectively), nitrite oxide scavenging activity (44.01%), SOD-like activity (78.05%), reducing power activity (1.08 OD 700 nm ), and ferrous ion-chelating activity (65.33%). Additionally, KGE had higher elastase, collagenase, and tyrosinase inhibition activities than KGW. These results suggest that KGE can be used as a bioactive and functional material in the food industry.

Effect of hydrothermal processing on ginseng extract

BackgroundPanax ginseng Meyer is cultivated because of its medicinal effects on the immune system, blood pressure, and cancer. Major ginsenosides in fresh ginseng are converted to minor ginsenosides by structural changes such as hydrolysis and dehydration. The transformed ginsenosides are generally more bioavailable and bioactive than the primary ginsenosides. Therefore, in this study, hydrothermal processing was applied to ginseng preparation to increase the yields of the transformed ginsenosides, such as 20(S)-Rg3, Rk1, and Rg5, and enhance antioxidant activities in an effective way. MethodsGinseng extract was hydrothermally processed using batch reactors at 100–160°C with differing reaction times. Quantitative analysis of the ginsenoside yields was performed using HPLC, and the antioxidant activity was qualitatively analyzed by evaluating 2,2'-azino-bis radical cation scavenging, 2,2-diphenyl-1-picrylhydrazyl radical scavenging, and phenolic antioxidants. Red ginseng and sun ginseng were prepared by conventional steaming as the control group. ResultsUnlike steaming, the hydrothermal process was performed under homogeneous conditions. Chemical reaction, heat transfer, and mass transfer are generally more efficient in homogeneous reactions. Therefore, maximum yields for the hydrothermal process were 2.5–25 times higher than those for steaming, and the antioxidant activities showed 1.6–4-fold increases for the hydrothermal process. Moreover, the reaction time was decreased from 3 h to 15–35 min using hydrothermal processing. ConclusionTherefore, hydrothermal processing offers significant improvements over the conventional steaming process. In particular, at temperatures over 140°C, high yields of the transformed ginsenosides and increased antioxidant activities were obtained in tens of minutes.

Coalescence of functional gold and monodisperse silver nanoparticles mediated by black Panax ginseng Meyer root extract.

A rapid biological synthesis of multifunctional gold nanoparticle (AuNp) and monodisperse silver nanoparticle (AgNp) was achieved by an aqueous extract of black Panax ginseng Meyer root. The physicochemical transformation into black ginseng (BG) greatly enhanced the pharmacological activities of white ginseng and its minor ginsenoside content. The optimal temperature conditions and kinetics of bioreduction were investigated. Formation of BG-AuNps and BG-AgNps was verified by ultraviolet–visible spectrophotometry at 548 and 412 nm, respectively. The biosynthesized BG-AgNps were spherical and monodisperse with narrow distribution, while BG-AuNps were icosahedral-shaped and moderately polydisperse. Synthesized nanoparticles exhibited long-term stability in buffers of pH 7.0–8.0 and biological media (5% bovine serum albumin) at an ambient temperature and at 37°C. BG-AgNps showed effective antibacterial activity against Escherichia coli and Staphylococcus aureus. BG-AuNps and BG-AgNps demonstrated increased scavenging activity against 2,2-diphenyl-1-picrylhydrazyl free radicals. In addition, BG-AuNps and BG-AgNps were nontoxic to HaCaT and MCF-7 cells; the latter showed no cytotoxicity at concentrations lower than 10 µg/mL. At higher concentrations, BG-AgNps exhibited apparent apoptotic activity in MCF-7 breast cancer cell line through reactive oxygen species generation and nuclear fragmentation.

Purification and characterization of a novel ginsenoside Rc-hydrolyzing β-glucosidase from Armillaria mellea mycelia.

Ginsenosides are the principal compounds responsible for the pharmacological effects and health benefits of Panax ginseng root. Among protopanaxadiol (PPD)-type ginsenosides, minor ginsenosides such as ginsenoside (G)-F2, G-Rh2, compound (C)-Mc1, C-Mc, C-O, C-Y, and C-K are known to be more pharmacologically active constituents than major ginsenosides such as G-Rb1, G-Rb2, G-Rc, and G-Rd. A novel ginsenoside Rc-hydrolyzing β-glucosidase (BG-1) from Armillaria mellea mycelia was purified as a single protein band with molecular weight of 121.5 kDa on SDS-PAGE and a specific activity of 17.9 U mg−1 protein. BG-1 concurrently hydrolyzed α-(1 → 6)-arabinofuranosidic linkage at the C-20 site or outer β-(1 → 2)-glucosidic linkage at the C-3 site of G-Rc to produce G-Rd and C-Mc1, respectively. The enzyme also hydrolyzed outer and inner glucosidic linkages at the C-3 site of G-Rd to produce C-K via G-F2, and inner glucosidic linkage at the C-3 site of C-Mc1 to produce C-Mc. C-Mc was also slowly hydrolyzed α-(1 → 6)-arabinofuranosidic linkage at the C-20 site to produce C-K with reaction time prolongation. Finally, the pathways for formation of C-Mc and C-K from G-Rc by BG-1 were G-Rc → C-Mc1 → C-Mc and G-Rc → G-Rd → G-F2 → C-K, respectively. The optimum reaction conditions for C-Mc and C-K formation from G-Rc by BG-1 were pH 4.0–4.5, temperature 45–60 °C, and reaction time 72–96 h. This is the first report of efficient production of minor ginsenosides, C-Mc and C-K from G-Rc by β-glucosidase purified from A. mellea mycelia.

Evaluation of ginsenoside bioconversion of lactic acid bacteria isolated from kimchi

BackgroundPanax ginseng is a physiologically active plant widely used in traditional medicine that is characterized by the presence of ginsenosides. Rb1, a major ginsenoside, is used as the starting material for producing ginsenoside derivatives with enhanced pharmaceutical potentials through chemical, enzymatic, or microbial transformation. MethodsTo investigate the bioconversion of ginsenoside Rb1, we prepared kimchi originated bacterial strains Leuconostoc mensenteroides WiKim19, Pediococcus pentosaceus WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49 and analyzed bioconversion products using LC-MS/MS mass spectrometer. ResultsL. mesenteroides WiKim19 and Pediococcus pentosaceus WiKim20 converted ginsenoside Rb1 into the ginsenoside Rg3 approximately five times more than Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49. L mesenteroides WIKim19 showed positive correlation with β-glucosidase activity and higher transformation ability of ginsenoside Rb1 into Rg3 than the other strains whereas, P. pentosaceus WiKim20 showed an elevated production of Rb3 even with lack of β-glucosidase activity but have the highest acidity among the five lactic acid bacteria (LAB). ConclusionGinsenoside Rg5 concentration of five LABs have ranged from ∼2.6 μg/mL to 6.5 μg/mL and increased in accordance with the incubation periods. Our results indicate that the enzymatic activity along with acidic condition contribute to the production of minor ginsenoside from lactic acid bacteria.

Fermentative transformation of ginsenoside Rb1 from Panax ginseng C. A. Meyer to Rg3 and Rh2 by Lactobacillus paracasei subsp. tolerans MJM60396

Lactic acid bacteria (LAB) were screened for ginsenoside transforming activity using crude ginseng extract. Thin-layer chromatography analysis of fermented ginseng extract showed that LAB strain MJM60396 possessed higher ginsenoside transformation ability than other strains. It converted major ginsenosides into minor ginsenosides such as Rg3 and Rh2. MJM60396 also showed high β-glucosidase activity. Strain MJM60396 was identified as Lactobacillus paracasei subsp. tolerans based on 16S rRNA gene sequence. To delineate the pathway involved in the production of the minor ginsenosides Rg3 and Rh2, strain MJM60396 was incubated with pure ginsenoside Rb1. HPLC analysis revealed the appearance of Rg3 and Rh2 peak from the incubation mixture containing Rb1 and strain MJM60396. Furthermore, β-glucosidase enzyme was prepared from strain MJM60396. To achieve its maximum activity, we optimized the pH and temperature conditions. Cell-free β-glucosidase enzyme hydrolyzed ginsenoside Rb1 through the following pathway: ginsenoside Rb1 → Rd → Rg3 → Rh2. This is the first report on the transformation of ginsenosides Rb1 to Rg3 and Rh2 by a Lac. paracasei subsp. tolerans strain. Our results indicate that Lac. paracasei subsp. tolerans MJM60396 has the potential to be used for preparing ginsenosides Rg3 and Rh2 as nutraceuticals.

Fermented Ginseng Contains an Agonist of Peroxisome Proliferator Activated Receptors α and γ.

Peroxisome proliferator activated receptor (PPAR) is a nuclear receptor that is one of the transcription factors regulating lipid and glucose metabolism. Fermented ginseng (FG) is a ginseng fermented by Lactobacillus paracasei A221 containing minor ginsenosides and metabolites of fermentation. DNA microarray analysis of rat liver treated with FG indicated that FG affects on lipid metabolism are mediated by PPAR-α. To identify a PPAR-α agonist in FG, PPAR-α transcription reporter assay-guided fractionation was performed. The fraction obtained from the MeOH extract of FG, which showed potent transcription activity of PPAR-α, was fractionated by silica gel column chromatography into 16 subfractions, and further separation and crystallization gave compound 1 together with four known constituents of ginseng, including 20(R)- and 20(S)-protopanaxadiol, and 20(R)- and 20(S)-ginsenoside Rh1. The structure of compound 1 was identified as 10-hydroxy-octadecanoic acid by (1)H- and (13)C-NMR spectra and by EI-MS analysis of the methyl ester of 1. Compound 1 demonstrated much higher transcription activity of PPAR-α than the other isolated compounds. In addition, compound 1 also showed 5.5-fold higher transcription activity of PPAR-γ than vehicle at the dose of 20 μg/mL. In the present study, we identified 10-hydroxy-octadecanoic acid as a dual PPAR-α/γ agonist in FG. Our study suggested that metabolites of fermentation, in addition to ginsenosides, contribute to the health benefits of FG.

Biotransformation of ginsenoside Rb1 to ginsenoside C-K by endophytic fungus Arthrinium sp. GE 17-18 isolated from Panax ginseng.

This research aimed to isolate β-glycosidase-producing endophytic fungus in Panax ginseng to achieve biotransformation of ginsenoside Rb1 to ginsenoside C-K. Of these 15 β-glucosidase-producing endophytic fungus isolated from ginseng roots, a β-glucosidase-producing endophytic fungi GE 17-18 could hydrolyse major ginsenosides Rb1 to minor ginsenoside C-K with metabolic pathways: ginsenoside Rb1→ginsenoside Rd→ginsenoside F2→ginsenoside C-K. Phylogenetic analysis of ITS gene sequences indicated that the strain GE 17-18 belongs to the genus Arthrinium and is most closely related to Arthrinium sp. HQ832803.1. This is the first study to provide information of cultivable β-glycosidase-producing Endophytic fungus in Panax ginseng. The strain GE 17-18 has potential to be applied on the preparation for minor ginsenoside C-K in pharmaceutical industry.

Tartaric acid induced conversion of protopanaxadiol to ginsenosides Rg3 and Rg5 and their in situ recoveries by integrated expanded bed adsorption chromatography.

Panax ginseng has been applied in traditional Chinese medicine for over 2000 years. It is still one of the most popular herbs in recent decades. The prescribed ginseng-containing medicines consist of protopanaxadiol and protopanaxatriol ginsenosides, which are the major constituents of the herb. Minor ginsenosides at low levels in the herb, such as Rg3 and Rg5 , have attracted more rising attention than the major ones. The existing approaches to prepare Rg3 and Rg5 usually rely on either steamed red ginseng as the source or chemical/enzymatic conversion of protopanaxadiol to the targets. It is still highly desirable to effectively achieve such minor components. In this paper, a method integrated extraction of protopanaxadiol and conversion of it to Rg3 and Rg5 has been proposed. Protopanaxadiol was extracted and simultaneously converted to Rg3 and Rg5 by d,l-tartaric acid. The targets were absorbed by resins on expanded bed adsorption chromatography and were then separated from other ginsenosides in different stages. Compared with conventional methods, the developed process has advantages in shortening time consumption and improving the conversion ratio of protopanaxadiol, which is promising in directly achieving Rg3 and Rg5 from P. ginseng.

Viscozyme® L를 이용한 인삼유래 사포닌으로부터 Minor Ginsenoside F2의 생산

Viscozyme® L를 이용한 인삼유래 사포닌으로부터 Minor Ginsenoside F2의 생산

MRM-based strategy for the homolog-focused detection of minor ginsenosides from notoginseng total saponins by ultra-performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry

A validated MRM-based strategy was established for targeted detection of minor ginsenosides from NGTS by using a LC-Q-Trap/MS.

Microbial conversion of major ginsenosides in ginseng total saponins by Platycodon grandiflorum endophytes

BackgroundIn this study, we screened and identified an endophyte JG09 having strong biocatalytic activity for ginsenosides from Platycodon grandiflorum, converted ginseng total saponins and ginsenoside monomers, determined the source of minor ginsenosides and the transformation pathways, and calculated the maximum production of minor ginsenosides for the conversion of ginsenoside Rb1 to assess the transformation activity of endophyte JG09. MethodsThe transformation of ginseng total saponins and ginsenoside monomers Rb1, Rb2, Rc, Rd, Rg1 into minor ginsenosides F2, C-K and Rh1 using endophyte JG09 isolated by an organizational separation method and Esculin-R2A agar assay, as well as the identification of transformed products via TLC and HPLC, were evaluated. Endophyte JG09 was identified through DNA sequencing and phylogenetic analysis. ResultsA total of 32 β-glucosidase-producing endophytes were screened out among the isolated 69 endophytes from P. grandiflorum. An endophyte bacteria JG09 identified as Luteibacter sp. effectively converted protopanaxadiol-type ginsenosides Rb1, Rb2, Rc, Rd into minor ginsenosides F2 and C-K, and converted protopanaxatriol-type ginsenoside Rg1 into minor ginsenoside Rh1. The transformation pathways of major ginsenosides by endophyte JG09 were as follows: Rb1→Rd→F2→C-K; Rb2→C-O→C-Y→C-K; Rc→C-Mc1→C-Mc→C-K; Rg1→Rh1. The maximum production rate of ginsenosides F2 and C-K reached 94.53% and 66.34%, respectively. ConclusionThis is the first report about conversion of major ginsenosides into minor ginsenosides by fermentation with P. grandiflorum endophytes. The results of the study indicate endophyte JG09 would be a potential microbial source for obtaining minor ginsenosides.

Identification of mountain-cultivated ginseng and cultivated ginseng using UPLC/oa-TOF MSE with a multivariate statistical sample-profiling strategy

BackgroundMountain-cultivated ginseng (MCG) and cultivated ginseng (CG) both belong to Panax ginseng and have similar ingredients. However, their pharmacological activities are different due to their significantly different growth environments. MethodsAn ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS)-based approach was developed to distinguish MCG and CG. Multivariate statistical methods, such as principal component analysis and supervised orthogonal partial-least-squares discrimination analysis were used to select the influential components. ResultsUnder optimized UPLC-QTOF-MS/MS conditions, 40 ginsenosides in both MCG and CG were unambiguously identified and tentatively assigned. The results showed that the characteristic components of CG and MCG included ginsenoside Ra3/isomer, gypenoside XVII, quinquenoside R1, ginsenoside Ra7, notoginsenoside Fe, ginsenoside Ra2, ginsenoside Rs6/Rs7, malonyl ginsenoside Rc, malonyl ginsenoside Rb1, malonyl ginsenoside Rb2, palmitoleic acid, and ethyl linoleate. The malony ginsenosides are abundant in CG, but higher levels of the minor ginsenosides were detected in MCG. ConclusionThis is the first time that the differences between CG and MCG have been observed systematically at the chemical level. Our results suggested that using the identified characteristic components as chemical markers to identify different ginseng products is effective and viable.

A Distinctive Pattern of Beauveria bassiana-biotransformed Ginsenoside Products Triggers Mitochondria/FasL-mediated Apoptosis in Colon Cancer Cells.

Ginseng is one of the most commonly used adaptogens. Transformation into the minor ginsenosides produces compounds with more effective action. Beauveria bassiana, a teleomorph of Cordyceps bassiana, is a highly efficient producer of mammalian steroids and produces large amounts of sugar-utilizing enzymes. However, the fermentation of steroid glycosides in ginseng with B. bassiana has never been studied. Thus, we evaluated the bioconversion of the major ginsenosides in white ginseng by B. bassiana. Interestingly, B. bassiana increased the total amount of protopanaxadiols and hydrolyzed Rb1 into minor ginsenosides, exhibiting high levels of Rd and Rg3, as well as moderate levels of Rb2 and Rc analyzed by high-performance liquid chromatography coupled with evaporative light-scattering detection. The β-glucosidase activity was highly increased, which led to the selective elimination of sugar moiety at the 20-C position of Rb1 to Rd, followed by Rg3. Rb2 and Rc accumulated because of the minimal activities of α-L-arabinopyranosidase and α-L-arabinofuranosidase, respectively. The fermentation product exerted dose-dependent cytotoxicity in HCT-15 cells, which are resistant to ginseng. The product, but not white ginseng, exhibited apoptotic effects via the Fas ligand and caspase 8/9. This study demonstrates for the first time that the B. bassiana-fermented metabolites have potent apoptotic activity in colon cancer cells, linking to a therapeutic use.

Production of Ginsenoside F2 by Using Lactococcus lactis with Enhanced Expression of β-Glucosidase Gene from Paenibacillus mucilaginosus.

This study aimed to produce a pharmacologically active minor ginsenoside F2 from the major ginsenosides Rb1 and Rd by using a recombinant Lactococcus lactis strain expressing a heterologous β-glucosidase gene. The nucleotide sequence of the gene (BglPm) was derived from Paenibacillus mucilaginosus and synthesized after codon optimization, and the two genes (unoptimized and optimized) were expressed in L. lactis NZ9000. Codon optimization resulted in reduction of unfavorable codons by 50% and a considerable increase in the expression levels (total activities) of β-glucosidases (0.002 unit/mL, unoptimized; 0.022 unit/mL, optimized). The molecular weight of the enzyme was 52 kDa, and the purified forms of the enzymes could successfully convert Rb1 and Rd into F2. The permeabilized L. lactis expressing BglPm resulted in a high conversion yield (74%) of F2 from the ginseng extract. Utilization of this microbial cell to produce F2 may provide an alternative method to increase the health benefits of Panax ginseng.