Motlhatlego et al. BMC Complementary Medicine and Therapies (2020) 20:358 BMC Complementary https://doi.org/10.1186/s12906-020-03139-4 Medicine and Therapies RESEARCH ARTICLE Open Access Inhibitory effect of Newtonia extracts and myricetin-3-o-rhamnoside (myricitrin) on bacterial biofilm formation Katlego E. Motlhatlego1,2, Muna Ali Abdalla1,3* , Carmen M. Leonard4, Jacobus N. Eloff1 and Lyndy J. McGaw1 Abstract Background: Diarrhoea is a major health issue in both humans and animals and may be caused by bacterial, viral and fungal infections. Previous studies highlighted excellent activity of Newtonia buchananii and N. hildebrandtii leaf extracts against bacterial and fungal organisms related to diarrhoea-causing pathogens. The aim of this study was to isolate the compound(s) responsible for antimicrobial activity and to investigate efficacy of the extracts and purified compound against bacterial biofilms. Methods: The acetone extract of N. buchananii leaf powder was separated by solvent-solvent partitioning into eight fractions, followed by bioassay-guided fractionation for isolation of antimicrobial compounds. Antibacterial activity testing was performed using a broth microdilution assay. The cytotoxicity was evaluated against Vero cells using a colorimetric MTT assay. A crystal violet method was employed to test the inhibitory effect of acetone, methanol: dichloromethane and water (cold and hot) extracts of N. buchananii and N. hildebrandtii leaves and the purified compound on biofilm formation of Pseudomonas aeruginosa, Escherichia coli, Salmonella Typhimurium, Enterococcus faecalis, Staphylococcus aureus and Bacillus cereus. Results: Myricetin-3-o-rhamnoside (myricitrin) was isolated for the first time from N. buchananii. Myricitrin was active against B. cereus, E. coli and S. aureus (MIC = 62.5 μg/ml in all cases). Additionally, myricitrin had relatively low cytotoxicity with IC50 = 104 μg/ml. Extracts of both plant species had stronger biofilm inhibitory activity against Gram-positive than Gram-negative bacteria. The most sensitive bacterial strains were E. faecalis and S. aureus. The cold and hot water leaf extracts of N. buchananii had antibacterial activity and were relatively non-cytotoxic with selectivity index values of 1.98–11.44. Conclusions: The purified compound, myricitrin, contributed to the activity of N. buchananii but it is likely that synergistic effects play a role in the antibacterial and antibiofilm efficacy of the plant extract. The cold and hot water leaf extracts of N. buchananii may be developed as potential antibacterial and antibiofilm agents in the natural treatment of gastrointestinal disorders including diarrhoea in both human and veterinary medicine. Keywords: Newtonia, Fabaceae, Diarrhoea, Antimicrobial, Cytotoxicity, Biofilm formation * Correspondence: [email protected] 1Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa 3Department of Food Science and Technology, Faculty of Agriculture, University of Khartoum, 13314 Khartoum North, Sudan Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Motlhatlego et al. BMC Complementary Medicine and Therapies (2020) 20:358 Page 2 of 10 Background avoid antibiotic activity, the organic and aqueous ex- Diarrhoea is a neglected disease responsible for over tracts as well as the purified compound were investi- 700,000 deaths annually of children under the age of five gated for their ability to inhibit biofilm formation. worldwide [1]. Ciprofloxacin has been used for the treat- ment of gastrointestinal infections, such as diarrhoea, for Methods decades [2]. The over-prescribing and incorrect use of Plant material and extraction antibiotics commonly used to treat diarrhoea and other The plant species were collected from labelled trees in infections have led to global antibiotic resistance against the Lowveld National Botanical Garden in Nelspruit, several microbes. This has greatly impacted on the effi- Mpumalanga, South Africa in December 2014. Voucher cacy of most available antibacterial drugs [3]. Antibiotics specimens (PRU 122347 for N. hildebrandtii and PRU are less effective when biofilms form because of the rela- 122348 for N. buchananii) were prepared and lodged in tive impermeability of biofilms, the variable physiological the H.G.W.J. Schweickerdt Herbarium at the University status of microorganisms, subpopulations of persistent of Pretoria (South Africa) for reference purposes. strains, the presence of variations of phenotypes and the The collected plant material was dried at room expression of genes involved in the general stress re- temperature in a well-ventilated room and ground to a sponse [4]. Consequently, biofilms are a recognized fine powder in a Macsalab Mill (Model 2000 LAB Eriez). source of recurrent, persistent or sporadic bacterial One gram of each plant part (leaves and stems, and in infections [5, 6]. the case of N. buchananii also the seeds and seedpods The treatment of infection has become difficult be- combined) was separately extracted in 10 ml of acetone, cause the biofilm mode of microbial growth has in- 1:1 MeOH-DCM (technical grade, Merck), cold distilled creased the survival strategies and resistance levels of water, or boiling distilled water in polyester centrifuge microbes to drugs [3]. tubes. The tubes were vigorously shaken for 30 min on Many drugs have been developed that were originally an orbital shaker, then centrifuged at 4000 x g for 10 sourced from natural products [7]. Plants contain a var- min. The supernatant was filtered through Whatman iety of secondary metabolites such as alkaloids, flavo- No.1 filter paper before it was transferred into pre- noids, glycosides, phenols, saponins, steroids, terpenoids weighed glass containers. This was repeated thrice on and tannins [8]. These metabolites may have individual the same plant material and the solvent was removed by bioactivity, or may act together synergistically to disrupt evaporation under a stream of air in a fume hood at growth or pathogenic pathways of disease-causing or- room temperature to yield the dried crude extract. For ganisms [9]. isolation purposes, a similar extraction method was Some natural products are known to inhibit biofilm followed where 300 g of N. buchananii leaf material was formation or preformed biofilms [3]. Plants are therefore extracted in 3000 ml of acetone (technical grade, Merck) a potential source of novel antibiofilm agents [4, 10, 11] in a 5 L glass bottle. The bottle was vigorously shaken worthy of further investigation. and left overnight and the supernatant was filtered A wide variety of medicinal plants are used in south- through Whatman No.1 filter paper before it was trans- ern Africa to treat gastrointestinal ailments as well as ferred into pre-weighed glass containers. The extraction other infections. Two of these species include Newtonia yield was calculated as follows: hildebrandtii (Vatke) Torre and Newtonia buchananii (Baker) G.C.C. Gilbert & Boutiqu of the family Fabaceae, Plant crude extraction yield ðÞ% which are used for the treatment of skin conditions and Mass of dried extractðÞ g ¼ Â 100 wounds and for an upset stomach [12, 13]. In a previous Mass of plant powder extractedðÞ g study, the acetone and dichloromethane:methanol (1:1) extracts of the leaves and stems of the two species had very good antibacterial activity against a range of bacter- Antimicrobial assay ial species with minimum inhibitory concentration The antimicrobial activity of the water extracts was de- (MIC) values as low as 0.02 mg/ml [14]. This motivated termined against the following bacteria: Staphylococcus the present study where the isolation of compounds re- aureus (ATCC 29213), Bacillus cereus (ATCC 21366), sponsible for this promising activity was undertaken. Enterococcus faecalis (ATCC 29212), Escherichia coli Additionally, hot and cold water extracts of the two spe- (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) cies were included to more closely replicate the trad- and Salmonella enterica subsp. enterica serovar Typhi- itional methods of preparation and to provide evidence murium (ATCC 39183). The antimicrobial activity was to support their use in traditional medicine against evaluated in terms of minimum inhibitory concentration stomach upsets. As biofilms are an important mechan- (MIC) using a rapid broth microdilution