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Anti-mycobacterial, -oxidative, -proliferative and -inflammatory activities of dichloromethane leaf extracts of Gymnosporia senegalensis (Lam.) Loes
South African Journal of Botany 114 (2018) 217–222
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Anti-mycobacterial, -oxidative, -proliferative and -inflammatory activities of dichloromethane leaf extracts of Gymnosporia senegalensis (Lam.) Loes M.E. Makgatho a,⁎, W. Nxumalo b, L.A. Raphoko b a b
Department of Pathology and Medical Sciences, University of Limpopo, South Africa Department of Chemistry, University of Limpopo, South Africa
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i n f o
Article history: Received 6 September 2017 Received in revised form 28 October 2017 Accepted 3 November 2017 Available online xxxx Edited by J Van Staden Keywords: Gymnosporia senegalensis DPPH Dichloromethane Cell proliferation Nitric oxide Tumour necrosis factor-alpha Antioxidant activity Mycobacterium tuberculosis
a b s t r a c t A crude dichloromethane leaf extract of Gymnosporia senegalensis and its six fractions were tested for anti-mycobacterial activity using a fluorescent microplate assay against the H37Rv strain of Mycobacterium tuberculosis as well as antioxidant activity using the cell free based assays, DPPH and ferric ion reducing power as well as a colorimetric assay for measuring nitric oxide production in RAW 264.7 cells. The in vitro anti-inflammatory potential of fractions was screened by measuring tumor necrosis-alpha production in RAW 264.7 cells. The test agents exhibited anti-mycobacterial activity in the following sequence; SP2-1F5 N crude extract N SP2-1F2 N SP2-1F6 N SP2-1F1 N SP2-1F3 N SP2-1F4 with minimum inhibitory values ranging from 21.4 to 54.2 μg/mL. Fractions SP2-1F1, SP2-1F3 and SP2-1F5 exhibited significant antioxidant activities at concentrations between 125 μg/ mL and 500 μg/mL employing the cell free-based and colorimetric assays. The crude extract of the plant leaf also exhibited ferric ion reducing power at 250 and 500 μg/mL. Fractions SP2-1F1, SP2-1F3 and SP2-1F5 showed noticeable tumor necrosis factor-alpha production in RAW cell at 125 to 500 μg/mL. The laboratory activities conducted in the present study presumably indicate that the leaf extract of Gymnosporia senegalensis contain chemical entities that exhibit in vitro inhibition of mycobacterium growth, production of nitric oxide and tumor necrosis factor-alpha in a mouse macrophage cell line as well as increased radical scavenging potential. Further laboratory studies are to be conducted in pursuance of the active chemical ingredients of leafs of this plant species. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Escalating levels of Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) are a challenging public health concern especially in low income countries (Velayati et al., 2013). The emergence of drug resistance and human immunodeficiency virus (HIV) co-infection exacerbates the situation (WHO, 2015). Active tuberculosis is perpetuated by harmful, proinflammatory immune mechanisms that respond to stimulation and activation of alveolar macrophages by microbial signals (Cooper, 2009; Ferrara et al., 2009; Phillips and Ernst, 2012; Zumla et al., 2013; Pai et al., 2016). Typical biological mediators that drive this inflammatory paradigm are; interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α) and nitric oxide (Flynn and Chan, 2001; Ahmad, 2011; Zuñiga et al., 2012; Amaral et al., 2016). The use of alternative strategies such as complementary medicinal products to treat TB and alleviate the concomitant complications should be explored (Green ⁎ Corresponding author at: Department of Pathology and Medical Sciences, School of Health Care Sciences, Faculty of Health Sciences, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa. E-mail address: [email protected] (M.E. Makgatho).
https://doi.org/10.1016/j.sajb.2017.11.002 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
et al., 2010; Nguta et al., 2016). Bioactive metabolites from medicinal plants have provided a rich source for the development of new drugs against infectious diseases and modulations of the immune system (Cragg and Newman, 2013; Nguta et al., 2015). A number of research groups in South Africa are on a venture to identify and extract novel anti-mycobacterial compounds (Lall et al., 1999; Lall and Meyer, 1999; Eldeen and Van Staden, 2008; McGaw et al., 2008; Green et al., 2010; Labuschagné et al., 2012; Mulaudzi et al., 2012; Aro et al., 2016; Komape et al., 2017). Gymnosporia senegalensis (Lam.) Loes, alternatively named Maytenus senegalensis (Lam.) Excell is a small tree or tall shrub of the family Celastracae widely distributed in Arabia, Africa, Afghanistan and India (Hedberg et al., 1982; Gessler et al., 1995; Sosa et al., 2007; Malebo et al., 2015). African traditional healers use the root and stem bark to treat a number of ailments like malaria, rheumatism, dysmenorrhoea, chest pains, wounds, chronic illness, dyspepsia eye infection and diarrhoea (El Tahir et al., 1999; Hussein et al., 1999; Da Silva et al., 2010; Ahmed et al., 2013). The root, stem bark and leaf extracts of Gymnosporia senegalensis have been shown to exhibit anti-inflammatory activity in animal models of experimental inflammation like Croton oil-induced ear oedema and carrageenan-induced paw oedema (Sosa
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et al., 2007; Da Silva et al., 2017). A number of researchers have documented the antimicrobial activities of extracts from various parts (stem, leaf, bark and root) of Gymnosporia senegalensis (Matu and Van Staden, 2003; Clarkson et al., 2004; Malebo et al., 2015. Most importantly, Lall and Meyer demonstrated the antimycobacterial activity of acetone and water leaf extracts of this plant against a resistant strain of Mycobacterium tuberculosis (Lall and Meyer, 1999). In the present study, crude dichloromethane (DCM) leaf extract of Gymnosporia senegalensis and its six fractions were screened for activity against the H37Rv strain of Mycobacterium tuberculosis and antioxidant activity using the cell free based DPPH and ferric reducing power assays. DCM crude leaf extract fractions with substantive antioxidant activity were further assayed for their effect on nitric oxide and TNF-α production in a murine macrophage RAW 264.7 cell line.
2. Materials and methods Unless otherwise indicated, all chemicals and reagents were obtained from Sigma-Aldrich (Johannesburg, South Africa).
2.1. Plant material collection and preparation of extracts Gymnosporia senegalensis leaves were supplied by traditional healers in Tzaneen, Limpopo Province (South Africa) deposited in the University of Limpopo Herbarium with voucher number 1145. The leaves were cleaned and dried in an oven at 37 °C for 7 days. Dichloromethane (500 mL) was added to a 20 g dried powder from the leaves of Gymnosporia senegalensis in a 1000 mL volumetric flask and the resulting mixture stirred at room temperature for 24 h. The mixture was filtered to remove undissolved material and the filtrate washed twice with 10 mL DCM. The solvent was removed in a rotary evaporator to give 0.6412 g of crude extract. The crude extract was then subjected to preparative thin layer chromatography (prep-TLC), eluting with ethyl acetate-hexane (1:4) to give six fractions.
2.2. Anti-mycobacterial screening of test material A broth micro dilution technique that allows numerous drug concentrations to be screened on a single 96-well microtitre plate in order to determine minimum inhibitory concentration (MIC) was used in this study (Collins and Franzblau, 1997). A culture supernatant of mutant Mycobacterium tuberculosis strain was grown to OD600 of 0.6–0.7 and diluted 100× in GAST/Fe medium. The culture suspension (100 μL) and two-fold dilutions of test material (0.2–125 μg/mL) in triplicates were added to 96 well plates to a final volume of 200 μL. Control wells contained culture and medium only. The plates were incubated at room temperature and fluorescence intensity measured at day 7 employing a Fluorostar Optima microplate reader (BMG Labtech, Thermofisher Scientific, USA) at 484 nm. MIC was expressed as the lowest agent concentration inhibiting 90% and 99% microbial growth.
2.4. Determination of the ferric ion reducing power The ferric ion reducing power of the different DCM extracts were determined. Various concentrations (16–500 μg/mL) of the extracts in deionised water (100 μL) were prepared. A blank was prepared without adding extract, while ascorbic acid was used as the reference standard. The test agents were mixed with phosphate buffer (250 μL) (pH 7.4 and concentration 0.2 M) together with potassium ferri-cyanide (250 μL) and incubated at 50 °C for 20 min. After incubation, aliquots of trichloroacetic acid (250 μL) were added to the mixture and centrifuged at 3000 rpm for 10 min. The supernatant (250 μL) was mixed with distilled water (250 μL) and freshly prepared ferric chloride solution (50 μL). The absorbance of the samples was measured at 700 nm. Test material which has reduction potential (i.e., react with potassium ferri-cyanide (Fe3 +) to form potassium ferro-cyanide (Fe2 +), which then reacts with ferric chloride to form a ferric-ferrous complex that has an absorption maximum at 700 nm). Increased absorbance of the reaction mixture indicates increase in reducing power which was measured at 700 nm using a microtiter-plate multimode detector (Promega-Glomax Multi detection system, Promega, South Africa). 2.5. RAW 264.7 cell culture The murine macrophage cell line, RAW 264.7 was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 10% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. 2.5.1. Measurement of cell proliferation using the WST-8 assay The cell suspension (2 × 106 cells/mL) was incubated with or without test material (16–500 μg/mL) for 48 h in a 5% CO2 incubator and proliferation measured using the WST-8 assay according to the manufacturer's instruction (Cayman Chemicals, Johannesburg, South Africa). 2.5.2. Determination of NO production Nitric oxide production by the cell line was determined by measurement of nitrite levels in the culture medium. Cells were incubated for 24 h with or without test agents (16–500 μg/mL) and nitric oxide measured as nitrite levels in the cell culture supernatant using the Griess reagent (Bogdan, 1998). After incubation, 100 μL of the culture supernatant was added to 100 μL of the Griess reagent (0.1% NED, 1% sulphalinamide, and 2.5% phosphoric acid) and incubated at room temperature for 10 min in dark condition. The absorbance was read at 540 nm using microplate reader (Beckman Coulter, Johannesburg, South Africa). 2.5.3. Measurement of TNF-α production by RAW cells After treatment of cells with or without plant extracts (16–500 μg/ mL) for 24 h, TNF-α production in culture supernatants was measured using a Human TNF alpha ELISA kit (Biocom Africa Pty Ltd., Centurion, South Africa) according to the manufacturer's instructions. 3. Data analysis Data of experiments (repeats of 3–4 tests in triplicate) was analysed using GraphPad Prism Software Version 10.00. Results are expressed as
2.3. Determination of antioxidant activity by DPPH method The antioxidant activity of the six DCM leaf extract of G. senegalensis (16–500 μg/mL) and the antioxidant standards were assessed on the basis of radical scavenging effect of the stable 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical. One hundred microliter of a 0.2% solution of DPPH radical in methanol was mixed with 100 μL of plant extracts. After mixing, they were left for 30 min at room temperature. The DPPH radical inhibition was measured at 540 nm using Beckman Coulter Du® 730 Life Science UV–Visible spectrophotometer (Moein and Moein, 2010). Tests were carried out in duplicate and Ascorbic acid (Vitamin C) was used as positive control.
Table 1 Minimum inhibitory concentrations (MIC90 and MIC99) of DCM leaf extracts of Gymosporia senegalensis against the H37Rv strain of Mycobacterium tuberculosis. Sample name
MIC90 (μg/mL)
MIC99 (μg/mL)
SP2-IC (DCM crude) SP2-1F1 SP2-1F2 SP2-1F3 SP2-1F4 SP2-1F5 SP2-1F6
36.8 48.6 43.3 50 54.2 21.4 44.2
47.7 70.1 52.2 73.7 86 27.4 59.2
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Fig. 1. Free radical scavenging activity of DCM leaf extracts of Gymnosporia senegalensis as measured by DPPH assay. Results are expressed as mean percentage radical scavenging activity by the test agents (16–500 μg/mL) plus SEM of 3–4 experiments conducted in triplicates. *The result shows statistical significance.
mean percentage of the variable ± SEM (Standard deviation). A p ≤ 0.05 was considered significant. 4. Results 4.1. Anti-mycobacterial activity of leaf fractions against H37Rv strain Mycobacterium tuberculosis Table 1 shows the minimum inhibitory concentrations (MIC90/99) of leaf extracts as measured employing the green fluorescence protein microplate assay (GFPMA). The plant leaf crude extracts and its six fractions inhibited growth of the M. tuberculosis H37Rv strain with MIC90 and MIC99 values ranging from 21 to 54 μg/mL and 27 and 86 μg/mL respectively. The overall anti-mycobacterial activity of test agents exhibited activity in the following sequence as order of strength; SP2-1F5 N DCM crude N SP2-1F2 N SP2-1F6 N SP2-1F1 N SP2-1F3 N SP2-1F4 with MIC90 concentration of between 21.4 to 54.2 μg/mL.
reducing power. The leaf extract fractions, SP2-1F1, SP2-1F3 and SP21F5 showed substantial (p ≤ 0.05) antioxidant activity at concentrations between 125 and 500 μg/mL as shown in Figs. 1 and 2. Moreover, the DCM crude extract also exhibited significant (p ≤ 0.05) ferric reducing power at 250 and 500 μg/mL. Other test agents failed to show any antioxidant activity (p ≥ 0.05). These three fractions were then further screened for growth inhibitory potential as well as NO and TNF-α production in RAW 264.7 cells. 4.3. Cytotoxic activity of test agents against RAW 264.7 cells The results of the anti-proliferative activity of test agents are shown in Fig. 3. Test agent SP2-1F3 showed significant activity at a concentration of 62 μg/mL and above while fractions SP2-1F1 and SP2-1F5 inhibited cell growth at concentrations from 125 to 500 μg/mL. 4.4. Production of nitric oxide by RAW cells
4.2. Antioxidant activity of leaf fractions using cell free- based assays The radical species scavenging activity of the plant test material was determined using two cell free based assays, DPPH and ferric ion
Nitric oxide production was measured as nitrite levels in cell culture supernatants and measured using the Griess reagent. Fig. 4 shows that fractions SP2-1F1 and SP2-1F5 showed significant activity at 125 to
Fig. 2. Ferric ion reducing activity of DCM leaf extract of G. senegalensis. Results are presented as mean ferric ion reducing power by the leaf extracts (16–500 μg/mL) plus SEM of 3–4 assays done triplicates. *The result is statistically significant.
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Fig. 3. Inhibition of cell proliferation by leaf extract of G. senegalensis in RAW 264.7 cells. Results are expressed as mean percentage cell growth inhibition by the test agents (16–500 μg/mL) plus SEM of 3–4 experiments conducted in triplicate. *Result is statistically significant.
500 μg/mL while agent SP2-1F3 showed reduction of NO production at concentrations between 62 and 500 μg/mL. 4.5. Production of TNF-α by RAW cells Fig. 5 shows that all three fractions; SP2-1F1, SP2-1F3 and SP2-1F5 inhibited TNF-α production by RAW cells from 125 to 500 μg/mL. 5. Discussion and conclusions Active tuberculosis in patients is promoted by an inflammatory cellmediated immunity that, if not under proper surveillance may lead to; granuloma formations (to contain macrophages infected with bacilli particles), its lysis and initiation of extra-pulmonary tuberculosis (Phillips and Ernst, 2012). It is thus essential to develop anti-mycobacterial treatment regimens that not only inhibit growth of the bacteria, but also mediate or limit the inflammatory response that it does not proceed to a chronic phase. Our study explored the potential of DCM fractions of Gymnosporia senegalensis to inhibit the growth of a strain of Mycobacterium tuberculosis. The fractions, in addition to their DCM crude extract, exhibited on the overall in vitro anti-mycobacterial activities (MIC90) of between 21
and 54 μg/mL. This finding is commendable as compared to observations documented in the study conducted by Lall and Meyer, (1999). In their study, they used acetone and water for extract preparation and anti-mycobacterial activity was only noticeable at 1 mg/mL. Moreover, the team employed agar plate broth dilution method and the radiometric assay (BACTEC TB-460 assay) for anti-mycobacterial screening purposes different from the technique used in the current study. Different species of Gymnosporia are used in traditional medicine as anti-inflammatory and analgesic mixtures administered orally or topically (Neuwinger, 2000). The current study evaluated the effect of test agents on anti-oxidative activity (radical scavenging activity) and NO production by RAW cells, inhibition of cell growth and production of TNF-α. Three of the fractions; SP2-1F1, SP2-1F3 and SP2-1F5 exhibited significant anti-oxidative (125–500 μg/mL), cell growth inhibitory (62–500 μg/mL) for the various fractions and anti-inflammatory (TNFα production) activities (125–500 μg/mL). Phytochemical analysis of M. senegalensis leaf identified alkaloids, alkenes and alkanols, terpenes, steroids and phenols compounds which exhibit an anti-inflammatory activity in biological systems (Mueller and Mechler, 2005). Another study found that the leaf extract of this plant species also reduces carrageen-induced paw edema in mice (Da Silva et al., 2010). South American species of Gymnosporia have already
Fig. 4. Inhibition of nitric oxide production by test agents in RAW 264.7 cells. Results are expressed as the mean percentage of inhibition of NO production by test agents (16–500 μg/mL) plus SEM of 3–4 experiments conducted in triplicate. *Results are statistically significant.
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Fig. 5. Effects of leaf extract fractions on the production of TNF-α IN RAW 264.7 cells. Results are expressed as mean percentage inhibition of TNF-α production by test agents (16–500 μg/ mL) plus SEM of 3–4 experiments conducted in triplicates. *Results are significant.
been evaluated for anti-inflammatory activity (Kimura et al., 2000; Jorge et al., 2004; Santos et al., 2007). Maytenoic acid, pristimerin, lupenone, β-amyrin and β-sitosterol have been identified from stem and roots of G. senegalensis as chemicals entities promoting the antimicrobial anti-inflammatory properties of this plant. These mentioned bioactive elements of this plant have not yet been isolated in leaves of G. senegalensis. Further studies are necessitated to identify the chemical constituents of the plant and evaluate their anti-mycobcaterial and immune-regulatory properties. Abbreviations DPPH ELISA NED RPMI TLC WHO WST-8
diphenyl-1-picrylhydrazyl enzyme-linked immunosorbent assay naphthyl ethylenediamine Roswell Park Memorial Institute thin layer chromatography World Health Organization 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H–tetrazolium monosodium salt
Conflict of interest We hereby declare that there are no conflicts of interest. Acknowledgements We are thankful to Prof TM Matsebatlela for providing us with the RAW 264.7 murine cell line. We also acknowledge the University of Cape Town Medical School and Prof Digby Warner for the Mtb screening assays. Funding This work was supported by the University of Limpopo [grant number 1706-3642] and National Research Foundation (NRF) [grant number TTK1205280912]. Author contributions MME and NW conceived and designed the experiments. MME, NW and RLA conducted the laboratory work. MME and NW analysed the data. MME and NW contributed laboratory reagents and equipment. MME and NW wrote the paper. All authors read and approved the final article.
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Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Anti-mycobacterial, -oxidative, -proliferative and -inflammatory activities of dichloromethane leaf extracts of Gymnosporia senegalensis (Lam.) Loes M.E. Makgatho a,⁎, W. Nxumalo b, L.A. Raphoko b a b
Department of Pathology and Medical Sciences, University of Limpopo, South Africa Department of Chemistry, University of Limpopo, South Africa
a r t i c l e
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Article history: Received 6 September 2017 Received in revised form 28 October 2017 Accepted 3 November 2017 Available online xxxx Edited by J Van Staden Keywords: Gymnosporia senegalensis DPPH Dichloromethane Cell proliferation Nitric oxide Tumour necrosis factor-alpha Antioxidant activity Mycobacterium tuberculosis
a b s t r a c t A crude dichloromethane leaf extract of Gymnosporia senegalensis and its six fractions were tested for anti-mycobacterial activity using a fluorescent microplate assay against the H37Rv strain of Mycobacterium tuberculosis as well as antioxidant activity using the cell free based assays, DPPH and ferric ion reducing power as well as a colorimetric assay for measuring nitric oxide production in RAW 264.7 cells. The in vitro anti-inflammatory potential of fractions was screened by measuring tumor necrosis-alpha production in RAW 264.7 cells. The test agents exhibited anti-mycobacterial activity in the following sequence; SP2-1F5 N crude extract N SP2-1F2 N SP2-1F6 N SP2-1F1 N SP2-1F3 N SP2-1F4 with minimum inhibitory values ranging from 21.4 to 54.2 μg/mL. Fractions SP2-1F1, SP2-1F3 and SP2-1F5 exhibited significant antioxidant activities at concentrations between 125 μg/ mL and 500 μg/mL employing the cell free-based and colorimetric assays. The crude extract of the plant leaf also exhibited ferric ion reducing power at 250 and 500 μg/mL. Fractions SP2-1F1, SP2-1F3 and SP2-1F5 showed noticeable tumor necrosis factor-alpha production in RAW cell at 125 to 500 μg/mL. The laboratory activities conducted in the present study presumably indicate that the leaf extract of Gymnosporia senegalensis contain chemical entities that exhibit in vitro inhibition of mycobacterium growth, production of nitric oxide and tumor necrosis factor-alpha in a mouse macrophage cell line as well as increased radical scavenging potential. Further laboratory studies are to be conducted in pursuance of the active chemical ingredients of leafs of this plant species. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Escalating levels of Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) are a challenging public health concern especially in low income countries (Velayati et al., 2013). The emergence of drug resistance and human immunodeficiency virus (HIV) co-infection exacerbates the situation (WHO, 2015). Active tuberculosis is perpetuated by harmful, proinflammatory immune mechanisms that respond to stimulation and activation of alveolar macrophages by microbial signals (Cooper, 2009; Ferrara et al., 2009; Phillips and Ernst, 2012; Zumla et al., 2013; Pai et al., 2016). Typical biological mediators that drive this inflammatory paradigm are; interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α) and nitric oxide (Flynn and Chan, 2001; Ahmad, 2011; Zuñiga et al., 2012; Amaral et al., 2016). The use of alternative strategies such as complementary medicinal products to treat TB and alleviate the concomitant complications should be explored (Green ⁎ Corresponding author at: Department of Pathology and Medical Sciences, School of Health Care Sciences, Faculty of Health Sciences, University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa. E-mail address: [email protected] (M.E. Makgatho).
https://doi.org/10.1016/j.sajb.2017.11.002 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
et al., 2010; Nguta et al., 2016). Bioactive metabolites from medicinal plants have provided a rich source for the development of new drugs against infectious diseases and modulations of the immune system (Cragg and Newman, 2013; Nguta et al., 2015). A number of research groups in South Africa are on a venture to identify and extract novel anti-mycobacterial compounds (Lall et al., 1999; Lall and Meyer, 1999; Eldeen and Van Staden, 2008; McGaw et al., 2008; Green et al., 2010; Labuschagné et al., 2012; Mulaudzi et al., 2012; Aro et al., 2016; Komape et al., 2017). Gymnosporia senegalensis (Lam.) Loes, alternatively named Maytenus senegalensis (Lam.) Excell is a small tree or tall shrub of the family Celastracae widely distributed in Arabia, Africa, Afghanistan and India (Hedberg et al., 1982; Gessler et al., 1995; Sosa et al., 2007; Malebo et al., 2015). African traditional healers use the root and stem bark to treat a number of ailments like malaria, rheumatism, dysmenorrhoea, chest pains, wounds, chronic illness, dyspepsia eye infection and diarrhoea (El Tahir et al., 1999; Hussein et al., 1999; Da Silva et al., 2010; Ahmed et al., 2013). The root, stem bark and leaf extracts of Gymnosporia senegalensis have been shown to exhibit anti-inflammatory activity in animal models of experimental inflammation like Croton oil-induced ear oedema and carrageenan-induced paw oedema (Sosa
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et al., 2007; Da Silva et al., 2017). A number of researchers have documented the antimicrobial activities of extracts from various parts (stem, leaf, bark and root) of Gymnosporia senegalensis (Matu and Van Staden, 2003; Clarkson et al., 2004; Malebo et al., 2015. Most importantly, Lall and Meyer demonstrated the antimycobacterial activity of acetone and water leaf extracts of this plant against a resistant strain of Mycobacterium tuberculosis (Lall and Meyer, 1999). In the present study, crude dichloromethane (DCM) leaf extract of Gymnosporia senegalensis and its six fractions were screened for activity against the H37Rv strain of Mycobacterium tuberculosis and antioxidant activity using the cell free based DPPH and ferric reducing power assays. DCM crude leaf extract fractions with substantive antioxidant activity were further assayed for their effect on nitric oxide and TNF-α production in a murine macrophage RAW 264.7 cell line.
2. Materials and methods Unless otherwise indicated, all chemicals and reagents were obtained from Sigma-Aldrich (Johannesburg, South Africa).
2.1. Plant material collection and preparation of extracts Gymnosporia senegalensis leaves were supplied by traditional healers in Tzaneen, Limpopo Province (South Africa) deposited in the University of Limpopo Herbarium with voucher number 1145. The leaves were cleaned and dried in an oven at 37 °C for 7 days. Dichloromethane (500 mL) was added to a 20 g dried powder from the leaves of Gymnosporia senegalensis in a 1000 mL volumetric flask and the resulting mixture stirred at room temperature for 24 h. The mixture was filtered to remove undissolved material and the filtrate washed twice with 10 mL DCM. The solvent was removed in a rotary evaporator to give 0.6412 g of crude extract. The crude extract was then subjected to preparative thin layer chromatography (prep-TLC), eluting with ethyl acetate-hexane (1:4) to give six fractions.
2.2. Anti-mycobacterial screening of test material A broth micro dilution technique that allows numerous drug concentrations to be screened on a single 96-well microtitre plate in order to determine minimum inhibitory concentration (MIC) was used in this study (Collins and Franzblau, 1997). A culture supernatant of mutant Mycobacterium tuberculosis strain was grown to OD600 of 0.6–0.7 and diluted 100× in GAST/Fe medium. The culture suspension (100 μL) and two-fold dilutions of test material (0.2–125 μg/mL) in triplicates were added to 96 well plates to a final volume of 200 μL. Control wells contained culture and medium only. The plates were incubated at room temperature and fluorescence intensity measured at day 7 employing a Fluorostar Optima microplate reader (BMG Labtech, Thermofisher Scientific, USA) at 484 nm. MIC was expressed as the lowest agent concentration inhibiting 90% and 99% microbial growth.
2.4. Determination of the ferric ion reducing power The ferric ion reducing power of the different DCM extracts were determined. Various concentrations (16–500 μg/mL) of the extracts in deionised water (100 μL) were prepared. A blank was prepared without adding extract, while ascorbic acid was used as the reference standard. The test agents were mixed with phosphate buffer (250 μL) (pH 7.4 and concentration 0.2 M) together with potassium ferri-cyanide (250 μL) and incubated at 50 °C for 20 min. After incubation, aliquots of trichloroacetic acid (250 μL) were added to the mixture and centrifuged at 3000 rpm for 10 min. The supernatant (250 μL) was mixed with distilled water (250 μL) and freshly prepared ferric chloride solution (50 μL). The absorbance of the samples was measured at 700 nm. Test material which has reduction potential (i.e., react with potassium ferri-cyanide (Fe3 +) to form potassium ferro-cyanide (Fe2 +), which then reacts with ferric chloride to form a ferric-ferrous complex that has an absorption maximum at 700 nm). Increased absorbance of the reaction mixture indicates increase in reducing power which was measured at 700 nm using a microtiter-plate multimode detector (Promega-Glomax Multi detection system, Promega, South Africa). 2.5. RAW 264.7 cell culture The murine macrophage cell line, RAW 264.7 was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 10% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. 2.5.1. Measurement of cell proliferation using the WST-8 assay The cell suspension (2 × 106 cells/mL) was incubated with or without test material (16–500 μg/mL) for 48 h in a 5% CO2 incubator and proliferation measured using the WST-8 assay according to the manufacturer's instruction (Cayman Chemicals, Johannesburg, South Africa). 2.5.2. Determination of NO production Nitric oxide production by the cell line was determined by measurement of nitrite levels in the culture medium. Cells were incubated for 24 h with or without test agents (16–500 μg/mL) and nitric oxide measured as nitrite levels in the cell culture supernatant using the Griess reagent (Bogdan, 1998). After incubation, 100 μL of the culture supernatant was added to 100 μL of the Griess reagent (0.1% NED, 1% sulphalinamide, and 2.5% phosphoric acid) and incubated at room temperature for 10 min in dark condition. The absorbance was read at 540 nm using microplate reader (Beckman Coulter, Johannesburg, South Africa). 2.5.3. Measurement of TNF-α production by RAW cells After treatment of cells with or without plant extracts (16–500 μg/ mL) for 24 h, TNF-α production in culture supernatants was measured using a Human TNF alpha ELISA kit (Biocom Africa Pty Ltd., Centurion, South Africa) according to the manufacturer's instructions. 3. Data analysis Data of experiments (repeats of 3–4 tests in triplicate) was analysed using GraphPad Prism Software Version 10.00. Results are expressed as
2.3. Determination of antioxidant activity by DPPH method The antioxidant activity of the six DCM leaf extract of G. senegalensis (16–500 μg/mL) and the antioxidant standards were assessed on the basis of radical scavenging effect of the stable 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical. One hundred microliter of a 0.2% solution of DPPH radical in methanol was mixed with 100 μL of plant extracts. After mixing, they were left for 30 min at room temperature. The DPPH radical inhibition was measured at 540 nm using Beckman Coulter Du® 730 Life Science UV–Visible spectrophotometer (Moein and Moein, 2010). Tests were carried out in duplicate and Ascorbic acid (Vitamin C) was used as positive control.
Table 1 Minimum inhibitory concentrations (MIC90 and MIC99) of DCM leaf extracts of Gymosporia senegalensis against the H37Rv strain of Mycobacterium tuberculosis. Sample name
MIC90 (μg/mL)
MIC99 (μg/mL)
SP2-IC (DCM crude) SP2-1F1 SP2-1F2 SP2-1F3 SP2-1F4 SP2-1F5 SP2-1F6
36.8 48.6 43.3 50 54.2 21.4 44.2
47.7 70.1 52.2 73.7 86 27.4 59.2
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Fig. 1. Free radical scavenging activity of DCM leaf extracts of Gymnosporia senegalensis as measured by DPPH assay. Results are expressed as mean percentage radical scavenging activity by the test agents (16–500 μg/mL) plus SEM of 3–4 experiments conducted in triplicates. *The result shows statistical significance.
mean percentage of the variable ± SEM (Standard deviation). A p ≤ 0.05 was considered significant. 4. Results 4.1. Anti-mycobacterial activity of leaf fractions against H37Rv strain Mycobacterium tuberculosis Table 1 shows the minimum inhibitory concentrations (MIC90/99) of leaf extracts as measured employing the green fluorescence protein microplate assay (GFPMA). The plant leaf crude extracts and its six fractions inhibited growth of the M. tuberculosis H37Rv strain with MIC90 and MIC99 values ranging from 21 to 54 μg/mL and 27 and 86 μg/mL respectively. The overall anti-mycobacterial activity of test agents exhibited activity in the following sequence as order of strength; SP2-1F5 N DCM crude N SP2-1F2 N SP2-1F6 N SP2-1F1 N SP2-1F3 N SP2-1F4 with MIC90 concentration of between 21.4 to 54.2 μg/mL.
reducing power. The leaf extract fractions, SP2-1F1, SP2-1F3 and SP21F5 showed substantial (p ≤ 0.05) antioxidant activity at concentrations between 125 and 500 μg/mL as shown in Figs. 1 and 2. Moreover, the DCM crude extract also exhibited significant (p ≤ 0.05) ferric reducing power at 250 and 500 μg/mL. Other test agents failed to show any antioxidant activity (p ≥ 0.05). These three fractions were then further screened for growth inhibitory potential as well as NO and TNF-α production in RAW 264.7 cells. 4.3. Cytotoxic activity of test agents against RAW 264.7 cells The results of the anti-proliferative activity of test agents are shown in Fig. 3. Test agent SP2-1F3 showed significant activity at a concentration of 62 μg/mL and above while fractions SP2-1F1 and SP2-1F5 inhibited cell growth at concentrations from 125 to 500 μg/mL. 4.4. Production of nitric oxide by RAW cells
4.2. Antioxidant activity of leaf fractions using cell free- based assays The radical species scavenging activity of the plant test material was determined using two cell free based assays, DPPH and ferric ion
Nitric oxide production was measured as nitrite levels in cell culture supernatants and measured using the Griess reagent. Fig. 4 shows that fractions SP2-1F1 and SP2-1F5 showed significant activity at 125 to
Fig. 2. Ferric ion reducing activity of DCM leaf extract of G. senegalensis. Results are presented as mean ferric ion reducing power by the leaf extracts (16–500 μg/mL) plus SEM of 3–4 assays done triplicates. *The result is statistically significant.
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Fig. 3. Inhibition of cell proliferation by leaf extract of G. senegalensis in RAW 264.7 cells. Results are expressed as mean percentage cell growth inhibition by the test agents (16–500 μg/mL) plus SEM of 3–4 experiments conducted in triplicate. *Result is statistically significant.
500 μg/mL while agent SP2-1F3 showed reduction of NO production at concentrations between 62 and 500 μg/mL. 4.5. Production of TNF-α by RAW cells Fig. 5 shows that all three fractions; SP2-1F1, SP2-1F3 and SP2-1F5 inhibited TNF-α production by RAW cells from 125 to 500 μg/mL. 5. Discussion and conclusions Active tuberculosis in patients is promoted by an inflammatory cellmediated immunity that, if not under proper surveillance may lead to; granuloma formations (to contain macrophages infected with bacilli particles), its lysis and initiation of extra-pulmonary tuberculosis (Phillips and Ernst, 2012). It is thus essential to develop anti-mycobacterial treatment regimens that not only inhibit growth of the bacteria, but also mediate or limit the inflammatory response that it does not proceed to a chronic phase. Our study explored the potential of DCM fractions of Gymnosporia senegalensis to inhibit the growth of a strain of Mycobacterium tuberculosis. The fractions, in addition to their DCM crude extract, exhibited on the overall in vitro anti-mycobacterial activities (MIC90) of between 21
and 54 μg/mL. This finding is commendable as compared to observations documented in the study conducted by Lall and Meyer, (1999). In their study, they used acetone and water for extract preparation and anti-mycobacterial activity was only noticeable at 1 mg/mL. Moreover, the team employed agar plate broth dilution method and the radiometric assay (BACTEC TB-460 assay) for anti-mycobacterial screening purposes different from the technique used in the current study. Different species of Gymnosporia are used in traditional medicine as anti-inflammatory and analgesic mixtures administered orally or topically (Neuwinger, 2000). The current study evaluated the effect of test agents on anti-oxidative activity (radical scavenging activity) and NO production by RAW cells, inhibition of cell growth and production of TNF-α. Three of the fractions; SP2-1F1, SP2-1F3 and SP2-1F5 exhibited significant anti-oxidative (125–500 μg/mL), cell growth inhibitory (62–500 μg/mL) for the various fractions and anti-inflammatory (TNFα production) activities (125–500 μg/mL). Phytochemical analysis of M. senegalensis leaf identified alkaloids, alkenes and alkanols, terpenes, steroids and phenols compounds which exhibit an anti-inflammatory activity in biological systems (Mueller and Mechler, 2005). Another study found that the leaf extract of this plant species also reduces carrageen-induced paw edema in mice (Da Silva et al., 2010). South American species of Gymnosporia have already
Fig. 4. Inhibition of nitric oxide production by test agents in RAW 264.7 cells. Results are expressed as the mean percentage of inhibition of NO production by test agents (16–500 μg/mL) plus SEM of 3–4 experiments conducted in triplicate. *Results are statistically significant.
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Fig. 5. Effects of leaf extract fractions on the production of TNF-α IN RAW 264.7 cells. Results are expressed as mean percentage inhibition of TNF-α production by test agents (16–500 μg/ mL) plus SEM of 3–4 experiments conducted in triplicates. *Results are significant.
been evaluated for anti-inflammatory activity (Kimura et al., 2000; Jorge et al., 2004; Santos et al., 2007). Maytenoic acid, pristimerin, lupenone, β-amyrin and β-sitosterol have been identified from stem and roots of G. senegalensis as chemicals entities promoting the antimicrobial anti-inflammatory properties of this plant. These mentioned bioactive elements of this plant have not yet been isolated in leaves of G. senegalensis. Further studies are necessitated to identify the chemical constituents of the plant and evaluate their anti-mycobcaterial and immune-regulatory properties. Abbreviations DPPH ELISA NED RPMI TLC WHO WST-8
diphenyl-1-picrylhydrazyl enzyme-linked immunosorbent assay naphthyl ethylenediamine Roswell Park Memorial Institute thin layer chromatography World Health Organization 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H–tetrazolium monosodium salt
Conflict of interest We hereby declare that there are no conflicts of interest. Acknowledgements We are thankful to Prof TM Matsebatlela for providing us with the RAW 264.7 murine cell line. We also acknowledge the University of Cape Town Medical School and Prof Digby Warner for the Mtb screening assays. Funding This work was supported by the University of Limpopo [grant number 1706-3642] and National Research Foundation (NRF) [grant number TTK1205280912]. Author contributions MME and NW conceived and designed the experiments. MME, NW and RLA conducted the laboratory work. MME and NW analysed the data. MME and NW contributed laboratory reagents and equipment. MME and NW wrote the paper. All authors read and approved the final article.
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