Preclinical safety evaluation of the aqueous extract from Mangifera indica Linn. (Anacardiaceae): genotoxic, clastogenic and cytotoxic assessment in experimental models of genotoxicity in rats to predict potential human risks Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Gustavo Roberto Villas Boas, João Marcos Rodrigues Lemos, Matheus William de Oliveira, Rafael Claudino dos Santos, Ana Paula Stefanello da Silveira, Flávia Barbieri Bacha, Caren Naomi Aguero Ito, Ediane Bortolotte Cornelius, Fernanda Brioli Lima, Andrea Marisa Sachilarid Rodrigues, Nathália Belmal Costa, Felipe Francisco Bittencourt, Fernando Freitas de Lima, Marina Meirelles Paes, Priscila Gubert, Silvia Aparecida Oesterreich Abstract Ethnopharmacological relevance Medicinal plants widely used by the population contain significant concentrations of biologically active compounds and, although they have proven pharmacological properties, can cause DNA damage and develop fatal diseases. Aim of the study: The present study aimed to evaluate the genotoxic, cytotoxic potential and clastogenic effects of the aqueous extract from Mangifera indica leaves (EAMI) on rats submitted to experimental genotoxicity models and through the SMART test performed in Drosophila melanogaster. Material and methods The comet assay and the micronucleus test were performed on peripheral and bone marrow blood, respectively, of Wistar rats, orally treated with EAMI at doses of 125, 250, 500 and 1000 mg/kg/bw for 28 days. In the SMART test, the standard cross between three mutant D. melanogaster strains was used. Larvae were treated with EAMI at different concentrations, and the wings of adult flies were evaluated for the presence/frequency of mutant spots and compared to the negative control group. Results Phytochemical analysis of EAMI indicated high levels of flavonoids. The tests performed in rats showed that EAMI did not present significant genotoxic or clastogenic effects. The results showed a critical dose-dependent cytoprotective effect exerted by EAMI. This result was attributed to the high content of polyphenols and flavonoids. The biotransformation metabolites of EAMI did not present genotoxic activity, as demonstrated by the SMART test. Conclusions These results are relevant since they provide safety information about a plant species of great therapeutic, economical, nutritious and ethnopharmacological value for the population. Graphical abstract

Comprehensive HPTLC fingerprinting as a tool for a simplified analysis of purity of ginkgo products Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Débora Arruda Frommenwiler, Anthony Booker, Roser Vila, Michael Heinrich, Eike Reich, Salvador Cañigueral Abstract Ethnopharmacological relevance Herbal medicinal products based on ginkgo leaf refined dry extract (GBE) are an European development from the Eastern Asia traditionally used species Ginkgo biloba L. Nowadays, ginkgo products have increased the presence in the market, mainly as dietary supplements. Its adulteration with rutin and quercetin or herbal extracts rich in these compounds is a common practice. Tests featuring assays and detection of adulterants need to be performed on top of other existent methods (e.g. identification test). This may increase the costs of evaluating the quality of ginkgo products. Aim of the study To prove that comprehensive HPTLC fingerprinting can provide information beyond identification of ginkgo products, avoiding additional chromatographic tests for detection of adulterations. Materials and methods The information contained in the fingerprint obtained by HPTLC analysis of flavonoids was used for identification and for detection of adulterants, as well as to verify the limits of rutin and quercetin, which are normally determined by HPLC and used for detection of adulterants. For this purpose, peak profiles were generated from HPTLC chromatogram images. USP-HPLC methods were used for quantification of total flavonoids and testing the limits of rutin and quercetin. HPLC data were used to support the validity of the HPTLC method. An additional reversed phase HPTLC method was developed as a possible confirmatory method for the quercetin limit test. Results The proposed HPTLC method uses a particular sequence of detections, resulting in a number of images, which are later interpreted in a certain order. It is able to identify ginkgo products, to detect adulterants (rutin, quercetin, sophora fruit and flower bud, and buckwheat), and, using peak profiles generated from the chromatogram images prior to and after derivatisation, to evaluate the limits of rutin and quercetin. Forty-eight out of fifty-nine ginkgo dietary supplements analysed contained one or more adulterants. Furthermore, results of the HPTLC and HPLC limit tests for rutin and quercetin were in agreement in 98% of the cases. Finally, a decision tree showing the sequence of interpretation of the fingerprints obtained with the different detections after a single HPTLC analysis is included to help the analyst to evaluate whether samples have the correct identity and whether they contain or not adulterants. Conclusion A single HPTLC analysis is able to provide information on identity and purity of the products. This simplifies the analytical workflow and reduces the number of analyses prescribed in the USP powdered ginkgo extract monograph. Graphical abstract

α-Glucosidase inhibitory activity and in vivo antihyperglycemic effect of secondary metabolites from the leaf infusion of Ocimum campechianum mill Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Javier A. Ruiz-Vargas, Dulce L. Morales-Ferra, Guillermo Ramírez-Ávila, Alejandro Zamilpa, Elizabeth Negrete-León, Juan José Acevedo-Fernández, Luis M. Peña-Rodríguez Abstract Ethnopharmacological relevance Wild basil (Ocimum campechianum Mill.), an aromatic herb of the Lamiaceae family known as “albahaca de monte” (Spanish) or “x'kakaltun” (Mayan) in Yucatan, is used in Mayan traditional medicine to treat diabetes, as well as to alleviate fever symtoms, stomach pain, conjunctivitis, and various skin affections. Aim of the study To isolate and identify the bioactive metabolites responsible for the α-glucosidase inhibitory activity previously detected in the leaf infusion O. campechianum. Materials and methods The bioassay-guided fractionation and purification of the lyophilized infusion was carried out using a liquid-liquid partition procedure, followed by successive chromatographic purifications of the semipurified fractions. The isolated metabolites were identified by comparing their spectroscopic data with those reported in the literature. The liophylized infusion, together with the semipurified fractions, and the pure metabolites were tested for their antioxidant and α-glucosidase inhibitory activities, as well as their antihyperglycemic effect. Results Chromatographic purification of the semipurified fractions led to the isolation of the polymethoxylated flavones 5-demethyl nobiletin (1) and 5-demethyl sinensetin (2), together with luteolin (3), methyl rosmarinate (4) and rosmarinic acid (5). Metabolites 4 and 5 appear to be responsible for the α-glucosidase inhibitory activity and the antihyperglycemic effect detected in the lyophilized infusion. A Lineweaver-Burk double reciprocal plot confirmed that the higher α-glucosidase inhibitory activity of 4 is of non-competitive nature. Both 4 and 5 caused a decrease in blood glucose higher than that caused by acarbose, a result that appears to be related to their strong α-glucosidase inhibitory activity. Even though flavonoids 1–3 did not show a good inhibition of α-glucosidase, these products decreased blood glucose in the in vivo model, suggesting a different antihyperglycemic mechanism. Conclusions The results confirm both the traditional use of O. campechianum and the importance of the leaf infusion as a potential source of antihyperglycemic agents. The isolation of 5-demethyl nobiletin (1) and 5-demethyl sinensetin (2) from O. campechianum and other Ocimum spp. suggests that polymethoxyflavones can be considered chemotaxonomical markers for the genus. Graphical abstract

Bioactivity-guided isolation of flavonoids from Urtica dioica L. and their effect on endometriosis rat model Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Mert Ilhan, Zulfiqar Ali, Ikhlas A. Khan, Hakkı Taştan, Esra Küpeli Akkol Abstract Ethnopharmacological relevance Urtica dioica L. has been used traditionally for centuries. U. dioica leaves and roots are used as a blood purifier, emmenagogue, and diuretic, as well as to treat menstrual hemorrhage, rheumatism, and eczema. The present study aimed to evaluate the activity of U. dioica L. aerial parts in endometriosis rat model. Materials and methods To evaluate the effects of the plant in endometriosis, n-hexane, ethyl acetate (EtOAc), and methanol (MeOH) extracts were prepared from the aerial parts of the plant and utilized in a rat surgical endometriosis model. In this model, adhesion scores of endometriotic implants and the spherical volumes of ectopic uterine tissues were evaluated. In addition to these parameters, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and interleukin-6 (IL-6) levels of the peritoneal fluids were evaluated. Furthermore, histopathological studies were conducted on the endometriotic tissues. Results Post-treatment implant volumes and adhesion scores were significantly reduced in the reference and the MeOH extract treated groups. Significant differences were found between the peritoneal TNF-α, VEGF, and IL-6 levels of MeOH extract treated group and those of control group. Moreover, histopathological findings supported the biological activity results. Furthermore, isolation studies were conducted on the MeOH extract, which showed prominent activity in the rat endometriosis model. Rutin (1), isoquercetin (2), the mixture of kaempferol-3-O-rutinoside (nicotiflorin) (3a) and isorhamnetin-3-O-rutinoside (narcissin) (3b) (3), the mixture of kaempferol-3-O-glucoside (astragalin) (4a) and isorhamnetin-3-O-glucoside (4b) (4) were isolated from the active fraction. Conclusions The present study demonstrated that aerial parts of U. dioica exhibited promising activity in the endometriosis rat model due to its flavonoids. Graphical abstract

Uncovering pharmacological mechanisms of Zhi-Zi-Hou-Po decoction in chronic unpredictable mild stress induced rats through pharmacokinetics, monoamine neurotransmitter and neurogenesis Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Hang Xing, Xiaoxu Zhang, Nannan Xing, Huaidong Qu, Kuo Zhang Abstract Ethnopharmacological relevance Zhi-Zi-Hou-Po decoction (ZZHPD), a classical Chinese prescription, has been reported to improve depressive behaviors in clinic. However, definite pharmacological effects and mechanisms of ZZHPD on monoaminergic system and hippocampal neurogenesis are ambiguous. It need to be further illuminated. Aim of the study Our study is designed to reveal pharmacological mechanisms of ZZHPD on depression through pharmacokinetics, monoamine neurotransmitters and neurogenesis. Materials and methods Chronic unpredictable mild stress (CUMS) is used to establish rats model of depression. Then, the antidepressant effects of ZZHPD are evaluated by detecting body weight, sucrose preference and forced swimming test. The regulatory functions of ZZHPD on monoaminergic system are assessed by measuring monoamine neurotransmitters, neurotransmitter precursor substances, synthesized rate-limiting enzymes and transporters. Finally, potential molecular mechanism of ZZHPD on hippocampal neurogenesis is evaluated by investigating newborn immature neuron and newborn mature neuron. Results Our results show that ZZHPD remarkably normalizes CUMS-induced decline in weight gain, decrease of sucrose consumption rate in sucrose preference test and increase of immobility time in forced swimming test. Moreover, ZZHPD significantly reverses CUMS-induced reduction of 5-hydroxytryptamine (5-HT), dopamine (DA), tryptophan (Trp), tyrosine (Tyr), tryptophan hydroxylase2 (TPH2) and tyrosine hydroxylase (TH), whereas decreases level of serotonin transporter (SERT) in CUMS-induced rats. Finally, ZZHPD obviously improves CUMS-induced decrease of newborn immature neuron and newborn mature neuron in dentate gyrus of hippocampus. Conclusion This study demonstrates that ZZHPD can alleviate CUMS-induced depression-like behaviors. It is probably attributed to the fact that ZZHPD could enhance monoaminergic system and hippocampal neurogenesis. Our findings provide the new perspectives on molecular targets of ZZHPD, and it will facilitate its clinical application. Graphical abstract

Evaluation of antitrypanosomal activity of Pterocarpus santalinoides L’H'erit ex DC hydroethanol leaf extract in rats experimentally infected with Trypanosoma brucei Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Chukwunonso F. Obi, Terry A. Nzeakor, Michael I. Okpala, Ikenna O. Ezeh, Lotanna G. Nwobi, Martin O. Omeje, Romanus C. Ezeokonkwo Abstract Ethnopharmacological relevance The leaves of Pterocarpus santalinoides are used alone or with other plants by traditional healers in some Southern Nigerian and Ivorian rural villages to treat blood parasitic infections including trypanosomiasis and malaria. However, their efficacy and safety remains doubtful. Aim To evaluate the antitrypanosomal activity of Pterocarpus santalinoides hydroethanol leaf extract (PSELE) in vitro and in vivo. Materials and methods The phytochemical and acute toxicity studies of PSELE were performed using standard methods. Eleven different concentrations of the extract were screened for in vitro antitrypanosomal activity. For the in vivo study, twenty-five female albino rats were randomly assigned into five groups of five rats each. Group A was the uninfected and untreated group while groups B - E were infected intraperitoneally with 106 trypanosomes. Group C rats were treated once on day 11 post infection (PI) with 7 mg/kg diminazene aceturate while groups D and E rats were also treated on same day with 200 mg/kg and 400 mg/kg PSELE respectively for seven days. The level of parasitaemia (LOP), survivability, packed cell volume (PCV), total leucocyte count (TLC), total erythrocyte count (TEC), body weight and rectal temperature were used to assess the antitrypanosomal effect of PSELE. Results Phytochemical analysis revealed the presence of flavonoids, tannins, carbohydrates and reducing sugar. No sign of toxicity or mortality was observed following acute toxicity study at a single dose of 2000 mg/kg. The LC50 of PSELE was 0.0625 mg/ml. Parasitaemia was first detected on day 8 and all infected rats became parasitaemic on day 10 (PI). PSELE significantly reduced (p < 0.05) LOP, improved PCV, TEC and prolonged survival time of the rats compared with the infected untreated group B. Conclusion It was therefore concluded that PSELE is safe, possesses some antitrypanosomal activity and may serve as a lead for the development of an effective alternative antitrypanosomal drug. Graphical abstract

Tocolytic activity assessment of the methanol leaf extract of Justicia flava Vahl (Acanthaceae) on mouse myometrial contractility and preliminary mass spectrometric determination of secondary metabolites Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Enitome E. Bafor, Faith Ukpebor, Osemelomen Omoruyi, Ejiro Ochoyama, Glory Omogiade, Jude Ekufu, RuAngelie Edrada-Ebel Abstract Ethnopharmacological relevance The leaves of Justicia flava are traditionally used in the South of Nigeria to prevent preterm births. Aim of the study: In this study, the activity of the methanol leaf extract of J. flava (JF) was investigated on uterine contractility in non-pregnant and pregnant isolated mouse tissues. Material and methods The effects on spontaneous, oxytocin, and KCl-induced contractions were determined. The effects in calcium-free media were also determined. Possible mechanisms of activity were investigated using receptor and channel modulators. Mass spectrometric analysis was additionally performed on the leaf extract to identify secondary metabolites. Results JF was observed to inhibit spontaneous, oxytocin and high KCl-induced uterine contractility. JF also inhibited contractions in Ca2+-free media. JF was found to exert its inhibitory effect via interaction with inositol triphosphate and ryanodine receptors and also through modulation of K+- channels. Lignans and alkaloids were identified with the lignans being the most abundant in JF. Conclusion JF has been shown to potently inhibit uterine contractions in non-pregnant and pregnant isolated mouse uterus. The inhibitory activity of JF has been shown to occur via blockade of extracellular and intracellular calcium entry and these effects may be due to the lignans identified in - JF. JF has therefore been shown in this study to be a lead plant in the discovery of new drugs with uterine inhibitory activity. Graphical abstract

Study of the aqueous extract of Aloe vera and its two active components on the Wnt/β-catenin and Notch signaling pathways in colorectal cancer cells Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Chang Peng, WeiJia Zhang, Cong Dai, Wa Li, Xue Shen, YueMei Yuan, Li Yan, Wei Zhang, MeiCun Yao Abstract Ethnopharmacological relevance Aloe vera (L.) Burm. f. (Aloe vera) is a common Traditional Chinese Medicine (TCM) recorded in Pharmacopoeia of the People's Republic of China (version 2015). It has been traditionally used for treatment of constipation. Aloe verarequires much attention for its safety evaluation because several studies have reported the association between oral consumption of Aloe vera and the development of colorectal cancer (CRC). However the material basis and molecular mechanism are.still less well elucidated. Although Wnt/β-catenin and Notch signaling pathway have been known to be closely related to the initiation and development of CRC, the impacts of Aloe vera on these cancerous pathways have not been completely determined yet. Aim of this study Hence, this study aimed to study the impacts of Aloe vera on the Wnt/β-catenin and Notch signaling pathway, as well as proliferation of CRC cells. Materials and methods Firstly, the effects of Aloe vera aqueous extract and its two active components (aloin and aloesin) on the Wnt/β-catenin and Notch signaling pathway were studied by luciferase reporter, RT-qPCR, western blotting and immunofluorescence assays, respectively. Furthermore, RNA sequencing analysis (RNA-seq) was then performed to verify their regulatory activities on the Wnt-related and Notch-related genes expression. Finally, their impacts on RKO cell proliferation and cell cycle phase were also evaluated via MTT assay and cell cycle analysis. Results Our results indicate that the aqueous extract of Aloe vera and its active component aloin activated the Wnt/β-catenin pathway and inhibited the Notch signaling pathway only in the presence of Wnt3a. While aloesin was characterized to directly activate the Wnt/β-catenin pathway and inhibit the Notch pathway independent of Wnt3a. Within 24h, the Aloe vera extract and its two components were failed to affect the proliferation or cell cycle phase of RKO cells. Nevertheless, in the presence of Wnt3a, the aqueous extract of Aloe vera with the concentration of 33.3 μg/ml start to promote the cell proliferation of RKO cells after 48h incubation. Conclusion In conclusion, this study showed that Aloe vera extract and its active component aloin activated the Wnt/β-catenin pathway and inhibited the Notch pathway in the presence of Wnt3a. While another active component, aloesin, activated the Wnt/β-catenin pathway and inhibited the Notch signaling pathway independent of Wnt3a. Given that Wnt/β-catenin and Notch pathway are closely associated with the progression of CRC, these findings would be helpful to better understand the colonic carcinogenicity of Aloe vera. Graphical abstract

Rhodojaponin II attenuates kidney injury by regulating TGF-β1/Smad pathway in mice with adriamycin nephropathy Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Yue Qiu, Junfei Zhou, Hanqi Zhang, Haofeng Zhou, Hui Tang, Chuntao Lei, Chen Ye, Chaoqun You, Yu Chen, Yumei Wang, Jing Xiong, Hua Su, Guangmin Yao, Chun Zhang Abstract Ethnopharmacological relevance Rhododendron molle G. Don (Ericaceae) (RM) is a natural medicinal plant. Its root extracts have been applied in clinic and proved to be effective in chronic glomerulonephritis and rheumatoid arthritis in China. Surprising, little is understood about the key compound of RM and the exact mechanisms underlying its treatment on kidney diseases. In this study, we will explore whether rhodojaponin II (R–II), as the important compound of RM, also exerts the major effect. Materials and methods Mouse model of focal segmental glomerulosclerosis was induced by single dose of adriamycin injection. Induced adriamycin nephropathy (ADRN) mice were treated individually with RM root extract (5 mg/kg, n = 5), RM root extract (60 mg/kg, n = 5), R–II (0.04 mg/kg, n = 6) or captopril (30 mg/kg, n = 5) for five weeks. Podocyte marker (nephrin and podocin) expressions were examined by immunohistochemical staining and Western Blot analysis. Fibronectin level was evaluated by immunohistochemical staining and Western Blot analysis. Interstitial infiltrated inflammatory cells (CD4+ T cells, CD8+ T cells, and CD68+ macrophages) were examined with immunohistochemical staining. The expressions of NF-ĸB p-p65 and TGF-β1/Smad pathway associated key proteins, such as TGF-β1, Smad3, phosphorylated-Smad3 (p-Smad3), and Smad7, were analyzed respectively by Western Blot analysis. Results RM root extract (5 mg/kg) and its important compound R–II (0.04 mg/kg) significantly ameliorated proteinuria, podocyte injury, and glomerulosclerosis, meanwhile, they hampered interstitial fibrosis in mice with ADRN. R–II significantly reduced NF-ĸB p65 phosphorylation, interstitial infiltrated CD4+ T cells, CD8+ T cells, and CD68+ macrophages, at the same time, down-regulated TGF-β1 and p-Smad3 protein expressions in mice with ADRN. Conclusion RM root extract, R–II, could effectively ameliorate proteinuria and kidney injury in ADRN, related to its anti-inflammatory effects, as well as suppression of TGF-β1/Smad signaling pathway. Graphical abstract

Influence of organic anion transporter 1/3 on the pharmacokinetics and renal excretion of ginkgolides and bilobalide Publication date: 28 October 2019 Source: Journal of Ethnopharmacology, Volume 243 Author(s): Peter Yaro, Jing Nie, Mingcheng Xu, Kui Zeng, Houhong He, Jianbiao Yao, Ruwei Wang, Su Zeng Abstract Ethnopharmacological relevance The major terpene lactones of ginkgo biloba extract (GBE) include ginkgolide A, B, C and bilobalide are used for the protection of cardiovascular, cerebrovascular and neurodegenerative diseases. Terpene lactones are orally bioavailable and predominantly eliminated via the renal pathway. However, information on the transporters involved in the pharmacokinetics (PK) and renal excretion of terpene lactones is limited. Aim of the study The objective of this study is to assess the role of OAT1/3 which are important transporters in the human kidney in the PK and renal excretion ginkgolide A, B, C and bilobalide. Materials and methods Uptake of ginkgolide A, B, C and bilobalide in Madin-Darby Canine Kidney (MDCK) and human embryonic kidney 293 (HEK293) cells overexpressing OAT1 or OAT3, respectively were studied. To verify the result from in vitro cell models, the studies on PK, kidney accumulation and urinary excretion of ginkgolide A, B, C and bilobalide were carried out in rats. Results The result showed that ginkgolide A, B, C and bilobalide are low-affinity substrates of OAT1/3. Following co-administration with probenecid, a typical inhibitor of OAT1/3, the rat plasma concentrations of ginkgolide A, B, C and bilobalide increased significantly. AUC showed a significant increase in the probenecid-treated rats compared to control rats (893.48 vs. 1123.85, 314.91 vs. 505.74, and 2724.97 vs. 3096.40 μg/L*h for ginkgolide A, B and bilobalide, respectively), while the clearance of these compounds significantly decreased. The accumulation of ginkgolide A, B and bilobalide in the kidney of the probenecid-treated rats was reduced by 1.8, 2.4, and 1.5-fold, respectively; further reducing the cumulative urinary recovery of these compounds. Conclusion The findings indicated that ginkgolide A, B and bilobalide are excreted via OAT1/3-mediated transport in the kidney and OAT1/3 inhibitor significantly influence the PK ginkgolides and bilobalide. Graphical abstract