Qi Chang

Antitumor activity and mechanisms of action of total glycosides from aerial part of Cimicifuga dahurica targeted against hepatoma

BackgroundMedicinal plant is a main source of cancer drug development. Some of the cycloartane triterpenoids isolated from the aerial part of Cimicifuga dahurica showed cytotoxicity in several cancer cell lines. It is of great interest to examine the antiproliferative activity and mechanisms of total triterpenoid glycosides of C. dahurica and therefore might eventually be useful in the prevention or treatment of Hepatoma.MethodsThe total glycosides from the aerial part (TGA) was extracted and its cytotoxicity was evaluated in HepG2 cells and primary cultured normal mouse hepatocytes by an MTT assay. Morphology observation, Annexin V-FITC/PI staining, cell cycle analysis and western blot were used to further elucidate the cytotoxic mechanism of TGA. Implanted mouse H22 hepatoma model was used to demonstrate the tumor growth inhibitory activity of TGA in vivo.ResultsThe IC50 values of TGA in HepG2 and primary cultured normal mouse hepatocytes were 21 and 105 μg/ml, respectively. TGA induced G0/G1 cell cycle arrest at lower concentration (25 μg/ml), and triggered G2/M arrest and apoptosis at higher concentrations (50 and 100 μg/ml respectively). An increase in the ratio of Bax/Bcl-2 was implicated in TGA-induced apoptosis. In addition, TGA inhibited the growth of the implanted mouse H22 tumor in a dose-dependent manner.ConclusionTGA may potentially find use as a new therapy for the treatment of hepatoma.

Neuroprotective effects of Total Saikosaponins of Bupleurum yinchowense on corticosterone-induced apoptosis in PC12 cells

ETHNOPHARMACOLOGICAL RELEVANCEnThe root of Bupleurum yinchowense Shan et Y. Li, a well-known medicinal plant in China, was originally documented in the Shennongs Herbal, which is the oldest Chinese materia medica monographs. It has the action of soothing liver and relieving constraint for improving symptoms of emotional instability such as depression, anxiety and phobia. The in vivo experiment of our previous study has showed an efficacy of Total Saikosaponins (TSS) from Bupleurum yinchowense in acute stress and chronic unpredictable mild stress models. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of TSS on corticosterone-induced apoptosis in PC12 cells. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of TSS.nnnMATERIALS AND METHODSnThe PC12 cells were treated with 250 μM corticosterone in the absence or presence of different concentrations of TSS for 24 h, then the cell viability, lactate dehydrogenase (LDH) release, Hoechst 33342 and propidium iodide (PI) double staining and the DNA fragmentation of the apoptotic PC12 cells were determined. The mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca(2+) ([Ca(2+)]i) concentration and western blot analysis of caspase-3, glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible proteins 153 (GADD-153), X-box DNA-binding protein-1 (XBP-1), Bax, Bcl-2 were investigated.nnnRESULTSnPretreatment of PC12 cells with TSS (3.125, 6.25, 12.5, 25 μg/ml) partly reversed corticosterone-induced neurotoxicity in a dose dependent manner. TSS (25 =g/ml) reversed the increase of dead cells in the Hoechst 33342 stain, the accumulation in LDH leakage and the number of TUNEL positive cells induced by corticosterone to PC12 cells. Moreover, the cytoprotection of TSS was proved to be associated with the homeostasis of intracellular Ca(2+), the stabilization of ER stress via the down-regulation of GRP78, GADD-153, XBP-1, and the restoration of mitochondrial function, which included mPTP, MMP and caspase-3 activity. Furthermore, TSS (25 μg/ml) markedly ameliorated up-regulation of Bax and down-regulation of Bcl-2 in corticosterone-induced PC12 cells.nnnCONCLUSIONnThe result depicted that antidepressant-like effect of TSS in vivo may be associated with the cytoprotection of neuron, and the neuroprotective mechanisms were correlated with inhibiting the ER stress and the mitochondrial apoptotic pathways.

The effect of Acorus gramineus on the bioavailabilities and brain concentrations of ginsenosides Rg1, Re and Rb1 after oral administration of Kai-Xin-San preparations in rats

AIM OF THE STUDYnTo investigate the effect of Acorus gramineus (AG), a supposed delivering servant according to traditional Chinese medicine principles governing multi-herb formula preparation and formulation, on facilitating the uptake of ginsenosides Rg1, Re and Rb1 to the brain after oral administration of Kai-Xin-San (KXS) preparations.nnnMATERIALS AND METHODSnGinseng extracts or KXS with or without AG were administered to rats for pharmacokinetic study and mice for behaviour tests at a dose of 3 g ginseng per kg. The concentrations of ginsenosides in plasma and brain were determined by an LC-MS/MS method, whilst the effects of preparations on spatial learning were evaluated using the Morris water maze test.nnnRESULTSnKXS in the presence of AG tended to significantly reverse the learning impairment induced by scopolamine. The presence of AG in the KXS formula led to increases in the initial absorption rate and extent of Rg1 and Re in terms of Cmax1 and AUC(0-3h) compared to KXS without AG. Although KXS were found to increase the bioavailabilities and brain concentrations of ginsenosides relative to ginseng extract, the brain-to-plasma AUC(0-12h) ratios appeared not to be affected.nnnCONCLUSIONSnThe results suggested that the presence of AG in the KXS formula promoted the initial absorption of ginsenosides Rg1 and Re in the gastrointestinal tract, but unlikely affected the brain-to-plasma AUC ratios.

Neuroprotective effect of water extract of Panax ginseng on corticosterone-induced apoptosis in PC12 cells and its underlying molecule mechanisms

ETHNOPHARMACOLOGICAL RELEVANCEnThe root of Panax ginseng C.A. Meyer (Family Araliaceae) is an important medicinal plant which has been employed as a panacea for more than 2,000 years in China. It has the actions of invigorating primordial qi, recovering pulse and desertion, engendering liquid, and calming spirit. The water extract of Panax ginseng (WEG) has been used to treat kinds of central nervous system disorders, such as depression, insomnia, Alzheimer׳s disease and Parkinson׳s disease. Our previous work has demonstrated that WEG possessed antidepressant-like activities in both acute and chronic stress models of depression. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of WEG on corticosterone-induced apoptosis. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of WEG.nnnMATERIALS AND METHODSnThe PC12 cells were treated with 250 μmol/L corticosterone in the absence or presence of WEG for 24h, then 3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) detection, Hoechst33342 staining and TUNEL staining were investigated to confirm the neuroprotection of WEG. Then, mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca(2+) ([Ca(2+)]i), reactive oxygen species (ROS) concentration, and the expression level of glucocorticoid receptor (GR), heat shock protein 90 (Hsp90), histone deactylase 6 (HDAC6), glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible protein 153 (GADD153), X-box DNA-binding protein-1 (XBP-1), caspase-12, cytochrome C, inhibitor of caspase-activated deoxyribonuclease (ICAD), caspase-3 and caspase-9 were assessed by Western Blot analysis to understand the molecule mechanisms of neuroprotection of WEG.nnnRESULTSnWEG partly reversed corticosterone-induced damage in PC12 cells, which increased cell viability, decreased LDH release, and attenuated corticosterone-induced apoptosis as compared with the corticosterone-treated group. Mechanistically, compared with the corticosterone-treated group, WEG strongly attenuated [Ca(2+)]i overload and ROS level, and restored mitochondrial function, including mPTP and MMP. Furthermore, WEG strongly up-regulated the expression of GR and HDAC6, and down-regulated the expression of Hsp90, cytochrome C, ICAD, caspase-3, caspase-9 as well as endoplasmic reticulum (ER) stress-related proteins, such as GADD153, GRP78, XBP-1, and caspase-12.nnnCONCLUSIONnWEG possessed neuroprotection against corticosterone-induced damage in PC12 cells, and the underlying molecule mechanisms was depended on the intervening of HDAC6 and HSP90 of the GR-related function proteins, and subsequent restoration of ER and mitochondria functions.

Different pharmacokinetics of the two structurally similar dammarane sapogenins, protopanaxatriol and protopanaxadiol, in rats

Dammarane Sapogenins (DS), with main ingredients of protopanaxatriol (PPT, 33%) and protopanaxadiol (PPD, 16%), is an alkaline hydrolyzed product of ginsenosides and had significant activities in improving learning and memory and decreasing chemotherapy-induced myelosuppression. In the present study, the pharmacokinetics and oral bioavailabilities of PPT and PPD were investigated when a single dose of DS was administrated orally (75 mg/kg) and intravenously (i.v., 30 mg/kg) to rats. Their in vitro stabilities in the GI tract were also investigated. PPT and PPD concentrations were measured by LC-MS. The results showed that PPT was eliminated rapidly from the body with an average t1/2, λz value of 0.80 h and CL of 4.27 l/h/kg after i.v. administration, while PPD was eliminated relatively slowly with a t1/2, λz of 6.25 h and CL of 0.98l/h/kg. After oral administration, both PPD and PPT could be absorbed into the body, but their systemic exposures were quite different. PPT was absorbed into the body quickly, with a Tmax of 0.58 h and a Cmax of 0.13 μg/ml, while PPD was absorbed relatively slowly with a Tmax of 1.82 h and a Cmax of 1.04 μg/ml. The absolute bioavailabilities of PPT and PPD were estimated as 3.69% and 48.12%, respectively. The stability test found that PPT was instable in the stomach with 40% degradation after 4h incubation at 37°C, both in pH1.2 buffer and in the stomach content solution. The instability in the stomach might be one of the reasons for PPTs poor bioavailability.

Identification and characterization of potent CYP2D6 inhibitors in lotus leaves

ETHNOPHARMACOLOGICAL RELEVANCEnThe herb of lotus (Nelumbo nucifera) leaves is a commonly used traditional Chinese herbal medicine that is utilized for the treatment of sunstroke, to assuage thirst, and to cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the herb exhibits various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-HIV, anti-microbial, and anti-hypoglycemic activities. Currently, the herb is becoming more popular in China as a tea drink or as a main ingredient of some herbal formulations, which implies that the herb and/or its products are now more likely to be concurrently administered with conventional medicines for losing body weight and reducing blood lipids. However, its potential inhibitory effect on human cytochrome P450 (CYP) has not been systemically investigated to date. The present study was performed to assess the potential inhibitory effects of lotus leaf alcoholic extract (LAE), its major fractions, and its main compounds on five CYP isoenzymes (CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in vitro.nnnMATERIAL AND METHODSnFive probe substrates were incubated with human liver microsomes in the presence or absence of the LAE, the alkaloid fraction (AF), the flavonoid fraction (FF), or the individual aporphine alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and 2-hydroxy-1-methoxyaporphine (HMA). After the incubation, the relative metabolites of the substrates were analyzed using LC-MS/MS.nnnRESULTSnThe results showed that the LAE strongly inhibited CYP2D6 with an IC50 value of 12.05µg/mL and weakly inhibited other isoenzymes. In addition, FF was found to weakly inhibit CYP2D6, whereas AF exerted a markedly higher inhibitory effect on CYP2D6 activity with an IC50 value of 0.96µg/mL. The three aporphine alkaloids isolated from the AF (NF, N-NF, and HMA) significantly inhibited CYP2D6 with IC50 values of 3.78, 3.76, and 3.15µM, respectively. Their Lineweaver-Burk plots and Dixon plots showed that NF, N-NF, and HMA competitively inhibited CYP2D6 activity with Ki values of 1.88, 2.34, and 1.56µM, respectively.nnnCONCLUSIONnThe study revealed that the alkaloid compounds in lotus leaves exert a potent inhibitory effect on CYP2D6 isoenzyme. The possible drug interactions of the leaves and their preparations with conventional medicines should thus be taken into account.

Antidepressant-like effects of total saikosaponins of Bupleurum yinchowense in mice

Bupleurumxa0is known traditionally to effectively treat depressive-like disorders in East Asia. Pharmacological studies have been carried out on several species ofxa0Bupleurum, such asBupleurum chinensexa0DC. andxa0Bupleurum falcatumxa0L.xa0Bupleurum yinchowense, the same genus ofxa0Bupleurumxa0R. H. Shan and Yin Li, is abundantly distributed in the Northwest of China and is widely used in folk medicine, but few reports explore its antidepressant-like activities. In the present study, it was observed that the total saikosaponins isolated fromxa0B. yinchowensexa0(75 and 150 mg/kg) significantly reduced the immobility time in the forced swim and tail suspension test in mice after 7-day treatment. In the chronic mild stress model, chronic treatment with the total saikosaponins isolated fromxa0B. yinchowensexa0(50 and 100 mg/kg) increased the sucrose intake in the sucrose preference test and decreased the latency to feed in the novelty-suppressed feeding (NSF) test. In addition, the total saikosaponins isolated fromxa0B. yinchowensexa0augmented the diminished monoamine neurotransmitter concentrations (5-HT, DA and NE) in the prefrontal cortex induced by chronic mild stress. Taken together, our results suggest that the total saikosaponins isolated fromxa0B. yinchowensexa0exerts an antidepressant-like action by modulating the 5-HT, DA and NE levels in the prefrontal cortex. n n xa0 n n Key words:xa0Chronic mild stress, mice, antidepressant, neurotransmitter,xa0Bupleurum yinchowense.

Cajaninstilbene acid protects corticosterone-induced injury in PC12 cells by inhibiting oxidative and endoplasmic reticulum stress-mediated apoptosis.

It has been reported that high corticosterone level could damage the normal hippocampal neurons both in vitro and in vivo. Furthermore, high concentration of corticosterone induced impair in PC12 cells has been widely used as in vitro model to screen neuroprotective agents. Cajaninstilbene acid (CSA), a natural stilbene isolated from Cajanus cajan leaves, has various activities. In present study, we investigated the effect of CSA on corticosterone-induced cell apoptosis and explored its possible signaling pathways in PC12 cells. We demonstrated that pretreatment with CSA at the concentrations of 1-8 μmol/L remarkably reduced the cytotoxicity induced by 200 μmol/L of corticosterone in PC12 cells by MTT, and further confirmed the neuroprotection by Hoechst 33342 and PI double staining and lactate dehydrogenase release (LDH) assay at the concentration of 8 μmol/L. Moreover, the cytoprotection of CSA was proved to be associated with the homeostasis of intracellular Ca(2+), relieving corticosterone-induced oxidative stress by decreasing the contents of ROS and malondialdehyde (MDA), increasing the activities of superoxide dismutase (SOD) and catalase (CAT), and the stabilization of ER stress via down-regulating the expression of ER chaperone protein glucose-regulated protein 78 (GRP78), ER stress associated transcription factor C/EBP homologous protein (CHOP/GADD153), and the X box-binding protein-1 (XBP-1), as well as the expression of ER stress-specific protein caspase-12 and its downstream protein caspase-9. Considering all the findings, it is suggested that the neuroprotective activity of CSA against the impairment induced by corticosterone in PC12 cells was through the inhibition of oxidative stress and ER stress-mediated pathway.

Pharmacokinetic mechanism of enhancement by Radix Pueraria flavonoids on the hyperglycemic effects of Cortex Mori extract in rats

ETHNOPHARMACOLOGICAL RELEVANCEnDiabetes mellitus, characterized by abnormal blood glucose evaluation, is a serious chronic disease. In the treatment of the disease, α-glycosidase inhibitors play an important role for controlling the postprandial blood glucose level. Cortex Mori, a traditional Chinese herbal medicine, has a long history of use for the treatment of headaches, cough, edema and diabetes. Modern pharmacological studies have shown that the herb has beneficial effects on the suppression of postprandial blood glucose levels by inhibiting α-glycosidase activity in the small intestine. 1-Deoxynojirimycin (DNJ), the main active ingredient of this herb, is recognized as a potent α-glycosidase inhibitor. Our previous studies have shown that the hypoglycemic effect of Cortex Mori extract (CME) was significantly improved when giving CME in combination with Radix Pueraria flavonoids (RPF). In the present study, the pharmacokinetics and intestinal permeability of DNJ were comparatively investigated in rats after being given orally or by intestinal perfusion with CME alone or in CME-RPF pairs, to explore the mechanism of this synergistic effect.nnnMATERIALS AND METHODSnThe role of RPF on the plasma and urine concentrations of DNJ from CME orally administered was investigated. Four groups of rats received a single oral dose of either CME or CME-RPF, at DNJ equivalent doses of 20 and 40mg/kg, respectively. After dosing, plasma and urine were collected and assayed by LC/MS/MS. In addition, another two groups of rats were used for small intestinal perfusion with CME or CME-RPF at DNJ concentration of 10µM.nnnRESULTSnCompared to the data when dosing with CME alone, the Cmax of DNJ were decreased from 5.78 to 2.94µg/ml (p<0.05) and 10.66 to 5.35µg/ml (p<0.01); Tmax were delayed from 0.40 to 0.55h and 0.35 to 0.50h (p<0.05); and MRT were significantly prolonged from 1.14 to 1.72h (p<0.05) and 0.95 to 1.62h (p<0.01), after dosing with CME-RPF at DNJ doses of 20 and 40mg/kg, respectively. In addition, the urinary recovery of DNJ over the first 4h after dosing significantly decreased from 48.76% to 33.86%. Effective permeability (Peff) of DNJ was decreased from 7.53×10(-3) to 3.09×10(-3)cm/s (p<0.05) when RPF was added to CME, when it was evaluated using the rat intestinal perfusion model.nnnCONCLUSIONSnAll the above results demonstrate that RPF was able to suspend and delay the absorption of DNJ, but did not affect the total amount of DNJ in the body. The resulting higher concentration of DNJ in the small intestine produced a relatively stronger effect of depressing the elevation of the postprandial blood glucose level. These findings support the important role of RPF in the application of CME on blood glucose control.

Identification of in vivo components in rats after oral administration of lotus leaf flavonoids using ultra fast liquid chromatography with tandem mass spectrometry

The herb of lotus (Nelumbo nucifera) leaves is the traditional Chinese medicine He Ye, which is commonly used to treat sunstroke, assuage thirst, and cure both diarrhea and fever in China. Lotus leaves are rich in flavonoids, which exhibit various biological activities. However, the in vivo components, including parent compounds and their metabolites after consumption of the leaves have not been investigated extensively. In the present study, a method based on ultra fast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) was established to identify the in vivo components in rats after oral administration of a lotus leaf flavonoid extract. Plasma and urine samples were collected before and after dosing and treated by liquid–liquid extraction with ethyl acetate, and followed by UFLC-MS/MS assay. Q1 (first quadrupole) full scan combined with multiple reaction monitoring (MRM) survey scan were used for the detection of parent flavonoids and their metabolites. MRM-information dependent acquisition (IDA) of enhanced product ions (MRM-IDA-EPI) was used for the structural identification of detected components. A total of thirty-seven components were identified, including quercetin-3-O-glucuronide, quercetin-3-O-glucoside, quercetin-3-O-galacoside, quercetin and kaempferol, as well as their methylation, glucuronidation, and sulfonation metabolites. The result may help better understand the pharmacological activities of the traditional Chinese medicine He Ye.