• Journal of Ginseng Research
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• v.46 no.4
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• pp.505-514
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• 2022

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

• Journal of Ginseng Research
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• v.21 no.2
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• pp.125-132
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• 1997

Our previous study showed that in vivo treatment of spontaneously hypertensive rats (SHR) with protopanaxatriol ginsenosides (PPT) reduces the blood pressure and inhibits the con- tractions induced by endothelium-derived contracting factor (prostaglandin endoperoxide ($PGH_2$) and superoxide anion) in aorta isolated from SHR. The aim of the present study is to examine whether PPT improves endothelial functions in the isolated thoracic aorta of SHR in vitro. Treatments of aortic rings with PPT, purified ginsenoside $Rg_1$ ($Rg_1$) or indomethacin normalized endotheliuln-dependent relaxation to acetylcholine, but not with protopanaxadiol ginsenosides (PPD) and purified ginsenoside Rb1 (Rb1). The effects of PPT were dose-dependent. PGH,- and oxygen free radical-inducted contractions in rat aorta without endothelium were inhibited by PPT or $Rg_1$, but not by PPD or $Rb_1$. Contractions induced by PGF2$\alpha$, U-46619, a stable thromboxane A2 agonist or KCI (60 mM) were not inhibited by PPT, $Rg_1$ or $Rb_1$. These findings demonstrate that PPT but not PPD scavenges the oxygen-derived free radicals and/or antagonize the effects of $PGH_2$ in the vascular smooth muscle and may explain the hypotensive effect of ginseng in the SHR.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.2
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• pp.113-118
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• 2012

Ginsenosides are low molecular weight glycosides found in ginseng that exhibit neuroprotective effects through inhibition of $N$-methyl-D-aspartic acid (NMDA) receptor channel activity. Ginsenosides, like other natural compounds, are metabolized by gastric juices and intestinal microorganisms to produce ginsenoside metabolites. However, little is known about how ginsenoside metabolites regulate NMDA receptor channel activity. In the present study, we investigated the effects of ginsenoside metabolites, such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT), on oocytes that heterologously express the rat NMDA receptor. NMDA receptor-mediated ion current ($I_{NMDA}$) was measured using the 2-electrode voltage clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, PPT, but not CK or PPD, reversibly inhibited $I_{NMDA}$ in a concentration-dependent manner. The $IC_{50}$ for PPT on $I_{NMDA}$ was $48.1{\pm}4.6\;{\mu}M$, was non-competitive with NMDA, and was independent of the membrane holding potential. These results demonstrate the possibility that PPT interacts with the NMDA receptor, although not at the NMDA binding site, and that the inhibitory effects of PPT on $I_{NMDA}$ could be related to ginseng-mediated neuroprotection.

• Journal of Ginseng Research
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• v.38 no.1
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• pp.66-72
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• 2014

Panax ginseng is one of the most important medicinal plants in Asia. Triterpene saponins, known as ginsenosides, are the major pharmacological compounds in P. ginseng. The present study was conducted to evaluate the changes in ginsenoside composition according to the foliation stage of P. ginseng cultured in a hydroponic system. Among the three tested growth stages (closed, intermediate, and opened), the highest amount of total ginsenoside in the main and fine roots was in the intermediate stage. In the leaves, the highest amount of total ginsenoside was in the opened stage. The total ginsenoside content of the ginseng leaf was markedly increased in the transition from the closed to intermediate stage, and increased more slowly from the intermediate to opened leaf stage, suggesting active biosynthesis of ginsenosides in the leaf. Conversely, the total ginsenoside content of the main and fine roots decreased from the intermediate to opened leaf stage. This suggests movement of ginsenosides during foliation from the root to the leaf, or vice versa. The difference in the composition of ginsenosides between the leaf and root in each stage of foliation suggests that the ginsenoside profile is affected by foliation stage, and this profile differs in each organ of the plant. These results suggest that protopanaxadiol- and protopanaxatriol(PPT)-type ginsenosides are produced according to growth stage to meet different needs in the growth and defense of ginseng. The higher content of PPT-type ginsenosides in leaves could be related to the positive correlation between light and PPT-type ginsenosides.

• Natural Product Sciences
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• v.20 no.1
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• pp.58-64
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• 2014

In this study, edible protopanaxadiol saponin enriched fraction were prepared by ultrafiltration (UF). Ginseng extract was prepared from mixtures of ginseng main root and rootlet (root: rootlet = 4 : 6). UF system was used the four-piston Diaphragm pump equipped with 5 kDa pore size Hydrosart Cassette made by regenerated cellulose acetate (CA) or 3 kDa pore size Hollow Fiber cartridge made by polyethersulfone (PES). Total ginsenoside contents of concentrated fraction by UF system was found to higher, compared to before those of untreated method. Especially, processing of UF showed the increase of PPD-type ginsenoside, while PPT-type ginsenoside was gradually decreased by both 3 kDa and 5 kDa membrane. After removal of 80% water by the 5 kDa Hydrosart Cassette and by 3 kDa Hollow Fiber cartridge, ginsenoside Rb1 content was higher 37.2 mg/g and 25.3 mg/g than 20.8 mg/g in untreated process. The ratio of Rb1 to Rg1 (Rb1/Rg1) and PPD- to PPT- type ginsenoside (PPD/PPT) were higher in inner fluid of ginseng extract after UF by 3 kDa cartridge (47.1 and 23.5, respectively) and 5 kDa Cassette (25.3 and 11.9, respectively) than those of before UF (5.7 and 3.7, respectively). PPD-type ginsenoside enriched fraction by UF system could be developed as a new ginseng material in food and cosmetic industrials.

• Journal of Ginseng Research
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• v.35 no.3
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• pp.375-383
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• 2011

In the previous report, we have demonstrated that ginsenoside Rc, one of major ginsenosides, is a major component for the restoration for normal growth of worms in cholesterol-deprived medium. In the present study, we further investigated the roles of minor ginsenosides, such as ginsenoside $Rh_1$ and $Rh_2$, ginsenoside metabolites such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT) and ginsenoside epimers such as 20(R)- and 20(S)-ginsenoside $Rg_3$ in cholesterol-deprived medium. We found that ginsenoside $Rh_1$ almost restored normal growth of worms in cholesterol-deprived medium in F1 generation. However, supplement of ginsenoside $Rh_2$ caused a suppression of worm growths in cholesterol-deprived medium. In addition, CK and PPD also slightly restored normal growth of worms in cholesterol-deprived medium but PPT not. In experiments using ginsenoside epimers, supplement of 20(S)- but not 20(R)-ginsenoside $Rg_3$ in cholesterol-deprived medium also almost restored worm growth. These results indicate that the absence or presence of carbohydrate component at backbone of ginsenoside, the number of carbohydrate attached at carbon-3, and the position of hydroxyl group at carbon-20 of ginsenoside might plays important roles in restoration of worm growth in cholesterol-deprived medium.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.255-261
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• 2018

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as memory enhancement and tumor inhibition. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues linked to APPT, but not other glycoside residues. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer sugar at C-6 but not the inner glucose at C-6 or the glucose at C-20. Thus, the combined use of DT-bgl and PF-bgl is expected to increase the biotransformation of PPT-type ginsenosides to APPT. We optimized the ratio of PF-bgl to DT-bgl, the concentrations of substrate and enzyme, and the reaction time to increase the biotransformation of ginsenoside Re and PPT-type ginsenosides in Panax ginseng leaf extract to APPT. DT-bgl combined with PF-bgl converted 1.0 mg/ml PPT-type ginsenosides in ginseng leaf extract to 0.58 mg/ml APPT without other ginsenosides, with a molar conversion of 100%. We achieved the complete biotransformation of ginsenoside Re and PPT-type ginsenosides in ginseng leaf extract to APPT by the combined use of two ${\beta}$-glycosidases, suggesting that discarded ginseng leaves can be used as a source of the valuable ginsenoside APPT. To the best of our knowledge, this is the first quantitative production of APPT using ginsenoside Re, and we report the highest concentration and productivity of APPT from ginseng extract to date.

• Journal of Ginseng Research
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• v.40 no.1
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• pp.47-54
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• 2016

Background: The roots of Panax ginseng contain noble tetracyclic triterpenoid saponins derived from dammarenediol-II. Dammarene-type ginsenosides are classified into the protopanaxadiol (PPD) and protopanaxatriol (PPT) groups based on their triterpene aglycone structures. Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) are critical for the production of PPD and PPT aglycones, respectively. CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes PPT production from PPD in P. ginseng. Methods: We constructed transgenic P. ginseng lines overexpressing or silencing (via RNA interference) the CYP716A53v2 gene and analyzed changes in their ginsenoside profiles. Result: Overexpression of CYP716A53v2 led to increased accumulation of CYP716A53v2 mRNA in all transgenic roots compared to nontransgenic roots. Conversely, silencing of CYP716A53v2 mRNA in RNAi transgenic roots resulted in reduced CYP716A53v2 transcription. HPLC analysis revealed that transgenic roots overexpressing CYP716A53v2 contained higher levels of PPT-group ginsenosides ($Rg_1$, Re, and Rf) but lower levels of PPD-group ginsenosides (Rb1, Rc, $Rb_2$, and Rd). By contrast, RNAi transgenic roots contained lower levels of PPT-group compounds and higher levels of PPD-group compounds. Conclusion: The production of PPD- and PPT-group ginsenosides can be altered by changing the expression of CYP716A53v2 in transgenic P. ginseng. The biological activities of PPD-group ginsenosides are known to differ from those of the PPT group. Thus, increasing or decreasing the levels of PPT-group ginsenosides in transgenic P. ginseng may yield new medicinal uses for transgenic P. ginseng.

• Journal of Ginseng Research
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• v.43 no.2
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• pp.261-271
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• 2019

Background: Protopanaxatriol (PPT) is an aglycone of ginsenosides, which has high medicinal values. Production of PPT from natural ginseng plants requires artificial deglycosylation procedures of ginsenosides via enzymatic or physicochemical treatments. Metabolic engineering could be an efficient technology for production of ginsenoside sapogenin. For PPT biosynthesis in Panax ginseng, damarenediol-II synthase (PgDDS) and two cytochrome P450 enzymes (CYP716A47 and CYP716A53v2) are essentially required. Methods: Transgenic tobacco co-overexpressing P. ginseng PgDDS, CYP716A47, and CYP716A53v2 was constructed via Agrobacterium-mediated transformation. Results: Expression of the three introduced genes in transgenic tobacco lines was confirmed by Reverse transcription-polymerase chain reaction (RT-PCR). Analysis of liquid chromatography showed three new peaks, dammarenediol-II (DD), protopanaxadiol (PPD), and PPT, in leaves of transgenic tobacco. Transgenic tobacco (line 6) contained $2.8{\mu}g/g$ dry weight (DW), $7.3{\mu}g/g$ DW, and $11.6{\mu}g/g$ DW of PPT, PPD, and DD in leaves, respectively. Production of PPT was achieved via cell suspension culture and was highly affected by auxin treatment. The content of PPT in cell suspension was increased 37.25-fold compared with that of leaves of the transgenic tobacco. Transgenic tobacco was not able to set seeds because of microspore degeneration in anthers. Transmission electron microscopy analysis revealed that cells of phloem tissue situated in the center of the anther showed an abnormally condensed nuclei and degenerated mitochondria. Conclusion: We successfully achieved the production of PPT in transgenic tobacco. The possible factors deriving male sterility in transgenic tobacco are discussed.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.430-433
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• 2008

A high performance liquid chromatography (HPLC) method has been successfully developed to identify and quantify major ginsenosides in Korean ginseng seeds and roots. Using gradient elution of acetonitrile and water without buffer, the 6 major ginsenosides ($Rb_1,\;Rb_2$, Rc, Rd, Re, and $Rg_1$) were identified. Compared with ginseng roots, the amount of ginsenoside Re and Rd in ginseng seeds were significantly higher than those in ginseng roots (p<0.05). In ginseng seeds, the content of protopanaxtriol (PPT) was higher than that of protopanaxdiol (PPD) and the ratio of PPT and PPD was approximately 2.2 : 1. However, the content of PPT was lower than that of PPD in ginseng roots. It should be mentioned that both content of PPT and PPD in ginseng seeds were much higher than those in ginseng roots.