In vitro antimycobacterial and cytotoxic data on medicinal plants used to treat tuberculosis

Data in Brief Volume 7, June 2016, Pages 1124-1130 open access Data Article Author links open overlay panelJoseph M.NgutaabReginaAppiah-OpongaAlexander K.NyarkoaDorothyYeboah-ManucPhyllis G.A.AddodIsaac D.OtcherecAbenaKissi-Twuma Show more https://doi.org/10.1016/j.dib.2016.03.088Get rights and content Open Access funded by Bill & Melinda Gates Foundation Under a Creative Commons license Refers to Joseph M. Nguta, Regina Appiah-Opong, Alexander K. Nyarko, Dorothy Yeboah-Manu, Phyllis G.A. Addo, Isaac Otchere, Abena Kissi-Twum Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana b Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Nairobi, Kenya c Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana d Department of Animal Experimentation, Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana Antimycobacterial and cytotoxic activity of selected medicinal plant extracts Journal of Ethnopharmacology, Volume 182, 22 April 2016, Pages 10-15 PDF (270KB) Abstract This article contains data on in vitro antimycobacterial activity and cytotoxicity of hydroethanolic crude extracts from five selected medicinal plant species traditionally used to treat tuberculosis in Ghanaian ethnomedicine, see “Medicinal plants used to treat TB in Ghana” [1]. The interpretation and discussion of these data and further extensive insights into drug discovery against tuberculosis from natural products of plant biodiversity can be found in “Antimycobacterial and cytotoxic activity of selected medicinal plant extracts” [2]. Previous article in issueNext article in issue Keywords in vitro antimycobacterialin vitro cytotoxicityCrude extractsTuberculosisMedicinal plantsMTS assayMABA assay Specification table Subject area Pharmacology and Toxicology More specific subject area Drug discovery against tuberculosis Type of data Tables How data was acquired in vitro antimycobacterial data was acquired using Microplate alamar blue assay (MABA), while cytotoxicity data was generated using MTS assay, with absorbance being read at 490 nm using Infinite M200 ProTMplate reader (Tecan, Austria, GmbH) Data format Analyzed Experimental factors Test samples extracted with 80% ethanol by cold maceration, concentrated and lyophilized. Test crude extracts were dissolved in distilled water. Positive controls were prepared according to manufacturer׳s instructions Experimental features MABA was utilized to generate MIC values while MTS assay was used to evaluate cytotoxicity. Data source location Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana and Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Nairobi, Kenya Data accessibility Data within this article Value of the data • This data provides a comprehensive in vitro antimycobacterial activity of hydroethanolic total extracts from five selected medicinal plants which can serve as a benchmark for other researchers. • This data set includes in vitro cytotoxicity values from five selected ethnobotanicals and reference anti-TB drugs which can be used as a basis for further research. • This data may provide insights for future drug development against tuberculosis. • The data set can be used to guide further isolation of anti-TB compounds. • This data set can be used for external validation of data acquired from similar experiments. 1. Data Data on comparative antimycobacterial activity of total hydroethanolic extracts derived from five medicinal plant species is presented (Table 1). Data on in vitro cytotoxic activity of tested plant species and positive control drugs, isoniazid, rifampicin and ethambutol against MRC-5, human fetal lung fibroblast cell line (ATCC® CCL-171™) is shared in Table 2. Table 1. Comparative minimum inhibitory concentration (MIC in mg/mL) values of selected plant species against pathogenic and non-pathogenic mycobacterial strains. Solanum torvum Chenopodium ambrosioides Dissotis rotundifolia Zingiber officinale Aloe vera var. barbadensis Isoniazid Rifampicin Ethambutol M. tb H37Rv(ATCC® 27294™) 1.25 >10 5 10 5 0.00003 0.00003 0.002 M. smegmatis(ATCC® 19420™) 2.5 10 10 10 10 0.002 0.002 0.00025 M. tb; Strain H37Ra(ATCC® 25177™) 0.1563 5 >10 2.5 2.5 0.00008 0.0005 0.0005 Table 2. Cytotoxic activity of selected medicinal plant species against human fetal lung fibroblast cell line, MRC-5 (ATCC® CCL-171™). Plant species Concentration (mg/mL) Absorbance 1 Absorbance 2 Absorbance 3 Mean Corrected mean Standard deviation % cell viability Blank Negative control Solanum torvum 0 0.2509 0.2509 0.2509 0.2509 0.1597 0.000 100.00 0.0865 0.2578 0.625 0.4418 0.4496 0.4434 0.4449 0.2076 0.004 130.01 0.0969 0.2687 1.25 0.4168 0.3984 0.3960 0.4037 0.0531 0.0110 33.27 0.0950 0.2651 2.5 0.6005 0.6030 0.5825 0.5953 0.1527 0.0110 95.64 0.0814 0.2674 5 0.9941 0.9438 0.9356 0.9578 0.3567 0.0320 223.38 0.0985 0.2696 10 1.4339 1.5394 1.4953 1.4895 0.1869 0.053 117.05 0.0980 0.2625 Chenopodium ambrosioides 0 0.2509 0.2509 0.2509 0.2509 0.1597 0.000 100.00 0.0895 0.1989 0.625 0.2614 0.2596 0.1789 0.2333 0.1011 0.0470 63.31 0.0957 0.2469 1.25 0.2678 0.2920 0.2117 0.2572 0.1209 0.0410 75.68 0.1566 0.2556 2.5 0.2947 0.3078 0.1921 0.2649 0.1324 0.0630 82.88 0.0912 0.2578 5 0.2986 0.3264 0.2005 0.2752 0.1232 0.066 77.12 0.1003 0.2372 10 0.1801 0.2119 0.1384 0.1768 −0.0162 0.0370 −10.14 0.091 0.2253 Dissotis rotundifolia 0 0.2509 0.2509 0.2509 0.2509 0.1597 0.000 100.00 0.087 0.2023 0.625 0.2994 0.3332 0.3428 0.3251 0.0762 0.0230 47.74 0.1063 0.2603 1.25 0.3127 0.3252 0.3503 0.3294 0.0150 0.019 9.39 0.1 0.2690 2.5 0.2960 0.3342 0.3421 0.3241 −0.0405 0.025 −25.36 0.09 0.2728 5 0.4618 0.4840 0.5069 0.4842 −0.0603 0.023 −37.74 0.1042 0.2676 10 0.7912 0.7025 0.8740 0.7892 −0.2213 0.0860 −138.5 0.0876 0.2699 Zingiber officinale 0 0.2509 0.2509 0.2509 0.2509 0.1597 0.000 100.00 0.0979 0.0912 0.625 0.2692 0.2796 0.2649 0.2712 0.1322 0.008 82.80 0.0892 0.1390 1.25 0.3164 0.3387 0.2655 0.3069 0.1732 0.038 108.43 0.0880 0.1337 2.5 0.1681 0.1577 0.1858 0.1705 0.0216 0.014 13.55 0.0788 0.1489 5 0.1874 0.2249 0.2719 0.2281 0.0311 0.042 19.45 0.0879 0.1970 10 0.3833 0.4711 0.4020 0.4188 0.1590 0.0460 99.56 0.0942 0.2598 Aloe vera var. barbadensis 0 0.2509 0.2509 0.2509 0.2509 0.1597 0.000 100.00 0.0888 0.0912 0.625 0.2902 0.2886 0.2770 0.2853 0.1269 0.007 79.44 0.0854 0.1584 1.25 0.2863 0.2773 0.2625 0.2754 0.1389 0.0120 86.95 0.0916 0.1365 2.5 0.3027 0.2891 0.2806 0.2908 0.1258 0.0110 78.77 0.0812 0.1650 5 0.1907 0.1852 0.1938 0.1899 −0.0148 0.004 −9.27 0.0845 0.2047 10 0.1964 0.2024 0.2019 0.2002 −0.0319 0.003 −19.95 0.0893 0.2321 Isoniazid 0 0.3547 0.3547 0.3547 0.3547 0.1427 0.000 100.00 0.2136 0.2120 0.625 0.4501 0.3552 0.3296 0.3783 0.1569 0.0630 109.95 0.2117 0.2214 1.25 0.3441 0.3282 0.3565 0.3429 0.1214 0.0140 85.097 0.2064 0.2215 2.5 0.3815 0.3882 0.3476 0.3724 0.1533 0.0220 107.45 0.2097 0.2191 5 0.3327 0.4934 0.3431 0.3897 0.1748 0.0900 122.51 0.2279 0.2149 10 0.4652 0.3455 0.3621 0.3909 0.1701 0.0650 119.22 0.2303 0.2208 Rifampicin 0 0.3547 0.3547 0.3547 0.3547 0.1427 0.000 100.00 0.2139 0.2120 0.625 0.4120 0.3482 0.3352 0.3651 0.1468 0.041 102.897 0.2089 0.2183 1.25 0.3450 0.4193 0.3511 0.3718 0.0936 0.041 65.592 0.2098 0.2782 2.5 0.3597 0.3825 0.3529 0.3650 0.1033 0.0160 72.413 0.2085 0.2617 5 0.3514 0.3610 0.3770 0.3631 0.1268 0.013 88.881 0.2070 0.2363 10 0.3859 0.3830 0.4586 0.4092 0.1606 0.043 112.52 0.2319 0.2486 Ethambutol 0 0.3547 0.3547 0.3547 0.3547 0.1427 0.000 100.00 0.2025 0.2120 0.625 0.3909 0.3756 0.3550 0.3738 0.1557 0.0180 109.133 0.2128 0.2181 1.25 0.3380 0.3549 0.3442 0.3457 0.1231 0.009 86.265 0.18112 0.2226 2.5 0.3498 0.3287 0.3309 0.3365 0.1249 0.0120 87.503 0.2132 0.2116 5 0.3447 0.3696 0.3254 0.3466 0.1354 0.0220 94.861 0.1739 0.2112 10 0.3511 0.3396 0.3304 0.3404 0.1037 0.0100 72.647 0.2096 0.2367 2. Experimental design, materials and methods 2.1. Antibiotics and chemicals Antimycobacterial reference standards [(ethambutol hydrochloride, European pharmacopoeia (EP) reference standard (EMB); Isoniazid >99%, (INH) and rifampicin 95% (RIF)] were all obtained from Sigma-Aldrich (St. Louis, MO) and stock solutions at 1000 μg/mL filter sterilized and stored at −20 °C until use. Middlebrook 7H9 broth supplemented with 0.05% (vol/vol) Tween 80 (Sigma); 10% OADC (oleic acid, albumin, dextrose, and catalase); Becton-Dickinson; 0.1% casitone and 0.5% glycerol was used to prepare working solutions at the following concentrations: RIF and EMB, 16 μg/mL; INH, 1.6 μg/mL and crude extracts, 10,000 μg/mL. A mixture of 10% Tween 80 and 10× Alamar blue dye (Alamar Biosciences/Accumed, Westlake, OH), at the ratio of 1:1 was prepared and stored at 4 °C for up to 1 week after sterilization by filtration. 2.2. Collection and preparation of plant materials The rhizomes of Zingiber officinale and the leaves of Chenopodium ambrosioides, Dissotis rotundifolia, Aloe vera var. barbadensis and Solanum torvum were collected from the eastern region of Ghana, identified and prepared for bioassays as earlier described [1,2]. 2.3. Antimycobacterial assay 2.3.1. Mycobacterial strains and growth conditions Non-pathogenic fast growing mycobacterial strain, Mycobacterium smegmatis (ATCC®19420™), non-pathogenic slow growing strain, Mycobacterium tuberculosis; Strain H37Ra (ATCC® 25177™) and slow growing pathogenic laboratory strain, Mycobacterium tuberculosis subsp.tuberculosis (ATCC® 27294™) were obtained from American Type Culture Collection (Manassas, VA 20108, USA). The mycobacterial strains were maintained on Lowenstein–Jensen slopes and cultured on enriched media comprising of Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with 0.05% (vol/vol) Tween 80 (Sigma); 10% (vol/vol) OADC (oleic acid, albumin, dextrose, catalase; Difco), 1.0 g of casitone (Difco) per liter and 0.2% (vol/vol) glycerol (Sigma Chemical Co., Saint Louis, MO). The enriched culture medium was referred to as 7H9GC-Tween. Cultures were incubated at an aerobic atmosphere at 37 °C. 2.3.2. Microplate alamar blue assay (MABA) Mycobacterial strains were maintained on Lowenstein Jensen slopes, while the test inoculum was prepared in 7H9GC-tween broth, adjusted to a no. 1 McFarland tube standard, to give approximately 3.0×108 cells and further diluted at the ration of 1:10 in the enriched broth (7H9GC-Tween) for anti-TB bioassays [2,3]. The assay was conducted as earlier described by Nguta et al. [2] and Palomino et al. [3]. Briefly, 100 μL of the enriched broth was dispensed in each well of a sterile flat-bottom 96-well plate, followed by serial two-fold dilutions of the crude extracts. The positive control drugs were prepared directly in the microtiter plate. One hundred microliters (100 μL) of the prepared inoculum was added to each well. A sterile control comprising of the medium only and a growth control consisting of cells and growth medium were also included for each experiment. Evaporation during the incubation period was minimized by addition of sterile water to all perimeter wells of the 96-well microtiter plate. The covered microtiter plates were sealed in plastic bags and incubated at 37 °C under a normal atmosphere. After 7 days of incubation for M. tuberculosis H37Ra and M. tuberculosis subsp. tuberculosis, and 48 h of incubation for M. smegmatis, 30 μL of a mixture of 10% tween 80 and alamar blue solution in a ratio of 1:1 was added to each well, and the plate incubated overnight. A color change from blue to pink indicated mycobacterial growth, and the minimum inhibitory concentration (MIC) was taken as the highest dilution of the crude extract or positive control drug that prevented color change from blue to pink. The drug and crude extract concentration ranges used for the experiments were as follows: for positive controls, INH, 0.025–1.6 μg/mL, EMB and RIF, 0.25–16 μg/mL and for crude extracts, 10,000–19.5 μg/mL. All the tests were run in triplicate in a biosafety level three (BSL-3) laboratory. 2.4. in vitro cytotoxic evaluation 2.4.1. Chemicals and reagents Chemicals and reagents for cytotoxic investigations were procured from Promega (USA), Sigma (USA) and Gibco (USA). 2.4.2. MRC-5 cell lines and culture conditions Human fetal lung fibroblast cell line, MRC-5 (ATCC® CCL-171™) was procured from the American Type Culture Collection (ATCC) (Manassas, VA 20108, USA). Cultures for cytotoxic evaluation were maintained in a phenol red free Dulbecco׳s modified essential medium/Ham׳s 12 nutrient mixture (DMEM/F12), (Gibco), supplemented with 1% (vol/vol) antibiotic [(2 mM L-glutamine, 100 U/mL Penicillin and 100 μg/mL Streptomycin; (Gibco)] and 5% [(vol/vol) fetal calf serum (JS Bioscience, Australia)]. MRC-5 fibroblast cells were incubated at 37 °C in a humidified 5% carbon dioxide incubator. The culture medium was removed from the flask and the cells were rinsed three times with sterile Hank׳s Balanced Salt Solution (HBSS), (Gibco), once the cells reached confluence. Trypsin/EDTA (Gibco, USA), was used to enzymatically remove the confluent layers, followed by suspension in culture medium. A light microscope (Leitz Wetzlar, Germany) was used to determine cell numbers after vital staining with trypan blue (0.4% (wt/vol); Sigma, USA). 2.4.3. Preparation of test extracts Total (crude) extracts were suspended in enriched phenol free Dulbecco׳s modified essential medium/Ham׳s 12 nutrient mixture (DMEM/F12), (Gibco) at the concentration of 10 000 μg/mL and dispersed by ultrasonic vibration for 15 min. Crude extracts were stirred on a vortex for one minute before every use to ensure uniform suspension. 2.4.4. MTS assay in vitro cytotoxicity assay was performed using the Promega CellTiter 96 AQueous Non-Radioactive Cell Proliferation (MTS) assay as earlier described [2,4]. The procedure for cytotoxic evaluation was adopted from manufacturer׳s instructions and previously published papers ([5,6]). Total extracts were suspended in enriched Dulbecco׳s modified essential medium/Ham׳s/12 nutrient mixture, followed by serial dilution across 96-well microtiter plates (100 μL), and incubation at 37 °C with 5% carbon dioxide (CO2) for a period of 24 h in a humidified CO2 incubator. Four (4 h) hours prior to the end of each experiment, an MTS mixture (20 μL/well) was added to each of the test wells of the 96-well microtiter plate. The plates were then placed on a microwell plate reader (Tecan, Austria, GmbH), shaken for 10 s and the absorbance of the formazan product read at 490 nm after the completion of each exposure period. Each experiment was run in triplicate. For each experiment, two internal controls were set: (i) IC100 consisting of medium only and (ii) an IC0 consisting of cells only. Background absorbance due to the non-specific reaction between the MTS reagent and the crude extracts was deducted from exposed cell values [7]. Conflict of interest declaration We declare no conflict of interest. Acknowledgments Authors are thankful to the Bill and Melinda Gates Foundation (BMGF, Grant No. OPP52155) for funding the study through Noguchi Memorial Institute for Medical Research postdoctoral program on infectious diseases. We also acknowledge Mr. Heron Blogogyee of Centre for Scientific Research into Plant Medicine (CSRPM-Mampong) for assistance in plant collection, identification and preparation. Appendix A. Supplementary material Download Word document (45KB)Help with doc files Supplementary material References [1] J.M. Nguta, R. Appiah-Opong, A.K. Nyarko, D. Yeboah-Manu, P.G.A. Addo Medicinal plants used to treat TB in Ghana Int. J. Mycobacteriol., 4 (2015), pp. 116-123 ArticlePDF (2MB)CrossRefView Record in Scopus [2] J.M. Nguta, R. Appiah-Opong, A.K. Nyarko, D. Yeboah-Manu, P.G.A. Addo, I.D. Otchere, A. Kissi-Twum Antimycobacterial and cytotoxic activity of selected medicinal plant extracts J. Ethnopharmacol., 182 (2016), pp. 10-15 ArticlePDF (270KB)View Record in Scopus [3] J.C. Palomino, A. Martin, M. Camacho, H. Guerra, J. Swings, F. Portaels Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis Antimicrob. Agents Chemother., 46 (2002), pp. 2720-2722 CrossRefView Record in Scopus [4] Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay Technical Bulletin #TB169, Promega Corporation, Madison, USA (2005) [5] S. Bakand, A. Hayes, C. Winder, C. Khalil, B. Markovic, In vitro cytotoxicity testing of airborne formaldehyde collected in serum-free culture media. Toxicol. Ind. Health 21 (2005a) 147–154. [6] S. Bakand, C. Winder, C. Khalil, A. Hayes, Toxicity assessment of industrial chemicals and airborne contaminants: transition from in vivo to in vitro test methods: A review. Inhal. Toxicol. 17 (2005b) 775–787 [7] A.J. Hayes, B. Markovic Toxicity of Australian essential oil Backhousia citriodora (Lemon myrtle). Part 1. Antimicrobial activity and in vitro cytotoxicity Food Chem. Toxicol., 40 (2002), pp. 535-543 ArticlePDF (125KB)View Record in Scopus © 2016 The Authors. Published by Elsevier Inc.