Antimycoplasmal activity of some plant species from northern Nigeria compared to the currently used therapeutic agent

Abstract

Context: Mycoplasma spp. are obligate parasites of humans and animals. But due to the special requirements needed to culture Mycoplasma in the laboratory, little or no research has been done to evaluate the efficacy of medicinal plants on the organism.

Objective: To screen medicinal plants traditionally used to treat infections for possible antimycoplasmal and cytotoxic activities.

Materials and methods: Acetone extracts of 21 Nigerian medicinal plants were analyzed for antimycoplasmal and cytotoxicity activities using the metabolic inhibition and colorimetric methods, respectively. The extract with the best antimycoplasmal activities was also analyzed for its phytochemical constituents using the desktop method.

Results: Calotropis procera (Aiton) R.Br (Asclepiadaceae) extract had the best antimycoplasmal effect with a minimum inhibitory concentration (MIC) of 80 µg/mL and minimum mycoplasmacidal concentration (MMC) of 160 µg/mL. This extract contained saponins, tannins, cardiac glycosides, alkaloids, and flavonoids. The extract of Vernonia amygdalina Delile (Compositae) was the most cytotoxic with median lethal concentration (LC₅₀) of approximately 17 µg/mL, and that of Anacardium occidentale L. (Anacardiaceae) was the least cytotoxic with an LC₅₀ of approximately 1919 µg/mL.

Discussion: Calotropis procera is a promising plant for an alternative antimycoplasmal agent because the crude acetone extract had a higher mycoplasmacidal activity than the conventional drug tylosin, which is currently used in treatment of the disease in Nigeria.

Conclusion: The crude extract of Calotropis procera is worth investigating for the development of a potent agent against cattle Mycoplasma, which has long defied solution by conventional chemotherapy.

Keywords::

• Anacardium occidentale
• Calotropis procera
• CBPP
• cytotoxicity
• Vernonia amygdalina
• Mycoplasma
• MIC
• MMC

Introduction

Mycoplasmosis represents a group of disease conditions caused by Mycoplasma spp. These are Gram-positive, prokaryotic, wall-less, and self-replicating bacteria belonging to the class Mollicutes, order Mycoplasmatales, and family Mycoplasmataceae. The bacterium can be cultured in a defined medium called PPLO (pleuropneumonia-like organism medium), and is responsible for many disease conditions involving respiratory, genitourinary, articular, and ocular systems in man and animals.

The emergence of antibiotic resistance in both Gram-positive and Gram-negative bacteria has also been observed in Mycoplasma (Hannan, 2000), and this has heightened interest in medicinal plants as a potential source of unique antibacterial compounds (Cowan, 1999).

There are many published articles on the activities of various medicinal plant extracts on bacteria, but, to the best of our knowledge, there was no such information of activity against the Mycoplasma organism. The only information concerning the ethnomedical treatment of the disease condition was gathered from oral tradition that was never published. For instance, the nomads from the northern part of Nigeria sometimes treated their cattle for contagious bovine pleuropneumonia (CBPP) using extracts from the plant Boswellia dalzielli Hutch. (Burseraceae) (Nwude, 1997). Even the work of Nfi et al. (2001) in Cameroon and of Alawa et al. (2002) in Nigeria, which documented the ethnoveterinary plants used by the indigenous people to treat their livestock, made no mention of any particular plant used against mycoplasmosis. Although Alawa et al. (2002) reported that some plants were used to treat pneumonia and other respiratory diseases, infertility, mastitis, swollen joint, and retained placenta, mycoplasmosis was not specifically mentioned. This may be owing to difficulties in diagnosing the disease by pastoralists, and hence the lack of medications reported for treatment of the disease.

We have evaluated the antimicrobial (antimycoplasmal) activities of some indigenous plants from Nigeria against Mycoplasma mycoides subsp. mycoides SC, and to the best of our knowledge this is the first report of this activity.

Materials and methods

Plant collection, drying, and storage

On the basis of published ethnobotanical data (Abdullahi et al., 2003), the leaves of 20 and the seeds of one [Allium sativum L. (Liliaceae)] Nigerian medicinal plants traditionally known for their antibacterial and or anti-respiratory tract infection activities were collected from Zaria in Kaduna State, Nigeria, between February 2007 and March 2007. The plants were identified by Mallam Musa Abdullahi at the Herbarium Section of the Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria, where a voucher specimen for each species was deposited (Table 1). The leaves of each plant were dried in the sun at a very mild ambient temperature, then ground in a mill (IKA® Werke M20) into a fine powder and stored in closed containers in the dark until used.

Table 1.  Antimycoplasmal and cytotoxic activities of plant extracts.

Plant extract	Family	VS No.	% Yield	MIC (µg/mL)	LC50 (µg/mL)	MoS
Terminalia mollis	Combretaceae	901452	4.06	160 ± 0	203.6	1.27
Combretum micranthum	Combretaceae	900257	7.5	310 ± 0	807.27	2.61
Combretum molle	Combretaceae	900191	13.96	160 ± 0	95.89	0.6
Terminalia macroptera	Combretaceae	7394	14.22	420 ± 0.18	75.6	0.18
Combretum ghasalense	Combretaceae	6949	8.62	310 ± 0	75.6	0.25
Guiera senegalensis	Combretaceae	1823	9.54	160 ± 0	203.66	1.27
Terminalia laxiflora	Combretaceae	2981	13.98	630 ± 0	150.73	0.24
Terminalia avicennioides	Combretaceae	900239	18.5	310 ± 0	99.5	0.32
Anogeissus leiocarpus	Combretaceae	167	15.15	160 ± 0	95.89	0.6
Terminalia catapa	Combretaceae	1556	8.74	310 ± 0	23.83	0.08
Terminalia brownii	Combretaceae	406	4.14	420 ± 0.18	23.8	0.06
Combretum sericeum	Combretaceae	900135	12.2	310 ± 0	75.6	0.25
Occimum gratissimum	Lamiaceae	1285	6.2	160 ± 0	23.8	0.15
Vernonia amygdalina	Compositae	675	6.72	310 ± 0	16.82	0.05
Psidium guajava	Myrtaceae	3252	12.96	310 ± 0	203.7	0.66
Calotropis procera	Asclepiadaceae	900219	6.14	80 ± 0	203.7	2.55
Anacardium occidentale	Anacardiaceae	184	9.48	310 ± 0	1918.9	6.19
Cassia alata	Caesalpinoieae	2421	8.9	310 ± 0	108.7	0.36
Adansonia digitata	Bombacaceae	7071	7.68	310 ± 0	95.8	0.31
Agava sisalana	Agavaceae	1821	1.53	520 ± 0.18	NT	NT
Allium sativum	Liliaceae	2196	0.9	310 ± 0	NT	NT

Note. VS No., voucher specimen number; NT, not tested; MoS, margin of safety.

Extraction procedure

The dried, finely ground leaves of each of the species were extracted using only acetone (GR), based on the data that this is the best extractant when investigating plants for antimicrobial activities (Eloff, 1998a; Kotze & Eloff, 2002). A suspension of 1:10 w/v was prepared and shaken vigorously with the aid of a laboratory shaker (Labotec®) for up to 2 h and then allowed to settle before the supernatant was collected by simple filtration through Whatman No. 1 filter paper. This process was repeated twice by adding half the original volume of fresh acetone to the sediments and shaking for an hour so as to exhaustively extract the plant material. The filtrates were collected and combined. Each filtrate was concentrated and dried under a stream of air (fast-moving fan) at room temperature in low light intensity, and the percentage yields from the individual plant species were determined. The extracts were stored in closed containers in the dark and then dissolved in acetone to make a working solution of 10,000 µg/mL prior to use.

Antimycoplasmal assays

Mycoplasma medium or simply pleuropneumonia-like organism (PPLO) basal medium (Difco Laboratory, Detroit, USA) was prepared according to the standard method (Thiaucourt & Di-Maria, 1992). This medium is effective for in vitro antimicrobial susceptibility testing (Hannan, 2000). Filter-sterilized amoxicillin–clavulanic acid was added at a concentration of 5 × 10⁵ iu/L growth medium. One percent glucose and phenol red, which served as indicators for detection of the presence of Mycoplasma growth, was added to an aliquot of the enriched PPLO medium (Muraina et al., 2009).

A two-fold dilution of each plant extract at a starting concentration of 10,000 µg/mL was carried out with 100 μL of PPLO medium in sterile 96-well microtiter plates. Thereafter, 100 μL of 1:10 dilution of 48 h actively growing Mycoplasma mycoides subsp. mycoides (T1/44 strains obtained from the Department of Veterinary Tropical Diseases, University of Pretoria, South Africa) broth culture containing approximately 6 × 10¹² cfu/mL was added to each well. The antibiotic tylosin, at a starting concentration of 1280 µg/mL, was also serially diluted and served as the positive drug control; half-strength acetone was used as the negative control (tylosin was used as positive drug control because it is the drug of choice for treatment of the disease in Nigeria). To determine the minimum inhibitory concentration (MIC), a widely used method of two-fold serial microplate dilution was used with tetrazolium violet as an indicator of growth (Eloff, 1998b). This method did not work well, and a new method was developed (Muraina et al., 2009). The microplate was covered with thin aluminum foil to prevent light-associated damage to plant extracts and then incubated at 37°C for 24–36 h before the results were read by observation of color change of the reaction mixture from pinkish-red to yellow, indicating the presence of growth of metabolically active organisms (Muraina et al., 2009). The minimum mycoplasmacidal concentration (MMC) of extracts with the lowest MICs was assessed by inoculating different concentrations of the extracts on PPLO agar and incubating for 3–10 days under a 5% carbon dioxide atmosphere to detect growth.

Cytotoxicity assays

The cytotoxicity of each plant extract was measured by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphynyltetrazolium bromide] assay (Mosmann, 1983), using the African green monkey kidney (Vero) cell line. Aliquots of 200 μL of logarithmically growing Vero cells at a density of 0.5–10 × 10³ cells/mL were seeded into wells of 96-well microtiter plates. Different concentrations of each extract (i.e. 1, 10, 100, and 1000 µg/mL) were prepared by extracting in dimethylsulfoxide (DMSO) and diluted by cell culture growth medium (minimum essential medium (MEM) + 0.1% gentamicin + amphotericin B + fetal calf serum) being added to the cells in quadruplicate. The growth medium and DMSO were used as negative controls, while berberine chloride (Sigma) was used as a cytotoxic drug positive control. The microtiter plates were incubated for 5 days in a 5% CO₂ atmosphere at 37°C, after which 30 μL of a 5000 µg/mL MTT solution in phosphate buffered saline (PBS) was added to all wells. The plates were further incubated for 4 h at 37°C before the growth medium was gently aspirated from each well. Without allowing the cells to dry out, 50 μL of DMSO was added to each well to dissolve the blue formazan crystals formed by actively dividing cells. The absorbance was measured using an enzyme linked immunosorbent assay (ELISA) microtiter plate reader (Versamax; Molecular Devices) at a wavelength of 570 nm. The 50% lethal concentration (LC₅₀) for each extract on Vero cells was determined by plotting the graph of mean absorbance against mean log of concentration.

Phytochemical analysis

The desktop method of Trease and Evans (1996) was used to analyze the phytochemical constituents of the plant extract that had the best antimycoplasmal activity.

Results

The percentage yield of each extract is shown in Table 1. The acetone extract yields varied from 0.9 to 18.5%. Yields of acetone extracts of 27 different members of the Combretaceae family varied between 2.6 and 22.6%, and the yields for three Terminalia species varied between 8.2 and 22.6% (Eloff, 1998a). The yield is determined by the age of the leaves, with young leaves containing fewer insoluble fibers, but mainly by the composition of the metabolites (Kotze & Eloff, 2002). If there is a high concentration of sugars and amino acids, non-polar extractants would not extract these metabolites and lead to a lower yield.

The values of the minimum inhibitory concentration (MIC) of each extract against Mycoplasma are shown in Table 1. From the results, nearly all the plant extracts had good antimycoplasmal activity according to a classification by Aligiannis et al. (2001), where those extracts with values equal to or less than 500 µg/mL, between 600 and 1500 µg/mL, and equal to or greater than 1600 µg/mL are regarded as good, weak, and poor inhibitors, respectively.

Based on the LC₅₀ values for each extract (Table 1), the most cytotoxic extract among the plant species investigated was Vernonia amygdalina (Compositae), with a value as low as 16.8 µg/mL. When compared with the value of the positive control cytotoxic drug (berberine chloride), with an LC₅₀ of 6.2 µg/mL, V. amygdalina extract was three times less toxic. On the other hand, the extract of Anacardium occidentale (Anacardiaceae), which was the least cytotoxic extract with an LC₅₀ of 1919 µg/mL, was about 310 times less toxic than berberine.

The result of phytochemical analysis for the extract of Calotropis procera (Asclepiadaceae) revealed the presence of alkaloids, tannins, saponins, cardiac glycosides, and flavonoids.

Discussion

From our findings, the plant extracts of Terminalia mollis (Combretaceae), Combretum molle (Combretaceae), Occimum gratissimum (Lamiaceae), Guiera senegalensis (Combretaceae), Anogeissus leiocarpus (Combretaceae), and Calotropis procera had good antimycoplasmal activity. C. procera had the best activities, with an MIC of 80 µg/mL. This was followed by O. gratissimum and G. senegalensis with MIC values of 160 µg/mL. It was also found that C. procera possessed the best MMC value of 160 µg/mL, which was better than that of the conventional (refined) pharmaceutical drug tylosin with an MMC value of 320 µg/mL. It is possible that the antimicrobial action of the extract of C. procera could be attributed to 3-O-acetyl-calotropin, which is a powerful bacteriolytic agent present in the latex (Mueen-Ahmed et al., 2005). Also, the presence of active antimicrobial compounds in the extract of Anogeissus leiocarpus (3,4,3′-tri-O-methylflavellagic acid and its glucoside) as identified by Adigun et al. (2000) could be responsible for the good antimycoplasmal activities of this plant extract. The antimycoplasmal activity of the C. procera effect may be explained by the presence of alkaloids, tannins, and flavonoids in the acetone extract of C. procera. Compounds in these classes show antibacterial activities (Silva et al., 1996; Cowan, 1999).

The good activity shown by many of the plant extracts (81% of the extracts) against Mycoplasma, at a threshold value of 500 µg/mL, indicates that Mycoplasma organisms are more susceptible to plant extracts than other bacteria (47 and 61% for Staphylococcus aureus and Escherichia coli, respectively, from a previous study; Muraina et al., 2008). This observation may be connected to the anatomical structure of Mycoplasma, which lacks a rigid cell wall. This feature may enable potent extracts to penetrate and diffuse into the organism through the semipermeable cell membrane, thereby causing inhibition of growth, or death, of the organism.

It is surprising that bitter leaf (Vernonia amygdalina), which is used as a food (vegetable) by the Nigerian populace, was found to be the most cytotoxic among the plant extracts tested. The processing of the plant before use as food, which sequentially involves soaking in water for a long period, washing several times with plenty of water, and drying before cooking, may explain the detoxification of the plant.

Interestingly, the extract of Calotropis procera had a better antimycoplasmal activity of 80 µg/mL and was less cytotoxic (LC₅₀ = 0.204 mg/mL).

Conclusion

The extract of Calotropis procera, which was less cytotoxic and had good MIC and better MMC activities than the conventional antimycoplasmal drug tylosin, should be explored further for the development of an antimycoplasmal agent from a natural source for the treatment of CBPP, which has defied solution by conventional therapy. Isolating the active compounds from C. procera and determining in vivo toxicicty and efficacy appear to be the logical next steps.

Declaration of interest

The support of the Executive Director, National Veterinary Research Institute, Vom, made it possible for the senior author to carry out this research in the Phytomedicine Programme of the University of Pretoria, South Africa (www.up.ac.za/phyto). The National Research Foundation, South Africa, provides the funds for this research.