Combinational Anti-tumor Effects of Chemicals from Paeonia lutea Leaf Extract in Oral Squamous Cell Carcinoma Cells.

AIM: We identified chemical components that exhibited antitumor activity against oral squamous cell carcinoma (OSCC) cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis and cell cycle of OSCC cells. MATERIALS AND METHODS: Using high-performance liquid chromatography, nuclear magnetic resonance spectroscopy and electrospray ionization-mass spectrometry, we identified the main chemical components of the methanol extracts from Paeonia lutea. We investigated the pharmaceutical effects of those components on the proliferation, apoptosis, and cell cycle of an OSCC cell line, SAS, using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and caspase assays, as well as flow cytometry cell cycle analysis. We also examined the effects of those components on the mitogen-activated protein kinase signal transduction pathway by western blotting. Finally, the effects on normal human epidermal keratinocyte cells were also examined in similar experiments. RESULTS: Three chemicals have been identified in P. lutea leaves using high performance liquid chromatography: gallic acid methyl ester (GAME), pentagalloyl glucose (PGG) and paeoniflorin (PF). Both GAME and PGG significantly suppressed cell proliferation, and their combined effects were synergistic, while the effect of PF was minimal. However, those chemicals did not induce apoptosis. Cell cycle and western blotting analysis showed that the suppressive effects on cell proliferation resulted from G2 arrest and the suppression of phosphorylation of Akt/PKB. No effect was identified on normal human epidermal keratinocyte cells. CONCLUSION: These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.

Methanol and Butanol Extracts of Paeonia lutea Leaves Repress Metastasis of Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is one of the most common cancers of the head and neck region worldwide and is generally treated surgically in combination with radiotherapy and/or chemotherapy. However, anticancer agents have numerous serious side effects, and alternative, less toxic agents that are effective as chemotherapeutics for SCC are required. The Paeoniaceae family is widely used in traditional Chinese medicine. We examined methanol and butanol extracts of Paeonia lutea (P. lutea) leaves for their potential as an anticancer agent. Both extracts decreased the proliferation of SCC cells, induced apoptotic cell death, and modulated migration, adhesion, chemotaxis, and haptotaxis in an extracellular matrix- (ECM-) dependent manner due to altered expression of several integrin subunits. Subsequently, SCC cells were subcutaneously transplanted into athymic nude mice; the extracts reduced the metastasis of SCC cells but had little effect on the volume of the primary tumor or survival or body weight of the mice. The results suggest that the extracts may hold promise for preventing cancer metastasis.

Vascular effects and safety of supplementation with shark liver oil in middle-aged and elderly males.

Shark liver oil (SLO) has long been used as a traditional health food, with a particular benefit for vascular health, in Japan. The aim of this study was to assess the effect of dietary supplementation with SLO on arterial stiffness and peripheral microvascular function in otherwise healthy middle-aged and older males with slightly increased arterial stiffness. A randomized, double-blind, placebo-controlled, parallel study design was used to assign 41 healthy males with a mean age of 59.0±4.0 years (range, 45-69 years) to either SLO (n=21) or placebo (n=20) treatment for eight weeks. The effects on arterial stiffness and peripheral microvascular function were assessed by the cardio-ankle vascular index (CAVI) and by measurement of hand blood flow to cutaneous tissues using a laser Doppler perfusion imaging (LDPI) technique, respectively. Although the magnitude of the changes in the CAVI value during the eight-week intervention for the SLO group did not significantly differ from that for the placebo group, the changes in the CAVI value for the former group were significantly associated (r=0.575, P<0.01) with age. It was also found that the LDPI values at week 8 were significantly lowered (P<0.05) compared with the baseline values in the placebo group, while no change was observed in the SLO group, resulting in a significant difference in the changes between the two groups (P=0.002). Neither SLO supplementation-related adverse side-effects nor any abnormal changes in routine laboratory tests, including lipid profiles and anthropometric and haemodynamic parameters, were observed throughout the intervention. SLO may have the potential to safely improve vascular health in middle-aged and elderly males.

Potential anti-osteoporotic effects of herbal extracts on osteoclasts, osteoblasts and chondrocytes in vitro.

BACKGROUND: Osteoporosis (OP) is one of the most serious diseases in the modern world, and OP patients frequently suffer from fragility fractures in the hip, spine and wrist, resulting in a limited quality of life. Although bisphosphonates (BPs) are the most effective class of anti-bone-resorptive drugs currently available and the most commonly prescribed for the clinical treatment of OP, they are known to cause serious side effects such as bisphosphonate-related osteonecrosis of the jaw. Novel therapeutic materials that can replace the use of BPs have therefore been developed. METHODS: We commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only in OP, but also in oral and skeletal diseases. In the present study, we report on 3 Chinese medical herbal extracts from the root barks of Melia azedarach, Corydalis turtschaninovii, and Cynanchum atratum. RESULTS: All of these extracts inhibited osteoclast proliferation and induced apoptosis by up-regulation of caspase activity and increase of mitochondrial pro-apoptotic proteins expression. Furthermore, the extracts enhanced differentiation, but did not affect proliferation of both osteoblasts and chondrocytes. The osteo-inducible effect was also observed in cultured primary bone marrow cells. CONCLUSIONS: Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects in OP. In this study, we elucidate the potency of these herbal extracts as novel candidates for OP therapy.

Root bark extracts of Juncus effusus and Paeonia suffruticosa protect salivary gland acinar cells from apoptotic cell death induced by cis-platinum (II) diammine dichloride.

Cis-platinum (II) diammine dichloride (CDDP) is a platinum-based anticancer agent, and is often used for chemotherapy for malignant tumors, albeit CDDP has serious side-effects, including xerostomia (dry mouth). Since patients with xerostomia have reduced quality of life, it is urgent and important to identify nontoxic and natural agents capable of reducing the adverse effect of chemotherapy on salivary gland function. Therefore, we commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only on xerostomia, but also on oral diseases. In the present study, we report on two Chinese medical herbal extracts from the root barks of Juncus effusus and Paeonia suffruticosa. The two extracts showed a protective effect in NS-SV-Ac cells from the cytotoxicity and apoptosis caused by CDDP. The effect was dependent on the p53 pathway, protein kinase B/Akt 1 and mitochondrial apoptosis-related proteins (i.e. Bcl-2 and Bax), but was not dependent on nuclear factor κB. Notably, the apoptosis-protective effect of the extracts was not observed in adenocystic carcinoma cell lines. Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects on xerostomia. Thus, in the present study, we elucidated the potency of these herbal extracts as novel candidates for xerostomia to improve the quality of life of patients undergoing chemotherapy.

The potential anticancer activity of extracts derived from the roots of Scutellaria baicalensis on human oral squamous cell carcinoma cells.

Various herb products derived from plants have potent biological effects including anticancer activity. In the present study, the antitumor activity of a herbal product derived from the Scutellaria baicalensis (S. baicalensis) was examined, using in vitro assays in a human oral squamous cell carcinoma (OSCC) cell line. Results showed that S. baicalensis root extract at the concentration of 100 µg/ml inhibited monolayer- and anchorage-independent growth in human OSCC cell lines, while not affecting the adhering abilities of cells. This suggested that it did not alter the expression of any of the adhesion receptors that mediate cell-extracellular matrix (ECM) interactions. The S. baicalensis root extract demonstrated potent cytostatic and apoptotic effects due to the downregulation of the cyclin-dependent kinase 4 expression and its partner cyclin D1, resulting in G1 arrest and poly (ADP-ribose) polymerase (PARP) cleavage. Additionally, the S. baicalensis root extract was found to have blocked vascular endothelial growth factor (VEGF)-induced migration and tube formation in human endothelial cells. Taken together, these results demonstrate that as a herbal product, the S. baicalensis root extract is a potential inhibitor of tumori- and angiogenesis and may be valuable in the development of pharmaceutical medications for the treatment of oral squamous cell carcinoma.

The root bark of Paeonia moutan is a potential anticancer agent in human oral squamous cell carcinoma cells.

Currently there is growing use of complementary and alternative anticancer medicines worldwide, and considerable interest in finding anticancer drugs among Chinese medicinal herbs. The aim of this study was to determine the antitumor activity of the root bark of Paeonia moutan (RBPM) in human squamous cell carcinoma (OSCC) cells. Cell lines derived from human oral squamous cell carcinoma (HSC2, 3, 4, SAS) were tested with different concentrations of RBPM (1-100 µg/ml) using a series of in vitro assay systems. RBPM at a concentration of 100 µg/ml inhibited monolayer and anchorage-independent growth, and interrupted coordinated migration. RBPM activated the phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase AKT in 30 min; then, at a later stage (after 6 hours) exhibited potent cytostatic, pro-apoptotic effects through the down-regulation of the expression of cyclin-dependent kinase 4 and its partner cyclin D1, and poly(ADP-ribose) polymerase cleavage. We found direct evidence that RBPM induces apoptotic cell death via DNA fragmentation. Taken together, the antitumor activity of RBPM was demonstrated through antiproliferative and apoptotic effects.

Anti-inflammatory effects of seeds of the tropical fruit camu-camu (Myrciaria dubia).

The methanolic extract of seeds of the tropical fruit camu-camu was screened for its anti-inflammatory activity in carrageenan-induced paw edema model mice. The extract significantly suppressed both the formation of edema in mice by oral administration and the release of nitric oxide from macrophage-derived RAW 264.7 cells in vitro. Based on the results of a spectroscopic analysis, the active compound was identified by in vivo bioassay-guided fractionation to be 3ß-hydroxy-lup-20(29)-en-28-oic acid, betulinic acid, known as an anti-inflammatory triterpenoid. These findings suggest that camu-camu seed extract is a potentially useful material as a source of betulinic acid and as a functional food for prevention of immune-related diseases.

Banana peel extract suppressed prostate gland enlargement in testosterone-treated mice.

A methanol extract of banana peel (BPEx, 200 mg/kg, p.o.) significantly suppressed the regrowth of ventral prostates and seminal vesicles induced by testosterone in castrated mice. Further studies in the androgen-responsive LNCaP human prostate cancer cell line showed that BPEx inhibited dose-dependently testosterone-induced cell growth, while the inhibitory activities of BPEx did not appear against dehydrotestosterone-induced cell growth. These results indicate that methanol extract of banana peel can inhibit 5alpha-reductase and might be useful in the treatment of benign prostate hyperplasia.

Suppressive effects of Anoectochilus formosanus extract on osteoclast formation in vitro and bone resorption in vivo.

Anoectochilus formosanus, a plant native to Taiwan, is used as a folk medicine. It was found that oral administration of A. formosanus extract (AFE) (500 mg/kg) for 4 weeks suppressed bone weight loss and trabecular bone loss in ovariectomized mice, an experimental model of osteoporosis. Although AFE at 12.5 and 25 mug/ml inhibited osteoclast formation in co-culture of osteoblasts and bone marrow cells, AFE did not inhibit the formation of osteoclast progenitor cells and preosteoclast cells in bone marrow cells and RAW264 cells. However, AFE (at 12.5 and 25 microg/ml) decreased RANKL expression. These results suggested that AFE might suppress the bone loss caused by estrogen deficiency through suppression of RANKL expression required for osteoclast formation.

Suppressive effects of astaxanthin against rat endotoxin-induced uveitis by inhibiting the NF-kappaB signaling pathway.

We investigated the effects of astaxanthin (AST), a carotenoid, on endotoxin-induced uveitis (EIU), and over the course of the disease measured the expression of inflammatory cytokines and chemokines in the presence or absence of AST. EIU was induced in male Lewis rats by footpad injection of lipopolysaccharide (LPS). The animals were randomly divided to 12 groups with eight animals in each. Immediately after the inoculation, AST (1, 10, or 100 mg kg(-1)) was injected intravenously. Aqueous humour was collected at 6, 12 and 24 hr after LPS inoculation and the number of infiltrating cells in the anterior chamber was counted. In addition, we assayed the concentration of protein, nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2). Immunohistochemical staining with a monoclonal antibody against activated NF-kappaB was performed in order to evaluate the effects of AST on NF-kappaB activation. Rats injected with AST showed a significant decrease in the number of infiltrating cells in the anterior chamber and additionally there was a significantly lower concentration of protein, NO, TNF-alpha and PGE2 in the aqueous humour. Moreover, even early stages of EIU were suppressed by injection of AST. The number of activated NF-kappaB-positive cells was lower in iris-ciliary bodies treated with 10 or 100 mg kg(-1) AST at 3 hr after LPS injection. These results suggest that AST reduces ocular inflammation in eyes with EIU by downregulating proinflammatory factors and by inhibiting the NF-kappaB-dependent signaling pathway.

The effects of Ginkgo biloba extract on lipopolysaccharide-induced inflammation in vitro and in vivo.

PURPOSE: Ginkgo biloba extract (GBE) contains many different flavone glycosides and terpenoides. Several previous studies have demonstrated that GBE exhibits a wide variety of biological activities, including an antioxidant action, on which we focused our attention. The aim of the present study was to investigate the efficacy of GBE on endotoxin induced uveitis in rats. The anti-inflammatory potency of GBE in vivo was compared with that of prednisolone. In addition, we also investigated nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha) and the expression of iNOS in a mouse macrophage cell line (RAW 264.7) treated with GBE in vitro to clarify the anti-inflammatory effect. METHODS: EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS inoculation, either 1, 10 or 100 microg of GBE were injected intravenously. 24hr later, the aqueous humor was collected from both eyes, and the number of infiltrating cells, protein concentration and NO level in the aqueous humor was determined. The RAW 264.7 cells were pretreated with various concentrations of GBE for 24hr and subsequently incubated with LPS for 24hr. Levels of NO, PGE2 and TNF-alpha were determined by enzyme-linked immunosorbent assay. The expression of iNOS protein was analyzed by Western blotting method. RESULTS: GBE treatment in vivo decreased the concentrations of protein and NO in the aqueous humor of EIU rats. The anti-inflammatory effect of 1 mg GBE was as strong as that of same dose prednisolone. It also significantly reduced the concentration of PGE2, TNF-alpha and NO production in the medium of RAW 264.7 cells compared to that of the LPS group in vitro. The expression of iNOS protein in the 1000 microg ml(-1) of GBE treated cells decreased significantly. CONCLUSION: The present results indicate GBE suppresses the inflammation of EIU by blocking the iNOS protein expression and its anti-inflammatory effect on eye is comparable with the effect of prednisolone used in similar doses.

[Importance of "health foods", EPA and DHA, for preventive medicine].

The "lifestyle-related disease" has been increasing in Japan as the population advances in age and the food culture becomes westernized. Although prevention, treatment and therapy for this disease have been attempted using certain kinds of food and nutritive elements, so-called "health foods" such as DHA and EPA, which are mostly contained in fish oil, have been a special focus within these attempts. There have been many reports regarding the pharmacological functions and the mechanisms of DHA and EPA. Also, in the past few years, it has become possible to produce ingestible DHA and EPA oils, oils for chemical compounds, oils for animal feed, and highly purified DHA and EPA for medical and pharmaceutical use. EPA ethyl ester has a wide market as a preventive medicine in Japan. Initially in 1990, this medicine was administered in cases of arterisclerosis obliterans, using its anti-platelet aggregation ability. Four years later, in 1994, its effectiveness in triglyceride reduction was recognized, and its application was extended to cases of hyperlipidemia, which has remarkably broadened its market. Clinical studies with DHA have shown improvement in senile dementia (cerebral thrombosis, Alzheimer's disease), atopic dermatitis, and the ability to focus on moving objects, as well as control of aggressiveness against others caused by stress, and prevention of hyperlipidemia, hypertension, and cancer.

Effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo.

PURPOSE: Astaxanthin (AST) is a carotenoid that is found in marine animals and vegetables. Several previous studies have demonstrated that AST exhibits a wide variety of biological activities including antioxidant, antitumor, and anti-Helicobacter pylori effects. In this study, attention was focused on the antioxidant effect of AST. The object of the present study was to investigate the efficacy of AST in endotoxin-induced uveitis (EIU) in rats. In addition, the effect of AST on endotoxin-induced nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor (TNF)-alpha production in a mouse macrophage cell line (RAW 264.7) was studied in vitro. METHODS: EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). AST or prednisolone was administered intravenously at 30 minutes before, at the same time as, or at 30 minutes after LPS treatment. The number of infiltrating cells and protein concentration in the aqueous humor collected at 24 hours after LPS treatment was determined. RAW 264.7 cells were pretreated with various concentrations of AST for 24 hours and subsequently stimulated with 10 microg/mL of LPS for 24 hours. The levels of PGE2, TNF-alpha, and NO production were determined in vivo and in vitro. RESULTS: AST suppressed the development of EIU in a dose-dependent fashion. The anti-inflammatory effect of 100 mg/kg AST was as strong as that of 10 mg/kg prednisolone. AST also decreased production of NO, activity of inducible nitric oxide synthase (NOS), and production of PGE2 and TNF-alpha in RAW264.7 cells in vitro in a dose-dependent manner. CONCLUSIONS: This study suggests that AST has a dose-dependent ocular anti-inflammatory effect, by the suppression of NO, PGE2, and TNF-alpha production, through directly blocking NOS enzyme activity.