Comparative Study for the Volatile Oil Constituents and Antimicrobial Activity of Rhanterium epapposum Oliv . Growing in Qassim , Saudi Arabia

Background: Rhanterium epapposum is an herbaceous plant widely distributed in the Gulf region and used by Bedouins as antiseptic for wounds, skin infections and in gastrointestinal disturbances. Aim: The study aimed to compare the results obtained from volatile oil analysis of R. epapposum growing in Buraydah, Qassim with reported data of the same plant growing in Riyadh “Al-Majmaah” and Northern border region of Saudi Arabia. Both cold and hot extracts of the R. epapposum were used to find the best extraction method to be adopted as an antimicrobial agent. Methods: Volatile oils were distillated using Clevenger apparatus and analyzed by GC-MS. The plant powder was extracted by cold maceration and hot contentious extraction methods. Furthermore, antimicrobial activity was conducted using the agar diffusion method. Results: The hydro-distillation of R. epapposum growing in Qassim region yielded 0.5 % w/w of the total volatile oils. Moreover, forty-three compounds of 97.68% of the volatile oil components were identified while modephene, caryophyllene, linalyl acetate and epizonarene were the major components of volatile oils. Nevertheless, the volatile oils diversity and concentrations were found to be different in R. epapposum growing in Buraydah, as compared with the plant growing in Riyadh and the northern border region of Saudi Arabia which almost due to the differences in the environmental condition. Among all extracts, ethyl acetate hot extract showed the best inhibition to bacterial strains while fugal strain Candida albicans growth was better inhibited by hot n-hexane extract. Conclusion: Volatile oils were active against all microbial strains. Hot extracts were more active against bacterial strains while the opposite effects were found against Candida albicans which was generally inhibited by the cold extracts.


INTRODUCTION
2][3][4] R. epapposum is known for its insecticide effect 5 and used in traditional medicine for the treatment of gastrointestinal disorders and skin infections. 6he volatile oil constituents of R. epapposum collected from many eastern countries such as Saudi Arabia 7,8 and Iran 9 have been previously measured.The common essential oil constituents in R. epapposum were the oxygenated monoterpenes such as Limonene, Linalool, geraniol and α-Terpineol, 7 non-oxygenated terpenes such as α-phellandrene and sesquiterpenoid essential oils such as α-cadinol, 7,9 Sterols and triterpenes, carotenoids, alkaloids, coumarins, tannins, flavonoids, polyuronides and saponins have also been identified in petroleum ether, methanol and aqueous extracts of R. epapposum.The antimicrobial 10 and wound healing activities 11 for the extracts as well as volatile oils of R. epapposum have been reported.R. epapposum honeybees (Spring Lena honey) has been shown a strong inhibition toward different microbial strains such as Staphylococcus aureus and Streptococcus mutans Gram-positive bacteria in addition to Gramnegative strains such as Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. 12

R. epapposum ethaol extract was reported to have
Pharmacognosy Journal, Vol 11, Issue 1, Jan-Feb, 2019 antioxidant activity which attributed to its total phenolic constituents. 13lso, the anti-inflammatory effect of R. epapposum has been measured in addition to their total flavonoids and phenolic constituents.The study revealed that this plant extract could be used for treatment of rheumatism and other inflammatory disorders. 14Furthermore, R. epapposum extract was exhibit cytotoxic effect against leukemia CCRF-CEM cell line. 15he present study was planned to investigate the volatile oil constituents and design the best extraction method for the maximal antimicrobial activity of R. epapposum growing in Buraydah, Qassim region.In addition, the volatile oil constituents have been compared with the recently reported data for the volatile oils isolated from the same plant growing in Riyadh and the northern border region.

Plant materials
The plant was collected in April 2017, during the flowering stage from central Buraydah city, in Qassim and was identified as Rhanterium epapposum family Asteraceae by the agricultural experimental station in Unaizah.A voucher specimen of the plant under a number of 77 is deposited at herbarium of College of Pharmacy, Qassim University.The whole plant materials were spread in an open-air area until completely dry; then the dried plant materials were ground to a coarse powder before use.

Hydro-distillation of volatile oils
An amount of 250 g of coarse dried plant material was placed in a 500 ml round bottom flask connected with a Clevenger distillation apparatus; 16 the distilled water containing volatile oils (distillate) was collected and extracted three times with chloroform.The chloroform layer containing volatile oils was dried with anhydrous sodium sulfate to remove all water and then the solvent was evaporated with a rotary evaporator under reduced pressure at room temperature to get the volatile oil.

Gas chromatography-mass spectroscopy analysis (GC-MS)
The GC-MS analysis was carried out on a Trace 1300 GC, Tsq DUO Triple Quadrupole MS with a column TG 5MS (30m × 0.25mm, 0.25μm).Helium was used as a carrier gas at a flow rate of 1.2 ml/min.Split/Splitless (S/SL) injector was used with 250°C injector temperature.1.0μl sample injection volume was utilized.Ion source temperature was maintained at 200°C.The oven temperature was programmed initially at 40°C for 4 min, then programmed to increase to 250°C at a rate of 10°C/min ending with a 5 min isothermal at 280°C.Total run time was 27.0 min.The MS transfer line was maintained at a temperature of 250°C.TSQ DUO Triple Quadrupole MS detector (mass range from m/z 45 to 600) was used for analysis.The mass spectra of the components were matched with the data available in the National Institute of Standards and Technology (NIST) library (Table 1).

Extraction Method Hot extraction
An amount of 200 g of the dried plant material were extracted by continuous Soxhlet extraction apparatus 17 using n-hexane, chloroform, ethyl acetate and ethanol, respectively.After 10 refluxes, each extract was dried under vacuum at 40°C and the extractive values were calculated as grams of dried extracts obtained from hot extraction procedure per 100 gm of the dried plant powders (Table 2).

Cold Extraction
An amount of 200 g of dried plant materials was transferred to a 1000 ml conical flask.500 ml of n-hexane was used to extract the plant constituents at room temperature. 18After 24 h stirring, the extract was filtered and dried under vacuum at room temperature.The residue of the plant materials left after removal of n-hexane extract was subjected to extraction by chloroform, ethyl acetate and ethanol, in sequence using the same previous manner used in n-hexane extract.The extractive values of cold extracts were calculated as grams of dried extracts obtained from the cold extraction procedure per 100 gm of the dried plant powders (Table 2).

Determination of antimicrobial activity
The antimicrobial screening of R. epapposum volatile oils, as well as cold and hot extracts, was performed using agar diffusion method described by Cooper and Woodman. 19The microbial suspensions of the gramnegative Escherichia coli, gram-positive Staphylococcus aureus and Candida albicans yeast were prepared, incubated for few minutes' till they showed turbidity/growth comparable to 0.5 McFarland tube as a reference.Sterile swaps were smeared on the agar surface, incubated for few minutes.Then 50μl of the test sample was micropipetted to the designed cups.Positive control drug disc (10μg/ml amoxycillin, gentamycin and clarimazole) were placed onto the top of the inoculated agar plate which were then incubated.[22]

RESULTS AND DISCUSSION
Volatile oil constituents of R.epapposum growing in Qassim region R. epapposum is a wildly herbaceous plant, wide-distributed in deserts of Saudi Arabia, particularly the middle and northern region of the kingdom.The GC-MS analysis of the volatile oils of R. epapposum growing in Qassim yielded 43 volatile oil constituents as it is shown in Table 1.These volatile constituents have been previously isolated from a huge number of aromatic plants.Oxygenated and non-oxygenated monoterpene and sesquiterpene hydrocarbons were represented in the volatile oil constituents of R. epapposum.Limonene was the only non-oxygenated monoterpene compound with a percentage of 1.64 % of total volatile oil amount.On the other hand, eight oxygenated monoterpene compounds were detected which are Linalool, Linalyl acetate, α-Terpinyl acetate, Terpinen-4-ol, L-α-Terpineol, Nerol, Geraniol and Citronellyl acetate; they collectively represent 22.7 % of the total R. epapposum volatile oils.
In addition, 14 non-oxygenated sesquiterpene compounds were detected in the volatile oil which was evaluated to be 43.5 % of the total volatile oils (Table 1).Out of the forty-three volatile oil compounds, there are 18 compounds of oxygenated sesquiterpenes which are represented by 28 % of the total volatile oil.The GC-MS analysis showed that mode-phene, caryophyllene, linalyl acetate and epizonarene were the major compounds among all other volatile constituents of R. epapposum growing in Qassim, with a percentage of 10.51 %, 8.59 %, 8.46 % and 5.43 %, respectively (Table 1).
The GC-MS analysis results were used to investigate the variation in the volatile oils production and diversity in the R. epapposum plant according to their climate growing area.Comparing the present results with the most recent reported data for the volatile oil constituents of R. epapposum growing in Al-Majmaah region "Riyadh" (165 km away from Buraydah, the place where the R. epapposum plant was collected and used in the current study) 8 and northern border region of Saudi Arabia, 7 indicates that the difference is marked not only in the concentration of particular compounds but also in their diversity, for instance, out of 43 volatile components of R. epapposum growing in Buraydah (Qassim), only 14 compounds were present in the same plant growing in Riyadh (Al-Majmaah) and/or northern border region.Furthermore, the concentrations of these 14 compounds were completely different from the R. epapposum volatile oil compounds collected from those places. 7,8n addition, the most abundant constituents of the volatile oils of R. epapposum growing on Qassim were not detected during the analysis of volatile oils of R. epapposum growing in Riyadh (Al-Majmaah) and the northern border region.For instance, modephene (10.51%),Linalyl acetate (8.46%) and epizonarene (5.43%) that have been identified in large proportion in R. epapposum growing on Qassim, were not identified in the analysis of volatile oils of R. epapposum growing in Riyadh and northern border region (Table 1). 7,8tractive values and antimicrobial activity of

R. epapposum extracts
The extractive values obtained from both cold and hot extraction methods are summarized in Table 2.Among all solvents, chloroform produced the highest amount of extract 2.8 gm (cold) and 1.39 gm (hot) per 100 gram of the dried plant powder.The extractive values also showed that the extractive cold materials were almost Twice the materials obtained from hot extraction method (Table 2).The antimicrobial activity of R. epapposum, as well as its honey, is well established literatures reported previously. 8,10,12In addition, R. epapposum has a wound healing activity that may be attributed to its antimicrobial effect. 11Therefore, we extracted R. epapposum herb with different solvents and by two different methods (cold and hot methods) to investigate the best solvent and better method to extract the antimicrobial constituents of R. epapposum.In this regard, three microbial stains represent the Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (E.coli) in addition to fungal strain (Candida albicans) were used to determine the antimicrobial effect of the R. epapposum fractions (Table 2).The result shown in Table 2 indicates that the n-hexane fraction was active against Candida albicans fungus strain with 15 mm and 25 mm inhibi-

Table1: The Volatile Oil Contents of Rhanterium epapposum Olive herb.
RPPercentage was calculated according to the relative area (peak area relative to the total peaks area).*Essential oils detected in R. epapposum growing in Riyadh (Al-Majmaah) and/or northern border region of Saudi Arabia.