Ethnopharmacology

Cardioprotective effects induced by hydroalcoholic extract of leaves of Alpinia zerumbet on myocardial infarction in rats Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Emanuel Tenório Paulino, Amanda Karine Barros Ferreira, Jessyka Carolina Galvão da Silva, Cintia Danielli Ferreira Costa, Salete Smaniotto, João Xavier de Araújo-Júnior, Edeíldo Ferreira Silva Júnior, Janaína Herbele Bortoluzzi, Êurica Adélia Nogueira Ribeiro Abstract Ethnopharmacology relevance The leaves of Alpinia zerumbet is used in folk medicine in Brazil to treat hypertension. However, the cardioprotective effect of this plant has not been studied yet. Aim of this study To evaluate the cardioprotective effects of the hydroalcoholic extract of the leaves of Alpinia zerumbet (AZE) against isoproterenol (ISO)-induced myocardial infarction in rats. Material and methods Rats were pretreated orally with AZE (300 mg/kg) for 30 days prior to ISO-induced myocardial infarction. The rats were sacrificed and hearts were collected and homogenized for biochemical analysis. At the end of the experiment, cardiac marker enzyme levels, histological and morphometric parameters, and hemodynamic measurements were assessed. Phytochemical compounds were verified by gas chromatography-mass spectrometry (GC-MS). Results Rats administered with ISO showed a significant increase in cardiac marker enzymes, i.e., in creatine kinase-NAC (CK-NAC) and CK-MB. Triphenyltetrazolium chloride (TTC) staining exhibited an increase in infarct areas. In the animals treated with ISO induced a significant increase in heart rate. Pretreatment with AZE significantly inhibited these effects of ISO. Moreover, biochemical findings were supported by histopathological observations. The GC-MS analyses of AZE identified volatile oils, kavalactones, and phytosterols. Conclusions Haemodynamic, biochemical alteration and histopathological results suggest a cardioprotective protective effect of oral administration of AZE in isoproterenol induced cardiotoxicity. Graphical abstract

Cytotoxicity and antimitotic activity of Rhinella schneideri and Rhinella marina venoms Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Sara Abdelfatah, Xiaohua Lu, Guillermo Schmeda-Hirschmann, Thomas Efferth Abstract Ethnopharmacological relevance Rhinella schneideri and Rhinella marina are toad venoms distributed in different parts of the world, including Brazil, Columbia and amazon. Venoms extracted from different species have many clinical applications such as antimicrobial cardiotonics and treatment of cancer. Aim of the study; In this study, we aim to investigate the effect of venoms extracted from R. schneideri and R. marina on cancer cells and verify possible mechanism of action. Material and method Cytotoxicity analyses was performed using the resazurin reduction assay, where different concentrations of venoms were tested against sensitive CCRF-CEM and P-gp overexpressing ADR/CEM5000 leukemia cells. Programmed cell death was investigated using the flow cytometric annexin V/propidium iodide apoptosis assay. Furthermore, we analyzed flow cytometric cell cycle analyses of CCRF-CEM cells. Effect on tubulin formation was tested using molecular docking and fluorescence microscopy of U2OS-GFP-α-tubulin osteosarcoma cells treated for 24 h with venoms. Results Cytotoxicity assays revealed a strong activity towards wild-type CCRF-CEM cells (IC50 values of 0.202 ± 0.005 μg/ml and 0.18 ± 0.007 μg/ml for R. schneideri and R. marina, respectively) and multidrug-resistant CEM/ADR5000 cells (IC50 0.403 ± 0.084 μg/ml and 0.32 ± 0.077 μg/ml for R. schneideri and R. marina, respectively). The venoms induced apoptosis as major mechanism of cell death. The venoms induced strong G2/M cell arrest in CCRF-CEM cells. We suggested tubulin as a major target for the venoms. In silico molecular docking of the major constituents of the venoms, i.e. bufalin, marinobufagin, telocinbufagin, hellebrigenin, showed strong binding affinities to tubulin. This result was verified in vitro. The venoms dysregulated microtubule arrangement of U2OS cells expressing GFP-labeled tubulin. Toxicity predictions by QSAR methodology highlighted the toxic features of bufadienolides. Conclusion Our study demonstrated the importance of toad venoms as source of cytotoxic compounds that may serve as lead compounds for the development of novel anticancer drugs. Graphical abstract

In vitro and in vivo antiplasmodial activity of extracts and isolated constituents of Alstonia congensis root bark Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): R. Kanyanga Cimanga, S. Lumpu Nsaka, M. Ehata Tshodi, B. Maya Mbamu, C. Munduku Kikweta, F. Bool-Miting Makila, Paul Cos, Louis Maes, Arnold J. Vlietinck, Vassiliki Exarchou, Emmy Tuenter, Luc Pieters Abstract Ethnopharmacological relevance An aqueous decoction of root bark of Alstonia congensis Engl. (Apocynaceae) is used in several African countries to treat various ailments including malaria. Materials and methods Extracts of different polarity and isolated constituents were tested in vitro for their antiplasmodial activity against the chloroquine-resistant strain Plasmodium falciparum K1 and the chloroquine-sensitive strain P. falciparumNF54A19A, as well as for their cytotoxic effects againt MRC-5 cells (human lung fibroblasts). Extracts and fractions were evaluated in vivo against the chloroquine-resistant strain P. yoelii N67 and the chloroquine-sensitive strain P. berghei berghei ANKA. Results The aqueous extract, the 80% methanol extract and the alkaloid-enriched extract exhibited strong antiplasmodial activity against P. falciparum K1 with IC50 values < 10 µg/ml and against P. falciparum NF54 A19A with IC50values < 0.02 µg/ml. In vivo against P. yoelii N67, at the highest oral dose of 400 mg/kg body weight, all extracts produced 70–73% chemosuppression, while against P. berghei berghei, more than 75% chemosuppression was observed. With regard to the isolated constituents, boonein was inactive in vitro against P. falciparum K-1 (IC50 > 64 µM), while echitamine, 6,7-seco-angustilobine B and β-amyrin exhibited moderate activity (IC50< 30 µM). Against P. falciparum NF54 A19A, boonein was inactive (IC50 > 64 µM), while echitamine, 6,7-secoangustilobine and β-amyrin showed moderate IC50 values of 11.07, 21.26 and 40.70 µM, respectively. Conclusion These results demonstrated that all extracts from A. congensis root bark possessed antiplasmodial activity in vitro and in vivo. They can be used as raw materials for the preparation of ameliorated remedies for the treatment of uncomplicated malaria. The observed antiplasmodial activity may be due in part to the presence of indole alkaloids. Graphical abstract

Flavonoids of Tripodanthus acutifolius inhibit TNF–α production in LPS–activated THP–1 and B16–F10 cells Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): L. Apaza T, Andreea Madalina Serban, A.H. Cabanillas, A. Villacampa, Angel Rumbero Abstract Ethnopharmacological relevance T. acutifolius is an endemic species from South America which has been used in traditional medicine since ancient times due to its biological properties, including its anti–inflammatory effects. Aim of the study: The aim of the article is to investigate the inhibitory activity of T. acutifolius over TNF–α production in THP–1 and B16–F10 cells. To achieve this, phytochemical analysis has been used to determine the compounds present in the species with anti–inflammatory effects. Materials and methods Leaves and stems of T. acutifolius were extracted with n–heptane, dichloromethane, methanol and water. The resulting extracts were analysed in THP–1 and B16–F10 cells by measuring their inhibitory capacity over the production of TNF–α stimulated with LPS. Results The guided–bioassay led to the isolation of 6,2',4'–trimethoxyflavone (1), 5,3',4'–trihydroxy–6,7,8–trimethoxyflavone (2), (E)–2',4'–dihydroxy–6'–methoxy–chalcone (3) and 5,4'–dihydroxy–6,7,8–trimethoxyflavone (4) from the dichloromethanic extract. Compounds showed an inhibitory activity of TNF–⍺ production in THP–1 cells, with IC50 of 2.38 ± 0.02 μM, 12.36 ± 0.17 μM, 1.12 ± 0.01 μM and 8.09 ± 0.04 μM, respectively. In addition, the compounds showed an inhibitory activity of TNF–⍺ production in B16–F10 cells with IC50 of 1.32 ± 0.03 μM, 5.63 ± 0.09 μM, 0.60 ± 0.02 μM and 3.77 ± 0.15 μM, respectively. Conclusions We identified 3 flavones (6,2',4'–trimethoxyflavone, 5,3',4'–trihydroxy–6,7,8–trimethoxyflavone, 5,4'–dihydroxy–6,7,8–trimethoxyflavone) and a chalcone ((E)–2',4'–dihydroxy–6'–methoxy–chalcone) present in the leaves and stems of T. acutifolius. These compounds are an alternative for the treatment of immune–mediated inflammatory disorders, acting as negative modulators over the TNF–α production. Graphical abstract

Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) powdered leaves are effective in treating anxiety symptoms: A phase-2, randomized, placebo-controlled clinical trial Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Fabio Carmona, Fernando Saraiva Coneglian, Priscila Alves Batista, Davi Casale Aragon, Mateus Andrea Angelucci, Edson Zangiacomi Martinez, Ana Maria Soares Pereira Abstract Ethnopharmacological relevance Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) is a plant traditionally used as medicine for anxiety symptoms. This activity was confirmed in preclinical studies. However, its efficacy was never studied in human clinical trials. Aim of the study We aimed to test the hypothesis that the herbal medicine of A. polystachya is superior to placebo for the treatment of anxiety-related symptoms in adults after 8 weeks. Patients and methods This was a randomized, double-blind, placebo-controlled, phase-2 clinical trial. Fifty-four adults with self-reported anxiety symptoms were randomly allocated to receive either capsules containing A. polystachya powdered leaves (300 mg, twice a day) or placebo (maltodextrin), for 8 weeks. The intensity of anxiety symptoms was assessed by the Hamilton Anxiety Ranking Scale (HAM-A) at baseline and after 2, 4 and 8 weeks. All analyses were adjusted for physical activity (assessed by the International Physical Activity Questionnaire [IPAQ], short version) and gender. Results We confirmed the presence of acteoside (chromatographic analysis) and carvone and limonene (gas chromatography) as major constituents in our plant material. Only patients that received A. polystachya experienced a significant decrease in their HAM-A scores, with none or mild side-effects. Conclusion Administration of powdered leaves of A. polystachya, rich in acteoside, carvone and limonene, to adults with anxiety symptoms was significantly superior to placebo in decreasing HAM-A scores after 8 weeks. This finding confirms the ethnopharmacological use of this plant for anxiety symptoms. Graphical abstract

Ginsenoside Rg3 upregulates myotube formation and mitochondrial function, thereby protecting myotube atrophy induced by tumor necrosis factor-alpha Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Sang-Jin Lee, Ju Hyun Bae, Hani Lee, Hyunji Lee, Jongsun Park, Jong-Sun Kang, Gyu-Un Bae Abstract Ethnopharmacological relevance Ginsenoside Rg3 from Panax ginseng has reported to have multiple pharmacological activities including anti-diabetics, anti-inflammation and anti-cancer. However, the effect of ginsenoside Rg3 on myogenic differentiation and muscle atrophy is unknown. Aim to the study In this study, we investigated the myogenic effect and underlying molecular mechanisms of ginsenoside Rg3 on myotube atrophy induced by tumor necrosis factor-α (TNF-α). Materials and methods C2C12 myoblasts were induced to differentiate for one day followed by the treatment of TNF-α along with vehicle or ginsenoside Rg3 for additional 2 days and subjected to immunoblotting, immunocytochemistry, quantitative RT-PCR and biochemical analysis for mitochondrial function. Results Ginsenoside Rg3 promotes myogenic differentiation and multinucleated myotube formation through Akt activation in a dose-dependent manner, without any cytotoxicity. Ginsenoside Rg3 treatment restores myotube formation and increases myotube diameters under TNF-α-treated conditions. Ginsenoside Rg3 enhances Akt/mTOR (mammalian target of rapamycin) signaling that in turn stimulates muscle-specific gene expression such as myosin heavy chain (MHC) and Myogenin, and suppresses the expression of muscle-specific ubiquitin ligases. In addition, ginsenoside Rg3 in TNF-α-treated myotubes significantly inhibits the production of mitochondrial ROS and restores mitochondrial membrane potential (MMP) and ATP contents. Furthermore, ginsenoside Rg3 upregulates the activities and expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and the mitochondrial biogenetic transcription factors, nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor A (Tfam) in TNF-α-induced myotube atrophy. Conclusions This study provides a mechanistic insight into the effect of ginsenoside Rg3 on myogenic differentiation and myotube atrophy, suggesting that ginsenoside Rg3 has a promising potential as a therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy. Graphical abstract

A network pharmacology integrated pharmacokinetics strategy for uncovering pharmacological mechanism of compounds absorbed into the blood of Dan-Lou tablet on coronary heart disease Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Mingya Ding, Wenfang Ma, Xiaoyan Wang, Shujing Chen, Shuhan Zou, Jinna Wei, Yuqiao Yang, Jin Li, Xuejing Yang, Hui Wang, Yuhong Li, Qilong Wang, Haoping Mao, Xiu-mei Gao, Yan-xu Chang Abstract Ethnopharmacological relevance Dan-Lou tablet (DLT) is developed from the traditional Chinese medicine (TCM) formula Gualou Xiebai Baijiu Tang which has been used for at least 2000 years in China. DLT has been widely used in clinical practice to treat cardiovascular diseases. Aim of the study This study aimed to uncover the pharmacological mechanism of the compounds absorbed into the blood of Dan-Lou tablet (DLT) on coronary heart disease (CHD) using a network pharmacology integrated pharmacokinetics strategy. Materials and methods A rapid and sensitive method was developed for the simultaneous determination of the six compounds (puerarin, formononetin, calycosin, paeoniflorin, cryptotanshinone and tanshinone IIA) in rat plasma by liquid chromatography tandem mass spectrometry (LC-MS/MS). Then, the pharmacology network was established based on the relationship between five compounds absorbed into the blood targets (puerarin, formononetin, calycosin, cryptotanshinone and tanshinone IIA) and CHD targets. Results The intra-and inter-day precision were less than 11% and the accuracy ranged from 88.2% to 112%, which demonstrated that the LC-MS/MS method could be used to evaluate the pharmacokinetic feature of the six compounds in rats after oral administration of DLT. The pathway enrichment analysis revealed that the significant bioprocess networks of DLT on CHD were positive regulation of estradiol secretion, negative regulation of transcription from RNA polymerase II promoter, lipopolysaccharide-mediated signaling pathway and cytokine activity. Conclusion The proposed network pharmacology integrated pharmacokinetics strategy provides a combination method to explore the therapeutic mechanism of the compounds absorbed into the blood of multi-component drugs on a systematic level. Graphical abstract

Anti-inflammatory and phytochemical evaluation of Combretum aculeatum Vent growing in Sudan Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Kamal M. Hamad, Manal M. Sabry, Sabah H. Elgayed, Abdel-Rahman El Shabrawy, Ahlam M. El-Fishawy, Gehad A. Abdel Jaleel Abstract Ethnopharmacological relevance Combretum aculeatum Vent was traditionally used in Sudan, Eretria and Ethiopia as anti-inflammatory in case of skin inflammation, catarrh, wounds, scorpion stings and snake bites. Nevertheless, there is no scientific information regarding this activity. Aim of study The present study aimed to evaluate the phytochemical constituents and the scientific basis for the traditional use of Combretum aculeatum Vent through studying its anti-inflammatory properties for the first time to illustrate the putative mechanisms behind this bioactivity. Materials and methods the ethanolic extract was partitioned by petroleum ether, methylene chloride, ethyl acetate, and n-butanol saturated with water. The petroleum ether fraction was saponified and the saponifiable and unsaponifiable fractions were analyzed on GC/MS. The different fractions were subjected to phytochemical investigation to isolate pure compounds. In-vivo anti-inflammatory activity of the ethanolic extract was evaluated using carrageenan induced rat paws edema method at doses of 200, 400 and 600 mg/kg and proved based on histopathological and biochemical parameters. Results Five known compounds were isolated for the first time from the aerial parts of Combretum aculeatum Vent: quercetin, vitexin, isorhamnetin 3-O-β-glucoside, isovitexin and rutin, in addition to two previously isolated ones: β-sitosterol and its glucoside. The ethanolic extract evidenced in-vivo anti-inflammatory activity by oral intake of 400 mg/kg of the ethanolic extract significantly (P ≥ 0.05) decreased the paw edema (only 32±1.9% increase in paw weight after 4 h) compared to indomethacin (28.6±2.5%). Moreover, it significantly suppressed the serum malondialdehyde (MDA) and nitric oxide (NO) and increased the GSH to be 11.76±0.85, 5.13±0.62 μmol/mL and 5.66±0.28 μM/mL, respectively. It diminished the serum cytokines TNF-α, IL-6 and IL-1β levels to be 39.1±1.2, 32.6±1.1 and 37.5±1.2 pg/mL, respectively. Results are accompanied by histopathological examination. Conclusion Overall, the results herein presented significant anti-inflammatory properties traditionally ascribed to Combretum aculeatum Vent. Moreover, the biochemical mechanisms associated to this action were highlighted, introducing new prospects for the development of effective anti-inflammatory herbal medicinal products. Graphical abstract

Dangguibuxue decoction abolishes abnormal accumulation of erythroid progenitor cells induced by melanoma Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Chengyin Li, Fenglin Zhu, Chong Xu, Ping Xiao, Junsong Wen, Xia Zhang, Bin Wu Abstract Ethnopharmacologic relevance Dangguibuxue decoction (DGBX), is a well-known traditional Chinese medicine that contains two types of materials used to treat anemia. In this study, we aimed to explore the effect and mechanism of DGBX on abolishing erythroid progenitor cell (Ter119+CD71+) accumulation induced by melanoma. Materials and methods B16/F10 melanoma cells were used to establish transplanted and metastatic melanoma models. DGBX or normal saline were administered intragastrically daily after the models were established. Tumor sizes and metastatic nodules were observed after tumor cell inoculation. To further test the function of DGBX on erythroid progenitor cell (EPC) accumulation and immunosuppressive abilities, the percentage of EPCs in the blood, and spleen were quantified with flow cytometry. The proportion of CD8+ T cells and related functional mediators, IFN-γ and TNF-α,were also quantified with flow cytometry. To further strengthen our in vivo observations, DGBX serum was prepared from the rats three days after DGBX was administered. Liquid chromatography-mass spectrometry was carried out to control the quality of the experiments. B16/F10 melanomacells were cultured with DGBX serum, and proliferation and apoptosis were observed with the CCK8 assay and AnnexinV/7AAD staining, respectively. EPCs were isolated from B16/F10-bearing mice and cultured under erythroid differentiation conditions. EPCs were treated with DGBX serum, and mature red cell proportions and cell denucleations were tested with flow cytometry and Giemsa staining of the cultured EPCs. Flow cytometry and qPCR were used to analyze the effects of DGBX on the expression of key molecules involved in erythroid development and to explore the mechanism by which DGBX relieves abnormal EPC accumulation. Results DGBX treatments significantly reduced B16 melanoma tumor sizes and metastatic nodules. Most importantly, our study strongly suggested that DGBX could alleviate anemia, and systematically enhance anti-tumor immune responses by reducing abnormal EPC accumulation. Moreover, DGBX serum treatments had no direct effect on tumor cell proliferation and apoptosis, but could promote EPCs to differentiate into mature red blood cells, in vitro. Mechanistically, at least in part, DGBX relieved abnormal EPC accumulation by altering the “master switch” transcription factors, Pu.1 and Gata-1. Conclusions DGBX significantly alleviates abnormal tumor-induced EPC accumulation, inhibits B16 melanoma progression, and enhances anti-tumor immune responses. Graphical abstract

Lychnophora pinaster ethanolic extract and its chemical constituents ameliorate hyperuricemia and related inflammation Publication date: 5 October 2019 Source: Journal of Ethnopharmacology, Volume 242 Author(s): Camila Martins de Sá Müller, Grazielle Brandão Coelho, Marcela Carolina de Paula Michel Araújo, Dênia Antunes Saúde-Guimarães Abstract Ethnopharmacological relevance Lychnophora pinaster, known as “Brazilian arnica” is used in folk medicine as alcoholic extract to treat inflammation, pain, rheumatism and bruises. Aim of the study Evaluate the effects of the Lychnophora pinaster's ethanolic extract and its chemical constituents on inflammation and hyperuricemia. Materials and methods Ethanolic and hexanic extracts were obtained from the aerial parts of L. pinaster. Sesquiterpene E-lychnophoric acid was isolated from hexanic extract and identified by RMN, GC/MS and IR. In vivo anti-hyperuricemic and anti-inflammatory effects of the ethanolic extracts from L. pinaster (40, 125, 375 mg/kg), E-lychnophoric acid and other constituents previous isolated from L. pinaster and identified in the ethanolic extract by HPLC/UV/DAD (rutin, quercetin and vitexina flavonoids, caffeic, cinnamic and chlorogenic acids, lupeol and stigmasterol, at dose of 15 mg/kg) were assayed by experimental model of oxonate-induced hyperuricemia in Swiss mice, liver xanthine oxidase (XOD) inhibition and by MSU-induced paw edema in mice. Results Ethanolic extract and all its components presented anti-hyperuricemic activity by inhibiting the hepatic xanthine oxidase activity. Ethanolic extract and its chemical constituents, except quercetin and vitexin, were able to reduce paw edema size induced by urate crystals. Hypouricemic and anti-inflammatory results obtained for the ethanolic extract (40, 125, 375 mg/kg) and E-lychnophoric acid (15 mg/kg) were similar those obtained for standard drugs, allopurinol (10 mg/kg) and indomethacin (3 mg/kg). Conclusion Ethanolic extract and E-lychnophoric, chlorogenic, cinnamic and caffeic acids, rutin, lupeol and stigmasterol presented anti-inflammatory and anti-hyperuricemic actvities. These compounds are responsible for the activities presented by the ethanolic extract of L. pinaster. Ethanolic extract and its chemical constituents can be considered promising agents in the therapeutic of inflammation, hyperuricemia and gout. Graphical abstract