Antimicrobial activity, phytochemical screening of crude extracts, and essential oils constituents of two Pulicaria spp. growing in Sudan (2024)

The search for substances with highly antimicrobial activity has been one of the most intensive field of research to minimize the risk of infectious diseases that caused by bacteria, fungi, viruses, and parasites, which are pathogenic to humans. Plants extracts are still themajor sources of many therapeutic agents including antimicrobial agents for the treatment of infectious diseases^1,2.

The family Asteraceae includes about 100genera, and 2300 species. The genus Pulicaria is one of these genera, and it includes 100 species distributed worldwide³. Seven species of the genus Pulicaria have been reported in Sudan, namely; P. attenuata, P. crispa, P. dysenterica var. stenophylla, P. grantii, P. petiolaris, P. undulata, and P. vulgaris⁴. Only three species of the genus Pulicaria have been found in Khartoum State, which are P. crispa, P. grantii, and P. undulata⁵.

Pulicaria crispa (Forsk.) Oliv. (synonym Francoeuria (Forsk.)) and Pulicaria undulata (L.) C.A.Mey., are two wild aromatic plants growing in Sudan. Their local names are "alrabul", and "altager", and these plants contain plenty of compounds with medicinal importance⁶. P. crispa and P. undulata are annual herbs or sometimes perennial sub-shrubs, with small yellow flowers containing essential oil characterized by a strong aromatic odor. These plants are one of the most widespread desert plants growing wild in Sudan, Saudi Arabia, Kuwait, Iran, Iraq, Southern Egypt, Afghanistan, Pakistan, India, and parts of north & west tropical Africa^7,8,9.

Different Pulicaria species have been traditionally used in several countries to repel insects, to treat back pain, to treatintestinal disorders, to treat inflammation, and to reduce influenza, and common cold symptoms. Pulicaria species contain many bioactive compounds such as monoterpenes, sesquiterpene acetylenes, flavonoids, isocomene, alkaloids, glycosides, comarins, and tannins^1,6.

Many studies have reported that, P. undulata have been used traditionally in Sudan against alopecia, as a tea substitute, asan antispasmoic, as an ingredient of local perfumes. In addition, the plant has been used in folk medicine in many countries as anantiepileptic, asgalactagogue, and as insect repellent (farmers are used to but it inside the vegetables packing containers). Also it has been reported that the essential oil of P. undulata has been used in the preparation of tonics, as sedative, and as anantibacterialagent^{9,10,11,12,13}.

A group of researcher reported that P. crispa was found to have many folkloric medicinal uses in many countries. It has been used for many years in conventional medicine for the cure of heart diseases due to its antioxidative nature, also it has been used by the people of Sudan, Southern Egypt and Saudi Arabia to treat inflammation, asanantimicrobialagent, as aninsect repellent, for the treatment of colds, coughs, colic, excessive sweating, and as carminative^9,14,15.

The antimicrobial activity of P. crispa and P. undulata, methanolic crude extracts and essential oils were studied against pathogenic bacteria, and fungi. A preliminary phytochemical screening of the crude extracts, and the constituents of the essential oils have also been investigated in the present study.

The yield percentages of thecrude methanolic extracts and theessential oils

Data in Table 1 show the yield percentages of the crude methanolicextracts, and theessential oils. The crudemethanolic extraction yieldof P. crispa and P. undulata was 22.6% and 23%, respectively, (w/w) on dry weightsbases. 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 weightsbases. 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 species¹⁴. Reported that themethanolic crudeextract of P. crispa yielded 27% crude extract¹⁶. 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 thedifferences in environmental condition.

Antimicrobial activity of thecrude methanolic extracts and theessential 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 (19mm), and moderate activity against B. subtilis (16mm), E. coli (15mm), and P. aeruginosa (15mm). P. undulata methanolic crude extract showed high activity against B. subtilis (18mm), and moderate activity against E. coli (16mm), P. aeruginosa (16mm), and S. aureus (15mm), which is also similar in activity to Tetracycline. Regarding the essential oil of P. crispa, it shows high activity against S. aureus (18mm), which is higher than positive controls; also it showed moderate activity against B. subtilis (17mm), P. aeruginosa (17mm), and E. coli (16mm). P. crispa essential oil showed high activity against S. aureus (18mm). Regarding the essential oil of P. undulata, it showed high activity against B. subtilis (25mm), P. aeruginosa (24mm), and was found to be moderately active against E. coli (17mm) and S. aureus (17mm), even though it is higher in activity than tetracycline (16mm). Generally the essential oil of P. undulata showed high activity against B. subtilis, and P. aeruginosa compared to positive controls.

Antifungal activity of the methanoliccrude extracts, and theessential 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 23mm, P. undulata methanolic crude extract 21mm, P. crispa methanolic crude extract 20mm, and P. crispa essential oil 19mm, While the extracts were found to be relatively less active against A. niger; P. undulata essential oil 22mm, P. crispa essential oil 21mm, P. undulata methanolic crude extract 20mm, and P. crispa methanolic crude extract 19mm, which was lower than positive controls (Nystatin (26mm) and Clotrimazole (34mm).

Minimum inhibitory concentrations (MIC) of P.crispa and P.undulata methanolic crude extracts, and essential oils

The results of MIC presented in Table 4 showed that all microorganisms were very susceptible to the minimum inhibitory concentration of P. crispa essential oil (6.25mg/ml) except for B. subtilis the MIC value was 25mg/ml, similarly all microorganisms were susceptible to minimum inhibitory concentration of P. undulata essential oil (6.25mg/ml), except for E. coli and B. subtilis with MIC value (50mg/ml). Regarding P. crispa methanolic crude extract the minimum inhibitory concentration was 6.25mg/ml for P. aeruginosa and A. niger, 25mg/ml for E. coli and S. aureus, 50mg/ml for C. albicans and 100mg/ml for B. subtilis. While, minimum inhibitory concentration of P. undulata methanolic crude extract was 6.25mg/ml for A. niger and C. albicans, 12.5mg/ml for P. aeruginosa and S. aureus, the MIC was 50mg/ml for E. coli and 100mg/ml for B. subtilis.

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 crudeextracts 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.

GC–MS analysis of theessential oils resulted in identification of twenty-eight constituents in P. crispa essential oil, andforty-five constituents in P. undulata essential oil. The main constituents of the essentialoil of P. crispa were 1,4-ditert-butylbenzene (22.81%), caryophyllene (13.19%), carvone (11.80%), neryl (s)- 2-methylbutanoate (10.33%). In addition, the main constituents of the essential oilof P. undulata werecamphor (44.48%), thymyl acetate (10.31%), bicycle (3.46%), and azulenol (3.40%), other minor constituents have been identified in the essential oils of P. crispa and P. undulata. Bothlinalool, and camphor are presented in the essential oil of P. crispa and P. undulata. Result of P. undulata essential oil constituents agrees with those obtained by²¹ of P. undulata collected from Yemen in linalool, camphor, and thymol.

The bactericidal properties of P. undulata essential oil were due to the presence of thymol, and thymol derivatives, which were found to have a significant antimicrobial activity²².

This study was carried out at theMedicinal and Aromatic Plants and Traditional Medicine Research Institute, National Center for Research, Khartoum, Sudan.

Plant material

Plants were selected randomly followed by antimicrobial assays. Aerial parts of P. crispa and P. undulatawere collected during the flowering stage in July 2015 from different locationsin Khartoum State, Sudan. The plants materials were taxonomically authenticated at theherbarium of Medicinal and Aromatic Plants and Traditional Medicine Research Institute, P. crispa voucher specimen number is W-1995-41-MAPTRI-H,and P. undulata voucher specimen number is A-1995-126-MAPTRI-H.

Preparation of theplant materials

P. crispa and P. undulata, plants parts were freed from dust, and foreign material, then dried indoors at room temperature for three days, powdered, then kept in plastic containers at room temperature until used.

Preparation of the crude extracts

P. crispa and P. undulata, methanolic crude extracts were prepared by maceration of thedried powdered plants materials in organic solvent(methanol). Twenty grams of each plant sample were extracted using 50ml of absolute methanol as solvent. The mixture was allowed to stand for 72h 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 theweight of thesample multiplied by 100. The extracts were stored at 4°C until used²³.

Preparation of the essential oils

P. crispa and P. undulata essential oils were prepared by hydro-distillation ofthe dried powdered plants materials in water. 100grams of each sample were submitted to hydro-distillation 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 used¹⁶_.

Antimicrobial activity screening of the methanoliccrude extracts, and theessential oils of P.crispa and P.undulata, microorganisms

The antimicrobial activity of P. crispa and P. undulata methanolic crude extracts, and theessential oils were evaluated by disc diffuison method using ATCC (American Type Culture Collection), and NCTC (National Collection of Type Cultures) strains. The strains werefour 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 themicroorganism culture

All thetest microorganisms were inoculated on blood agar, and nutrient agar plates. Thebacterial strains were incubated at 37ºC for 24h, and thefungal strains were incubated at 37°C for 48h 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 methanoliccrude extracts, andthe 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 theplants extracts, and performed by using Mueller Hinton agar (MHA). The experiment was carried out according to the National Committee for Clinical Laboratory Standards²⁴. Thebacterial suspension was diluted with a sterile physiological solution to 108CFU/ml. 100µl of thebacterial suspension were swabbed uniformly on the surface of MHA and the inoculum was allowed to dry for five minutes. Thesterilized filter paper discs (Whatman No. 2, England) were placed individually on the surface of the MHA and impregnated with 20µl of samples solution. The inoculated plates with bacteria were incubated at 37°C for 24h in the inverted position and 48h for the fungal strains. The diameters (mm) of the inhibition zones were measured; thevalues of the antimicrobial activity were expressed as the mean of inhibition zones (mm) with three replicates for each treatment. Gentamicin, Tetracycline, Clotrimazole and Nystatin served as positive controls. The results of the diameters of the zones of inhibitions of theextracts were interpreted as sensitive; (> 18mm), intermediate (14–17mm), and resistant (< 14mm)^17,18.

Determination of theminimum inhibitory concentration (MIC)

The principle of the agar plate dilution is the inhibition of growth on the surface of the agar by the plant extracts incorporated into the medium was used to determine the minimum inhibitory concentration (MIC) according to²⁵. The MICs of the extracts were used in concentrations (6.25–100mg/ml). Agars were prepared in the series of increasing concentrations of the plant extract. The bottom of each plate was marked off into four segments. The organisms tested were growing in broth overnight to contain 108CFU/ml. Loop-full of diluted culture is spotted with a standard loop that delivers 0.001ml on the surface of the segment and incubation for 24h or 48h at 37°C. The MIC were determined visually in the agar as the lowest concentrations of the extracts inwhich no bacterial/fungal growth was visible. The MICs values were express as mg/ml.

Phytochemical screening of the methanolic crude extracts

Themethanolic crude extracts were subjected to qualitative examination for the detection of various phytochemical constituents (saponins, comarins, alkaloids, anthraquinones, tannins, flavonoids, sterols, and triterpenes) using standard procedures^26,27,28.

Essential oils analysis by thegas chromatography-mass spectrometry (GC–MS)

P. crispa and P. undulata, essential oils were analyzed using Shimadzu Gas Chromatography MassSpectrometry Apparatus (Japan) (GC.MS- QP2010 Ultra). Analysis was carried out on a Varian 3400 system equipped with a DB-5 fused silica column (30m length × 0.25mm diameter, 0.25μm film thickness). Helium was used as the carrier gas (1.2ml/min), and the program used was four minutes isothermal at 35 °C , following by 40–240 °C at the rate of 4 °C/min, then held at 260 °C, for three minutes, the injection temperature was 250 °C. The components of the essential oils were identified by library searches²⁹, based on comparing their retention indices, and mass spectra with those obtained from authentic samples, and/or the NIST/NBS, Wiley libraries, and the literature.

Statistical analysis

The collected data were subjected to theanalysis of variance (ANOVA), and themeans were separated using the least significant difference (LSD) at p ≤  0.05 and at p ≤  0.01.

Authors and Affiliations

1. Department of Horticulture, Faculty of Agriculture, University of Khartoum, Shambat, 13314, Khartoum, Sudan

   Ebtihal Alsadig Ahmed Mohamed&Ali Mahmoud Muddathir

2. Horticultural Crops Research Center, Agricultural Research Corporation, Shambat 30, Khartoum, Sudan

   Ebtihal Alsadig Ahmed Mohamed

3. Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Centre for Research, 2404, Khartoum, Sudan

   Magda Abker Osman

Contributions

E.A.A.M. was responsible for the conceptualization, and thedesigning of the research work, responsible for the elaboration of the research project, performed the research work, technical work (laboratory), responsible for the interpretation of thedata, wrote, and revised the manuscript. A.M.M. supervised the research project, and revised the manuscript. M.A.O. contributed inthe conceptualization, and thedesigning of the research work, contributed in the supervision of the research project, performed the statistical analysis, contributed in thedata interpretation, and revised the manuscript. All authors reviewed the manuscript.

Corresponding author

Correspondence to Ebtihal Alsadig Ahmed Mohamed.

Competing interests

The authors declare no competing interests.

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