Comparative Study of Antioxidant Activity of Different Extracts and Essential oil Compositions Obtained from Artemisia Sieberi Besser Using Two Different Methods

Introduction

Artemisia sieberi Besser, belongs to the Asteraceae family, is a widely distributed plant in Iran [1-2]. In Iranian folk medicine, Artemisia sieberi is introduced to be useful for spasmolytic effects [3-4], vermicide and also for strengthening stamina and relieving cold symptoms. A. sieberi oil like some other species of Artemisia has potential to be used as an insecticide [5]. Animal dermatophytosis and clinical improvement with 3% solution of A. sieberi oil have been observed which is in comparison with clotrimazole [6]. Antidiabetic, antimalarial and antimicrobial effects of this plant have been reported previously [7-9]. As mentioned above, because of its medicinal properties, this plant is undoubtly a good candidate for evaluating phytochemicals or biological activities. A couple of studies in the literature have reported composition of the essential oil of Artemisia sieberi, extracted by hydrodistillation method and also extraction using supercritical carbon dioxide from different locations of Iran [10-15]. It is clear that production of secondary metabolites in plants is firmly depended on ecological conditions [16] and, in addition, yield of oil extraction and the chemical composition of the obtained essential oil may change as the method of extraction changes. According to the above descriptions and lack of enough reports about phytochemical and biological properties of this plant in the literature, this research focuses on comparative study on the chemical constituents of volatile oil obtained by SDE and general hydrodistillation extraction methods from A. siberi growing wild in Kashan, central Iran. Antioxidant activities of water, ethanol and chloroform extracts of this plant have also been assessed.

Experimental

Plant materials

The plants were collected from Karkas Mountains of Kashan, Iran, at an altitude about 1800 m in April 2010. The voucher specimens of the plant were confirmed in the Herbarium of Research Center of Barij Essence Pharmaceutical Company of Kashan (code: 1_108).

Essential oil isolation

The aerial parts of examined plants were dried in shadow at room temperature. Hydro-distillation and simultaneous water steam distillation-organic solvent extraction (SDE) methods [17] were used in this study. The extracted oils were then dehydrated by anhydrous sodium sulphate, to yield 0.3% and 1.13% of brown-red oil respectively.

 GC-Mass analysis

The isolated Essential oils were analyzed using GC/FID for quantitative analysis and GC/MS to qualitative analysis on a HEWLETT-PACKARD 6890 gas chromatograph coupled with a mass detector (HEWLETT-PACKARD model 6973 HP). HP-Innowax capillary column (60 m × 0.32 mm, 0.50 mm film thickness) was used as the oil analyser. The mass spectra were obtained by electron ionization at 70

eV. The oven temperature was programmed as follows: 80ºC (3 min), 80-230ºC (3ºC /min), 230ºC (5 min), 230-250ºC (3ºC /min) and 250 º C (10 min).

The injection temperature was 250 °C. The carrier gas (helium) flow rate was 1 ml/min. The sample (1 µl) was injected with a split ratio of 1/90. Retention indices were calculated for all components using a homologous series of n-alkanes injected in conditions equal to those of the samples. Identification of components of Eos was based on retention indices (RI) relative to n-alkanes and computer matching with the Wiley275.L and Wiley7n.L libraries. Also, comparisons of the fragmentation pattern of the mass spectra were made with data published in the literature [18].

Preparation of extracts

Thirty grams of the powdered aerial parts of the plant were soxhlet-extracted with 300 ml solvent. Solvent removal using rotary evaporation and drying the residue using a vacuum oven at 50 °C yielded 14.0% w/w of water, 14.0% w/w of ethanol and 9.5% w/w of chloroform extracts with the colours brown, green and light green respectively. Extracts were kept in dark at 4 °C until be tested.

Antioxidant activity

For evaluating antioxidant activities of samples, DPPH bleaching and total phenol measuring assays were used.

DPPH bleaching assay

In this assay the antioxidant activity was determined by measuring DPPH free radical scavenging ability. To plot BHT standard curve, absorbance of DPPH methanol solution in the presence of various concentrations of BHT after 40 min in 517 nm was measured and inhibition of DPPH free radical in percent (I %) was calculated as follows:  I % = (Ablank-Asample/Ablank) ×100 (Eq. A)  Where Ablank is the absorbance of the control reaction, containing all reagents except the test compound. Inhibition percentage of the samples (1 mg/ml of water, ethanol and chloroform extracts) and BHT standard were calculated similarly and all tests were carried out in triplicate to improve accuracy. Ability of DPPH radical scavenging was calculated as %I.

Total phenol measuring

Total phenolic constituents of the aforesaid extracts of A. sieberi were determined based on the absorbance values of the extract reacted with Folin-Ciocalteu reagent and compared the results with gallic acid as standard solution [19] with slight modifications. This assay was carried out in order to obtain the total phenolic contents of the extracts according to the µg gallic acid equivalent (GAE) per 1mg of extract. For this purpose, a solution containing 1 mg crude extract in ethanol was exposed to 1 ml of Folin-Ciocalteu reagent in the presence of 3 ml of a 2% Na2CO3 in a 50 ml volumetric balloon. The mixture was allowed to stand for 2 h with intermittent shaking (roughly every 30 minutes). Absorbance was measured at 760 nm. The same procedure was repeated for different concentrations of standard gallic acid  solutions (0–1000 mg per 0.1 ml). The equation for the standard curve obtained was found to be like eq. (1). Absorbance = 0.0012 × Gallic acid (µg) + 0.0033       (1) Gallic acid equivalent of the samples was calculated as μg of gallic acid per mg of sample.

Results and Discussion

The yield of hydrodistillated essential oil (HDEO) was 0.3% for Artemisia sieberi Besser from Kashan in comparison with 1.13% which obtained from SDE prepared essential oil (SDEO) which is almost 4 times more than that of HDEO. Compositions of the essential oils were evaluated using GC-MS analysis. The major components in the HDEO were found to be verbenol (16.09 %), myristicin (13.76%,), E-epoxy ocimen (9.77%) and α-phellandren-8-ol (7.44%). These are very different from the other reported components detected in A. siberi of other locations (10,11,13) these components are also , very different from those of SDEO with E-epoxy ocimen (14.02 %), verbenol (13.93%), artemiseole (10.25%) and camphor (8.89%) as the majors. As can be seen, although total number of components detected in both mentioned essential oils were almost equal, their types and percentages were very different (Table 1);

Table 1: essential oils compositions of Artemisia sieberi Besser aerial parts from Kashan, Iran 

 

 

for example some compounds such as lavandulol (0.78%), cis chrysanthenol (1.83%), p- cymen-8-ol (0.92%), cis carvone oxide (1.65%) caryophyllene oxide (1.17%) and 6,10,14-trimethyl 2-pentadecanone, (1.35%) detected in HDEO, were not seen in SDEO. On the other hand, verbenene (1.36%), 1, 8 cineole (0.14%), β –thujone (4.69%), trans-chrysanthenyl acetate (1.10%) and 2E-hexenyl butanoate (2.62%) which were found in SDEO, were not seen in HDEO. Compounds such as myristicin and verbenol observed in the mentioned plant have been reported to be the most important contributors in insecticiding effects of the essential oil of Artemisia species. Comparing these results, it can be revealed that method of essential oil isolation has a significant role on extraction yield, type and amount of extracted compounds. Antioxidant activities, based on DPPH free-radical assay,  for water, ethanol and chloroform extracts (Table 2) exhibit

Table 2: Extraction yields and antioxidant activity of extracts of Artemisia siberi from Kashan, Iran

 

Sample	Yield of extaction	DPPH %I	Totalphenolics:Gallicacid eq (µg/mg sample)
Water extract	14.0%	69.00 ± 0.40	66.14 ± 0.91
Ethanol  extract	14.0%	55.73 ± 1.00	59.61± 0.54
Choloroform extract	9.5%	13.47 ± 0.92	36.30 ± 0.39
BHT	–	87.10 ± 0.82	–

 

higher activity of  water extract in the order of water> ethanol > chloroform. Total phenolic contents of these extracts also obey this order, but the differences are not as significant as antioxidant activities. Considering these findings and the yields of extraction, it seems that water as a safe and eco-friendly solvent, which is used locally in domestic consumptions, is the best solvent for extracting bioactive materials as far as antioxidant activity is concerned.

Conclusion

According to these results it needs to point out that the extraction solvent and method has generally a significant influence on properties and amount of active components of the plant extracts. In addition, as it is observed in this research, the yield of essential oil extraction for SDE method is much higher than general hydrodistillation technique; therefore, it can be concluded that the use of this especial techniques can be useful particularly for plants with low-content essential oil, such as Artemisia species.

References

1. Mozaffarian V. A dictionary of Iranian plants names, Tehran, Iran: FarhangMoaser; 1998.
2. Ghahraman A. Color Flora of Iran, Tehran, Iran: Forest and Rangelands Resarch Institute; 1997.
3. Zargari A. Medicinal Plants, 6th ed. Tehran, Iran: Tehran University Press; 1996.
4. Mirza M, Sefidkon F & Ahmadi L. Natural Essence, Extraction, Identification, Tehran, Iran: Research Institute of Forest and Rangelands; 1998.
5. Negahban M, Moharramipour S, Sefidkon F. Insecticidal activity of essential oil from Artemisia sieberi Beser against three stored-product insects, J Stored Prod Res, 2007; 43:123-128.
6. Khosravi A.R, Mahmoudi M & Shirani D. Evaluation of the use of Artemisia sieberi essence for treatment of cats and dogs with dermatophytosis, J Facul Vet Med, 2003; 58: 293-5.
7. Mahboubi M, Farzin N. Antimicrobial activity of Artemisia sieberi essential oil from central Iran, Iran J Microbiol, 2009; 1(2): 43-48.
8. Irshad F, Manis K & Aburjai T, Antidiabetic effect of eesential oil from Artemisia sieberi growing in Jordan, Pak. J. Biol. Sci, 2010;13(9): 423-430.
9. Nahrevanian H, Sheykhkanlooye Milan B, Kazemi M, Hajhosseini, R, Soleymani Mashhadi S & Nahrevanian S. Antimalarial effects of Iranian flora Artemisia sieberi on Plasmodium berghei In Vivo in mice and phytochemistry analysis of its herbal  extracts, Malar Res Treat, 2012; Article ID 727032, 8 pages.
10. Weyerstahl S, SchneiderS, Marschall H and Rustaiyan A, The essential oil of Artemisia sieberi Bess. Flavor Frag J, 1993; 8:139-145.
11. Sefidkon F, Jalili A & Mirhaji T, Essential oil composition of three Artemisia sp. from Iran. Flavor Frag J, 2002; 7:150-152.
12. Azarnivand H. Investigation of botanical and ecological characteristics of Artemisia sieberi and Artemisia oucheri in the Southern Aspect of Alborz, M.Sc. Thesis, Tehran University, Iran; 2003.
13. Farzaneh M, Ghorbani-Ghouzhdi H, Ghorbani M & Hadian J,  Composition and antifungal activity of essential oil of Artemisia sieberi bess. on soil-borne phytopathogens, Pak. J. Biol. Sci, 2006;  9:1979-1982.
14. Ghaemi E.O,  Heshmati G.A,  Behmanesh B, Ahmadi  A.R, Rezaei M.B,  Bakhshandeh Nosrat S & Mazandarani M. Chemical composition and antibacterial activity from essential oil of Artemisia sieberi Besser subsp. Sieberi in North of Iran, Asian J Plant Sci, 2007; 6(3): 562-564.
15.  Ghasemi E, Yamini Y,  Bahramifar N, Sefidkon F, Comparative analysis of the oil and supercritical CO 2 extract of Artemisia sieberi, J Food Eng, 2007; 79(1): 306-311.
16. Omidbeigi  R, Production and Processing of Medicinal Plants, 1st Edn., Razavy Ghods Astan Press, Mashad, pp: 347; 2005.
17. Filek G, Bergamini M & Lindner W, Steam distillation-solvent extraction, a selective sample enrichment technique for gas chromatographic-electron-capture detection of organochlorine compounds in milk powder and other milk products. J. Chromatogr. A, 1995; 712: 355-364.
18.   Adams R P, Identification of EO components by gas chromatography/mass spectroscopy, Carol Stream IL: Allured Publishing Co; 2001.
19. Slinkard K, Singleton VL, Total phenol analysis: automation and comparison with manual methods, Am J Enol Viticult, 1997; 28: 49-55.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.