Antimicrobial activity, phytochemical screening of crude extracts, and essential oils constituents of two Pulicaria spp. growing in Sudan

The search for plant extracts with highly antimicrobial activity has been increased nowadays. This study evaluated the antimicrobial activity of Pulicaria crispa (Forsk.) Oliv., and Pulicaria undulata (L.) C.A.Mey., which have been used traditionally in Sudan as insect replants. The antimicrobial activity was evaluated against six pathogenic microorganisms, four bacteria (two Gram-positive; Bacillus subtilis and Staphylococcus aureus, two Gram-negative; Escherichia coli, and Pseudomonas aeruginosa), and two fungi (Aspergillus niger and Candida albicans) using disc diffusion method. The extraction of the crude extracts was done by maceration. The essential oils were extracted by hydro-distillation. Phytochemical screening was done using reference method. Essential oils were analyzed using Gas Chromatography Mass Spectrometry. The results indicated that all used the microorganisms were sensitive to the plants extracts. Results of the preliminary phytochemical screening showed the presence of saponins, comarins, tannins, sterols, and triterpenes, and absence of alkaloids, anthraquinones, and flavonoids. Twenty-eight and forty-five constituents were identified in P. crispa and P. undulata, essential oils, respectively. The main constituents in the essential oil of P. crispa were 1,4-ditert butylbenzene (22.81%), caryophyllene (13.19%), carvone (11.80%), and neryl(s)-2-methylbutanoate (10.33%), and for P. undulata were camphor (44.48%), and thymyl acetate (10.31%). Data from this study could be used for developing of natural bioactive agents to improve human health.


Results and discussion
The yield percentages of the crude methanolic extracts and the essential oils. Data in Table 1 show the yield percentages of the crude methanolic extracts, and the essential oils. The crude methanolic extraction yield of P. crispa and P. undulata was 22.6% and 23%, respectively, (w/w) on dry weights bases. The essential oils of both species obtained by hydro-distillation from whole plants of P. crispa and P. undulata was 0.1% and 0.4%, respectively, (v/w) on dry weights bases. The odor of the aerial parts (stems, leaves, and flowers) of P. undulata was sharper than that of P. crispa, due to the percentages, and essential oils constituents of the two species 14 . Reported that the methanolic crude extract of P. crispa yielded 27% crude extract 16 . Stated that the essential oil extracted from P. crispa grown wild in Egypt reach to 0.23%. These differences in yield percentages of crude extract and essential oils could by refer to the differences in environmental condition.
Antimicrobial activity of the crude methanolic extracts and the essential oils of P. crispa and P. undulata. The extracts of the studied plants exhibited varying degrees of inhibition activity against the tested bacteria (Table 2); and the results were expressed in terms of the diameter of the growth-inhibition zone (clear zones). The results clearly showed that tested bacteria were susceptible to the four extracts. There were significant differences (p < 0.05) in mean diameter inhibition zone between the four extracts. P. crispa methanolic crude extract showed high activity against S. aureus (19 mm), and moderate activity against B. subtilis (16 mm), E. coli (15 mm), and P. aeruginosa (15 mm). P. undulata methanolic crude extract showed high activity against B. subtilis (18 mm), and moderate activity against E. coli (16 mm), P. aeruginosa (16 mm), and S. aureus (15 mm), which is also similar in activity to Tetracycline. Regarding the essential oil of P. crispa, it shows high activity against S. aureus (18 mm), which is higher than positive controls; also it showed moderate activity against B. subtilis (17 mm), P. aeruginosa (17 mm), and E. coli (16 mm). P. crispa essential oil showed high activity against  (24 mm), and was found to be moderately active against E. coli (17 mm) and S. aureus (17 mm), even though it is higher in activity than tetracycline (16 mm). Generally the essential oil of P. undulata showed high activity against B. subtilis, and P. aeruginosa compared to positive controls. Antifungal activity of the methanolic crude extracts, and the essential oils of P. crispa and P. undulata was presented in Table 3. There were significant differences (p < 0.05) in mean diameter inhibition zone between the four extracts. All the extracts showed activity against the two fungal microorganisms. However, P. undulata essential oil showed the highest activity against the two fungi compared to the other extracts. Generally the extracts showed high activity against C. albicans; P. undulata essential oil 23 mm, P. undulata methanolic crude extract 21 mm, P. crispa methanolic crude extract 20 mm, and P. crispa essential oil 19 mm, While the extracts were found to be relatively less active against A. niger; P. undulata essential oil 22 mm, P. crispa essential oil 21 mm, P. undulata methanolic crude extract 20 mm, and P. crispa methanolic crude extract 19 mm, which was lower than positive controls (Nystatin (26 mm) and Clotrimazole (34 mm). Table 4 showed that all microorganisms were very susceptible to the minimum inhibitory concentration of P. crispa essential oil (6.25 mg/ml) except for B. subtilis the MIC value was 25 mg/ml, similarly all microorganisms were susceptible to minimum inhibitory concentration of P. undulata essential oil (6.25 mg/ml), except for E. coli and B. subtilis with MIC value (50 mg/ ml). Regarding P. crispa methanolic crude extract the minimum inhibitory concentration was 6.25 mg/ml for P. aeruginosa and A. niger, 25 mg/ml for E. coli and S. aureus, 50 mg/ml for C. albicans and 100 mg/ml for B. subtilis. While, minimum inhibitory concentration of P. undulata methanolic crude extract was 6.25 mg/ml for A. niger and C. albicans, 12.5 mg/ml for P. aeruginosa and S. aureus, the MIC was 50 mg/ml for E. coli and 100 mg/ ml for B. subtilis.

Minimum inhibitory concentrations (MIC) of P. crispa and P. undulata methanolic crude extracts, and essential oils. The results of MIC presented in
Preliminary phytochemical screening of the crude extracts of P. crispa and P. undulata. Data presented in Table 5 show the preliminary phytochemical examination of the methanolic crude extracts of P. crispa and P. undulata, which were rich in sterols, and terpenes, tannins, comarins, saponins. At the same time data confirm the absence of alkaloids, flavonoids, and anthraquinones. These results are in line with the findings of previous researches [16][17][18][19][20] . These groups of phytochemicals might be responsible for the observed antimicrobial activity P. crispa and P. undulata.
The chemical constituents of P. crispa and P. undulata essential oils. The hydro-distillation of the dry aerial parts of P. crispa, and P. undulata grown in Sudan gave yellow-colored essential oils. The percentage composition, and identification of each Pulicaria species essential oil are listed in Tables 6 and 7. Table 3. Antifungal activity of P. crispa and P. undulata methanolic crude extracts, and the essential oils against the fungal microorganisms. (*) Positive control. Means separated by least significant difference (LSD) test at p < 0.05. Means followed by a similar letter(s) in the same column are not significantly different at p ≤ 0.05 according to the least significant difference test. Preparation of the crude extracts. P. crispa and P. undulata, methanolic crude extracts were prepared by maceration of the dried powdered plants materials in organic solvent (methanol). Twenty grams of each plant sample were extracted using 50 ml of absolute methanol as solvent. The mixture was allowed to stand for 72 h at room temperature with daily filtration using a standard filter paper (Whatman No. 2, England). The solvent was evaporated under reduced pressure to dryness using rotary evaporator, then the crude extracts have left to dry at room temperature for three days. The yield percentages were determined by dividing the weight of extract by the weight of the sample multiplied by 100. The extracts were stored at 4 °C until used 23 .
Preparation of the essential oils. P. crispa and P. undulata essential oils were prepared by hydro-distillation of the dried powdered plants materials in water. 100 grams of each sample were submitted to hydrodistillation for four hours using Clevenger-type apparatus (Duran West Germany). The obtained essential oils were calculated as a relative's percentage (v/w), and dried over anhydrous sodium sulfate, filtered, and stored at 4 °C until used 16 .  Antimicrobial activity screening of the methanolic crude extracts, and the essential oils of P. crispa and P. undulata, microorganisms. The antimicrobial activity of P. crispa and P. undulata methanolic crude extracts, and the essential oils were evaluated by disc diffuison method using ATCC (American Type Culture Collection), and NCTC (National Collection of Type Cultures) strains. The strains were four bacterial strains; two Gram-positive (Bacillus subtilis (NCTC 8236) and Staphylococcus aureus (ATCC 25923)), two Gram-negative (Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853)), and two fungi (Aspergillus niger (ATCC9763) and Candida albicans (ATCC7596)).
Preparation of the microorganism culture. All the test microorganisms were inoculated on blood agar, and nutrient agar plates. The bacterial strains were incubated at 37 ºC for 24 h, and the fungal strains were incubated at 37 °C for 48 h in the inverted position, incubated aerobically, and the obtained growth were then stored in the refrigerator at 4 °C till used.
Determination of antimicrobial activity of the methanolic crude extracts, and the essential oils of P. crispa, and P. undulata, by disc diffuison method. The paper disc diffusion method was used to screen the antimicrobial activity of the plants extracts, and performed by using Mueller Hinton agar (MHA). The experiment was carried out according to the National Committee for Clinical Laboratory Standards 24 . The bacterial suspension was diluted with a sterile physiological solution to 108 CFU/ml. 100 µl of the bacterial suspension were swabbed uniformly on the surface of MHA and the inoculum was allowed to dry for five minutes.