Medicinal Plants and their collection: The herbals used in this study were Including Artemisia vulgaris, Silybum marianum, Ramhormaz Ocimum basilicum, Abadan Ocimum basilicum, Glycyrrhiza glabra and Oliveria decumbens. During the flowering season, between December and April 2019, samples were gathered from several areas in Khuzestan (Table 1). The samples were thoroughly cleaned to remove any unusual flora, dust, or other pollutants.
Table 1
Scientific name
|
Plant family
|
Collection site
|
Altitude (m)
|
Collection time
|
Extracted part
|
Oliveria decumbens Vent.
|
Apiaceae
|
Shushtar
|
165
|
February
|
Flowers and flowering branches
|
Glycyrrhiza glabra
|
Fabaceae
|
Behbahan
|
300
|
February
|
Root
|
Ramhormoz Ocimum basilicum
|
Lamiaceae
|
Ramhormoz
|
100
|
March
|
Purple mass
|
Abadan Ocimum basilicum
|
Lamiaceae
|
Abadan
|
120
|
March
|
Purple mass
|
Silybum marianum
|
Asteraceae
|
Hamidiyeh
|
120
|
December
|
seed
|
Artemisia vulgaris
|
Asteraceae
|
Izeh
|
300
|
April
|
flowering branches
|
Essential oil extraction: Essential oils were extracted by hydro-steam distillation using the Clevenger equipment from fresh, clean, weighed aerial parts of flowers, flowering branches, seeds, and rhizomes (Table 1) and collected and stored in dark sterile vials (13). Briefly, 100 to 150 g of each plant were placed in a distillation flask (1 L), which was linked to a steam generator through a glass tube and to a condenser. This was recovered in a funnel tube. Essential oil aromatic molecules were released from the plant material and evaporated into hot steam. The heated steam forced the plant material to release the essential oil without burning it. The steam containing the essential oil was then sent through a cooling system to condense it. The steam was applied for 3 hours. The essential oil was extracted once the recovered mixture had been settled. The produced essential oil was dried by filtering the supernatant essential oil through anhydrous Na2SO4. Following that, the essential oil was gathered in tighter vials and refrigerated. Several dilutions of the oils were done using dimethyl sulfoxide (DMSO) for the antibacterial activity test.
Ethanolic extract preparation: Samples were washed, air dried for 7–8 days, and ground into powder before being put in the flask of the Soxhlet apparatus for extraction using ethanol with increasing order of polarity to extract the phytoconstituents individually at 20oC for 3–4 hours. The extracts were then filtered using Whatman No.1 filter sheets. Following that, decreased pressure was used to evaporate and dry the filtrates, which were then kept at -20oC in labeled, sterile, screw-capped vials.
Microorganisms and their maintenance: 12 Standard strains, including Staphylococcus aureus (S. aureus) PTCC 1113, Escherichia coli (E. coli) PTCC 1533, Bacillus cereus PTCC 1015, staphylococcus epidermidis PTCC 1435, Pseudomonas aeruginosa PTCC 1558, Helicobacter pylori PTCC 5211, Acinetobacter baumannii PTCC 1855, Enterococcus faecalis PTCC 1237, Enterobacter cloacae PTCC 1003, Shigella dysenteriae PTCC 1188, Klebsiella pneumoniae PTCC 1290 and Corynebacterium diphtheria ATCC 27010 obtained from the Persian Type Culture Collection. The subculture of bacteria using a panel of laboratory control strains obtained from the Persian Type Culture Collection. All bacteria were stored in trypticase soy broth containing 25% (v/v) glycerol for the time of the investigation.
Antimicrobial activity of plants: The best factor for evaluation of the antimicrobial activity of plants and comparison of their antimicrobial effect is a measurement of minimum inhibitory concentration (MIC). In this study, the modified E-test was used (14). A 5% concentration of each plant extracts and essential oils was prepared and diluted using the double dilution method. Then the 108 CFU/ml suspension of each bacteria was impregnated on the plate of sterile Mueller-Hinton agar (MHA, Merck) media by sterile cotton swab and permitted to stay in contact for 1 minute. Eight blank sterile paper discs were put on culture medium. Ten microliters of each dilution were impregnated in each disc. The plates were incubated at 37°C for 24 hours (15). All tests were done fourfold and the mean of results was calculated.