Inhibition of matrix metalloproteinases related to metastasis by diosgenyl and pennogenyl saponins

Ethnopharmacological relevance

Diosgenyl and pennogenyl saponins isolated from Rhizoma Paridis, showed pro-apoptosis and immunoregulation with antitumor activity in cultured cells and animal systems.

Aim of the study

To evaluate their anti-metastatic mechanism on cancer cells and discuss their structure-activity relationship on anti-tumor effect.

Materials and methods

This research used the wound healing and migration assay to detect their anti-invasive effect on B16 melanoma cells. Through the gelatin zymography assay, immunofluorescence analysis and western blot, saponins exhibited different levels of protein expression inhibition of MMP-1, -2, -3, -9 and -14.

Results

Through the analysis, diosgenyl and pennogenyl saponins inhibited the metastasis of B16 melanoma cells. Diosgenyl saponins also showed strong suppression of enzyme activity of MMP-2 and -9. Different saponins exhibited different levels of inhibition on MMP expression.

Conclusions

17-α OH increases the sensitivity of diosgenyl saponins to the membrane-bound protease which can stimulate proMMP-2 activation, but it also decreases the anti-metastatic activity of diosgenyl saponin. Furthermore, their combination might provide a potential therapeutic modality for metastasis.     

Phaleria macrocarpa (Scheff.) Boerl fruit aqueous extract enhances LDL receptor and PCSK9 expression in vivo and in vitro

Ethnopharmacological relevance

Phaleria macrocarpa (Scheff.) Boerl (Pm) has been shown to reduce cholesterol level in vitro and in vivo experiment.

Aim of the study

This study investigated the effects of Pm fruit on weight control and mechanistic basis of its anti-hypercholesterolemic effect in both in vivo and in vitro.

Materials and method

In the in vivo study, thirty six male Sprague Dawley were randomized to six groups. Five groups were induced into hypercholesterolemia by giving 3% cholesterol enriched-diet for 52 days while one group acted as control. The rats were then treated with Pm extract (0, 20, 30 and 40 mg/ml) or simvastatin for 84 days. The following parameters were determined: (1) body weight, (2) blood lipid profile (total cholesterol, triglyceride, HDL and LDL) and (3) hepatic LDL receptor (160 kDa and 120 kDa) and PCSK9 proteins. In the in vitro study, HepG2 cells were cultured in serum-free RPMI supplemented with 0.2% BSA with or without LDL and in the presence of Pm extract (0, 0.1, 2, 40 and 1000 μg/ml) or simvastatin (4.60 μg/ml) for 24 h. The abundance of both LDL receptor and PCSK9 proteins and mRNA were investigated.

Results

Pm extract significantly (P < 0.05) reduced body weight gain, total cholesterol, triglycerides, HDL LDL levels and upregulated hepatic LDL receptor as well as PCSK9 proteins of hypercholesterolemic rats. These results were supported by studies in HepG2 cells whereby Pm extract also significantly upregulated both LDL receptor and PCSK9 at protein and mRNA levels.

Conclusion

This study enhances the potential usage of Pm fruit for controlling the body weight of obese people and for treating hypercholesterolemia.     

The methanolic extract of Boesenbergia rotunda (L.) Mansf. and its major compound pinostrobin induces anti-ulcerogenic property in vivo: possible involvement of indirect antioxidant action

Ethnopharmacological relevance

Boesenbergia rotunda (L) Mansf. has been used for the treatment of gastrointestinal disorders including peptic ulcer. In the current study we aimed to investiagte the anti-ulcer activities of methanolic extract of B. rotunda (MEBR) and its main active compound, pinostrobin on ethanol-induced ulcer in rats. The possible involevement of lipid peroxidation, nitric oxide, cyclooxygenases and free radical scavenging mechanisms also has been investigated.

Materials and methods

Pinostrobin was isolated form the rhizomes of B. rotunda. Ulcer index, gastric juice acidity, mucus content, gross and histological gastric lesions and thiobarbituric acid reactive substances (TBARS) were evaluated in ethanol-induced ulcer in vivo. The effect of pinostrobin into lipopolysaccharide/interferon-γ stimulated rodent cells, COX-1 and COX-2 activities were done in vitro.

Results

Pre-treatment with MEBR, pinostrobin or omeprazole protected the gastric mucosa as seen by reduction in ulcer area and mucosal content, reduced or absence of submucosal edema and leucocytes infiltration. Pinostrobin significantly (p<0.05) lowered the elevated TBARS level into gasteric homogenate. Pinostrobin did not produced significant in vitro inhibition of NO from LPS/IFN-γ activated rodent cells without affecting the viability of these cells. Further, the compound did bot revleaed inhibitory effects on both COX- 1& 2 enzymes. The antioxidant assays also exhibited non significance in vitro.

Conclusion

Thus it can be concluded that MEBR possesses anti-ulcer activity, which could be attributed to indirect anti-oxidant mechanism of pinostrobin but not to the intervention with nitric oxide and COX inflammation pathways.     

Toona sinensis (leaf extracts) inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells

Aim of the study

Toona sinensis is well known as a traditional Chinese medicine; also, it has been shown to exhibit anticancer and anti-inflammatory effects. This study was aimed at evaluating the anti-angiogenesis effect of the aqueous extracts of Toona sinensis (TS extracts) or gallic acid, a major component of TS extracts, against both VEGF-induced EA.hy 926 and human umbilical vein endothelial cells (HUVECs).

Materials and methods

Anti-proliferative activity of TS extracts or gallic acid, was determined against EA.hy 926 and HUVECs by trypan blue exclusion method. Invasion, tube formation and chick chorioallantoic membrane assay were carried out to determine the in vitro and in vivo anti-angiogenic effects.

Results

Non-cytotoxic concentration of TS extracts (50-100 μg/mL) and gallic acid (5 μg/mL) inhibited the proliferation of VEGF-stimulated EA.hy 926 and HUVECs. Inhibitory effects of TS extracts and gallic acid on angiogenesis were assessed by VEGF-induced migration/invasion and capillary-like tube formation by EA.hy 926 and HUVECs. Additionally, gelatin zymography assays showed that TS extracts and gallic acid suppressed the activity of metalloproteinase (MMP)-9 and MMP-2 activated by VEGF. In vivo, TS extracts and gallic acid strongly suppressed neovessel formation in the chorioallantoic membrane of chick embryos. Flow cytometry analyses and Western blot demonstrated that treatment with TS extracts and gallic acid induced G₀/G₁ arrest in VEGF-stimulated EA.hy 926 cells via a reduction in the amounts of cyclin D1, cyclin E, CDK4, hyperphosphorylated retinoblastoma protein (pRb), VEGFR-2, and eNOS.

Conclusions

These results support an anti-angiogenic activity of Toona sinensis that may contribute critically to its cancer and inflammation chemopreventive potentials.     

In vitro anti-inflammatory and anti-cancer activities of Cuscuta reflexa Roxb

Aim of the study

To determine anti-inflammatory and anti-cancer activities of Cuscuta reflexa in cell lines (in vitro).

Materials and methods

Anti-inflammatory activity of the water extract was analysed in vitro using lipopolysaccharide (LPS) induced inflammatory reactions in murine macrophage cell line RAW264.7. The expression of COX-2 and TNF-α genes involved in inflammation was analysed by SQ RT-PCR. EMSA was conducted to analyse the influence of the extract on NF-κB signalling. Anti-cancer activity was analysed on Hep3B cells by MTT assay, DAPI staining, annexin V staining and SQ-RT PCR analysis of BAX, Bcl-2, p53 and survivin.

Results

The extract down regulated LPS induced over expression of TNF-α and COX-2 in RAW264.7 cells; blocked NF-κB binding to its motifs and induced apoptosis in Hep3B cells as evidenced from MTT, DAPI staining and annexin V staining assays. The extract up regulated pro-apoptotic factors BAX and p53, and down regulated anti-apoptotic factors Bcl-2 and survivin.

Conclusions

The study showed that Cuscuta reflexa inhibits LPS induced inflammatory responses in RAW264.7 cells through interplay of TNF-α, COX-2 and NF-κB signalling. It induced apoptosis in Hep3B cells through the up regulation of p53, BAX and down regulation of Bcl-2 and survivin.     

Dragon's Blood extract has antithrombotic properties, affecting platelet aggregation functions and anticoagulation activities

Ethnopharmacological relevance

Dragon's Blood from Dracaena cochinchinensis (Lour.) S.C. Chen (Yunnan, China), as a traditional Chinese medicinal herb, was shown to have certain antithrombotic effects. A new preparation process was used to extract effective components from Dragon's Blood. A 95% ethanol extract A (EA) and a precipitate B (PB) fraction were obtained and compared. Reliability of the preparation process was validated by pharmacodynamic experiments.

Materials and methods

A rat/mouse thrombosis and blood stasis model was developed for this study, and EA and PB effects on thrombosis, platelet functions and blood coagulation activities were analyzed.

Results

It was observed that the EA fraction had significantly better inhibitory effects than the PB fraction on thrombosis (p < 0.05), platelet aggregation function (p < 0.01) and anticoagulation activity (p < 0.05-0.01).

Conclusions

The results obtained here showed that EA fraction from Dragon's Blood contained pharmacologically effective compounds with antithrombotic effects, partially improving platelet function and anticoagulation activity.     

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.     

Pharmacological properties of Anagallis arvensis L. ("scarlet pimpernel") and Anagallis foemina Mill. ("blue pimpernel") traditionally used as wound healing remedies in Navarra (Spain)

Ethnopharmacological relevance

: Anagallis arvensis and Anagallis foemina are traditionally used in Navarra (Spain) for dermatological purposes regarding wound healing properties. In some cases they are also used to threat internal infections although they are known to be toxic at high doses.

Aim of study

: Due to lack of studies, we decided to evaluate the potential of the plants as wound healing remedies measuring antimicrobial and anti-inflammatory properties using in vitro procedures.

Materials and methods

Antimicrobial effects were studied against four bacteria and one fungus. Anti-inflammatory properties were measured in terms of COX-1 and -2 inhibition as well as superoxide radical scavenging capacity.

Results

Both species exerted antimicrobial and anti-inflammatory effects. The methanolic extract obtained from Anagallis arvensis seemed to produce the highest inhibition in Candida albicans (MIC = 0.31 mg/ml). Inhibition of COX-1 and -2 was also stronger for methanolic extracts whereas aqueous were revealed as better free radical scavengers.

Conclusions

The study reveals that both species posses antimicrobial and anti-inflammatory activities related to their ethnomedicinal uses.     

The protective role of Tongxinluo on blood–brain barrier after ischemia–reperfusion brain injury

Ethnopharmacological relevance

Tongxinluo (TXL), a renowned traditional Chinese medicine, consists of several different kinds of ingredients and has been widely used to treat myocardial infarction and ischemic stroke. However, the underlying neuroprotective mechanisms are not fully understood.

Aim of the study

We focus on the effect of TXL on blood–brain barrier (BBB) including edema formation and tight junction (TJ) protein rearrangement, and inflammatory response after transient middle cerebral artery occlusion (tMCAO). We further explore the protective mechanism of TXL on ischemia-induced BBB damage.

Materials and methods

Adult CD1 male mice (n=168) were randomly divided into TXL pre-treatment group, TXL pre-post treatment group, TXL post-treatment group, control group and sham group. Mice in TXL pre-treatment group were given TXL solution by 1 g/kg/day orally for 7 days before tMCAO. Mice in pre-post treatment group were continuously given TXL 7 days before and 14 days after tMCAO. Mice in TXL post-treatment group were given TXL solution immediately after tMCAO. Rotarod test and neurological severity scores were evaluated at 1–14 days following tMCAO. Brains were harvested for examining infarct volume, edema formation, and immunofluorescent staining at 1 and 3 days after tMCAO. Cytokines IL-6, IL-1β and TNF-α mRNA expression, and BBB permeability were further examined by RT-PCR and immunostaining.

Results

TXL pre-post treatment improved neurobehavioral outcomes and reduced infarct volume compared to the control (p<0.05). Meanwhile, hemispheric swelling, Evans blue and IgG protein extravasation reduced, while TJ protein expression up-regulated in pre-post treatment group (p<0.05). Further study indicated that infarct volume was smaller and BBB damage was less severe in TXL pre-post treatment group compared to TXL pre-treatment alone. It was noted that fewer myeloperoxidase (MPO) positive cells and less cytokines IL-6, IL-1β and TNF-α expression in pre-post treatment group compared to the control group (p<0.05).

Conclusions

TXL pre-treatment and pre-post treatment effectively protected the brain from BBB disruption via alleviating inflammatory response. Moreover, pre-post treatment has better outcomes, suggesting that continuous administration of TXL before and throughout ischemia period is necessary because of multiple functions of TXL.     

Anti-angiogenic effects of the fruit of Alpinia oxyphylla

Aim of study

The fruit of Alpinia oxyphylla, an herb commonly used in East Asian medicine, is variously used for the treatment of cancer and inflammatory conditions, which may possibly be mediated through anti-angiogenesis. This study aims to check for anti-angiogenic functions in the herb.

Materials and methods

The 95% ethanol extract and four subsequent fractions (n-hexane, ethyl acetate, n-butanol and aqueous fractions) of the fruit of A. oxyphylla were tested on zebrafish model by quantitative endogenous alkaline phosphatase assay; then the active fractions were further tested on wild type and Tg(fli1a:EGFP)y1 zebrafish embryos and human umbilical vein endothelial cells and tumor cell lines for the anti-angiogenic effects.

Results

The n-hexane and ethyl acetate fractions showed anti-angiogenic potentials in both in vivo and in vitro models.

Conclusions

The use of A. oxyphylla for cancer and inflammation diseases may be partly due to its effects against vessel formation.