Transcriptome-Wide Identification and Integrated Analysis of a UGT Gene Involved in Ginsenoside Ro Biosynthesis in Panax ginseng.

Panax ginseng as a traditional medicinal plant with a long history of medicinal use. Ginsenoside Ro is the only oleanane-type ginsenoside in ginseng, and has various pharmacological activities, including anti-inflammatory, detoxification, and antithrombotic activities. UDP-dependent glycosyltransferase (UGT) plays a key role in the synthesis of ginsenoside, and the excavation of UGT genes involved in the biosynthesis of ginsenoside Ro has great significance in enriching ginsenoside genetic resources and further revealing the synthesis mechanism of ginsenoside. In this work, ginsenoside-Ro-synthesis-related genes were mined using the P. ginseng reference-free transcriptome database. Fourteen hub transcripts were identified by differential expression analysis and weighted gene co-expression network analysis. Phylogenetic and synteny block analyses of PgUGAT252645, a UGT transcript among the hub transcripts, showed that PgUGAT252645 belonged to the UGT73 subfamily and was relatively conserved in ginseng plants. Functional analysis showed that PgUGAT252645 encodes a glucuronosyltransferase that catalyzes the glucuronide modification of the C3 position of oleanolic acid using uridine diphosphate glucuronide as the substrate. Furthermore, the mutation at 622 bp of its open reading frame resulted in amino acid substitutions that may significantly affect the catalytic activity of the enzyme, and, as a consequence, affect the biosynthesis of ginsenoside Ro. Results of the in vitro enzyme activity assay of the heterologous expression product in E. coli of PgUGAT252645 verified the above analyses. The function of PgUGAT252645 was further verified by the result that its overexpression in ginseng adventitious roots significantly increased the content of ginsenoside Ro. The present work identified a new UGT gene involved in the biosynthesis of ginsenoside Ro, which not only enriches the functional genes in the ginsenoside synthesis pathway, but also provides the technical basis and theoretical basis for the in-depth excavation of ginsenoside-synthesis-related genes.

Complex components of Shengmai formula interact with organic cation transporter 2 (OCT2) in MDCK cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai formula (SMF) is a well-known Chinese herbal compound preparation, which is utilized extensively for the treatment of myocardial ischemia, arrhythmia and other life-threatening conditions. Our previous researches have shown that some of the active ingredients in SMF can interact with organic anion transport polypeptide 1B1 (OATP1B1), breast cancer resistance protein (BCRP) and organic anion transporter 1 (OAT1), etc. Organic cation transporter 2 (OCT2) is a highly expressed uptake transporter in the kidney, and its interaction with the major active components in SMF remains unclear. AIM OF THE STUDY: We purposed to explore OCT2-mediated interactions and compatibility mechanisms of the main active compounds in SMF. MATERIALS AND METHODS: Fifteen active ingredients of SMF, including ginsenoside Rb1, Rd, Re, Rg1, Rf, Ro and Rc, methylophiopogonanone A and B, ophiopogonin D and D', schizandrin A and B, schizandrol A and B, were selected to investigate OCT2-mediated interactions in Madin-Darby cacine kidney (MDCK) cells stably expressing OCT2. RESULTS: Among the above 15 main active components, only ginsenosides Rd, Re and schizandrin B could significantly inhibit the uptake of 4-(4-(dimethylamino)styryl)-N-methyl pyridiniumiodide (ASP+), a classical substrate of OCT2. Ginsenoside Rb1 and methylophiopogonanone A can be transported by MDCK-OCT2 cells, and their uptake was significantly reduced when OCT2 inhibitor decynium-22 was added. Ginsenoside Rd could remarkably reduce the uptake of methylophiopogonanone A and ginsenoside Rb1 by OCT2, ginsenoside Re only decreased the uptake of ginsenoside Rb1, while schizandrin B had no effect on the uptake of both. CONCLUSIONS: OCT2 mediates the interaction of the major active components in SMF. Ginsenosides Rd, Re and schizandrin B are the potential inhibitors of OCT2, while ginsenosides Rb1 and methylophiopogonanone A are the potential substrates of OCT2. There is an OCT2-mediated compatibility mechanism among these active ingredients of SMF.

Corrigendum to "Ginsenoside Ro, an oleanolic saponin of Panax ginseng, exerts anti-inflammatory effect by direct inhibiting toll like receptor 4 signaling pathway" [J Ginseng Res 46 (2022) 156-166].

[This corrects the article DOI: 10.1016/j.jgr.2021.05.011.].

Ginsenoside Ro, an oleanolic saponin of Panax ginseng, exerts anti-inflammatory effect by direct inhibiting toll like receptor 4 signaling pathway.

BACKGROUND: Panax ginseng Meyer (P. ginseng), a herb distributed in Korea, China and Japan, exerts benefits on diverse inflammatory conditions. However, the underlying mechanism and active ingredients remains largely unclear. Herein, we aimed to explore the active ingredients of P. ginseng against inflammation and elucidate underlying mechanisms. METHODS: Inflammation model was constructed by lipopolysaccharide (LPS) in C57BL/6 mice and RAW264.7 macrophages. Molecular docking, molecular dynamics, surface plasmon resonance imaging (SPRi) and immunofluorescence were utilized to predict active component. RESULTS: P. ginseng significantly inhibited LPS-induced lung injury and the expression of pro-inflammatory factors, including TNF-α, IL-6 and IL-1ß. Additionally, P. ginseng blocked fluorescence-labeled LPS (LPS488) binding to the membranes of RAW264.7 macrophages, the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Furthermore, molecular docking demonstrated that ginsenoside Ro (GRo) docked into the LPS binding site of toll like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex. Molecular dynamic simulations showed that the MD2-GRo binding conformation was stable. SPRi demonstrated an excellent interaction between TLR4/MD2 complex and GRo (KD value of 1.16 × 10-9 M). GRo significantly inhibited LPS488 binding to cell membranes. Further studies showed that GRo markedly suppressed LPS-triggered lung injury, the transcription and secretion levels of TNF-α, IL-6 and IL-1ß. Moreover, the phosphorylation of NF-κB and MAPKs as well as the p65 subunit nuclear translocation were inhibited by GRo dose-dependently. CONCLUSION: Our results suggest that GRo exerts anti-inflammation actions by direct inhibition of TLR4 signaling pathway.

The dual roles of ginsenosides in improving the anti-tumor efficiency of cyclophosphamide in mammary carcinoma mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyclophosphamide (CTX) is a first line chemotherapeutic agent, but often limited for its unstable therapeutic effect and serious side effects. Ginsenosides could facilitate the anti-tumor efficiency of CTX, including benefiting therapeutic effect and decreasing side effects. AIM OF THE STUDY: To investigate the potential mechanism of ginsenosides on benefiting the anti-tumor efficiency of CTX. MATERIALS AND METHODS: Mammary carcinoma mice were applied to investigate the anti-tumor efficiency and potential mechanism of combinational treatment of ginsenosides and CTX. Therapeutic effect was evaluated based on survival rate, tumor burden, tumor growth inhibition rate, and apoptosis and histological changes of tumor tissues. Anti-tumor immunity was studied by measuring serum level of anti-tumor cytokines. Gut mucositis, one of lethal side effects of CTX, was evaluated by diarrhea degree, gut permeability and tight junction proteins expressions. Gut microbial diversity was analyzed by 16S rRNA gene sequencing, and fecal transplant and antibiotics sterilized animals were performed to evaluate the therapeutic effect of gut microbiota on tumor suppression. RESULTS: Ginsenosides facilitated the therapeutic effect of CTX in mice, which manifested as prolonged survival rate, decreased tumor burden, as well as enhanced tumor growth inhibition rate and apoptosis. The favoring effect was related to elevation of anti-tumor immunity which manifested as the increased anti-tumor cytokines (INF-γ, IL-17, IL-2 and IL-6). Further studies indicated the elevation was ascribed to ginsenosides promoted reproduction of gut probiotics including Akkermansia, Bifidobacterium and Lactobacillus. Moreover, co-administration of ginsenosides in mice alleviated CTX-induced gut mucositis, including lower gut permeability, less diarrhea, less epithelium damage and higher tight junction proteins. Further researches suggested the alleviation was related to ginsenosides activated Nrf2 and inhibited NFκB pathways. CONCLUSION: Ginsenosides show dual roles to facilitate the anti-tumor efficiency of CTX, namely promote the anti-tumor immunity through maintaining gut microflora and ameliorate gut mucositis by modulating Nrf2 and NFκB pathways.

Spectrum-effect relationship between UPLC fingerprints and anti-lung cancer effect of Panax ginseng.

OBJECTIVES: Lung cancer has the highest mortality rate among the various types of cancer. Panax ginseng (C. A. Mey). is a popular anti-cancer herbal supplement. The quality control of ginseng is crucial to ensure its clinical efficacy. This study aimed to establish new quality control methods for ginseng and to identify its main active components responsible for lung cancer treatment. METHODS: Ultra-high-performance liquid chromatography (UPLC) was used to establish fingerprints of 18 batches of ginseng. CCK-8 test was performed to evaluate the inhibitory activity of ginseng on Lewis lung cancer (LLC) cells. The spectrum-effect relationship analysis of ginseng was assessed by canonical correlation analysis (CCA) and bioactivity validation. KEY FINDINGS: Six common peaks were identified and the variation coefficients were determined. The 18 batches of ginseng inhibited the proliferation of LLC cells to different degrees, showing different half maximal inhibitory concentration (IC50 ) values. Spectrum-effect relationship analysis showed that ginsenoside Ro is the main anti-proliferative constituent of LLC cell. CONCLUSIONS: Spectrum-effect relationship is suitable for quality control of ginseng used for lung cancer. It is also effective in discovering the active ingredients related to the clinical efficacy of traditional Chinese medicine.

Quality control of red ginseng based on the ratio of ginsenosides Ro/Re / 药学学报

Ginsenoside Ro decreased measures of inflammation, aging, oxidants and thrombus formation in a previous study. To measure ginsenoside Ro content in red ginseng from different years, an optimized extraction method was developed to determine ginsenoside Rg1, Re, Rb1 and Ro content by HPLC in 43 batches of red ginseng from different origins, growing years and manufacturers. The results indicate that the best extraction method was to ultrasonify a 1 g sample in 70% methanol for 50 min. The total running time of the optimized gradient was 50 min using a C18 core-shell column and was half the time described in the Chinese Pharmacopoeia, 2015 edition. The separation resolution of all of targeted compounds was greater than 1.6. The peak shape of ginsenoside Ro was optimal when the mobile phase consisted of acetonitrile and water with 0.1% phosphoric acid. The content of ginsenoside Ro was in the range of 0.11% to 0.43%, and the average content was 0.26%, which was higher than that of ginsenoside Rg1 and Re. The ratio of ginsenoside Ro and Re as a threshold could be used to discriminate red ginseng from different growing years; in addition, 100%, 94.4% and 46.6% of red ginseng from six, five and four years exceeded the threshold of 1.3. Our optimized analytical method for characterization of red ginseng is convenient and shortens the assay time.

Anewoleanolicacid-typesaponinfromrootsofPanaxginseng / 中草药

Objective To systematically investigate the chemical constituents of the roots of Panaxginseng. Methods Mild cold-soaked extraction by 70% aqueous ethanol, successive solvent extraction by ethyl acetate and n-butanol, column chromatography by D101 macroporous absorption resin and reversed-phase silica gel, and semi-preparative HPLC, were used for compounds isolation and purification, while high-resolution mass spectrometry, 1D and 2D NMR data were analyzed for compounds identification. Results A new oleanolic acid tetraglycoside (1) and 19 known ginsenosides (2-20) were isolated and identified. Compound 1 was identified as oleanolic acid 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside, named ginsenoside Ro1 (1). The 19 known ginsenosides were notoginsenoside FP1 (2), ginsenoside Re3 (3), notoginsenoside Rt (4), 20-O-glucosyl ginsenoside Rf (5), ginsenoside Re2 (6), ginsenoside Rg2 (7), ginsenoside Ra2 (8), ginsenoside Rb1 (9), ginsenoside Rc (10), ginsenoside Ra1 (11), malonylginsenoside Rb1 (12), malonylfloralginsenoside Rd5 (13), malonylginsenoside Rc (14), ginsenoside Ro (15), ginsenoside Rd (16), ginsenoside F2 (17), 20(R)-ginsenoside Rh2 (18), ginsenoside F3 (19) and ginsenoside F1 (20). Conclusion Compound1is a new compound. Compound 2, notoginsenoside FP1, is isolated from this plant for the first time.

Natural medicines for the treatment of fatigue: Bioactive components, pharmacology, and mechanisms.

It is a common phenomenon that people are in a sub-health condition and facing "unexplained fatigue", which seriously affects their health, work efficiency and quality of life. Meanwhile, fatigue is also a common symptom of many serious diseases such as HIV/AIDS, cancer, and schizophrenia. However, there are still no official recommendations for the treatment of various forms of fatigue. Some traditional natural medicines are often used as health care products, such as ginseng, Cordyceps militaris (L.ex Fr.Link) and Rhodiola rosea L., and these have been reported to have specific anti-fatigue effects with small toxic and side effects and rich pharmacological activities. It may be promising treatment strategy for sub-health. In this review, we first outline the generation of fatigue. Furthermore, we put emphasis on the anti-fatigue mechanism, bioactive components, and clinic trials of natural medicines, which will contribute to the development of potential anti-fatigue agents and open up novel treatments for sub-health.

Inhibitory Effects of Ginsenoside Ro on the Growth of B16F10 Melanoma via Its Metabolites.

Ginsenoside Ro (Ro), a major saponin derived and isolated from Panax ginseng C.A. Meyer, exerts multiple biological activities. However, the anti-tumour efficacy of Ro remains unclear because of its poor in vitro effects. In this study, we confirmed that Ro has no anti-tumour activity in vitro. We explored the anti-tumour activity of Ro in vivo in B16F10 tumour-bearing mice. The results revealed that Ro considerably suppressed tumour growth with no significant side effects on immune organs and body weight. Zingibroside R1, chikusetsusaponin IVa, and calenduloside E, three metabolites of Ro, were detected in the plasma of Ro-treated tumour-bearing mice and showed excellent anti-tumour effects as well as anti-angiogenic activity. The results suggest that the metabolites play important roles in the anti-tumour efficacy of Ro in vivo. Additionally, the haemolysis test demonstrated that Ro has good biocompatibility. Taken together, the findings of this study demonstrate that Ro markedly suppresses the tumour growth of B16F10-transplanted tumours in vivo, and its anti-tumour effects are based on the biological activity of its metabolites. The anti-tumour efficacy of these metabolites is due, at least in part, to its anti-angiogenic activity.

Inhibitory effects of thromboxane A2 generation by ginsenoside Ro due to attenuation of cytosolic phospholipase A2 phosphorylation and arachidonic acid release.

BACKGROUND: Thromboxane A2 (TXA2) induces platelet aggregation and promotes thrombus formation. Although ginsenoside Ro (G-Ro) from Panax ginseng is known to exhibit a Ca2+-antagonistic antiplatelet effect, whether it inhibits Ca2+-dependent cytosolic phospholipase A2 (cPLA2α) activity to prevent the release of arachidonic acid (AA), a TXA2 precursor, is unknown. In this study, we attempted to identify the mechanism underlying G-Ro-mediated TXA2 inhibition. METHODS: We investigated whether G-Ro attenuates TXA2 production and its associated molecules, such as cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS), cPLA2α, mitogen-activated protein kinases, and AA. To assay COX-1 and TXAS, we used microsomal fraction of platelets. RESULTS: G-Ro reduced TXA2 production by inhibiting AA release. It acted by decreasing the phosphorylation of cPLA2α, p38-mitogen-activated protein kinase, and c-Jun N-terminal kinase1, rather than by inhibiting COX-1 and TXAS in thrombin-activated human platelets. CONCLUSION: G-Ro inhibits AA release to attenuate TXA2 production, which may counteract TXA2-associated thrombosis.

Inhibitory Effects of Ginsenoside Ro on Clot Retraction through Suppressing PI3K/Akt Signaling Pathway in Human Platelets.

Glycoprotein IIb/IIIa (αIIb/ß3) is the most abundant integrin on platelet surfaces, which is involved in interaction between platelets, and triggers an intracellular signaling cascade, platelet shape changes, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibronectin and fibrinogen to αIIb/ß3 and clot retraction. We found that G-Ro inhibited thrombin-induced platelet aggregation dose-dependently and attenuated the fibronectin-, and fibrinogen-binding to αIIb/ß3 through the dephosphorylation of phosphoinositide 3-kinase p85 and Akt, which influence clot retraction, reflecting the intensification of thrombus. We observed that G-Ro is involved in αIIb/ß3 in human platelets. These results suggest that G-Ro is beneficial, inhibiting fibronectin adhesion, fibrinogen binding, and clot retraction. Therefore, G-Ro in Panax ginseng may prevent platelet aggregation-mediated thrombotic disease.

Promoting effect of Ginsenoside Ro on differentiation of THP-1-derived DC / 中国免疫学杂志

Objective:To investigate the promoting effect of Ginsenoside Ro on the differentiation of THP-1-derived dendritic cells (DCs) induced by GM-CSF and IL-4.Methods: Sensitive leukemia-derived DC cell line was screened first.Then,the selected sensitive cell line THP-1 was stimulated to differentiate into DCs by cytokines (GM-CSF and IL-4) and small(5 μmol/L),middle(10 μmol/L),and large (20 μmol/L) dose of Ginsenoside Ro respectively.The expressions of CD1a,MHCⅡ and CD86 of leukemia-derived DCs were detected by flow cytometry.In addition,the transcription levels of CD1a,CD86 and MHCⅡ of leukemia-derived DCs were detected by RT-PCR.ELISA was used to measure the protein levels of TNF-α and IL-6 in the culture supernatant.Results: THP-1 was the sensitive leukemia cell line which could be induced to differentiate into DCs by cytokines.Compared with cytokine stimulation alone,the expression of CD1a,MHCⅡ and CD86 in leukemia-derived cells was significantly increased after the stimulation of Ginsenoside Ro combined with cytokine(P<0.05).The CD1a,CD86 and MHCⅡ mRNA expression was significantly increased after the treatment of Ginsenoside Ro combined with cytokine(P<0.05).Moreover,the protein levels of TNF-α and IL-6 in culture supernatant were significantly increased (P<0.05) after the stimulation of Ginsenoside Ro in combination with cytokines.Conclusion: Ginsenoside Ro can significantly promote the differentiation of leukemia-derived DCs.

Vasodilator-stimulated phosphoprotein-phosphorylation by ginsenoside Ro inhibits fibrinogen binding to αIIb/ß3 in thrombin-induced human platelets.

BACKGROUND: Glycoprotein IIb/IIIa (αIIb/ß3) is involved in platelet adhesion, and triggers a series of intracellular signaling cascades, leading to platelet shape change, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibrinogen to αIIb/ß3. METHODS: We investigated the effect of G-Ro on regulation of signaling molecules affecting the binding of fibrinogen to αIIb/ß3, and its final reaction, clot retraction. RESULTS: We found that G-Ro dose-dependently inhibited thrombin-induced platelet aggregation and attenuated the binding of fibrinogen to αIIb/ß3 by phosphorylating cyclic adenosine monophosphate (cAMP)-dependently vasodilator-stimulated phosphoprotein (VASP; Ser157). In addition, G-Ro strongly abrogated the clot retraction reflecting the intensification of thrombus. CONCLUSION: We demonstrate that G-Ro is a beneficial novel compound inhibiting αIIb/ß3-mediated fibrinogen binding, and may prevent platelet aggregation-mediated thrombotic disease.

Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint.

Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47(PHOX) (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.

Antioxidative properties of ginsenoside Ro against UV-B-induced oxidative stress in human dermal fibroblasts.

Ginsenoside Ro (Ro), an oleanolic acid-type ginsenoside, exhibited suppressive activities on reactive oxygen species (ROS) and matrix metalloproteinase-2 (MMP-2) elevation in UV-B-irradiated fibroblasts. Ro could overcome the reduction of the total glutathione (GSH) contents in UV-B-irradiated fibroblasts. Ro could not interfere with cell viabilities in UV-B-irradiated fibroblasts. Collectively, Ro possesses a potential skin anti-photoaging property against UV-B radiation in fibroblasts.

Ginsenoside Ro suppresses interleukin-1ß-induced apoptosis and inflammation in rat chondrocytes by inhibiting NF-κB.

This study investigated effects of Ginsenoside Ro (Ro) on interleukin-1ß (IL-1ß)-induced apoptosis and inflammation in rat chondrocytes. The rat chondrocytes were co-treated with IL-1ß (10 ng·kg(-1)) and Ro (50, 100 and 200 µmol·L(-1)) for 48 h. Chondrocytes viability was detected by the MTT assay and Annexin V-FITC/PI dual staining assay. Caspase 3 activity was measured by using caspase 3 colorimetric assay kit. Apoptosis related proteins Bax, Bad, Bcl-xL, PCNA, p53 and phospho-p53, along with inflammation related protein MMP 3, MMP 9 and COX-2, and the expression of phospho-NF-κB p65 were assayed by western blotting analyses. Ro could improve IL-1ß-induced chondrocytes viability. Ro could suppress IL-1ß-induced apoptosis by inhibiting levels of Bax and Bad, decreasing p53 phosphorylation and promoting the expression of Bcl-xL and PCNA. Ro inhibited caspase 3 activity. IL-1ß-induced inflammation and matrix degration were also alleviated by Ro with down-regulating the expression of MMP 3, MMP 9 and COX-2. Moreover, Ro inhibited NF-κB p65 phosphorylation induced by IL-1ß. In conclusion, these results suggested Ro exerted anti-apoptosis and anti-inflammation in IL-1ß-induced rat chondrocytes, which might be related to NF-κB signal pathway. Therefore, we propose that Ro might be a potential novel drug for the treatment of osteoarthritis.

Upregulation of heme oxygenase-1 by ginsenoside Ro attenuates lipopolysaccharide-induced inflammation in macrophage cells.

BACKGROUND: The beneficial effects of ginsenoside species have been well demonstrated in a number of studies. However, the function of ginsenoside Ro (GRo), an oleanane-type saponin, has not been sufficiently investigated. Thus, the aim of the present study was to investigate the anti-inflammatory effects of GRo in vitro using the Raw 264.7 mouse macrophage cell line treated with lipopolysaccharide (LPS), and to clarify the possible mechanism of GRo involving heme oxygenase-1 (HO-1), which itself plays a critical role in self-defense in the presence of inflammatory stress. METHODS: Raw 264.7 cells were pretreated with GRo (up to 200µM) for 1 h before treatment with 1 µg/mL LPS, and both cell viability and inflammatory markers involving HO-1 were evaluated. RESULTS: GRo significantly increased cell viability in a dose dependent manner following treatment with LPS, and decreased levels of reactive oxygen species and nitric oxide. GRo decreased inflammatory cytokines such as nitric oxide synthase and cyclooxygenase-2 induced by LPS. Moreover, GRo increased the expression of HO-1 in a dose dependent manner. Cotreatment of GRo with tin protoporphyrin IX, a selective inhibitor of HO-1, not only inhibited upregulation of HO-1 induced by GRo, but also reversed the anti-inflammatory effect of GRo in LPS treated Raw 264.7 cells. CONCLUSION: GRo induces anti-inflammatory effects following treatment with LPS via upregulation of HO-1.

Ginsenoside Ro suppresses interleukin-1β-induced apoptosis and inflammation in rat chondrocytes by inhibiting NF-κB / 中国天然药物

This study investigated effects of Ginsenoside Ro (Ro) on interleukin-1β (IL-1β)-induced apoptosis and inflammation in rat chondrocytes. The rat chondrocytes were co-treated with IL-1β (10 ng·kg(-1)) and Ro (50, 100 and 200 μmol·L(-1)) for 48 h. Chondrocytes viability was detected by the MTT assay and Annexin V-FITC/PI dual staining assay. Caspase 3 activity was measured by using caspase 3 colorimetric assay kit. Apoptosis related proteins Bax, Bad, Bcl-xL, PCNA, p53 and phospho-p53, along with inflammation related protein MMP 3, MMP 9 and COX-2, and the expression of phospho-NF-κB p65 were assayed by western blotting analyses. Ro could improve IL-1β-induced chondrocytes viability. Ro could suppress IL-1β-induced apoptosis by inhibiting levels of Bax and Bad, decreasing p53 phosphorylation and promoting the expression of Bcl-xL and PCNA. Ro inhibited caspase 3 activity. IL-1β-induced inflammation and matrix degration were also alleviated by Ro with down-regulating the expression of MMP 3, MMP 9 and COX-2. Moreover, Ro inhibited NF-κB p65 phosphorylation induced by IL-1β. In conclusion, these results suggested Ro exerted anti-apoptosis and anti-inflammation in IL-1β-induced rat chondrocytes, which might be related to NF-κB signal pathway. Therefore, we propose that Ro might be a potential novel drug for the treatment of osteoarthritis.

The involvement of ß-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng.

Panax species are the most popular medicinal herbs. The root of these plants contains pharmacologically active triterpene saponins, also known as ginsenosides, compounds that are divided into dammarane- and oleanane-type triterpenes. Two CYP716A subfamily genes (CYP716A47 and CYP716A53v2) were recently characterized, encoding an enzyme catalyzing the hydroxylation of dammarane-type triterpenes in Panax ginseng. Herein, we report that one CYP716A subfamily gene (CYP716A52v2) isolated from P. ginseng encodes a ß-amyrin 28-oxidase, which is suggested to modify ß-amyrin into oleanolic acid, a precursor of an oleanane-type saponin (mainly ginsenoside Ro) in P. ginseng. The ectopic expression of both PNY1 and CYP716A52v2 in recombinant yeast resulted in erythrodiol and oleanolic acid production, respectively. In vitro enzymatic activity assays biochemically confirmed that CYP716A52v2 catalyzed the oxidation of ß-amyrin to produce oleanolic acid, and the chemical structure of the oleanolic acid product was confirmed using gas chromatography-mass spectrometry (GC/MS). Transgenic P. ginseng plants were generated via Agrobacterium tumefaciens-mediated transformation: the overexpression of CYP716A52v2 greatly increased the content of oleanane-type ginsenoside (ginsenoside Ro), whereas RNA interference against CYP716A52v2 markedly reduced it. Furthermore, the levels of other dammarene-type ginsenosides were not affected in these transgenic lines. These results indicate that CYP716A52v2 is a ß-amyrin 28-oxidase that plays a key role in the biosynthesis of oleanane-type triterpenes in P. ginseng.