Hepatoprotective activity of the edible snail (Bellamia bengalensis) flesh extract in carbon tetrachloride induced hepatotoxicity in rats

Ethnopharmacological relevance

In the folk-traditional medicine, snails were used to purify blood, boost immune system, prevent conjunctivitis and to treat liver problems.

Objectives

To evaluate the hepatoprotective activity of the edible snail (Bellamia bengalensis) flesh extract in male Wistar rats treated with carbon tetrachloride as an hepatotoxicant.

Materials and methods

Live adult Bellamia bengalensis was collected commercially from the Kolkata market. Aqueous flesh extract (BBE) was prepared in 0.9% saline and expressed in terms of wet weight basis. The aqueous flesh extract was administered orally (1, 2 g kg⁻¹ day⁻¹) to male rats for 12 days. Liv52 was used as positive control. 24 h after administration of extract, the rats were given a single oral dose of CCl₄ (1.25 ml kg⁻¹), except vehicle control rats. After 24 h of CCl₄ administration, all the animals were sacrificed to collect the blood and liver tissue.

Results

BBE (1 and 2 g kg⁻¹ day⁻¹, p.o. × 12 days) significantly prevented CCl₄ induced elevation of SGOT, SGPT, γGT, ACP, ALP, bilirubin, LDH and CCl₄ induced decrease in total protein, triglyceride level in male Wistar rats. BBE treated rat liver anti-oxidant parameters (LPO, SOD, GSH, CAT, GPx) were significantly antagonized for the pro-oxidant effect of CCl₄. Histopathological studies also supported the protective effect of BBE.

Conclusion

This study validated the folk and traditional use of snail in liver disorder through CCl₄-induced rat experimental model.     

A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CCl4-injured rats orally administered rhubarb extract

Aim of the study

The present study comparatively investigated the tissue distributions of rhubarb anthraquinone derivatives (AQs) to examine whether they undergo different uptakes in normal or CCl₄-induced liver-damaged rats, to explore possible reasons for the different toxicities of AQs in pathological model rats and normal rats at the tissue distribution level.

Materials and methods

The total rhubarb extract (14.49 g kg⁻¹ of body weight per day based on the quantity of crude material) was administrated orally to normal and model rats for 12 weeks. The concentrations of free AQs in tissues were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS). After drug withdrawal for 4 weeks, tissue distributions were again determined.

Results

The five free AQs—aloe-emodin, rhein, emodin, chrysophanol and physcion—were detected in the liver, kidney and spleen, while only rhein, aloe-emodin and emodin reached the quantitative limit. The tissue distributions of rhein (p < 0.001), aloe-emodin (p < 0.001) and emodin (p < 0.05) in normal rats were higher than those in model rats with rhein > aloe-emodin > emodin in kidney and spleen tissues and aloe-emodin > rhein > emodin in liver tissues. Free AQs were not detected in the tissues after drug withdrawal for 4 weeks.

Conclusions

These results suggest that the tissue toxicity of AQs in normal animals is higher than that in pathological model animals with little accumulative toxicity of rhubarb. The results are concordant with the traditional Chinese theory of You Gu Wu Yun recorded first in Su Wen, a classical Chinese medical treatise.     

Action mechanism of Zuo Gui Yin Decoction's promotion on estradiol production in rats during the peri-menopausal period

Aim of the study

Although Zuo Gui Yin Decoction has long been used in Traditional Chinese Medicine to treat menopausal symptoms, the underlying mechanism(s) by which these effects are induced remains to be defined. The aim of this study was to investigate the action mechanism of Zuo Gui Yin Decoction on estradiol production in the rat ovary during peri-menopause.

Materials and methods

The peri-menopausal animal model was established by natural aging. Peri-menopausal rats were treated by intragastric administration (ig) with low (13.78 g kg⁻¹), middle (20.67 g kg⁻¹) or high (31 g kg⁻¹) dose of Zuo Gui Yin Decoction per day for 8 weeks. At the 8th weekend, the rats were sacrificed for sampling. Estradiol (E₂) levels in rats’ serum were evaluated by radioimmunoassay (RIA). RT-PCR, in situ hybridization and immunohistochemistry were used to determine mRNA and protein expression of relevant genes.

Results

Medium- and high-dose of Zuo Gui Yin Decoction could significantly increase serum estradiol concentration, ovarian CYP19 mRNA levels, and P450_arom protein expression in rats during peri-menopause. Zuo Gui Yin Decoction at three different dosages all could promote FSHR expression and the effect of low-dose was the greatest. Zuo Gui Yin Decoction could elevate LRH-1 and ER_α expression in a dose dependent manner.

Conclusions

Taken collectively, these findings suggest that Zuo Gui Yin Decoction could promote estradiol production in rat serum during peri-menopausal period through ovarian ER_α → LRH-1 → CYP19 pathway as well as the ovarian classical FSHR → CYP19 mechanism.     

Toxic effects caused by rhubarb (Rheum palmatum L.) are reversed on immature and aged rats

Aim of the study

Rhubarb is generally used to people of broad age, but diverse responses of people at different age to rhubarb have been little clarified. In this study, an attempt was made to access the safety of rhubarb to both immature and aged rats to provide some references for its clinical usage.

Materials and methods

The total extract of rhubarb was administered intragastricly to both immature and aged rats once a day and lasted for 5 weeks. Then histopathologic and biochemical examinations were performed.

Results

No death was observed in immature rat groups, while 23.3% (21/90) subjects in aged rat groups died and most of the death cases were observed in the high-dosage (40 g kg⁻¹ of body weight per day od, counted on the quantity of crude material) group. The death rate between aged and immature rats was found of significantly statistical difference. Dosage-dependent histopathologic changes in kidney were observed in all the rhubarb-treated rats, principally involving the proximal tubules. Kidney changes in aged rats were severer than those observed in immature ones. Hepatic cells necrosis was occasionally observed in the middle- and high-dosage aged rat groups and minimal biliary hyperplasia was found in all the rhubarb-treated aged rats. Increased incidences of activated Kupffer cells and lymphocytic infiltration were found in all the rhubarb-treated rats. And dosage-dependent increase of interleukin 6 (IL-6) and notable increase of IL-8 was found in aged rat groups.

Conclusions

The immature and aged rats showed reversed responses to the toxic potential of rhubarb extract. Elderly subjects were susceptible to the toxicity of high-dosage rhubarb, which drove rigorous consideration on rational use of rhubarb to aged people.     

Effects of HuangKui capsules on glibenclamide pharmacokinetics in rats

Ethnopharmacology

The main components of HuangKui capsules’ are the total flavonoids extracted from the flowers of Abelmoschus manihot L medic. They have been widely used to treat chronic glomerulonephritis, diabetic nephropathy and nephrotic syndrome. The combination of HuangKui capsules and glibenclamide is a possible therapy for patients with diabetes mellitus and diabetic nephropathy. However, there is no report about effects of HuangKui capsules on the glibenclamide pharmacokinetics till now.

Aim of the study

This study was aimed investigating the effect of HuangKui capsules on pharmacokinetics of glibenclamide in rats.

Material and methods

Eight rats were administered with an oral dose of HuangKui capsules (0.75 g kg⁻¹) once daily for 10 consecutive days. All the rats were administered orally with the glibenclamide (1 mg kg⁻¹) before the first time and after the last time given HuangKui capsules. LC-MS/MS was utilized to determine the concentration of glibenclamide in rat plasma and to calculate the corresponding pharmacokinetic parameters. The statistical differences of the two cycles were evaluated by paired-samples t-test.

Results

In the rats treated with HuangKui capsules and glibenclamide, the t_1/2, the time point of maximum plasma concentration (T_max) of glibenclamide increased obviously (p < 0.05) compared with the glibenclamide alone, while maximum plasma concentrations (C_max), area under the plasma concentration-time curve (AUC_(0−t)) decreased significantly (p < 0.05). There was no significant difference between other parameters.

Conclusion

HuangKui capsules can reduce the absorption of glibenclamide and accelerate the metabolism of glibenclamide.     

Oral bioavailability and gender-related pharmacokinetics of celastrol following administration of pure celastrol and its related tablets in rats

Ethnopharmacological relevance

Celastrol is a natural compound extracted from the traditional Chinese medicinal herb, Thunder God Vine (TGV). Owing to its potential anti-inflammatory and antitumor effects, celastrol has been considered as a promising candidate for drug development.

Aim of the study

To establish a sensitive LC–MS/MS method to investigate the pharmacokinetic properties of celastrol in rats. Key pharmacokinetic issues of celastrol including oral bioavailability, comparative pharmacokinetics between pure compound and tablet preparation, as well as gender-related pharmacokinetic difference are to be addressed for the first time.

Materials and methods

Sprague–Dawley rats were administrated an intravenous dose (100 μg kg⁻¹) of pure celastrol and an oral dose (1000 μg kg⁻¹) of pure celastrol and TGV tablets (corresponding to 534 μg kg⁻¹ of celastrol), respectively. At different time points, the concentration of celastrol in rat plasma was determined by a sensitive and well-validated LC–MS/MS method. Main pharmacokinetic parameters including area under the plasma concentration–time curve (AUC), maximal plasma concentration (Cmax), the time for maximal concentration (Tmax) and mean residence time (MRT) were estimated by Drug and Statistic1.0 pharmacokinetic software (Chinese Pharmacological Association, Anhui, PR China). Statistical analysis was performed using two one-side t test with p-values less than 0.05 as the level of significance.

Results

The standard curve of celastrol showed good linearity in the concentration range of 0.11∼54.3 ng mL⁻¹ in our current method, with acceptable selectivity, precision, recovery, and stability. The oral absolute bioavailability of celastrol significantly increased from 17.06% for pure celastrol to 94.19% for TGV tablets containing equivalent celastrol. After oral administration of TGV tablets, the Cmax and AUC values of celastrol in female rats were (32.03±8.41) μg L⁻¹ and (379.49±118.19) μg h L⁻¹, which were significantly higher (p<0.01) than that in males with the values of (14.31±7.33) μg L⁻¹ and (188.17±92.33) μg h L⁻¹.

Conclusion

Celastrol administered orally in the rat was poorly absorbed into the systemic circulation. However, the poor absorption of celastrol could be greatly improved when celastrol-containing TGV tablets orally administered, and thereby the oral bioavailability of celastrol was significantly increased. As for gender difference, female rats showed significantly better absorption of celastrol than males.     

Effect of ginkgolide B on striatal extracellular amino acids in middle cerebral artery occluded rats

Ethnopharmacological relevance

Ginkgo biloba leaves are traditionally used in China for its health-promoting properties. There is substantial experimental evidence to support the view that Ginkgo biloba extracts have neuroprotective properties under conditions such as hypoxia/ischemia.Although a number of studies have investigated that ginkgolide B, a purified terpene lactone component extracted from Ginkgo biloba leaves, is available “platelet activating factor (PAF) receptors antagonist”, “antioxidant” with a variety of actions, very little has been performed to explore the effect of ginkgolide B on extracellular amino acids in experimental animal of focal cerebral ischemia/reperfusion. In this study, the effect of ginkgolide B on the striatal extracellular levels of glutamate (Glu), aspartic acid (Asp), glycine (Gly) and γ-aminobutyric acid (GABA) was evaluated in rats undergone middle cerebral artery occlusion (MCAO) for 1 h followed by 23 h reperfusion.

Materials and methods

The Sprague-Dawley (SD) rats received intraperitoneal injections of ginkgolide B dissolved at a dose of 10 mg kg⁻¹ d⁻¹, 20 mg kg⁻¹ d⁻¹, or normal saline (NS) of same volume 3 d before the middle cerebral artery occlusion model establishment. Extracellular concentrations of glutamate, aspartic acid, glycine and GABA in striatum were monitored using in vivo microdialysis and analyzed using high-performance liquid chromatography. Excitotoxic index (EI) was calculated. Twenty-four hours after MCAO, the cerebral infarct volume was detected on 2,3,5-triphenyltetrazolium chloride-stained coronal sections.

Results

The result showed that administration of ginkgolide B (10 or 20 mg kg⁻¹) before ischemia reduced the ischemia-induced elevation of levels of glutamate, aspartic acid and glycine, increased the elevation of extracellular GABA, decreased the excitotoxic index and diminished the volume of cerebral infarction, although a clear concentration-response relationship was not found.

Conclusions

The present work provides the first evidence that ginkgolide B protects against cerebral ischemic injury by inhibiting excitotoxicity by modulating the imbalance of excitatory amino acids versus inhibitory amino acids, which may support the traditional use of Ginkgo biloba leaves for the treatment of stroke.     

Ginseng total saponins enhance neurogenesis after focal cerebral ischemia

Ethnopharmacological relevance

Ginseng, the root of Panax ginseng C.A. Meyer, is one of the most commonly used healing herbs for stroke and chronic debilitating conditions in China. Ginsenosides are the main active principles for ginseng's efficacy, but the mechanisms have not been fully clarified.

Aim of the study

To test the hypothesis whether or not the administration of Ginseng total saponins (GTS) can enhance neurogenesis after focal cerebral ischemia, and thereby improve neurological deficits.

Materials and methods

Male Wistar rats received intraperitoneal injections of GTS dissolved at a dose of 25 mg kg⁻¹ d⁻¹ or normal saline (NS) of same volume 3 days before the permanent middle cerebral artery occlusion (MCAO) model establishment until the animals were killed at the time points of 1 d, 3 d, 7 d and 14 d. The neurological function was assessed blindly. BrdU immunostaining and double staining were performed by following the 3-steps method.

Results

(A) GTS-treated rats have better neurological scores compared with those in NS group at 14 d time point (p < 0.05); (B) the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in GTS group were significantly higher than those in NS group in the ipsilateral subventricular zone and in the ipsilateral infarct area after MCAO, respectively (p < 0.05 or p < 0.01); (C) the increase of the number of BrdU⁺/NeuN⁺ cells highly correlated with the decrease of neurological scores. Coefficient correlation r = −0.828 (p < 0.01).

Conclusion

GTS can improve neurological deficits after focal cerebral ischemia by inducing endogenous neural stem cells activation and thereby enhance adult central nervous system regeneration.     

Effect of tongxinluo on vasoconstriction induced by the chronic injury of the adventitia in the rat carotid artery

Objective

Tongxinluo (TXL) is a traditional Chinese medicine that is developed on the meridian theory of traditional Chinese medicine, with the function of alleviating the angina. The present study was undertaken to explore the molecular mechanism of TXL in treating the pectoris angina through observing the effectiveness of TXL superfine powder on the vasoconstriction and the activation of RhoA/Rho-kinase pathway induced by the injury of the adventitia.

Methods

36 male Wistar Kyoto rats were assigned to 3 treatments (n = 12): vehicle, TXL (400 mg kg⁻¹ day⁻¹) and fasudil (15 mg kg⁻¹ day⁻¹). After 1 week of treatment, adventitia injury was induced by positioning a silicone collar around the right carotid artery for 1 week. Blood flow and vascular reactivity to serotonin were determined 1 week after injurying, the both sides of carotids were harvested for morphometry, Western blotting analysis and RT-PCR analysis.

Results

Adventitia injury leaded to histological changes of vasoconstriction with the lumen cross-sectional area of 44.7% (p < 0.001) decreasing and the media diameter of 62.31% (p < 0.001) increasing, accompanying by the reduction of the blood flow and the increase of vascular reactivity sensitivity to serotonin. Treatment with both TXL superfine powder and fasudil can prevent the development of vasoconstriction, improve the carotid blood flow and normalize the vascular hypersensitivity to serotonin. Adventitia injuring of the rat carotid increased the expression of Rho-kinase mRNA and p-MYPT1^Thr696 protein by 1.78-fold (p < 0.05) and >2-fold respectively (p < 0.05). TXL reduced the expression of Rho-kinase mRNA and p-MYPT1^Thr696 protein by 54.2% (p < 0.05) and 57.1% (p < 0.05) respectively in collared arteries. Fasudil restrained the p-MYPT1^Thr696 protein expression by 63.8% (p < 0.05) in collared arteries, did not affect the collar-induced the increase of Rho-kinase mRNA expression (p > 0.05).

Conclusions

Treatment with TXL, similar to that with fasudil, can effectively prevent collar-induced vasoconstriction and vascular hyperreactivity to serotonin through inhibiting the RhoA/Rho-kinase pathway.     

Tannin-rich fraction from Terminalia catappa inhibits quorum sensing (QS) in Chromobacterium violaceum and the QS-controlled biofilm maturation and LasA staphylolytic activity in Pseudomonas aeruginosa

Aim of the study

The study aimed to test the activity of Terminalia catappa L. against bacterial quorum sensing (QS) in order to provide a potential scientific basis for the traditional use of leaf extracts of this plant as an antiseptic.

Materials and methods

The anti-QS activity of the methanolic leaf extract of Terminalia catappa was detected through the inhibition of the QS-controlled violacein pigment production in Chromobacterium violaceum. Fractions resulting from size-exclusion chromatography were assayed. The most active fraction was characterized through qualitative phytochemical detection methods. The effect of this fraction on known QS-controlled phenotypes in test strains was assessed.

Results

The fraction with the highest activity (labeled as TCF12) was characterized to be tannin-rich. It specifically inhibited QS-controlled violacein production in Chromobacterium violaceum with 50% reduction achieved at 62.5 μg mL⁻¹ without significantly affecting growth up to about 962 μg mL⁻¹. The assessment of its effects on LasA activity of Pseudomonas aeruginosa ATCC 10145 found that the production of this virulence determinant is reduced in a concentration dependent manner with about 50% reduction at 62.5 μg mL⁻¹. Furthermore, it was found that TCF12 was able to inhibit the maturation of biofilms of Pseudomonas aeruginosa, a phenotype that has also been known to be QS-regulated.

Conclusion

Therefore, tannin-rich components of Terminalia catappa leaves are able to inhibit certain phenotypic expression of QS in the test strains used.     

Comparison of toxic reaction of Tripterygium wilfordii multiglycoside in normal and adjuvant arthritic rats

Aim of the study

Tripterygium wilfordii multiglycoside (GTW), an authorized Chinese patent drug, is used for treatment of rheumatoid arthritis and other immune disease. This study was to determine whether GTW induced different toxic reactions in adjuvant arthritis rats (AA rats) compared to those in normal rats.

Materials and methods

To prepare arthritic rat model, male Sprague-Dawley (SD) rats were immunized by injecting complete Freund's Adjuvant into right hind footpad. And then, GTW was given to rats intragastrically at dosage of 7 or 105 mg kg⁻¹ day⁻¹ from day 15 to day 28 after immunization. Routine clinical parameters and histopathologic changes of liver, kidney and testis were examined. Metabolic profiling in serum of groups was analyzed by LC-MS. A principal component analysis (PCA) and partial-least-squares discriminate analysis (PLS-DA) were carried out combined with mass spectrometry (MS) data set. All the quantitative data were performed by two-way ANOVA analysis following Student's t-test.

Results and conclusions

Treatment with GTW at both doses could diminish the right and left hind paws swelling. There was slight lipoid degeneration in hepatic tissue of normal rats treated by high dose of GTW, but there were not distinctly pathological changes in hepatic tissue of AA rats treated by GTW. Compared normal rats administered with GTW, no statistically significant difference in the serum alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN) levels were observed. However, the serum aspartate aminotransferase (AST) level was significant decreased in AA rats under exposure GTW compared with normal rats in the same conditions (p < 0.05), which indicated that GTW could offer a different liver toxic reaction in normal and AA rats. The metabolic analysis showed that a clear separation of PCA and PLS-DA score spot in normal rats, but not separation was seen in AA rats perturbed with low dosage GTW. The result indicated low dosage GTW might arouse a general toxic in normal rats but not in AA rats. The biomarker analysis showed that the level of lysophosphatidylcholines (LPCs) was down-regulated, but the level of ursodeoxycholic acid (UDCA) and chenodexycholic acid (CDCA) was up-regulated in AA rats compared with normal rats under exposure GTW. According to pathway analysis of metabolic markers, we conceived that LPC, UDCA and CDCA were the critical intermediates of choline and fatty acid metabolism. And the lipid metabolism was a correlative outcome of GTW induced toxicity in the liver in physiological condition animals. Taken together, GTW could induce different toxic reactions between normal and AA rats, and the lipid metabolism might be part of the mechanism for the hepatic lipidosis or the other liver injury.     

Xiao-Ban-Xia-Tang inhibits cisplatin-induced pica by down regulating obestatin in rats

Ethnopharmacological relevance

Xiao-Ban-Xia-Tang (XBXT), a traditional Chinese herbal medicine, has been used in China for more than 2000 years, and proved to be effective in various cases of vomiting in the clinic.

Objective

To investigate the inhibitive effect of XBXT on cisplatin-induced pica behaviour and its effective mechanism on obestatin, CCK and CGRP in the pica model of rat.

Materials and Methods

The inhibitive effect of XBXT was investigated in the pica model of rats induced by cisplatin (3 mg kg⁻¹, i.p.) in 72 h observation, the expression of obestatin in the area postrema and ileum was measured by immunohistochemistry and PCR, and the levels of CCK and CGRP in blood were measured by Elisa.

Results

The weight of kaolin eaten in rats induced by cisplatin was significantly reduced by pretreatment with XBXT in a dose-dependent manner during the 0-24 h and 24-72 h periods (P < 0.05). XBXT exhibited effective dose-dependent (P < 0.05) inhibition on the increase of expression levels of obestatin in both the ileum and area postrema, and markedly suppressed the increase levels of CCK and CGRP in blood induced by cisplatin in a dose-dependent manner (P < 0.05).

Conclusions

XBXT has good activity against cisplatin-induced eating kaolin in rats possibly by inhibiting central or peripheral increase of obestatin, or the levels of CCK and CGRP in blood.     

Inonotus obliquus extracts suppress antigen-specific IgE production through the modulation of Th1/Th2 cytokines in ovalbumin-sensitized mice

Ethnopharmacological relevance

Chaga mushroom (Inonotus obliquus, IO) has been used as a folk remedy for cancer, digestive system diseases, and other illnesses in Russia and Eastern Europe.

Aim of the study

In the present study, we investigated the immunomodulating effects of IO through in vivo and ex vivo studies.

Materials and methods

Serum immunoglobulins (IgE, IgG₁, and IgG_2a) and cytokines (interleukin (IL)-4, interferon (IFN)-γ, and IL-2) were measured in concanavalin A (ConA)-stimulated splenocytes and CD4⁺ T cells. The nitric oxide (NO) secretion of lipopolysaccharide (LPS)-stimulated peritoneal macrophages was also measured after oral administration of 50, 100, or 200 mg kg⁻¹ d⁻¹ IO hot water extract (IOE) to ovalbumin (OVA)-sensitized BALB/c mice.

Results

We found that the OVA-induced increase in serum IgE and IgG_2a was significantly suppressed when IOE was orally administered after the second immunization with OVA. ConA stimulation in spleen cells isolated from OVA-sensitized mice treated with 100 mg kg⁻¹ IOE resulted in a 25.2% decrease in IL-4 production and a 102.4% increase in IFN-γ, compared to the controls. Moreover, IL-4, IFN-γ, and IL-2 were significantly reduced after ConA stimulation in isolated CD4⁺T cells. We also determined that IOE inhibits the secretion of NO from LPS-stimulated peritoneal macrophages ex vivo.

Conclusions

We suggest that IO modulates immune responses through secretion of Th1/Th2 cytokines in immune cells and regulates antigen-specific antibody production.     

Exploring Tagetes erecta Linn flower for the elastase, hyaluronidase and MMP-1 inhibitory activity

Ethnopharmacological relevance

Traditionally Tagetes erecta Linn flower is claimed to treat skin diseases like sores, burns, wounds, ulcers, eczema and several other skin ailments. The aim of the present experiment was to evaluate the anti-wrinkle potential of standardized flower extract of Tagetes erecta.

Materials and methods

The Tagetes erecta extract and fractions were screened for hyaluronidase, elastase and matrix metalloproteinase (MMP-1) inhibitory activity compared with the activity of standard oleanolic acid. Syringic acid and β-amyrin were obtained from the extract and quantified through RP-HPLC. Also the compounds were evaluated for anti-wrinkle activity.

Results

The methanol extract showed significant (^aP < 0.05) hyaluronidase and elastase inhibition with IC50 of 11.70 ± 1.79 μg mL⁻¹ and 4.13 ± 0.93 μg mL⁻¹ respectively and better MMP-1 inhibition compared to standard oleanolic acid. The isolated compounds syringic acid and β-amyrin found to inhibit enzymes comparable to oleanolic acid. The RP-HPLC analysis revealed that good amounts of syringic acid and β-amyrin (2.30%, w/w and 0.06%) are present in Tagetes erecta.

Conclusion

Tagetes erecta flower showed effective inhibition of hyaluronidase, elastase and MMP-1. Therefore, this experiment further rationalizes the traditional uses of this plant, which may be useful as an anti-wrinkle agent.     

The pharmacokinetics and oral bioavailability studies of columbianetin in rats after oral and intravenous administration

Ethnopharmacological relevance

The roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan (RAP) has been used as Traditional Chinese medicine to treat rheumatic disease in China since ancient times, but its action mechanisms was not well understood. Columbianetin is one of the main active constituents isolated from RAP, which has been shown to have various biological activities, but the absorption characteristics and oral bioavailability dose proportionality of columbianetin in vivo were not studied.

Materials and methods

Male Sprague Dawley rats (210–230 g) received either an intravenous (i.v. 5, 10 and 20 mg kg⁻¹) or oral (5, 10 and 20 mg kg⁻¹) dose of columbianetin. The levels of columbianetin in plasma were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The simple liquid–liquid extraction with ethyl acetate was used for sample preparation. Osthole was selected as internal standard (IS).

Results

The chromatographic separation was accomplished on a C₁₈ column at a flow rate of 1 mL min⁻¹, where water–methanol was used as mobile phase. The calibration curve of the method was linear in the concentration range of 0.05–2000 μg mL⁻¹. The intra and inter-day accuracy for columbianetin in rat plasma samples were within 8% and the variation was less than 8.3%. This method was suitable for the determination and pharmacokinetic study of columbianetin in rat plasma after both intravenous and oral administration. The results indicated that maximum plasma concentrations(C_max) for the columbianetin (17–42 μg mL⁻¹) were achieved at 0.3–0.5 h post-oral dosing and the apparent volume of distribution (V/F) ranged from 0.38 to 0.44 L. Absolute bioavailability of columbianetin was assessed to be 81.13±45.85, 81.09±33.63 and 54.30±23.19%, respectively. Terminal elimination half-life (T_1/2) of the columbianetin after oral dosing was 60–90 min and were 2.5–3.3 fold longer than those observed for the i.v. dosing.

Conclusions

The pharmacokinetic properties of columbianetin in rat after oral administration were characterized as rapid oral absorption, quick clearance and good absolute bioavailability. The bioavailability of columbianetin ranged from 54 to 81% for 5, 10 and 20 mg kg⁻¹ oral doses. The bioavailability of columbianetin is independent of the doses studied. Columbianetin showed dose proportionality over the dose range 5–20 mg kg⁻¹. The results clearly demonstrated that columbianetin was one of the material bases of RAP. Furthermore, an HPLC method was demonstrated in this study for the research of traditional Chinese medicine.     

Astragaloside IV attenuates inflammatory cytokines by inhibiting TLR4/NF-кB signaling pathway in isoproterenol-induced myocardial hypertrophy

Ethnopharmacological relevance

Astragaloside IV (As IV) is one of the main effective components isolated from the traditional Chinese medical herb Astragalus membranaceus. The protective effect of Astragalus membranaceus on myocardial hypertrophy has been extensively proved. To test the hypothesis that Astragaloside IV can ameliorate the myocardial hypertrophy and inflammatory effect induced by β-adrenergic hyperactivity, we carried out in vivo and in vitro experiments.

Material and methods

In in vivo study, the isoproterenol (Iso) (5 mg kg⁻¹ d⁻¹) was used as a model of myocardial hypertrophy by intraperitoneal injection. SD rats were randomly assigned to following six groups: A: the control; B: Iso group; C: Iso plus As IV 20 mg kg⁻¹ d⁻¹; D: Iso plus As IV 40 mg kg⁻¹ d⁻¹; E: Iso plus As IV 80 mg kg⁻¹ d⁻¹; F: Iso plus Propranolol 40 mg kg⁻¹ d⁻¹. In in vitro study, cultured neonatal rat cardiomyocytes were pretreated with As IV (3, 10, 30 μ mol L⁻¹), Propranolol (2 μ mol L⁻¹) and BAY11-7082 (5 μ mol L⁻¹) for 30 min, and then incubated with Iso (10 μ mol L⁻¹) for 48 h. For the rats in each group, the heart mass index (HMI) and the left ventricular mass index (LVMI) were measured. To measure the transverse diameter of left ventricular myocardial cells (TDM), the hematoxylin-eosin (HE) staining method was applied. In addition, the volume and the total protein content of cardiomyocytes were measured, the mRNA expression of ANP and TLR4 were quantified by RT-PCR, the protein expression of TLR4, IκBα and p65 were quantified by Western blot, and the level of TNF-α and IL-6 were measured by ELISA.

Results

In vivo: Comparing the Iso group to the control, the HMI, LVMI, TDM were significantly increased; the protein expression of TLR4 and p65 were increased, while the IκBα were decreased; the expression of ANP, TLR4 mRNA, and TNF-α, IL-6 in serum were significantly increased. These changes could be partly prevented by As IV and Pro. In vitro: the over-expression of the cell size, total protein content could remarkably down-regulated by As IV and Pro, and the results of RT-PCR, Western blot and ELISA were similar to those of in vivo.

Conclusions

The results of these studies indicate that Astragaloside IV has good protective effect on myocardial hypertrophy induced by isoproterenol. More specifically, the cardioprotection is related to inhibiting the TLR4/NF-кB signaling pathway and the attenuating inflammatory effect.     

Antioxidant and hepatoprotective activity of Fagonia schweinfurthii (Hadidi) Hadidi extract in carbon tetrachloride induced hepatotoxicity in HepG2 cell line and rats

Ethnopharmacological relevance

The whole plant of Fagonia schweinfurthii (Hadidi) Hadidi (Family: Zygophyllaceae) is used in variety of diseases including hepatic ailments in deserts and dry areas of India.

Aim of the study

To evaluate antioxidant and hepatoprotective activity of ethanolic extract from Fagonia schweinfurthii (Hadidi) Hadidi (FSEE) in carbon tetrachloride (CCl₄) induced hepatotoxicity in HepG2 cell line and rats.

Materials and methods

In vitro antioxidant activity was determined by DPPH, ABTS radicals and hydrogen peroxide methods. In vitro cytotoxicity and hepatoprotective potential of FSEE were evaluated using HepG2 cells. Based on the cytotoxicity assay, FSEE (50, 100, 200 µg/ml) was assessed for hepatoprotective potential against CCl₄ induced toxicity in HepG2 cell line by monitoring cell viability, aspartate aminotransferase (AST), alanine aminotransaminase (ALT), lactate dehydrogenase (LDH) leakage, lipid peroxidation (LPO) and glutathione level (GSH). Further, in vivo hepatoprotective activity of FSEE was evaluated against CCl₄ induced hepatotoxicity in male Wistar albino rats. Rats were pre-treated with FSEE (200 mg, 400 mg kg⁻¹ day⁻¹ p.o.) for 7 days followed by a single dose of CCl₄ (1.0 ml/kg, i.p.) on 8th day. Silymarin was used as positive control. After 24 h of CCl₄ administration, various biochemical parameters like aspartate aminotransferase (AST), alanine aminotransaminase (ALT), alkaline phosphatase (ALP), total bilirubin (TB) and total protein (TP) levels were estimated in serum. The antioxidant parameters like superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione (GSH) content and malondialdehyde (MDA) level in the liver homogenate were determined. Histopathological changes in the liver of different groups were also studied.

Results

The FSEE possessed strong antioxidant activity in vitro. The results indicated that CCl₄ treatment caused a significant decrease in cell viability. The CCl₄-induced changes in the HepG2 cells were significantly ameliorated by treatment of the FSEE. FSEE significantly prevented CCl₄ induced elevation of AST, ALT, ALP, TB, and CCl₄ induced decrease in total protein in rats. FSEE treated rat liver anti-oxidant parameters (SOD, CAT, MDA and GSH,) were significantly antagonized for the pro-oxidant effect of CCl₄. Histopathological studies also supported the protective effect of FSEE.

Conclusion

The results of this study revealed that FSEE has significant hepatoprotective activity. This effect may be due to the ability of the extract to inhibit lipid peroxidation and increase in the anti-oxidant enzymatic activity.     

Mechanisms of vasorelaxation induced by Ziziphora clinopodioides Lam. (Lamiaceae) extract in rat thoracic aorta

Aim of the study

Ziziphora clinopodioides Lam. (ZC) is widely used in Uyghur folk medicine for the treatment of hypertension diseases in Xinjiang, an autonomous region of China. To provide pharmacological basis for this traditional use, we explored the vasodilating effects of ZC and investigated the underlying mechanisms.

Materials and methods

Activity of hexane (ZCHE), dichloromethane (ZCDE) and aqueous (ZCAE) extracts of ZC were evaluated on isolated rat aortic rings pre-contracted with phenylephrine (PE) or high KCl. The mechanisms were evaluated on ZCDE, the most potent extract.

Results

ZCDE-induced relaxation in endothelium-intact aortic rings pre-contracted with phenylephrine (PE, 10⁻⁶ M) or high KCl (6 × 10⁻² M), with respective EC₅₀ values of 0.27 ± 0.03 and 0.34 ± 0.04 g/l. Mechanic removal of the endothelium did not significantly modify ZCDE-induced relaxation. In endothelium-denuded aorta pre-contracted with PE (10⁻⁶ M), the vasorelaxant effect of ZCDE was significantly decreased by 4-amino-pyridine (10⁻³ M), but not by glibenclamide (10⁻⁴ M), iberiotoxin (3 × 10⁻⁸ M) and thapsigargin (10⁻⁷ M). In Ca²⁺ free solution, ZCDE significantly inhibited extracellular Ca²⁺-induced contraction in high KCl and PE pre-contracted rings. Additionally ZDCE inhibited the intracellular Ca²⁺ release sensitive to PE (10⁻⁶ M).

Conclusions

The results demonstrate that ZDCE exhibits endothelium-independent vasodilating properties that are mediated by inhibition of extracellular Ca²⁺ influx through voltage- and receptor-operated Ca²⁺ channels (VDDCs and ROCCs), by inhibition of Ca²⁺ release from intracellular stores, and also by the opening of voltage-dependent K⁺ channels.     

Aqueous extract of Astragali Radix ameliorates proteinuria in adriamycin nephropathy rats through inhibition of oxidative stress and endothelial nitric oxide synthase

Aim of the study

To investigate the effects of aqueous extract of Astragali Radix (ARE) on the oxidative stress status and endothelial nitric oxide synthase level in adriamycin (ADR) nephropathy rats.

Materials and methods

ADR nephropathy rats were randomly treated with ARE (2.5 g/kg/d, n = 6, ARE group), or benazepril (10 mg/kg/d, n = 6, angiotensin-converting enzyme inhibitor (ACEI) group) for ten weeks. Serum urea nitrogen, creatinine, albumin, total protein, cholesterol and 24-h urinary protein concentration were determined. Renal cortex catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA) activities, and 24-h urinary NO₃⁻/NO₂⁻ excretion were determined by chromatometry. Renal cortex cyclic guanosine monophosphate (cGMP) level was measured by enzyme immunoassay and eNOS expression was determined by immunohistochemistry.

Results

ARE and ACEI treatments could remarkably reduce more 24 h urinary protein excretion than that in ADR group (88.32 ± 9.96 mg, 81.78 ± 16.28 mg vs. 153.91 ± 28.63 mg, P < 0.01), and there was no difference between ARE and ACEI group. Renal cortex CAT, GSH-Px activities in ARE and ACEI group were significantly higher than ADR group, and renal cortex SOD activity in ARE group was higher than ADR group. Renal cortex MDA activity, cGMP level, and glomerular and tubular eNOS expression in ARE and ACEI group were lower than that in ADR group, and 24-h urinary NO₃⁻/NO₂⁻ excretion in ARE group was lower than ADR group. Renal cortex MDA content (r = 0.895, P < 0.01), cGMP content (r = 0.666, P < 0.01) and eNOS expression in glomerulus (r = 0.910, P < 0.01) were strongly positively associated with 24 h urinary protein excretion. And renal cortex SOD content was negatively associated with 24 h urinary protein excretion (r = −0.861, P < 0.01).

Conclusions

ARE may ameliorate the proteinuria by suppressing the over expression of eNOS, and inhibiting the oxidative injury in ADR nephropathy rats.