P iodonitrotetrazolium chloride

HPLC-grade acetonitrile was purchased from Fisher Scientific (Lisbon, Portugal). Standards of gallic acid and catechin were acquired from Sigma (St. Louis, MO, USA), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene from Alfa Aesar (Ward Hill, MA, USA), and phenolic compounds from Extrasynthese (Genay, France). Muller–Hinton broth (MHB), tryptic soy broth (TSB), blood agar with 7% sheep blood, and MacConkey agar plates were obtained from Biomerieux (Marcyl’Etoile, France). The dye p-iodonitrotetrazolium chloride (used as a microbial growth indicator) was provided by Sigma-Aldrich (St. Louis, MO, USA), while propanediol, citric acid, and triethanolamine were from Sigma (St. Louis, MO, USA). Ethanol and all other solvents and chemicals were of analytical grade and provided by Merck (Darmstadt, Germany). A Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA) was used to treat water.

Tryptone Soya broth was purchased from HIMEDIA® (Himedia Laboratories Pvt Ltd, Mumbai, INDIA; p-Iodonitrotetrazolium chloride (INT) from SIGMA® (Sigma- Aldrich®, St Louis, USA); pantoprazole (lyophilized powder for i.v injection; Batch No. JKJ 3534D and manufactured by Sun Pharmaceutical Industries Ltd, Halol-Baroda Highway, Halol-389350, Gujarat, India.), was sourced from a local pharmacy. Ethanol (absolute) was purchased from Fluka Chemie GmbH (Sigma-Aldrich®, Zwijndrecht, Netherlands), while dimethyl sulfoxide (DMSO) was purchased from Sigma® (Poole, Dorset, UK). Cyclophosphamide, NEOPHOS 500® (CIPLA Ltd, MIDC Boisar, INDIA) was purchased from a local Pharmacy in Dar es Salaam, Tanzania. Escherichia coli (ATCC 25922), Salmonella typhi (NCTC 8385), Vibrio cholerae (clinical isolate), and Klebsiella pneumoniae (clinical isolate) were obtained from the Department of Microbiology and Immunology, Muhimbili University of Health and Allied Sciences (MUHAS). The brine shrimp eggs were bought from Aquaculture Innovations (Grahamstown 6140, South Africa) and sea salt was prepared locally by evaporating water collected from the Indian Ocean, along the Dar es Salaam Coast.

Determination of MIC was performed according to a method described by Eloff [76 (link)], with some modifications by Magangana et al. [45 (link)]. Briefly, the microorganisms used in this study consisted of two Gram-negative bacteria, Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883, and two Gram-positive bacteria, Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600. Streptomycin (100 µg/mL; Sigma Aldrich) was used as a positive control and bacteria-free broth, 70% (v/v) ethanol, and sterile distilled water were used as negative controls. The final concentration of peel extracts ranged from 19.53 to 2500 µg/mL, while Streptomycin concentrations ranged from 0.20 to 25 µg/mL in the respective wells. The plates were left for 18 h at 37 °C for incubation purposes. After this period, 40 µL of p-iodonitrotetrazolium chloride (Sigma-Aldrich, Darmstadt, Germany) was added to the well plates to show bacterial growth. The results were expressed in terms of the MIC value, with this test performed in triplicate.

The reference antibiotics tested in the present work were: chloramphenicol (CHL), tetracycline (TET), ciprofloxacin (CIP), streptomycin (STR), erythromycin (ERY) and kanamycin (KAN) obtained from Sigma-Aldrich (St. Quentin Fallavier, France). p-Iodonitrotetrazolium chloride ≥ 97% (INT, Sigma-Aldrich) was used as microbial growth indicator meanwhile phenylalanine-arginine-β-naphthylamide (PAßN) (Sigma-Aldrich) was used as efflux pump inhibitor (EPI).

The chemicals used in antimicrobial assays were chloramphenicol ≥98 % (Sigma-Aldrich, St-Quentin-Fallavier, France) as reference antibiotic and p-Iodonitrotetrazolium chloride ≥97 % (INT, Sigma-Aldrich) as microbial growth indicator (Eloff 1998 (link); Mativandlela et al. 2006 (link)). Phenylalanine-Arginine-β-Naphthylamide (PAβN; Sigma-Aldrich) was used as efflux pumps inhibitor (EPI). Natural products (Figs. 1, 2) used in the study were obtained from the chemical bank of the natural products research laboratory of the Chemistry Department, University of Nairobi, Kenya. Isolation and identification of the compounds in study were previously reported from the following plants; a number of Rumex species including; Rumex dentatus, R. abyssinicus, R. usambarensis, R. bequaertii, R. ruwenzoriensis, R. crispus; Plumbago zeylanica,Myrsine africana, Maesa lanceolata, Rapanea melanphloes, Aloe saponaria, Erythrina abyssinica, Polygonum senegalense, Psiadia punctulata, Dodonaea angustifolia and Senecio roseiflorus.

Ciprofloxacin (BDH Chemicals, England) and oxytetracyclin (BDH Chemicals, England) were used as reference antibiotics. P-iodonitrotetrazolium chloride (Sigma-Aldrich, Germany) was used as microbial growth indicator.

Soft agar assays were performed in 35 mm dishes, which were layered first with 0.5% agar (Sigma Aldrich, Germany) in cell culture medium, followed by 0.35% agar in cell culture medium containing 5000 cells. Colonies were stained with p-iodonitrotetrazolium chloride (1 mg/mL, Sigma Aldrich, Germany), incubated overnight at 37 °C, and photographed.