Peonidin-3-O-glucoside and Resveratrol Increase the Viability of Cultured Human hFOB Osteoblasts and Alter the Expression of Genes Associated with Apoptosis, Osteoblast Differentiation and Osteoclastogenesis.

High-throughput RNA-sequencing can determine the impact of nutrients and their combinations on gene transcription levels in osteocytes, and clarify the biological pathways associated with their impact on bone tissues. Previously, we reported that resveratrol (RES) and peonidin-3-O-glucoside (POG) increased osteoblastogenesis, as well as reduced osteoclastogenesis in transgenic teleost fish models. Here, we perform whole-genome transcriptomic profiling of osteoblasts treated with POG or RES to provide a comprehensive understanding of alterations in gene expression and the molecular mechanisms involved. Cultured human fetal osteoblastic hFOB 1.19 cells were treated with the test compounds, and then RNA was used to prepare RNA-seq libraries, that were sequenced using a NovaSeq 6000. Treatment with POG or RES increased osteoblast proliferation and reduced apoptosis. Transcriptomic profiling showed that of the 29,762 genes investigated, 3177 were differentially expressed (1481 upregulated, 1696 downregulated, FDR ≤ 0.05) in POG-treated osteoblasts. In the RES-treated osteoblasts, 2288 genes were differentially expressed (DGEs, 1068 upregulated, 1220 downregulated, FDR ≤ 0.05). Ingenuity® Pathway Analysis (IPA) of DGEs from RES or POG-treated osteoblasts revealed significant downregulation of the apoptosis, osteoarthritis and HIF1α canonical pathways, and a significant reduction in Rankl mRNA expression. The data suggest that RES and POG have both anabolic and anticlastogenic effects.

Peonidin-3-O-glucoside and cyanidin increase osteoblast differentiation and reduce RANKL-induced bone resorption in transgenic medaka.

Experimental and clinical studies suggest a positive impact of anthocyanins on bone health; however, the mechanisms of anthocyanins altering the differentiation and function of osteoblasts and osteoclasts are not fully understood. This work demonstrates that dietary anthocyanins and resveratrol increased proliferation of cultured human hFOB 1.19 osteoblasts. In addition, treatment of serum starvation of hFOB osteoblasts with anthocyanins and resveratrol at 1.0 µg/ml reduced apoptosis, the Bax/Bcl-2 ratio, p53, and HDAC1 expression, but increased SIRT1/3 and PGC1α mRNA expression, suggesting mitochondrial and epigenetic regulation. In Sp7/osterix:mCherry transgenic medaka, peonidin-3-O-glucoside and resveratrol increased osteoblast differentiation and increased the expression of Sp7/osterix. Cyanidin, peonidin-3-O-glucoside, and resveratrol also reduced RANKL-induced ectopic osteoclast formation and bone resorption in col10α1:nlGFP/rankl:HSE:CFP medaka in doses of 1-4 µg/ml. The results indicate that both cyanidin and peonidin-3-O-glucoside have anabolic effects on bone, increasing osteoblast proliferation and differentiation, mitochondrial biogenesis, and by altering the osteoblast epigenome. Cyanidin and peonidin-3-O-glucoside also reduced RANKL-induced bone resorption in a transgenic medaka model of bone resorption. Thus, peonidin-3-O-glucoside and cyanidin appear to both increase bone formation and reduce bone loss, suggesting that they be further investigated as potential treatments for osteoporosis and osteomalacia.

Fractions of Boswellia Serrata Suppress LTA4, LTC4, Cyclooxygenase-2 Activities and mRNA in HL-60 Cells and Reduce Lung Inflammation in BALB/c Mice.

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 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.

Medicinal plants used in the treatment of tuberculosis - Ethnobotanical and ethnopharmacological approaches.

Tuberculosis is a highly infectious disease declared a global health emergency by the World Health Organization, with approximately one third of the world's population being latently infected with Mycobacterium tuberculosis. Tuberculosis treatment consists in an intensive phase and a continuation phase. Unfortunately, the appearance of multi drug-resistant tuberculosis, mainly due to low adherence to prescribed therapies or inefficient healthcare structures, requires at least 20 months of treatment with second-line, more toxic and less efficient drugs, i.e., capreomycin, kanamycin, amikacin and fluoroquinolones. Therefore, there exists an urgent need for discovery and development of new drugs to reduce the global burden of this disease, including the multi-drug-resistant tuberculosis. To this end, many plant species, as well as marine organisms and fungi have been and continue to be used in various traditional healing systems around the world to treat tuberculosis, thus representing a nearly unlimited source of active ingredients. Besides their antimycobacterial activity, natural products can be useful in adjuvant therapy to improve the efficacy of conventional antimycobacterial therapies, to decrease their adverse effects and to reverse mycobacterial multi-drug resistance due to the genetic plasticity and environmental adaptability of Mycobacterium. However, even if some natural products have still been investigated in preclinical and clinical studies, the validation of their efficacy and safety as antituberculosis agents is far from being reached, and, therefore, according to an evidence-based approach, more high-level randomized clinical trials are urgently needed.

Isolation and identification of three new chromones from the leaves of Pimenta dioica with cytotoxic, oestrogenic and anti-oestrogenic effects.

CONTEXT: Pimenta dioica (L.) Merr. (Myrtaceae) is used in Costa Rican traditional medicine for women's health. Our previous work showed that P. dioica extracts were oestrogenic. OBJECTIVES: This work identifies phytochemicals from P. dioica that are responsible for the plant's oestrogen-like activities. MATERIALS AND METHODS: P. dioica leaves were collected in Costa Rica in 2005. Fractions resulting from chromatographic separation of a methanol extract were tested at 50 µg/mL in a competitive oestrogen receptor-binding assay. Active compounds were isolated by HPLC and identified by NMR and MS. Pure compounds were tested at 1 µM in the oestrogen-responsive SEAP reporter gene assay. The effects on cell viability, cytotoxicity and apoptosis were investigated in breast cancer (MCF-7 and SK-BR3) and gastric cancer (AGS and NCI-N87) cell lines using the ApoTox-Glo and Caspase-Glo assays and qPCR. RESULTS: Quercitrin and three new chromones, including a 2-phenoxychromone, 6,8-di-C-methylcapillarisin (1) were isolated and identified. Compound 1 caused a 6.2-fold increase in SEAP expression at 1 µM (p < 0.05). This activity was blocked by the ER antagonist ICI 182,780. Compound 2 caused a 6.0-fold increase in SEAP, inhibited the growth of MCF-7, AGS and NCI-N87 cells (IC50 54.27, 38.13 and 51.22 µg/mL, respectively), and induced apoptosis via caspase 8 and increased the Bax/Bcl-2 mRNA ratio in MCF-7 cells. Compound 3 was anti-oestrogenic in MCF-7 cells. DISCUSSION AND CONCLUSIONS: Compounds from P. dioica have oestrogenic, anti-oestrogenic and cytotoxic effects that may explain the ethnomedical use of this plant.

RETRACTED: Medicinal plants used in the treatment of tuberculosis - Ethnobotanical and ethnopharmacological approaches.

Tuberculosis is a highly infectious disease declared a global health emergency by the World Health Organization, with approximately one third of the world's population being latently infected with Mycobacterium tuberculosis. Tuberculosis treatment consists in an intensive phase and a continuation phase. Unfortunately, the appearance of multi drug-resistant tuberculosis, mainly due to low adherence to prescribed therapies or inefficient healthcare structures, requires at least 20months of treatment with second-line, more toxic and less efficient drugs, i.e., capreomycin, kanamycin, amikacin and fluoroquinolones. Therefore, there exists an urgent need for discovery and development of new drugs to reduce the global burden of this disease, including the multi-drug-resistant tuberculosis. To this end, many plant species, as well as marine organisms and fungi have been and continue to be used in various traditional healing systems around the world to treat tuberculosis, thus representing a nearly unlimited source of active ingredients. Besides their antimycobacterial activity, natural products can be useful in adjuvant therapy to improve the efficacy of conventional antimycobacterial therapies, to decrease their adverse effects and to reverse mycobacterial multi-drug resistance due to the genetic plasticity and environmental adaptability of Mycobacterium. However, even if some natural products have still been investigated in preclinical and clinical studies, the validation of their efficacy and safety as antituberculosis agents is far from being reached, and, therefore, according to an evidence-based approach, more high-level randomized clinical trials are urgently needed.

Menoprogen, a TCM Herbal Formula for Menopause, Increases Endogenous E2 in an Aged Rat Model of Menopause by Reducing Ovarian Granulosa Cell Apoptosis.

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.

Combination studies of Eucalyptus torelliana F. Muell. leaf extracts and clarithromycin on Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacillus that is associated with the development of gastritis and peptic ulcer disease (PUD). In Nigeria, leaf extracts of Eucalyptus torelliana F. Muell. are used in traditional medicine to treat PUD and other gastrointestinal ailments. The additive and synergistic effects of E. torelliana leaf extracts, in combination with clarithromycin, were investigated using two types of H. pylori strains (ATCC 43629, ATCC 43579) and four clinical isolates of H. pylori (Ed, A2, G1-1, 5514) in the checkerboard assay and the fractional inhibitory concentration (FIC) index. A time-kill study was also performed on the strain ATCC 43579. The results showed that the E. torelliana extract inhibited the growth of all H. pylori strains, and the addition of one of the isolated active compounds, namely compound 2 (a substituted pyrenyl ester) enhanced the activity of clarithromycin. The minimum inhibitory concentration values of clarithromycin and the botanical compound were reduced twofold (from 0.125 to 0.0625 µg/mL and > 100 to 50 µg/mL respectively). A 100% reduction in CFU/mL of H. pylori ATCC 43579 was observed with the combination of 0.25 µg/mL clarithromycin and 100 µg/mL and 200 µg/mL compound 2 after 3 h of exposure. The results of the investigation showed that the combination of botanical compounds and antibiotics may be beneficial in the treatment of H. pylori infections.

In vitro susceptibility of Mycobacterium tuberculosis to extracts of Eucalyptus camaldulensis and Eucalyptus torelliana and isolated compounds.

CONTEXT: Eucalyptus camaldulensis Dehnh. (Myrtaceae) and Eucalyptus torelliana F. Muell are used in Nigerian traditional medicine for the treatment of cough associated with tuberculosis (TB) and other respiratory infections. OBJECTIVE: Hexane, chloroform, methanol extracts, and isolated compounds of E. camaldulensis and E. torelliana were screened for activity against Mycobacterium tuberculosis H37Rv (MtbH37Rv) to authenticate the traditional use of these plants. MATERIALS AND METHODS: The microplate alamar blue assay (MABA) method was used to investigate the anti-M. tuberculosis activities. Bioassay-guided fractionation of the hexane extract of E. torelliana leaf was performed, and isolated compounds were characterized by MS, 1D- and 2D-NMR. RESULTS: The extracts inhibited the growth of MtbH37Rv [minimum inhibitory concentration (MIC) 4-64 µg/mL]. Spectroscopic characterization led to the identification of two compounds, hydroxymyristic acid methylester (1) and a substituted pyrenyl ester, a sterol (2). Compounds 1 and 2 had MIC of 49.45 and 46.99 µg/mL; IC(50) >100 and 38.21 µg/mL; selectivity index (SI) >2.02 and 0.81, respectively, and a minimum bactericidal concentration (MBC) of 62.50 µg/mL. DISCUSSION AND CONCLUSIONS: The anti-TB activities of these plants on M. tuberculosis H37Rv support their use in traditional medicine for the treatment of coughs associated with TB and reveals the presence of anti-Mtb active compounds in the plants. These findings not only demonstrate a new potential area of therapeutic value of E. camaldulensis and E. torelliana, but also illustrate the role of esters as anti-Mtb active principles in ethnobotanical preparations and as lead compounds in the development of new and effective anti-Mtb drugs.

Fukinolic acid derivatives and triterpene glycosides from black cohosh inhibit CYP isozymes, but are not cytotoxic to Hep-G2 cells in vitro.

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 (IC(50)) ranging from 21.9 microg/ml to 65.0 microg/ml. Isolation of the active chemical constituents, showed that the triterpene glycosides were weakly active (IC(50) 25-100 microM), while fukinolic acid and cimicifugic acids A and B strongly inhibited all CYP isozymes (IC(50) 1.8-12.6 microM). None of the extracts inhibited the growth of Hep-G2 cells in concentrations up to 50 microg/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.