Protective effect of Icariin on the early stage of experimental diabetic nephropathy induced by streptozotocin via modulating transforming growth factor β1 and type IV collagen expression in rats

Ethnopharmacological relevance

Icariin is a major constituent of flavonoid isolated from the plant Herba epimedii, which is used as a traditional Chinese medicine to nourish the kidney and reinforce yang. Therefore, the pharmacological studied of its diabetic nephropathy effect was undertaken to validate its traditional use.

Materials and methods

Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (40 mg/kg i.v.). Sustained blood glucose levels (>16.7 mmol/l) were considered as diabetic and selected for experimentation. Thirty rats were randomly divided into three groups: control, diabetic, diabetic + Icariin (80 mg/kg, i.g.) administered 8 weeks from 5th to 12th week. Experiment was carried out at the beginning of 13th week. All rats were anaesthetized and then were killed to remove kidneys. Blood glucose, serum creatinine (Cr), blood urea nitrogen (BUN), the activity of superoxide dismutase (SOD), malondialdehyde (MDA) and hydroxyproline (Hyp) in the kidney tissue were measured. Glomerular morphology was observed by light microscopy. Immunohistochemistry and Western blot were employed to determine the proteins levels of TGF-β₁ and type IV collagen.

Results

The enhancement of Cr and BUN was found in model group, which was significantly attenuated by treatment with Icariin. Meanwhile, elevated MDA and Hyp levels in renal tissue as well as decreased SOD activities in renal tissue were significantly remitted by Icariin. The renal pathological changes in Icariin treatment group were ameliorated. Furthermore, the Icariin decreased the expression of TGF-β₁ and collagen IV protein.

Conclusions

Icariin can evidently relieve renal damage in rats with diabetic nephropathy induced by STZ, which might be related to modulating the expression of collagen IV and TGF-β₁ protein.     

Protective effect of Heliotropium foertherianum (Boraginaceae) folk remedy and its active compound, rosmarinic acid, against a Pacific ciguatoxin

Ethnopharmacological relevance

Terminalia pallida is an evergreen endemic tree, mentioned in Ayurveda as the fruits of Terminalia pallida are excellent in cardioprotective property. Tribal people use Terminalia pallida fruit for the treatment of diabetes and this plant widely used in many other disorders.

Aim of study

The present investigation was to evaluate the antioxidant, biochemical profile and histological studies of qualitatively standardized ethanolic extract of Terminalia pallida fruits (TpFE) against isoproterenol-induced myocardial infarction in rats.

Materials and methods

TpFE was standardized by high performance liquid chromatography (HPLC) and mass spectroscopy (MS). Rats were pretreated orally with different doses of TpFE (100, 300, and 500 mg kg⁻¹ body weight) and cardioprotective positive control gallic acid (GA) for 30 days prior to isoproterenol (ISO) induced myocardial infarction. The rats were sacrificed, hearts were collected and homogenized for biochemical analysis. The effects on total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C) and very low density lipoprotein cholesterol (VLDL-C), high density lipoprotein cholesterol (HDL-C), lipid peroxidation (LPO) marker, malondialdehyde (MDA), creatine kinase (CK), lactate dehydrogenase (LDH), alanine transaminase (ALT), aspartate transaminase (AST), catalase (CAT), glutathione peroxidase (GPx), sodium potassium (Na⁺/K⁺), calcium (Ca²⁺) and magnesium (Mg²⁺) adenosine triphosphatases (ATPases) were estimated in heart tissue homogenate.

Results

Rats administered with ISO showed a significant increase in TC, TG, LDL-C, VLDL-C, and MDA and a significant decrease in HDL-C, cardiac marker enzymes – CK, LDH, ALT and AST. ISO significantly reduced antioxidants – CAT, GPx, and membrane bound enzymes – Na⁺/K⁺, Ca²⁺ and Mg²⁺ ATPases. Pretreatment with TpFE (100, 300, and 500 mg kg⁻¹ bw) and GA (15 mg kg⁻¹ bw) for a period of 30 days significantly inhibited the effects of ISO. Moreover, biochemical findings were supported by histopathological observations.

Conclusion

The present study provide evidence for the first time, that TpFE pretreatment ameliorated myocardial injury in ISO-induced myocardial infarcted rats and exhibited cardioprotective activity.     

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.     

Immunosuppressive effects of an ethyl acetate extract from Urtica dentata Hand on skin allograft rejection

Aim of the study

To investigate the immunosuppressive effects of HPLC qualitied ethyl acetate extract (EAE) from Urtica dentate Hand on skin allograft rejection in a murine model.

Materials and methods

Allo-skin transplantation model was established by placing skin allograft of C57BL/6 mice in the wound bed which was on the back of Balb/c mice. We used FACS to study the effects of EAE on dendritic cells (DCs) maturation and CD4⁺CD25⁺T regulatory cells (Tregs) differentiation. We also studied spleen lymphocyte proliferation and T-bet gene expression in DCs. Concentration of Th1/Th2 cytokines was monitored as markers of Th1/Th2 responses by ELISA.

Results

A significant prolongation of skin allografts survival was observed as a dose-dependent manner in the animals treated with EAE. By FACS, we found that treatment with EAE (200 mg kg⁻¹) resulted in an immature statement of DCs and stimulated the differentiation of CD4⁺CD25⁺Tregs. Additionally, the expression of T-bet gene and the proliferation of spleen lymphocytes were efficiently abated in EAE treated mice. Comparing to the model control, EAE-treated recipients showed a significant down-regulation (P < 0.01) of Th1 cytokines (IL-2, IFN-γ) and an obviously increase (P < 0.01) of Th2 cytokine (IL-10) in the serum, which presented in a dose-related way.

Conclusions

The anti-allograft rejection effect of EAE by enhancing CD4⁺CD25⁺Tregs differentiation and sustaining DCs immaturation makes EAE to be a possible choice for treating autoimmune diseases in a way of inducing a stable immunological tolerance state.     

Effect of Astragali-Cordyceps Mixtura on TGF-β/Smad signal pathway in the lung of asthma airway remodeling

Aim of the study

We try to find out the influence of traditional Chinese Medicine Astragali-Cordyceps Mixtura (ACM) on TGF-β/Smad signal pathway in the lung of asthma airway remodeling.

Materials and methods

Mice were sensitized and challenged by OVA to establish a model of asthma. To assess the effects of ACM on the mice, animals of the ACM groups were treated with ACM. Data were achieved by using techniques as follow: counting cell number of BALF, assaying the amount of collagen deposition by Masson's staining, performing RT-PCR and immunohistochemistry for mRNA and protein expression of TGF-β1, Smad3 and Smad7.

Results

The depositions of collagen in airway wall greatly increased at the model group compared with that of the normal group. In contrast, these decreased at the ACM groups. As compared with the control group, TGF-β1 expression also decreased at both mRNA and protein level at the ACM-M group versus increased both at the model group. Whereas, Smad7 significantly decreased only at the model group and partly restored at the ACM-M group.

Conclusions

ACM greatly improves the symptoms of asthma airway remodeling by inhibiting the expression of TGF-β1 and upregulating the amount of Smad7.     

Inhibitory effects of citrus extracts on the experimental pulmonary fibrosis

Aim of the study

A Chinese herbal formula, Hu-qi-yin possessed an anti-pulmonary fibrosis effect. Pericarp of Citrus reticulata, one of the herbal drugs contained in this formula showed the most potent inhibitory activity on the proliferation of human embryonic lung fibroblasts (HELF). The present study was designed to clarify the active principles responsible for the activity and further explore the anti-pulmonary fibrosis effect in vivo.

Materials and methods

The water, 75% ethanol and flavonoids-enriched extracts of Citrus reticulata were prepared and screened for their anti-proliferation activity using HELF culture system. The ethanol extract was further administered orally at doses of 100 and 200 mg/(kg day) to bleomycin (BLM)-induced pulmonary fibrosis rats. The analyses of the rat body weight, hydroxyproline levels in serum and lung, scores of alveolitis and fibrosis, as well as the expression of transforming growth factor-β₁ (TGF-β₁) at the protein and the messenger ribonucleic acid (mRNA) levels in lung were performed.

Results

The ethanol extract showed the strongest inhibitory activity on HELF proliferation. Further research using BLM-induced rat model revealed that the ethanol extract at the doses of 100 and 200 mg/(kg day) caused a marked increase of body weight at first 7 days, significantly lowered the hydroxyproline levels in lung, greatly improved the pathologic scores, as well as inhibited the overexpressions of TGF-β₁ protein and mRNA.

Conclusions

The results suggest that the ethanol extract of Citrus reticulata has anti-pulmonary fibrosis effects and might have a great potential for the treatment of fibrosis of lung.     

Protective effect of Shenfu injection on thromboangiitis obliterans model rats

Ethnopharmacological relevance

Thromboangiitis obliterans (TAO) or Buerger's disease is a non atherosclerotic, segmentar inflammatory vasculitis that is incurable at present. Shenfu injection (SFI), a traditional Chinese formulation, have been confirmed to produce protective influences on several organs and limb during ischemia and reperfusion (IR) injury in rats. However, the effects of SFI on TAO remain unclear.

Materials and Methods

Adult male Sprague Dawley rats were randomly divided into sham operated group, TAO model group, SFI 2.5 mg/kg (low dose), 5 mg/kg (medium dose) and 10 mg/kg (high dose) groups (n = 8). Rats were intravenously administered SFI 2.5, 5 and 10 mg/kg or saline once per day for 15 days. TAO model was prepared by injecting sodium laurate into the femoral artery of rats. Then we examined the changes of pathological signs, pathologic grading of thrombus, the indexes of hematology, the contents of thromboxane B2 (TXB2), 6-keto-prostaglandin F_lα (6-K-PGF_1α) in plasma following SFI or saline treatment.

Results

More pathological signs of lesions, higher grades of pathological thrombosis, increased blood platelet counts, the increase in the TXB2 and TXB2/6-K-PGF_1α ratio, as well as the decrease of 6-K-PGF_1α in TAO model group were shown in present experiments; SFI treatment significantly improved the pathological signs of lesions induced by sodium laurate injection, reduced the numbers of thrombus formation, blood platelet counts, the TXB2 and TXB2/6-K-PGF_1α ratio but increased the 6-K-PGF_1α compared with TAO model group. However, there were no significant alterations in the counts of red blood cell, leucocyte and neutrophil among these groups.

Conclusions

Our preliminary findings first indicated that SFI can produce significant therapeutic effects on experimental Buerger's disease model rats in a dose independent manner. The underlying mechanisms may be due to its modifying hematology, inhibiting platelet aggregation and enhancing anti-thrombotic function of vessel endothelia.     

Preliminary study of antidiabetic activity of the methanolic leaf extract of Axonopus compressus (P. Beauv) in alloxan-induced diabetic rats

Ethnopharmacological relevance

Axonopus Compressus is commonly used by the people of Southern Nigeria to treat different ailment such as common cold and diabetes. This study therefore, evaluated the anti-diabetic effect of the methanolic leaf extract of the plant.

Materials and methods

Diabetes was induced in the rats by intraperitoneal (i.p.) injection of alloxan monohydrate at the dose of 180 mg/kg. Three test doses of the extract (250, 500 and 1000 mg/kg) administered per os through gastric gavage to the rats were used in the study. The activity was compared to a standard reference drug (glibenclamide, 2 mg/kg) and a negative control. Blood from the tail snip was used to measure the effects of the extract and drug at 0, 1, 3 and 6 h using autoanalyzer (AccuCheck Active^®) glucose kit.

Results

Methanolic leaf extract of Axonopus compressus at all the doses (250, 500 and 1000 mg/kg) used caused a respective time dependent and significant (p < 0.0001) reduction (by 31.5%, 19.8% and 24.5%) of the blood glucose levels in the diabetic rats when compared to the negative control group at the 6th hour. However, the reference drug (glibenclamide, 2 mg/kg) decreased the blood glucose levels by 69.9% and the tween 20 solution (negative control) increased the blood glucose level by 15.2% at the 6th hour. Moreso, the extract at the different test doses caused various degrees of reduction of the blood glucose levels of the test rats at 1st, 3rd and 6th hours when compared to the negative control rats.

Conclusion

The findings suggest that Axonopus compressus may possess antidiabetic property.     

Vasorelaxant and hypotensive effects of a hydroalcoholic extract from the fruits of Nitraria sibirica Pall. (Nitrariaceae)

Ethnopharmacological relevance

Fruits of Nitraria sibirica Pall. are traditionally used in Uighur medicine to treat hypertension. This study aimed to support that folk use by defining their vasoactive and hypotensive properties.

Materials and methods

The vasorelaxant activity and the underlying mechanisms of a hydroalcoholic extract from the fruits of Nitraria sibirica Pall. (NSHE) were evaluated on thoracic aortic rings isolated from Wistar rats. In addition, the acute hypotensive effect of NSHE was assessed in anesthetized spontaneously hypertensive rats (SHR) and in their normotensive control Wistar Kyoto (WKY) rats.

Results

NSHE (0.1–10 g/l) was clearly more effective to induce vasodilation of phenylephrine- (PE, 1 μM) than high KCl- (60 mM) pre-contracted aortic rings with respective E_max values of 82.9 ± 2.2% and 34.8 ± 3.6%. The removal of endothelium almost abolished the relaxant effect of the extract. In addition, pre-treatment with N^w-nitro-l-arginine-methyl ester (l-NAME, 100 μM), atropine (1 μM) or charybdotoxin (30 nM) plus apamin (30 nM), respective blockers of nitric oxide (NO) synthase, muscarinic receptors and endothelium-derived hyperpolarizing factor (EDHF), significantly reduced the observed effect of NSHE. By contrast, the cyclooxygenase (COX) inhibitor indomethacin (10 μM) or the K⁺ channels blockers glibenclamide (10 μM), iberiotoxin (30 nM) and 4-amino-pyridine (4-AP, 1 mM) failed to modify the vasodilation. Finally, the acute intravenous injection of NSHE (1, 5, 10, 20 mg/kg) induced an immediate and transient hypotensive effect in anesthetized SHR and in WKY rats.

Conclusions

This experimental animal study suggests that hydroalcoholic extract from the fruits of Nitraria sibirica Pall. induces vasorelaxation through an endothelium-dependent pathway involving NO synthase (NOS) activation, EDHF production and muscarinic receptor stimulation. Additionally, our results determine that this vasorelaxant effect is translated by a significant hypotensive effect.     

Paeoniflorin,the main active constituent of Paeonia lactiflora roots,attenuates bleomycin-induced pulmonary fibrosis in mice by suppressing the synthesis of type I collagen

Ethnopharmacological relevance

In the theory of traditional Chinese medicine, pulmonary fibrosis (PF) belongs to pulmonary arthralgia, which means blood stasis in lung tissue. The roots of Paeonia lactiflora Pall are usually used to relieve the symptoms of this disease by promoting blood circulation and removing blood stasis. Paeoniflorin, the main active ingredient of P. lactiflora, may have anti-PF potential.

Aim of study

This study aimed to investigate the effects and underlying mechanisms of paeoniflorin on bleomycin (BLM)-induced PF in mice.

Materials and Methods

The PF model was established in mice by an intratracheal instillation of BLM. Paeoniflorin (25, 50, 100 mg/kg) and prednisone (6 mg/kg), as a positive control, were orally administered for consecutive 21 days. Histopathological changes were evaluated by hematoxylin and eosin stain and Masson's trichrome stain. The content of hydroxyproline was detected by using kits. The contents of type I collagen, TGF-β1 and IFN-γ were detected by ELISA. The levels of α-SMA, Smad4, Smad7 and the phosphorylations of Smad2/3 were detected by western blot. The mRNA expressions of MMP-1 and TIMP-1 were detected by RT-PCR.

Results

In mice treated with BLM, paeoniflorin (50 mg/kg) significantly prolonged the survival periods, attenuated infiltration of inflammatory cells, interstitial fibrosis, and deposition of extracellular matrix in lung tissues. It also decreased the contents of hydroxyproline (a marker of collagens), type I collagen and α-SMA (an indicator of myofibroblasts) in lung tissues of mice. Paeoniflorin down-regulated the expressions of TGF-β1, Smad4 and the phosphorylations of Smad2/3, while up-regulated the expression of Smad7 in lung tissues. Moreover, paeoniflorin increased the content of IFN-γ. But, it only slightly affected mRNA expressions of MMP-1 and TIMP-1 in lung tissues of mice.

Conclusions

Paeoniflorin attenuates PF by suppressing type I collagen synthesis via inhibiting the activation of TGF-β/Smad pathway and increasing the expression of IFN-γ.     

Evaluation of diuretic activity of Amaranthus spinosus Linn. aqueous extract in Wistar rats

Ethnopharmacological relevance

Traditional Siddha medicine literature claims that the Amaranthus spinosus Linn. (family: Amaranthaceae) whole plant possesses diuretic property.

Aim of the study

To evaluate the diuretic potential of Amaranthus spinosus aqueous extract (ASAE) in rats.

Material and methods

Different concentrations of ASAE (200, 500, 1000, 1500 mg/kg), thiazide (10 mg/kg) and vehicle were orally administered to rats (n = 6 animals per group) and their urine output was collected after 24 h. Volume, pH, Na⁺, K⁺ and Cl⁻ concentrations of urine were estimated.

Results

ASAE produced increase in Na⁺, K⁺, Cl⁻ excretion, caused alkalinization of urine, showed strong saluretic activity and carbonic anhydrase inhibition activity. These effects were observed predominantly at 500 mg/kg dose and there was no dose–response relationship.

Conclusion

Our study strongly suggests that the Amaranthus spinosus is acting as a thiazide like diuretic with carbonic anhydrase inhibitory activity which restates the claim as diuretic herb in Siddha medicine.     

Cardioprotective effects of aqueous Schizandra chinensis fruit extract on ovariectomized and balloon-induced carotid artery injury rat models: effects on serum lipid profiles and blood pressure

Aim of the study

The fruit from Schizandra chinensis, a member of the Magnoliaceae family, has been used to treat menopause-related symptoms. We have previously reported that an aqueous extract of Schizandra chinensis fruit (ScEx) caused vascular relaxation via the production of endothelial nitric oxide. Estrogen-like molecules are known to play a protective role in cardiovascular diseases through several mechanisms, but the cardioprotective effects of ScEx have not been clearly demonstrated. Therefore, we investigated the vasculoprotective effects of ScEx on ovariectomized (OVX) and balloon-induced carotid artery injury rat models.

Materials and methods

An aqueous extract of Schizandra chinensis (ScEx) was examined for its cardioprotective effects. To test the arterial response to injury, we applied the balloon-induced carotid artery model to OVX Sprague-Dawley (SD) rats. Rats were subcutaneously administered vehicle, 17β-estradiol (E2; 0.02 or 0.2 mg/kg/day), or ScEx (0.2 or 2.0 mg/kg/day) over the course of the study. Vessel morphology was assessed two weeks after injury. To identify the cardioprotective effects after ScEx treatment, we measured serum lipid profiles and blood pressure levels in the OVX- and sham-operated normotensive and spontaneously hypertensive rats (SHR). Serum lipid profiles were measured in OVX rats after five weeks of treatment with vehicle, E2 (0.5 mg/kg/day), or ScEx (0.5 or 5.0 mg/kg/day). Tail systolic blood pressure in OVX SHR was measured weekly.

Results

In the balloon-induced carotid artery injury model, treatment with E2 (0.2 mg/kg/day) or ScEx (2.0 mg/kg/day) reduced the intimal area and the intima-to-media ratio compared to control animals. Injection of ScEx or E2 reduced body weight gain but did not inhibit the decrease in uterine weight. Treatment with ScEx (5.0 mg/kg/day) or E2 (0.5 mg/kg/day) in OVX SD rats reduced total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), TC/high-density lipoprotein cholesterol (HDL-C), and TC-(HDL-C)/HDL-C compared to control animals. In OVX rats, treatment with ScEx or E2 also significantly reduced LDL-C compared with the OVX control rats, and systolic blood pressure was significantly attenuated compared to OVX control and the sham control rats.

Conclusions

ScEx treatment restored endothelial function in rats that underwent balloon-induced carotid artery injury, and it reduced serum cholesterol levels in OVX rats. Similar to E2, ScEx exihibited hypotensive effects in OVX SHR. Therefore, ScEx and E2 exhibited similar cardioprotective effects, thereby suggesting that ScEx is a potential candidate to replace estradiol in the prevention and treatment of cardiovascular diseases.     

Effect on blood pressure of a dietary supplement containing traditional medicinal plants of Côte d'Ivoire

Ethnopharmacological relevance

A medicinal composition containing salt (sodium chloride) is given as a traditional dietary supplement to hypertensive patients (TDSHP) in Côte d’Ivoire. It consists of whole plant of Bidens pilosa (Asteraceae) and fresh leaves of Moringa oleifera (Moringaceae).

Aim of the study

The aim of this study was to establish the scientific basis for the use of this traditional recipe rich in sodium chloride in hypertension settings.

Materials and methods

We used a total aqueous extract of this traditional dietary supplement containing medicinal plants (Bidens pilosa, Moringa oleifera) and salt (sodium chloride). Experiment was carried out to evaluate its effect on arterial blood pressure of rabbits. The experimental device used for recording blood pressure in rabbits is based on the principle of Ludwig mercury manometer.

Results

TDSHP between 5 × 10⁻⁸ and 5 × 10⁻² mg/kg caused a dose-dependent hypotension. TDSHP elicited drops in blood pressure ranging between 7.14 ± 4 and 100 ± 7.5%, compared to normal blood pressure of rabbits. Fifty percent effective dose of TDSHP was 3.95 × 10⁻⁴ mg/kg. Similarly as the hypotension induced by acetylcholine, the one caused by TDSHP at dose of 3.95 × 10⁻⁴ mg/kg in rabbit was progressively inhibited by atropine, dosed between 5 × 10⁻⁴ to 5 × 10⁻² mg/kg. The percentage drop of recorded blood pressure ranged from 50.3 ± 1.87 to 3.71 ± 1.09% compared to the normal value of blood pressure. In the presence of atropine, TDSHP effect was partially inhibited. The same increasing doses of TDSHP reduced significantly the increase of blood pressure induced by adrenaline dosed at 4.76 × 10⁻⁴ mg/kg from 89.3 ± 2.19 to 1.19 ± 0.59%.

Conclusion

The consumption of this traditional dietary supplement is justified in hypertensive patients according to its composition and its ability to reduce blood pressure has been demonstrated experimentally. TDSHP should not be considered as an antihypertensive drug, it remains to us a salt substitute to be taken with moderation with strict adherence to the traditional dose.     

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.     

Baicalein, isolated from Scutellaria baicalensis, protects against endothelin-1-induced pulmonary artery smooth muscle cell proliferation via inhibition of TRPC1 channel expression

Ethnopharmacological relevance

We investigated the antiproliferative effects of baicalein, isolated from Scutellaria baicalensis (Huang-qin), on ET-1-mediated pulmonary artery smooth muscle cells (PASMCs) proliferation and the mechanisms underlying these effects.

Materials and methods

Intrapulmonary artery smooth muscle cells were isolated and cultured from female Sprague-Dawley rats and used during passages 3-6. The proliferation of PASMCs was quantified by cell counting and XTT assay. The protein expression of TRPC1 and PKCα were determined by western blotting. The cell cycle pattern was assayed by flow cytometry. The intracellular calcium concentrations ([Ca²⁺]_i) were measured using the fluorescent indicator fura-2-AM and flow cytometry.

Results

Baicalein (0.3-3 μM) inhibited PASMCs proliferation, promoted cell cycle progression, enhanced [Ca²⁺]_i levels, increased capacitative Ca²⁺ entry (CCE), upregulated the canonical transient receptor potential 1 (TRPC1) channel and membrane protein kinase Cα (PKCα) expression induced by ET-1 (0.1 μM). The PKC activator PMA (1 μM) reversed the inhibitory effects of baicalein on ET-1-induced upregulation of TRPC1 expression and S phase accumulation, while the PKC inhibitor chelerythrine (1 μM) potentiated baicalein-mediated G₂/M phase arrest and TRPC1 channel inhibition.

Conclusion

Our findings suggest that baicalein protects against ET-1-induced PASMCs proliferation via modulation of the PKC-mediated TRPC channel.     

Double directional adjusting estrogenic effect of naringin from Rhizoma drynariae (Gusuibu)

Ethnopharmacological relevance

Chinese traditional medicine Rhizoma drynariae (Gusuibu) is widely used for clinically treating osteoporosis and bone non-union. Naringin and its active metabolite naringenin are the main active ingredients of Rhizoma drynariae total flavonoids.

Aim of the study

The purpose of this paper is to confirm estrogenic and anti-estrogenic activity of naringin and naringenin, and provide the basic data to further study for the dose-effect relationship and the mechanism for Rhizoma drynariae in treatment of osteoporosis and other estrogen deficiency-related diseases.

Materials and methods

Naringin was extracted from Rhizoma drynariae. Naringin and its metabolin naringenin were tested estrogenic and anti-estrogenic activities through the experiment of cell proliferation and uterus weight gain in mice. Their estrogen-receptor binding abilities were tested by yeast two-hybrid experiment and nuclear receptor cofactor assays (RCAS) experiment, and their possible binding sites for ERβ were performed by computer aided molecular docking technology.

Results

Naringin and naringenin showed significant effects on the proliferation of estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen. Induction increased proliferation as the drug concentration, and the strongest proliferation appeared at a concentration of 8.6 × 10⁻⁵ M. When estradiol (10⁻¹⁰ M) and the different concentrations of naringin or naringenin were treated at the same time, naringin and naringenin could result in antagonistic effects on estradiol-induced MCF-7 cell proliferation, but they did not significantly affect proliferation of estrogen-insensitive ER(−) MDA-MB-231 cells. Naringin and naringenin exhibited higher binding capacity to estrogen receptor β (ERβ) than estrogen receptor α (ERα) in yeast two-hybrid experiments and nuclear receptor cofactor assays (RCAS) experiment. Docking simulation between naringin/naringenin and ERβ were performed, and the corresponding binding free energies of naringin-receptor and naringenin-receptor docked complexes were −7.95 and −10.45 kcal/mol. Hydrogen bonds were found between naringin and the amino acid residues Lys304 and His308. The oxygen atom (O11) of naringenin formed hydrogen bond to Arg346, and there may be hydrophobic space interactions between phenyl group (C13-C18) of naringenin and the amino acid residues Leu298, Met336, Met340, Phe356, Ile376 and Leu380.

Conclusions

Naringin and naringenin revealed a double directional adjusting function of estrogenic and anti-estrogenic activities. Both of them showed estrogenic agonist activity at low concentration or lack of endogenous estrogen. On the other hand, they also acted as estrogenic antagonists at high concentrations or too much endogenous estrogen. They produced estrogenic and anti-estrogenic effects primarily through selectively binding with ERβ, which could prevent and treat osteoporosis with the mechanism of estrogenic receptor agitation. This paper confirmed the estrogenic and anti-estrogenic activity of naringin and naringenin, and further studies were still essential to study their dose-effect relationship and the anti-osteoporosis mechanism for Rhizoma drynariae in the treatment of osteoporosis and other estrogen deficiency-related diseases.     

Aqueous fraction from Costus spiralis (Jacq.) Roscoe leaf reduces contractility by impairing the calcium inward current in the mammalian myocardium

Ethnopharmacological relevance

Brazilian folk medicine uses infusion of Costus spiralis leaf to help people to treat arterial hypertension and syndromes of cardiac hyperexcitability.

Aim of the study

Evaluate the aqueous fraction (AqF) effect on atrial contractility and investigate its mechanism of action.

Materials and methods

The AqF effect on the cardiac contractility was studied on isolated electrically driven guinea pig left atria. Atropine and tetraethylammonium (TEA) were employed to investigate whether potassium contributes for the inotropic mechanism of the AqF. The role of calcium in this effect was also studied. This was done by analysing the AqF effect on the Bowditch's phenomenon, as well as by studying whether it could interfere with the concentration-effect curve for CaCl₂, isoproterenol, and BAY K8644. Mice isolated cardiomyocytes were submitted to a whole-cell patch-clamp technique in order to evaluate whether the L-type calcium current participates on the AqF effect. Furthermore, the intracellular calcium transient was studied by confocal fluorescence microscopy.

Results

AqF depressed the atrial contractile force. It was the most potent fraction from C. spiralis leaf (EC₅₀ = 305 ± 41 mg/l) (crude extract: EC₅₀ = 712 ± 41; ethyl acetate: EC₅₀ = 788 ± 121; chloroform: EC₅₀ = 8948 ± 1346 mg/l). Sodium and potassium content in the AqF was 0.15 mM and 1.91 mM, respectively. Phytochemical analysis revealed phenols, tannins, flavones, xanthones, flavonoids, flavonols, flavononols, flavonones, and saponins. Experiments with atropine and TEA showed that potassium does not participate of the inotropic mechanism of AqF. However, this fraction decreased the force overshoot characteristic of the Bowditch's phenomenon, and shifted the concentration-response curve for CaCl₂ (EC₅₀ from 1.12 ± 0.07 to 7.23 ± 0.47 mM) indicating that calcium currents participate on its mechanism of action. Results obtained with isoproterenol (1-1000 pM) and BAY K8644 (5-2000 nM) showed that AqF abolished the inotropic effect of these substances. On cardiomyocytes, 48 mg/l AqF reduced (∼23%) the L-type calcium current density from −6.3 ± 0.3 to −4.9 ± 0.2 A/F (n = 5 cells, p < 0.05) and reduced the intracellular calcium transient (∼20%, 4.7 ± 1.2 a.u., n = 42 cells to 3.7 ± 1.00 a.u., n = 35 cells, p < 0.05). However, the decay time of the fluorescence was not changed (control: 860 ± 32 ms, n = 42 cells; AqF: 876 ± 26 ms, n = 35 cells, p > 0.05).

Conclusions

The AqF of C. spiralis leaf depresses myocardial contractility by reducing the L-type calcium current and by decreasing the intracellular calcium transient. Despite the lack of data on the therapeutic dose of AqF used in folk medicine, our results support, at least in part, the traditional use of this plant to treat cardiac disorders.     

The effects of Polygonum cuspidatum extract on wound healing in rats

Aim of the study

Polygonum cuspidatum has long been used as a traditional medicine inducing wound healing. In this study, the extract from the Chinese medicinal herb Polygonum cuspidatum was investigated on its wound healing activity, in order to obtain an accurate elucidation of its traditional use value.

Materials and methods

After creating wound healing model on the back of rats, the extract from the Chinese medicinal herb Polygonum cuspidatum was applied. Wound healing rates were calculated at 3, 7, 14, and 21 days after the wounding, and tissues were harvested at 1, 3, 7, 14 and 21 days for histological and immunohistochemistry analysis. The stages of wound granulation tissues were evaluated histopathologically. The expression of TGF-β1 was determined by immunohistochemically.

Results

Wound healing rates were significantly higher at 3, 7, 14 and 21 days in the extract group than in the control (p < 0.05). Histological results showed more well-organized bands of collagen, more fibroblasts and hair follicle and less inflammatory cells in the extract group. The immunohistochemical results revealed that TGF-β1 increased in the extract group on day 1, 3 and 7 post-wounding (p < 0.05).

Conclusion

The present study has shown that the extract from the Chinese medicinal herb Polygonum cuspidatum possesses wound healing activity, and thus provided the evidence for its traditional use value.     

Endothelium-independent relaxation and contraction of rat aorta induced by ethyl acetate extract from leaves of Morus alba (L.)

Aim of the study

Based on screening for vasoactive traditional Chinese medicinal herbs, the present study was performed to investigate the vasoactive effects of an ethyl acetate extract from leaves of Morus alba (L.) (ELM) on rat thoracic aorta and the mechanisms underlying these effects.

Materials and methods

Isolated rat thoracic rings were mounted in an organ bath system and the effects of ELM on their responses were evaluated.

Results

ELM (0.125–32.000 g/l) induced a concentration-dependent relaxation (P < 0.01 vs. control) both in endothelium-intact and -denuded aortas precontracted by high K⁺ (6 × 10⁻² M) or 10⁻⁶ M phenylephrine (PE). In endothelium-denuded aortas, ELM at the EC₅₀ concentration reduced Ca²⁺-induced contraction (P < 0.01 vs. control) after PE or KCl had generated a stable contraction in Ca²⁺-free solution. And after incubation with verapamil, ELM induced contraction in endothelium-denuded aortas precontracted by PE (P < 0.01 vs. control); this was abolished by ruthenium red (P < 0.01 vs. ELM-treated endothelium-denuded group; P > 0.05 vs. control), but not by heparin (P > 0.01 vs. ELM-treated endothelium-denuded group; P < 0.01 vs. control).

Conclusions

The results showed that ELM had dual vasoactive effects, and the relaxation was greater than the contraction. The relaxation was mediated by inhibition of voltage- and receptor-dependent Ca²⁺ channels in vascular smooth muscle cells, while the contraction occurred via activation of ryanodine receptors in the sarcoplasmic reticulum.