Multitargeted inhibition of hepatic fibrosis in chronic iron-overloaded mice by Salvia miltiorrhiza

Ethnopharmacological relevance

Salvia miltiorrhiza (SM, also known as Danshen) is a well-known Chinese medicinal herb, which has shown hepatoprotective effects with anti-fibrotic, anti-oxidative, anti-inflammatory and anti-apoptotic properties. To explore the effects and potential mechanism of SM against hepatic fibrosis induced by chronic iron overload in mice.

Materials and methods

Sixty male mice were randomized into five groups (n=12 in each group): control (saline), iron overload, iron overload with low-dose SM (3 g/kg/day), iron overload with high-dose SM (6 g/kg/day) and iron overload with deferoxamine (100 mg/kg/day) groups. The iron overload model was established by intraperitoneal injection with iron dextran at 50 mg/kg body weight/day, and the entire course lasted for 7 weeks. The major constituents of SM injection were quantified by high performance liquid chromatography. Changes of hepatic iron, hydroxyproline (Hyp), glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were assayed by standard procedures. Protein expression levels of type I collagen, type III collagen, tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α) were analyzed by immunohistochemistry, and mRNA levels of transforming growth factor-β (TGF-β), matrix metal proteinase-9 (MMP-9) and caspase-3 were detected by RT-PCR. Morphological changes were observed with Prussian blue, Masson's trichrome and hematoxylin–eosin staining.

Results

Treatment of chronic iron-overloaded mice with SM dose-dependently ameliorated changes in hepatic morphology and coefficient, reduced iron deposition and Hyp content, suppressed overexpression of type I collagen and type III collagen, downregulated expression of TGF-β mRNA, and upregulated expression of MMP-9 mRNA in the liver. Moreover, SM treatment contributed to decreased MDA content, increased SOD activity and GSH content, while it reduced expression of TNF-α, IL-1α and caspase-3.

Conclusions

SM displayed anti-fibrotic activity in the liver induced by chronic iron overload, which may be attributed to multitargeted inhibition of iron deposition and collagen accumulation, as well as oxidative stress, inflammation and apoptosis.     

Renoprotection of Danshen Injection on streptozotocin-induced diabetic rats,associated with tubular function and structure

Ethnopharmacological relevance

Danshen Injection, the aqueous extracts of Radix Salvia miltiorrhiza (S. miltiorrhiza), is one of the most commonly used traditional Chinese herbs in chronic renal failure treatment. In present study, the mechanism of the renoprotective effect of Danshen Injection was analyzed on streptozocin (STZ)-induced diabetic rats.

Materials and methods

Diabetic experimental model was established in male Sprague-Dawley (SD) rats by intraperitoneal injection of STZ. Rats with blood glucose concentration of higher than 300 mg/dl were intraperitoneally administered with Danshen Injection at a dose of 0.78 ml/kg day. The blood glucose, 24 h urinary protein excretion, serum creatinine (sCr), blood urea nitrogen (BUN), advanced glycation end products (AGEs), lipid peroxide (LPO), antioxidant enzyme of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), transforming growth factor-β1 (TGF-β1), and histomorphological changes in kidney of diabetic rats were analyzed during the course of Danshen Injection administration, as well as the tubular function index of albumin reabsorption of fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA).

Results

The intraperitoneal administration of Danshen Injection could ameliorate the physiological dysfunctions of increased 24 h urinary protein excretion((48.21±8.04)%), sCr((39.4±3.7)%), and BUN((43.37±6.74)%), alleviate the ultrastructural abnormalities of hypertrophy, matrix expansion, and fibrosis in glomerulus, decrease the TGF-β1 expression, AGEs and LPO accumulation, and increase the activity of SOD and GSH-Px in kidney of diabetic rats, but did not significantly influence the blood glucose. Besides these, the Danshen Injection administration also partly restored the decrease of megalin expression in tubules and reabsorptive function of FITC-BSA, in diabetic rats.

Conclusion

The renoprotection of Danshen Injection on diabetic rats was associated with the preservation of tubular function and structure from the hyperglycemia induced toxicities of inappropriate cytokines secretion, oxidative stress, advanced glycation stress, and megalin expression deletion.     

Inhibitory effects of salvianolic acid B on CCl4-induced hepatic fibrosis through regulating NF-κB/IκBα signaling

Ethnopharmacological relevance

Hepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. Salvianolic acid B (SA-B) is one of water soluble compounds derived from Salvia miltiorrhiza Bunge (Danshen in Chinese) widely used for chronic liver diseases. In this study we investigated the protective effects of SA-B on CCl₄-induced hepatic fibrosis.

Materials and methods

Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl₄). Rats were divided into four groups, including normal controls (N group), model (M group), low SA-B of 10 mg/kg body weight (L group), or high SA-B of 20 mg/kg body weight (H group). After 6 weeks, macroscopic features of the liver and weight ratio of liver to body were measured. Liver fibrosis of the rats was evaluated by HE and Massion staining. Activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were checked with automated biochemistry analyzer. Serum levels of hyaluronic acid (HA), type IV collagen (IV-C), Laminin (LN) and procollagen III peptide (PIIIP) were detected by radioimmunoassay (RIA). The expression of NF-κB and IκBα was detected by western blotting.

Results

SA-B was shown to reduce CCl₄-induced hepatic fibrosis in rats. The serum levels of ALT, AST, and TBIL were significantly lower in the SA-B treatment groups than in the M group. Compared the M group, the serum levels of HA, LN, IV-C and PIIIP were decreased markedly after treatment with SA-B, especially in the H group. Treatment with SA-B at 10–20 mg/kg (L and N groups, respectively) dose-dependently decreased the expression of NF-κB in the nucleolus and increased the expression levels of NF-κB and IκBα protein in the cytoplasm compared to that of the M group.

Conclusions

This study reveals that SA-B could prevent the progression of liver angiogenesis and alleviate liver fibrosis possibly by regulating the expression of NF-κB and IκBα.     

Acute toxicity and sub-chronic toxicity of steroidal saponins from Dioscorea zingiberensis C.H.Wright in rodents

Ethnopharmacological relevance

Steroidal saponins from Dioscorea zingiberensis are widely used in China for curing cardiovascular diseases. However, there was little toxicological information available on them.

Aim of the study

The study evaluated potential toxicity of the steroidal saponins and analyzed the metabolites in rats.

Materials and methods

For the acute study, the steroidal saponins were administered to kunming mice in single doses of 112.5–9000 mg/kg given by gavage. General behavior adverse effects, mortality and liver histopathological changes were examined. For the sub-chronic toxicity study, Sprague–Dawley rats were gavaged at doses of 127.5, 255 and 510 mg/kg/day for 30 days, then examined the biochemical and hematological parameters. Metabolites in serum were analyzed by HPLC–MS.

Results

The steroidal saponins caused dose-dependent general behavior adverse effects, mortality and liver histopathological changes in the acute toxicity study. In the sub-chronic toxicity study, 510 mg/kg/day of steroidal saponins increased total bilirubin (TBIL) in serum and decreased protein content in liver significantly. The metabolic process of TBIL in liver includes TBIL intaking, conjugated bilirubin forming, conjugated bilirubin excreting to biliary passage. Treatment with high dose of the steroidal saponins in vivo may lead to vacuolization of the cytoplasm of hepatocytes and canalicular cholestasis. In all doses used in the experiment, the steroidal saponins decreased aspartate aminotransferase (GOT), alanine aminotransferase (ALT) and alkaline phosphatase (AKP) in serum and increased reduced glutathione hormone (GSH), glutathione reductase (GR) and glutathione S-transferases (GST) in liver. Diosgenin was the main metabolite in serum.

Conclusions

The steroidal saponins did not show any sign of toxicity up to oral dose of 562.5 mg/kg in mice. No significant changes of biochemical and hematological parameters in rats (except at 510 mg/kg/day), it was concluded that the steroidal saponins did not appear to have significant toxicity in their traditional uses.     

The protective effect of Zizyphus jujube fruit on carbon tetrachloride-induced hepatic injury in mice by anti-oxidative activities

Aim of the study

The study was aimed to investigate the protective effect against hepatic injury induced by CCl₄ for the ethanolic extract of FZJ.

Materials and methods

The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected as biomarker in blood of hepatic injury. Product of lipid peroxidation (MDA), activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and reduced glutathione (GSH) contents were evaluated for oxidative stress in hepatic injury. Moreover, histopathological observation was assayed at the degree of hepatic injury.

Results

After administrated the FZJ, the dose of 200 mg/kg significantly decreased ALT and AST, and attenuated histopathology of hepatic injury, and ameliorated the oxidative stress in hepatic tissue. Partly assayed indexes were ameliorated after administrated FZJ at the dose of 100 mg/kg.

Conclusion

These results indicated that hepatic protective effects of FZJ were very relevant to modulate the oxidative stress in hepatic injury.     

Evaluation of hepatoprotective effect of Zhi-Zi-Da-Huang decoction and its two fractions against acute alcohol-induced liver injury in rats

Aim of the study

To investigate the hepatoprotective effect of Zhi-Zi-Da-Huang decoction (ZZDHD) and its two fractions (one is extracted with diethyl ether as a solvent from the water extract and is called ZD-DE for short, the other is the remained aqueous fraction after extracted with diethyl ether and is abbreviated as ZD-AQ) against acute alcohol-induced liver injury in rats.

Materials and methods

Animals were administered orally with alcohol 6 g/kg at 2 h after the doses of ZZDHD and two fractions everyday for eight consecutive days except rats in normal group. The protective effect was evaluated by biochemical parameters including aspartate transaminase (AST), alanine transferase (ALT), reduced glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD). The biochemical observations were supplemented by histopathological examination. HPLC-UV was used for phytochemical analysis of the ZZDHD and its two fractions.

Results

The high dose of ZZDHD exhibited a significant protective effect by reversing the biochemical parameters and histopathological changes. ZD-DE and ZD-AQ demonstrated different protective action in biochemical examination. Partly assayed indexes were ameliorated after administrated the media dose of ZZDHD. HPLC analysis indicated that ZZDHD contained flavonoids, anthraquinones and iridoids, which might be the active chemicals.

Conclusions

This study demonstrated the hepatoprotective activity of ZZDHD thus scientifically supported the usage of this formula.     

Polyprenols from Taxus chinensis var. mairei prevent the development of CCl4-induced liver fibrosis in rats

Ethnopharmacological relevance

The aim of this study was to investigate the anti-fibrotic effects and the possible underlying mechanisms of taxus polyprenols (TPs) isolated from the needles of Taxus chinensis var. mairei.

Materials and methods

The animals were randomly divided into normal control with vehicles only (olive oil), rat model given CCl₄ only, CCl₄+low TPs (48 mg/kg), CCl₄+medium TPs (120 mg/kg), CCl₄+high TPs (300 mg/kg), and CCl₄+Polyene phosphatidylcholine (PP, 120 mg/kg). The rat model of liver fibrosis was induced by subcutaneous injection of 40% (v/v) of CCl₄ diluted in olive oil (3 mL/kg body weight) twice per week for 8 weeks. Liver histopathological study was performed. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and albumin (ALB) of the serum were determined for evaluating the liver function. In order to reveal the possible mechanisms of the anti-fibrotic effects, oxidative stress level, hepatic collagen metabolism, and hepatic stellate cells (HSCs) activation were investigated. Furthermore, the mRNA expression of the fibrotic-related factors was measured by the quantitative real-time RT-PCR.

Results

TPs successfully attenuated liver injury induced by CCl₄ shown by histopathological sections of livers and improved liver function as indicated by decreased ALT, AST and ALP levels and increased ALB levels in serum of the rats. TPs significantly increased the hepatic Cu/Zn SOD and GSH-Px activities along with GSH content while a remarkable decrease in MDA content. Both immunohistochemical staining and mRNA expression levels of α-SMA indicated a profound suppression of HSCs activation. Furthermore, it significantly inhibited the mRNA expression of the pro-fibrotic cytokines Col α1(I), Col α1(Ш), MMP-2, TIMP-1, TIMP-2, PDGF-β, TGF-β1, CTGF and TNF-α and restored the hepatoprotective factor HGF.

Conclusion

These results suggest that the protective effects of TPs in chronic CCl₄-induced liver fibrosis might be related with the reduction of oxidative damage, the inhibition of HSCs activation, the down-regulation of pro-fibrogenic stimuli and the protection of hepatocytes.     

Effects of ethanolic dried leaf extract of Lecaniodiscus cupanioides on antioxidant enzymes and biochemical parameters in rats

Ethnopharmacological relevance

Lecaniodiscus cupanioides is widely used in West African folk medicine for the treatment of inflammatory conditions, fevers and bacterial infections.

Aim of the study

To evaluate the potential toxic effects of the ethanolic dried leaf extract of Lecaniodiscus cupanioides (LC) on antioxidant enzymes in selected organs and biochemical parameters.

Materials and Methods

Crude ethanolic extract of Lecaniodiscus cupanioides dried leaves was prepared. A 90-day sub-chronic toxicity study was conducted using albino rats. Reconstituted Lecaniodiscus cupanioides was administered at a dosage of 400, 800 and 1600 mg/kg (high dose) with a control group receiving 10 ml/kg orally. Histopathological studies of major organs and blood chemistry analysis were performed on blood obtained via cardiac puncture after euthanization. Selected organs (liver, kidney and brain) were harvested for antioxidant and histopathological assessments.

Results

The extract produced significant (p<0.05) increases in the weights of liver, kidney and brain at 800 mg/kg and 1600 mg/kg compared to the control. Biochemical analysis showed significant increase in Alanine transferase (ALT) at 800 mg/kg and 1600 mg/kg. Assay for antioxidant enzymes showed a reversible decrease in the activity of Catalase (CAT), Superoxide dismutase (SOD) and Glutathione (GSH) with an increase in Malondialdehyde (MDA) at 800 mg/kg and 1600 mg/kg Lecaniodiscus cupanioides. Histopathological study showed reversible congestion in the brain, liver, and kidney at 800 mg/kg and 1600 mg/kg.

Conclusion

Findings in this study reveal that the ethanolic dried leaf extract of Lecaniodiscus cupanioides has the potential for inhibiting in vivo antioxidant enzymes activity and causing hepatotoxicity after prolonged exposure.     

Preventive and therapeutic effects of Danhong injection on lipopolysaccharide induced acute lung injury in mice

Ethnopharmacological relevance

Danhong injection (DHI), a Chinese Materia Medica standardized product extracted from Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese), has been reported to have anti-inflammatory, anti-oxidative and anti-fibrinolytic properties, which is used extensively for the treatment of cardiovascular diseases in clinic.

Aim of this study

The present study aimed to investigate the preventive and therapeutic effects of DHI on lipopolysaccharide (LPS) induced acute lung injury (ALI) in mice.

Materials and methods

Lung injury was induced by intranasal instillation with 10 μg LPS. Mice were randomly divided into four groups:Control group; LPS group; LPS+5 ml/kg DHI group and LPS+10 ml/kg DHI group. The effects of DHI on LPS-induced neutrophils influx, inflammatory cytokines release, protein leakage, myeloperoxidase (MPO) and superoxide dismutase (SOD) activities, malondialdehyde (MDA) level were examined. In addition, the NF-κB activation in lung tissues was detected by Western blot.

Results

In LPS challenged mice, DHI significantly reduced the infiltration of activated neutrophils and decreased the levels of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). DHI also inhibited protein extravasation in BALF, attenuated edema and the pathological changes in the lung. In addition, DHI markedly prevented LPS-induced elevation of MDA and MPO levels, as well as reduction of SOD activity. Further study demonstrated that DHI effectively inhibited the NF-κB activation in lung tissues.

Conclusion

DHI has been demonstrated to protect mice from LPS induced acute lung injury by its anti-inflammatory and anti-oxidant activities.     

Neuroprotective effect of calycosin on cerebral ischemia and reperfusion injury in rats

Ethnopharmacological relevance

Radix Astragali has been commonly used as traditional herbal medicine in China for reinforcing vital energy, strengthening superficial resistance and promoting the discharge of pus and the growth of new tissue.

Aim of the study

The present study was to investigate the neuroprotective effect of calycosin isolated from the roots of Radix Astragali on cerebral ischemic/reperfusion injury.

Materials and methods

After 24 h of reperfusion following ischemia for 2 h induced by middle cerebral artery occlusion (MCAO), Sprague-Dawley rats were intragastrically administered different doses of calycosin (7.5, 15, 30 mg/kg, respectively). Neurological deficit, infarct volume, histopathology changes and some oxidative stress markers were evaluated after 24 h of reperfusion.

Results

Treatment with calycosin significantly ameliorated neurologic deficit and infarct volume after cerebral ischemia reperfusion. Calycosin also reduced the content of malondialdehyde (MDA), protein carbonyl and reactive oxygen species (ROS), and up-regulated the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) in a dose-dependent manner. Moreover, calycosin can also inhibit the expression of 4-Hydroxy-2-nonenal (4-HNE).

Conclusion

These results suggest that calycosin has a neuroprotective effect against cerebral ischemia/reperfusion injury. The mechanism might be attributed to its antioxidant effects.     

Hepatoprotective effect of Trichosanthes cucumerina Var cucumerina L. on carbon tetrachloride induced liver damage in rats

Ethnopharmacological relevance

Different parts of the plant Trichosanthes cucumerina Var cucumerina L. (cucurbitaceae) are used to treat liver disorders, traditionally. It is one among the constituents in various Ayurvedic formulations used for the treatment of liver disorders and other diseases.

Aims of study

The aim of the present study is to evaluate the protective effect of Trichosanthes cucumerina against experimentally induced liver injury.

Materials and methods

The methanolic extract of whole plant of Trichosanthes cucumerina (TCME) was evaluated for the hepatoprotective activity against carbon tetrachloride (CCl₄) induced hepatotoxicity in rats. Various biochemical parameters like alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), total protein (TP) and albumin (ALB) levels were estimated in serum as well as the glutathione (GSH) and malondialdehyde (MDA) levels in the liver were determined. Histopathological changes in the liver of different groups were also studied.

Results

The pre-treatment of TCME at dose levels of 250 and 500 mg/kg b.w.p.o. had controlled the raise of AST, ALT, ALP, TB and MDA levels and the effects were comparable with standard drug (silymarin 100 mg/kg b.w.p.o.). The GSH, TP and ALB levels were significantly increased in the animals received pre-treatment of the extract. The animals received pre-treatment of the extract shown decreased necrotic zones and hepatocellular degeneration when compared to the liver exposed to CCl₄ intoxication alone. Thus the histopathalogical studies also supported the protective effect of the extract.

Conclusion

This study demonstrates the hepatoprotective activity of Trichosanthes cucumerina and thus scientifically supports the usage of this plant in various Ayurvedic preparations and traditional medicine for treatment of liver disorders.     

Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicin-induced liver injury in mice

Ethnopharmacological relevance

Yulangsan polysaccharide (YLSPS) is often used in popular folk medicine in the Guangxi Zhuang Autonomous Region of China as a chief ingredient of Millettia pulchra, which is used as an hepatic protection, anti-aging and memory improving agent. In this study, the hepatoprotective effects of YLSPS against isoniazid (INH) or rifampicin and isoniazid (RFP+INH)-induced liver injury were investigated in mice.

Materials and methods

The liver injury was induced by intragastric administration of INH or RFP+INH daily for 10 days. During the experiment, the model group received INH or RFP+INH only, and the normal control group received an equal volume of saline. Treatment groups received not only INH or RFP+INH but also the corresponding drugs, DDB (200 mg/kg/day) or YLSPS (100, 200, and 400 mg/kg/day) 2 h after the administration of INH or RFP+INH.

Results

Analysis experiments showed that YLSPS significantly alleviated liver injury as indicated by the decreased levels of ALT and AST and the increased levels of SOD, GSH and GSH-Px. Moreover, YLSPS could effectively reduce the pathological tissue damage. The research on the mechanisms underlying the hepatoprotective effect showed that YLSPS was able to reduce lipid peroxidation and activate the anti-oxidative defense system.

Conclusion

Our results show that YLSPS is effective in attenuating hepatic injury in the INH or RFP+INH-induced mouse model, and could be developed as a new drug for treatment of liver injury.     

Hepatoprotective effects of Coptidis rhizoma aqueous extract on carbon tetrachloride-induced acute liver hepatotoxicity in rats

Aim of the study

Coptidis rhizoma (CR, Chinese name is Huanglian) has been used in treating infectious and inflammatory diseases for two thousand years in Traditional Chinese Medicine (TCM). Its related pharmacological basis for the therapeutics has been studied intensively, but CR can also be used for vomiting of “dampness-heat type or acid regurgitation” due to “liver-fire attacking stomach” in TCM, whose symptoms seem to link the hepatic and biliary disorders, yet details in the therapies of liver diseases and underlying mechanism(s) remain unclear. To clarify this ethnopharmacological relevance, hepatoprotective effect of Coptidis rhizoma aqueous extract (CRAE) and its possible mechanism were studied in rats intoxicated with carbon tetrachloride (CCl₄) in the present study.

Materials and methods

Sprague–Dawley (SD) rats aged 7 weeks old were intraperitoneally injected with CCl₄ at a dose of 1.0 ml/kg as a 50% olive oil solution. The rats were orally given the CRAE at doses of 400, 600, 800 mg/kg and 120 mg/kg berberine body weight (BW) after 6 h of CCl₄ treatment. At 24 h after CCl₄ injection, samples of blood and liver were collected and then biochemical parameters and histological studies were carried out.

Results

The results showed that CRAE and berberine inhibited significantly the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and increased the activity of superoxide dismutase (SOD). Observation on the hepatoprotective effect of berberine was consistent to that of CRAE.

Conclusion

The study is the first time to demonstrate that CRAE has hepatoprotective effect on acute liver injuries induced by CCl₄, and the results suggest that the effect of CRAE against CCl₄-induced liver damage is related to antioxidant property.     

Investigation of the absorbed and metabolized components of Danshen from Fuzheng Huayu recipe and study on the anti-hepatic fibrosis effects of these components

Ethnopharmacology

Fuzheng Huayu recipe (FZHY) was formulated on the basis of Chinese medicine theory in treating liver fibrosis. It has a significant efficacy against liver fibrosis caused by chronic hepatitis B, with the action mechanisms of inhibition of hepatic stellate cell activation, protection of hepatocyte oxidative injury and regulations of hepatic matrix remodeling etc.

Aim of the study

To identify the absorbed components and metabolites of Danshen in FZHY in rat serum, and find their active components for anti-liver fibrosis.

Material and methods

A valid high performance liquid chromatography–electrospray ionization ion trap mass spectrometry (HPLC-ESI/MSⁿ) method was established to investigate the absorbed and metabolized compounds of Danshen in FZHY in rat serum after oral administration. Mass spectra were acquired in both negative and positive modes. Otherwise, to evaluate the anti-hepatic fibrosis efficacies of absorbed and metabolized compounds, the LX-2 cell line of hepatic stellate cell (HSC), which was crucial cellular basis of fibrogenesis, was cultured and incubated with absorbed compounds, the cytotoxicity was determined with the cellomics Multiparameter Cytotoxicity Kit 1 by High Content Screening (HCS), the cell proliferation was assayed with EdU-DNA incorporation, and the cell activation was analyzed through α-smooth muscle actin (α–SMA) expression with high content screening technology.

Results

More than 11 compounds and 2 metabolites from Danshen were identified in the serum after oral administration of FZHY by comparing their mass spectra and retention behavior with reference compounds or literature data. Among these compounds, there were no obvious changes in nuclear morphology, membrane permeability with blow 96 μM of six polar compounds treatment in comparison with control cells, respectively. And the salvianolic acid B (6 μM, 48 μM), caffeic acid (6 μM, 48 μM) and rosmarinic acid (48 μM) could obviously inhibit LX-2 cells proliferation, down-regulate α–SMA expression.

Conclusion

The results proved that the established method could be applied to analyze the absorbed into blood compounds of Danshen after oral administration FZHY. These absorbed compounds included 11 compounds and 2 metabolites of Danshen. Among them, the salvianolic acid B, caffeic acid and rosmarinic acid were the effective components of FZHY to anti-hepatic fibrosis effects.     

Insulin releasing and alpha-glucosidase inhibitory activity of ethyl acetate fraction of Acorus calamus in vitro and in vivo

Ethnopharmacological relevance

The radix of Acorus calamus L. (AC) is widely used in the therapy of diabetes in traditional folk medicine of America and Indonesia, and we previously reported the insulin sensitizing activity of the ethyl acetate fraction of AC (ACE).

Aim of the study

To investigate the insulin releasing and alpha-glucosidase inhibitory activity of ACE in vitro and in vivo.

Materials and methods

Insulin releasing and alpha-glucosidase inhibitory effects of different fractions from AC were detected in vitro using HIT-T15 cell line and alpha-glucosidase enzyme. Furthermore, effects of ACE orally on serum glucose were detected in fasted and glucose/amylum challenged normal mice.

Results

AC and ACE increased insulin secretion in HIT-T15 cells as gliclazide did. As in vivo results, ACE (400 and 800 mg/kg) significantly decreased fasting serum glucose, and suppressed the increase of blood glucose levels after 2 g/kg glucose loading in normal mice. In addition, ACE as a mixed-type inhibitor inhibited alpha-glucosidase activity in vitro with an IC₅₀ of 0.41 μg/ml, and 100 mg/kg of it clearly reduced the increase of blood glucose levels after 5 g/kg amylum loading in normal mice.

Conclusions

Apart from its insulin sensitizing effect, ACE may have hypoglycemic effects via mechanisms of insulin releasing and alpha-glucosidase inhibition, and thus improves postprandial hyperglycemia and cardiovascular complications.     

Investigation of antihyperglycaemic activity of aqueous and petroleum ether extract of stem bark of Pongamia pinnata on serum glucose level in diabetic mice

Aim of the study

The aim of the study was to evaluate the antihyperglycaemic activity of aqueous (PPSB-AQE) and petroleum ether (PPSB-PEE) extract of stem bark Pongamia pinnata in alloxan induced diabetic mice.

Materials and methods

Diabetes was induced in mice by alloxan (80 mg/kg, i.v.). After acute and subacute treatment serum glucose was determined. OGTT was performed in PPSB-PEE pretreated animals.

Results

PPSB-PEE (25, 50, 100, 200 and 400 mg/kg) showed significant reduction in serum glucose level in acute and subacute studies. The antihyperglycaemic effects of PPSBPE (100, 200 and 400 mg/kg) showed onset at 2 h and peak effect at 6 h and the effect was sustained until 24th h with 400 mg/kg. In subacute study, antihyperglycaemic effect was observed on 21st day. In PPSBPE treated mice the body weight was not reduced in contrast to that in vehicle group. In OGTT, increased glucose utilization was observed.

Conclusions

It is concluded that PPSB-PEE but not PPSB-AQE showed antihyperglycaemic activity.     

Aqueous extract of Artemisia capillaris exerts hepatoprotective action in alcohol–pyrazole-fed rat model

Ethnopharmacologic relevance

Artemisia capillaris, also called “InJin” in Korean, has been widely used to treat various hepatic disorders in traditional Oriental medicine.

Aims

The purpose of this study is to evaluate the hepatoprotective effect of Artemisia capillaris (aqueous extract, WAC) on alcoholic liver injury.

Materials and methods

Liver injury was induced by oral administration of 30% alcohol (10 mL/kg, twice per day) plus pyrazole (PRZ, 30 mg/kg) with/without WAC (50, 100 mg/kg, orally once per day) or silymarin (50 mg/kg) for 10 days. The hepatoprotective effects were assessed by observing histopathological changes, hepatic transaminase enzymes, hepatic oxidation and antioxidant parameters, inflammatory cytokines, and alcohol metabolic enzymes in serum and hepatic gene expression level, respectively.

Results

Alcohol–PRZ treatment drastically increased the serum levels of aspartate transaminase (AST), alanine transaminase (ALT), and malondialdehyde (MDA) levels in serum and liver tissues while these changes were significantly ameliorated by WAC administration (p<0.05 or 0.01). The prominent microvesicular steatosis and mild necrosis in hepatic histopathology were induced by alcohol–PRZ treatment, but notably attenuated by WAC administration. Moreover, the alcohol–PRZ treatment-induced depletions of the antioxidant components including glutathione content, total antioxidant capacity (TAC), activities of glutathione peroxidase (GSH-Px), reductase (GSH-Rd), catalase, and superoxide dismutase (SOD) were significantly ameliorated by WAC administration (p<0.05, except GSH-Rd). These results were in accordance with the modulation of NF-E2-related factor (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Alcohol–PRZ treatment increased the levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) in hepatic tissues. However they were significantly normalized by WAC administration (p<0.05 or 0.01). In addition, WAC administration significantly attenuated the alterations of aldehyde dehydrogenase (ALDH) level in serum and hepatic gene expressions of ALDH and alcohol dehydrogenase (ADH).

Conclusions

These results support the relevance in clinical use of Artemisia capillaris for alcohol-associated hepatic disorders. The underlying mechanisms may involve both enhancement of antioxidant activities and modulation of proinflammatory cytokines.     

The protective effects of Acanthopanax senticosus Harms aqueous extracts against oxidative stress: Role of Nrf2 and antioxidant enzymes

Ethnopharmacological relevance

Acanthopanax senticosus (Rupr.et Maxim.) Harms, classified into the family of Araliaceae, is used in a variety of diseases in traditional Chinese system of medicine including hypertension, ischemic heart disease and hepatitis.

Materials and methods

Different doses (75 mg/kg, 150 mg/kg and 300 mg/kg) of aqueous extracts of Acanthopanax senticosus Harms were evaluated for the antioxidant activity against oxidative stress in mice induced by tert-butyl hydroperoxide (t-BHP) through observating histopathology of the liver and detecting antioxidant enzyme activity, concentration of antioxidant, and related gene and protein expression.

Results

Acanthopanax senticosus Harms aqueous extracts (ASE) attenuated the morphological injury of liver induced by t-BHP and increased the activity of antioxidant enzymes and the ratio of GSH/GSSG in serum and liver homogenates. Medium and high doses of ASE also elevated the gene expression of NF-E2-related factor-2 (Nrf2), but not CuZnSOD, MnSOD, catalase (CAT), glutathione peroxidase (GPx) and GCLC. Protein expression results showed that Nrf2 and the antioxidant enzymes were all increased significantly by medium and high doses of ASE.

Conclusion

The present results indicated that ASE protect against oxidative stress which may be generated via the induction of Nrf2 and related antioxidant enzymes.     

Fuzheng Huayu inhibits carbon tetrachloride-induced liver fibrosis in mice through activating hepatic NK cells

Aim of the study

Fuzheng Huayu (FZHY) is a Chinese compound herbal preparation which consists of six Chinese herbs. This study examines the preventative effects of FZHY on liver fibrosis induced by carbon tetrachloride (CCl₄) and explores its possible mechanisms of action.

Materials and methods

Liver fibrosis was induced in male C57BL/6N mice by injecting a 10% CCl₄ solution intraperitoneal twice a week for six weeks. After 6 weeks of treatment, serum ALT and AST assay, liver tissue histological examination and immunostaining were carried out to examine the liver function and fibrosis degree. The expression levels of alpha-smooth muscle actin (SMA) were measured by quantitative real-time PCR and western blot. Hepatic natural killer (NK) cells were isolated from liver and evaluated by FACS.

Results

Upon pathological examination, the FZHY-treated mice showed significantly reduced liver damage. The expression of α-SMA increased markedly upon treatment with CCl₄ and the increase was reversed by FZHY treatment. FZHY treatment also enhanced the activation of hepatic NK cells and the production of interferon-gamma (IFN-γ). The protective effects of FZHY were reversed in the mice that were depleted of NK cells by anti-ASGM-1 Ab treatment.

Conclusions

FZHY can efficiently inhibit CCl₄-induced liver fibrosis. Furthermore, the depletion of NK cells attenuates the protective effects of FZHY. We conclude that FZHY could be an effective drug for liver fibrosis, and its mechanism of action involves the activation of hepatic NK cells.