Antidiarrheal and antispasmodic activities of Trillium govanianum rhizomes extract: involvement of calcium channel blockade.

The antidiarrheal effect of methanolic extract of Trillium govanianum Wall. ex D. Don (Melanthiaceae alt. Trilliaceae) was studied at doses of 12.5, 25, and 50 mg/kg in different animal models of diarrhea including castor oil (6 mL/kg), magnesium sulfate (2 gm/kg), sodium picosulfate (2 mL/kg) and lactitol (0.25 mL/kg). The antispasmodic effect of T. govanianum was studied on isolated rabbit's jejunum, using acetylcholine as tissue stabiliser and verapamil as calcium channel blocker. T. govanianum attenuated the diarrhea by producing a significant decrease in the number and weight of stool, and an increase in stool latency time. T. govanianum completely inhibited both spontaneous as well as high potassium induced contractions of isolated rabbit's jejunum, which was analogous to verapamil. Moreover, T. govanianum produced a right shift in calcium concentration response curve, confirming its calcium channel blocking activity. These findings provide scientific ground to its medicinal use in diarrhea and gut spasms.

Anti-allergic activities of Umbelliferone against histamine- and Picryl chloride-induced ear edema by targeting Nrf2/iNOS signaling in mice.

BACKGROUND: The current study was aimed to investigate the anti-allergic activities of the Umbelliferone (UMB) against the acute Histamine and chronic Picryl chloride (PiCl)-induced allergy in mice. UMB is a coumarin derivative (isolated from Angelica decursiva) found in various parts of the plants such as flowers, roots and, stems isolated from the plants of Umbelliferae family. METHODS: The UMB (1, 10, 50 mg/kg) was administered intraperitoneally (i.p) half an h before or 2 h after the induction of allergic ear edema. The acute ear edema was induced by histamine (intradermally, i.d), while the chronic ear edema was induced by painting the PiCl (sensitized with the toluene) on the ear. The antioxidants and oxidative stress markers were assessed. The histological changes were assessed using Hematoxylin and eosin (H and E) and giemsa staining. The immunohistochemistry studies were performed to assess the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) and inducible nitric oxide synthase (iNOS). The data was analyzed using one-way ANOVA tests followed by Tukey's test with p < 0.05 was chosen as criteria for statistical significance. RESULTS: UMB treatment markedly reduced the allergic ear edema and ear weight compared to the negative control. Furthermore, the UMB attenuated the oxidative stress markers, while induced the antioxidants enzymes. Similarly, the UMB treatment significantly attenuated the serum immunoglobulin E (IgE) level. The UMB treatment markedly improved the histological parameters using H and E staining and Giemsa staining. The UMB administration induced the Nrf2 expression, while attenuated the iNOS expression. Furthermore, the computational analysis was performed to assess the interaction of the UMB with the various protein targets and to determine the mechanism of interaction with the target proteins. CONCLUSION: In conclusion, the UMB treatment significantly alleviated the allergic symptoms, attenuating the oxidative stress, improved the histological features using in vivo and computational approaches.

Evaluation of the anti-diarrheal effects of the whole plant extracts of Cuscuta reflexa Roxb in pigeons.

BACKGROUND: Cuscuta reflexa (dodder) belonging to the family Convolvulaceae has many ethno-medicinal uses such as antidiarrheal and antiemetic. This plant has been employed to treat diarrhea, where the antidiarrheal use of this plant is well established in different communities around the world without scientific bases. In addition, the antibacterial, anthelmintic, anticholinergic, and antihistaminic effects of this parasitic vine are partly responsible for the folkloric antidiarrheal use of this plant. In the present study, the antidiarrheal activity of C. reflexa was evaluated in pigeons (Columba livia) using the juice (JCR), aqueous (CRAE), and methanol (CRME) extracts. METHODS: The antidiarrheal effect of C. reflexa was evaluated using different reported research models, with few modifications. In pigeons, diarrhea was induced by administration of castor oil (6 mL/kg, PO), ampicillin (250 mg/kg, IP), magnesium sulfate (2 gm/kg, PO), and cisplatin (6 mg/kg, IV). In these experiments, loperamide (2 mg/kg, IM) was used as a positive control, whereas JCR (1 mL/kg (1%) and 1 mL/kg (2%), CRAE (50, 100 and 200 mg/kg) and CRME (50, 100 and 200 mg/kg) were administered intramuscularly at different doses into each pigeon in the test groups. RESULTS: In addition to cisplatin-induced diarrhea, all paradigms tested gave significant results (P < 0.01). The JCR, at different doses, exhibited a significant (p < 0.01) a dose-dependent antidiarrheal effect on both the frequency and the onset of diarrhea. Similarly, CRAE and CRME, at doses of 100 and 200 mg/kg, showed considerable (p < 0.001) inhibition against the onset and frequency of diarrhea. On the other hand, JCR, CRAE, and CRME exerted significant effects (p < 0.001) on the percentage inhibition (PI) of diarrhea and gastrointestinal charcoal transit in a dose-dependent manner. In this respect, the maximum PI (p < 0.01) of JCR, CRAE, and CRME in different experimental paradigms was 43.13, 49.14, and 55.99 %, respectively. CONCLUSIONS: Taken all together, results from this study reveal that the juice, aqueous, and methanol extract of C. reflexa exhibit significant anti-motility and anti-secretory potential. These findings may explain the medicinal use of C. reflexa in folk medicine as an antidiarrheal medicinal plant.

Therapeutic potential of curcumin for multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS), characterized by demyelination, neuronal injury, and breaching of the blood-brain barrier (BBB). Epidemiological studies have shown that immunological, genetic, and environmental factors contribute to the progression and development of MS. T helper 17 (Th17) cells are crucial immunological participant in the pathophysiology of MS. The aberrant production of IL-17 and IL-22 by Th17 cells crosses BBB promotes its disruption and interferes with transmission of nerve signals through activation of neuroinflammation in the CNS. These inflammatory responses promote demyelination through transcriptional activation of signal transducers and activators of transcription-1 (STAT-1), nuclear factor kappa-B (NF-κB), matrix metalloproteinases (MMPs), interferon ϒ (IFNϒ), and Src homology region 2 domain-containing phosphatase-1 (SHP-1). B cells also contribute to disease progression through abnormal regulation of antibodies, cytokines, and antigen presentation. Additionally, oxidative stress has been known as a causative agent for the MS. Curcumin is a hydrophobic yellowish diphenolic component of turmeric, which can interact and modulate multiple cell signaling pathways and prevent the development of various autoimmune neurological diseases including MS. Studies have reported curcumin as a potent anti-inflammatory, antioxidant agent that could modulate cell cycle regulatory proteins, enzymes, cytokines, and transcription factors in CNS-related disorders including MS. The current study summarizes the reported knowledge on therapeutic potential of curcumin against MS, with future indication as neuroprotective and neuropharmacological drug.

Phytochemical analysis and effects of Pteris vittata extract on visual processes.

The present study was designed to explore the possible effects of Pteris vittata on visual sensitivity, ERG waves, and other components of the visual system. Electrophysiological techniques including electroretinography (ERG) were used in the present study. The phytochemical composition of the extract was investigated using liquid chromatography-mass spectrometry (LC-MS) techniques. The results indicated that the extract significantly augmented dark- and light-adapted ERG b-wave amplitude. Furthermore, these findings showed that P. vittata extract does not have Gamma-aminobutyric acid receptor antagonistic activity but may function as a retinal neural antagonist in bullfrog retina. P. vittata extract improved the visual sensitivity by 0.8 log unit of light intensity, and reduced the regeneration time for rhodopsin. The six main peaks obtained through LC-MS were identified as flavonoids. Based on these results, it was concluded that P. vittata extract or its constituents may be used to treat eye diseases.

Separation of triterpenoid saponins from the root of Bupleurum falcatum by counter current chromatography: the relationship between the partition coefficients and solvent system composition.

A separation method using counter current chromatography coupled with an evaporative light-scattering detection system was developed to purify five triterpenoid saponins from the roots of Bupleurum falcatum. The methanol extract was loaded onto a Diaion® HP20 column and fractionated by a methanol and water gradient elution. The saikosaponin-enriched fraction was obtained by elution with 100% methanol. The two-phase solvent systems used for separation were composed of chloroform/methanol/isopropanol/water at a volume ratio of 60:60:1:60 and 6:6:1:6. The relationship between the isopropanol ratio of each phase and the partition coefficients of the target compounds was investigated by calculating partition coefficient by high-performance liquid chromatography and measuring the accurate composition of each phase by (1) H NMR spectroscopy. Each fraction obtained was collected and dried, which yielded the following five saikosaponins from 700 mg of injected sample: saikosaponin B1 (8.7 mg), saikosaponin A (86 mg), saikosaponin B3 (17 mg), saikosaponin B2 (41 mg), and saikosaponin C (33 mg). Saikosaponin A showed the most potent cytotoxicity against human cancer cells (gastric cancer, AGS cells; breast cancer, MCF-7 cells; and hepatoma, HepG2 cells) after 24 h. The IC50 values for the above three cell types were 34.6, 33.3, and 23.4 µmol/L, respectively.

Anti-inflammatory properties of samidin from Seseli resinosum through suppression of NF-κB and AP-1-mediated-genes in LPS-stimulated RAW 264.7 cells.

Seseli is a herb widely used for its anti-inflammation, anti-flatulence and various other healing properties. In the present study, we investigated the effects of samidin on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The results demonstrated that samidin significantly inhibited the production of nitric oxide, as well as the gene expression levels of inducible nitric oxide synthase and cyclooxygenase-2. The results from an electrophoretic mobility shift assay illustrated that samidin significantly suppressed NF-κB and AP-1 DNA-binding affinity. In addition, both the NF-κB subunit p65 and the AP-1-related c-jun were markedly inhibited by samidin. The time course experiment demonstrated that samidin showed significant inhibitory effect on p38 and JNK activation. Furthermore, tumor necrosis factor-α mRNA level were remarkably down-regulated by samidin in LPS-stimulated macrophages based on quantitative-real-time polymerase chain reaction. Our results suggested that samidin has a potential to be developed as a therapeutic agent for various inflammatory diseases.

Anti-hyperalgesic and anti-allodynic activities of capillarisin via suppression of inflammatory signaling in animal model.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia capillaris has widespread traditional and pharmacological applications such as analgesic, anti-inflammatory, anti-pyretic, enhance immunity and anti-tumor activity properties. To evaluate the pharmacological activities of this plant, capillarisin, one of the potent constituent of Artemisia capillaris was studied based on anti-hyperalgesic and anti-allodynic effects with detailed mechanism. It can be assumed that measurement of anti-nociceptive effects of capillarisin is one of the parameter for the evaluation of this herb. Capillarisin has extensive pharmacological properties and has been considered to have promising ant-inflammatory and anti-nociceptive activities. The aim of the current study is to investigate the effect of capillarisin and underlying molecular mechanisms of action in preventing acute and subchronic inflammatory pain. MATERIALS AND METHODS: The inflammatory pain was induced after 40 min or 1h of administration of vehicle, 70% EtOH extract of Artemisia capillaris (100mg/kg) or capillarisin (20 and 80 mg/kg) by intraplantar (i.p.l.) injections of CFA and carrageenan in ICR mice, respectively. Mechanical hyperalgesia and allodynia were evaluated in both acute and subchronic models. Further analysis was performed in CFA-induced mice exploring various molecular and signaling pathways such as NF-κB, AP-1, and ERK-CREB involved in the persistent pain sensations. RESULTS: In acute model, mechanical hyperalgesia and allodynia were evaluated after every 2h until 6h of CFA and after 4h of carrageenan injections. Whereas, in subchronic inflammatory pain model, mechanical hyperalgesia and paw edema were measured after 4h of CFA injection and every day after 4h of daily treatment until 5 days with interval of day four in order to assess the tolerance effect of capillarisin. Further analysis was performed in CFA-induced mice exploring various molecular and signaling pathways such as NF-κB, AP-1 and ERK-CREB involved in the persistent of pain sensations. Pre-treatment of capillarisin strongly inhibited NF-κB mediated genes (iNOS, COX-2), involved in pain. The plasma leading nitrite production was significantly reduced by capillarisin. Moreover, i.p. administration of capillarisin markedly suppressed the adenosine 5׳-triphosphate (ATP) in plasma and substance P in CFA-induced paw tissue. CONCLUSIONS: The present study indicates that capillarisin possessed promising anti-hyperalgesic and anti-allodynic effects through the inhibition of various inflammatory pain signaling, suggesting that capillarisin constitutes a significant component for the treatment of inflammatory pain.

Ginsenosides from Korean red ginseng inhibit matrix metalloproteinase-13 expression in articular chondrocytes and prevent cartilage degradation.

Among the mammalian matrix metalloproteinases (MMPs), MMP-1, -3 and -13 are collagenases. Particularly, MMP-13 is important for the degradation of major collagens in cartilage under certain pathological conditions such as osteoarthritis. To establish a potential therapeutic strategy for cartilage degradation disorders, the effects of 11 ginseng saponins (ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rg5, Rk1 and F4) on MMP-13 induction were examined in a human chondrocyte cell line, SW1353. Among these, several saponins including ginsenoside Rc, Rd, Rf, Rg3 and F4 were found to inhibit MMP-13 expression in IL-1ß-treated SW1353 cells at non-cytotoxic concentrations (1-50 µM). The most prominent inhibitors were ginsenosides F4 and Rg3. Ginsenoside F4 inhibited MMP-13 expression 33.5% (P<0.05), 57.9% (P<0.01) and 90.0% (P<0.01) at 10, 30 and 50 µM, respectively. Significantly, ginsenoside F4 was found to strongly inhibit activation of p38 mitogen-activated protein kinase in signal transduction pathways (86.6 and 100.0% inhibition at 30 and 50 µM, P<0.01). The MMP-13 inhibitory effect was also supported by the finding that ginsenosides F4 and Rg3 reduced glycosaminoglycan release from IL-1α-treated rabbit joint cartilage culture to some degree. Taken together, these results indicate that several ginsenosides inhibit MMP-13 expression in IL-1ß-treated chondrocytes. Ginsenoside F4 and Rg3 blocked cartilage breakdown in rabbit cartilage tissue culture. Thus, it is suggested that certain ginsenosides have therapeutic potential for preventing cartilage collagen matrix breakdown in diseased tissues such as those found in patients with arthritic disorders.

Application of stepwise gradients in counter-current chromatography: a rapid and economical strategy for the one-step separation of eight coumarins from Seseli resinosum.

The targeted purification of compounds with a broad polarity range from traditional medicinal plants is a big challenge for counter-current chromatography (CCC). Gradient elution was introduced in CCC to address this problem. However, once a suitable solvent system is selected, the separation process requires optimization of operational parameters. The present study was conducted to optimize various operational parameters to integrate the flow rate and solvent gradients for the rapid isolation of eight coumarins from Seseli resinosum in a single run. An increase in the system temperature from 15°C to 35°C increased the stationary phase retention and solubility of the sample, whereas the operation time and viscosity of the system were decreased. The high purity of each compound was ensured by collecting the fractions from the main peaks while all the shoulder peaks were mixed and separated under the same conditions with semi-preparative CCC. GC-FID was used to analyze the components of each phase, which was prepared without presaturation to save the time and solvent consumption. Finally, eight coumarins were purified, including (1) d-laserpitin, (2) (3'S,4'S)-3'-angeloyloxy-4'-hydroxy-3',4'-dihydroseselin, (3) (+)-samidin, (4) (3'S,4'S)-3'-acetoxy-4'-angeloyloxy-3',4'-dihydroseselin, (5) deltoin (6), calipteryxin, (7) (3'S,4'S)-3',4'-disenecioyloxy-3',4'-dihydroseselin, and (8) (-)-anomalin. The present technique has successfully accomplished the goal of one-step separation of these compounds with high purity and recovery in an economic and time efficient manner.

Mechanism underlying anti-hyperalgesic and anti-allodynic properties of anomalin in both acute and chronic inflammatory pain models in mice through inhibition of NF-κB, MAPKs and CREB signaling cascades.

The numerous mediators of pain and inflammation are products of injury-induced gene expression that lead to changes in the nervous system and immune responses. These multiple molecules and mechanisms suggest novel strategies that could be used for analgesic drug development. The present study investigated the possible anti-hyperalgesic effects of anomalin in complete Freund's adjuvant (CFA)-induced acute and chronic inflammatory pain models. Acute pretreatment of mice with anomalin (10 and 50mg/kg, i.p.) produced a significant anti-nociceptive effect against CFA- and carrageenan-induced mechanical hyperalgesia and allodynia. In a chronic pain model, administration of anomalin inhibited CFA-induced hyperalgesia, and it did not cause any apparent toxicity. Another set of experiments observed that anomalin inhibited CFA- and carrageenan-induced paw edema in acute and chronic models. To elucidate the molecular mechanism underlying the anti-nociceptive effect of anomalin, the various pain signaling pathways [NF-κB, cAMP response element-binding protein (CREB), and mitogen activated protein kinase (MAPKs)/AP-1] that are involved were examined. Intraperitoneal (i.p.) pretreatment of anomalin exhibited potent inhibitory effects on direct mediators of hyperalgesia (iNOS and COX-2). The release of CFA-induced plasma nitrite and paw tissue hyperalgesic cytokine (TNF-α) was reduced remarkably. In addition, the adenosine 5'-triphosphate (ATP) in plasma and substance P (SP) in paw tissue were markedly suppressed by anomalin. These results demonstrate that anomalin exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of the NF-κB, CREB, and MAPKs/AP-1 signaling pathways.

Molecular mechanism of capillarisin-mediated inhibition of MyD88/TIRAP inflammatory signaling in in vitro and in vivo experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia capillaris Thunberg (Compositae) have been used as traditional medicine as a diuretic, liver protective agent, and for amelioration of inflammatory and analgesic disorders. The present study was carried out to establish the scientific rationale for treating inflammation and to find active principles from A. capillaris. The aim of the present study is to investigate the possible anti-inflammatory mechanism of the major component (capillarisin) isolated from A. capillaris via inhibition of MyD88/TIRAP inflammatory signaling both in vitro and in vivo models. MATERIALS AND METHODS: The nitrite, PGE(2), and TNF-α productions were evaluated by Griess reagent and ELISA kits. The protein and mRNA expression levels were investigated by Western blot and RT-PCR. The NF-κB and AP-1 DNA-binding was performed by electrophoretic mobility shift assay. The CFA- and carrageenan-induced paw edema was performed in ICR mice in which 20 and 80 mg/kg body weight of capillarisin was administered intraperitoneally (i.p.). RESULTS: The results demonstrated that pretreatment with capillarisin effectively inhibited the LPS-induced activation of NF-κB, Akt, and MAP kinase-activated inflammatory genes, which is mediated by MyD88 and TIRAP. Treatment with capillarisin reduced the mRNA and protein levels of iNOS and COX-2 in RAW 264.7 cells as assessed by RT-PCR and Western blot. Capillarisin suppressed LPS-induced inhibitory kappa kinase (IKK) phosphorylation and the degradation of inhibitory kappa B (IκBα) and prevented the nuclear translocation of p65 and p50. Capillarisin also exhibited a promising inhibitory effect on the LPS-induced NF-κB and AP-1 DNA binding activity based on an electrophoretic mobility shift assay. The LPS-induced activation of p-JNK, p-p38, p-ERK, and p-Akt was significantly inhibited. In addition, the TNF-α level in the media was effectively reduced by capillarisin. In vivo experimental analysis revealed that capillarisin (20 and 80 mg/kg, i.p.) inhibited complete Freund's adjuvant (CFA)-and carrageenan-induced paw edema, nitrite production in plasma, and TNF-α, a pro-inflammatory cytokine production. CONCLUSION: The results presented here demonstrate that capillarisin has consistent anti-inflammatory properties and acts by inhibiting inflammatory mediators in in vitro and in vivo experimental models, and suggest its potential utility in the control of inflammatory disorders.

State-of-the-art separation of ginsenosides from Korean white and red ginseng by countercurrent chromatography.

Ginseng (Panax ginseng C. A. Meyer) has been one of the most popular herbs used for nutritional and medicinal purposes by the people of eastern Asia for thousands of years. Ginsenosides, the mostly widely studied chemical components of ginseng, are quite different depending on the processing method used. A number of studies demonstrate the countercurrent chromatography (CCC) separation of ginsenosides from several sources; however, there is no single report demonstrating a one-step separation of all of these ginsenosides from different sources. In the present study, we have successfully developed an efficient CCC separation methodology in which the flow-rate gradient technique was coupled with a new solvent gradient dilution strategy for the isolation of ginsenosides from Korean white (peeled off dried P. ginseng) and red ginseng (steam-treated P. ginseng). The crude samples were initially prepared by extraction with butanol and were further purified with CCC using solvent gradients composed of methylene chloride-methanol-isopropanol-water (different ratios, v/v). Gas chromatography coupled with flame ionization detector was used to analyze the components of the two-phase solvent mixture. Each phase solvent mixture was prepared without presaturation, which saves time and reduces the solvent consumption. Finally, 13 ginsenosides have been purified from red ginseng with the new technique, including Rg1, Re, Rf, Rg2, Rb1, Rb2, Rc, Rd, Rg3, Rk1, Rg5, Rg6, and F4. Meanwhile, eight ginsenosides have been purified from white ginseng, including Rg1, Re, Rf, Rh1, Rb1, Rb2, Rc, and Rd by using a single-solvent system. Thus, the present technique could be used for the purification of ginsenosides from all types' ginseng sources. To our knowledge, this is the first report involving the separation of ginsenoside Rg2 and Rg6 and the one-step separation of thirteen ginsenosides from red ginseng by CCC.

Rescue of PINK1 protein null-specific mitochondrial complex IV deficits by ginsenoside Re activation of nitric oxide signaling.

PINK1, linked to familial Parkinson's disease, is known to affect mitochondrial function. Here we identified a novel regulatory role of PINK1 in the maintenance of complex IV activity and characterized a novel mechanism by which NO signaling restored complex IV deficiency in PINK1 null dopaminergic neuronal cells. In PINK1 null cells, levels of specific chaperones, including Hsp60, leucine-rich pentatricopeptide repeat-containing (LRPPRC), and Hsp90, were severely decreased. LRPPRC and Hsp90 were found to act upstream of Hsp60 to regulate complex IV activity. Specifically, knockdown of Hsp60 resulted in a decrease in complex IV activity, whereas antagonistic inhibition of Hsp90 by 17-(allylamino) geldanamycin decreased both Hsp60 and complex IV activity. In contrast, overexpression of the PINK1-interacting factor LRPPRC augmented complex IV activity by up-regulating Hsp60. A similar recovery of complex IV activity was also induced by coexpression of Hsp90 and Hsp60. Drug screening identified ginsenoside Re as a compound capable of reversing the deficit in complex IV activity in PINK1 null cells through specific increases of LRPPRC, Hsp90, and Hsp60 levels. The pharmacological effects of ginsenoside Re could be reversed by treatment of the pan-NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) and could also be reproduced by low-level NO treatment. These results suggest that PINK1 regulates complex IV activity via interactions with upstream regulators of Hsp60, such as LRPPRC and Hsp90. Furthermore, they demonstrate that treatment with ginsenoside Re enhances functioning of the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis in PINK1 null neurons by restoring NO levels, providing potential for new therapeutics targeting mitochondrial dysfunction in Parkinson's disease.

Rational development of a selection model for solvent gradients in single-step separation of ginsenosides from Panax ginseng using high-speed counter-current chromatography.

A new model of solvent gradients selection was rationally developed for the preparative separation of target compounds. The solvent gradients were selected based on a three-stage screening process where stationary phase retention was ensured by introducing a new parameter termed as the phase ratio. The phase ratio was calculated after mixing the upper phase of a solvent system with the lower phase of a different solvent system (1:1, v/v). The developed model was applied to the one-step separation of eight ginsenosides from Panax ginseng. Three gradients were selected on the basis of new model and eight ginsenosides, Rb(1), Rb(2), Rc, Rd, Re, Rg(1), Rf, and Rh(1), were efficiently separated by high-speed counter-current chromatography coupled with evaporative light scattering detector. The structures of all compounds were characterized by electrospray-ionization mass spectrometry and nuclear magnetic resonance spectroscopy.

Development of a rapid and convenient method to separate eight ginsenosides from Panax ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detection.

Ginsenosides exhibit diverse biological activities and are major well-known components isolated from the radix of Panax ginseng C.A. Meyer. In the present work, a rapid and facile method for the separation and purification of eight ginsenosides from P. ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detector (HSCCC-ELSD) was successfully developed. The crude samples for HSCCC separation were first purified from ginseng extract using a macroporous resin; the extract was loaded onto a Diaion-HP20 column and fractionated by methanol and water gradient elution. The ginsenosides-protopanaxadiol (PPD) and protopanaxatriol (PPT) fractions were subsequently eluted with 65 and 80% methanol and water gradient elution, respectively. Furthermore, these two fractions were separated by HSCCC-ELSD. The two-phase solvent system used for separation was composed of chloroform/methanol/water/isopropanol at a volume ratio of 4:3:2:1. Each fraction obtained was collected and dried, yielding the following eight ginsenosides: Rg(1), Re, Rf, Rh(1), Rb(1), Rc Rb(2) and Rd. The purity of these ginsenosides was greater than 97% as assessed by HPLC-ELSD, and their structures were characterized by electrospray-ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. This is the first report regarding the separation of the ginsenosides Rh(1), Rb(2) and Rc from P. ginseng by HSCCC.