An-Gong-Niu-Huang Wan protects against cerebral ischemia induced apoptosis in rats: Up-regulation of Bcl-2 and down-regulation of Bax and caspase-3

Ethnopharmacological relevance

The An-Gong-Niu-Huang Wan (AGNH), a Chinese traditional medicine, has been used for treatment of cerebral diseases for centuries in China and other Asian countries, and is approved by the State Food and Drug Administration of China for the treatment of stroke. The aim of present study is to test the neuroprotective effects of AGNH on cerebral ischemia in rats and to explore the underlying mechanisms.

Materials and methods

75 Male Sprague-Dawley rats were randomly divided into 5 groups: sham, ischemia–reperfusion (I/R), and I/R plus 0.065 g/kg/d AGNH, 0.125 g/kg/d AGNH and 0.25 g/kg/d AGNH. Cerebral ischemia was induced by 1.5 h of middle cerebral artery occlusion (MCAO). Neurological functional deficits were evaluated according to Zea longa?s score, cerebral infarct area was measured by tetrazolium staining. Cell injury and apoptosis were assessed by Nissl staining and DNA fragmentation assay. The expression of Bax, Bcl-2 and caspase-3 were analyzed by Western blot.

Results

Rats subjected to MCAO exhibited worsened neurological score, infarct area, cell damage and apoptosis. These were all attenuated by AGNH (0.125 and 0.25 g/kg/d). Moreover, AGNH reversed cerebral ischemia induced decreases in Bcl-2 expression and increases in Bax and caspase-3 expression.

Conclusions

These results suggest that AGNH exerts neuroprotective effects, and the neuroprotection is likely to relate to depressed Bax/Bcl-2 ratio and caspase-3 level, leading to inhibition of apoptotic cell death.     

Xiao-Xu-Ming decoction preserves mitochondrial integrity and reduces apoptosis after focal cerebral ischemia and reperfusion via the mitochondrial p53 pathway

Ethnopharmacological relevance

Xiao-Xu-Ming decoction (XXMD) has been used to treat stroke and other neurological diseases for more than 1000 years. The purpose of this study was to investigate the effects of XXMD on mitochondrial damage and apoptosis after cerebral ischemia and reperfusion.

Materials and methods

Male Sprague-Dawley rats were randomly divided into 3 groups: sham, cerebral ischemia and reperfusion (I/R), and cerebral ischemia and reperfusion plus XXMD (60 g/kg/day) (XXMD60). Focal cerebral ischemia and reperfusion models were induced by middle cerebral artery occlusion. Cerebral ischemic injury was evaluated by hematoxylin and eosin staining. Ultrastructural features of mitochondria in the penumbra of the ischemic cortex were analyzed by transmission electron microscopy. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining and cleaved caspase 3 immunohistochemistry. Proteins in the mitochondrial p53 pathway were detected by western blot and immunofluorescence.

Results

The results showed that XXMD treatment markedly attenuated ischemic changes, preserved mitochondrial integrity, and significantly reduced apoptosis. In addition, we found that XXMD treatment reduced p53 and Bax levels and increased Bcl-2 levels in mitochondrial fractions. XXMD significantly blocked the release of cytochrome c and Smac/Diablo from mitochondria, and inhibited activation of caspase 9 and caspase 3 in cytoplasmic fractions. Increased expression of c-IAP1 was observed in the XXMD60 group.

Conclusions

The findings demonstrated that XXMD protected mitochondria from ischemic injury and inhibited apoptosis. The mitochondrial p53 pathway could be partially involved in the protective effects.     

Neuroprotective effects of Fructus Chebulae extracts on experimental models of cerebral ischemia

Objective

To investigate the neuroprotective effects of Fructus Chebulae extract using both in vivo and in vitro models of cerebral ischemia.

Methods

As an in vitro model, oxygen glucose deprivation followed by reoxygenation (OGD-R) and hydrogen peroxide (H₂O₂) induced cellular damage in rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effects of extract of Fructus Chebulae. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to calculate cell survival. For in vivo, occlusion of left middle cerebral artery on rats was carried out as a focal cerebral ischemic model.

Results

Fructus Chebulae extract increases the PC12 cell survival against OGD-R and H₂O₂ by 68% and 91.4% respectively. Fructus Chebulae also decreases the cerebral infarct volume by 39% and extent of hemisphere swelling from 17% in control group to 10% in Fructus Chebulae treated group.

Conclusion

Fructus Chebulae, as a traditional medicine, can rescue the neuronal cell death against ischemia related damage. The possible mechanism for the neuroprotection might be the inhibition of oxidative damages occurring after acute phase of cerebral ischemia.     

Neuroprotective activity of Matricaria recutita Linn against global model of ischemia in rats

Ethnopharmacological relevance

Traditionally, the whole plant is used for various diseases, including neuronal disorders.

Aim of the study

To evaluate the neuroprotective effect of Matricaria recutita L. against global cerebral ischemia/reperfusion (I/R) injury-induced oxidative stress in rats.

Materials and methods

Neuroprotective activity was carried out by global cerebral ischemia on Sprague–Dawley rats by bilateral carotid artery (BCA) occlusion for 30 min followed by 60 min reperfusion. The antioxidant enzymatic and non-enzymatic levels were estimated along with cerebral infarction area and histopathological studies.

Results

The Matricaria recutita L. methanolic extract showed dose-dependent neuroprotective activity by significant decrease in lipid peroxidation (LPO) and increase in the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and total thiol levels in extract treated groups as compared to ischemia/reperfusion group. Cerebral infarction area was significantly reduced in extract treated groups as compared to ischemia/reperfusion group.

Conclusion

The methanolic extract of Matricaria recutita L. showed potent neuroprotective activity against global cerebral ischemia/reperfusion injury-induced oxidative stress in rats.     

Inhibitory effect of Thymus caramanicus Jalas on hyperglycemia-induced apoptosis in in vitro and in vivo models of diabetic neuropathic pain

Ethnopharmacological relevance

Since Thymus caramanicus Jalas is used as a folk medicine for the treatment of rheumatism, skin disorders, bacterial infections and diabetes and it contain antioxidant agents, we decided to investigate the possible effects of Thymus caramanicus Jalas (TCJ) extract on in vitro and in vivo models of diabetic neuropathy.

Materials and methods

The high glucose-induced cell injury in Pheochromocytoma (PC12) cells and streptozotocin-induced diabetic rats were used. Tail-flick and rotarod treadmill assessments were used to determine nociceptive threshold and motor coordination. Cell viability was determined by MTT assay test. Western blotting was performed to measurement of apoptosis markers.

Results

The data showed that elevation of glucose consecutively increases functional cell injury and apoptosis. Furthermore, diabetic rats developed thermal hyperalgesia and motor deficit. Activated caspase 3, cytochrome c release and Bax/Bcl-2 ratio were significantly increased in high glucose-treated PC12 cells and in spinal cord of diabetic animals. TCJ extract (60 and 80 µg/ml) attenuates high glucose-induced PC12 cells damage and apoptosis. In diabetic animals, TCJ extract at daily doses of 100 and 150 mg/kg ameliorated hyperalgesia and suppressed spinal apoptosis.

Conclusion

The data indicate that TCJ extract has neuroprotective effects against high glucose-induced neural damage. These protective effects are mediated, at least in part, through attenuation of neural apoptosis and suggest therapeutic potential of TCJ extract in amelioration of diabetic neuropathy.     

Chrysanthemum morifolium Ramat (CM) extract protects human neuroblastoma SH-SY5Y cells against MPP-induced cytotoxicity

Ethnopharmacological relevance

Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used in Korean and Chinese traditional herbal medicines with numerous therapeutic applications.

Aim of the study

To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract against 1-methyl-4-phenylpridinium ions (MPP⁺), Parkinsonian toxin through oxidative stress and impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms.

Materials and methods

The effects of CM against MPP⁺-induced cytotoxicity and neuronal cell viability, oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were evaluated by using SH-SY5Y neuroblastoma cells.

Results

CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and PARP proteolysis.

Conclusion

These results demonstrate that CM possesses potent neuroprotective activity and therefore, might be a potential candidate in neurodegenerative diseases such as Parkinson's disease.     

Total saponins of Panax Notoginseng modulate the expression of caspases and attenuate apoptosis in rats following focal cerebral ischemia-reperfusion

Ethnopharmacological relevance

Total saponins of Panax notoginseng (TSPN), main constituents extracted from Panax Notoginseng, a highly valued traditional Chinese medicine, have been shown to be an effective agent on cerebral infarction.

Aim of the study

The effects of TSPN on apoptosis and expressions of caspase-1, caspase-3 and caspase-8 were studied after cerebral ischemia for 2 h followed by reperfusion for 46 h in rats.

Materials and methods

Rats were subjected to transient middle cerebral artery occlusion (MCAO) model using the intraluminal thread. TSPN was administered intraperitoneally at 5 min before and 12 h, 24 h and 36 h after MCAO, respectively.

Results

TSPN (at the dose of 25 mg/kg) significantly attenuated TUNEL-positive cells and reduced the expression of caspase-1 and caspase-3 compared to the model group, while it had no obvious effect on the expression of caspase-8.

Conclusions

The neuroprotective effect of TSPN on focal ischemia may be related to inhibition of apoptosis and caspases activation.     

Neuroprotective effects of Dioscorea opposita on scopolamine-induced memory impairment in in vivo behavioral tests and in vitro assays

Ethnopharmacological relevance

Plants belong to the genus Dioscorea have long been used as edible tuber crops in many tropical and subtropical areas and as a traditional herbal medicine in oriental countries including China, Japan and Korea.

Aim of the study

In this study, in vivo and in vitro tests were carried out to evaluate the cognitive enhancing effects of CHCl₃-soluble extract from Dioscorea opposita against scopolamine-induced amnesic mice and glutamate- and H₂O₂-treated cortical neurons of rats.

Materials, methods and results

Acute treatment (200 mg/kg body weight, p.o.) and 10 days’ daily administration (50 mg/kg body weight, p.o.) of CHCl₃-soluble extract showed significant spatial learning and memory improvement on mice. Furthermore, the neuroprotective effects on glutamate- and H₂O₂-induced neurotoxicity in primary cultured cortical neurons of rats were assessed. Pretreatment with the extract was found to impart significant protection against neurotoxicity.

Conclusions

These in vivo and in vitro results suggest that the Dioscorea opposita has neuroprotective effects on memory impairment related neurodegenerative diseases.     

Hexane extract from Polygonum multiflorum attenuates glutamate-induced apoptosis in primary cultured cortical neurons

Ethnopharmacological relevance

Polygonum multiflorum has traditionally had wide use as an anti-aging treatment in East Asian countries. We investigated the neuroprotective effects of Polygonum multiflorum against glutamate-induced neurotoxicity with a focus on the anti-apoptotic mechanism in primary cultured cortical neurons.

Material and methods

Cell viability, cytotoxicity, morphological, flow cytometry, Western blot, and caspase activity assays were performed for examination of the neuroprotective effects of active hexane extract from Polygonum multiflorum (HEPM).

Results

Pretreatment with HEPM resulted in significantly decreased glutamate-induced neurotoxicity in a concentration-dependent manner and also resulted in drastically inhibited glutamate-induced apoptosis. Treatment with HEPM resulted in decreased expression of glutamate-induced death receptor (DR)4, and enhanced expression of glutamate-attenuated anti-apoptotic proteins, including Bcl-2, XIAP, and cIAP-1, and slightly reduced glutamate-induced cleavage of Bid. In addition, treatment with HEPM resulted in suppressed glutamate-induced activation of caspase-8, caspase-9, and caspase-3, and, subsequently, decreased degradation of poly(ADP-ribose) polymerase, β-catenin, and phospholipase Cγ1 protein, which are downstream targets of activated caspase-3.

Conclusions

The results of this study demonstrated that HEPM exerts a neuroprotective effect against glutamate-induced neurotoxicity via inhibition of apoptosis. This protection may be mediated through suppression of DR4 and up-regulation of Bcl-2, XIAP, and cIAP-1, as well as inhibition of caspase activation, resulting in prevention of apoptosis of cortical neurons.     

Anti-tumor activity and relative mechanism of ethanolic extract of Marsdenia tenacissima (Asclepiadaceae) against human hematologic neoplasm in vitro and in vivo

Ethnopharmacological relevance

Marsdenia tenacissima, a traditional Chinese medicinal herb, endemic to Yunnan Province is widely used to treat cough, asthma, expectorant, esophageal cancer, gastric cancer, lung cancer, and hepatocellular carcinoma. The aim of this study was to evaluate in vitro and in vivo anti-hematologic neoplasm potential of the ethanolic extract of this herb (crude ethanolic extract of Marsdenia tenacissima, CME) and by using different assays to elucidate its possible mechanism of action.

Materials and methods

The cytotoxicity of CME on tumor cells and peripheral blood mononuclear cells (PBMCs) was evaluated using MTT and apoptosis assays. We also tested the effect of CME on colony formation inhibition and cell cycle distribution of tumor cells. The protein expressions of Cyclin D1, Bax, Bcl-2, caspase-3 and caspase-9 were detected through Western blotting. In vivo anti-tumor effect was evaluated by measuring tumor volume changes, measuring tumor weight, evaluation of tumor microvessel density (MVD) and TUNEL staining by using immunohistochemistry staining in tumor models of nude mice.

Results

Marsdenia tenacissima ethanolic extract exhibited effects of proliferation inhibition and induction of apoptosis on human hematologic neoplasm tumor cells in vitro, as well as hematologic neoplasm growth in vivo.

Conclusion

This study clearly indicated that the ethanolic extract of Marsdenia tenacissima displayed strong anti-tumor effects against hematologic neoplasm cells and could induce tumor cells apoptosis in vitro and in vivo, and also had a significant anti-angiogenic effect in vivo against tumor cell apoptosis. Its multi-mechanism of action might be associated with the cell cycle (G0/G1) arrest, induction of apoptosis through up-regulation protein expressions of Bax, caspase-9 and caspase-3 genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes, a decrease in tumor microvessel density and an increase of TUNEL-positive cells in vivo. These findings provided the molecular theoretical basis of clinical application.     

Memory improvement in ibotenic acid induced model rats by extracts of Scutellaria baicalensis

Ethnopharmacological relevance

Scutellaria baicalensis Georgi (Labiatae) extracts have been used as traditional Korean medicine, to treat cerebral ischemia in addition to bacterial infection and inflammatory diseases.

Aim of the study

The improvement effect on learning and memory by the administration of Scutellaria baicalensis extracts was evaluated and the underlying mechanisms were investigated.

Materials and methods

Memory behavior was tested by the passive avoidance test and Y-maze test. We also investigated the cells expressing neuronal markers related to memory processes by immunofluorescence staining analysis in memory deficient animal model (Ibo model) rats and in hippocampal progenitor cells.

Results

We found neuronal cells immunoreactive to choline acetyltransferase (ChAT), a marker for cholinergic neurons were increased in the hippocampus, while cells producing GABA and glutamate were not after 30 mg/kg Scutellaria baicalensis administration. Futhermore, Scutellaria baicalensis extracts enhanced the survival of a hippocampal progenitor cell line, HiB5 and its differentiation to ChAT immunoreactive cells. The increased expression of memory related neurotransmitter, NMDA receptor and a reduction of activated microglia in the hippocampus were also observed in the Ibo model when administrated Scutellaria baicalensis extracts.

Conclusions

These results imply that Scutellaria baicalensis has significant neuroprotective effects in the Ibo model.     

In vivo and in vitro anti-inflammatory activity of Lentinus polychrous extract

Ethnopharmacological relevance

Lentinus polychrous is a Thai local edible mushroom, traditionally used for the treatments of fever and inflammation due to snake or scorpion envenomation.

Aim of study

The present study aimed to investigate an anti-inflammatory effect of Lentinus polychrous mycelial extract (LPME) both in vitro and in vivo.

Materials and methods

The cytotoxicity and suppressive effects of LPME on nitric oxide production, intracellular O₂⁻ production, pro-inflammatory mediator expression, TNF-α production were determined by using LPS-activated RAW 264.7 cells. In addition, Anti-inflammatory effect of LPME was evaluated by using carageenan-induced paw edema in rats.

Results

The LPME exhibited cytotoxicity with 50% inhibitory concentration (IC₅₀) of 280.25±10.10 μg/ml and significantly suppressed the productions of NO and intracellular O₂⁻ with dose-dependent manner. LPME decreased the expressions of iNOS, IL-1β, IL-6, TNF-α and COX-2 and significantly decreased the TNF-α production in LPS-activated macrophage with dose-dependent manners. Moreover, LPME showed significant suppressive effect on paw edema in rats.

Conclusion

The results clearly revealed that the LPME inhibited NO and pro-inflammatory productions by down-regulating the gene expressions of pro-inflammatory mediators leading to the decrease paw edema in rat which support the traditional use.     

Protective effects of Gastrodia elata Blume on MPP-induced cytotoxicity in human dopaminergic SH-SY5Y cells

Aim of the study

Gastrodia elata (GE) Blume (Orchidaceae) has been traditionally used as a folk medicine in Oriental countries since centuries for their variety of therapeutic benefits. This study is an attempt to investigate the protective effects of GE extract against MPP⁺-induced cytotoxicity in human dopaminergic SH-SY5Y cells and explore the neuroprotective mechanisms involved.

Materials and methods

Human dopaminergic SH-SY5Y cells were used to demonstrate the protective effects of GE against multiple parameters such as MPP⁺-induced cell viability, oxidative damage, expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase proteolysis.

Results

GE effectively attenuated the cytotoxicity and improved cell viability in a dose-dependent manner. GE was effective in inhibiting both, the increased production of reactive oxygen species (ROS) and increase in Bax/Bcl-2 ratio, cleaved caspase-3 and PARP proteolysis.

Conclusion

Data from this study suggests the protective effects of GE on MPP⁺-induced cytotoxicity in dopaminergic cells, which may be ascribed to its significant anti-oxidative and anti-apoptotic properties, thus, GE might prove to be a valuable therapeutic agent for the treatment of various neurodegenerative diseases including progressive Parkinson's disease (PD).     

Traditional Chinese herb Dihuang Yinzi (DY) plays neuroprotective and anti-dementia role in rats of ischemic brain injury

Aim of the study

Traditional Chinese herb Dihuang Yinzi (DY) is well known to treat neurological diseases by traditional Chinese medical practitioners. This study is to elucidate its neuroprotective and anti-dementia role in ischemic brain injury.

Materials and methods

The effects of DY on the pathohistological changes, lactate dehydrogenase (LDH) release, Morris water maze task, expression of synaptophysin (SYP) and extracellular signal-regulated protein kinase (ERK) of hippocampi of rats with ischemic brain injury were investigated.

Results

This study showed that DY not only significantly decreased the number of TUNEL-positive cells but also reduced the LDH release of hippocampus of model rat. Morris water maze test showed that the ability of learning and memory of rats dramatically impaired after ischemic brain injury. However, DY ameliorated the impairment of learning and memory of ischemic rats. Furthermore, western blotting and immunohistochemical data showed that the expression of extracellular regulated protein and synaptophysin, which correlates with synaptic formation and function, decreased after ischemic insult. However, DY inhibited the reduction of ERK an SYP expression in a dose-dependent way.

Conclusion

These results suggest that DY possesses neuroprotective and anti-dementia properties, at least in part, by preventing the loss of neural cells and synapses in ischemic brain injury.     

The anti-tumor activity of Antrodia salmonea in human promyelocytic leukemia (HL-60) cells is mediated via the induction of G1 cell-cycle arrest and apoptosis in vitro or in vivo

Ethnopharmacological relevance

The medicinal mushroom Antrodia salmonea has been used as a traditional Chinese medicine and has demonstrated antioxidant and anti-inflammatory effects.

Materials and methods

In the present study, we examined the anti-tumor activity of the fermented culture broth of Antrodia salmonea (AS) in vitro and in vivo and revealed its underlying molecular mechanism of action.

Results

Treatment of human promyelocytic leukemia (HL-60) cells with AS (50–150 μg/mL) significantly reduced cell viability and caused G₁ arrest via the inhibition of cell-cycle regulatory proteins, including cyclin D1, CDK4, cyclin E, cyclin A, and phosphorylated retinoblastoma protein (p-Rb). Furthermore, AS treatment induced apoptosis, which was associated with DNA fragmentation, followed by a sequence of events, including intracellular ROS generation; mitochondrial dysfunction; Fas ligand activation; cytochrome c release; caspase-3, -8, -9, and PARP activation; and Bcl-2/Bax dysregulation. The results of the in vitro study suggested that AS-induced apoptosis in HL-60 cells was mediated by both the mitochondrial and death receptor pathways. Furthermore, we found that AS treatment was effective in delaying tumor incidence in HL-60 xenografted nude mice and reducing tumor burden.

Conclusions

To the best of our knowledge, this is the first report confirming the anti-tumor activity of this potentially beneficial mushroom against human promyelocytic leukemia.     

In vitro and in vivo safety evaluation of Acer tegmentosum

Ethnopharmacological relevance

Acer tegmentosum, which contains salidroside and tyrosol, has been used for the treatment of hepatic disorders in eastern Asia. However, little is known about its safety.

Aim of the study

To determine the safety of Acer tegmentosum, we evaluated its acute oral toxicity and genotoxicity profiles.

Materials and methods

Salidroside and tyrosol present in Acer tegmentosum were quantified using high-performance liquid chromatography. Acute oral toxicity testing of Acer tegmentosum was performed in rats. Genotoxicity of Acer tegmentosum was assessed by bacterial reverse mutation, chromosomal aberration, and bone marrow micronucleus tests. All the tests were conducted in accordance with the good laboratory practices.

Results

The amounts of salidroside and tyrosol in Acer tegmentosum were found to be 85.01±1.21 mg/g and 3.12±0.04 mg/g, respectively. In the bacterial reverse mutation test, Acer tegmentosum increased the number of revertant Salmonella typhimurium TA98 colonies, regardless of metabolic activation by S9 mixture. In contrast, Acer tegmentosum application did not significantly increase the number of chromosomal aberrations in Chinese hamster ovary (CHO)-K1 cells and micronucleated polychromatic erythrocytes in mice. In the acute oral toxicity test, the median lethal dose (LD₅₀) of Acer tegmentosum was found to be >2000 mg/kg in rats.

Conclusion

Take together, Acer tegmentosum exhibits mutagenicity, which was evident from the bacterial reverse mutation test. Further studies are needed to identify the components responsible for such an effect and the underlying mechanisms.     

Cinchonain Ib isolated from Eriobotrya japonica induces insulin secretion in vitro and in vivo

Aims of the study

Eriobotrya japonica leaves had been used traditionally for the treatment of diabetes mellitus by immersing the dried leaves in a hot water drink. Few studies have shown the hypoglycemic effect of Eriobotrya japonica using crude alcoholic extract and isolated methanolic compounds. These studies proposed that the mechanism of action could be by stimulating the β-islets of Langerhans to secrete insulin, however with no scientific evidence.

Methods

Eriobotrya japonica water extract (EJWE) and the compounds derived from it: cinchonain Ib, procyanidin B-2, chlorogenic acid and epicatechin, were tested for their effects on insulin secretion from INS-1 cells and following oral administration in rats.

Results

The present study showed that EJWE increased significantly (p < 0.05) insulin secretion from INS-1 cells in dose-dependent manner. Oral administration of EJWE at 230 mg/kg to rats, however, decreased plasma insulin level for as long as 240 min post-administration and caused a transient drop of blood glucose at 15 and 30 min post-administration. On the other hand, cinchonain Ib enhanced significantly (p < 0.05) insulin secretion from INS-1 cells, whereas epicatechin inhibited significantly (p < 0.05) insulin secretion from INS-1 cells. In addition, cinchonain Ib enhanced significantly (150%: p < 0.05) plasma insulin level in rats for as long as 240 min after 108 mg/kg oral administration but did not induce any change in blood glucose level.

Conclusion

These data indicate that cinchonain Ib has an insulinotropic effect and suggest the possible use of cinchonain Ib for managing type 2 diabetes.     

Mirabijalone E: A novel rotenoid from Mirabilis himalaica inhibited A549 cell growth in vitro and in vivo

Ethnopharmacological relevance

The roots of Mirabilis himalaica have been used in Tibetan folk medicine for treatment of uterine cancer, nephritis edematous, renal calculus and arthrodynia. In our previous work, the ethanol extract of roots had shown potent cytotoxicity against human cancer cells. However, no information is available on the antitumor effect of Mirabilis himalaica. The aim of the present study was to investigate the active constituents guided by bioassay and evaluate the related antitumor efficacy in vitro and in vivo.

Materials and methods

The active subextract (ethyl acetate) was subjected to successive chemical separation using a combination of silica gel, LH-20 chromatography and semi-preparative HPLC. The structures were determined by spectroscopic analysis techniques such as nuclear magnetic resonance (NMR) and mass spectrometry. Three human cancer cell lines, A549, HepG2 and HeLa were used for in vitro cytotoxicity evaluation of all isolated compounds by MTT-assay. Then, the potent and novel compound mirabijalone E was employed to the mechanism study againstA549 cells. BrdU immunofluorescence, soft agar assay and cell cycle analysis were employed to detect the cell proliferation effects. Annexin V-FITC/PI staining assay was used for examining apoptotic effects. Expression levels of apoptosis-related proteins were determined by western blot assay. in vivo tumorigenic assay was used to evaluate the xenograft tumor growth treated with mirabijalone E.

Results

One new rotenoid compound, mirabijalone E, together with eight known rotenoids was isolated from Mirabilis himalaica. Mirabijalone E, 9-O-methyl-inone B, boeravinone C and boeravinone H exhibited cytotoxicity against A 549 and HeLa cells. Further study on mirabijalone E was carried out in vitro and in vivo. Mirabijalone E inhibited A549 cells growth in a time and dose-dependent manner, which arrested cell cycle in S phase. Mechanistically, mirabijalone E treatment resulted in the increase of Bax expression level, the decrease of Bcl-2 level and the activation of caspase-3, which suggested the activation of apoptosis cascades. Consequently, the xenograft treated with mirabijalone E showed markedly suppressed tumor growth.

Conclusions

The result suggested that mirabijalone E, together with active compounds, 9-O-methyl-4-hydroxyboeravinone B, boeravinone C and boeravinone H could be a promising candidate for cancer therapy.