Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Hyal-1 inhibitors from the leaves of Phyllanthus muellerianus (Kuntze) Excell John Nii Addotey a , Isabelle Lengers b , Joachim Jose b , Andreas Hensel a,* a University of Münster, Institute of Pharmaceutical Biology and Phytochemistry, Corrensstrasse 48, D-48149, Münster, Germany b University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, Corrensstrasse 48, D-48149, Münster, Germany ARTICLE INFO Keywords: Phyllanthus muellerianus Chebulanin Ellagitannin Hyaluronidase-1 Inflammation Wound-healing ABSTRACT Ethnopharmacological relevance: Leaves and twigs from Phyllanthus muellerianus Kuntze Excell are known to exert anti-inflammatory and antipyretic properties as well as wound healing properties. During a wide screening for human hyaluronidase-1 inhibitors from natural sources leaf extracts from P. muellerianus turned out to show basic anti-hyaluronidase activity. A detailed investigation of this effect should rationalize the potential anti- inflammatory activity of the extract for improved wound healing. Aim of the study: The following study aimed to characterize the anti-Hyal-1 activity of the extract from P. muellerianus and to pinpoint the responsible natural products responsible for this bioactivity. Materials and methods: Using cell surface displayed human Hyal-1 on Escherichia coli, the activity of inhibitors was determined by the stains-all assay method. A hydroalcoholic extract PWE from P. muellerianus was subjected to bioactivity-guided fractionation. Active compounds were characterized by means of mass spectrometry and NMR. Results: PWE exerts a concentration dependent inhibition of Hyal-1 with an IC 50 of 80 μg/mL. Bioassay-guided fractionation revealed 13 compounds from the two most active fractions, mainly ellagitannins and flavonoid glycosides. The most activeHyal-1 inhibitor was found to be the ellagitannin chebulanin 10 (IC 50 132 μM). This represents the first description of chebulanin in P. muellerianus. Conclusions: The hydroalcoholic extract of P. muellerianus, as well as several subfractions obtained during bioassay-guided fractionation showed strong activity against Hyal-1. The main activity can be correlated to the ellagitanin chebulanin. Additionally, also synergistic effects are observed, indicating that the traditional use of aqueous extracts of P. muellerianus is justified, rather than the use of the isolated tannins. The traditional use of the plant as an anti-inflammatory agent for improved wound-healing can be rationalized by the anti-Hyal-1 activities of its constituents. 1. Introduction The leaves from Phyllanthus muellerianus (Kuntze) Exell from the plant family Euphorbiaceae are traditionally used for wound healing in Western Africa (Agyare et al., 2009). Also infections, associated with inflammation and fever have been reported to be treated with aqueous extracts from the leaves (Boakye et al., 2018). The leave extract exerts also antiplasmodial effects (Ndjonka et al., 2012). An aqueous extract from P. muellerianus stem bark has been proven to show antimicrobial activity against Streptococcus and Clostridium species (Brusotti et al., 2011) due to the presence of the alkaloid Nitidine (Cesari et al., 2015). From the phytochemical point of view the leaves are characterized by the presence of the ellagitaninns geraniin, corilagin, and furosin, the flavonoids quercetin-3-O-β-D-glucoside (isoquercitrin), kaempferol-3- O-β-D-glucoside (astragalin), quercetin-3-O-D-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid). Further on brevifolin car- boxylic acid (Agyare, 2010) and the phytosterols 3-friedelanone and β- sitosterol have been described for the herbal material (Saleem et al., 2009). During in vitro investigations for rationalizing the traditional use for wound healing, tests on human keratinocytes indicate that an aqueous leaf extract from P. muellerianus increases the cellular proliferation, the mitochondrial activity and the terminal differentiation of keratinocytes https://doi.org/10.1016/j.jep.2019.03.022 Received 20 November 2018; Received in revised form 5 March 2019; Accepted 7 March 2019 Abbreviations: COSY, correlated spectroscopy; HA, hyaluronic acid; HHDP, hexahydroxydiphenoyl moiety, 6,6′-dicarbonyl-2,2′,3,3′,4,4′-hexahydroxybiphenyl; HMBC, heteronuclear multi-bond correlation; HR-ESI, High resolution electrospray ionization; HSQC, heteronuclear single-quantum correlation; Hyal, human hyaluronidase; E. coli, Escherichia coli. * Corresponding author. E-mail address: ahensel@uni-muenster.de (A. Hensel). Journal of Ethnopharmacology 236 (2019) 326–335 Available online 12 March 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved. T