Discrimination of Baphicacanthis Cusiae Rhizoma et radix and its adulterant species and establishment of an assay method for quality control.

BACKGROUND: Baphicacanthis Cusiae Rhizoma et Radix, commonly known as Nan-Ban-Lan-Gen (NBLG), is an essential traditional Chinese medicine that possesses diverse bioactivities, particularly noteworthy for its antiviral properties. Although NBLG has been listed in the Chinese Pharmacopoeia as an independent Chinese medicine, the establishment of a comprehensive quality standard for NBLG remains elusive. The absence of assay for marker compound in its quality standards has led to the lack of corresponding quality control measures for NBLG-containing preparations, and its discrimination from adulterant species in the market which thereby can significantly impact the efficacy and safety of NBLG-containing products. METHODS: Ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was employed for comprehensive profiling of the chemical constituents of NBLG, the stem of Baphicacanthus cusia (Nees) Bremek (NBLJ), and the roots of Isatis indigotica Fort. (Bei-Ban-Lan-Gen, BBLG). Additionally, multivariate analysis was conducted to compare the chemical components of NBLG with those of NBLJ and BBLG. Furthermore, we established an optimized and validated HPLC method to obtain the fingerprint of NBLG and quantify the content of 2-benzoxazolinone and acteoside in the samples. RESULTS: A total of 73 compounds belonging to six classes were assigned in NBLG, with alkaloids being the most abundant and diverse species. High compositional similarities with significant differences in content were observed between NBLG and NBLJ. Moreover, the chemical profile of BBLG markedly differed from that of NBLG. An informative high performance liquid chromatography (HPLC) fingerprint of NBLG comprising seven characteristic peaks that can be used for quality assessment was established. Notably, we propose a quality control standard for NBLG, stipulating that the limit of content in dry weight for both 2-benzoxazolinone and acteoside should not be less than 0.010%. CONCLUSION: This study provides the most comprehensive chemical information to date on NBLG, offering valuable insights into its authentication and quality control. Our findings highlight the importance of comprehensive chemical profiling to differentiate potential substitutions and adulterations of herbal medicines, particularly when the original source is scarce or unavailable. These results can aid in the development of quality control measures for NBLG-containing preparations, ensuring their safety and efficacy.

Comprehensive chemical study on different organs of cultivated and wild Sarcandra glabra using ultra-high performance liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS).

To illuminate the similarities and differences between wild and cultivated Sarcandra glabra (S. glabra), we performed a comprehensively study on 26 batches of cultivated S. glabra and 2 batches of wild S. glabra. Chemical constituents and distribution characteristics of roots, stems and leaves in both wild and cultivated S. glabra were investigated through UHPLC-TOF-MS method. The result revealed that there were significant differences between roots, stems and leaves in S. glabra. And the chemical contents in the root part were less or even absence than those in leaf and stem, which suggested the root organ could be excluded as medicine. Meanwhile, the chemical contents of stems and leaves in cultivated S. glabra was sightly higher than that of wild samples. Therefore, cultivated S. glabra may have a high potential for substitution of wild S. glabra without affecting its pharmaceutical properties. In summary, our study could provide important information to the molecular basis for quality control of S. glabra.

Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca2+-dependent mechanism.

Resistance of cancer cells to chemotherapy is a significant clinical concern and mechanisms regulating cell death in cancer therapy, including apoptosis, autophagy or necrosis, have been extensively investigated over the last decade. Accordingly, the identification of medicinal compounds against chemoresistant cancer cells via new mechanism of action is highly desired. Autophagy is important in inducing cell death or survival in cancer therapy. Recently, novel autophagy activators isolated from natural products were shown to induce autophagic cell death in apoptosis-resistant cancer cells in a calcium-dependent manner. Therefore, enhancement of autophagy may serve as additional therapeutic strategy against these resistant cancers. By computational docking analysis, biochemical assays, and advanced live-cell imaging, we identified that neferine, a natural alkaloid from Nelumbo nucifera, induces autophagy by activating the ryanodine receptor and calcium release. With well-known apoptotic agents, such as staurosporine, taxol, doxorubicin, cisplatin and etoposide, utilized as controls, neferine was shown to induce autophagic cell death in a panel of cancer cells, including apoptosis-defective and -resistant cancer cells or isogenic cancer cells, via calcium mobilization through the activation of ryanodine receptor and Ulk-1-PERK and AMPK-mTOR signaling cascades. Taken together, this study provides insights into the cytotoxic mechanism of neferine-induced autophagy through ryanodine receptor activation in resistant cancers.

[Analysis of alcohol extract and water extract of Polygonum capitatum by UPLC-Q-TOF-MS].

In this study, we used Ultra Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry(UPLC-TOF-MS)to identify the chemical constituents in both ethanol and water extract of Polygonum capitatum. A Waters ACQUITY UPLC BEH C18 column(2.1 mm×100 mm,1.7 µm) was used for separation. The mobile phase was consisted of(A) 0.10% formic acid in water and(B)0.10% formic acid in acetonitrile, and the flow rate was 0.35 mL•min⁻¹. ESI source in negative ion mode was used for MS detection. Structural identification was carried out according to the accurate mass and matching with database. The results showed that flavonoids, polyphenols and lignans were the main components in both extracts. However, the chemical compositions of both extracts were different, e.g. there are less hydrolyzable tannins, loss of ellagic acid and more anthocyanins in ethanol extract. In a conclusion, this study provides an important scientific basis for identifying the active ingredients in P. capitatum, which also help to reveal the pharmacological effect of P. capitatum.

New Immunosuppressive Sphingoid Base and Ceramide Analogues in Wild Cordyceps.

A comprehensive identification of sphingoid bases and ceramides in wild Cordyceps was performed by integrating a sequential chromatographic enrichment procedure and an UHPLC-ultrahigh definition-Q-TOF-MS based sphingolipidomic approach. A total of 43 sphingoid bases and 303 ceramides were identified from wild Cordyceps, including 12 new sphingoid base analogues and 159 new ceramide analogues based on high-resolution MS and MS/MS data, isotope distribution, matching with the comprehensive personal sphingolipid database, confirmation by sphingolipid standards and chromatographic retention time rule. The immunosuppressive bioassay results demonstrated that Cordyceps sphingoid base fraction exhibits more potent immunosuppressive activity than ceramide fraction, elucidating the immunosuppressive ingredients of wild Cordyceps. This study represented the most comprehensive identification of sphingoid bases and ceramides from a natural source. The findings of this study provided an insight into therapeutic application of wild Cordyceps.

[Effects of Acupuncture Stimulation of Bilateral "Hegu" (LI 4) and "Taichong" (LR 3) on Learning-memory Ability, Hippocampal AP 42 Expression and Inflammatory Cytokines in Rats with Alzheimer's Disease].

OBJECTIVE: To observe the effect of acupuncture stimulation of bilateral "Hegu" (LI 4) and "Taichong" (LR 3, the so-called "Four Gate Points") on learning-memory ability, hippocampal interleukin-1 (IL-1) P and IL-2 and amyloid beta (Abeta) 42 levels in Alzheimer's disease (AD) rats,so as to reveal its underlying mechanism in improving AD. METHODS: Male SD rats were randomly divided into sham operation, model, medication and acupuncture groups (n = 12 rats in each group). The AD model was created by microinjection of streptozotocin (10 pL, 3 mg/kg) into the lateral ventricle (repeated the microinjection once two days later). Bilateral LR 3 and LI 4 were punctured with filiform needles and stimulated manually, once a day, 6 days a week for 4 weeks. The rats of the medication group were intragastric perfusion of Donepezil HOI (0.045 mg/kg), once a day for 4 weeks. The learning-memory ability was detected by Morris water maze swimming tests. The immunoactivity of hippocampal Abeta 42 was detected by immunohistochemistry, and the contents of IL-1 P and IL-2 in the hippocampus tissue were determined by ELISA. RESULTS: After modeling, the average escape latency of Morris water navigation task was significantly increased, and the target-platform crossing times of space probe trials were significantly reduced in the model group (P<0.05), suggesting a g-memory ability. After acupuncture intervention, the increased escape latency and the decreased target-platform crossing times were reversed, suggesting an improvement of the learning-memory. The hippocampal Abeta 42 immunoactivity and IL-1 beta content were significantly higher in the model group than in the sham operation group (P<0.05), but the hippocampal IL-2 content was markedly decreased in the model group (P<0. 05). Following the interventions, the increased Abeta 42 expression and IL-1 beta contents, and the decreased IL-2 contents in the hippocampus were also reversed in both the acupuncture and medication groups (P<0.05). CONCLUSION: Acupuncture may improve learning-memory ability in AD rats, which may be associated with its effects in reducing hippocampal Abeta13 42 expression and IL-1beta content and in up-regulating IL-2 level.

[Identification of Official Rhubarb Samples by Using PLS and Terahertz Time-Domain Spectroscopy].

The development of terahertz technology is attracting broad intention in recent years. The quality identification is important for the quality control of Chinese medicine production. In the present work, terahertz time-domain spectroscopy (THz-TDS) combined with partial least squares (PLS) were used for the identification model building and studied based on 41 official and unofficial rhubarb samples. First, the THz-TDS spectra of rhubarb samples were collected and were preprocessed by using chemometrics methods rather than transformed to absorption spectra. The identification models were then established based on the processed terahertz time domain spectra. The spectral preprocessing methods include Savitzky-Golay (S-G) first derivative, detrending, standard normal transformation (SNV), autoscaling, and mean centering. The identification accuracy of 90% was accomplished by using proper pretreatment methods, which was higher than the classified accuracy of 80% without any preprocessing for the time domain spectra. The component number of the PLS model was evaluated by leave-one-out cross-validation (LOOCV). The minimum values of the root-mean squared error of cross-validation (RMSECV) and root-mean squared error of prediction (RMSEP) were 0.076 6 and 0.169 0 by using mean centering method, respectively. The results of this work showed that the combination of terahertz time domain spectroscopy technology with chemometrics methods, as well as PLS can be applied for the recognition of genuine and counterfeit Chinese herbal medicines, as well as official and unofficial rhubarbs. The advantage of using terahertz time domain spectra directly with no transformation into absorption spectra is: (1) the thickness of samples could not be considered in the model establishment, and (2) the spectral processing was simplified. The proposed method based on the combination of THz-TDS and chemometrics proved to be rapid, simple, non-pollution and solvent free, suitable to be developed as a promising tool for quality control of many other Chinese herbal medicines.

Chemical profiling and cytotoxicity assay of bufadienolides in toad venom and toad skin.

ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom and toad skin have been widely used for treating various cancers in China. Bufadienolides are regarded as the main anticancer components of toad venom, but the difference on composition and anticancer activities of bufadienolides between toad venom and toad skin remains unclear. METHODS: Fractions enriched with free and conjugated bufadienolides were prepared from toad venom and toad skin. Bufadienolides in each fraction were comprehensively profiled by using a versatile UHPLC-TOF-MS method. Relative contents of major bufadienolides were determined by using three bufogenins and one bufotoxin as marker compounds with validated UHPLC-TOF-MS method. Furthermore, cytotoxicity of the fractions was examined by MTT assay. RESULTS: Two fractions, i.e., bufogenin and bufotoxin fractions (TV-F and TV-C) were isolated from toad venom, and one bufotoxin fraction (TS-C) was isolated from toad skin. Totally 56 bufadienolides in these three fractions were identified, and 29 were quantified or semi-quantified. Bufotoxins were identified in both toad venom and toad skin, whereas bufogenins exist only in toad venom. Bufalin-3-conjugated bufotoxins are major components in toad venom, whereas cinobufotalin and cinobufagin-3-conjugated bufotoxins are main bufotoxins in toad skin. MTT assay revealed potent cytotoxicity of all the fractions in an order of TV-F>TV-C>TS-C. CONCLUSIONS: Our study represents the most comprehensive investigation on the chemical profiles of toad venom and toad skin from both qualitative and quantitative aspects. Eight bufotoxins were identified in toad skin responsible for the cytotoxicity for the first time. Our research provides valuable chemical evidence for the appropriate processing method, quality control and rational exploration of toad skin and toad venom for the development of anticancer medicines.

Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers.

Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK.

Characterization of oxygenated metabolites of ginsenoside Rb1 in plasma and urine of rat.

Oxygenated metabolites have been suggested as the major circulating metabolites of ginsenosides. In the current study, 10 oxygenated metabolites of ginsenoside Rb1 in plasma and urine of rat following iv dose were characterized by comparison with chemically synthesized authentic compounds as quinquenoside L16 (M1 and M2), notoginsenoside A (M3), ginsenoside V (M4 and M7), epoxyginsenoside Rb1 (M5 and M9), notoginsenoside K (M6), and notoginsenoside C (M8 and M10), 9 of which were detected as in vivo metabolites for the first time. After oral administration of ginsenoside Rb1, M3, M4, and M7 were observed as major circulating metabolites and presented in the bloodstream of rat for 24 h. Characterization of the exact chemical structures of these circulating metabolites could contribute greatly to our understanding of chemical exposure of ginsenosides after consumption of ginseng products and provide valuable information for explaining multiple bioactivities of ginseng products.

Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy.

Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington's disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

Catechins and procyanidins of Ginkgo biloba show potent activities towards the inhibition of ß-amyloid peptide aggregation and destabilization of preformed fibrils.

Catechins and procyanidins, together with flavonoid glycosides and terpene trilactones, are three important categories of components in the standard extract of Ginkgo biloba leaves (EGb761). In this research, catechins and proanthocyanidins were found to exist in both the extract of Ginkgo leaves and Ginkgo products. By comparing with reference compounds, six of them were identified as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin and procyanidins B1 and B3. The activities of these polyphenols in the inhibition of Aß42 aggregation and the destabilization of preformed fibrils were evaluated using biochemical assays, which showed that all six of the polyphenols, as well as a fraction of the extract of Ginkgo biloba leaves (EGb) containing catechins and procyanidins, exerted potent inhibitory activities towards Aß42 aggregation and could also destabilize the performed fibrils. Catechins and procyanidins can therefore be regarded as the potent active constituents of EGb761 in terms of their inhibition of Aß42 aggregation and destabilization of the fibrils. Although quantitative mass spectroscopic analysis revealed that the catechins and procyanidins are only present in low concentrations in EGb761, these components should be studied in greater detail because of their potent inhibitory effects towards Aß42 aggregation and their ability to destabilize preformed fibrils, especially during the quality control of Ginkgo leaves and the manufacture of Ginkgo products.

Quantitative analysis of the flavonoid glycosides and terpene trilactones in the extract of Ginkgo biloba and evaluation of their inhibitory activity towards fibril formation of ß-amyloid peptide.

The standard extract of Ginkgo biloba leaves (EGb761) is used clinically in Europe for the symptomatic treatment of impaired cerebral function in primary degenerative dementia syndromes, and the results of numerous in vivo and in vitro studies have supported such clinical use. The abnormal production and aggregation of amyloid ß peptide (Aß) and the deposition of fibrils in the brain are regarded as key steps in the onset of Alzheimer's Disease (AD), and the inhibition of Aß aggregation and destabilization of the preformed fibrils represent viable approaches for the prevention and treatment of AD. Flavonoid glycosides and terpene trilactones (TTLs) are the two main components of EGb761 which represent 24 and 6% of the overall content, respectively. In our research, seven abundant flavonoid glycosides 1-7 were isolated from the extract of Ginkgo biloba leaves and characterized by spectroscopic analysis. Furthermore, an ultra-high performance liquid chromatography method was established for the simultaneous quantification of these seven flavonoids. The inhibitory activities of these flavonoids, as well as four TTLs, i.e., ginkgolides A, B, and C and bilobalide (compounds 8-11), were evaluated towards Aß42 fibril formation using a thioflavin T fluorescence assay. It was found that three flavonoids 1, 3 and 4 exhibited moderate inhibitory activities, whereas the other four flavonoids 2, 5, 6 and 7, as well as the four terpene trilactones, showed poor activity. This is the first report of the inhibition of Aß fibril formation of two characteristic acylated flavonoid glycosides 6, 7 in Ginkgo leaves, on the basis of which the structure-activity relationship of these flavonoids 1-7 was discussed.

Transformation of ginsenosides from notoginseng by artificial gastric juice can increase cytotoxicity toward cancer cells.

Multicomponent metabolic profile of notoginseng saponins in artificial gastric juice was qualitatively and quantitatively investigated, showing that ginsenosides were transformed via multiple pathways including deglycosylation, dehydration, hydration, and oxygenation. A total of 83 metabolites was identified by using UPLC-Q-TOF-MS, among which 16 new dammarane glycosides were further characterized by comparing with synthesized authentic compounds. Transformation time-course of notoginseng saponins in artificial gastric juice was quantitatively measured for the first time, showing rapid degradation of primary ginsenosides and concomitant formation of deglycosylation, hydration, and dehydration products. It was further demonstrated that the resultant metabolites exhibited enhanced cytotoxicity toward cancer cells. The extensive metabolism of ginsenosides within a transit time span in stomach, together with the formation of metabolites with diversified chemical structures possessing enhanced biological activities, indicated an important role of transformation in gastric juice in the systematic effects of ginsenosides.

Total ginsenosides of Radix Ginseng modulates tricarboxylic acid cycle protein expression to enhance cardiac energy metabolism in ischemic rat heart tissues.

To elucidate the underlying mechanism of cardio-protective activity of the total ginsenosides (TGS) of Radix Ginseng, proteomic analysis using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF-MS techniques was employed for identifying the underlying targets of TGS on improvement of the energy metabolism of isolated rat heart tissues perfused in Langendorff system under ischemia-reperfusion injury conditions. The image analysis results revealed 11 differentially expressed proteins in the TGS-treated heart tissues; these proteins, including LDHB and ODP-2, were found to be closely related to the function of tricarboxylic acid (TCA) cycle that plays pivotal roles in cardiac energy metabolism. It is thus concluded that improvement of cardiac energy metabolism via activating proteins in TCA cycle could be the major action pathway and targets of TGS activity against rat heart tissue injury.

Formation and conformation of baicalin-berberine and wogonoside-berberine complexes.

It is well-known that baicalin-berberine complex (1) precipitates in the water decoction of numerous Chinese Medicinal formulae containing Radix Scutellariae and Rhizoma Coptidis or Cortex Phellodendri. In the current study, ionic interaction between the carboxylate ion of baicalin and the quaternary ammonium ion of berberine was revealed to be responsible for the formation of 1 and wogonoside-berberine (2) by using FAB-MS and NMR titration experiments. In addition, nuclear Overhauser effect spectroscopy (NOESY) correlations observed in 1 and 2 suggested quite different conformation of the two complexes, which was further supported by the fact that the [α](D) of the canadine obtained by reduction of 1 is of an opposite sign to that obtained from 2. Partition coefficients (n-octanol/water) determination demonstrated 12-20 times larger partition coefficient of each complex (1, 2) than that of each single compound (baicalin, wogonoside, and berberine), indicating the significant role of the formation of the complex in the bioavailability enhancement of these pharmacologically active constituents.

Inhibition of P-glycoprotein leads to improved oral bioavailability of compound K, an anticancer metabolite of red ginseng extract produced by gut microflora.

Ginsenosides are hydrolyzed extensively by gut microflora after oral administration, and their metabolites are pharmacologically active against lung cancer cells. In this study, we measured the metabolism of various ginsenosides by gut microflora and determined the mechanisms responsible for the observed pharmacokinetic behaviors of its active metabolite, Compound K (C-K). The results showed that biotransformation into C-K is the major metabolic pathway of ginsenosides after the oral administration of the red ginseng extract containing both protopanaxadiol and protopanaxatriol ginsenosides. Pharmacokinetic studies in normal mice showed that C-K exhibited low oral bioavailability. To define the mechanisms responsible for this low bioavailability, two P-glycoprotein (P-gp) inhibitors, verapamil and cyclosporine A, were used, and their presence substantially decreased C-K's efflux ratio in Caco-2 cells (from 26.6 to <3) and significantly increased intracellular concentrations (by as much as 40-fold). Similar results were obtained when transcellular transport of C-K was determined using multidrug resistance 1 (MDR1)-overexpressing Madin-Darby canine kidney II cells. In MDR1a/b(-/-) FVB mice, its plasma C(max) and AUC(0-24h) were increased substantially by 4.0- and 11.7-fold, respectively. These increases appear to be due to slower elimination and faster absorption of C-K in MDR1a/b(-/-) mice. In conclusion, C-K is the major active metabolite of ginsenosides after microflora hydrolysis of primary ginsenosides in the red ginseng extract, and inhibition/deficiency of P-gp can lead to large enhancement of its absorption and bioavailability.

The in vitro structure-related anti-cancer activity of ginsenosides and their derivatives.

Panax ginseng has long been used in Asia as a herbal medicine for the prevention and treatment of various diseases, including cancer. The current study evaluated the cytotoxic potency against a variety of cancer cells by using ginseng ethanol extracts (RSE), protopanaxadiol (PPD)-type, protopanaxatriol (PPT)-type ginsenosides fractions, and their hydrolysates, which were prepared by stepwise hydrolysis of the sugar moieties of the ginsenosides. The results showed that the cytotoxic potency of the hydrolysates of RSE and total PPD-type or PPT-type ginsenoside fractions was much stronger than the original RSE and ginsenosides; especially the hydrolysate of PPD-type ginsenoside fractions. Subsequently, two derivatives of protopanaxadiol (1), compounds 2 and 3, were synthesized via hydrogenation and dehydration reactions of compound 1. Using those two derivatives and the original ginsenosides, a comparative study on various cancer cell lines was conducted; the results demonstrated that the cytotoxic potency was generally in the descending order of compound 3 > 20(S)-dihydroprotopanaxadiol (2) > PPD (1) > 20(S)-Rh2 > 20(R)-Rh2 ≈ 20(R)-Rg3 ≈ 20(S)-Rg3. The results clearly indicate the structure-related activities in which the compound with less polar chemical structures possesses higher cytotoxic activity towards cancer cells.

Ginseng protects rodent hearts from acute myocardial ischemia-reperfusion injury through GR/ER-activated RISK pathway in an endothelial NOS-dependent mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng (Panax ginseng C.A. Meyer) is widely used in Asian communities for treating cardiovascular diseases. However, the mechanism by which it protects the myocardium in ischemia-reperfusion (I/R) injury remains unclear. In this study, we aim to investigate whether a standardized ginseng extract (RSE) protects rodent hearts against I/R injury and if glucocorticoid and/or estrogen receptor-mediated activation of Akt and Erk1/2 (the reperfusion injury salvage kinase pathway, RISK) and subsequent nitric oxide (NO) synthesis signaling are involved in this effect. MATERIALS AND METHODS: Rats or gene-deleted mice were subjected to 30 min ischemia by occluding the left anterior descending coronary artery and 90 min reperfusion. Infarct size, serum level of creatine kinase (CK), lactate dehydrogenase (LDH), and NO, expression and phosphorylation of glucocorticoid receptor (GR), estrogen receptor (ER), phosphatidylinositol-3 kinase (PI3K), Akt, NO synthase (NOS), extracellular signal-regulated kinase (Erk) 1/2, p38, and c-Jun NH2 terminal kinases (JNK) were examined in rat or mice treated with or without RSE in the absence or presence of pharmacological inhibitors. RESULTS: RSE significantly reduced infarct size in a dose-dependent manner and reduced the incidence of arrhythmia, increased serum NO production, reduced serum activities of creatine kinase and lactate dehydrogenase. The infarct size reduction effect of RSE was abolished by RU468 (an inhibitor of GR), tamoxifen (an inhibitor of ER), LY294002 (an inhibitor of PI3K), Akt inhibitor IV (an inhibitor of Akt protein kinase), U0126 (an inhibitor of Erk1/2) and NG-nitro-l-arginine methyl ester hydrochloride (an inhibitor of NOS), but not actinomycin D (an inhibitor of transcription process). RSE also significantly increased the activation of GR/ER, PI3K-Akt-eNOS cascades and Erk1/2 signaling in rat heart. However, RSE did not markedly reduce infarct size in endothelium NOS(-/-) mice. This differs from its effect in inducible NOS(-/-) and wild type mice, suggesting that endothelium NOS is required for the beneficial effect of RSE on the heart. CONCLUSION: Our findings showed for the first time that RSE protects hearts subjected to acute I/R injury and the infarct size reduction effect of RSE is associated with GR and/or ER-mediated Akt and Erk1/2 activation in an endothelium NOS-dependent manner.

Total ginsenosides increase coronary perfusion flow in isolated rat hearts through activation of PI3K/Akt-eNOS signaling.

BACKGROUND: Ginseng is the most popular herb used for treatment of ischemic heart diseases in Chinese community; ginsenosides are considered to be the major active ingredients. However, whether ginsenosides can enhance the coronary artery flow of ischemic heart and, if so, by what mechanisms they do this, remains unclear. METHODS: Isolated rat hearts with ischemia/reperfusion injury in Langendorff system were employed for examining the effect of total ginsenosides (TGS) on coronary perfusion flow (CPF). In addition, human aortic endothelial cells (HAECs) were used for mechanistic study. Levels of various vasodilative molecules, intracellular calcium concentration ([Ca²+](i)), and expressions and activation of proteins involving regulation of nitric oxide (NO) signaling pathways in heart tissues and HAECs were determined. RESULTS: TGS dose-dependently and significantly increased CPF and improved systolic and diastolic function of the ischemia/reperfused rat heart, while inhibitors of NO synthase (NOS), soluble guanylate cyclase (sGC), heme oxygenase (HO), cyclooxygenase (COX), and potassium channel abolished the vasodilation effect of TGS. Positive control verapamil was effective only in increasing CPF. TGS elevated levels of NO and 6-keto-prostaglandin F1α, a stable hydrolytic product of prostacyclin I2 (PGI2), in both coronary effluents and supernatants of HAECs culturing medium, and augmented [Ca²+](i) in HAECs. TGS significantly up-regulated expression of phosphoinositide 3-kinase (PI3K) and phosphorylations of Akt and endothelial NOS (eNOS) as well. CONCLUSIONS: TGS significantly increased CPF of ischemia/reperfused rat hearts through elevation of NO production via activation of PI3K/Akt-eNOS signaling. In addition, PGI2, EDHF and CO pathways also partially participated in vasodilation induced by TGS.