Icariin enhances youth-like features by attenuating the declined gut microbiota in the aged mice.

We previously reported the neuroprotective effects of icariin in rat cortical neurons. Here, we present a study on icariin's anti-aging effect in 24-month aged mice by treating them with a single daily dose of 100 mg/kg of icariin for 15 consecutive days. Icariin treatment improved motor coordination and learning skills while lowered oxidative stress biomarkers in the serum, brain, kidney, and liver of the aged mice. In addition, icariin improved the intestinal integrity of the aged mice by upregulating tight junction adhesion molecules and the Paneth and goblet cells, along with the reduction of iNOS and pro-inflammatory cytokines (IL-1ß, TNF-α, IL-2 and IL-6, and IL-12). Icariin treatments also significantly upregulated aging-related signaling molecules, Sirt 1, 3 & 6, Pot1α, BUB1b, FOXO1, Ep300, ANXA3, Calb1, SNAP25, and BDNF in old mice. Through gut microbiota (GM) analysis, we observed icariin-associated improvements in GM composition of aged mice by reinstating bacteria found in the young mice, while suppressing some bacteria found in the untreated old mice. To clarify whether icariin's anti-aging effect is rooted in the GM, we performed fecal microbiota transfer (FMT) from icariin-treated old mice to the old mice. FMT-recipients exhibited similar improvements in the rotarod score and age-related biomarkers as observed in the icariin-treated old mice. Equal or better improvement on the youth-like features was noticed when aged mice were FMT with feces from young mice. Our study shows that both direct treatments with icariin and fecal transplant from the icariin-treated aged mice produce similar anti-aging phenotypes in the aged mice. We prove that GM plays a pivotal role in the healing abilities of icariin. Icariin has the potentials to be developed as a medicine for the wellness of the aged adults.

Saponins isolated from Radix polygalae extent lifespan by modulating complement C3 and gut microbiota.

With the increase in human lifespan, population aging is one of the major problems worldwide. Aging is an irreversible progressive process that affects humans via multiple factors including genetic, immunity, cellular oxidation and inflammation. Progressive neuroinflammation contributes to aging, cognitive malfunction, and neurodegenerative diseases. However, precise mechanisms or drugs targeting age-related neuroinflammation and cognitive impairment remain un-elucidated. Traditional herbal plants have been prescribed in many Asian countries for anti-aging and the modulation of aging-related symptoms. In general, herbal plants' efficacy is attributed to their safety and polypharmacological potency via the systemic manipulation of the body system. Radix polygalae (RP) is a herbal plant prescribed for anti-aging and the relief of age-related symptoms; however, its active components and biological functions remained un-elucidated. In this study, an active methanol fraction of RP containing 17 RP saponins (RPS), was identified. RPS attenuates the elevated C3 complement protein in aged mice to a level comparable to the young control mice. The active RPS also restates the aging gut microbiota by enhancing beneficial bacteria and suppressing harmful bacteria. In addition, RPS treatment improve spatial reference memory in aged mice, with the attenuation of multiple molecular markers related to neuroinflammation and aging. Finally, the RPS improves the behavior and extends the lifespan of C. elegans, confirming the herbal plant's anti-aging ability. In conclusion, through the mouse and C. elegas models, we have identified the beneficial RPS that can modulate the aging process, gut microbiota diversity and rectify several aging-related phenotypes.

Mushroom polysaccharides and jiaogulan saponins exert cancer preventive effects by shaping the gut microbiota and microenvironment in ApcMin/+ mice.

The incidence of colorectal cancer (CRC) is alarming among younger peoples. While no effective chemopreventive drug available in the market, researchers have been searching for alternative strategies against CRC that are in demand. Therefore, we tested the cancer-preventive properties of Ganoderma lucidum (Lingzhi) polysaccharides (GLP), along with the saponins extracted from Gynostemma pentaphyllum (GpS), an herbal tea with prebiotic-like effects. Here, we report that saponins from Gynostemma pentaphyllum (GpS) and polysaccharides from Ganoderma lucidum (GLP together with GpS) profoundly improved the inflamed gut barrier of ApcMin/+ mice by reducing polyps, shifting colonic M1 to M2 macrophages, positively reverting E-cadherin/N-cadherin ratio, and downregulating oncogenic signaling molecules. The treatments also markedly promoted short-chain fatty acids (SCFAs)-producing bacteria and abridged sulfate-reducing bacteria in a time-dependent manner. G-protein coupled-receptors were significantly stimulated in the treated mice, accompanied by the modulated expressions of histone deacetylases, anti-cancer gut hormone PYY, and PPAPγ. These findings suggest that some of the herbal medicinal foods could modulate the relationship between the host and the gut microbiota (GM) to exert their beneficial properties to the host. Our study also implicates that these dietary mushroom polysaccharides and the Gp saponins have the potential to be developed as new preventive medicines against CRC.

Polarity-Tuning Derivatization-LC-MS Approach for Probing Global Carboxyl-Containing Metabolites in Colorectal Cancer.

Carboxyl-containing metabolites (CCMs) widely exist in living systems and are the essential components for life. Global characteristics of CCMs in biological samples are critical for the understanding of physiological processes and the discovery for the onset of relevant diseases. However, their determination represents a challenge due to enormous polarity differences, structural diversity, high structural similarity, and poor ionization efficiency in mass spectrometry. Herein, 5-(diisopropylamino)amylamine (DIAAA) derivatization coupled with liquid chromatography-mass spectrometry (LC-MS) was developed for mapping the CCMs. With this methodology, the sensitivity was significantly enhanced. More importantly, the hydrophobicity of polar CCMs, amino acids, TCA cycle intermediates, and short-chain fatty acids and the hydrophilicity of low-polar CCMs, long-chain fatty acids, and bile acids were significantly increased, resulting in a remarkable separation efficiency for which 68 CCMs can be simultaneously determined. Furthermore, the polarity-tuning effect was confirmed to be induced by the different impacts of aliphatic chains and nitrogen atom in DIAAA, the latter existing as a cation in the acidic mobile phase, using different derivatization reagents. Finally, this derivatization method was utilized to hunt for the potential biomarkers in colorectal cancer (CRC) patients and 52 CCMs, related with several key metabolic pathways, including amino acids metabolism, TCA cycle, fatty acid metabolism, pyruvate metabolism, and gut flora metabolism were identified. This innovative polarity-tuning derivatization-LC-MS approach was proved to be a valuable tool for probing global metabolome with high separation efficiency and sensitivity in various biological samples.

The Protective Effects of Icariin against the Homocysteine-Induced Neurotoxicity in the Primary Embryonic Cultures of Rat Cortical Neurons.

Icariin, an ingredient in the medicinal herb Epimedium brevicornum Maxim (EbM), has been considered as a potential therapeutic agent for neurodegenerative diseases such as Alzheimer's disease (AD). Hyperhomocysteinaemia is a risk factor for AD and other associated neurological diseases. In this study we aim to investigate whether icariin can reverse homocysteine (Hcy)-induced neurotoxicity in primary embryonic cultures of rat cortical neurons. Our findings demonstrated that icariin might be able restore the cytoskeleton network damaged by Hcy through the modulation of acetyl-α-tubulin, tyrosinated-α-tubulin, and phosphorylation of the tubulin-binding protein Tau. In addition, icariin downregulated p-extracellular signal-regulated kinase (ERK) which is a kinase targeting tau protein. Furthermore, icariin effectively restored the neuroprotective protein p-Akt that was downregulated by Hcy. We also applied RT² Profiler PCR Arrays focused on genes related to AD and neurotoxicity to examine genes differentially altered by Hcy or icariin. Among the altered genes from the arrays, ADAM9 was downregulated 15 folds in cells treated with Hcy, but markedly restored by icariin. ADAM family, encoded α-secreatase, plays a protective role in AD. Overall, our findings demonstrated that icariin exhibits a strong neuroprotective function and have potential for future development for drug treating neurological disorders, such as AD.

Kaempferol acts on bile acid signaling and gut microbiota to attenuate the tumor burden in ApcMin/+ mice.

Emerging evidence points to a strong association between the bile acid (BA)-gut microbiota (GM) axis, and the risk of colorectal cancer (CRC). Kaempferol, a common polyphenol in the daily diet, shows various pharmacological activities. However, it remains unclear about the effect of kaempferol on the CRC development and the BA-GM homeostasis. Here, we found kaempferol effectively reduced tumor burden, restored the damaged intestinal barrier and downregulated antigen Ki67 and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) expressions in ApcMin/+ mice. For BA metabolism, kaempferol reversed the decreasing trend in chenodesoxycholic acid (CDCA) and 12α-hydroxylated BAs by increasing the sterol 27-hydroxylase (CYP27A1) and sterol 12α-hydroxylase (CYP8B1) expressions, and upregulated FXR expression. Importantly, molecular docking analysis revealed a direct interaction between kaempferol and farnesoid X receptor (FXR), the mater regulator of BA signaling. For GM analysis, we found higher abundances of species with anticancer properties and lower abundances of species associated with inflammation, obesity, and metabolic disorders in kaempferol-treated groups. Moreover, the gut of kaempferol-treated mice was predominantly colonized by short-chain fatty acid (SCFA) and lactic acid producing bacteria. Based on the PICRUSt-predicted pathways of our GM dataset, we demonstrated that kaempferol downregulated secondary BA synthesis pathways, increased G protein-coupled receptor activity and decreased NOD-like receptor activity, affecting cell differentiation, proliferation, survival, and apoptosis. Collectively, these results reveal that kaempferol effectively attenuates the tumor burden in ApcMin/+ mice by modulating the BA signaling and GM homeostasis.

The prebiotic and anti-fatigue effects of hyaluronan.

Hyaluronan (HA) is a mucopolysaccharide that naturally exists in all living organisms as the main component of the extracellular matrix. Over the last 30 years, HA has been used as the main ingredient in cosmetic products, eye drops, and medicinal products. It is also taken orally as a health supplement. However, the physiological effect of the ingested HA is not clear. In the current study, the interaction between HA and gut microbiota, and the potential prebiotic effects were investigated. HA was used to treat the C57BL/6 mice for 15 consecutive days, then fecal genomic DNA was extracted from fecal samples for 16S rRNA amplicon sequencing. The results showed that HA could significantly change the composition of gut microbiota (GM), e.g., increased the relative abundance of beneficial bacteria, including short-chain fatty acids (SCFAs)-producing bacteria and xylan/cellulose-degrading bacteria, whereas decreased the relative abundance of potential pathogens including sulfate-reducing bacteria (SRB), inflammation and cancer-related bacteria. The rotarod test was used to evaluate the anti-fatigue effects of HA in C57BL/6 mice. The results showed that HA could lengthen the mice's retention time on the accelerating rotarod. HA increased the concentration of glycogen and superoxide dismutase (SOD) in mice's muscle and liver, whereas decreased the serum concentration of malondialdehyde (MDA). Moreover, the metabolic products of Desulfovibrio vulgaris (MPDV), the model SRB bacteria, showed cytotoxic effects on H9c2 cardiomyocytes in a dosage-dependent manner. MPDV also caused mitochondrial damage by inducing mitochondrial fragmentation, depolarization, and powerless ATP production. Taken together, we show that HA possesses significant prebiotic and anti-fatigue effects in C57BL/6 mice.

Far infrared irradiation suppresses experimental arthritis in rats by down-regulation of genes involved inflammatory response and autoimmunity.

Introduction: Far-infrared radiation (FIR) is widely used in the treatment of various diseases such as insomnia and cardiovascular risk. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease in which the therapeutic potential of FIR in RA is unclear. Objectives: To determine the therapeutic potential and mechanistic actions of FIR in treatment of RA. Methods: Adjuvant-induced arthritis (AIA) rat models were established to assess the therapeutic potency of FIR in RA treatment. The scoring parameters such as arthritis score, swelling of the hind paw, spleen and thymus indices, micro-CT analysis indices were adopted to estimate the beneficial effects of FIR during RA treatment in AIA model. PCR gene expression arrays were used to analyze inflammatory and autoimmune genes expression profiles in rat synovium. The inflammatory and immunity genes profiling was further analyzed through transcription factor prediction using PROMO. A signaling network map of possible molecular circuits connecting the identified differential genes to the RA's pathogenesis was constructed based on extensive literature reviews, and the major signaling pathways were validated by Western blotting. Results: Thirty minutes of FIR treatment significantly improved the symptoms of AIA in rats. Gene expression profiling indicated that 27 out of 370 genes were down-regulated by FIR. AP-1, CEBPα, CEBPß, c-Fos, GR, HNF-3ß, USF-1, and USF-2 were predicted as key transcription factors that regulated the identified differential genes. In addition, MAPK, PI3K-Akt, and NF-κB signaling are the major molecular pathways down-regulated by FIR treatment. Conclusion: FIR may provide beneficial effects on the AIA rat model of arthritis by suppression of the MAPK, PI3K-Akt and NF-κB signaling pathways. Therefore, we believe that FIR may provide an alternative non-pharmacological and non-surgical therapeutic approach for the treatment of RA.

Poria cocos polysaccharides exert prebiotic function to attenuate the adverse effects and improve the therapeutic outcome of 5-FU in ApcMin/+ mice.

BACKGROUND: As a first-line chemotherapeutic agent, 5-fluorouracil (5-FU) exhibits many side effects, weakening its efficacy in cancer treatment. In this study, we hypothesize that Poria cocos polysaccharides (PCP), a traditional Chinese herbal medicine with various bioactivities and prebiotic effects, might improve the therapeutic effect of 5-FU by restoring the homeostasis of the gut microenvironment and the commensal gut microflora. METHODS: ApcMin/+ mice were employed to evaluate the anti-cancer effect of 5-FU in conjunction with PCP treatment. Body weight and food consumption were monitored weekly. Polyp count was used to assess the anti-cancer effect of PCP and 5-FU. Expressions of mucosal cytokines and gut epithelial junction molecules were measured using qRT-PCR. 16S rRNA gene sequencing of fecal DNAs was used to evaluate the compositional changes of gut microbiota (GM). Transplantation of Lactobacillus johnsonii and Bifidobacterium animalis were performed to verify the prebiotic effects of PCP in improving the efficacy of 5-FU. RESULTS: The results showed that PCP treatment alleviated the weight loss caused by 5-FU treatment and reduced the polyp burden in ApcMin/+ mice. Additionally, PCP treatment eased the cytotoxic effects of 5-FU by reducing the expressions of pro-inflammatory cytokines, increasing the anti-inflammatory cytokines; and significantly improving the gut barriers by enhancing the tight junction proteins and associated adhesion molecules. Furthermore, 16S rRNA gene sequencing data showed that PCP alone or with 5-FU could stimulate the growth of probiotic bacteria (Bacteroides acidifaciens, Bacteroides intestinihominis, Butyricicoccus pullicaecorum, and the genera Lactobacillus, Bifidobacterium, Eubacterium). At the same time, it inhibited the growth of potential pathogens (e.g., Alistipes finegoldii, Alistipes massiliensis, Alistipes putredinis., Citrobacter spp., Desulfovibrio spp., and Desulfovibrio desulfuricans). Moreover, the results showed that transplantation of L.johnsonii and B.animalis effectively reduced the polyp burden in ApcMin/+ mice being treated with 5-FU. CONCLUSION: Our study showed that PCP could effectively improve the anti-cancer effect of 5-FU by attenuating its side effects, modulating intestinal inflammation, improving the gut epithelial barrier, and modulating the gut microbiota of ApcMin/+ mice.

Chemical and DNA authentication of taste variants of Gynostemma pentaphyllum herbal tea.

Gynostemma pentaphyllum Makino (Gp) was once used as a sweetener in Japan and is now widely consumed as an herbal tea worldwide for lowering cholesterol levels. Two taste variants, bitter and sweet, of Gp exist in the commercial market, but they cannot be differentiated morphologically nor by existing chemical analytical methods. This has been creating a problem in quality control of Gp products. In the present study, using HPLC-DAD and HPLC-ESI-MS analysis, we found that the Gp saponins, not flavonoids, from the sweet and bitter variants have distinctly different profiles. In addition, the two variants share only 69.01% homology in the ribosomal ITS-1 region, suggesting a phylogenic gap between these two variants. The combinations of chemical profiling and phylogenic analysis clearly confirm, for the first time, the distinction between these two taste variants. This information has direct application in the authentication and quality assessment of the various Gynostemma tea products.

Vorinostat targets UBE2C to reverse epithelial-mesenchymal transition and control cervical cancer growth through the ubiquitination pathway.

Vorinostat is a histone deacetylase inhibitor (HDACi) that was demonstrated in our previous study to inhibit the proliferation, migration, and invasion of cervical cancer cells by regulating the PI3K/Akt signaling pathway. However, the molecular mechanism of vorinostat in cervical cancer treatment remains to be further elucidated. A nude mouse xenograft model was established to analyze the antitumor effect of vorinostat in vivo. The combination of iTRAQ-based proteomics and parallel reaction monitoring (PRM) technology has proven to be an efficient and reliable method to identify potential targets for cancer chemotherapy. In this study, 254 differentially expressed proteins in vorinostat-treated cervical cancer cells, among which 180 were upregulated and 74 were downregulated, were identified by using an iTRAQ-based proteomic strategy. Subsequent bioinformatic and PRM analysis of these differentially expressed proteins indicated that UBE2C is a promising target of vorinostat in the inhibition of cervical cancer cell proliferation. We confirmed that the expression of endogenous UBE2C in cervical cancer cell lines was significantly higher than that in normal cervical epithelial cell lines. Additionally, we found that vorinostat downregulated the expression of UBE2C, SQSTM1/p62, N-cadherin, vimentin and upregulated E-cadherin in SiHa and HeLa cells. Our results also showed that vorinostat can downregulate the expression of SQSTM1/p62, N-cadherin, and vimentin during the treatment of cervical cancer cells by regulating UBE2C, while upregulating the expression of E-cadherin. In conclusion, vorinostat reverses epithelial-mesenchymal transition by targeting UBE2C and controls the proliferation of cervical cancer cells through the ubiquitination pathway. UBE2C can be used as a promising target for the development of vorinostat treatment strategies.

The role of traditional Chinese herbal medicines in cancer therapy--from TCM theory to mechanistic insights.

Traditional Chinese medicine-based herbal medicines have gained increasing acceptance worldwide in recent years and are being pursued by pharmaceutical companies as rich resources for drug discovery. For many years, traditional Chinese medicines (TCM) have been applied for the treatment of cancers in China and beyond. Herbal medicines are generally low in cost, plentiful, and show very little toxicity or side effects in clinical practice. However, despite the vast interest and ever-increasing demand, the absence of strong evidence-based research and the lack of standardization of the herbal products are the main obstacles toward the globalization of TCM. In recent years, TCM research has greatly accelerated with the advancement of analytical technologies and methodologies. This review of TCM specifically used in the treatment of cancer is divided into two parts. Part one provides an overview of the philosophy, approaches and progress in TCM-based cancer therapy. Part two summarizes the current understanding of how TCM-derived compounds function as anticancer drugs.

Far infrared radiation induces changes in gut microbiota and activates GPCRs in mice.

Far infrared radiation (FIR) has been widely used to treat chronic diseases and symptoms; however, the underlying mechanism remains unclear. As gut microbiota (GM) markedly impact the host's physiology, making GM a potential target for the therapeutic evaluation of FIR. C57BL/6J mice were exposed to five times of 2 min-FIR exposure on the abdomen, with a two-hour interval of each exposure within one day. Fecal samples were collected on day one and day 25 after the FIR/control treatment, and the extracted fecal DNAs were evaluated using ERIC-PCR and 16S amplicon sequencing. Host's G-protein coupled receptors (GPCR) were analyzed using qRT-PCR. FIR induced immediate changes in the GM composition. A prompt and significant (p < 0.05) reduction in the abundance of phylum Deferribacteres (comprised of several pathogens) was observed in the FIR-irradiated mice compared to the control group. Contrarily, FIR exposure induced beneficial genera such as Alistipes, Barnesiella, and Prevotella. The gut of FIR-irradiated mice was predominated by short-chain fatty acids (SCFAs) producers. Also, FIR stimulated the expression of SCFAs-sensing receptors, GPCR 41, 43, and 109 in the gut epithelial barrier. These findings provide the first-hand evidence in which the beneficial effects of FIR radiation might be partially through the modulation of GM.

Fructo-oligosaccharides from Morinda officinalis remodeled gut microbiota and alleviated depression features in a stress rat model.

BACKGROUND: Inulin-type fructo-oligosaccharides (FOSs) purified from Morinda officinalis How., an effective oral antidepressant for mild to moderate depression, have a largely unknown efficacy and poor bioavailability. PURPOSE: Therefore, the microbiota-gut-brain axis was used to investigate the antidepressive properties of FOSs at the interface of the gut microbiota (GM). STUDY DESIGN AND METHODS: FOSs was introduced via intragastric gavage to rats exposed to chronic unpredictable mild stress (CUMS), and the antidepressive effects were investigated through behavioral tests, intestinal morphology and corticosterone levels. Bacterial genomic DNA was extracted from feces, and the GM was profiled for using enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis, partial least squares-discriminant analysis (PLS-DA) and 16S rRNA gene pyrosequencing. RESULTS: It was observed that FOSs alleviated depression-like behaviors and repaired intestinal epithelia damages. FOSs treatment lowered corticosterone levels in the plasma and urine of the model rats. Moreover, the GM compositions of normal and model rats were distantly clustered and were mainly related to the disappearance of beneficial bacteria (e.g., Acinetobacter, Barnesiella, Coprococcus, Dialister, Lactobacillus, and Paenibacillus) and appearance of depression-associated bacteria (e.g., Anaerostipes, Oscillibacter, Proteobacteria, and Streptococcus) in depressive rats. Interestingly, the dysbiosis in depressive rats' gut was reinstated with FOSs treatments. Notably, FOSs promoted the abundance of the bacterial phylum Cyanobacteria, a group of bacteria known for the secretion of pharmacologically important metabolites, such as H2S, that exhibit antidepressant-like properties. Apparently, FOSs-induced modulation of GM was more antidepressive compared to a component of FOSs, degrees of polymerization (DP) 5, and fluoxetine, the standard antidepressant drug. CONCLUSION: In conclusion, this study implied that antidepressant efficacy of FOSs was inseparable from and strongly associated with the modulation of the host' s GM.

Corrigendum to "Lycium Berry Polysaccharides Strengthen Gut Microenvironment and Modulate Gut Microbiota of the Mice".

[This corrects the article DOI: 10.1155/2020/8097021.].

Patchouli Essential Oil and Its Derived Compounds Revealed Prebiotic-Like Effects in C57BL/6J Mice.

Pogostemon cablin (Blanco) Benth (PC) is a Chinese medicinal plant traditionally used for the treatment of gastrointestinal symptoms. To investigate the prebiotic effect of patchouli essential oil (PEO) and its derived compounds through the modulation of gut microbiota (GM). C57BL/6J mice were treated with the PEO and three active components of PEO, i.e. patchouli alcohol (PA), pogostone (PO) and ß-patchoulene (ß-PAE) for 15 consecutive days. Fecal samples and mucosa were collected for GM biomarkers studies. PEO, PA, PO, and ß-PAE improve the gut epithelial barrier by altering the status of E-cadherin vs. N-cadherin expressions, and increasing the mucosal p-lysozyme and Muc 2. Moreover, the treatments also facilitate the polarization of M1 to M2 macrophage phenotypes, meanwhile, suppress the pro-inflammatory cytokines. Fecal microbial DNAs were analyzed and evaluated for GM composition by ERIC-PCR and 16S rRNA amplicon sequencing. The GM diversity was increased with the treated groups compared to the control. Further analysis showed that some known short chain fatty acids (SCFAs)-producing bacteria, e.g. Anaerostipes butyraticus, Butytivibrio fibrisolvens, Clostridium jejuense, Eubacterium uniforme, and Lactobacillus lactis were significantly enriched in the treated groups. In addition, the key SCFAs receptors, GPR 41, 43 and 109a, were significantly stimulated in the gut epithelial layer of the treated mice. By contract, the relative abundance of pathogens Sutterlla spp., Fusobacterium mortiferum, and Helicobacter spp. were distinctly reduced by the treatments with PEO and ß-PAE. Our findings provide insightful information that the microbiota/host dynamic interaction may play a key role for the pharmacological activities of PEO, PA, PO, and ß-PAE.

Gynostemma pentaphyllum saponins induce melanogenesis and activate cAMP/PKA and Wnt/ß-catenin signaling pathways.

BACKGROUND: Melanogenesis is a physiological process of melanin production in response to UV exposure, which is modulated through multi-signaling pathways including cAMP/PKA, Wnt/ß-catenin and MAPK signaling cascades. HYPOTHESIS/PURPOSE: The present study aims to investigate the molecular mechanism of hyperpigmentation induced by Gynostemma pentaphyllum saponins. STUDY DESIGN/METHODS: In this study, we investigated the melanogenic effects of triterpenoid saponins of Gynostemma pentaphyllum (GpS), a medicinal plant. Two mouse melanogenic cell lines B16 and B16F10 were employed for the current study. RESULTS: The results showed that non-toxic doses of GpS markedly increased melanin formation in both B16 and B16F10 cells. Western blot analysis showed that GpS treatment significantly up-regulated the expression levels of the key melanogenic proteins, including tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), TRP-1 and TRP-2 in a dose-dependent manner. The phospho-CREB, which is the downstream target of PKA is also elevated upon GpS treatment. We further observed that H89, a PKA inhibitor, attenuated the GpS induced tyrosinase activity, melanin content, the expression of phospho-CREB. In addition to the cAMP/PKA signaling pathway, GpS treatment also up-regulated the ß-catenin of the Wnt signaling pathway which is involved in the transcriptional activation of MITF in melanogensis. We further demonstrated that treatment with GpS markedly enhance mRNA expression of MITF, along with the downstream target molecules, TYR, TRP-1 and TRP-2. Knock-down MITF with siMITF inhibited the expression of MITF mRNA by 63%, and the melanin content was reduced 70% in the siMITF-transfected cells compared to untransfected or scramble siRNA control cells. CONCLUSION: These findings demonstrated strong melanogenic activities of GpS, and the MITF is essential for the melanogenesis stimulated by GpS.

MAK-4 and -5 supplemented diet inhibits liver carcinogenesis in mice.

BACKGROUND: Maharishi Amrit Kalash (MAK) is an herbal formulation composed of two herbal mixtures, MAK-4 and MAK-5. These preparations are part of a natural health care system from India, known as Maharishi Ayur-Veda. MAK-4 and MAK-5 are each composed of different herbs and are said to have maximum benefit when used in combination. This investigation evaluated the cancer inhibiting effects of MAK-4 and MAK-5, in vitro and in vivo. METHODS: In vitro assays: Aqueous extracts of MAK-4 and MAK-5 were tested for effects on ras induced cell transformation in the Rat 6 cell line assessed by focus formation assay. In vivo assays: Urethane-treated mice were put on a standard pellet diet or a diet supplemented with MAK-4, MAK-5 or both. At 36 weeks, livers were examined for tumors, sera for oxygen radical absorbance capacity (ORAC), and liver homogenates for enzyme activities of glutathione peroxidase (GPX), glutathione-S-transferase (GST), and NAD(P)H: quinone reductase (QR). Liver fragments of MAK-fed mice were analyzed for connexin (cx) protein expression. RESULTS: MAK-5 and a combination of MAK-5 plus MAK-4, inhibited ras-induced cell transformation. In MAK-4, MAK-5 and MAK4+5-treated mice we observed a 35%, 27% and 46% reduction in the development of urethane-induced liver nodules respectively. MAK-4 and MAK4+5-treated mice had a significantly higher ORAC value (P < 0.05) compared to controls (200.2 +/- 33.7 and 191.6 +/- 32.2 vs. 152.2 +/- 15.7 ORAC units, respectively). The urethane-treated MAK-4, MAK-5 and MAK4+5-fed mice had significantly higher activities of liver cytosolic enzymes compared to the urethane-treated controls and to untreated mice: GPX(0.23 +/- 0.08, 0.21 +/- 0.05, 0.25 +/- 0.04, 0.20 +/- 0.05, 0.21 +/- 0.03 U/mg protein, respectively), GST (2.0 +/- 0.4, 2.0 +/- 0.6, 2.1 +/- 0.3, 1.7 +/- 0.2, 1.7 +/- 0.2 U/mg protein, respectively) and QR (0.13 +/- 0.02, 0.12 +/- 0.06, 0.15 +/- 0.03, 0.1 +/- 0.04, 0.11 +/- 0.03 U/mg protein, respectively). Livers of MAK-treated mice showed a time-dependent increased expression of cx32. CONCLUSION: Our results show that a MAK-supplemented diet inhibits liver carcinogenesis in urethane-treated mice. The prevention of excessive oxidative damage and the up-regulation of connexin expression are two of the possible effects of these products.

Ginsenosides Rb3 and Rd reduce polyps formation while reinstate the dysbiotic gut microbiota and the intestinal microenvironment in ApcMin/+ mice.

Studies showed that manipulation of gut microbiota (GM) composition through the treatment of prebiotics could be a novel preventive measure against colorectal cancer (CRC) development. In this study, for the first time, we assessed the non-toxic doses of the triterpene saponins (ginsenoside-Rb3 and ginsenoside-Rd) - as prebiotics - that effectively reinstated the dysbiotic-gut microbial composition and intestinal microenvironment in an ApcMin/+ mice model. Rb3 and Rd effectively reduced the size and the number of the polyps that accompanied with the downregulation of oncogenic signaling molecules (iNOS, STAT3/pSTAT3, Src/pSrc). Both the compounds improved the gut epithelium by promoting goblet and Paneth cells population and reinstating the E-cadherin and N-Cadherin expression. Mucosal immunity remodeled with increased in anti-inflammatory cytokines and reduced in pro-inflammatory cytokines in treated mice. All these changes were correlating with the promoted growth of beneficial bacteria such as Bifidobacterium spp., Lactobacillus spp., Bacteroides acidifaciens, and Bacteroides xylanisolvens. Whereas, the abundance of cancer cachexia associated bacteria, such as Dysgonomonas spp. and Helicobacter spp., was profoundly lower in Rb3/Rd-treated mice. In conclusion, ginsenosides Rb3 and Rd exerted anti-cancer effects by holistically reinstating mucosal architecture, improving mucosal immunity, promoting beneficial bacteria, and down-regulating cancer-cachexia associated bacteria.

Triterpenoid herbal saponins enhance beneficial bacteria, decrease sulfate-reducing bacteria, modulate inflammatory intestinal microenvironment and exert cancer preventive effects in ApcMin/+ mice.

Saponins derived from medicinal plants have raised considerable interest for their preventive roles in various diseases. Here, we investigated the impacts of triterpenoid saponins isolated from Gynostemma pentaphyllum (GpS) on gut microbiome, mucosal environment, and the preventive effect on tumor growth. Six-week old ApcMin/+ mice and their wild-type littermates were fed either with vehicle or GpS daily for the duration of 8 weeks. The fecal microbiome was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and 16S rRNA gene pyrosequencing. Study showed that GpS treatment significantly reduced the number of intestinal polyps in a preventive mode. More importantly, GpS feeding strikingly reduced the sulfate-reducing bacteria lineage, which are known to produce hydrogen sulfide and contribute to damage the intestinal epithelium or even promote cancer progression. Meanwhile, GpS also boosted the beneficial microbes. In the gut barrier of the ApcMin/+ mice, GpS treatment increased Paneth and goblet cells, up-regulated E-cadherin and down-regulated N-cadherin. In addition, GpS decreased the pro-oncogenic ß-catenin, p-Src and the p-STAT3. Furthermore, GpS might also improve the inflamed gut epithelium of the ApcMin/+ mice by upregulating the anti-inflammatory cytokine IL-4, while downregulating pro-inflammatory cytokines TNF-α, IL-1ß and IL-18. Intriguingly, GpS markedly stimulated M2 and suppressed M1 macrophage markers, indicating that GpS altered mucosal cytokine profile in favor of the M1 to M2 macrophages switching, facilitating intestinal tissue repair. In conclusion, GpS might reverse the host's inflammatory phenotype by increasing beneficial bacteria, decreasing sulfate-reducing bacteria, and alleviating intestinal inflammatory gut environment, which might contribute to its cancer preventive effects.