Abstract
Background
A great revival of scientific interests in drug discovery has been witnessed in recent years from medicinal plants for health maintenance. The aim of this work was to investigate three Nigerian medicinal plants collected in Nigeria for their in vitro antiplasmodial and antimicrobial activities.
Materials and Methods
Extracts obtained from parts of Persea americana, Jatropha podagrica and Picralima nitida and their fractions were evaluated for in vitro antiprotozoal and antimicrobial activity.
Result
The methanol extract of P. nitida demonstrated activity against chloroquine-sensitive and chloroquine-resistant P. falciparum clones with IC50 values of 6.3 and 6.0 µg/mL, respectively. Methanol and chloroform extracts of P. americana seed showed antifungal activity against Cryptococcus neoformans IC50 less than 8 and 8.211 µg/mL respectively. Finally, the petroleum ether extract of P. americana had activity against methicillin-resistant Staphylococcus aureus (MRSA) with an IC50 value of 8.7 µg/mL.
Conclusion
The study revealed the antibacterial and antiplasmodial activities of the plants extracts at the tested concentrations.
Keywords: Antifungal, Antibacterial, Persea americana, Picralima nitida, Jatropha podagrica, Plasmodium falciparum
Introduction
Parasitic diseases such as malaria have a high mortality rate having a significant impact in developing countries and affecting several hundred millions of people worldwide. Malaria is one of the most important parasitic diseases in the world and is a major global health problem affecting over one hundred countries with disease prevalence escalating at an alarming rate, particularly in the last two decades. Rapid development of resistance by Plasmodium falciparum to the conventional drugs such as chloroquine necessitates the search for new antimalarials (Iwu et al., 1994; Wolf, 2002; Guerin et al., 2002; Fournet and Munoz, 2002). Malaria, a devastating infectious disease caused by highly adaptable protozoan parasites of the genus Plasmodium, has impacted on humans for more than 4000 years, causing illness and an estimated 1.5–2.5 million deaths each year. Malaria is endemic throughout the tropics, especially in sub-Saharan Africa and the developing world, threatening about 40 % of the world's population. Although four Plasmodium parasite species can infect humans, Plasmodium falciparum causes the majority of illnesses and deaths. Severe malaria, defined as acute malaria with major signs of organ dysfunction or high levels of parasitemia, predominantly affects children and pregnant women (Piece and Miller, 2009; Rosenthal, 2008; White, 2008).
Chemotherapy is still at the forefront in the fight against malaria due to the unavailability of effective vaccines. Numerous drugs have been developed for the treatment of uncomplicated malaria, for example, mefloquine, primaquine, quinidine, proguanil (Genton, 2008; Vekemans and Ballon, 2005). There is still need to search for newer and novel antimalarial agents from natural products via ethnopharmacological approach.
Similarly, an increasing number of multidrug-resistant microbial pathogens have become a serious problem particularly during the last decade and provide the impetus for the search and discovery of novel antibacterial and antifungal agents active against these pathogens (Liu and Balasubramanian, 2001).
Picralima nitida is a tree or shrub belonging to the family Apocynaceae. It is widely distributed in high deciduous forest of West and Central Africa. It is employed in African traditional medicine as antipyretic, antimalarial, aphrodisiac and for GIT disorders (Wosu and Ibe, 1989).
Jatropha podagrica Hook is a shrub grown in West Africa gardens for its showy red flowers. It is known locally in south western Nigeria as lapalapa funfun. Jatropha species are found in Africa, Asia and Latin America where they are used in folk medicine to treat various diseases including parasitic skin infections, hepatitis and sexually transmitted diseases (Aiyelaagbe et al., 2002; Sanni et al., 1988; Schmeda-Hirschmann et al., 1992). Various medicinal and pesticidal properties including antibacterial (pneumonia), antitumor and insect antifeedant have been attributed to this plant (Odebiyi, 1980). Phytochemicals in different parts of the plants includes flavonoids, steroids, alkaloids and diterpenoids have been isolated and characterized (Aiyelaagbe et al., 2007, 1998; Das and Venkatail, 2006).
Persea americana Mill commonly known as ‘avocado pear’ is a medium-sized, single-stemmed, terrestrial, erect, perennial, deciduous, evergreen tree of 15 – 20 m in height. The leaves and other morphological parts of P. americana possess medicinal properties, and are widely used in traditional medicines of many African countries as antitussive, antimicrobial, antidiabetic, antiparasitic, anti allergic, antihypertensive, analgesic and anti-inflammatory remedies (Adeyemi et al., 2002; Antia et al., 2005; Adeboye et al., 1999; Owolabi et al., 2005).
In this study, extracts of different parts of P. americana were evaluated for the first time for antifungal and antibacterial activities. The methanol extract and fractions of P. nitida against two strains of Plasmodium falciparum were determined.
Materials and Methods
Plant materials
All Plant material (Table 1) was collected from Ovia North east, Owan east and Benin City, all in Edo State, Nigeria between January and June, 2013. They were identified and authenticated by Mr. Ugbogu O. A. and Shasanya O. S. of the Forest Research Institute of Nigeria (FRIN), Ibadan where voucher specimens is deposited in the Herbarium (Table 1).
Table 1.
Plants collection, parts tested and voucher number
| Plant | Voucher speciment no. |
Locality | Family | Part tested | Extraction yield | |
| Picralima nitida Durand | FHI109572 | Benin City | Apocynaceae | stem | 11.25 | |
| PNS1 | 2.23 | |||||
| PNS2 | 1.45 | |||||
| PNS3 | 2.23 | |||||
| PNS4 | 1.08 | |||||
| PNS5 | 3.45 | |||||
| Persea americana Mill | FHI 107767 | Ovia north east local | Lauraceae | Root, seed | 40.68 ( R) | |
| PASC | government area, | 5.78 | ||||
| PARP | Edot State | 2.34 | ||||
| PAS | 12.85 | |||||
| Jatropha podagrica Hook | FHI 93265 | Owan east local | Euphorbiaceae | fruit | 7.02% | |
| government area, | ||||||
| Edo State | ||||||
Preparation of extracts
The powdered (100 g) material of each sample was extracted with 500 ml methanol for 48 hr ( 3 X ) by cold maceration, filtered and the filtrate evaporated to dryness to obtained crude extract of P. nitida (PNS), J. podagrica fruit (JPF), P. americana root (PAR), P. americana seed (PAS ) and chloroform fraction of P. americana seed (PASC). The dried extracts and fractions were each screened for antiplasmodial and antimicrobial activities. The fractions PN1, PN2, PN3, PN4 and PN5 are hexane: ethylacetate 50 %, ethylacetate 100 %, hexane: ethylacetate 80 %, methanol 100 % and alkaloidal mixture respectively, were obtained from the vacuum liquid chromatography (VLC) with normal silica and the solvents.
Antimicrobial testing
In vitro antimicrobial activity against a panel of microorganisms, including fungi: Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), C. neoformans (ATCC 90113) and Aspergillus fumigatus (ATCC 204305); and bacteria: Staphylococcus aureus (ATCC 29213), methicillin-resistant S. aureus (MRSa) (ATCC 33591), Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27853) and Mycobacterium intracellulare (ATCC 23068), was determined using modified versions of the CLSI/NCCLS methods (NCCL, 2000; NCCL, 2002). M. intracellulare and A. fumigatus was tested using an Alamar Blue method (Franzblau et al., 1998). All organisms were obtained from the American Type Culture Collection (Manassas, VA). Samples, dissolved in DMSO, were serially diluted in saline and transferred in duplicate to 96 well micro plates. Susceptibility testing was performed for all organ cate to 96-well flat bottom micro plates. Microbial inocula were prepared by correcting the OD630 of microbe suspensions in incubation broth to afford final target inocula. Controls [fungi: amphotericin B; bacteria: ciprofloxacin (ICN Biomedicals, OH)] were included in each assay. All plates were read at 530 or 544(ex)/590(em) nm (M. intracellulare and A. fumigatus) prior to and after incubation. Percent growth was plotted versus test concentration to afford the IC50 using XLFit (Alameda, CA).
Antimalarial/Parasite LDH Assay
The in vitro antimalarial assay procedure utilized was an adaptation of the parasite lactate dehydrogenase (pLDH) assay developed by Makler et al., 1993. The assay was performed in a 96-well microplate and included two P. falciparum clones [Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine-resistant)]. In primary screening the crude plant extracts were tested, in duplicate, at a single concentration of 15.9 íg/mL only on the chloroquine-sensitive (D6) strain of P. falciparum. The extract showing >50 % growth inhibition of the parasite was subjected to screening. For bioassay-guided fractionation, the column fractions were also tested only at single concentration. The pure compounds were subjected to additional testing for determination of IC50 values. The standard antimalarial agents chloroquine and artemisinin were used as positive controls, with DMSO (0.25 %) as the negative (vehicle) control. The selectivity indices (SI) were determined by measuring the cytotoxicity of samples on mammalian cells (VERO; monkey kidney fibroblast). All experiments were carried out in duplicate.
Results
Air-dried parts of the plants tested were pulverized, extracted and the percentage yield calculated as shown in Table 1. The percentage yield of 11.25, 3.45, 40.68 and 12.85 were obtained for P. nitida, PNS1, PNS5, P. americana root and PAS, respectively.
The antibacterial and antifungal activities of P. americana and Jatropha podagrica against a panel of organisms are shown in Tables 2 and 3. The results showed that the plant extracts were effective against both bacterial and fungal organisms. The plant extracts showed different antimicrobial activity (IC50 values) to the test organisms. The methanol extract of Persea americana seed (PAS) showed activity against all the bacterial and fungal isolates. A very good activity was observed against C. neoformans at IC50 value of 8 µg/mL. The chloroform fraction (PASC) also exhibited high inhibitory activity against C. neoformans (Tables 2 and 3).
Table 2.
Percent Inhibitions of Persea americana and Jatropha podagrica against bacteria and fungi
| Test organism | % inhibition @ 50µg/mL | |||
| PARP | PASC | PAS | JPF | |
| C.albicans | 29 | 55 | 52 | 0 |
| C. glabrata | 16 | 49 | 40 | 8 |
| C. krusei | 37 | 68 | 58 | 17 |
| A. fumigatus | 13 | 96 | 12 | 6 |
| C.neoformans | 45 | 100 | 100 | 82 |
| S. aureus | 35 | 47 | 21 | 22 |
| MRSA | 84 | 97 | 14 | 10 |
| E.coli | 24 | 40 | 33 | 23 |
| P. aeruginosa | 6 | 8 | 2 | 38 |
| M.intracellulare | 0 | 54 | 0 | 0 |
Table 3.
Antimicrobial activities of extract and fractions of selected plants
| Test organism | IC50 (µg/mL) of extracts and fractions | |||||
| PARP | PASC | PAS | JPF | Amphotericin B |
Ciprofloxacin | |
| C.albicans | >200 | 32.2 | 157.4 | >200 | 0.27 | NT |
| C. glabrata | 150.9 | 30.6 | 116.5 | >200 | 0.39 | NT |
| C. krusei | 142.2 | 15.5 | 71.1 | >200 | 0.65 | NT |
| A. fumigatus | >200 | 14.7 | 195.1 | >200 | 1.18 | NT |
| C.neoformas | >200 | <8 | 8.2 | 16.9 | 0.24 | NT |
| S. aureus | 51.3 | 40.3 | >200 | >200 | NT | 0.12 |
| MRSA | 8.7 | 35.2 | >200 | >200 | NT | 0.10 |
| E.coli | >200 | >200 | >200 | >200 | NT | 0.006 |
| P. aeruginosa | >200 | 171.3 | >200 | 58.7 | NT | 0.09 |
| M.intracellulare | >200 | 95.7 | 186.1 | >200 | NT | 0.40 |
| NT: Not tested | ||||||
The antimalarial activity of Picralima nitida total extract and fractions against two strains of Plasmodium falciparum demonstrated high inhibitory activity against the two species (Table 4).
Table 4.
Activity of P. nitida extract and fractions against Plasmodium falciparum
| IC50 (µg/mL) of extracts and fractions | |||||
| Extract/fractions | P. falciparum | P. falciparum | P. falciparum W2 | P. falciparum | VERO |
| D6 IC50 | D6 SI | IC50 | W2 SI | IC50 | |
| PNS | 6331.3 | >7.5 | 6005.6 | >7.9 | >47600 |
| PNS1 | >35051.8 | >1.4 | 28376.9 | >1.7 | >47600 |
| PNS2 | >4760 | 1 | >47600 | 1 | >47600 |
| PNS 3 | >4760 | 1 | >4760 | 1 | >4760 |
| PNS4 | 16992.1 | >2.8 | 15902.6 | >3 | >4760 |
| PNS5 | >4760 | 1 | >4760 | 1 | >4760 |
Discussion
The current study was carried out on the extracts and fractions obtained from 3 plants used in Nigerian ethnomedicine for malaria and bacterial infections. The vacuum liquid chromatography of the P. nitida was done in line with standard operating procedures in pre-fractionation technique to separate the non-polar (n-hexane), intermediate (hexane: ethylacetate), and polar (ethylacetate: methanol) compounds. We have evaluated these extracts and fractions for activities against a panel of pathogenic fungi, bacteria, and the protozoa to explore the beneficial effects of these herbs. Traditional herbal practitioners in Nigeria have achieved success with the use of P. nitida as remedy against malaria. Here, we report the antiplasmodial activity of the crude extracts and VLC fractions on two strains of Plasmodium falciparum (P. falciparum D6 and P. falciparum W2). Primary antimalarial evaluation of the methanol extract of P. nitida showed 91% growth inhibition in P. falciparum D6 clone at a concentration of 15867 µg/ml. Further evaluation of the methanol extract of P. nitida (Table 2) revealed moderate dose-dependent activity with IC50 values of 6.33 mg/ml and 6.00 mg/ml against D6 and W2 clones with a selectivity index ranging from >7.5 and >7.9, respectively. The other fractions of PNS showed activity against the two strains of Plasmodium falciparum. PNS1 gave a strong activity at IC50 of 3.50 and 2.82 mg/ml for D6 and W2 respectively, at a concentration of 4.70 mg/ml, and a selectivity index of >1.4 and >1.7.
Extracts obtained from P. americana and J. podagrica were also evaluated for their in vitro antimicrobial activities, and the results in Tables 3 and 4.
The primary assay (Table 3) showed that the two plants had antifungal activity against Candida krusei (68 and 58%) and Candida albicans (55 and 52 % for JPF). These two organizms are known to contribute to opportunistic fungal infections. The antimicrobial results indicated that pet. ether extracts of P.americana root, chloroform fraction of P. americana seed and methanol fraction of P. americana seed showed strong antifungal activity against Candida krusei (IC50 142.20 mg/ml, 15.51mg/ml and 71.10 mg/ml respectively). Previous reports had indicated that P. americana is used for treatment of skin diseases related to fungal infections, such as white lesions of vulva, vitiligo, psoriasis, tinea capitis, and lichen amyloidosis. This investigation provides a significant experimental evidence for development of PA as a potential antifungal agent. As well, the fraction chloroform extract of P. americana seed, methanol fraction of PAS and methanol fraction of JPF showed strong activity against Cryptococcus neoformans at 100,100 and 82 % inhibition with IC50 values of < 8, 8.211 and 16.93 µg/ml respectively. Only the petroleum ether of P. americana root, showed low activity against C. neoformans at IC50 of > 200 mg/ml. PARP and PASC showed a significant activity against methicillin-resistant Staphylococcus aureus (IC50 8.695 mg/ml and 35.189 mg/ml respectively). All of the extracts were inactive against E. coli, PASC was the only extract active against Mycobacterium intracellulare 54% (IC50 95.93) and Aspergillus fumigatus 96% (IC50 14.728).
Extract of P. americana exhibited significant in vitro antimicrobial activity against of microorganisms, C. neoformans, methicillin-resistant S. aureus, E. coli, M. intracellulare, and A. fumigatus. The fractions of P. nitida, demonstrated strong in vitro antimalarial activities against chloroquine susceptible (D6) and resistant (W2) strains of Plasmodium falciparum with IC50s ranging from 120 – 270 ng/mL.
The in vitro antibacterial assay of JPF showed complete inactivity to bacteria. A cursory electronic literature search did not reveal any reports of significant antibacterial activity for the fruit of J. podagrica, though the root and stems were documented to possess antibacterial activity.
Conclusion
The results indicated promising antimicrobial and antiplasmodial actions of P. americana and P. nitida. Further evaluation of in vivo antimicrobial and antiplasmodial activities in an animal model is needed
Acknowledgement
This work was in part supported by a US-Senior Fulbright Award granted study at the University of Mississippi, USA, CIESCs for the Fulbright award, NIH, NIAID, Division of AIDS, Grant No. AI 27094 (antifungal) and the USDA Agricultural Research Service Specific Cooperative Agreement No.58-6408-1-603(antibacterial). Support of TETFUND2013, URPC VC23 and STEP-B is also acknowledged.
Conflict of interest: There is no declaration of interest
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