Chemical Profile and Biological Activities of Essential oil of Aerial parts of Artemisia monosperma Del. Growing in Libya

Background: From the bioactivity stand point Artemisia monosperma Del. is reputed to have antispasmodic and anthelmintic properties. Various types of secondary metabolites were reported in A. monosperma plants from different localities. Objective: The current study was planned aiming to investigate the influence of stage of development on the composition, antimicrobial, antiinflammatory and antioxidant activities of the essential oil derived from the aerial parts of the Libyan plants. Material and Methods: A. monosperma volatiles were hydro-distilled from aerial parts of Libyan plants, collected at different stages of growth: before flowering (A1), and at beginning (A2) and by the end (A3) of flowering stage. Yields ranged from 0.16-0.26 ml/100g fresh material (A3, highest). GC/FID and GC/MS analyses were performed. Results: Among 16-20 identified components (97.63-99.00% of total composition), 11 were common in all samples. A1 and A2 showed close amounts of hydrocarbons (63.56 and 66.55%), but lesser than A3 (88.36%); monoterpenoids were mainly represented by sabinene (13.15-22.85%), β-pinene (9.00-24.03%) and β-cis-ocimene (3.73-12.92%); while sesquiterpenoids appeared absent. Among oxygenated components (11.29, 31.08 and 35.44 % in A3, A2 and A1), bornyl acetate was the major monoterpenoid (8.00-31.00%, highest in A1); and the sesquiterpenoid, β-eudesmol (8.01%) was detected in A2 only. Moreover, A2 demonstrated significant antifungal effect against Aspergillus fumigatus and Geotricum candidum (MIC 0.98 and 0.24 μg/ml). Conclusion: A3 exerted the highest anti-inflammatory activity as compared to the other volatiles. A1 restored the reduced blood GHS level in diabetic animals almost as efficiently as Vitamin E. The antioxidant activity of the volatiles is decreased during the flowering stage, being the highest before flowering (A1); this could be associated to the decreasing bornyl acetate content of the samples.


INTRODUCTION
Genus Artemisia (Asteraceae) comprises about 300 species of broad distribution all over the world. 1 Moreover, the essential oils derived from several members of this genus have acquired special importance due to their wide variety of applications in folk and modern medicine. 2,3The genus is represented in Libya by five species viz., A. arborescens, A. herba-alba, A. judaica, A. campestris and A. monosperma. 4The latter is a Saharo-Arabian element and has been reported from Gasar Gharian, Ghat area and Southern Tripolitania. 4A. monosperma is a green glabrous shrublet, 50-70 cm high, with ascending or diffuse stems ending in many flowered panicles; capitula are small (1mm in diameter) with 10-12 tubular florets; the flowering season extends from September to December in most areas. 4From the bioactivity stand point A. monosperma Del. is reputed to have antispasmodic, anthelmintic and anti-hypertensive properties; 5,6 its leaves are traditionally used in Jordan to induce abortion 7 and were found to possess antioxidant, 8 GC/FID Analysis Instrument and operating conditions A Shimadzu GC gas chromatograph (GC-17ver.3)coupled with FID detector was used, and detector temperature set at 240 o C. To obtain the same elution order as with GC/MS, simultaneous auto injection was carried out on a duplicate of the same operational conditions.

Identification of Components
This was performed via comparison of the retention times (Rt) and retention indices (RI, Kovat's indices) of the detected components with those of authentic samples.RIs were computed relative to those of a mixture of a continuous series of n-alkane hydrocarbons (C4-C28) analyzed under the same operating conditions as for the samples.5] Relative percentages of the identified components were calculated based on GC/FID peak areas without the use of correction factors.Results are compiled in Table 2, Figure 1 Voucher specimen 1192013 is kept in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

Oil samples
The essential oils were hydro-distilled from 500 g fresh plant samples in a Clavenger-type apparatus, isolated and dehydrated.Aliquots (5 µl, each) of the prepared oils were, separately, mixed with approximately 1 ml of CH 2 Cl 2 in auto sampler vials and saved for chromatographic investigation.For biological evaluation solutions (1mg/ml) were prepared in DMSO.

Experimental animals
Adult Sprague-Dawley rats weighing 160 ± 10 g were provided from the animal-breeding unit of National Research Center, El-Dokki, Giza, Egypt.They were kept under standard conditions with temperature at 23 ± 2 o C and a 12/12 hours light/dark cycle and allowed free access to food and water throughout the experiment.This study was conducted in accordance with the standard guidelines used in handling of the experimental animals and approved by the Institutional Animal Care and Use Committee (IACUC) (No. 9-031), College of Pharmacy, Cairo University.

Operating conditions
Injection volume, 1 µl of CH 2 Cl 2 solution of tested samples; oven temperature programming: initial temperature, 40 °C (isothermal for 3 min), increased (12°C/min) to 180 °C and hold for 5 min, then further increased to final temperature 240 °C (40°C/min) and maintained for 5 min; injector temperature, 240 °C and split ratio, set at 1:54; carrier gas, He at flow rate 0.9 ml/min; mass spectrometer, electronic ionization (EI) mode 70eV, scan range 40-500 and transfer line temperature, 230 °C.

Antimicrobial assay
The antibacterial activity of the essential oils was screened by the agar well-diffusion method, zone of inhibitions being measured in mm, as described by Holder and Boyce (1994). 26The essential oils were, separately, tested against the selected strains at concentration of 1mg/ml.DMSO was used as a negative control; meanwhile Penicillin G and Streptomycin (10 mg/ml) were used as a positive controls for bacterial strains and Amphotericin B as positive control for fungi.Experiments were carried out in triplicates.Bacterial cultures were incubated at 37 °C, for 24 h and the fungal ones at 25-30°C, for 3-7 days.Results are recorded (Table 3) as mean zone of inhibition in mm ± Standard deviation beyond well diameter (6 mm) produced on a range of environmental and clinically pathogenic microorganisms using (1 mg/ml) concentration of tested samples. 27Potencies relative to the appropriate antibiotic are represented by the histograms in (Figures 4-6).

Determination of minimal inhibitory concentrations (MIC)
Broth dilution method 28 was used for determination of minimum inhibitory concentrations (MIC) of the most active sample.Serial dilutions of each essential oil sample were prepared in DMSO and mixed with broth media in a 96-wells microtiter plate to achieve a final concentration range from 0.003-4 % v/v.The plates were then inoculated with standardized suspension containing 5 × 10 5 bacterial count per well.Optical density was measured at 600 nm after a specified incubation period by means of a JENWAY 6051 colorimeter (U.K.) and was used as a measure of bacterial growth. 29

Determination of Median Lethal dose (LD 50 )
The acute toxicity resulting from oral administration of the volatile oils of the aerial parts of A. monosperma Del. was investigated.In this respect, its median lethal dose (LD 50 ) was determined according to Karber (1931) 30 as follows: Preliminary experiments were carried out to determine the minimal dose that kills all animals (LD 100 ) and the maximal dose that fails to kill any animal.Several doses at equal logarithmic intervals were selected in between these two doses, and each was orally    administered to a group of experimental animals (n=6).The mice were observed for 24 h and the symptoms of toxicity as well as mortality rates in each group were recorded.

Evaluation of Anti-inflammatory activity
The acute anti-inflammatory activity was assessed in-vivo by means of the paw swelling, or foot edema method, which is convenient for assessing inflammatory responses to antigenic challenges and irritants.This was performed by examining the ability of the tested samples to reduce or prevent the development of carrageenan-induced paw swelling.This model is commonly used to assess the efficiency of nonsteroidal antiinflammatory drugs (NSAIDs) that inhibit prostaglandin production. 31

Experimental design
The method of Winter et al (1962) 32 was adopted.Carrageenan suspension (1% in sterile saline) was prepared, and placed in a refrigerator, at

Evaluation of the antioxidant activity
Glutathione (GSH) is an important antioxidant in plants, animals, fungi, and some bacteria, preventing damage to important cellular components caused by reactive oxygen species such as free radicals, peroxides, lipid peroxides and heavy metals. 33In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form (GSH) and less than 10% exists in the disulfide form (GSSG); and an increased GSSG-to-GSH ratio is indicative of oxidative stress. 34arious methods have been devised for the determination of glutathione in biological samples, including spectrophotometric-and high-performance liquid chromatography (HPLC)-based techniques.The most popular is the 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) spectrophotometric method. 35Blood glutathione was determined in Alloxandiabetic rats by adopting the Beutler et al (1963). 36The restoration of GSH level reduced due to oxidative stress induced by diabetes was taken as a measure of antioxidant activity.The principle of the assay depends on that protein and non-protein SH-groups react with Ellman's reagent [5, 5-dithiobis-(2-nitrobezoic acid)] to form the stable yellow colored 5-thio-nitrobenzoic acid, which can be measured at 412 nm.

Experimental design
Thirty-six adult male albino rats of the Sprague-Dawley strain (120-150 g) were used as experimental animals.Diabetes mellitus was induced by a single intra-peritoneal injection of Alloxan (150 mg/kg b. wt.) followed by an overnight fast. 37Hyperglycemia was assessed, after 72 h of diabetes induction, by measuring the blood glucose levels according to Trinder(1969). 38he rats were divided into 6 groups, each of 6 animals (n=6).The 1 st group received 1ml saline and was kept as a negative control (normal, non-diabetic).Diabetes was induced to the other 6 groups as previously indicated.
Group 2: (Diabetic control) was kept untreated.A1, A2 and A3: volatile oils of aerial parts before, during and at the end of the flowering stage (0.01mL/kg); S.E.= standard error; *Statistically significant from the control normal inflamed group at the corresponding time: P< 0.05.Statistical analysis was carried out using repeated measures one way ANOVA followed by Least significant test for multiple comparison.a % Oedema =wt of right paw-wt.ofleft paw x 100/wt of left paw; b %oedema inhibition (% of change )=(Dc-Dt)x100/ Dc ; Dc is the mean oedema in control rats; Mt is the mean Oedema in drug-treated animals.Median lethal dose LD 50 The 24 h LD 50 was approximately more than 0.05 ml /kg b. wt., of the essential oils of the aerial parts.These results showed that the essential oils are safe and non-toxic.

Anti-inflammatory activity
Results presented in Table 5 and Figure 7 revealed that the A 3 volatile sample (hydro-distilled from the aerial parts of the plant collected at the end of the flowering stage) exerted the highest anti-inflammatory activity as compared to the other volatiles obtained from earlier gathered plant material.The sample exhibited a significantly high potency ranging from (70.03-81.62%)relative to the reference NSAID Indomethacin; and its efficiency appeared the highest after 1 h of treatment.This remarkable effect could be correlated to its relatively high monoterpenoid content viz., β-Pinene (24.03%),Sabinene (17.10%), β-trans-Ocimene (10.98%) and β-cis-Ocimene (12.92 %) with reported anti-inflammatory activity. 44

Anti-oxidant activity
The results represented in Table 6 and Figure 8 revealed that the A 1 (volatiles hydro-distilled from plants collected before flowering) restored the reduced blood GHS level in diabetic animals almost as efficiently as Vitamin E (relative potencies 97.03 and 94.28 %, respectively) followed by A 2 and A 3 (88.79 and 67.44%).The antioxidant activity of the volatiles is thus decreased during the flowering stage, being the highest before flowering (A 1 ); this could be associated to the decreasing bornyl acetate content of the samples (31.00, 14.05 and 8.00% in A 1 , A 2 and A 3 , respectively).In fact, both borneol and bornyl acetate are considered as major contributors in the antioxidant activity of essential oils. 45,46e blood GSH level was recorded, and percentage change from control computed as follows: % Change = (G-G 0 ) × 100/G 0 ; where:G 0 is the GHS level in diabetic animals prior sample administration and G that measured after.

Yield and Composition of the Volatiles
The hydro-distilled volatiles of the aerial parts of A. monosperma, collected before flowering (A 1 ), and at the beginning (A 2 ) and end (A 3 ) flowering stage, were obtained as greenish yellow dextro-rotatory liquids with characteristic odour.Oil yields ranged from 0.16 to 0.26% v/w (on fresh weight basis), the highest being recorded for A 3 (Table 1).

Antimicrobial Activity
The antimicrobial activity of the different samples was evaluated against a set of microorganisms, including 4 fungal and 8 bacterial strains (Table 3 and 4) (Figures 4-6).The tested samples exhibited moderate to remarkable growth inhibitory potential against most of the tested strains as compared to the appropriate standard antibiotics.Yet, all were inactive against the filamentous fungus Syncephalastrum racemosum, the Grampositive bacterium Streptococcus pyogenes and the Gram-negative Proteus vulgaris.Sample A 2 demonstrated the highest activity against all affected microorganisms with best efficiency as antifungal, especially on Aspergillus fumigatus (potency, 89.87% relative to Amphotericin B).In all cases A 3 was the least active, except on Klebsiella pneumoniae on which it revealed a slightly higher effect than A 2 .The potentiality of the oil sample derived from aerial parts collected at the beginning of the flowering stage (sample A 2 ) may be attributed to its bornyl acetate content (31%). 43

CONCLUSION
The genotype, ontogenic development and environmental growth conditions of any plant species are known to have a great impact on the qualitative and quantitative composition of its constituents, thus resulting in different chemotypes.In this respect, it was here evident that the stage of development influenced the yield and composition of the hydro-distilled volatiles of the aerial parts of Artemisia monosperma and consequently affected its antimicrobial, anti-inflammatory and antioxidant potencies.The latter could not be exclusively correlated to the efficiency of a specific constituent but rather to a synergistic effect of all components.Meanwhile, the difference in composition observed in comparison with samples obtained from other localities may be referred to a number of extrinsic factors which affect growth conditions and consequently the production of secondary metabolites.

Figure 1 :
Figure 1: Chromatograms 1, 2 and 3 representing the GC-FID of the essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage of A. monosperma, respectively.

Figure 2 :
Figure 2: Histogram representing the relative percentages of the different classes of constituents in the essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage of A. monosperma, respectively.

Figure 3 :
Figure 3: Histogram representing the relative percentages of the major components detected in the essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage of A. monosperma, respectively.

Figure 4 :
Figure 4: Histogram representing the antifungal activity of essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage of A. monosperma, respectively; expressed as potencies as compared to standard Amphotericin B.

Figure 5 :
Figure 5: Histogram representing the antibacterial activity of essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage respectively of aerial parts of A .monosperma, expressed as potencies as compared to standard Gentamycin.
4ºC for an overnight, to allow complete hydration of the polysaccharide; then injected (100 µl) into the sub-plantar tissue of the right hind paw of Sprague-Dawley male albino rats (120-150 g) to induce edema.Meanwhile, the left hind paw was injected with 100µl saline.The animals were randomized into 5 groups (n=6); one hour before carrageenan injection, they received (by oral administration, p.o.) appropriate doses of the vehicle, tested samples and standard drug, as follows: Group 1 (negative control): received the vehicle (saline, 1 ml /Kg b. wt.).Groups 2, 3 and 4: received the volatile oil samples (A 1 -A 3 0.01 mL/kg b.wt., each).Group 5 (positive control): received the standard anti-inflammatory drug, Indomethacin (20 mg/kg b. wt.)The hind paw diameter was measured by means of Vernier caliper.Measurements were carried out immediately after carrageenan injection, and at selected time intervals (1 h, 2 h, 3 h and 4 h) after drug or samples administration.Experiments were carried out in triplicates.The percentage edema was recorded and percentage edema inhibition calculated as follows: % Edema inhibition = (M 0 -M t )/ M 0 × 100, where: M 0 = Mean paw diameter of control group at a given time; M t = Mean paw diameter of treated (extract or standard) group at the same time.
GHS: reduced glutathione; A1, A2 and A3: volatile oil of aerial parts before, during and at the end of the flowering stage; * Statistically significant for control group at P< 0.01; Statistical analysis was carried out using repeated measures one way ANOVA followed by Least significant test for multiple comparison.a % Change from control=(Mc-Mt)x100/ Mc ; Mc is the mean change in control rats; Mt is the mean change in drug-treated animals.b %Relative potency calculated as regard to standard drug.

Figure 6 :
Figure 6: Histogram representing the antibacterial activity of essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage of A. monosperma, respectively; expressed as potencies as compared to standard Ampicillin.

Figure 7 :
Figure 7: Histogram representing the antiinflamatory activity of essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage of A. monosperma, respectively; expressed as potencies as compared to standard indomethacin.

Figure 8 :
Figure 8: Histogram representing the antioxidant activity of essential oils (A1, A2 and A3) samples collected before flowering, and at the beginning and end of the flowering stage of A. monosperma respectively, as potencies as compared to standard vitamin E.

Table 2 : Constituents identified by GC/FID and GC/MS analyses in the essential oil samples of the aerial parts of A. monosperma.
*: Retention indices relative to n-alkanes on SLB-5ms column; RI**: Retention indices reported on non polar column; A1, A2 & A3: samples collected before flowering, and at the beginning and end of the flowering stage.

Table 3 : Antimicrobial activity of essential oil samples of the aerial parts of A. monosperma, expressed as diameter of zone of inhibition in mm.
A1, A2 & A3: samples collected before flowering, and at the beginning and end of the flowering stage; NA: no action

Table 4 : Antimicrobial Activity of A2 oil sample* expressed as MICs (µg/ml)
A2 sample of aerial parts collected at the beginning of the flowering stage; NA: no action.

Table 6 : Effect of volatile oil samples (A1-A3) of the aerial parts of A. monosperma on GHS blood level in Alloxan-induced diabetic rats, as compared to Vitamin E.
A total of 16-20 components were identified; 11, including 9 non-oxygenated, were common in all the samples.Identified constituents represented 97.63-99.00% of the total composition of the analyzed oils.Samples were noticeably enriched in hydrocarbons; A 1 and A 2 showed appreciable and close amounts of these constituents (63.56 and 66.55%) although distinctly lower than in A 3 (88.36%).Monoterpenoids dominated the group, while sesquiterpenoids were missing and aromatics represented by p-cymene only.Sabinene (13.15-22.85%)andβ-pinene(9.00-24.03%)weredetected in appreciable amounts in all samples and p-cymene mainly in A 2 (11.20%).Oxygenated components were detected in lesser amounts(11.29,31.08 and 35.44 % in A 3 , A 2 and A 1 , respectively) being gradually decreased during flowering (highest % in A 1 , before flowering).They include esters, alcohols, ketones and phenols.They are mostly terpenoids with few aromatics viz., eugenol methyl ether, methyl iso-eugenol, 7-methyl-1-naphthol and capillin.The bicylic monoterpenoid ester, bornyl acetate was the major identified oxygenated compound in all the samples (8.00-31.00%)withhighest amount in A 1 decreasing to about the quarter by time (least inA 3