Neuroprotective effects of Buyang Huanwu decoction against hydrogen peroxide induced oxidative injury in Schwann cells

Ethnopharmacological relevance

Buyang Huanwu decoction (BYHWD) is a traditional Chinese medicine and can be used to promote peripheral nerve regeneration. However the regenerative mechanism of BYHWD remains unclear. The objective of this study was to investigate the protective mechanisms of BYHWD in Schwann cells damaged by hydrogen peroxide (H₂O₂).

Materials and methods

Schwann cells which were derived from neonatal sciatic nerves of rats were used in subsequent experiments. Schwann cells were injured by various concentrations of H₂O₂ (0.25, 0.5 and 1 mM final concentration). BYHWD (600 μg/ml final concentration) was added to the medium either simultaneously or 1 h later after the addition of H₂O₂. Subsequently, methyl thiazolyl tetrazolium (MTT) assay was performed. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were also examined after 12 h. The expression of Caspase 3 and the concentration of intercellular Ca²⁺ ([Ca²⁺]i) were also determined.

Results

Among three concentrations of H₂O₂, 0.5 mM H₂O₂ induced Schwann cells swelled and neuritis disappeared after 12 h. In the presence of BYHWD, MTT assay showed that more cells were viable in comparison with the H₂O₂ injury group. Moreover, the addition of BYHWD has also increased the SOD activity with decreased in MDA level. Furthermore, the concentration of [Ca²⁺]i and expression of Caspase 3 were decreased with the addition of BYHWD in culture.

Conclusions

Our results revealed that BYHWD protected Schwann cells from oxidative injury. The mechanism of BYHWD promoting neural regeneration possibly associated with its anti-oxidative activity.     

Standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal induces apoptosis via mitochondrial pathway in human cervical cancer HeLa cells

Ethnopharmacological relevance

Artemisia princeps Pampanini is widely used in Eastern traditional medicine for the treatment of circulatory disorders, such as, dysmenorrhea, hematuria, hemorrhoids, and inflammation, and is also used to treat chronic conditions, such as, cancers, ulcers, and digestive disorders.

Aim of the study

The purpose of this study is to investigate the effect of a standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal (FRAP) on the induction of apoptosis and the molecular mechanism involved in human cervical cancer HeLa cells.

Materials and methods

Human cervical cancer HeLa cells were treated with FRAP and apoptosis was detected by cell morphologic observation, annexin-V-PI staning and western blot analysis on the expression of protein associated with cell death.

Results

FRAP led to the cleavages of caspase-3, -8, and -9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD-fmk) significantly suppressed the FRAP-induced accumulation of annexin V positive cells. Furthermore, it was found that FRAP caused a loss of mitochondrial membrane potential (MMP) and the release of cytochrome c to the cytosol. Furthermore, the overexpression of Bcl-xL significantly prevented FRAP-induced apoptosis, MMP changes, and the activations of caspase-3, -8, and -9. Interestingly, pretreatment with caspase-8 inhibitor significantly reduced the FRAP-induced activation of caspase-3 but not that of caspase-9, whereas the caspase-3 inhibitor, z-DEVD-fmk, markedly attenuated the FRAP-induced activation of caspase-8. In BALB/c^nu/nu mice bearing a HeLa xenograft, FRAP dosed at 25 or 50 mg/kg significantly inhibited tumor growth.

Conclusion

Our results indicate caspase-mediated activation of the mitochondrial death pathway plays a critical role in the FRAP-induced apoptosis of HeLa cells and that FRAP inhibits the in vivo tumor growth of HeLa xenograft mice.     

Protective effect of 5-hydroxymethylfurfural derived from processed Fructus Corni on human hepatocyte LO2 injured by hydrogen peroxide and its mechanism

Aim of the study

The aim of the present study was to evaluate the putative protective effect of 5-hydroxymethylfurfural (5-HMF) derived from processed Fructus Corni on human hepatocyte cell line (LO2) injured by hydrogen peroxide in vitro and the mechanism of its protection.

Materials and methods

The percentage of cell viability was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The hepatocyte cell apoptosis and cell cycle were detected by flow cytometric analysis. The content of nitric oxide and caspase-3 activity were quantified spectrophotometrically by enzyme-linked immunoassay.

Result

The study showed that incubation with 5-HMF caused significant increase in the viability of LO2 cell, decrease of cell apoptosis and recovery of cell cycle in LO2 cell injured by hydrogen peroxide, which was accompanied with the decreased nitric oxide level and caspase-3 activity.

Conclusions

Our data suggest that 5-HMF protects LO2 cell against damage induced by hydrogen peroxide through inhibiting effect of cell apoptosis caused by promoting S phase to G2/M phase and the decreased caspase-3 activity and nitric oxide level. 5-HMF is one of the active principles in processed Fructus Corni.     

Gastroprotective and ulcer healing effects of essential oil from Hyptis spicigera Lam. (Lamiaceae)

Aim of the study

Tanshinone IIA (Tan IIA) is one of the key components of Salvia miltiorrhiza Bunge that has been widely used for various cardiovascular and cerebrovascular disorders in Asian countries. Many studies have reported that Tan IIA has antioxidative properties, but whether Tan IIA can rescue neurons from oxidative insult has never been reported. The present study was undertaken to evaluate the possible neuroprotective effects of Tan IIA on hydrogen peroxide (H₂O₂)-induced oxidative stress in rats.

Materials and methods

H₂O₂-induced cytotoxicity was evaluated by the cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and flow cytometry with PI staining. Calcium imaging experiments were carried out to measure intracellular free calcium concentration. Western blotting was used to determine the expression of Bax and Bcl-2 protein. Electrophysiological studies in hippocampal slices were performed to investigate the effect of Tan IIA on synaptic function and cognitive impairment caused by H₂O₂.

Results

It was found that pretreatment with Tan IIA protected primary rat cortical neurons against H₂O₂-induced cytotoxicity. Furthermore, Tan IIA markedly reduced the elevation of [Ca²⁺]_i evoked by H₂O₂. Western blot analysis indicated that pretreatment with Tan IIA prevented the increase in Bax/Bcl-2 ratio induced by H₂O₂. In addition, preincubation of Tan IIA 20 min prior to H₂O₂ exposure could reverse H₂O₂-induced hippocampal LTP impairment, but without significant alteration in basal synaptic transmission and LTP induction.

Conclusions

These findings demonstrate that Tan IIA might serve as a novel promising therapeutic agent for oxidative stress injury in neurodegenerative diseases.     

Eucommia ulmoides Oliv. Bark. protects against hydrogen peroxide-induced neuronal cell death in SH-SY5Y cells

Ethnopharmacological relevance

Eucommia ulmoides Oliv. Bark. (EUE), has commonly been used to fortify the muscles and lungs, lower blood pressure, prevent miscarriage, improve the tone of liver and kidneys, and promote longevity the traditional tonic medicines of Korea, China, and Japan.

Aim of the study

In this study, we investigated that the neuroprotective activities and possible mechanisms of EUE aqueous extract in hydrogen peroxide (H₂O₂)-induced neuronal cell death in human SH-SY5Y neuroblastoma cells.

Material and method

We examined the effects of EUE against H₂O₂-induced cytotoxicity, DNA condensation, the production of reactive oxygen species (ROS), loss of mitochondria membrane potential (MMP), the proteolysis of cleaved poly-ADP-ribose polymerase (PARP), and the expression of Bcl-2, Bcl-xL, cleaved caspase-3, and release of cytochrome c. Moreover, we attempted to determine whether EUE suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), and phosphoinositide 3-kinase (PI3K)/Akt.

Results

Pretreatment with EUE increased cell viability and inhibited cytotoxicity and DNA condensation. EUE also attenuated the increase in ROS production and MMP reduction. Western blot data revealed that EUE inhibited H₂O₂-induced up- or down-regulation of cleaved PARP, cleaved caspase-3, Bcl-2, and Bcl-xL. The EUE inhibited release of cytochrome c from mitochondria to the cytosol, and significantly attenuated H₂O₂-induced phosphorylation of JNK, p38 MAPK, ERK 1/2, and PI3K/Akt.

Conclusion

The potent neuroprotective capacity of EUE, shown in these experiments, may potentially be applied in the prevention or treatment of neurodegenerative diseases such as Alzheimer's disease (AD).     

Cytotoxic and apoptotic activity of Cheilanthes farinosa (Forsk.) Kaulf. against human hepatoma,Hep3B cells

Aim of the study

Cheilanthes farinosa (Forsk.) Kaulf., family: Adianthaceae, is a fern of immense medicinal properties used in ethno-medicine. The Gaddis tribe of Himachal Pradesh, India, has been using this fern to treat liver damage. Aim of the current study was to determine the apoptosis inducing and cytotoxic activity, if any, of this fern towards hepatic cancer cells.

Materials and methods

Water extract of the plant was used in the study. MTT assay was performed in hepatocellular carcinoma cell line, Hep3B as well as murine macrophage cell line, RAW264.7 to analyze the cytotoxic activity of the plant. Further, the apoptosis inducing action of water extract of the plant was evaluated using comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining.

Results

This plant was found to produce considerable cytotoxicity in hepatoma cell line, Hep3B without inducing substantial damage to non-cancerous cell line RAW264.7. In addition, this plant was found to induce apoptosis in Hep3B cells. This was substantiated by comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining for detecting early stage of apoptosis.

Conclusions

This investigation shows that the water extract of Cheilanthes farinosa has antiproliferative and apoptotic activity in human liver cancer cells and is not deleterious towards non-cancerous macrophage cell line.     

Anti-tumor effect of Shu-gan-Liang-Xue decoction in breast cancer is related to the inhibition of aromatase and steroid sulfatase expression

Ethnopharmacological relevance

Shu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula used to ameliorate the hot flushes in breast cancer patients, was reported to have anti-tumor effect on breast cancer. Estrogen plays a critical role in the genesis and evolution of breast cancer. Aromatase and steroid sulfatase (STS) are key estrogen synthesis enzymes that predominantly contribute to the high local hormone concentrations. The present study was to evaluate the anti-tumor effect of SGLXD on estrogen receptor (ER) positive breast cancer cell line ZR-75-1, and to investigate its underlying mechanisms both in vitro and in vivo.

Materials and methods

The anti-tumor activity of SGLXD in vitro was investigated using the MTT assay. The in vivo anti-tumor effect of SGLXD was evaluated in non-ovariectomized and ovariectomized athymic nude mice. The effect of SGLXD on enzymatic activity of aromatase and STS was examined using the dual-luciferase reporter (DLR) based on bioluminescent measurements. Aromatase and STS protein level were assessed using Western blot assay.

Results

SGLXD showed dose-dependent inhibitory effect on the proliferation of ZR-75-1 cells with IC₅₀ value of 3.40 mg/mL. It also suppressed the stimulating effect on cell proliferation of testosterone and estrogen sulfates (E₁S). Oral administration of 6 g/kg of SGLXD for 25 days resulted in a reduction in tumor volume in non-ovariectomized and ovariectomized nude mice. The bioluminescent measurements confirmed that SGLXD has a dual-inhibitory effect on the activity of aromatase and STS. Western blot assay demonstrated that the treatment of SGLXD resulted in a decrease in aromatase and STS protein levels both in vitro and in vivo.

Conclusion

Our results suggested that SGLXD showed anti-tumor effect on breast cancer cells both in vitro and in vivo. The anti-tumor activity of SGLXD is related to inhibition of aromatase and STS via decreasing their expression. SGLXD may be considered as a novel treatment for ER positive breast cancer.     

Immuno-enhancement effects of ethanol extract from Cyrtomium macrophyllum (Makino) Tagawa on cyclophosphamide-induced immunosuppression in BALB/c mice

Ethnopharmacological relevance

Cyrtomium macrophyllum (Makino) Tagawa has been traditionally used as a herbal medicine for the treatment of various infectious diseases such as tapeworm infestation, colds, and viral diseases. However, no systematic study of the immunity of Cyrtomium macrophyllum ethanol extracts (CM) has yet been reported. The present work evaluates these traits.

Materials and methods

120 male BALB/c mice were divided into 6 groups of 20 mice each: (1) normal group (sterile physiological saline), which served as a blank control; (2) model group (Cyclophosphamide, CY) group (sterile physiological saline), which served as a negative control; (3) low-dose CM (50 mg/kg BW); (4) intermediate-dose CM (100 mg/kg BW); (5) high-dose CM (200 mg/kg BW); (6) CM group (200 mg/kg BW). CY (0.2 ml) was administered via intraperitoneal injection. The other regimens were administered via gavage in 0.2 ml solution. Phytochemical of CM was characterized by HPLC–LTQ-Orbitrap. The acute toxicity effect of the ethanol extract of Cyrtomium macrophyllum was also investigated.

Results

The spleen and thymus indices of mice receiving low, intermediate, and high doses of CM recovered more quickly than those of CY mice, and they did so in a dose-dependent manner. These mice also showed higher T cell and B cell proliferation responses and macrophage function than those of CY mice, and their serum levels of interleukin-6 and interferon-γ had become normal. In acute toxicity test, CM exhibited no mortality and behavioral changes in mice. Quantitative phytochemical analysis showed flavonoids, polyphenols, and tannins to be the major compounds present in the extract, at 27.64%, 30.87%, and 11.22%, respectively. We found that 16 compounds were characterized by the interpretation of their mass spectra obtained by the MS/MS.

Conclusion

The current study demonstrates that Cyrtomium macrophyllum ethanol extract improved immune function in CY-treated mice.     

A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells

Ethnopharmacological relevance

Entada africana is a plant used in African traditional medicine for the treatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery.

Aims of the study

This study aimed at evaluating the anti-inflammatory activity of fractions of the stem bark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages model.

Materials and methods

The crude extract was prepared using the mixture CH₂Cl₂/MeOH (1:1, v/v) and fractionated by flash chromatography using solvents of increasing polarity to obtain five different fractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitric oxide (NO) production screened by Griess test. The most active fraction was further investigated for its effects on reactive oxygen species (ROS) production using flux cytometry, the expression of inducible nitric oxide synthase (iNOS), pro-and anti-inflammatory cytokines (IL1β, TNFα, IL6, IL10 and IL13) by RT-PCR, and the activity of the enzyme p38 MAPK kinase by enzyme-linked immunosorbent assay (ELISA).

Results

The fractions presented no significant effect on the viability of macrophages at 100 μg/ml after 24 h incubation. The CH₂Cl₂/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NO production with a half inhibition concentration (IC₅₀)=18.36 μg/ml, and showed the highest inhibition percentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 μg/ml. Ea5, as well as Baicalin significantly (P<0.05) inhibited the expression of TNFα, IL6 and IL1β mRNA, attenuated mRNA expression of inducible NO synthase in a concentration-dependent manner, stimulated the expression of anti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPK kinase.

Conclusion

The results of the present study indicate that the fraction Ea5 of Entada africana possesses most potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeutic agent in the treatment of inflammatory related diseases cause by over-activation of macrophages.     

The inhibitory activities of the components of Huang-Lian-Jie-Du-Tang (HLJDT) on eicosanoid generation via lipoxygenase pathway

Aim of the study

Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional Chinese medicine with anti-inflammatory use. In the present study, the effects of its component herbs and pure components were observed on eicosanoid generation to find out the contributory components and their precise targets on arachidonic acid (AA) cascade.

Materials and methods

By monitoring leukotriene B₄ (LTB₄), 5-hydroxyeicosatetraenoic acid (5-HETE), and 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT), we compared the effects of HLJDT, HLJDT free of one or two component herbs, and water extract of four single component herbs of HLJDT (Rhizoma coptidis, Radix scutellariae, Cortex phellodendri and Fructus gardeniae) on eicosanoid generation in rat elicited peritoneal macrophages. In addition, thirteen pure compounds from HLJDT (baicalin, baicalein, wogonoside, wogonin, berberine, magnoflorine, phellodendrine, coptisine, palmatine, jateorrhizine, crocin, chlorogenic acid, and geniposide) were tested in the macrophages. Furthermore, the efficacies of these thirteen compounds were evaluated on cell-free purified enzymes: leukotriene A₄ hydrolase (LTA₄H), 5-, 15-lipoxygenase (5-, 15-LO), and cyclo-oxygenase-1/2 (COX-1/2). Moreover, the possible synergetic effect on LO pathway derived LTB₄ generation between the active components was also tested in rat peritoneal macrophages.

Results

Our experiments showed that Rhizoma coptidis and Radix scutellariae were responsible for the suppressive effect of HLJDT on eicosanoid generation. Some of the pure components including baicalein, baicalin, wogonoside, wogonin, coptisine, and magnoflorine inhibited eicosanoid generation in rat macrophages via LO pathway of AA cascade. Further experiments on cell-free purified enzymes confirmed that Radix scutellariae derived baicalein and baicalin showed significant inhibition on 5-LO and 15-LO, while Rhizoma coptidis derived coptisine showed medium inhibition on LTA₄H. On the other hand, no significant inhibition of thirteen components on COX-1/2 was observed. Moreover, the slight synergetic inhibition on LTB₄ between baicalein and coptisine was proved in the rat peritoneal macrophages.

Conclusions

Baicalein and coptisine, the active components of HLJDT, for the first time are found to interfere with arachidonic acid cascade via inhibition on different points of LO pathway. This finding makes the mechanism of HLJDT clearer and achieves its safer therapeutic application.     

Cinobufacini,an aqueous extract from Bufo bufo gargarizans Cantor,induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells

Aim of the study

Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG₂ and Bel-7402.

Materials and methods

The effects of cinobufacini on cell proliferation of HepG₂ and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Δψm) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis.

Results

Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Δψm) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP).

Conclusion

The present study indicated that cinobufacini can induce apoptosis of HepG₂ and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.     

Eucommia ulmoides Oliv. bark. attenuates 6-hydroxydopamine-induced neuronal cell death through inhibition of oxidative stress in SH-SY5Y cells

Ethnopharmacological relevance

Eucommia ulmoides Oliv. Bark. (EUE) has commonly been used to fortify the muscles and lungs, lower blood pressure, prevent miscarriage, improve liver and kidney tone, and promote longevity as a traditional tonic medicine in Korea, China, and Japan.

Aim of the study

In this study, we investigated the mechanisms by which EUE protects neuronal cells from apoptosis induced by the Parkinson's disease (PD)-related neurotoxin, 6-hydroxydopamine (6-OHDA).

Materials and methods

We determined the neuroprotective effects of EUE on 6-OHDA-induced neuronal cell death, cytotoxicity, reactive oxygen species (ROS) production, and mitochondrial membrane dysfunction. Moreover, we examined whether EUE suppressed phosphorylation of c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3 beta (GSK-3β). Furthermore, the neuroprotective effects of EUE on 6-OHDA-induced activation of nuclear factor-kappa B (NF-κB) was studied in SH-SY5Y cells.

Results

Pretreatment of SH-SY5Y cells with EUE significantly reduced 6-OHDA-induced cell death and cytotoxicity. EUE inhibited 6-OHDA-induced generation of ROS, which conferred cytoprotection against 6-OHDA-induced oxidative injury. EUE treatment also strikingly inhibited 6-OHDA-induced mitochondrial dysfunction. In addition, EUE suppressed phosphorylation of JNK, PI3K/Akt, and GSK-3β. Furthermore, EUE blocked 6-OHDA-induced NF-κB nuclear translocation, an event downstream from JNK, PI3K/Akt, and GSK-3β phosphorylation. Moreover, chlorogenic acid (CGA), one of the active constituents of EUE, was also able to reduce 6-OHDA-induced toxicity in SH-SY5Y cells.

Conclusion

Taken together, these results suggest that EUE attenuates oxidative stress through activation of JNK, PI3K/Akt, GSK-3β, and NF-κB pathways, thereby protecting cells from neuronal cell death.     

Chemopreventive action of Lygodium flexuosum extract in human hepatoma PLC/PRF/5 and Hep 3B cells

Ethnopharmacological relevance

Lygodium flexuosum (Lygodiaceae), a medicinal fern used in Indian traditional medicine against liver disorders.

Aim of the study

The rationale of the study was to examine whether the n-hexane extract from plant Lygodium flexuosum affects apoptosis on human hepatoma PLC/PRF/5 and Hep 3B cells.

Materials and methods

Chemopreventive activity of the Lygodium flexuosum extract was determined by MTT assay, annexin-V FITC binding to phosphatidyl serine and cleavage of PARP. Subdiploid condition of cells treated with Lygodium flexuosum was analyzed by flow cytometry. Further, used transiently transfected NF-κB reporter in PLC/PRF/5 cells to evaluate the inhibitive effect of Lygodium flexuosum extract.

Results

Lygodium flexuosum extract inhibited the cell viability and induced apoptosis in hepatoma cells in a concentration dependent manner as evidenced by apoptotic changes such as flipping of phosphatidyl serine, cleavage of PARP. Cell cycle analysis showed the subG1 apoptotic population in cells treated with higher concentrations of the extract. When activated with exogenous TNF-α in transfected hepatoma cells it was observed that NF-κB dependent gene expression was inhibited by treatment with Lygodium flexuosum extract in PLC/PRF/5 cells dose-dependently.

Conclusions

This investigation suggests that the Lygodium flexuosum extract has antiproliferative and apoptotic activity in both cancer cells and has inhibitive role in TNF-α induced NF-κB activation in PLC/PRF/5 cells confirms the potential of the extract as a chemopreventive agent.     

Nauclea latifolia Smith (Rubiaceae) exerts antinociceptive effects in neuropathic pain induced by chronic constriction injury of the sciatic nerve

Ethnopharmacological relevance: The roots of Nauclea latifolia Smith (Rubiaceae) popularly known as “koumkouma” is used in traditional Cameroonian medicine as neuropathic pain remedy and for the treatment of headache, inflammatory pain and convulsion. This study was conducted to evaluate the antinociceptive effects of the alkaloid fraction isolated from Nauclea latifolia in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rat.     

Wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using RAW264.7 cells

Ethnopharmacological relevance

The root of Scutellaria baicalensis Georgi, also called Huangqin in China, is an herbal-based nutraceutical which is usually used in Chinese medicated diet (CMD). As an abundant ingredient in Huangqin, wogonoside is a flavonoid glycoside. The present work investigated the anti-inflammatory activities of wogonoside in lipopolysaccharides (LPS)-induced RAW264.7 cells.

Materials and methods

RAW264.7 cells were used. The inhibition of wogonoside against nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells were measured. Additionally, the effects of wogonoside on mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), TNF-α and IL-6 were also investigated.

Results and discussion

Wogonoside not only dose-dependently decreased the production of inflammatory mediators including NO and PGE₂ but also inhibited the release of pro-inflammatory cytokines including TNF-α and IL-6 in LPS-induced RAW264.7 cells. Furthermore, wogonoside possessed significantly in vitro inhibitory effects on the gene expression of iNOS, COX2, TNF-α and IL-6.

Conclusion

These results suggest that wogonoside may be used as a functional food component for prevention and treatment of inflammation.     

Abrogation of nimesulide induced oxidative stress and mitochondria mediated apoptosis by Fumaria parviflora Lam. extract

Ethanopharmacological relevance

Fumaria parviflora Lam. is used for treating aches and pains, diarrhea, fever, influenza and other complications. The herb mixed with honey is taken to prevent vomiting as per Ayurvedic text.

Aim of the study

In vivo studies were conducted to explore the hepatoprotective potential of Fumaria parviflora Lam. Fp extract against nimesulide induced oxidative stress and regulation of critical events in mitochondria mediated apoptosis.

Materials and methods

Group of Wistar rats were fed with nimesulide for 5 days (80 mg/kg/day, po), another group was pre-treated with Fp extract/silymarin (200 mg/kg/day, po) for 5 days followed by nimesulide exposure. Liver serum biomarkers and histopathology were done to assess hepatotoxicity caused by nimesulide. Antioxidant enzymes (SOD, LPO, GPx, GR) were assessed using biochemical assays as well as gene expression by RT-PCR. GSH content and ROS generation was also evaluated using flow cytometry. Key apoptotic markers like phosphatidyl serine externalization, Bax, Bcl-2 translocation, mitochondrial membrane potential, cytochrome c release, caspases (9/3) activation and DNA damage were also observed in all the groups to confirm involvement of mitochondrial pathway.

Results

Pre-treatment with Fp extract for 5 days significantly reduced the impact of nimesulide induced toxicity as evident from the serum biomarkers of liver damage and histopathology. It also modulated antioxidant enzymes mRNA expression as well as activity (SOD, glutathione peroxidase, glutathione reductase) and reduced lipid peroxidation during nimesulide toxicity. Nimesulide exposure decreased GSH content (92.9%) and increased reactive oxygen species (9.29 fold) which was attenuated in Fp treated rats. Fp pre-treatment significantly altered key apoptotic events like Bcl2 and Bax translocation, inhibited mitochondrial depolarization, prevented cytochrome c release, caspase-9/caspase-3 activation and DNA damage.

Conclusion

Our in vivo findings regarding protection accorded by Fp extract against nimesulide toxicity suggest that Fp not only reduced hepatotoxicity but attenuated critical control points of apoptotic cell death.     

Evaluating the anti-inflammatory potential of Tectaria cicutaria L. rhizome extract in vitro as well as in vivo

Ethnopharmacological relevance

The rhizome of Tectaria cicutaria has been used in the folklore system of Indian traditional medicine (Ayurveda) for the treatment of various disorders such as rheumatic pain, chest complaints, burns, sprain, poisonous bites, tonsilitis, toothache, gum complaints, cuts and wounds. The present work has for the first time tried to elucidate the anti-inflammatory potential of aqueous extract of Tectaria cicutaria rhizome (TCR_aq) in vitro as well as in vivo.

Materials and methods

Anti-inflammatory potential of TCR_aq was analyzed in vivo in carrageenan induced rat paw edema model. Serum antioxidant status in TCR_aq-treated as well as untreated control rodents was measured by oxygen radical absorbance capacity (ORAC) assay. In vitro experiments for analyzing the anti-inflammatory potential of TCR_aq were performed on murine macrophage cell line, RAW 264.7. Analysis of nitric oxide release in RAW 264.7 cells was done by Griess reaction. RT-PCR and western blotting experiment was performed to analyze the expression of iNOS. Expression of COX-2 and NFκB proteins was evaluated by western blotting.

Results

TCR_aq significantly reduced the paw volume in Sprague-Dawley rats at a dose of 200 mg/kg body weight, which was comparable with the standard diclofenac treatment. The rats treated with TCR_aq showed a significant increase in the serum antioxidant levels compared to the untreated control animals. TCR_aq was able to reduce the nitric oxide (NO) levels in RAW 264.7 cells that had been stimulated with lipopolysaccharide (LPS). This was accompanied by a corresponding decrease in iNOS expression at mRNA and protein level. Interestingly, TCR_aq was found to decrease the expression of COX-2 as well as the nuclear translocation of NFκB in RAW 264.7 cells.

Conclusion

Our study signifies the anti-inflammatory potential of Tectaria cicutaria and scientifically validates its traditional use in inflammatory conditions.     

Fructus panax ginseng extract promotes hair regeneration in C57BL/6 mice

Ethnopharmacological relevance

Radix panax ginseng (Panax ginseng C.A. Meyer, Araliaceae, RPG) has been documented to possess hair growth activity and widely used to treat alopecia, while no report has been issued to date on the effect of Fructus panax ginseng (FPG) on hair regeneration.

Materials and methods

To investigate the effects of FPG extract on the proliferation of human hair dermal papilla cells (DPCs) and on the promotion of hair regeneration in C57BL6 mice, cell proliferation was evaluated in cultured DPC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and measured the expressions of Bcl-2 and Bax by immunoblot assay. We also compared the effects of topical FPG extract (1 and 10 mg/ml, 100 μl/d) with the effects of minoxidil as a positive control (5%, 100 μl/d) or vehicle control (30% ethanol) on the depilation-induced hair cycling in 7 week-old-C57BL/6 mice.

Results

FPG extract significantly increased the proliferation of DPCs in dose and time dependent manners (P < 0.05, P < 0.01 and P < 0.001). FPG extract also enhanced Bcl-2 expression and decreased Bax expression compared with control (P < 0.01). Moreover, significant elongations of anagen phase during hair cycle after application of FPG were evaluated by photographical and histological observations.

Conclusions

FPG extract improves the cell proliferation of human DPCs through anti apoptotic activation. Topical administration of FPG extract might have hair regeneration activity for the treatment of hair loss.     

KIOM-79 inhibits high glucose or AGEs-induced VEGF expression in human retinal pigment epithelial cells

We evaluated whether KIOM-79, a mixture of extracts obtained from Puerariae lobata, Magnolia officinalis, Glycyrrhiza uralensis and Euphorbia pekinensis, could inhibit vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells cultured under high glucose (HG, 25mM) or S100b (a specific ligand of the receptor for advance glycation end products (RAGE), 5microg/ml). In this study, the effect of KIOM-79 on HG or S100b-induced VEGF expression was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, RT-PCR, ELISA, and Western blot on human RPE cells. The MTT assay (p<0.01) revealed that KIOM-79 (up to 1mg/ml) had no effect on cell growth. HG or S100b induced an increase in expression of VEGF at both mRNA and protein levels (p<0.05; p<0.01 versus control). The increase in VEGF expression by HG or S100b was dose- and time-dependently prevented by KIOM-79 (p<0.05 versus 25mM glucose; p<0.01 versus S100b). Also, KIOM-79 inhibited protein kinase C (PKC)-alpha/beta(alpha) and p38 mitogen-activated protein kinase (MAPK) activation. Our results demonstrate that KIOM-79 can inhibit VEGF expression via inhibition of the MAPK and PKC pathway.