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Black cohosh (Actaea racemosa L. [syn. Cimifuga racemosa L.]) extracts (BCE) are marketed worldwide for the management of menopausal symptoms. However, recently more than 75 cases of hepatotoxicity associated with black cohosh ingestion have been reported. While these cases have not been fully substantiated for causality, the data suggest that herb-drug interactions may be involved rather than a direct hepatotoxic event. This work describes the in vitro inhibition of four CYP450 enzymes (1A2, 2D6, 2C9, 3A4) by black cohosh extracts and identifies the active inhibitory constituents. Ethanol extracts (75 and 80% ethanol) and a 40% isopropanol extract induced a concentration-dependent inhibition of all CYP450 isozyme activities, with median inhibitory concentrations (IC50) ranging from 21.9 μg/ml to 65.0 μg/ml. Isolation of the active chemical constituents, showed that the triterpene glycosides were weakly active (IC50 25-100 μM), while fukinolic acid and cimicifugic acids A and B strongly inhibited all CYP isozymes (IC50 1.8-12.6 μM). None of the extracts inhibited the growth of Hep-G2 cells in concentrations up to 50 μg/ml. These data suggest that BCEs are not directly hepatotoxic, but may have the potential to induce herb-drug interactions, which may in turn explain the rare cases of hepatotoxicity observed in women using multiple medications and dietary supplements, including black cohosh.

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The effect of Menoprogen (MPG) on ovarian granulosa cell (GC) apoptosis was investigated in vitro and in vivo in an aged rat model of menopause. Intragastric administration of Menoprogen or estradiol valerate to 14-month-old senile female rats for eight weeks increased plasma E2 levels, as well as the weight of both ovarian and uterine tissues. Flow cytometric (FCM) analysis of isolated GCs from MPG-treated aged rats showed reductions in the G0/G1 ratio and apoptotic peaks. Isolated GCs also exhibited an increase in cell size and the number of cytoplastic organelles and intracellular gap junctions, the reappearance of secretory granules, and a lack of apoptotic bodies as determined by TEM. Results from a TdT-mediated dUTP nick end-labeling (TUNEL) assay revealed a reduction in TUNEL-positive GCs after MPG treatment. Immunohistochemical analysis showed a downregulation of proapoptotic Bax proteins and an upregulation of antiapoptotic Bcl-2 proteins. The addition of MPG-medicated serum to the media of cultured GCs also reduced cadmium chloride-induced apoptosis and downregulated caspase-3 protein expression. This work demonstrates that Menoprogen inhibits GC apoptosis in aged female rats and thereby increases E2 production. This represents a novel mechanism of action for this herbal medicine in the treatment of menopausal symptoms.

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Introduction: In previous work, we have shown the synergistic effects of combinations of vitamins A, D2, and D3 in reducing the proliferation of HCT-116 colon cancer cells. This combination also induced apoptosis and altered gene expression patterns as determined by transcriptomic profiling. Aims: The aims of this study were to investigate the effects of vitamins A and D in HCT-116 Crl:NU(NCr)-Foxn1nu mouse xenographs. Also, to determine potential mechanisms of action using miRNA-seq and correlated these data with results from RNA-seq.Methods: HCT-116 colon cancer cells were cultured and used for the xenograph study. Crl:NU(NCr)-Male Foxn1nu mice were injected subcutaneously with HCT-116-cells (1 X 106 in 100 µl 50% Matrigel and Sterile PBS), and after 7 days, were treated with vitamin A and D in feed for 21 days. The mice were imaged on day 28, sacrificed and the tumors were excised and measured. RNA was isolated from the HCT-116 cells and tumors, and RNA-seq and miRNA-seq were performed.Results: There was a concentration-dependent reduction in the HCT-116 colon cancer cell viability after treatment with vitamin A and D combinations. In Crl:NU(NCr)-Foxn1nu mice injected with HCT-116 colon tumor cells, treatment with vitamin A (25,000 IU) and vitamin D (4,000 IU) or vitamin A (35,000 IU) and vitamin D (5,000 IU) for 21 days significantly reduced tumor growth by ~38% and ~58%, respectively (p < 0.001). In the HCT-116 cells and excised tumors, treatment upregulated the expression of Bim, Bax, p53, and PTEN, and downregulated AXIN2, ID2 and DDX20 mRNAs, all well-known pro-apoptotic proteins, tumor suppressors, and molecules involved in the epithelial-mesenchymal transition. Ingenuity Pathway Analysis of miRNA-seq paired with RNA-seq showed correlations between miRNAs expression and expression of these genes. For example, miRNA-mRNA correlation pairing showed that treatment downregulated miR-30c-3p and miR-125b-3p which was associated with upregulated TP53. Upregulation of the tumor suppressor PTEN was associated with the downregulation of eleven miRNAs, including oncogenic miR17 and miR21.Conclusion: The results suggest that the combinations of vitamins A and D reduced the HCT-116 tumor burden in mice and altered the expression of miRNAs directly associated with genes in the apoptosis, tumor suppression, and epithelial-mesenchymal transition pathways. The data supports the hypothesis that vitamin A and D combinations impact multiple cancer signaling pathways and thus may be more effective in the treatment and prevention of cancer, as well as reducing cancer metastasis.   Keywords: apoptosis, cholcalciferol, colon cancer, DDX20, epithelial-mesenchymal transition, ergocalciferol, p53, RNA-seq, miRNA-seq, PTEN, synergism, transcriptome

Background: Purified fractions from a Boswellia serrata Roxb. Ex. Colebr. (Burseraceae) extract (ETOH and DCM) contain biologically active compounds that are well known for having inflammation inhibitory properties. In this work, the purified fractions were tested in-vitro for LTC4, LTA4 and COX-2 activities using ELISA and qPCR was performed to determine gene regulation in human leukemia (HL-60) Cells. Two D-imaging tomography was performed to determine the anti-inflammatory activities of the fractions in BALB/c mouse model of lung inflammation. Objective: To evaluate anti-inflammatory activities of bioactive compounds of Boswellia serrata purified fractions. Methods: In-vitro MTT assay was performed in HL-60 cell lines for measuring the toxicity/viability of the cells. ELISA tests were performed for evaluating LTA4, LTC4 and COX-2 activities. qPCR was performed to evaluate the expression of mRNA in HL-60 cells. In-vivo experiments were performed in OVA sensitized and challenged BALB/c mice at two doses of Boswellia serrata purified fraction containing 6% Boswellic acid of 50 and 100mg/kg body weight were given orally and the standard drug dexamethasone (DXA, 4 mg/kg body weight) and reduction in lung inflammation was assessed by using an IVIS Xenogen in-vivo fluorescence imaging system. Results: A purified fraction of Boswellia serrata ETOH extracts reduced leukotriene-C4-synthase activity by 52%, leuktotriene-A4-hydrolase activity by 22% and COX-2 activity by 99% with an IC50 of 12.5µg/ml. Intragastric administration of the purified fraction of Boswellia serrata at two doses of 50mg/kg b.w. and 100mg/kg b.w., respectively along with 2-3% HPMC resulted in a ~51% (P value <0.01) reduction in OVA induced lung inflammation in BALB/c mice as observed by imaging tomography. Treatment of the OVA challenged mice with a standard drug dexamethasone (DXA) reduced inflammation by ~66% with significant value (P<0.0001). Conclusion: The present study describes that Boswellia serrata ethanolic extracts purified fraction (ETOH-BS) possess significant anti-inflammatory activities in HL-60 and in BALB/c and further supports for its use as Ayurvedic medicines traditionally in the treatment of lung disorders including allergy and asthma.