• 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.

• Korean Journal of Pharmacognosy
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• v.26 no.4
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• pp.360-367
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• 1995

The metabolism of ginseng saponins by human intestinal bacteria was studied using human feces under anaerobic culture conditions. $Ginsenoside-Rb_1$, $-Rb_2$ and -Rc(protopanaxadiol type) were mainly metabolized to compound-K(C-K), $20-O-[{\alpha}-L-arabinopyranosyl(1{\rightarrow}6)-{\beta}-{_D}-glucopyranosyl]-20(S)-protopanaxadiol(compound-Y,\;C-Y)$, $20-O-[{\alpha}-L-arabinopyranosyl(1{\rightarrow}6)-{\beta}-{_D}-glucopyranosyll-20(S)-protopanaxadiol(ginsenosied-MC,{\;}MC)$, respectively, and $ginsenoside-Rg_1$ and -Re(protopanaxatriol type) to their aglycon, 20(S)-protopanaxatriol, though the pathway and rate of the metabolism were affected by fermentation medium. C-K was not decomposed any more, while C-Y and Mc were both gradually hydrolyzed to C-K.

• Proceedings of the Ginseng society Conference
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• 1993.09a
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• pp.74-83
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• 1993

In an attempt toward the synthesis of the difficulty accessible ginseng saponins the four dammarane glycosides identical to the natural $ginsenosides-Rh_2,$ - F2, compound K and chikusetsusaponin - LT8 have been prepared from betulafolienetriol(=dammar-24-ene-$3{\alpha},12{\beta}\;20(S)-triol).\;3-O-{\beta}-D-Glucopyranoside$ of 20(S) - protopanaxadiol $(=ginsenoside-Rh_2)$ have been obtained by the regio - and stereoselective glycosylation of the $12-O-acetyldammar-24-ene-3{\beta},\;12{\beta},$ 20(S)-triol. The 12-ketoderivative of 20(S)-protopanaxadiol has been used as aglycon in synthesis of chikusetsusaponin - LT8. Attempted regio - and stereoselective glycosylation of the less reactive tertiary C - 20 - hydroxyl group in order to synthesize the $20-O-{\beta}-D-glucopyranoside$ of 20(S)-protopanaxadiol(=compound K) using 3, 12 - di - O - acetyldammar - 24 - ene - $3{\beta},12{\beta},20(S)$-trial as aglycon was unsuccessful. Glycosylation of 3, 12 - diketone of betulafolienetriol followed by $NaBH_4$ reduction yielded the $20-O-{\beta}-D-glucopyranoside\;of\;dammar-24-ene-3{\beta},12{\alpha},$ 20(S)-triol, the $12{\alpha}-epimer$ of 20(S) - protopanaxadiol. Moreover, a number of semisynthetic ocotillol - type glucosides, analogs of natural pseudoginsenosides, have been prepared.

• Korean Journal of Pharmacognosy
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• v.28 no.1
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• pp.35-41
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• 1997

Following ginsenoside-Rb1-hydrolyzing assay, strictly anaerobic bacteria were isolated from human feces and identified as Prevotella oris. The bacteria hydrolyzed ginsenoside Rb1 and Rd to $20-O-{\beta}-D-glucopyranosyl-20(S)-protopanaxadiol$ (I), ginsenoside Rb2 to $20-O-[{\alpha}-L-arabinofuranosyl (1{\rightarrow}6)-{\beta}-D-glucopyranosyl] - 20(S)-protopanaxadiol$ (ll) and ginsenoside Rc to $20-O-[{\alpha}-L-arabinofuranosyl (1{\rightarrow} 6){\beta}-D-g1ucopyranosyl]-20(S)-protopanaxadiol$ (III) like fecal microflora, but did not attack ginsenoside Re nor Rgl (Protopanaxatriol-type). Pharmacokinetic studies of ginseng saponins was also performed using specific pathogen free rats and demonstrated that the intestinal bacterial metabolites I-111, 20(S)- protopanaxatriol(IV) and 20(S)-protopanaxadiol(V) were absorbed from the intestines to $blood(0.4-5.1\;{\mu}g/ml)$ after oral administration with total saponin(1 g/kg/day).

• 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 Microbiology and Biotechnology
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• v.30 no.10
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• pp.1559-1566
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• 2020

Compound K (C-K) is one of the most pharmaceutically effective ginsenosides, but it is absent in natural ginseng. However, C-K can be obtained through the hydrolysis of protopanaxadiol-type ginsenosides (PPDGs) in natural ginseng. The aim of this study was to obtain the high concentration of food-available C-K using PPDGs in Korean ginseng extract by an extracellular enzyme from Aspergillus niger KACC 46495. A. niger was cultivated in the culture medium containing the inducer carboxymethyl cellulose (CMC) for 6 days. The extracellular enzyme extracted from A. niger was prepared from the culture broth by filtration, ammonium sulfate, and dialysis. The extracellular enzyme was used for C-K production using PPDGs. The glycoside-hydrolyzing pathways for converting PPDGs into C-K by the extracellular enzyme were Rb1 → Rd → F2 → C-K, Rb2 → Rd or compound O → F2 or compound Y → C-K, and Rc → Rd or compound Mc1 → F2 or compound Mc → C-K. The extracellular enzyme from A. niger at 8.0 mg/ml, which was obtained by the induction of CMC during the cultivation, converted 6.0 mg/ml (5.6 mM) PPDGs in Korean ginseng extract into 2.8 mg/ml (4.5 mM) food-available C-K in 9 h, with a productivity of 313 mg/l/h and a molar conversion of 80%. To the best of our knowledge, the productivity and concentration of C-K of the extracellular enzyme are the highest among those by crude enzymes from wild-type microorganisms.

• Journal of Ginseng Research
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• v.47 no.5
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• pp.605-614
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• 2023

Ginsenosides are bioactive components of Panax ginseng with many functions such as anti-aging, anti-oxidation, anti-inflammatory, anti-fatigue, and anti-tumor. Ginsenosides are categorized into dammarane, oleanene, and ocotillol type tricyclic triterpenoids based on the aglycon structure. Based on the sugar moiety linked to C-3, C-20, and C-6, C-20, dammarane type was divided into protopanaxadiol (PPD) and protopanaxatriol (PPT). The effects of ginsenosides on skin disorders are noteworthy. They play antiaging roles by enhancing immune function, resisting melanin formation, inhibiting oxidation, and elevating the concentration of collagen and hyaluronic acid. Thus, ginsenosides have previously been widely used to resist skin diseases and aging. This review details the role of ginsenosides in the anti-skin aging process from mechanisms and experimental research.

• 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.30 no.4
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• pp.181-187
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• 2006

Protopanaxadiol (PPD) is a mixture of protopanaxadiol type saponins with a dammarane skeleton, from Korean red ginseng (Panax ginseng C.A. Meyer; Araliaceae). Korean ginseng is well-known herb to treat almost all kinds of diseases in Oriental medicine. This herb was particularly prescribed for treatment various inflammatory diseases, including rheumatoid arthritis, atherosclerosis, and diabetes mellitus, for centuries. To understand the efficacy of ginseng against inflammatory diseases, we aimed to show anti-inflammatory activities of the PPD in murine macrophage cell line, RAW264.7 cells using nitric oxide (NO) production assay and the expressions of pro-inflammatory cytokines, such as tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), interleukin-$1{\beta}$ (IL-$1{\beta}$), and IL-6, and monocyte chemotactic protein-1 (MCP-1). We found that PPD saponin significantly blocked LPS ($1{\mu}g/ml$)-induced NO production in a dose-dependent manner. In addition, PPD abrogated the expressions of LPS-induced pro-inflammatory cytokines, such as IL-$1{\beta}$ and MCP-1. Moreover, cyclooxygenase (COX)-2, a critical enzyme to produce prostaglandin E2 (PGE2), was significantly inhibited by PPD in LPS-activated RAW264.7 cells. Taken together, these results suggested that anti-inflammatory efficacy of Korean red ginseng on inflammatory diseases is, at least, due to the NO inhibitory activity and the inhibition of the expressional level of inflammatory cytokines and/or mediators.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.343-351
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• 2012

In this paper, the kinetics of a cloned special glucosidase, named ginsenosidase type III hydrolyzing 3-O-glucoside of multi-protopanaxadiol (PPD)-type ginsenosides, were investigated. The gene (bgpA) encoding this enzyme was cloned from a Terrabacter ginsenosidimutans strain and then expressed in E. coli cells. Ginsenosidase type III was able to hydrolyze 3-O-glucoside of multi-PPD-type ginsenosides. For instance, it was able to hydrolyze the 3-O-${\beta}$-D-(1${\rightarrow}$2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-${\beta}$-D-glucopyranosyl of gypenoside XVII to gypenoside LXXV. Similarly, the enzyme could hydrolyze the glucopyranosyls linked to the 3-O-position of Rb2, Rc, Rd, Rb3, and Rg3. With a larger enzyme reaction $K_m$ value, there was a slower enzyme reaction speed; and the larger the enzyme reaction $V_{max}$ value, the faster the enzyme reaction speed was. The $K_m$ values from small to large were 3.85 mM for Rc, 4.08 mM for Rb1, 8.85 mM for Rb3, 9.09 mM for Rb2, 9.70 mM for Rg3(S), 11.4 mM for Rd and 12.9 mM for F2; and $V_{max}$ value from large to small was 23.2 mM/h for Rc, 16.6 mM/h for Rb1, 14.6 mM/h for Rb3, 14.3 mM/h for Rb2, 1.81mM/h for Rg3(S), 1.40 mM/h for Rd, and 0.41 mM/h for F2. According to the $V_{max}$ and $K_m$ values of the ginsenosidase type III, the hydrolysis speed of these substrates by the enzyme was Rc>Rb1>Rb3>Rb2>Rg3(S)>Rd>F2 in order.