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Antimicrobial and antiprotozoal activities of twenty-four Nigerian medicinal plant extracts
South African Journal of Botany 117 (2018) 240–246
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Antimicrobial and antiprotozoal activities of twenty-four Nigerian medicinal plant extracts O.O. Ogbole a,⁎, P.A. Segun b, P.S. Fasinu c a b c
Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria Department of Pharmacognosy, Faculty of Pharmacy, Olabisi Onabanjo University, Sagamu Campus, Nigeria Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, Campbell University, Buies Creek, NC 27506, USA
a r t i c l e
i n f o
Article history: Received 10 November 2017 Received in revised form 4 May 2018 Accepted 29 May 2018 Available online xxxx Edited by A Moteetee Keywords: Antileishmanial Antimicrobial Antiplasmodial Antitrypanosomal Drug discovery Eleusine indica Macaranga barteri
a b s t r a c t Medicinal plants exploration has become an important tool in the discovery of bioactive natural substances capable of inhibiting the mechanisms of microbial and protozoal activity. The aim of this study was to determine the antimicrobial and antiprotozoal activities of medicinal plants extracts used for the treatment of various illnesses in southwestern Nigeria. Twenty-four medicinal plant extracts were screened for antimicrobial activity against three fungal and six bacterial strains; and antiprotozoal activities against chloroquine-sensitive strains of Plasmodium falciparum (antiplasmodial assay), a culture of Leishmania donovani promastigotes and axenic amastigotes (antileishmanial activity) and two-days old culture of Trypanosoma brucei brucei (antitrypanosomal assay). Five extracts exhibited strong antifungal activity against Cryptococcus neoformans, with Ricinodendron heudelotii (Baill.) Heckel, Terminalia ivorensis A.Chev and Macaranga barteri Müll. Arg, having an IC50 of 31.73, 32.10 and 75.63 μg/mL, respectively. Ten of the extracts were active against T. brucei, with Eleusine indica displaying the most significant activity (IC50 and IC90 of 8.26 and 10.14 μg/mL). None of the extracts displayed any significant antiplasmodial and antileishmanial activities. In addition, none of the extracts displayed cytotoxicity on transformed human monocytic (THP1) cells. The study revealed that M. barteri had the broadest spectrum of activity, with activity against C. neoformans, P. aeruginosa, vancomycin resistant Enterococci faecalis (VRE) and T. brucei. M. barteri could be exploited for broad spectrum antimicrobial and antitrypanosomal activity, while Eleusine indica could be subjected to bioassay guided fractionation and isolation of antitrypanosomal constituents. This study suggests that the evaluated plants are potential sources of novel anti-infective agents. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Infectious diseases are one of the most important factors responsible for the high rate of morbidity and mortality, especially in developing countries, where over 25% of global annual mortality are primarily caused by infectious diseases (Mahady, 2005). Meanwhile, secondary effects of infections are responsible for millions more deaths (Satcher, 1995; Chua and Gubler, 2013). Human African trypanosomiasis (HAT), Chagas diseases, leishmaniasis and malaria are infectious diseases caused by protozoans. HAT, Chagas diseases and leishmaniasis are classified as neglected tropical diseases (NTDs) by the World Health Organization (WHO) and they represent a major threat to the health and well-being of over a billion lives worldwide (WHO, 2010; Llurba-Montesino et al., 2015). Over the years, significant progress has been made in the field of microbiology and the control of microorganisms. In spite of this ⁎ Corresponding author. E-mail address: [email protected] (O.O. Ogbole).
https://doi.org/10.1016/j.sajb.2018.05.028 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
progress, sporadic episodes of epidemics triggered by drug-resistant microorganisms and pathogenic microbes remain a massive threat to public health. The relentless rise in antibiotic resistance, and emergence of new and chronic infectious diseases are factors that stimulate a continuous search for new anti-infective agents (Costa et al., 2016; Mahady, 2005; Al-Judaibi, 2014; Mehani et al., 2016). Plant-based traditional medicine represents the primary or the only veritable form of accessible primary health care in many parts of the developing world, especially for people living in rural Africa. The information about the healing properties of medicinal plants have been transmitted over the centuries among generations of human communities; the existence of traditional medicine depends on plant species diversity and the knowledge of their use as herbal medicine. Currently, the demand for the herbal drug treatment of various ailments is increasing and plants are being explored globally for the development of newer drugs (Silva and Fernandes, 2010; Mahomoodally, 2013). Considering the importance of medicinal plants for the discovery and identification of bioactive natural substances capable of inhibiting the
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mechanisms of microbial activity and resistance (Amoa-Bosompem, 2016; Nguyen, 2016), this work reports the evaluation of twenty-four extracts from medicinal plants utilized in ethnobotanical medicine in Nigeria (Ajaiyeoba et al., 2003; Osowole et al., 2005; Ogbole et al., 2010; Segun et al., 2018) against agents responsible for various microbial infections including malaria, leishmaniasis and trypanosomiasis.
2. Materials and methods 2.1. Plant collection Fresh leaves of the medicinal plants were collected from their various habitat at the onset of raining season in Ibadan, Nigeria between April and May 2015. The plants were identified and authenticated by Dr. Osiyemi of the Forestry and Research Institute of Nigeria (FRIN), Ibadan and assigned voucher specimen numbers. Voucher specimens were deposited at the Forest Herbarium, Ibadan (FHI).
2.2. Preparation and extraction of plant materials Each plant material was shade-dried at room temperature (27–33 °C) and pulverized. The dried powdered materials of each plant (100 g) were extracted by maceration into methanol (750 mL) and then filtered to obtain methanol extracts of each plant. The filtrate was concentrated with a rotary evaporator at 40 °C and the dried extracts were kept in a refrigerator until needed. Even though water is mostly used as solvent base for traditional herbal preparation, methanol was used in this study, mainly because of its amphiphilicity; it dissolves a wider range of compounds than water, including polar and to a large extent some non-polar compounds. Moreover, methanol easily evaporates so it is easier to separate from the extract than water.
2.3. Antimicrobial assay Antimicrobial susceptibility testing was carried out at the National Center for Natural Products Research, University of Mississippi, USA. The test organisms were obtained from the American Type Culture Collection (ATCC, Manassas, VA). These included the fungi: Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 90113 and Aspergillus fumigatus ATCC 204305; bacteria: Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 4352 and Vancomycin resistant Enterococci faecalis ATCC 29212 (VRE) The in vitro antimicrobial assay was done using a modified version of the CLSI methods as described below (Samoylenko et al., 2009). Extracts were dissolved in dimethyl sulfoxide (DMSO) and serially diluted in 20% DMSO/saline and then transferred, in duplicate, into 96-well flat bottom microplates. The final concentration of DMSO used in this assay was 0.1%, as concentration below 1% is not known to exert any observable toxic effect on cells (Timm et al., 2013). Microbial innocula were prepared in assay medium to afford target 106 cfu/mL, after addition to the samples. Growth, solvent and media controls were included in each test plate. Assay plates were read at 530 nm before and after incubation using the Biotek Power wave XS plate reader. Percentage growth was plotted against test concentration to afford the IC50 values (concentration that affords 50% growth relative to control). Crude plant extracts were tested in the primary assay at a single concentration of 200 μg/mL and percentage inhibitions were calculated, extracts with more than 50% inhibition were subjected to the secondary screening using serially diluted extract at concentration range of 200–8 μg/mL. Drug controls were included in the secondary assay; ciprofloxacin (ICN Biomedicals, Ohio) for bacteria and amphotericin B (ICN Biomedicals, Ohio) for fungi.
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2.4. Antiplasmodial assay Antiplasmodial activities were measured in vitro using the assay protocol based on a colorimetric method that determines the parasitic lactate dehydrogenase (pLDH) activity (Makler et al., 1993; Samoylenko et al., 2009). The assay was performed in 96-well microplate and included a chloroquine-sensitive strains of Plasmodium falciparum (D6). In the primary screening, the crude plant extracts were tested in duplicate, at a single concentration of 15.9 μg/mL on the chloroquine-sensitive (D6) strain of P. falciparum. DMSO and chloroquine were included as vehicle and drug control, respectively. 2.5. In vitro assay for antileishmanial activity Alamar blue assay was used to evaluate the antileishmanial activity of the plant extracts. The assay was carried out on a culture of Leishmania donovani promastigotes and axenic amastigotes (Mikus and Steverding, 2000). The promastigotes culture was maintained at 26 °C in Roswell Park Memorial Institute medium (RPMI 1640), pH 7.4 with 10% fetal bovine serum (FBS). The axenic amastigotes were cultured at 37 °C and 5% CO2 in RPMI-1640 supplemented with 4-morpholineethanesulfonic acid (MES) (4.88 g/L), L-glutamine (298.2 mg/L), adenosine (26.7 mg/L), folic acid (10.1 mg/L), BME vitamin mix, sodium bicarbonate (352.8 mg/L) and 10% FBS. The pH of the culture medium was 5.5. For the primary assay, plant extracts were tested at a single concentration of 20.0 μg/mL (in duplicate) on both the promastigotes and axenic amastigotes culture. The extracts that showed more than 90% inhibition of growth in primary screening were subjected to secondary screening for dose response analysis. In a 96-well microplate, appropriate dilutions of samples, were added to the Leishmania promastigotes/axenic amastigote culture (2 × 106 cell mL− 1). The extracts were tested at six concentrations ranging from 40 to 0.0128 μg/mL. The plates were incubated for 72 h at 26 °C and 37 °C, respectively, for promastigotes and axenic amastigotes. The growth of Leishmania promastigotes/amastigotes was determined. IC50 and IC 90 values were computed from the dose–response curves (Donega et al., 2014). 2.6. Antitrypanosomal assay Two-day old culture of Trypanosoma brucei, in the exponential growth stage, was diluted with Iscove's Modified Dulbecco's medium (IMDM) to obtain 5000 parasites/mL. The assays were conducted in 96-well microplates. Extract dilutions (1 mg/mL) were made in IMDM from the stock extracts (20 mg/mL) for the primary screening. Each well received 4 μL of diluted extract and 196 μL of the culture medium to obtain a final volume of 200 μL. Plates were incubated at 37 °C in 5% CO2 for 48 h. Ten microliter (10 μL) of alamar blue (AbD Serotec, catalog number BUF012B) was added into each well and plates were then incubated overnight. Standard fluorescence was measured on a Fluostar Galaxy fluorometer (BMG LabTechnologies) at 544 nm excitation and 590 nm emission. Pentamidine was included as the control drug (Table 4). Extracts with more than 90% inhibition of T. brucei growth in primary screening were subjected to secondary screening to evaluate dose–response analysis. They were screened at concentrations ranging from 10 to 0.4 μg/mL. IC50 and IC90 values were computed from dose response growth inhibition curve by XLfit version 5.2.2. (Jain et al., 2016). 2.7. Cytotoxicity assay The extracts were screened for cytotoxicity against transformed human monocytic (THP1) cells. Four-day old culture of THP1 cells was diluted with RPMI medium to 2.5 × 105 cells/mL. For transformation of the cells to adherent macrophages, phorbol 12-myristate 13-acetate (PMA) was added to the culture at 25 ng/mL concentration
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(Jain et al., 2012). The PMA treated THP1 cell culture was dispensed in 96 well plates with 200 μL culture (2.5 × 105 cells/mL) in each well and plates were incubated at 37 °C in 5% CO2 incubator overnight. Extracts were diluted in separate plates in RPMI medium. The medium in plates with THP1 cells was replaced with fresh medium. The diluted plant extracts were added to these plates. The plates were returned to the CO2 incubator for 48 h at 37 °C, 5% CO2. After 48 h, 10 μL of alamar blue solution was added to each well and the plates were incubated further overnight. Standard fluorescence was measured on a fluorometer at 544 nm excitation and 590 nm emission. Cytotoxicity screening was carried out only for active extracts, that have shown more than 90% inhibition in the primary assay.
and Terminalia ivorensis displaying percentage inhibition of 83%, 82% and 82% respectively, against P. aeruginosa in the primary antimicrobial assay. The extracts of Lagenaria breviflora and Secamone afzelii exhibited significant antimicrobial activity against the MRSA. Only M. barteri extract had activity against VRE, none of the extracts displayed any significant activity against K. pneumoniae, E. coli and two of the fungiC. albicans and A. fumigatus. Five extracts, namely, Lagenaria breviflora, Macaranga barteri, Ricinodendron heudelotii, Spondias mombin (l) and Spondias mombin (b) extracts had percentage inhibition greater than 80% against C. neoformans in the primary assay (Table 2). Active extracts were subjected to secondary assay and the result is displayed in Table 3. 3.2. Antiplasmodial assay
3. Results The ethnobotanical information on the various plants belonging to 18 families are summarized in Table 1. Twenty-four plant extracts were screened for their antimicrobial and antiplasmodial activity. Plant extracts with percentage inhibition greater than 50% against T. brucei brucei were subjected to the cytotoxicity and secondary assay. Extract of Macaranga barteri had a wider spectrum of activity than the rest of the plant extracts.
One strain of P. falciparum D6 (chloroquine-sensitive) was used in the primary screening, the antiplasmodial assay could not proceed beyond the primary screening since none of the extract exhibited significant inhibitory activity against the parasite, the result of the primary screening for antiplasmodial is presented in Table 2. The highest (47%) activity was displayed by S. afzelii. 3.3. Antileishmanial assay
3.1. Antimicrobial assay The antimicrobial assay was carried out against three fungi and six bacteria, with the extracts of Macaranga barteri, Entandrophragma utile
The primary assay carried out on L. donovani promastigotes and axenic amastigotes did not proceed beyond the primary assay since none of the extract had any significant activity on the protozoan.
Table 1 Ethnobotanical information on plants used in this study. S/no
Family
Plant
Local name (Yoruba)
Part used
Voucher no (FHI)
Ethnomedicinal uses
1.
Anacardiaceae
Spondias mombin L.
Iyeye
Leaves
63,948
2.
Anacardiaceae
Spondias mombin L.
Iyeye
Stembark
63,948
3.
Apocyanaceae
Picralima nitida (Stapf) T.Durand & H.Durand
Abeere
Seed
109,572
4.
Apocyanaceae
Secamone afzelii (Roem. & Schult.) K.Schum.
Ailu
Leaves
109,995
5.
Asteraceae
Acanthospermum hispidum DC
Dagunro gogoro
Leaves
110,050
6.
Boraginaceae
Heliotropium indicum L.
Apari igun
Leaves
110,156
7. 8.
Combretaceae Combretaceae
Terminalia ivorensis A.Chev Terminalia superba Engl. & Diels
Afara-dudu Afara
Stembark Stembark
105,432 109,800
9.
Cucurbitaceae
Tagiri
Leaves
109,040
10. 11. 12. 13.
Euphorbiaceae Euphorbiaceae Euphorbiaceae Euphorbiaceae
Agbasa Erinmado Iralodan Ajeofole
Leaves Stembark Stembark Leaves
107,230 110,573 109,985 109,041
Gonorrhea, cough, bronchitis (Adesegun et al., 2007). Stomach ulcer, wounds (Agyare et al., 2009). Bruises, boils, dislocations, burns (Iwu, 2014). Fever, dysentery, convulsions (Ngadjui et al., 2002).
14.
Fabaceae
Lagenaria breviflora (Benth.) Roberty (Syn: Adenopus breviflorus Benth) Macaranga barteri Müll.Arg. Ricinodendron heudelotii (Baill.) Heckel Bridelia ferruginea Benth. Croton gratissimus Burch. (Syn: Croton zambesicus Mull Arg) Mimosa pudica L.
Dysentery, hemorrhoids, gonorrhea, stomachache leucorrhoea, malaria (Ayoka et al., 2006, Diallo et al., 2006, Ruiz et al., 2011). Dysentery, hemorrhoids, gonorrhea, stomachache leucorrhoea, malaria (Ayoka et al., 2006, Diallo et al., 2006, Ruiz et al., 2011). Malaria, sexual impotence, dysmenorrhea, gastrointestinal disorder (Okokon et al., 2007). Diabetes, wounds, boils, malaria (Weniger et al., 2004, Abo et al., 2008, Zabri et al., 2008). Headaches, abdominal pains, convulsions, cough, jaundice, epilepsy. (Ganfon et al., 2012). Wounds, flatulence, inflammation, skin ulcers and conjunctivitis (Paul et al., 2015). Syphilis, burns, bruises, arthritis, hemorrhoids (Iwu, 2014). Diabetes mellitus, gastroenteritis, female infertility, abdominal pains (Kuete et al., 2010). Coccidiosis, measles (Olorunnisola et al., 2015).
Patanmo
Leaves
100,332
15. 16.
Lamiaceae Meliaceae
Efirin-odan Jebo
Leaves Stembark
108,121 86,848
Headache, migraine, insomnia, diarrhea, dysentery (Amalraj and Ignacimuthu, 2002). Malaria, wounds (Achenbach et al., 1992). Peptic ulcer (John and Onabanjo, 1990).
17. 18.
Nyctaginaceae Phytolaccaceae
Etipase-erinla Awopa
Root Leaves
109,603 109,603
19. 20. 21.
Poaceae Rubiaceae Rubiaceae
Gbegi Opepe Egbesi
Leaves Stembark Leaves
92,140 110,049 109,446
22.
Rutaceae
Eleusine indica (L.) Gaertn. Nauclea diderrichii (De Wild.) Merr. Sarcocephalus latifolius (Sm.) E.A.Bruce (Syn: Nauclea latifolia (Sm)) Clausena anisata (Willd.) Hook.f. ex Benth.
Atapari-obuko
Leaves
99,457
23. 24.
Sapindaceae Verbenaceae
Lecaniodiscus cupanioides Planch. ex Benth. Lippia multiflora Moldenke
Arika Eforomoba
Leaves Leaves
110,081 106,562.
Hoslundia opposita Vahl Entandrophragma utile (Dawe & Sprague) Sprague Boerhavia diffusa L. Petiveria alliacea L.
Ascites, wounds, jaundice (Rawat et al., 1997). Flu, venereal diseases, dysmonorrhoea and worm infections (Ayensu, 1981). Diuretic, anti-helminthic, cough (Iqbal and Gnanaraj, 2012). Stomach pains, fever, diarrhea (Lamidi et al., 1995). Malaria, fever, toothaches (Onyeyili et al., 2001). Epilepsy, malaria, wounds, arthritis, rheumatism (Ojewole, 2002). Boils, burns, wounds, fever (Yemitan and Adeyemi, 2005). hepatic insufficiency, fever (Abena et al., 2003).
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Table 2 Primary in vitro antimicrobial activity of methanol extracts of Nigerian medicinal plants. S/no
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Plant extracts
Primary assay (Percentage inhibition, %)
Acanthospermum hispidum Boerhavia diffusa Bridelia ferruginea Clausena anisata Croton gratissimus Eleusine indica Entandrophragma utile Heliotropium indicum Hoslundia opposita Picralima nitida Lagenaria breviflora Lecaniodiscus cupanioides Lippia multiflora Macaranga barteri Mimosa pudica Nauclea diderrichii Petiveria alliacea Ricinodendron heudelotii Sarcocephalus latifolius Secamone afzelii Spondias mombin (leaves) Spondias mombin (stem) Terminalia ivorensis Terminalia superba
P. falciparum D6Pinh
C. albicans
A. fumigatus
C. neoformans
MRSA
E. coli
P. aeruginosa
K. pneumoniae
VRE
12 33 31 29 29 37 39 34 43 43 28 33 41 36 35 23 20 28 11 47 24 22 36 32
0 0 0 0 0 0 0 0 4 0 0 0 1 5 0 0 0 0 0 0 0 7 0 0
8 0 1 0 1 1 4 4 0 1 6 5 5 4 8 3 0 6 3 3 0 5 2 3
1 0 2 0 28 5 25 3 5 5 1 95 0 97 0 0 2 96 0 0 74 85 92 0
3 1 44 0 6 0 0 0 0 0 54 8 4 9 4 4 6 10 3 92 0 3 2 0
13 22 1 9 8 7 25 0 11 0 8 34 4 36 17 24 9 36 14 4 20 31 45 19
2 2 0 0 0 1 82 0 0 4 11 0 0 83 1 0 0 0 0 6 0 0 0 82
0 12 15 0 0 0 16 0 0 0 0 34 11 36 0 3 0 39 0 0 7 32 44 19
8 8 4 1 8 5 0 1 7 8 8 10 7 78 11 2 3 14 7 9 1 6 8 2
The bold represent extracts with more than 50% inhibition in the primary assay.
3.4. Antitrypanosomal assay The result of this study shows that T. brucei brucei was sensitive to ten plant extracts, namely, Entandrophragma utile, Lecaniodiscus cupanioides, Lippia multiflora, Macaranga barteri, Nauclea diderrichii, Ricinodendron heudelotii, Spondias mombin, Terminalia ivorensis, Terminalia superba and Eleusine indica with more than 90% inhibition against T. brucei. Extracts from active plants were then subjected to the secondary screening to determine the IC50, result of the secondary assay is also displayed in Table 4. 3.5. Cytotoxicity assay Of the plants which inhibited growth of T. brucei brucei, none showed more than 50% inhibition on differentiated THP1 cells at 10 μg/mL concentration. The highest inhibition was 4% in the primary cytotoxicity assay hence secondary assay was not conducted (Table 4). 4. Discussion Plants secondary metabolism yields an immeasurable wealth of chemical structures which have been and will continue to be a source
Table 3 Result of secondary in vitro antimicrobial activity of the most active plant extracts showing the concentrations that inhibited 50% of the microorganism (IC50) (μg/mL). S/no Plant extract
C. neoformans
MRSA
P. aeruginosa VRE
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
– – 157.149 75.625 31.728 – 163.787 32.095 – 0.25 –
– 189.55 – – – 78.33 – – – – 0.11
167.456 – – 165.244 – – – – 170.39 – 0.09
Entandrophragma utile Lagenaria breviflora Lecaniodiscus cupanioides Macaranga barteri Ricinodendron heudelotii Secamone afzelii Spondias mombin (stem) Terminalia ivorensis Terminalia superba Amphotericin B Ciprofloxacin
– – 114.026 – – – – – –
of new drugs. In view of the fact that natural products have been a veritable source of anti-infective agents (Schmidt et al., 2012, 2015), twenty-four medicinal plants extracts were evaluated for various activities against infectious agents. Antimicrobial activity was detected in nine of the plant extracts with Cryptococcus neoformans being the most sensitive organism to the extracts, five of the extracts were active against C. neoformans at the initial concentration of 200 μg/mL (Table 2) and three extracts had IC50 less than 100 μg/mL in the secondary assay (Table 3). Cryptococcosis is a fungal infection caused by C. neoformans. Previously, there has been low global incidence of cryptococcosis indicating that the host immune response is sufficient to prevent demonstration in immunocompetent patients (Springer and Chaturvedi, 2010). However, the disease has been increasingly found in immunocompromised patients, especially HIV-infected individuals (Nasser et al., 2011; Spiliopoulou et al., 2012). Cryptococcal meningitis caused by C. neoformans is the fourth most commonly recognized cause of life-threatening infection among AIDS patients. HIV-associated cryptococcal meningitis has been reported in many areas of the world having high HIV sero-prevalence, especially in sub-Saharan Africa (Sen et al., 2017). Cryptococcosis is often fatal, it can be treated with amphotericin B and fluconazole, but even when treated, the global mortality rate remains 15–30%; fatally is up to 70% in sub-Saharan Africa (Park et al., 2009), thus novel and more effective anti-cryptococcal therapies are needed. It is interesting to note that five of the extracts strongly inhibited the growth of C. neoformans with Ricinodendron heudelotii, T. ivorensis and M. barteri having an IC50 of 31.73, 32.10 and 75.63 μg/mL, respectively. It has been stated that in order to develop a strong proof of concept for all antimicrobial bioassays, IC50 values should be below 100 μg/mL for plants extracts (Cos et al., 2006); these three extracts met this criterion and they would be further investigated to determine their anti-infective components. Another organism, P. aeruginosa was also sensitive to three of the plant extracts, that is, E. utile, M. barteri and T. superba. For the antiprotozoal assays, none of the extracts had significant activity on the P. falciparum strain with inhibitory activity lesser than 47%; L. donovani was also not sensitive to any of the extracts, while ten of the extracts exhibited strong inhibitory activity against T. brucei brucei. Trypanosoma brucei brucei is a parasitic protozoa belonging to the Trypanosoma genus; responsible for vector-borne disease of
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Table 4 In vitro antitrypanosomal activity of methanol extracts of Nigerian medicinal plants against Trypanosoma brucei. S/No
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Plant
Acanthospermum hispidum Boerhavia diffusa Bridelia ferruginea Clausena anisata Croton gratissimus Eleusine indica Entandrophragma utile Heliotropium indicum Hoslundia opposita Picralima nitida Lagenaria breviflora Lecaniodiscus cupanioides Lippia multiflora Macaranga barteri Mimosa pudica Nauclea diderrichii Petiveria alliacea Ricinodendron heudelotii Sarcocephalus latifolius Secamone afzelii Spondias mombin (leaves) Spondias mombin (stem) Terminalia ivorensis Terminalia superba Pentamidine
Primary assay (Percentage inhibition, %)
Secondary assay (μg/mL)
L. donovani
L. donovani AMAST
T. brucei
THP1
T. brucei IC50
T. brucei IC90
THP1 IC50/IC90
6 10 5 5 7 7 14 5 11 6 12 7 5 3 10 8 5 8 8 10 1 5 8 5 –
0 15 7 2 0 1 18 3 3 1 8 5 9 0 0 2 0 15 0 0 0 3 9 0 –
0 0 0 0 0 97 94 0 0 0 0 96 93 94 0 95 0 95 21 24 0 95 96 94 –
0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 –
– – – – – 8.26 12.79 – – – – 15.1 15.28 16.64 – 14.87 – 13.08 – – – 16.24 12.21 13.65 1.5
– – – – – 10.14 18.3 – – – – 18.42 19.15 19.3 – 18.69 – 18.04 – – – 18.95 17.25 18.63 1.56
– N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20
vertebrate animals. In humans, it causes Human African Trypanosomiasis (HAT), or sleeping sickness. In animals, it causes animal trypanosomiasis (WHO, 2017). HAT is a neglected disease endemic in subSaharan Africa, it is usually fatal if left untreated. Currently, drugs therapy for HAT are few and not very effective. Moreover, there has been an increase in parasite resistance towards these drugs; drug resistance threatens successful control of fatal sleeping sickness in man and hampers commercial livestock production in sub-Saharan Africa (Matovu et al., 2001). It has been stated that only one advance has been made into HAT treatment in the last 25 years. Therefore, there is an increasing need for development of new and more effective drugs to treat this disease (Danica and Mauro, 2017). Medicinal plants from this study could provide a veritable source of new drug lead for trypanosomiasis control with ten of them having over 90% inhibition against Trypanosoma brucei at a concentration of 20 μg/mL. The most active extract against T. brucei brucei was Eleusine indica with IC50 and IC90 of 8.26 and 10.14 μg/mL, respectively. To the best of our knowledge, no previous report exist on any antitrypanosomal activity of this plant, thus, the fractionation of this extract and evaluation of resulting bioactive fractions, with the aim of identifying its antitrypanosomal constituents and isolation of the relevant natural products is ongoing. Entandrophragma utile was also significantly active with IC50 of 12.79 μg/mL (Table 4). All the active extract had IC50 less than 20 μg/mL, which is remarkable, considering the fact that these are crude plant extracts. None of the active plant extracts was toxic to differentiated THP1 cells, the highest inhibition was 4% at 10 μg/mL concentration. Macaranga barteri had the broadest spectrum of activity against the microorganisms with noteworthy activity against C. neoformans, P. aeruginosa and VRE. In fact, it was the only extract that significantly inhibited VRE, and was still very active against T. brucei. Macaranga barteri belongs to the plant genus Macaranga (Family Euphorbiaceae) which has been widely reported for various antimicrobial activities (Lim et al., 2009; Uduak and Kola, 2010; Verma et al., 2013; Lim et al., 2014). Four of the plant extracts tested in this study belong to the Euphorbiaceae family. They are used traditionally in the treatment of ailments such as respiratory infections, venereal diseases, toothache, rheumatism, cough, ulcer and wounds (Oliver, 1960).
5. Conclusion Despite the great advances in modern medicine in recent years, plants still make important contribution to health care delivery especially in rural areas. Part of the reasons why plants remain a research focus for drug development is the fact that they are easily sourced and can be selected based on their ethno-medicinal use. Ten medicinal plants from ethnobotanical sources used in this study displayed significant antitrypanosomal activity (IC50 range 8.76–16.64 μg/mL), while three had significant activity against the fungus Cryptococcus neoformans, with IC50 below 100 μg/mL. The study justified the fact that ethno-medicine is still an authentic source of novel anti-infective agents. Abbreviations ATCC CFU DMSO FBS FRIN HAT IMDM MRSA NTDs pLDH PMA RPMI THP1 VRE WHO
American type culture collection colony forming unit dimethyl sulfoxide fetal bovine serum forestry and research institute of Nigeria human African trypanosomiasis Iscove's modified Dulbecco's medium methicillin-resistant Staphylococcus aureus neglected tropical diseases parasitic lactate dehydrogenase phorbol 12-myristate 13-acetate Roswell park memorial institute medium transformed human monocytic vancomycin resistant enterococci World Health Organization
Acknowledgments The authors wish to acknowledge the National Centre for Natural Products Research School of Pharmacy, University of Mississippi, for the in vitro assays.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Antimicrobial and antiprotozoal activities of twenty-four Nigerian medicinal plant extracts O.O. Ogbole a,⁎, P.A. Segun b, P.S. Fasinu c a b c
Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria Department of Pharmacognosy, Faculty of Pharmacy, Olabisi Onabanjo University, Sagamu Campus, Nigeria Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, Campbell University, Buies Creek, NC 27506, USA
a r t i c l e
i n f o
Article history: Received 10 November 2017 Received in revised form 4 May 2018 Accepted 29 May 2018 Available online xxxx Edited by A Moteetee Keywords: Antileishmanial Antimicrobial Antiplasmodial Antitrypanosomal Drug discovery Eleusine indica Macaranga barteri
a b s t r a c t Medicinal plants exploration has become an important tool in the discovery of bioactive natural substances capable of inhibiting the mechanisms of microbial and protozoal activity. The aim of this study was to determine the antimicrobial and antiprotozoal activities of medicinal plants extracts used for the treatment of various illnesses in southwestern Nigeria. Twenty-four medicinal plant extracts were screened for antimicrobial activity against three fungal and six bacterial strains; and antiprotozoal activities against chloroquine-sensitive strains of Plasmodium falciparum (antiplasmodial assay), a culture of Leishmania donovani promastigotes and axenic amastigotes (antileishmanial activity) and two-days old culture of Trypanosoma brucei brucei (antitrypanosomal assay). Five extracts exhibited strong antifungal activity against Cryptococcus neoformans, with Ricinodendron heudelotii (Baill.) Heckel, Terminalia ivorensis A.Chev and Macaranga barteri Müll. Arg, having an IC50 of 31.73, 32.10 and 75.63 μg/mL, respectively. Ten of the extracts were active against T. brucei, with Eleusine indica displaying the most significant activity (IC50 and IC90 of 8.26 and 10.14 μg/mL). None of the extracts displayed any significant antiplasmodial and antileishmanial activities. In addition, none of the extracts displayed cytotoxicity on transformed human monocytic (THP1) cells. The study revealed that M. barteri had the broadest spectrum of activity, with activity against C. neoformans, P. aeruginosa, vancomycin resistant Enterococci faecalis (VRE) and T. brucei. M. barteri could be exploited for broad spectrum antimicrobial and antitrypanosomal activity, while Eleusine indica could be subjected to bioassay guided fractionation and isolation of antitrypanosomal constituents. This study suggests that the evaluated plants are potential sources of novel anti-infective agents. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Infectious diseases are one of the most important factors responsible for the high rate of morbidity and mortality, especially in developing countries, where over 25% of global annual mortality are primarily caused by infectious diseases (Mahady, 2005). Meanwhile, secondary effects of infections are responsible for millions more deaths (Satcher, 1995; Chua and Gubler, 2013). Human African trypanosomiasis (HAT), Chagas diseases, leishmaniasis and malaria are infectious diseases caused by protozoans. HAT, Chagas diseases and leishmaniasis are classified as neglected tropical diseases (NTDs) by the World Health Organization (WHO) and they represent a major threat to the health and well-being of over a billion lives worldwide (WHO, 2010; Llurba-Montesino et al., 2015). Over the years, significant progress has been made in the field of microbiology and the control of microorganisms. In spite of this ⁎ Corresponding author. E-mail address: [email protected] (O.O. Ogbole).
https://doi.org/10.1016/j.sajb.2018.05.028 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
progress, sporadic episodes of epidemics triggered by drug-resistant microorganisms and pathogenic microbes remain a massive threat to public health. The relentless rise in antibiotic resistance, and emergence of new and chronic infectious diseases are factors that stimulate a continuous search for new anti-infective agents (Costa et al., 2016; Mahady, 2005; Al-Judaibi, 2014; Mehani et al., 2016). Plant-based traditional medicine represents the primary or the only veritable form of accessible primary health care in many parts of the developing world, especially for people living in rural Africa. The information about the healing properties of medicinal plants have been transmitted over the centuries among generations of human communities; the existence of traditional medicine depends on plant species diversity and the knowledge of their use as herbal medicine. Currently, the demand for the herbal drug treatment of various ailments is increasing and plants are being explored globally for the development of newer drugs (Silva and Fernandes, 2010; Mahomoodally, 2013). Considering the importance of medicinal plants for the discovery and identification of bioactive natural substances capable of inhibiting the
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mechanisms of microbial activity and resistance (Amoa-Bosompem, 2016; Nguyen, 2016), this work reports the evaluation of twenty-four extracts from medicinal plants utilized in ethnobotanical medicine in Nigeria (Ajaiyeoba et al., 2003; Osowole et al., 2005; Ogbole et al., 2010; Segun et al., 2018) against agents responsible for various microbial infections including malaria, leishmaniasis and trypanosomiasis.
2. Materials and methods 2.1. Plant collection Fresh leaves of the medicinal plants were collected from their various habitat at the onset of raining season in Ibadan, Nigeria between April and May 2015. The plants were identified and authenticated by Dr. Osiyemi of the Forestry and Research Institute of Nigeria (FRIN), Ibadan and assigned voucher specimen numbers. Voucher specimens were deposited at the Forest Herbarium, Ibadan (FHI).
2.2. Preparation and extraction of plant materials Each plant material was shade-dried at room temperature (27–33 °C) and pulverized. The dried powdered materials of each plant (100 g) were extracted by maceration into methanol (750 mL) and then filtered to obtain methanol extracts of each plant. The filtrate was concentrated with a rotary evaporator at 40 °C and the dried extracts were kept in a refrigerator until needed. Even though water is mostly used as solvent base for traditional herbal preparation, methanol was used in this study, mainly because of its amphiphilicity; it dissolves a wider range of compounds than water, including polar and to a large extent some non-polar compounds. Moreover, methanol easily evaporates so it is easier to separate from the extract than water.
2.3. Antimicrobial assay Antimicrobial susceptibility testing was carried out at the National Center for Natural Products Research, University of Mississippi, USA. The test organisms were obtained from the American Type Culture Collection (ATCC, Manassas, VA). These included the fungi: Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 90113 and Aspergillus fumigatus ATCC 204305; bacteria: Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 4352 and Vancomycin resistant Enterococci faecalis ATCC 29212 (VRE) The in vitro antimicrobial assay was done using a modified version of the CLSI methods as described below (Samoylenko et al., 2009). Extracts were dissolved in dimethyl sulfoxide (DMSO) and serially diluted in 20% DMSO/saline and then transferred, in duplicate, into 96-well flat bottom microplates. The final concentration of DMSO used in this assay was 0.1%, as concentration below 1% is not known to exert any observable toxic effect on cells (Timm et al., 2013). Microbial innocula were prepared in assay medium to afford target 106 cfu/mL, after addition to the samples. Growth, solvent and media controls were included in each test plate. Assay plates were read at 530 nm before and after incubation using the Biotek Power wave XS plate reader. Percentage growth was plotted against test concentration to afford the IC50 values (concentration that affords 50% growth relative to control). Crude plant extracts were tested in the primary assay at a single concentration of 200 μg/mL and percentage inhibitions were calculated, extracts with more than 50% inhibition were subjected to the secondary screening using serially diluted extract at concentration range of 200–8 μg/mL. Drug controls were included in the secondary assay; ciprofloxacin (ICN Biomedicals, Ohio) for bacteria and amphotericin B (ICN Biomedicals, Ohio) for fungi.
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2.4. Antiplasmodial assay Antiplasmodial activities were measured in vitro using the assay protocol based on a colorimetric method that determines the parasitic lactate dehydrogenase (pLDH) activity (Makler et al., 1993; Samoylenko et al., 2009). The assay was performed in 96-well microplate and included a chloroquine-sensitive strains of Plasmodium falciparum (D6). In the primary screening, the crude plant extracts were tested in duplicate, at a single concentration of 15.9 μg/mL on the chloroquine-sensitive (D6) strain of P. falciparum. DMSO and chloroquine were included as vehicle and drug control, respectively. 2.5. In vitro assay for antileishmanial activity Alamar blue assay was used to evaluate the antileishmanial activity of the plant extracts. The assay was carried out on a culture of Leishmania donovani promastigotes and axenic amastigotes (Mikus and Steverding, 2000). The promastigotes culture was maintained at 26 °C in Roswell Park Memorial Institute medium (RPMI 1640), pH 7.4 with 10% fetal bovine serum (FBS). The axenic amastigotes were cultured at 37 °C and 5% CO2 in RPMI-1640 supplemented with 4-morpholineethanesulfonic acid (MES) (4.88 g/L), L-glutamine (298.2 mg/L), adenosine (26.7 mg/L), folic acid (10.1 mg/L), BME vitamin mix, sodium bicarbonate (352.8 mg/L) and 10% FBS. The pH of the culture medium was 5.5. For the primary assay, plant extracts were tested at a single concentration of 20.0 μg/mL (in duplicate) on both the promastigotes and axenic amastigotes culture. The extracts that showed more than 90% inhibition of growth in primary screening were subjected to secondary screening for dose response analysis. In a 96-well microplate, appropriate dilutions of samples, were added to the Leishmania promastigotes/axenic amastigote culture (2 × 106 cell mL− 1). The extracts were tested at six concentrations ranging from 40 to 0.0128 μg/mL. The plates were incubated for 72 h at 26 °C and 37 °C, respectively, for promastigotes and axenic amastigotes. The growth of Leishmania promastigotes/amastigotes was determined. IC50 and IC 90 values were computed from the dose–response curves (Donega et al., 2014). 2.6. Antitrypanosomal assay Two-day old culture of Trypanosoma brucei, in the exponential growth stage, was diluted with Iscove's Modified Dulbecco's medium (IMDM) to obtain 5000 parasites/mL. The assays were conducted in 96-well microplates. Extract dilutions (1 mg/mL) were made in IMDM from the stock extracts (20 mg/mL) for the primary screening. Each well received 4 μL of diluted extract and 196 μL of the culture medium to obtain a final volume of 200 μL. Plates were incubated at 37 °C in 5% CO2 for 48 h. Ten microliter (10 μL) of alamar blue (AbD Serotec, catalog number BUF012B) was added into each well and plates were then incubated overnight. Standard fluorescence was measured on a Fluostar Galaxy fluorometer (BMG LabTechnologies) at 544 nm excitation and 590 nm emission. Pentamidine was included as the control drug (Table 4). Extracts with more than 90% inhibition of T. brucei growth in primary screening were subjected to secondary screening to evaluate dose–response analysis. They were screened at concentrations ranging from 10 to 0.4 μg/mL. IC50 and IC90 values were computed from dose response growth inhibition curve by XLfit version 5.2.2. (Jain et al., 2016). 2.7. Cytotoxicity assay The extracts were screened for cytotoxicity against transformed human monocytic (THP1) cells. Four-day old culture of THP1 cells was diluted with RPMI medium to 2.5 × 105 cells/mL. For transformation of the cells to adherent macrophages, phorbol 12-myristate 13-acetate (PMA) was added to the culture at 25 ng/mL concentration
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(Jain et al., 2012). The PMA treated THP1 cell culture was dispensed in 96 well plates with 200 μL culture (2.5 × 105 cells/mL) in each well and plates were incubated at 37 °C in 5% CO2 incubator overnight. Extracts were diluted in separate plates in RPMI medium. The medium in plates with THP1 cells was replaced with fresh medium. The diluted plant extracts were added to these plates. The plates were returned to the CO2 incubator for 48 h at 37 °C, 5% CO2. After 48 h, 10 μL of alamar blue solution was added to each well and the plates were incubated further overnight. Standard fluorescence was measured on a fluorometer at 544 nm excitation and 590 nm emission. Cytotoxicity screening was carried out only for active extracts, that have shown more than 90% inhibition in the primary assay.
and Terminalia ivorensis displaying percentage inhibition of 83%, 82% and 82% respectively, against P. aeruginosa in the primary antimicrobial assay. The extracts of Lagenaria breviflora and Secamone afzelii exhibited significant antimicrobial activity against the MRSA. Only M. barteri extract had activity against VRE, none of the extracts displayed any significant activity against K. pneumoniae, E. coli and two of the fungiC. albicans and A. fumigatus. Five extracts, namely, Lagenaria breviflora, Macaranga barteri, Ricinodendron heudelotii, Spondias mombin (l) and Spondias mombin (b) extracts had percentage inhibition greater than 80% against C. neoformans in the primary assay (Table 2). Active extracts were subjected to secondary assay and the result is displayed in Table 3. 3.2. Antiplasmodial assay
3. Results The ethnobotanical information on the various plants belonging to 18 families are summarized in Table 1. Twenty-four plant extracts were screened for their antimicrobial and antiplasmodial activity. Plant extracts with percentage inhibition greater than 50% against T. brucei brucei were subjected to the cytotoxicity and secondary assay. Extract of Macaranga barteri had a wider spectrum of activity than the rest of the plant extracts.
One strain of P. falciparum D6 (chloroquine-sensitive) was used in the primary screening, the antiplasmodial assay could not proceed beyond the primary screening since none of the extract exhibited significant inhibitory activity against the parasite, the result of the primary screening for antiplasmodial is presented in Table 2. The highest (47%) activity was displayed by S. afzelii. 3.3. Antileishmanial assay
3.1. Antimicrobial assay The antimicrobial assay was carried out against three fungi and six bacteria, with the extracts of Macaranga barteri, Entandrophragma utile
The primary assay carried out on L. donovani promastigotes and axenic amastigotes did not proceed beyond the primary assay since none of the extract had any significant activity on the protozoan.
Table 1 Ethnobotanical information on plants used in this study. S/no
Family
Plant
Local name (Yoruba)
Part used
Voucher no (FHI)
Ethnomedicinal uses
1.
Anacardiaceae
Spondias mombin L.
Iyeye
Leaves
63,948
2.
Anacardiaceae
Spondias mombin L.
Iyeye
Stembark
63,948
3.
Apocyanaceae
Picralima nitida (Stapf) T.Durand & H.Durand
Abeere
Seed
109,572
4.
Apocyanaceae
Secamone afzelii (Roem. & Schult.) K.Schum.
Ailu
Leaves
109,995
5.
Asteraceae
Acanthospermum hispidum DC
Dagunro gogoro
Leaves
110,050
6.
Boraginaceae
Heliotropium indicum L.
Apari igun
Leaves
110,156
7. 8.
Combretaceae Combretaceae
Terminalia ivorensis A.Chev Terminalia superba Engl. & Diels
Afara-dudu Afara
Stembark Stembark
105,432 109,800
9.
Cucurbitaceae
Tagiri
Leaves
109,040
10. 11. 12. 13.
Euphorbiaceae Euphorbiaceae Euphorbiaceae Euphorbiaceae
Agbasa Erinmado Iralodan Ajeofole
Leaves Stembark Stembark Leaves
107,230 110,573 109,985 109,041
Gonorrhea, cough, bronchitis (Adesegun et al., 2007). Stomach ulcer, wounds (Agyare et al., 2009). Bruises, boils, dislocations, burns (Iwu, 2014). Fever, dysentery, convulsions (Ngadjui et al., 2002).
14.
Fabaceae
Lagenaria breviflora (Benth.) Roberty (Syn: Adenopus breviflorus Benth) Macaranga barteri Müll.Arg. Ricinodendron heudelotii (Baill.) Heckel Bridelia ferruginea Benth. Croton gratissimus Burch. (Syn: Croton zambesicus Mull Arg) Mimosa pudica L.
Dysentery, hemorrhoids, gonorrhea, stomachache leucorrhoea, malaria (Ayoka et al., 2006, Diallo et al., 2006, Ruiz et al., 2011). Dysentery, hemorrhoids, gonorrhea, stomachache leucorrhoea, malaria (Ayoka et al., 2006, Diallo et al., 2006, Ruiz et al., 2011). Malaria, sexual impotence, dysmenorrhea, gastrointestinal disorder (Okokon et al., 2007). Diabetes, wounds, boils, malaria (Weniger et al., 2004, Abo et al., 2008, Zabri et al., 2008). Headaches, abdominal pains, convulsions, cough, jaundice, epilepsy. (Ganfon et al., 2012). Wounds, flatulence, inflammation, skin ulcers and conjunctivitis (Paul et al., 2015). Syphilis, burns, bruises, arthritis, hemorrhoids (Iwu, 2014). Diabetes mellitus, gastroenteritis, female infertility, abdominal pains (Kuete et al., 2010). Coccidiosis, measles (Olorunnisola et al., 2015).
Patanmo
Leaves
100,332
15. 16.
Lamiaceae Meliaceae
Efirin-odan Jebo
Leaves Stembark
108,121 86,848
Headache, migraine, insomnia, diarrhea, dysentery (Amalraj and Ignacimuthu, 2002). Malaria, wounds (Achenbach et al., 1992). Peptic ulcer (John and Onabanjo, 1990).
17. 18.
Nyctaginaceae Phytolaccaceae
Etipase-erinla Awopa
Root Leaves
109,603 109,603
19. 20. 21.
Poaceae Rubiaceae Rubiaceae
Gbegi Opepe Egbesi
Leaves Stembark Leaves
92,140 110,049 109,446
22.
Rutaceae
Eleusine indica (L.) Gaertn. Nauclea diderrichii (De Wild.) Merr. Sarcocephalus latifolius (Sm.) E.A.Bruce (Syn: Nauclea latifolia (Sm)) Clausena anisata (Willd.) Hook.f. ex Benth.
Atapari-obuko
Leaves
99,457
23. 24.
Sapindaceae Verbenaceae
Lecaniodiscus cupanioides Planch. ex Benth. Lippia multiflora Moldenke
Arika Eforomoba
Leaves Leaves
110,081 106,562.
Hoslundia opposita Vahl Entandrophragma utile (Dawe & Sprague) Sprague Boerhavia diffusa L. Petiveria alliacea L.
Ascites, wounds, jaundice (Rawat et al., 1997). Flu, venereal diseases, dysmonorrhoea and worm infections (Ayensu, 1981). Diuretic, anti-helminthic, cough (Iqbal and Gnanaraj, 2012). Stomach pains, fever, diarrhea (Lamidi et al., 1995). Malaria, fever, toothaches (Onyeyili et al., 2001). Epilepsy, malaria, wounds, arthritis, rheumatism (Ojewole, 2002). Boils, burns, wounds, fever (Yemitan and Adeyemi, 2005). hepatic insufficiency, fever (Abena et al., 2003).
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Table 2 Primary in vitro antimicrobial activity of methanol extracts of Nigerian medicinal plants. S/no
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Plant extracts
Primary assay (Percentage inhibition, %)
Acanthospermum hispidum Boerhavia diffusa Bridelia ferruginea Clausena anisata Croton gratissimus Eleusine indica Entandrophragma utile Heliotropium indicum Hoslundia opposita Picralima nitida Lagenaria breviflora Lecaniodiscus cupanioides Lippia multiflora Macaranga barteri Mimosa pudica Nauclea diderrichii Petiveria alliacea Ricinodendron heudelotii Sarcocephalus latifolius Secamone afzelii Spondias mombin (leaves) Spondias mombin (stem) Terminalia ivorensis Terminalia superba
P. falciparum D6Pinh
C. albicans
A. fumigatus
C. neoformans
MRSA
E. coli
P. aeruginosa
K. pneumoniae
VRE
12 33 31 29 29 37 39 34 43 43 28 33 41 36 35 23 20 28 11 47 24 22 36 32
0 0 0 0 0 0 0 0 4 0 0 0 1 5 0 0 0 0 0 0 0 7 0 0
8 0 1 0 1 1 4 4 0 1 6 5 5 4 8 3 0 6 3 3 0 5 2 3
1 0 2 0 28 5 25 3 5 5 1 95 0 97 0 0 2 96 0 0 74 85 92 0
3 1 44 0 6 0 0 0 0 0 54 8 4 9 4 4 6 10 3 92 0 3 2 0
13 22 1 9 8 7 25 0 11 0 8 34 4 36 17 24 9 36 14 4 20 31 45 19
2 2 0 0 0 1 82 0 0 4 11 0 0 83 1 0 0 0 0 6 0 0 0 82
0 12 15 0 0 0 16 0 0 0 0 34 11 36 0 3 0 39 0 0 7 32 44 19
8 8 4 1 8 5 0 1 7 8 8 10 7 78 11 2 3 14 7 9 1 6 8 2
The bold represent extracts with more than 50% inhibition in the primary assay.
3.4. Antitrypanosomal assay The result of this study shows that T. brucei brucei was sensitive to ten plant extracts, namely, Entandrophragma utile, Lecaniodiscus cupanioides, Lippia multiflora, Macaranga barteri, Nauclea diderrichii, Ricinodendron heudelotii, Spondias mombin, Terminalia ivorensis, Terminalia superba and Eleusine indica with more than 90% inhibition against T. brucei. Extracts from active plants were then subjected to the secondary screening to determine the IC50, result of the secondary assay is also displayed in Table 4. 3.5. Cytotoxicity assay Of the plants which inhibited growth of T. brucei brucei, none showed more than 50% inhibition on differentiated THP1 cells at 10 μg/mL concentration. The highest inhibition was 4% in the primary cytotoxicity assay hence secondary assay was not conducted (Table 4). 4. Discussion Plants secondary metabolism yields an immeasurable wealth of chemical structures which have been and will continue to be a source
Table 3 Result of secondary in vitro antimicrobial activity of the most active plant extracts showing the concentrations that inhibited 50% of the microorganism (IC50) (μg/mL). S/no Plant extract
C. neoformans
MRSA
P. aeruginosa VRE
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
– – 157.149 75.625 31.728 – 163.787 32.095 – 0.25 –
– 189.55 – – – 78.33 – – – – 0.11
167.456 – – 165.244 – – – – 170.39 – 0.09
Entandrophragma utile Lagenaria breviflora Lecaniodiscus cupanioides Macaranga barteri Ricinodendron heudelotii Secamone afzelii Spondias mombin (stem) Terminalia ivorensis Terminalia superba Amphotericin B Ciprofloxacin
– – 114.026 – – – – – –
of new drugs. In view of the fact that natural products have been a veritable source of anti-infective agents (Schmidt et al., 2012, 2015), twenty-four medicinal plants extracts were evaluated for various activities against infectious agents. Antimicrobial activity was detected in nine of the plant extracts with Cryptococcus neoformans being the most sensitive organism to the extracts, five of the extracts were active against C. neoformans at the initial concentration of 200 μg/mL (Table 2) and three extracts had IC50 less than 100 μg/mL in the secondary assay (Table 3). Cryptococcosis is a fungal infection caused by C. neoformans. Previously, there has been low global incidence of cryptococcosis indicating that the host immune response is sufficient to prevent demonstration in immunocompetent patients (Springer and Chaturvedi, 2010). However, the disease has been increasingly found in immunocompromised patients, especially HIV-infected individuals (Nasser et al., 2011; Spiliopoulou et al., 2012). Cryptococcal meningitis caused by C. neoformans is the fourth most commonly recognized cause of life-threatening infection among AIDS patients. HIV-associated cryptococcal meningitis has been reported in many areas of the world having high HIV sero-prevalence, especially in sub-Saharan Africa (Sen et al., 2017). Cryptococcosis is often fatal, it can be treated with amphotericin B and fluconazole, but even when treated, the global mortality rate remains 15–30%; fatally is up to 70% in sub-Saharan Africa (Park et al., 2009), thus novel and more effective anti-cryptococcal therapies are needed. It is interesting to note that five of the extracts strongly inhibited the growth of C. neoformans with Ricinodendron heudelotii, T. ivorensis and M. barteri having an IC50 of 31.73, 32.10 and 75.63 μg/mL, respectively. It has been stated that in order to develop a strong proof of concept for all antimicrobial bioassays, IC50 values should be below 100 μg/mL for plants extracts (Cos et al., 2006); these three extracts met this criterion and they would be further investigated to determine their anti-infective components. Another organism, P. aeruginosa was also sensitive to three of the plant extracts, that is, E. utile, M. barteri and T. superba. For the antiprotozoal assays, none of the extracts had significant activity on the P. falciparum strain with inhibitory activity lesser than 47%; L. donovani was also not sensitive to any of the extracts, while ten of the extracts exhibited strong inhibitory activity against T. brucei brucei. Trypanosoma brucei brucei is a parasitic protozoa belonging to the Trypanosoma genus; responsible for vector-borne disease of
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Table 4 In vitro antitrypanosomal activity of methanol extracts of Nigerian medicinal plants against Trypanosoma brucei. S/No
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Plant
Acanthospermum hispidum Boerhavia diffusa Bridelia ferruginea Clausena anisata Croton gratissimus Eleusine indica Entandrophragma utile Heliotropium indicum Hoslundia opposita Picralima nitida Lagenaria breviflora Lecaniodiscus cupanioides Lippia multiflora Macaranga barteri Mimosa pudica Nauclea diderrichii Petiveria alliacea Ricinodendron heudelotii Sarcocephalus latifolius Secamone afzelii Spondias mombin (leaves) Spondias mombin (stem) Terminalia ivorensis Terminalia superba Pentamidine
Primary assay (Percentage inhibition, %)
Secondary assay (μg/mL)
L. donovani
L. donovani AMAST
T. brucei
THP1
T. brucei IC50
T. brucei IC90
THP1 IC50/IC90
6 10 5 5 7 7 14 5 11 6 12 7 5 3 10 8 5 8 8 10 1 5 8 5 –
0 15 7 2 0 1 18 3 3 1 8 5 9 0 0 2 0 15 0 0 0 3 9 0 –
0 0 0 0 0 97 94 0 0 0 0 96 93 94 0 95 0 95 21 24 0 95 96 94 –
0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 –
– – – – – 8.26 12.79 – – – – 15.1 15.28 16.64 – 14.87 – 13.08 – – – 16.24 12.21 13.65 1.5
– – – – – 10.14 18.3 – – – – 18.42 19.15 19.3 – 18.69 – 18.04 – – – 18.95 17.25 18.63 1.56
– N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20 N20
vertebrate animals. In humans, it causes Human African Trypanosomiasis (HAT), or sleeping sickness. In animals, it causes animal trypanosomiasis (WHO, 2017). HAT is a neglected disease endemic in subSaharan Africa, it is usually fatal if left untreated. Currently, drugs therapy for HAT are few and not very effective. Moreover, there has been an increase in parasite resistance towards these drugs; drug resistance threatens successful control of fatal sleeping sickness in man and hampers commercial livestock production in sub-Saharan Africa (Matovu et al., 2001). It has been stated that only one advance has been made into HAT treatment in the last 25 years. Therefore, there is an increasing need for development of new and more effective drugs to treat this disease (Danica and Mauro, 2017). Medicinal plants from this study could provide a veritable source of new drug lead for trypanosomiasis control with ten of them having over 90% inhibition against Trypanosoma brucei at a concentration of 20 μg/mL. The most active extract against T. brucei brucei was Eleusine indica with IC50 and IC90 of 8.26 and 10.14 μg/mL, respectively. To the best of our knowledge, no previous report exist on any antitrypanosomal activity of this plant, thus, the fractionation of this extract and evaluation of resulting bioactive fractions, with the aim of identifying its antitrypanosomal constituents and isolation of the relevant natural products is ongoing. Entandrophragma utile was also significantly active with IC50 of 12.79 μg/mL (Table 4). All the active extract had IC50 less than 20 μg/mL, which is remarkable, considering the fact that these are crude plant extracts. None of the active plant extracts was toxic to differentiated THP1 cells, the highest inhibition was 4% at 10 μg/mL concentration. Macaranga barteri had the broadest spectrum of activity against the microorganisms with noteworthy activity against C. neoformans, P. aeruginosa and VRE. In fact, it was the only extract that significantly inhibited VRE, and was still very active against T. brucei. Macaranga barteri belongs to the plant genus Macaranga (Family Euphorbiaceae) which has been widely reported for various antimicrobial activities (Lim et al., 2009; Uduak and Kola, 2010; Verma et al., 2013; Lim et al., 2014). Four of the plant extracts tested in this study belong to the Euphorbiaceae family. They are used traditionally in the treatment of ailments such as respiratory infections, venereal diseases, toothache, rheumatism, cough, ulcer and wounds (Oliver, 1960).
5. Conclusion Despite the great advances in modern medicine in recent years, plants still make important contribution to health care delivery especially in rural areas. Part of the reasons why plants remain a research focus for drug development is the fact that they are easily sourced and can be selected based on their ethno-medicinal use. Ten medicinal plants from ethnobotanical sources used in this study displayed significant antitrypanosomal activity (IC50 range 8.76–16.64 μg/mL), while three had significant activity against the fungus Cryptococcus neoformans, with IC50 below 100 μg/mL. The study justified the fact that ethno-medicine is still an authentic source of novel anti-infective agents. Abbreviations ATCC CFU DMSO FBS FRIN HAT IMDM MRSA NTDs pLDH PMA RPMI THP1 VRE WHO
American type culture collection colony forming unit dimethyl sulfoxide fetal bovine serum forestry and research institute of Nigeria human African trypanosomiasis Iscove's modified Dulbecco's medium methicillin-resistant Staphylococcus aureus neglected tropical diseases parasitic lactate dehydrogenase phorbol 12-myristate 13-acetate Roswell park memorial institute medium transformed human monocytic vancomycin resistant enterococci World Health Organization
Acknowledgments The authors wish to acknowledge the National Centre for Natural Products Research School of Pharmacy, University of Mississippi, for the in vitro assays.
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