Berberine Induces Autophagic Cell Death by Inactivating the Akt/mTOR Signaling Pathway.

The incidence of skin cancer has been increasing over the past decades, and melanoma is considered highly malignant because of its high rate of metastasis. Plant-derived berberine, an isoquinoline quaternary alkaloid, has been reported to possess multiple pharmacological effects against various types of cancer cells. Therefore, we treated melanoma B16F10 cells with berberine to induce cell death and understand the cell death mechanisms. The berberine-treated cells showed decreased cell viability, according to berberine concentration. However, western blot analysis of apoptosis-related marker proteins showed that the expression of Bcl-2, an apoptosis inhibitory protein, and the Bcl-2/Bax ratio were increased. Therefore, by adding 3-methyladenine to the berberine-treated cells, we investigated whether the reduced cell viability was due to autophagic cell death. The results showed that 3-methyladenine restored the cell viability decreased by berberine, suggesting autophagy. To clarify autophagic cell death, we performed transmission electron microscopy analysis, which revealed the presence of autophagosomes and autolysosomes in the cells after treatment with berberine. Next, by analyzing the expression of autophagy-related proteins, we found an increase in the levels of light chain 3A-II and Atg12-Atg5 complex in the berberine-treated cells. We then assessed the involvement of the Akt/mTOR signaling pathway and found that berberine inhibited the expression of phosphorylated Akt and mTOR. Our data demonstrated that berberine induces autophagic cell death by inactivating the Akt/mTOR signaling pathway in melanoma cells and that berberine can be used as a possible target for the development of anti-melanoma drugs.

Antidepressant and Anti-Neuroinflammatory Effects of Bangpungtongsung-San.

Bangpungtongsung-san (BTS) is a traditional Korean medicine consisting of 18 herbs, some which have antidepressant effects. Here, we used an animal model of reserpine-induced depression and lipopolysaccharide (LPS)-stimulated BV2 microglia to assess the antidepressant and anti-neuroinflammatory effects of BTS. Aside from a control group, C57BL/6 mice were administered reserpine (0.5 mg/kg) daily for 10 days via intraperitoneal injection. BTS (100, 300, or 500 mg/kg), vehicle (PBS), or fluoxetine (FXT, 20 mg/kg) was administered orally 1 h before reserpine treatment. Following treatment, a forced swimming test (FST), tail suspension test (TST), and open field test (OFT) were performed, and immobility time and total travel distance were measured. Administration of BTS not only reduced immobility time in the FST and TST but also significantly increased the total travel distance in the OFT. Furthermore, reserpine-treated mice showed significantly elevated serum levels of corticosterone, a stress hormone; however, treatment with BTS significantly reduced corticosterone levels, similar to FXT treatment. Serotonin in reserpine-treated mice was significantly reduced compared to that in control mice, while BTS mice exhibited increased serotonin levels. BTS mice showed increased expression of brain-derived neurotrophic factor (BDNF) and a higher ratio of phosphorylated cAMP response element-binding protein (p-CREB) to CREB (p-CREB/CREB) in the hippocampus. Additionally, reserpine-treated mice exhibited significantly elevated mRNA levels of pro-inflammatory cytokines, but BTS mice showed reduced mRNA levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the hippocampus. To further demonstrate the anti-neuroinflammatory effects of BTS in vitro, we examined its anti-neuroinflammatory and neuroprotective effects in lipopolysaccharide (LPS)-stimulated BV2 microglia. BTS significantly reduced the levels of NO, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, TNF-α, IL-1ß, and IL-6 in a dose-dependent manner via a decrease in the expression of nuclear factor (NF)-κB p65. Furthermore, the neuroprotective factor heme oxygenase-1 (HO-1) was upregulated via the nuclear factor-E2-related factor 2 (NRF2)/CREB pathway. Taken together, our data suggest that BTS has considerable potential as an anti-neuroinflammation and antidepressant agent, as it has clear effects on depressive behaviors and associated factors caused by reserpine-induced depression.

Antineuroinflammatory and Neuroprotective Effects of Gyejibokryeong-Hwan in Lipopolysaccharide-Stimulated BV2 Microglia.

Microglia, the central nervous system's innate immune cells, mediate neuroinflammation and are implicated in a variety of neuropathologies. The present study investigated the antineuroinflammatory and neuroprotective effects of Gyejibokryeong-hwan (GBH), a traditional Korean medicine, in lipopolysaccharide- (LPS-) stimulated murine BV2 microglia. BV2 cells were pretreated with GBH, fluoxetine (FXT), or amitriptyline (AMT) for 1 h and then stimulated with LPS (100 ng/mL). The expression levels of nitric oxide (NO), cytokines, and chemokines were determined by the Griess method, ELISA, or real-time PCR. Western blotting was used to measure various transcription factors and mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt activity. GBH significantly reduced the levels of NO, inducible nitric oxide synthase (iNOS), cyclooxygenase- (COX-) 2, tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1ß, IL-6, macrophage inhibitory protein- (MIP-) 1α, macrophage chemoattractant protein- (MCP-) 1, and IFN-γ inducible protein- (IP-) 10, regulated upon activation normal T cell expressed sequence (RANTES) in a dose-dependent manner. Expression of nuclear factor- (NF-) κB p65 was significantly decreased and phosphorylation of extracellular signal-regulated kinase (Erk), c-Jun NH2-terminal kinase (JNK), and PI3K/Akt by GBH, but not p38 MAPK, was decreased. Furthermore, production of anti-inflammatory cytokine IL-10 was increased and Heme oxygenase-1 (HO-1) was upregulated via the nuclear factor-E2-related factor 2 (NRF2)/cAMP response element-binding protein (CREB) pathway, collectively indicating the neuroprotective effects of GBH. We concluded that GBH may suppress neuroinflammatory responses by inhibiting NF-κB activation and upregulating the neuroprotective factor, HO-1. These results suggest that GBH has potential as anti-inflammatory and neuroprotective agents against microglia-mediated neuroinflammatory disorders.

Antidepressant-Like and Neuroprotective Effects of Ethanol Extract from the Root Bark of Hibiscus syriacus L.

Hibiscus syriacus L. (Malvaceae) is an important ornamental shrub in horticulture and has been widely used as a medical material in Asia. The aim of this study was to assess the antidepressant and neuroprotective effects of a root bark extract of H. syriacus (HSR) and to investigate the underlying molecular mechanisms. Using an animal model of restraint stress, we investigated the effects of HSR on depressive-like behaviors and on the expression levels of serotonin, corticosterone, and neurotrophic factors in the brain. The mice were exposed to restraint stress for 2 h per day over a period of 3 weeks and orally treated with HSR (100, 200, or 400 mg/kg/day). We also examined the neuroprotective effect of HSR using corticosterone-treated human neuroblastoma SK-N-SH cells. The cells were incubated with the extract for 24 h, followed by corticosterone stimulation for 1 h, and then cell viability assay, cellular ATP assay, mitochondrial membrane potential (MMP) assay, cellular reactive oxygen species (ROS) assay, and western blotting were used to investigate the neuroprotective effects of HSR. Administration of HSR not only reduced the immobility times of the restraint-stressed mice in the forced swimming and tail suspension tests, but also significantly increased sucrose preference in the sucrose preference test. In addition, HSR significantly reduced the plasma levels of corticosterone and increased the brain levels of serotonin. The extract also increased the phosphorylation level of cyclic AMP response element-binding (CREB) protein and the expression level of brain-derived neurotrophic factor (BDNF). The in vitro assays showed that HSR pretreatment increased cell viability and ATP levels, recovered MMP, decreased ROS levels, and increased the expression of CREB and BDNF in corticosterone-induced neurotoxicity. Taken together, our data suggest that HSR may have the potential to control neuronal cell damage and depressive behaviors caused by chronic stress.

Antidepressant Effect of Fraxinus rhynchophylla Hance Extract in a Mouse Model of Chronic Stress-Induced Depression.

Prolonged exposure to stress can affect mood and cognition and lead to mood disorders. Research on stress-associated mood disorders is important in modern society as people are increasingly exposed to unavoidable stressors. We used a mouse model with 2 weeks of exposure to electric foot shock and restraint, to determine the effect of Fraxinus rhynchophylla Hance (FX) extract on chronic stress-induced depression. We measured the effect of FX extract using various physiological, behavioral, and biochemical measures. FX extract ameliorated chronic stress-induced body and relative liver weight loss and improved depressive-like behaviors in the open field and forced swim tests. In addition, plasma cortisol and serotonin levels in stress-induced mice following FX treatment were similar to normal mice, and the elevation of proinflammatory cytokines was prevented. Moreover, FX treatment increased the expression of phosphorylated cyclic adenosine-3',5'-monophosphate response element-binding protein (pCREB)/brain-derived neurotrophic factor (BDNF). Further experiments confirmed the efficacy of FX extract by showing similar results using esculin and esculetin, compounds extracted from FX. Taken together, these results indicate that FX extract has an antidepressant effect on chronic stress-induced depression by associating signaling with neuroinflammation and neurogenesis.

Antidepressant-Like Effects of Gyejibokryeong-hwan in a Mouse Model of Reserpine-Induced Depression.

Treatment with the antihypertensive agent reserpine depletes monoamine levels, resulting in depression. In the present study, we evaluated the antidepressant effects of Gyejibokryeong-hwan (GBH), a traditional Korean medicine, in a mouse model of reserpine-induced depression. Mice were treated with reserpine (0.5 mg·kg-1, i.p.) or phosphate-buffered saline (PBS, i.p., normal) once daily for 10 days. GBH (50, 100, 300, and 500 mg·kg-1), PBS (normal, control), fluoxetine (FXT, 20 mg·kg-1), or amitriptyline (AMT, 30 mg·kg-1) was administered orally 1 h prior to reserpine treatment. Mouse behavior was examined in the forced swim test (FST), tail suspension test (TST), and open-field test (OFT) following completion of the treatment protocol. Administration of GBH reduced immobility time in the FST and TST and significantly increased the total distance traveled in the OFT. Plasma serotonin levels were significantly lower in control mice than in normal mice, although these decreases were significantly attenuated to a similar extent by treatment with GBH, FXT, or AMT. Reserpine-induced increases in plasma corticosterone were also attenuated by GBH treatment. Moreover, GBH attenuated reserpine-induced increases in interleukin- (IL-) 1ß, IL-6, and tumor necrosis factor- (TNF-) α mRNA expression in the hippocampus. In addition, GBH mice exhibited increased levels of brain-derived neurotrophic factor (BDNF) and a higher ratio of phosphorylated cAMP response element-binding protein (p-CREB) to CREB (p-CREB/CREB) in the hippocampus. Our results indicated that GBH can ameliorate depressive-like behaviors, affect the concentration of mood-related hormones, and help to regulate immune/endocrine dysfunction in mice with reserpine-induced depression, likely via activation of the BDNF-CREB pathway. Taken together, these findings indicate that GBH may be effective in treating patients with depression.

Suppressive Effect of the n-Hexane Extract of Litsea japonica Fruit Flesh on Monosodium-Iodoacetate-Induced Osteoarthritis in Rats.

We examined the antiosteoarthritic effect of the n-hexane extract of Litsea japonica fruit flesh (LJF-HE) in a rat model of monosodium-iodoacetate- (MIA-) induced osteoarthritis. LJF-HE significantly reduced the difference in weight-bearing capabilities of the hind paws between healthy and MIA-treated rats. Histological examination of the knee joints indicated that LJF-HE suppressed cartilage and bone destruction. Additionally, there were decreases in the expression of matrix metalloproteinase-2 and metalloproteinase-9 and cyclooxygenase-2 in the joints. The serum levels of deoxypyridinoline (DPD) and osteocalcin, which are markers of bone metabolism, also decreased. Furthermore, LJF-HE significantly suppressed infiltration of inflammatory cells into the synovium and inhibited the expression of proinflammatory cytokines such as tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1, and IL-6 in the joints and serum. The serum levels of leukotriene B4 and lipoxygenase were also significantly lowered by LJF-HE. Finally, LJF-HE inhibited the production of nitric oxide, prostaglandin E2, IL-6, and TNF-α in lipopolysaccharide-activated macrophages, which might be associated with inhibited phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. Our data suggest that LJF-HE has an anti-inflammatory effect and may have potential as an antiosteoarthritic agent.

Administration of Wasabia koreana Ameliorates Irritable Bowel Syndrome-Like Symptoms in a Zymosan-Induced Mouse Model.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disease with complex pathophysiology involving the brain-gut axis. To assess the effects of Wasabia koreana (WK) on IBS, we employed a mouse model of colonic zymosan injection presenting with diarrhea-predominant IBS-like symptoms. Oral WK administration significantly diminished stool score, suppressed colon length and weight change, and minimized body weight loss without affecting food intake. In WK-treated mice, the submucosal thickening and epithelial lining of the colon were inhibited and were similar to those of naïve mice. Infiltration of mast cells into the colon and serum tumor necrosis factor-α levels were markedly suppressed. These effects were comparable to those of sulfasalazine, an anti-inflammatory drug. Furthermore, the number of visceral pain-related behaviors was significantly decreased, and locomotion activities measured in the elevated plus maze and open field tests were significantly increased by WK in a dose-dependent manner compared with amitriptyline, an antidepressant. These changes were accompanied by reduced FosB2 expression in the brain. Taken together, these data suggest that WK may have potential as a medicinal food for IBS by acting on inflammatory diarrhea and neural activity.

Gamisasangja-tang suppresses pruritus and atopic skin inflammation in the NC/Nga murine model of atopic dermatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gamisasangja-tang (GST) is a traditional herbal formula prescribed for patients with intractable pruritus in association with various inflammatory skin diseases. To evaluate the effects of GST on pruritic skin inflammation and investigate its cellular and molecular mechanisms. MATERIALS AND METHODS: We orally administered GST to NC/Nga (NC) mice, an animal model of atopic dermatitis. Scratching frequency and the dermatitis index were evaluated, and histological examination was performed using hematoxylin and eosin and toluidine blue staining. The levels of interleukin (IL)-31 and T-helper cell type 2 (TH2) cytokines were determined in both the dorsal skin and cultured splenocytes by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The serum levels of chemokines and immunoglobulin E (IgE) were determined by ELISA. Changes in the inflammatory cell population were analyzed by a hemocytometer. RESULTS: GST significantly lowered scratching frequency and inhibited increases in dermatitis index, thickness of epidermis/dermis and infiltration of chemokine (C-C motif) receptor 3 (CCR3)(+) and cluster of differentiation (CD)117(+)/FcεRIα (Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide)(+) cells in atopic skin. Both IL-31 mRNA expression and production were significantly reduced by GST, which was accomrease in the levels of IL-4, IL-5, and IL-13. Further, GST treatment suppressed the secretion of eotaxin, TARC (thymus and activation-regulated chemokine), IgE, and increases in the number of basophils and eosinophils in the blood. CONCLUSION: GST may have potential as an effective treatment for pruritic skin disease such as atopic dermatitis.

Oral administration of SSC201, a medicinal herbal formula, suppresses atopic dermatitis-like skin lesions.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, which requires safe and effective treatment. In this study, we evaluated the effects of SSC201, a herbal formulation consisting of Stemonae Radix, Spirodelae Herba, and Cnidii Fructus, on the development of AD induced by 2,4-dinitrochlorobenzene in the NC/Nga murine model. Oral administration of SSC201 significantly reduced the severity of dermatitis and the tendency of mice to scratch their lesions. SSC201 significantly reduced the thickening of the epidermis/dermis and the infiltration of T cells, eosinophils, and mast cells into the dermis. These results were supported by findings of reduced numbers of CD4(+), CCR3(+), and CD117(+)FcɛRIα(+) cells in the skin. Furthermore, SSC201 significantly decreased the number of CD4(+), CD8(+), and CD3(+)CD69(+) T cells in lymph nodes. SSC201 not only decreased the plasma levels of immunoglobulin E (IgE) and the numbers of IgE-producing B cells (B220(+)CD23(+)), but also reduced the number of eosinophils and the levels of eotaxin as well as concentrations of thymus and activation-regulated chemokine in the periphery. Splenic levels of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, were reduced, whereas the levels of IL-12, a Th1 cytokine, were increased. Taken together, our data suggest that SSC201 may be an effective therapeutic agent for the treatment of AD.

Eicosapentaenoic acid and docosahexaenoic acid suppress Th2 cytokine expression in RBL-2H3 basophilic leukemia cells.

It is known that the intake of omega-3 fatty acids, such as eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), is beneficial for preventing and/or treating allergic diseases. The pathogenesis of allergic diseases is associated with overactivation of Th2-skewed immunity. Basophils generate large amounts of Th2 cytokines such as interleukin (IL)-4 and IL-13, which are critically involved in allergic inflammation. We investigated how EPA and DHA affect Th2 cytokine expression in phorbol 12-myristate 13-acetate- and ionomycin (PI)-activated RBL-2H3 basophilic leukemia cells. EPA and DHA induced a dramatic decrease in the production of IL-4 and IL-13 and their transcription in a dose-dependent manner. Luciferase assays of RBL-2H3 cells stably expressing Il4 and Il13 promoter-reporter plasmids demonstrated a significant suppression of PI-induced promoter activation. Analysis of certain transcription factors revealed that nuclear expression of c-Fos and the mRNA expression were suppressed by EPA and DHA. Furthermore, they significantly inhibited the nuclear expression and translocation of nuclear factor of activated T cells (NF-AT)1. In contrast, the expression levels of nuclear factor kappa-B (NF-κB), GATA-binding proteins (GATAs), and CCAAT/enhancer binding protein alpha (C/EBPα) were not significantly affected by EPA and DHA. Phosphorylation of extracellular signal-related kinase was inhibited by EPA and DHA, and phosphorylation of p38 mitogen-activated protein kinase was decreased by DHA, but not by EPA. Taken together, our data suggest that EPA and DHA may suppress Th2-skewed allergic immune responses by inhibiting the expression of basophilic IL-4 and IL-13.

Pheophytin a and chlorophyll a identified from environmentally friendly cultivation of green pepper enhance interleukin-2 and interferon-γ in Peyer's patches ex vivo.

The oral consumption of capsicum has been reported to increase interleukin (IL)-2 and interferon (IFN)-γ production in Peyer's patches (PP); however, the active components responsible for these effects have not been completely identified. The beneficial biological effects of green peppers cultivated under environmentally friendly farming conditions (ECP), without the use of chemical pesticides, have rarely been compared with those of green peppers cultivated under conventional farming conditions (CCP). Oral administration of ECP extract significantly induced the production of IL-2 and IFN-γ in concanavalin A-treated cells from PP ex vivo; their levels were much higher than those in the CCP extract-treated group. A comparative analysis of the HPLC profiles indicated a 1.7-fold increase of a peak, named EF-1, at 415 nm in the ECP extract. The major component of EF-1 was identified as pheophytin a, which is a chlorophyll a molecule lacking a central Mg(2+) ion, as determined from NMR data. Intake of pheophytin a and chlorophyll a significantly increased IL-2 and IFN-γ production, and the percentage of IL-2- and IFN-γ-producing CD4+ T-cells in PP. Taken together, our data suggest that ECPs produce a higher content of pheophytin a than CCPs, and pheophytin a and chlorophyll a are immune-modulating components in green vegetables.

Omega-3 fatty acids suppress Th2-associated cytokine gene expressions and GATA transcription factors in mast cells.

Because the interaction between omega-3 fatty acids and mast cells has remained largely unknown in allergies, we investigated whether omega-3 fatty acids affect the activation of mast cells by examining Th2-associated cytokine production and possible molecular mechanisms. Alpha-linolenic acid and its metabolites including eicosapentaenoic acid and decosahexaenoic acid induced a dramatic decrease in the production of interleukin (IL)-4, IL-5 and IL-13 in a dose-dependent manner, as well as mRNA expression of their genes, in activated MC/9 mast cells and bone marrow-derived mast cells. The effects were comparable to those of cyclosporin A (1 µM), a well-known immunosuppressive agent. Nuclear expression of GATA binding protein-1 (GATA-1) and GATA binding protein-2 (GATA-2), essential transcription factors for mast cell activation, was also greatly suppressed. However, their mRNA expressions were not affected. In P815 mast cells, which do not express GATA-1, the suppressive effects on cytokines were abolished. On the contrary, omega-3 fatty acids had less significant effects on IL-4 and IL-5 and resulted in a slight decrease in IL-13 production in EL-4 T cells. Finally, oral administration of fish oil containing high level of omega-3 fatty acids significantly reduced the severity of dermatitis and the thickening of epidermis/dermis in a NC/Nga murine atopic model. The number of cells expressing CD117(+) and FcεRIα(+) was greatly decreased and GATA-1 expression in the cells was also diminished. Taken together, omega-3 fatty acids might target mast cells to a greater extent than T cells to suppress Th2 cytokine expression by inhibiting GATAs for alleviation of allergic disease.

Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13.

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a wellknown antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

Establishment of an in vitro test system to evaluate the down-regulatory activities of natural products on IL-4.

Interleukin-4 (IL-4), a representative TH2 cytokine, plays a pathologic role in the onset of various allergic diseases including atopic dermatitis, atopic rhinitis, and asthma. Several drug candidates that down-regulate IL-4 expression have been studied for their possible use as antiallergic agents in clinical settings. Therefore, an in vitro test to evaluate IL-4 promoter activities might be useful for selecting candidates of novel natural therapeutics. The promoter region (-741 to +56) of IL-4 was cloned upstream of a luciferase gene in the plasmid pGL4.14 with a hygromycin resistance gene as a selection marker to generate pGL4.14-IL-4. Treatment with PMA and A23187 highly increased luciferase activity by approximately 10-fold compared with the control in both EL-4 thymoma and RBL-2H3 cells transiently transfected with pGL4.14-IL-4, as well as in stable cell lines constantly expressing pGL4.14-IL-4. Cyclosporin A and dexamethasone, well-known anti-allergic agents, significantly down-regulated the activity in a dose-dependent manner. The feasibility of this system was evaluated by measuring the down-regulatory activities of various extracts from the TBRC plant library on PMA- and A23187-induced luciferase activities of IL-4 promoter, and by measuring IL-4 production in cultured cells using ELISA assays. The results of this study suggest that this primary screening system is simple and time-saving, and might be suitable for the selection of natural therapeutic candidates for allergic disease by measuring the down-regulatory effects of natural products on the IL-4 promoter.