Discovery of Potent Angiotensin-Converting Enzyme Inhibitors in Pomegranate as a Treatment for Hypertension.

Pomegranate (Punica granatum L.) is associated with numerous health benefits due to its high levels of antioxidant polyphenolic substances. Since pomegranate extract has been shown to inhibit angiotensin-converting enzyme (ACE), the potential inhibitory effect of most of its main constituents against ACE is unknown. Therefore, we tested the activities of 24 major compounds, the majority of which significantly inhibited ACE. Notably, pedunculagin, punicalin, and gallagic acid were the most effective ACE inhibitors with IC50 values of 0.91, 1.12, and 1.77 µM, respectively. As demonstrated in molecular docking studies, compounds block ACE by forming multiple hydrogen bonds and hydrophobic interactions with catalytic residues and zinc ions in ACE's C- and N-domains, consequently inhibiting ACE's catalytic activity. Also, the most active pedunculagin stimulated nitric oxide (NO) production, activated the endothelial nitric oxide synthase enzyme (eNOS), and significantly increased eNOS protein expression levels up to 5.3-fold in EA.hy926 cells. Furthermore, pedunculagin increased in cellular calcium (Ca2+) concentration promoted eNOS enzyme activation and reduced the production of reactive oxygen species (ROS). In addition, the active compounds improved glucose uptake in insulin-resistant C2C12 skeletal muscle cells in a dose-dependent manner. The results of these computational, in vitro, and cellular experiments provide further evidence to the traditional medicine that involves using pomegranates to treat cardiovascular diseases like hypertension.

Effects of Onchung-eum, an Herbal Prescription, on 5-Fluorouracil-Induced Oral Mucositis.

In most cancer patients, chemotherapy-induced oral mucositis (OM) is a frequent side effect, leading to low quality of life and delay in therapy. The aim of this study was to evaluate the effects of Onchung-eum, a well-known herbal prescription in traditional medicine comprising 8 herbs that has long been used for skin diseases, on 5-fluorouracil (5-FU)-induced OM in human pharyngeal cells and golden Syrian hamsters. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and reactive oxygen species production were measured in vitro. The effects of Onchung-eum on OM of hamster cheek pouches induced by 5-FU were evaluated histologically and using TUNEL assay. In addition, the expression of nuclear factor-κB, caspase-3, and pro-inflammatory cytokines were measured by immunoblotting and immunohistochemistry. Significantly increased cell viability was observed in the Onchung-eum-treated groups compared with the 5-FU-treated control group. In 500 and 1000 mg/kg Onchung-eum-treated groups, the damaged epithelial layers in the cheek pouches of hamsters were significantly recovered. Moreover, at all concentrations, cell death in the cheek pouches of hamsters in the Onchung-eum-treated groups significantly decreased. The expression of pro-inflammatory cytokines, nuclear factor-κB, and caspase-3 also significantly decreased in Onchung-eum-treated groups at 500 and 1000 mg/kg. In conclusion, this study revealed that Onchung-eum can be used to treat chemotherapy-induced OM. However, further studies are required to understand the underlying mechanisms.

Protective effect of Salvia miltiorrhiza Bunge on 5-fluorouracil-induced oral mucositis.

Oral mucositis is a common side-effect caused by chemotherapy or radiotherapy occurring in the majority of cancer patients and is characterized by inflammation and ulcers in the oral mucosa. In the present study, we examined the protective effects of Salvia miltiorrhiza Bunge (SM) on oral mucositis induced by 5-fluorouracil (5-FU) in human pharyngeal cells and golden Syrian hamsters. We investigated the proliferation and antioxidant abilities of SM using MTT, 2-diphenyl-1-picrylhydrazyl (DPPH) and reactive oxygen species (ROS) assays in vitro. Additionally, TUNEL assay was performed, and the expression levels of nuclear factor-κB (NF-κB), caspase-3 and proinflammatory cytokines were assessed by immunoblotting. The results showed that SM increased the cell proliferation rate in human pharyngeal cells up to 128.97±9.7% compared with this rate in the untreated cells and exerted protective effects on mucosal injury caused by 5-FU treatment. In addition, all concentrations of SM increased DPPH scavenging ability and blocked ROS generation in the treated cells. Taken together, following SM treatment, expression of NF-κB and cleaved caspase-3 were significantly decreased followed by inhibition of cell death. These data suggest that SM could be used for the prevention and treatment of oral mucositis caused by cancer therapies.

Effects of herbal Epimedium on the improvement of bone metabolic disorder through the induction of osteogenic differentiation from bone marrow-derived mesenchymal stem cells.

Herbal Epimedium (HE) has been commonly used as a tonic, antirheumatic agent and in the treatment of bone­associated diseases including osteoporosis. Treatment for osteoporosis is important to increase bone mass density and maintain to balance of bone remodeling. The present study was performed to investigate the effects of HE on mouse bone marrow mesenchymal stem cell (mBMMSC) proliferation and osteogenic differentiation, using MTT assays, proliferating cell nuclear antigen (PCNA) detection and apoptosis and differentiation assays. HE was demonstrated to inhibit the proliferation of mBMMSCs up to 45.43±3.33% and to decrease the level of PCNA expression compared with untreated cells. HE also induced late apoptosis at 24 and 48 h after treatment up to 71.93 and 67.03%, respectively, while only 14.93% of untreated cells exhibited apoptosis. By contrast, HE induced differentiation of mBMMSCs into an osteogenic lineage at the beginning of three weeks after commencement of treatment. This suggested that HE is a candidate as an inducer of osteogenesis from bone marrow mesenchymal stem cells, and additionally has potential for use in the treatment of bone metabolic disorders such as osteoporosis.

Astragalus membranaceus augment sperm parameters in male mice associated with cAMP-responsive element modulator and activator of CREM in testis.

Astragalus membranaceus BUNGE (AM; huáng qí) has been widely used as a medicinal herb for different kinds of diseases. AM treatment in vitro enhance sperm motility and ameliorates testicular toxicity, it has demonstrated the ability as a potential treatment for male infertility. In order to gain further insights on the molecular understanding of how AM enhances spermatogenesis, this study investigated whether AM has an affect on sperm parameters associated with cAMP response element modulator (CREM) and activator of CREM in testis (ACT) expression. Five-week-old male ICR mice were divided into four groups; control group and three different concentrations of AM treated groups. Each group was treated for 5 days a week for 5 weeks. Testis samples were collected for real time quantitative PCR and western blot analysis. Epididymis was taken out and used for sperm analysis using the computer assisted semen analysis (CASA) system. To facilitate expression of genes required for spermatogenesis, it is controlled by fine-tuning of CREM and its coactivator, ACT. AM treatment promotes CREM and ACT mRNA expression and also protein expression compared to control. AM enhances sperm values such as sperm count and motility compared to control. Overall, the study highlights, the ability of AM to increases CREM and ACT expression to facilitate sperm development and semen quality.

Trigonellae Semen Enhances Sperm Motility and the Expression of the Cation Sperm Channel Proteins in Mouse Testes.

Genetic defects during spermatogenesis can lead to a reduction in sperm motility and cause male infertility. The cation channels of sperm (CatSper) play a role in the regulation of hyperactivated sperm motility in mouse testes. The effect of Trigonellae Semen (TS) on the male reproductive system and CatSper protein in mouse testes during spermatogenesis was examined. C57BL/c mice were divided into the following five groups: normal, cyclophosphamide- (CP-) only treated (control group), and three groups treated with varying concentrations of TS with CP (100, 500, and 1000 mg/kg TS and 100 mg/kg CP). Real-time PCR, western blot analysis, and a testosterone immunoassay were performed to assess CatSper protein levels in the five groups. Additionally, sperm cell counts and motility were examined. Results indicate that sperm motility and sperm counts increased in the TS treated groups in a dose-dependent manner (p < 0.01). CatSper levels were also significantly higher in the TS treated groups compared to that of the control group (p < 0.001). Therefore, TS treatment could enhance sperm function by promoting spermatogenesis and the expression of CatSper proteins in mouse testes.

Effects of Jae-Seng Acupuncture Treatment on the Improvement of Nasolabial Folds and Eye Wrinkles.

The microneedle therapy system (MTS), a mechanical method involving making minute multiple holes in the skin, reportedly improves skin condition, such as by reducing flushing and melanin. A newly attempted bloodletting therapy, Jae-Seng Acupuncture, has several advantages over traditional mechanical punching methods because it allows the practitioner to regulate the depth and direction of needle stimulations and to choose whether to stimulate the muscle layers. This study was conducted to determine the efficacy of Jae-Seng Acupuncture in the treatment of nasolabial folds and eye wrinkles. The nasolabial folds and eye wrinkles of 107 patients ranging in age from their 20s to their 70s were subjected to DermaVision, a digital skin image analyzer, before the treatment and one to six months after treatment. Additionally, stimulation of the meridians, such as Taeyang, Tongjaryo, Chongmyong, Sungup, Sabaek, Yonghyang, Chichang, Taeyong, was performed to improve the function of the stomach, large intestine. Analyses of the images indicate that Jae-Seng Acupuncture improved nasolabial folds and eye wrinkles, suggesting that this technique is a safe and effective method for the improvement of facial skin conditions.

Panax ginseng induces the expression of CatSper genes and sperm hyperactivation.

The cation channel of sperm (CatSper) protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca²âº influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca²âº levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression.

Effects of low level laser therapy on ovalbumin-induced mouse model of allergic rhinitis.

Introduction. This study was designed to investigate the effects of low level laser therapy (LLLT) on experimental allergic rhinitis (AR) models induced by ovalbumin. Materials and Methods. AR was induced by 1% ovalbumin in mice. Twenty-four mice were divided into 4 groups: normal, control, low, and high dose irradiation. Low and high dose LLLT were irradiated once a day for 7 days. Total IgE, cytokines concentrations (IL-4 and IFN- γ ), and thymus and activation regulated chemokine (TARC) were measured. Histological changes in the nasal mucosal tissue by laser irradiation were examined. Results. LLLT significantly inhibited total IgE, IL-4, and TARC expression in ovalbumin-induced mice at low dose irradiation. The protein expression level of IL-4 in spleen was inhibited in low dose irradiation significantly. IL-4 expression in EL-4 cells was inhibited in a dose dependent manner. Histological damages of the epithelium in the nasal septum were improved by laser irradiation with marked improvement at low dose irradiation. Conclusion. These results suggest that LLLT might serve as a new therapeutic tool in the treatment of AR with more effectiveness at low dose irradiation. To determine the optimal dose of laser irradiation and action mechanisms of laser therapy, further studies will be needed.

Aconiti Lateralis Preparata Radix Activates the Proliferation of Mouse Bone Marrow Mesenchymal Stem Cells and Induces Osteogenic Lineage Differentiation through the Bone Morphogenetic Protein-2/Smad-Dependent Runx2 Pathway.

Mesenchymal stem cells have the capacity for self-renewal and under appropriate stimulation give rise to osteogenic, adipogenic, and chondrogenic lineages. To advance the clinical use of stem cell therapy, such as stem cell transplantation, it is important to find substances that promote endogenous stem cell proliferation and differentiation. We investigated whether medicinal herbs have the potential to promote stem cell proliferation and differentiation, using a cell cycle analysis and differentiation assay. We found that Aconiti Lateralis Preparata Radix (ALR) promoted the proliferation rate of mouse bone marrow mesenchymal stem cells (mBMMSCs) up to 122.24% compared to untreated cells. Fluorescence-activated cell sorter analysis showed that the percentage of cells in the G2/M phase increased to 17.33% in ALR-treated cells compared to 5.65% in normal cells. Signaling pathway analysis indicated that this was mediated through the extracellular signal-regulated kinase 1/2 pathway. A differentiation assay showed that ALR induced differentiation of mBMMSCs into an osteogenic lineage 2 weeks after treatment, whereas traditional osteogenic induction medium treatment did not promote differentiation for 3 weeks. This osteogenic differentiation was signaled by the bone morphogenetic protein-2/Smad-dependent Runx2 pathway. We found that ALR could promote mBMMSC proliferation and differentiation into the osteogenic lineage.

Inhibitory effect of Astragalus membranaceus root on matrix metalloproteinase-1 collagenase expression and procollagen destruction in ultraviolet B-irradiated human dermal fibroblasts by suppressing nuclear factor kappa-B activity.

OBJECTIVES: The root of Astragalus membranaceus, regarded as a tonic in traditional Korean medicine, has been prescribed for long periods to treat chronic illness by boosting the immune system. Ultraviolet (UV) irradiation causes damage to skin connective tissue by degrading collagen, which is a major structural component of the extracellular matrix. Such damage is considered to be a cause of the wrinkling observed in premature ageing of the skin. This study has investigated the photo-protective effect of A. membranaceus on UVB radiation-induced activation of nuclear factor kappa-B (NF-κB) activity in human dermal fibroblasts. METHODS: HS68 fibroblast cells cultured with various concentrations of A. membranaceus were exposed to UVB (40 mJ/cm²). Activation of NF-κB P65 and expression of matrix metalloproteinase-1 (MMP-1) and type 1 procollagen were measured by Western blotting. Translocation of NF-κB P65 and MMP-1 regulation were also examined by immunocytochemistry. KEY FINDINGS: Western blotting and immunocytochemistry results showed that A. membranaceus inhibited UVB-induced translocation of NF-κB P65 and MMP-1 expression. The data suggested that A. membranaceus restored type 1 procollagen synthesis by inhibiting NF-κB P65 activity and MMP-1 expression in UVB-exposed human dermal fibroblasts. CONCLUSION: A. membranaceus is a candidate for use in skin protection from UVB-induced skin inflammation and photoageing.

Astragalus membranaceus ameliorates reproductive toxicity induced by cyclophosphamide in male mice.

The root of Astragalus membranaceus B(UNGE) (AM) is a medicinal herb that has been capable of reducing the adverse effects of conventional chemotherapy. To investigate the effects of AM on cyclophosphamide (CP)-induced reproductive toxicity in mouse testes, 5-week-old male imprinting control region mice were divided into five groups; CP was treated on the first day of each week for 5 weeks (100 mg/kg, i.p.), and AM was treated for 5 days a week for 5 weeks. At the end of the treatment period, the testes were taken out, cleared of the adhering tissues, and weighed. Epididymis was taken out and used for sperm analysis. Testis samples were frozen for real-time quantitative PCR and Western blot analysis. AM treatment increased diminished relative testes weight, and sperm count and motility in mice treated with CP. CP treatment has detrimental effects on the expression of cAMP-responsive element modulator (CREM), a transcription factor that is highly expressed in male germ cells and is crucial to post-meiotic germ cell differentiation. AM restored CREM at both the mRNA and protein levels. AM has beneficial influences and appears able to ameliorate relative testes weight, sperm parameters, and CREM expression against CP-induced reproductive toxicity.

Cistanches herba induces testis cytotoxicity in male mice.

We investigated the effects of Cistanches herba (CH) on the male reproductive system in mice, assessing CREM gene expression and spermatogenesis. Our results demonstrate that CH treatment lead to a significant decrease in sperm count dose-dependently, 298.3 ± 48.9 vs. 296.6 ± 102.4 (250 mg/kg), 236.7 ± 75.1 (500 mg/kg), 223.0 ± 48.7 × 10(6) (1000 mg/kg), respectively. Additionally, serum testosterone levels decreased following CH treatment to as low as ~57% compared with the vehicle-treated group. CREM gene expression was also down-regulated following CH treatment and histological examination of the testicular seminiferous tubules showed severe damage on CH treatment. These results suggest that CH induces cytotoxicity in the male reproductive system, through the inhibition of spermatogenesis, testicular damage, and limited hormonal function.

Effects of Panax ginseng on glial cell-derived neurotrophic factor (GDNF) expression and spermatogenesis in rats.

Panax ginseng (PG) is a medicinal herb which has been used to improve male reproduction in traditional Korean medicine. This study investigated the effect of PG on spermatogenesis and the regulation of glial cell-derived neurotrophic factor (GDNF), which has a crucial role in spermatogonial stem cell maintenance. PG was administered to 8-week-old male Wistar rats (1.0 g/kg/day, p.o.) for 56 consecutive days, the sperm formation period of the rat. Sperm analysis, RT-PCR and western blot assays were then carried out. The PG-treated group had significantly enhanced sperm counts, GDNF mRNA level and protein level. These results suggest that PG induces spermatogenesis and GDNF activation in rat testes.

Inhibitory effects of Hoelen extract on melanogenesis in B16/F1 melanoma cells.

Melanin synthesis is regulated by melanogenic proteins, such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and TRP-2. The effects of Hoelen extract on melanogenesis were investigated in B16Fl murine melanoma cells. Specifically, tyrosinase activity, cell viability and melanin content were assayed, and western blotting and RT-PCR for tyrosinase, TRP-1 and TRP-2 conducted. The results show that Hoelen significantly inhibited melanin synthesis through inhibition of TRP-2 expression, while it did not affect tyrosinase activity or its expression. Taken together, RT-PCR results showed that the depigmentation effect of Hoelen may be due to inhibition of TRP-2 gene transcription. These results suggest that Hoelen may be a useful inhibitor for the attenuation of melanogenesis and hyperpigmentation in skin cells.

Cynomorium songaricum induces spermatogenesis with glial cell-derived neurotrophic factor (GDNF) enhancement in rat testes.

AIM OF THE STUDY: Cynomorium songaricum Ruprecht has been used in traditional Korean medicine to treat male infertility, including sexual dysfunction, by improving kidney function. Glial cell-derived neurotrophic factor (GDNF) produced by Sertoli cells induces the proliferation of undifferentiated spermatogonia. We investigated the effects of Cynomorium songaricum on sperm parameters and GDNF expression in rat testes. MATERIALS AND METHODS: Sperm analysis, RT-PCR, and Western blotting assays were performed after administration of CS to 8-week-old male Wistar rats for 56 consecutive days (1.0g/kg/day, p.o.), the period of sperm formation in the rat. RESULTS AND CONCLUSIONS: The CS-treated animals showed significant increases in epididymal sperm count and absolute testes weights compared to the control group. CS also increased the expression of GDNF at both the mRNA and protein levels. These results suggest that CS may improve male fertility by enhancing spermatogenesis and GDNF expression.

Treatment with Astragali radix and Angelicae radix enhances erythropoietin gene expression in the cyclophosphamide-induced anemic rat.

Anemia is a common cause of morbidity and disease and reduces the quality of life. This study examined the effect of a combination treatment (AAC) using Astragali radix (AMW) and Angelicae radix (AGW) in cyclophosphamide (CYP)-induced anemic rats on erythropoietin (EPO) expression and hematological parameters. Male 4-week-old Sprague-Dawley rats were divided into five groups with or without CYP-induced anemia and individual or the combined herbal treatments according to the experimental protocol. After treatment, reverse transcription-polymerase chain reaction was used to evaluate the effects of AAC on erythropoietin expression, and blood and serological parameters were measured. The EPO mRNA levels were lower in the CYP-treated group, compared to the normal group, and higher in the AAC-treated group. In the CYP-treated group, the serum iron concentration, total iron-binding capacity, and vitamin B(12) level were lower, but these were normal or almost normal in the AAC-treated group. The CYP-treated group gained less weight than the normal group, but weight gain was partially normalized in the AAC group. The feed efficiency ratio was lowest in the CYP group, but the differences were not significant. The numbers of red blood cells, white blood cells, and platelets, the hematocrit, and the hemoglobin level were measured. The results revealed a reduced number of blood cells in the CYP-treated group, whereas the AAC-, AMW-, and AGW-treated groups showed significantly enhanced blood cell numbers compared to the CYP-treated control group and the AAC-treated group. AAC enhanced EPO mRNA expression in the CYP-induced anemic rat and improved the hematological parameters and vitamin B(12) status.

Nelumbo nucifera semen extract improves memory in rats with scopolamine-induced amnesia through the induction of choline acetyltransferase expression.

Nelumbo nucifera semen (NNS) is a traditional herb with anti-diarrheal, anti-ganacratia, and tranquilizer-like pharmacological activities. In this study, we examined the anti-amnesic effect of NNS on rats with scopolamine-induced amnesia. Passive avoidance tests, acetylcholinesterase (ACHE) activity, and choline acetyltransferase (CHAT) expression were used to evaluate the NNS anti-amnesic effects. The rats were divided into five groups: the normal group, scopolamine-treated group (1mg/kg; control), NNS (1g/kg) and scopolamine (1mg/kg) co-treatment group, and the ARICEPT (1mg/kg) and scopolamine (1mg/kg) co-treatment group (positive control). The rats were administered the compounds orally for 14 days. The latency time of passive avoidance significantly increased by 54% in the NNS-treated group compared to the scopolamine-treated group. The ACHE activity in the NNS-treated group significantly decreased to 7.35% than that of the control group. CHAT-positive neurons increased by 51.02% in the NNS group compared to the control group. These results suggest that NNS extract improves scopolamine-induced dementia by inhibiting ACHE activity and inducing CHAT expression.

Novel effects of Nelumbo nucifera rhizome extract on memory and neurogenesis in the dentate gyrus of the rat hippocampus.

Memory enhancement is a matter of concern in general, and in particular to people suffering from cognitive dysfunction. In this study, we investigated the effect of Nelumbo nucifera rhizome extract on learning and memory function. A step-through passive avoidance test was performed with Wistar rats. In addition, immunohistochemistry was used to investigate cell proliferation and differentiation in the dentate gyrus of the hippocampus. The methanol extract of N. nucifera rhizome (MNR) resulted in significant improvements of memory functions and neurogenesis in the dentate gyrus. In the passive avoidance test, the retention time of MNR-treated rats was significantly longer than that of controls. Immunohistochemical analyses using BrdU, Ki-67, and DCX showed significantly increased cell proliferation and cell differentiation in the dentate gyrus. These results suggest that N. nucifera rhizome extract may improve learning and memory with enhancing neurogenesis in the DG of the hippocampus.

Cytoprotective effects of Morinda officinalis against hydrogen peroxide-induced oxidative stress in Leydig TM3 cells.

AIM: To investigate the antioxidant effects of Morinda officinalis (Morindae radix, MR) on H(2)O(2)-induced oxidative stress in cultured mouse TM3 Leydig cells. METHODS: We carried out 2,2-diphenyl-1-picrylhydrazyl free radical scavenging, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lipid peroxidation, testosterone enzyme immunoassay, superoxide dismutase (SOD), and catalase (CAT) assays in Leydig TM3 cells. RESULTS: MR showed a 47.8% 2,2-diphenyl-1-picrylhydrazyl radical scavenging effect in TM3 cells with no significant cytotoxicity. Oxidative stress was induced in TM3 cells with 100 micromol H(2)O(2), and treatment of the cells with 250 microg/mL MR showed the most significant protective effect (64%, P < 0.001) in the cell viability assay with a decreased lipid peroxidation level (1.75 nmol/mg protein, P < 0.05), increased testosterone production (43.5 pg/mL), and improvements in SOD activity (7.49 units of SOD/mg protein, P < 0.001) and CAT activity (74.6 units of CAT/mg protein, P < 0.001). CONCLUSION: These findings indicate that MR, as an antioxidant, protects functions of cultured mouse TM3 Leydig cells from H(2)O(2)-induced oxidative stress.