GC - MS analysis and antibacterial activities of some plants belonging to the genus Euphorbia on selected bacterial isolates

: Plant extracts have always been used as an alternative source of antimicrobial compounds. The recent spread of multi - drug - resistant bacteria and their increased treatment costs necessitated the study of alternative, cheap sources. The family Euphorbiaceae has over 300 genera and is widely used in traditional medicine. Euphorbia triacu - leata , E. fracti ﬂ exa , and E. inarticulata were selected to study the antibacterial activity of the methanolic extract against 13 Gram - positive Staphylococcus aureus strains ( including methicillin - resistant S. aureus ) and 2 Gram - nega - tive isolates, Escherichia coli and Klebsiella pneumoniae , by the Kirby Bauer Disc di ﬀ usion test. Paper discs with di ﬀ erent concentrations of the extracts ( 100, 50, and 25 µg mL − 1 ) were prepared, along with the methanol control and standard anti - biotic control. A gas chromatography - mass spectrometry ( GC/ MS ) analysis was done to study the phytochemical compo - nents present in the plant methanolic extracts. A total of 50 di ﬀ erent phytochemical compounds with antibacterial activity were detected by GC/MS analysis of the plants. Twenty - ﬁ ve compounds were detected in E. inarticulata , 24 in E. triacu - leata , and 21 in E. fracti ﬂ exa . Out of 37 compounds found in E. inarticulata and E. triaculeata , 12 ( 32.43% ) were common to both. Eleven ( 22% ) compounds were unique to E. inarticulata , while 9 ( 18% ) compounds were unique to E. triaculeata , and 13 ( 26% ) compounds were unique to E. fracti ﬂ exa . E. fracti - ﬂ exa showed the best antibacterial activity against MRSA and Gram - negative bacteria. It also showed higher unique compounds with antibacterial activity ( 26% ) , followed by E. inarticulata ( 11, 22% ) . This is the ﬁ rst GC/MS analysis and antimicrobial activity report of E. triaculeata and E. fracti ﬂ exa .


Introduction
Infectious diseases are a major public health problem in the world.They are one of the leading causes of death.The increased use of antibiotics in the past few years, both in appropriate and inappropriate ways, has led to increased antibiotic resistance.Bacteria are able to resist various antibiotics through horizontal gene transfer or mutation [1].It is expected that by 2050, 10 million lives will be lost per year, and a cumulative 100 trillion USD of economic output will be at risk due to the rise of drugresistant infections if we do not find proactive solutions now to slow down the rise of drug resistance [2].Methicillin-resistant Staphylococcus aureus (MRSA) is a recent problem that needs swift resolution.Although extensive research is carried out worldwide on drug discovery, very few new candidates are being discovered.The increasing failure of antimicrobials and antibiotic resistance shown by pathogenic microbial infectious agents has led to the screening of several medicinal plants for their potential antimicrobial activities [3].Adverse reactions shown by some drugs also encourage using local medicinal plants as possible candidates for alternative medicine [4].Traditional medicines have been the preferred choice in Saudi Arabia for minor ailments [5].
Euphorbiaceae is one of the largest families of angiosperms, with over 300 genera and 8,000 species, distributed mostly in subtropical and tropical regions [6].Euphorbiaceae represents one of the chemically most diverse angiosperms, with many economic and medicinal uses [6,7].
Euphorbia L. is the largest genus in the family, with more than 2,000 species [6].Euphorbia species is widely distributed in arid habitats of Africa and the Arabian Peninsula [8].They have been known as annual, biennial, or perennial herbs with thorns and milky latex.Previous studies on some Euphorbia species (E.granulata, E. helioscopia, E. hirta, E. inarticulata, and E. royleana) showed varied antimicrobial activities [9][10][11].Different phytochemicals have been found in Euphorbiaceae species mainly terpenoids, diterpenoids, flavonoids, alkaloids, tannins, etc. [12].
Not much information is found in the literature about the antimicrobial activities of many Euphorbia species.Furthermore, very little information is available about their activity against multi-drug-resistant bacteria.The spines on these Euphorbia species are particularly sharp and the latex is toxic in nature; hence it is extremely difficult to work with these plants.Many plants from this family are listed on the poisonous database of the United States Food and Drug Administration (USFDA 2019) [13].Therefore, this work aimed to study the antimicrobial activities of three Euphorbia species (Euphorbia triaculeata, E. fractiflexa, and E. inarticulate from Jazan province, southwestern Saudi Arabia) against Gram-positive, Gram-negative, drug-resistant, and drug-sensitive bacteria, and their phytochemical components.Gas chromatography mass spectrometry (GC/MS) analysis of their methanol extracts to examine their probable active compounds was also studied.This is the first GC/MS analysis and antimicrobial activity report of E. triaculeata and E. fractiflexa.

Collection, identification, and extraction of plant materials
The three Euphorbia species of this study are stem succulents, wildly growing in rocky habitats in Jazan province, southwestern Saudi Arabia.Stem branches of E. triaculeata Forssk., E. inarticulata Schweinf., and E. fractiflexa S.Carter & J.R.I.Wood were collected from different localities of rocky habitats, in Jazan province.The plants were identified, and a specimen was deposited in the Herbarium of the Department of Biology.The branches were thoroughly washed for 5 min with 2% of commercial sodium hypochlorite (NaOCl) and then five times with sterile distilled water.Samples were dried in an air oven at 55°C, powdered, and soaked in 60-80% (1:4 w/v) petroleum ether for 24 h; they were then filtered through Whatman paper No. 1. Plant materials were extracted with methanol (95%).Forty grams of the plant material were dissolved in 400 mL of 95% (1:10 w/v) methanol in a dark bottle of 1 L. The bottle was sealed and shaken for 1.5 h in a shaker water bath at 110 rpm and 30°C for 24 h at room temperature.This procedure was repeated five times.After 5 days, the contents of the bottle were filtered through filter paper Whatman No.1.The obtained solution of the plant methanol extract was evaporated to a thick mass in a shaker water bath at 50 rpm and 45°C and kept in a refrigerator till further analysis [14].

Microorganisms
Fifteen bacteria isolates, including 13 Gram-positive S. aureus (12 MRSA and 1 sensitive strain) and 2 Gram-negative bacteria (Klebsiella pneumonia and Escherichia coli), were included in the antimicrobial sensitivity testing.All microorganisms were obtained from the Biology Department, College of Science, Jazan University [15].The fresh culture was used for antimicrobial sensitivity testing by the Kirby Bauer Disc diffusion test [16].

Plant extract working solution
Different concentrations of plant methanolic extracts (100, 50, and 25 µg mL −1 ) and the blank methanol control were used.A total of 200 mg of the plant extract was dissolved in 20 mL of methanol (10 µg mL −1 ); from this, 10 mL was transferred to the next conical flask containing 10 mL of methanol (5 µg mL −1 ).After mixing thoroughly, 10 mL was transferred to the next flask containing 10 mL of methanol to give a 2.5 µg mL −1 concentration.About 10 mL from the last flask was discarded, and 10 mL of methanol was used as the blank.Different concentrations of the plant extracts and the blank were poured on sterile filter paper discs kept in different sterile Petri plates and allowed to dry in a bacteriological hood.These paper discs were used for the Kirby Bauer disc diffusion test.

Antibiotic sensitivity testing
Antibiotic sensitivity testing of the methanolic plant extract was carried out using Kirby Bauer's disc diffusion testing method on sterile Muller and Hinton agar plates [16].A 24 h fresh culture of the test isolates was used to prepare a saline suspension to match the 0.5 McFarland turbidity standard tubes (1.5 × 10 8 colony forming units/mL).Each isolate was spread on sterile MHA plates using sterile cotton swabs.Different concentration discs were placed on the MHA plates, and a standard antibiotic disc of 5 µg rifampicin was also placed along with it as a control for comparison.The plates were kept in an incubator at 37°C for 24 h.The zone of inhibition was measured using a zone-measuring ruler (Himedia, India) in millimeters (mm).All test procedures followed the recommended standards of the Clinical and Laboratory Standard Institute [17].All the tests were done in triplicates.

GC-MS
Methanol extracts of plants were analyzed using the GC-MS apparatus (model; QP2010 Ultra, Shimadzu Corporation, Kyoto, Japan), as described by Almalki et al. [11].The separation was achieved on the Rtx5MS capillary column (30 m length × 0.25 mm i.d.coated with a 0.25 μm film thickness stationary phase; Restek Corporation, USA).Helium was employed as the carrier gas at a constant linear velocity of 36.3 cm s −1 .A sample volume of 1.0 μL was injected using the AOC-20i + s auto-injector.The injection port was set at 290°C in a split-less mode.The temperature of the GC oven was programmed as follows: 5 min at 50°C, heated at 5°C min −1 to 310°C, and held for 10 min.The ion source temperature in the MS was set at 230°C and the interface at 280°C.A total ion chromatogram was created for the m/z range of 50-700.The GC peaks were identified by comparing their mass spectra with the database of the National Institute of Standards and Technology version 11.The relative amount of each component was calculated by comparing its peak area with the total area of peaks in the chromatogram.

Statistical analysis
Statistical analysis was carried out with SPSS V12 using appropriate analysis.Differences were found between the control and treated organisms.In triplicate, the results were interpreted as mean ± SEM (standard error of the mean) for each experiment or an average of three separate experiments (n = 3).The P-value obtained was statistically significant.

Results and discussion
Plants indigenous to a region have always been studied for their antimicrobial properties.They also give a natural alternative to chemotherapeutic agents.With the everincreasing burden of drug resistance and the need for newer antibiotic molecules, the race is always on to find a cure for multi-drug-resistant bacteria.MRSA is one of the drug-resistant bacteria causing problems worldwide.Considering this, antimicrobial sensitivity was carried out for methanolic extracts of three Euphorbiaceae plants, mainly E. triaculeata, E. inarticulata, and E. fractiflexa against 13 Gram-positive S. aureus, of which 12 isolates were MRSA and 2 Gram-negative bacteria, E. coli and K. pneumoniae.As not much information was available against these three plant species, GC/MS analysis was carried out to see the probable compounds present in the methanolic extract.A literature search was done on the compounds found by GC/MS analysis and previously reported antibacterial activity.

Antibacterial sensitivity testing
Table 1 shows the effect of E. triaculeata methanol extracts at concentrations of 2.5, 5, and 10 µg mL −1 on the growth of bacterial species by using the paper assay disc method.The data exhibited that E. triaculeata methanol extracts had antibacterial activities against S. aureus (R4), S. aureus (R6), S. aureus (R8), and S. aureus (R11).MIC was found to be 2.5 µg mL −1 against all four isolates.No antibacterial activity was seen against Gram-negative bacteria.Of the 12 MRSA isolates, the extract showed activity against 4 (33.33%)samples.No activity was observed against Gramnegative isolates.No previous literature was reported against this specie.
The data exhibited in Table 1 also indicate that E. inarticulata methanol extracts have antibacterial activities against S. aureus (R5), S. aureus (R10), and S. aureus (R11).Of the 12 MRSA isolates, the extract showed activity against 3 (25.00%)samples.MIC was found to be 5 µg mL −1 for isolate number 5 and 2.5 µg mL −1 against isolates 10 and 11.No activity was seen against Gram-negative bacteria.Only one report was found previously on the E. inarticulata plant [11], in which they reported activity against Gram-positive and Gram-negative bacteria.In their study, except for E. coli, all other isolates tested were different.They found that the methanolic extract was the best  extract to study the GC/MS analysis.The overall findings agreed with the previous literature.Being a strong polar solvent, methanol is considered highly efficient in extracting active compounds.
Different concentrations (2.5, 5, and 10 µg mL −1 ) of E. fractiflexa methanol extracts were used to evaluate their antibacterial activities against some Gram-positive and Gram-negative bacteria by using the paper assay disc method.Table 1 shows the inhibitory effect of the studied concentrations of the E. fractiflexa methanol extract against a sensitive strain of S. aureus, 12 strains of S. aureus (MRSA), K. pneumonia, and E. coli.The results indicated that the inhibitory effect of the E. fractiflexa methanol extract increased with increasing concentrations of E. fractiflexa methanol extracts.The extract at a concentration of 2.5 µg mL −1 showed antibacterial activity against S. aureus (s), S. aureus (R1), S. aureus (R3), S. aureus (R4), S. aureus (R5), S. aureus (R7), S. aureus (R10), and S. aureus (R12).The maximum inhibition zone for E. fractiflexa methanol extracts was recorded against S. aureus (s) (25.3 mm), followed by Staphylococcus aureus (R5) (21.6 mm) and S. aureus (R8) (21.3 mm).The methanolic extract of E. fractiflexa was shown to have a 100% antimicrobial effect on all 15 indicators, including the susceptible strain of S. aureus and all 12 strains of MRSA.It was also active against E. coli and K. pneumoniae.Figure 1 shows the antibiotic sensitivity testing of different methanolic extracts of the plants used.
Figure 2 shows the GC-MS chromatogram of the methanolic extract of E. inarticulata; 65 peaks were detected.Detected by utilizing total ion concentration vs. time (in minutes).As seen in Table 2, 25 phytochemical components were detected by the GC/MS analysis of methanol extracts of E. inarticulata showing antibacterial activity.Only one study on methanol extracts of E. inarticulata has been reported by Almalki et al. [11].The major difference between the two studies was that they did not study the effect of the plant extract on MRSA, while our study showed a high level of activity against MRSA.
As seen in Figure 3, the GC-MS chromatogram of the methanolic extract of E. triaculeata showed 49 peaks corresponding to different phytochemical compounds which were detected.A total of 24 phytochemical components (Table 3) were detected by GC/MS analysis of methanol extracts of E. triaculeata that showed antibacterial activity as previously reported by various studies.Though none of the previous literature was on E. triaculeata, the compounds were studied from various other plant sources.
As observed in the GC-MS chromatogram (Figure 4) of the methanolic extract of E. fractiflexa, 39 phytochemical components were detected.A total of 21 phytochemical components were detected by GC/MS analysis of methanol extracts of E. fractiflexa and they showed antibacterial activity as previously reported by various studies (Table 4).Though none of the previous literature was on E. fractiflexa, the compounds were studied from various other plant sources.
A total of 50 different phytochemical compounds were detected by GC/MS analysis of three plants.Twenty-five compounds were detected in E. inarticulata, 24 in E. triaculeata, and 21 in E. fractiflexa.Of the 37 compounds found in E. inarticulata and E. triaculeata, 12 (32.43%)compounds were common in both.Compounds 2, 8, and 11 were found in all three plants and showed antimicrobial activities, as reported by previous studies.Of the 41       As seen in Table 5, compound 2, furaneol (2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one), is an aroma molecule found in fruits.It is known for its antibacterial activity, as seen in previous studies [18].Compound 8, benzeneacetaldehyde, a phenolic compound, is also known to have antibacterial activity [19], and compound 11, n-hexadecanoic acid and a methyl ester of fatty acid, also known as palmitic acid, is known to have antibacterial activity [20].All three plants showed the presence of these three antibacterial compounds.β-Amyrin (compound 19), a triterpene, a known antibacterial agent, was found in E. inarticulata and E. fractiflexa, thereby indicating a strong antibacterial activity.Triterpenes have been shown to act as an efflux pump inhibitor and a growth inhibitor and cause cell membrane disruptions [21].Compound 22, glycerin, was also reported to have antibacterial activity and was found in the above two plant extracts [22].A total of 13 molecules present only in E. fractiflexa molecules (from 38 to 50 in Table 5) indicate a stronger antimicrobial response confirmed by 100% antimicrobial activity as seen in Table 3 against Gram-positive, including MRSA and Gram-negative bacteria.Further studies are required to analyze E. fractiflexa and study other extracts such as chloroform and acetone, as well as its application to control multi-drug-resistant bacteria.An overall positive indication toward action on MRSA has made this a very important study.

Conclusion
E. inarticulata, E. triaculeata, and E. fractiflexa showed varied antibacterial activities.E. fractiflexa showed the best antibacterial activity against multi-drug-resistant strains of S. aureus, MRSA, and Gram-negative bacteria.It also showed more unique compounds with antibacterial activity (26%), followed by E. inarticulata (11%, 22%).This is the first report on the antimicrobial activity of E. triaculeata and E. fractiflexa based on GC/MS analysis.More studies on extraction methods and antimicrobial activities of compounds are needed to better extract the active compounds found in the crude extract of the Euphorbiaceae family.

Table 1 :
Antibacterial activities of E. triaculeata, E. inarticulate, and E. fractiflexa extracts on bacterial isolates

Table 2 :
Phytochemical components identified in the methanol extracts of E. inarticulata by GC-MS analysis No. Name of the compound RT SI (%) MW (g mol −1 ) Peak area (%)

Table 3 :
Phytochemical components identified in the methanol extracts of E. triaculeata by GC-MS analysis No. Name of the compound RT SI (%) MW (g mol −1 ) Peak area (%)

Table 4 :
Phytochemical components identified in the methanol extracts of E. fractiflexa by GC-MS analysis