Anti-mycobacterial, Antimicrobial and Phytochemical Evaluation of Pulicaria crispa and Scoparia dulcis Plant Extracts

Aim: To examine the antimicrobial activity and evaluate the anti-mycobacterial potency of Pulicaria crispa and Scoparia dulcis whole plant extracts in solvents of different polarities (n-hexane, ethyl acetate and methanol. Study Design: Assessing the anti-microbial and anti-Mycobacterium tuberculosis activity of two Nigerian medicinal plants which have been reported according to folklore for treatment of various Original Research Article Adebiyi et al.; JAMPS, 7(4): 1-11, 2016; Article no.JAMPS.25199 2 ailments including respiratory tract infections. Place and Duration of Study: The experiment was conducted at the Chemistry Department of Ahmadu Bello University, Zaria, Nigeria and at the National Institute for Pharmaceutical Research and Development, Abuja, Nigeria between November 2014 and September 2015. Methodology: The standard pan sensitive tuberculosis reference strain (H37Rv), eleven bacterial and four fungal clinical isolates were used. Methanol, Ethyl Acetate and Hexane extracts of Scoparia dulcis and Pulicaria crispa (whole plants) were tested at 0, 20 and 40 μg/ml using the Microplate Alamar Blue Assay. The minimum inhibitory, minimum bactericidal and minimum fungicidal concentrations of each solvent extract were assessed. Phytochemical analysis was also performed. Results: Phytochemical compounds obtained in the methanol extract fractions where alkaloids, balsams, cardiac glycosides, glycosides, phenols, steroids and tannins in Scoparia dulcis (Linn), while extracts of Pulicaria crispa showed the presence of alkaloids, balsams, cardiac glycosides, flavonoids, glycosides, phenols, tannins and terpenoids. Saponins were detected in the n-hexane fractions for both plants but only appeared in the semi-polar fractions of Scoparia dulcis (Linn). Microplate Alamar Blue Assay (MABA) used for sensitivity study of Mycobacterium tuberculosis with 10 μg/ml rifampicin revealed that the n-hexane extract of Puliaria crispa and Scoparia dulcis gave 48.44±0.75, 12.14±0.02 mm zones of inhibition respectively, whereas the methanol and ethyl acetate extracts gave a 24.10±1.35 mm and 17.00±0.91 zones of inhibition for Puliaria crispa and Scoparia dulcis respectively in comparison to 33.70±0.64 mm obtained from the control. The minimal inhibitory content (MIC) of the methanol and n-hexane extracts for Pulicaria crispa were recorded at 8.01±1.70 and 10.03±1.33, while the MIC values for the ethyl acetate and n-hexane extracts of Scoparia dulcis was 12.03±0.86 and 20.40±0.24. The MIC value recorded for rifampicin was 0.38±1.40. Conclusion: The results obtained suggested that the studied plants possess anti-tuberculosis and selective antimicrobial activities with the major activity tailored to the phyto-constituents.


INTRODUCTION
The world is being ravaged by the spread of infectious diseases whereby the particular onslaught of drug resistant pathogens has diminished that effectiveness of regular antimicrobial treatment options. This has further been aggravated by the occurrence of opportunistic infections and a dwindling therapeutic spectrum alongside previously nonconceived side effects of synthetic drugs [1,2]. One of such infectious diseases also classified as an opportunistic infection in the case of HIV is tuberculosis. Tuberculosis is a bacterial disease caused by the Mycobacterium africanum, Mycobacterium bovis and Mycobacterium tuberculosis; the latter being the most potent. Mycobacterium tuberculosis is acid-fast, aerobic, non-capsulated bacilli with mold-like morphology and waxy cell walls that contain mycolic acid [3]. Their rate of replication is slower than most other bacteria and can stay dormant within their host for long periods of time, thereby requiring lengthy drug treatment regimens; a factor that maybe responsible for multi-drug resistant variants particular when patients fail to adhere to treatment plan. Drug-resistant strains of M. tuberculosis also arise from spontaneous chromosomal mutations at a predictable low frequency [4,5].
A report indicated that misuse of anti-TB drugs, such as monotherapy or the addition of single drugs to failing regimens, results in the emergence of resistant mutants. M. tuberculosis resistance to antibiotics; Rifampicin, in 40,000 studied tuberculosis cases across Africa was as a result of the multi-drug resistant strains of Mycobacterium tuberculosis [6]. Screening for alternative therapies, compounds or formulations with good prospects towards tuberculosis treatment warrants adequate consideration. The unique arsenal of bioactive compounds within certain plants is conceived as a cheaper alternative to synthetic drugs in the treatment of many different bacterial infections, mainly in developing countries [7,8]. Since the therapeutic information for a lot of such plants is based on folklore, there is a need to establish a scientific rationale for their use in treatment by performing in vivo and in vitro testing.
Studies indicate that extracts of Alpinia galanga were active against a pan sensitive strain of

MATERIALS AND METHODS
The whole plant Scoparia dulcis and Pulicaria crispa were collected at Samaru (9°45'0''N 8°23'0''E) and Bassawa (11°4'0''N 7°42'0''E) regions of Zaria locality in Kaduna state, Nigeria. The plants were identified at the herbarium of Department of Biological Science, Faculty of Science, Ahmadu Bello University, Zaria, Kaduna State, Nigeria, where voucher specimen numbers 1064 and 2583 were given and samples were deposited. The whole plant in each case were air-dried, powdered with the use of a mortar and pestle and then kept in airtight container until required for further laboratory analysis. Air-dried plant materials were pulverized into powder using a motar and pestle. 1kg each of ground plant samples were exhaustively extracted using maceration extraction method [14]. The marc was extracted successfully using 2.5L each of hexane, ethyl acetate and methanol to 1kg each of plant sample using batch extraction methods. The crude extracts were later concentrated to a minimum volume using rotary evaporator (Büchi Labortechnik AG, Switzerland) at 40°C and reduced pressure.
Stock solutions of 10 mg/ml of rifampicin were prepared by dissolving 0.1 g in 10 ml of methanol from which different concentrations were obtained and used for the susceptibility tests.

Phytochemical Screening
Phytochemical screening was carried out on the hexane, ethyl acetate and methanolic extracts for the qualitative determination of major constituents using methods previously described [14,15,16].

Antimicrobial Screening
All the Media were purchased from Sigma-Aldrich and were prepared in accordance with manufacturer instructions. The bacterial isolates were collected from Medical Microbiology Department of Specialist Teaching Hospital, University of Abuja, F.C.T., Nigeria on a slant Nutrient agar. The isolates were restored on Nutrient broth and confirmed using standard biochemical tests according to the Bergey's manual of Bacteriology [17]. While the collected fungal isolates were identified using fungi chrome test kits in Specialist Teaching Hospital, University of Abuja, F.C.T., Nigeria. The isolates were collected on a Potato dextrose agar slant and restored in Potato dextrose broth. Agar diffusion method was adopted from [18] was employed.

Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) was determined on the test organisms that were sensitive to the extracts and was done by broth dilution method [19]. Mueller Hinton broth was prepared, dispersed into test tubes and the broth was sterilized at 121°C for 15 smins, the broth was allowed to cool. Normal saline was prepared, 10 mls was dispersed into sterile test tube and the test microbes was inoculated and incubated at 39°C for 6hrs. Dilution of the test microbes was done in the normal saline until the turbidity marched that of the McFarland's standard scale by visual comparison at this point, this test microbes has a concentration of about 1.5x10 8 CFU/ml.
Two fold serial dilution of the extract in sterilized broth was made to obtain the concentration of 50.0 µg/ml, 25.0 µg/ml, 12.5 µg/ml, 6.25 µg/ml, and 3.2 µg/ml. The initial concentration was obtained by dissolving 6.0 mg of the extract in 10 mls of sterile broth. Having obtained the different concentration of the extracts in the sterile broth, was observed for turbidity (growth), the lowest concentration of the extract in the broth which shows no turbidity was recorded to the MIC.

Determination of Minimum Bactericidal and Fungicidal Concentrations
Minimum bactericidal concentration (MBC) and Minimum fungicidal concentration (MFC) were evaluated by plating the bacterial suspensions from individual well at the beginning and at the end of the experiments on Mueller Hinton agar medium for estimation of MBC [19]. The culture from MIC well was taken and streaked on the surface of fresh Mueller Hinton agar in a 90-mm plate with division and incubated at 37°C for 24 hours (bacteria) and 37°C for 48 hours (fungi) after which the plates of the medium was observed for colony growth, the MBC/MFC were the plates with lowest concentration of the extract without colony growth.

Microplate Alamar Blue Assay (MABA)
Stock solutions of the individual plant extracts were prepared in 0.05% DMSO (Dimethyl sulfoxide) diluted to the final concentration of 20 and 40 µg/ml in sterile distilled water as part of experimental standardization. The sensitivity of Mycobacterium tuberculosis strain to the various extracts was demonstrated by agar diffusion method [20]. This strain; H37Rv, is the most widely used reference strain as it retains its virulent ability following laboratory passaging and is sensitive to all first and second line drugs used in treating mycobacterium tuberculosis such including Isoniazid, Rifampicin, Kanamycin, Capreomicin, Ciprofloxacin, etc. A sterile cork borer of 7 mm diameter was used to bore holes into the inoculums seeded solidified nutrient agar. A 50 µl volume of each 20 and 40 µg/ml of the plant extracts was loaded into the labelled well in the prepared media plate using sterile pipette. The test was performed in triplicates and the plates were kept in a refrigerator for prediffusion of the sample and incubated at 37°C and 48 hours. Growth of Mycobacterium tuberculosis was observed after the incubation of 48 hr and the diameter of inhibition zone was measured subtracting the well size.

RESULTS AND DISCUSSION
The pharmacological activities of medicinal plants lie with the sundry of phytochemical compounds they possess, thus supporting their ethno-pharmacological use against different infectious diseases by traditional medicine practitioners in developing countries.
Susceptibility to common side effects associated with certain synthetic drugs is rare with use of phyto-medicines. Elucidating the presence of phytochemicals within targeted plants is a scientific first step aimed at unveiling which active compounds or synergy of compounds produce their antimicrobial activities thus making them effective drug candidates in addition to confirming the believe that local plants are the platform for traditional African medicine [21]. Table  1 represents the phytochemical composition from whole plants of Scoparia dulcis (Linn) and Pulicaria crispa. The qualitative tests carried out on the extracts showed most activity in the methanolic extract fractions whereby alkaloids, balsams, cardiac glycosides, glycosides, phenols, steroids and tannins in Scoparia dulcis (Linn). The methanolic extracts of Pulicaria crispa showed the presence of alkaloids, balsams, cardiac glycosides, flavonoids, glycosides, phenols, tannins and terpenoids. Saponins were detected in the nhexane fractions for both plants but only appeared in the semi-polar fractions of Scoparia dulcis (Linn). Phlobatanins and triterpenoids were absent in all extracted fractions of both plants. The presence of these secondary metabolites will enhance its therapeutic potential against several pathogens as well as its physiological activities [22,23]. For instance, plant glycosides and cardiac glycosides possess herpato-stimulatory activity thus making them useful for increasing heart muscles contractions as well as in cancer treatment [24,25]. In tuberculosis therapy, the aminoglycoside antibiotic; streptomycin and kanamycin, are of importance as these in combination with other similar second-line drugs is used for treating tuberculosis, meningitis, pneumonia and brucellosis [26,27]. The presence therefore of both glycosides and cardiac glycosides in the methanol extract of the studied plants gives a good indication that these plants may possess important bioactive compounds of different antibiotic class. Phenols are another very important class of plant secondary metabolites whose antiseptic properties have been exploited towards several antimicrobial activities and some polyhydroxyphenols have been shown to inhibit HIV. A number of antibiotics with strong antifungal potentials are phenolic in nature. This includes amphopterin B, nystatin, natamycin and griseofulvin; the latter is used to treat systemic infections whereby it acts by preventing the infestation of new tissue [28].
It was observed that none of the fractions contain cardenolides, phlobatanins, resins and volatile oil, thereby suggesting a limited antioxidant potential that if present would boost the host immunity against pathology induced free radical generation [29].
The highest recorded anti-mycobacterial activity was observed in the n-hexane extract of Pulicaria crispa (40 µg/ml) against H37Rv with a zone of   [30]. Ciproflaxin was used as standard for bacteria, ranging the value of zone of inhibition from 35 to 41 mm, while fluconazole was used as the control antifungal agent with a zone of inhibition ranging from 31 to 37 mm [31].
In the northern parts of Nigeria, traditional plants are consumed not only for forage but also in medicine, particularly in the treatment of dysentery, diarrhoea, stomach upsets, respiratory tract infections, certain viruses and haemorrhoids [32,33]. Scoparia dulcis is used mainly for the treatment of diabetes, as a birth control measure, with reports suggesting that it possesses bronchodilating and anti-asthmatic activity hence its use for treating respiratory infections within small communities [34,35]. With an increasing mortality rate each year due to different infectious disease including but not limited to upper respiratory tract infections, bacteremia, diarrhea and dysentery. Phytotherapeutic agents that show resistance to the causative microbial agents such as Methicillin Resistant Staphylococcus aureus, enteric bacteria (Enterococcus, Pseudomonas, Escherichia coli, etc) serves as a new frontier towards the treatment of a host of respiratory and digestive infections as the latter is most often the common route to outbreaks in developing countries. Data from Table 3 suggests that extracts of Pulicaria crispa proffer greater resistance to disease causing agents for most respiratory tract infections than that of Scoparia dulcis. However, solvent extracts of Scoparia dulcis gave more favourable results against agents that lead to stomach upsets than Pulicaria crispa. One more noticeable difference between the two plants is the degree of resistance that only the extracts of Pulicaria crispa confers to Neisseria gonorrheae which leads to venereal diseases, thus suggestive other applications of this plant in ethno-medicine. The findings from Tables 2 and 3 compliments the results given in Table 1 whereby other studies have shown that bioactive compounds of flavonoid and tannin origin are both active against Staphylococcus aureus, Escherichia coli and different strains of mycobacterium [36,37]. Other studies have implicated the compounds obtained from the saponin, terpenoid, flavonoid and tannin fractions as the driving force behind the reported activity against infectious disease causing agents like Staphylococcus aureus, Pseudomonas aeruginosa, Streptococus pyogenes as well as their activity against multi drug resistant strains of M. tuberculosis [38,39]. The success in treating diarrhoea and stomach upsets, attributed to Staphylococcus aureus and Salmonella spp infections could be due to the antibacterial effects of alkaloids, polyphenols, saponins and steroids [40]. Distorting the enzyme activity within bacteria is usually observed in the presence of phyto-tannins [32].  Table 3.

CONCLUSION
This study serves to authenticate the indigenous customs in developing countries in addition to contributing further depths to the growing literature on plant materials recognized as a reservoir of important novel anti-tuberculosis compounds. Furthermore, the awareness of the therapeutic effect of these plants in the treatment of tuberculosis seems to be well known in some Northern Nigerian cultures. The findings in this study have hence provided scientific support for the ethnomedical anti-TB activity of extracts of the whole plant of Scoparia dulcis and Pulicaria crispa, where the reactive solvent fractions were found to have a significantly adequate MIC values when compared to the other extracts using the standard drug (Rifampicin). The phytochemistry of the plant shows that the extracts contain steroids, saponin, alkaloid, terpenoids and tannin. Hence the constituent with the anti-TB activity can be drawn down to be from the above phyto-constituents.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.