ANTIOXIDANT AND ANTIMICROBIAL POTENTIAL OF OPOPANAX HISPIDUS(APIACEAE) EXTRACTS

The present study was aimed to investigate antioxidant and antimicrobial potential of methanol and ethyl-acetate extracts from dried aerial parts, inflorescences and fruits of Opopanax hispidus (Friv.) Griseb., fam. Apiaceae. The antioxidant potential was evaluated with the help of two in vitro antioxidant models – DPPH and ABTS assays and estimation of total phenolic and flavonoids using spectrophotometric methods. BHA and Vitamin C were used as standard and positive control for above models. Microdilution assay was used to evaluate antimicrobial potential for the most common human gastrointestinal pathogenic microbial strains. The results of DPPH and ABTS assay showed that the highest antioxidant activity have methanol (IC50=1.157 mg/ml) and ethyl-acetate (IC50=3.167 mg/ml) extracts from inflorescences. The highest value of total phenolic (89.95±0.005 mg GA/g) and total flavonoid (24.06 ± 0.004 mg Qu/g) was measured in inflorescences extracts also. Results indicate that both extracts (methanol and ethyl-acetate) of inflorescences have high amount of phenol and flavonoids, which could be responsible for its good antioxidant activity.The most susceptible were Listeria monocytogenes and Escherichia coli on ethyl-acetate extracts from fruits and inflorescence, respectively. This is the first record of antioxidant and antimicrobial activity of Opopanax hispidus from Serbia. It is also worth noting that these results validate the therapeutic use of the plant in traditional medicine.


INTRODUCTION
The genus Opopanax, family Apiaceae, consists of two species, O. chironium (L.) Koch, and O. hispidus (Friv.) Griseb, widely distributed in Balkan Peninsula and Aegean region (S. Italy and Sicilia) [1]. O. hispidus is found at only one locality in Serbia -Rujan Mountain, between Cer hill and the village of Mamince near Kosovska Mitrovica. This species is described in Red data book of flora of Serbia and qualified as critically endangered taxon [2].
The species O. hispidus is perennial plant 300 cm high with a stout, solid stem with branches verticillate or subopposite, offten very close below the terminal umbel. Leaves are 2-pinnate, with stellate hairs beneath. Lobes usually are 2-4 cm, ovate-lanceolate and hispid. Flowers are yellow and fruits are broadly elliptical, border wide and thin 7-9 mm [1].
As we know, this is the first report of antioxidant and antimicrobial activity of methanol and ethyl-acetate extracts from O. hispidus, the plant which may possess many pharmacognosy effects.

Plant material and preparing extracts
The aerial plant parts with flowers and fruits were collected in July 2010 at Rujan Mountain, Southeast Serbia. The plant material was collected and determined as Opopanax hispidus (Friv.) Griseb., by the author Ranđelović Vladimir. A voucher specimen (BEOU 16435) has been deposited at the Herbarium of the Institute of Botany and Botanical Garden "Jevremovac", Faculty of Biology, University of Belgrade.
Classical extraction was used for preparing extracts: 10g plant material (aerial parts, inflorescences and fruits separately) was dissolved with 100ml methanol or ethyl-acetate. After 24h in the dark, extracts were filtrated and solvent was evaporating with vacuum evaporator. The first and last hour extracts were in ultrasonic bath. Yield of dry extracts are shown in Table 1.

Determination of total phenolic content
The total phenolic content of extracts was determined by Folin-Ciocalteu method according to the procedure reported by Singleton et al. (1999) with some modifications [9]. Briefly, 300 µl of methanol and ethyl acetate extracts and 1500 µl of 1:10 Folin-Ciocalteau reagent were mixed and after 6 minutes in the dark were added 1200 µl of sodium carbonate (7.5%). After 2 h in the dark of incubation at room temperature, the absorbance at 740 nm was measured (Shimadzu, UV-Visible PC 1650 spectrophotometer). The total phenolic concentration was calculated from gallic acid (GA) calibration curve (10-100 mg/L). Data were expressed as gallic acid equivalents (GA)/g of extract averaged from 3 measurements.

Determination of flavonoid content
The total flavonoid content was evaluated using aluminium nitrate nonahydrate according to the procedure reported by Woisky and Salatino (1998) with some modifications [10]. The sample for determination was prepared by mixing a 600 µl of methanol and ethyl acetate extracts and 2580 µl of mixture (80% C 2 H 5 OH, 10% Al(NO 3 ) 3 x 9 H 2 O and 1M C 2 H 3 KO 2 ). After 40 min of incubation at room temperature, the absorbance at 415 nm was measured using Shimadzu, UV-Visible PC 1650 spectrophotometer. The total flavonoid concentration in different extracts was calculated from quercetin hydrate (Qu) calibration curve (10-100 mg/L) and expressed as quercetin equivalents (Qu)/g of dry extract. Measurements were done in triplicates.

Antioxidant activity
Antioxidant activity aerial parts, inflorescences and fruits of plant methanol and ethyl-acetate extracts had been tested by DPPH and ABTS assays. All results were obtained using Shimadzu, UV-Visible PC 1650 spectrophotometer. Data analysis was performed with OriginPro 8.0 software.
DPPH assay -For this assay was applied from Blois method [11]. Standard methanol solution contains 0.04 mg/mL DPPH. After shaking, 0.3 mL methanol (concentration 125, 500, 1000, 2000 and 5000 µl/ml) and ethyl-acetate solution of different extracts (concentration 2000, 3000, 5000, 8000 and 9000 µl/ml) and 2.7 mL DPPH radical solution, the reaction mixture had been incubated in the dark for 30min at room temperature. ABTS assay -A method of Miller and Rice-Evans with some modifications was used for this assay [12]. Reagans ABTS·+ was mixed with 2.46 mM potassium persulfate and as such a solution was standing in the dark 12-16h at room temperature. After that, 1ml ABTS·+ solution was diluted with 100-110 mL water to give an absorbance of 0.7±0.02 units at 734 nm using spectrophotometer. Methanol ant ethyl-acetate solutions of different extracts (concentration for tested extracts were been 1.5, 2 and 3 mg/mL) or standard solutions (concentration for BHA were been 0.10 mg/mL) 75 μL, were mixed with 3 mL of diluted ABTS solution. After 30 min incubation at 30ºC, the absorbance was measured at 734 nm. Modifications of this method are that the water was used as a blank. ABTS radical scavenging activity in aerial parts, inflorescences or fruits of plant extracts were calculated from the Vitamin C (VitC) calibration curve (0-2 mg/L) and expressed as Vitamin C (VitC)/g of extracts. All measurements were performed in triplicate and were expressed as average of three analyses ± standard deviation. Micro-well Dilution Assay -The inocula of the microbial strains were prepared from the overnight broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity (corresponding to 10 7 -10 8 CFU/ml, depending on genera -consensus standard by the NCCLS) [13]. A series of doubling dilutions of the tested extracts from O. hispidus -100 mg/ml in 30 % ethanol were prepared in a 96/well microtiter plate over the range of 0.1-50.0 mg/ml in inoculated Mueller-Hinton broth. The final volume was 100 μl and the final microbial concentration was 10 6 CFU/ml in each well. The plate was incubated for 24 h at 37 ºC for bacterial and 24 h at 25 ºC for yeast. All experiments were performed in triplicate. Two controls were included -medium with 30 % ethanol (negative control) and medium with Streptomycin, Chloramphenicol and Nystatin (positive control). Microbial growth was determined by adding 20 μl of 0.5 % triphenyl tetrazolium chloride (TTC) aqueous solution [14]. Minimal inhibitory concentration (MIC) was defined as the lowest concentration of the samples inhibiting visible growth (red colored pellet on the bottom of the wells after the addition of TTC). To determine MBC/MFC, the broth was taken from each well without visible growth and inoculated in Mueller-Hinton agar (MHA) for 24 h at 37 ºC for bacterial and 24 h at 25 ºC for yeast. Minimal bactericidal/fungicidal concentration (MBC/MFC) was defined as the lowest samples concentration killing 99.9 % of bacterial/fungal cells.

Total phenolic and flavonoid content
It has been shown that phenolic compounds represent a class of antioxidant agents which act as free radical terminators [15]. In other hand, flavonoids have antioxidant activity and their effects on human nutrition and health are considerable. The mechanisms of action of flavonoids are through scavenging or chelating process [16][17]. The results obtained for total phenolic and flavonoid content of O. hispidus extracts are presented in Table 2. The highest value of total phenolic (89.95 ± 0.005 mg GA/g) and total flavonoid (24.06 ± 0.004 mg Qu/g) was measured in inflorescences extracts. These results are in correlation with results for antioxidant activity, which is a confirmation of previous studies.

Antioxidant activity (DPPH and ABTS assay)
It was reported that the antioxidant constituents from natural, plant sources provide protection from damage caused by free radical induced oxidative stress [18][19].
The results of DPPH and ABTS assay showed that the highest antioxidant activity have methanol (IC 50 =1.157 mg/ml) and ethyl-acetate (IC 50 =3.167 mg/ml) extracts from inflorescences of O. hispidus in compare to aerial parts and fruits extracts. Methanol extracts possessed better antioxidant activity than ethyl-acetate ones.
So far, the resuts of antimicrobial effects of plant extracts are published for a number of Apiaceae species from Serbian flora in relation to medically significant bacteria and fungi [20][21][22][23]. There is no data on those activities on Opopanax species extracts.

CONCLUSION
From the results obtained in the present study, it can be concluded that methanol extract of O. hispidus show strong antioxidant activity by DPPH and ABTS assay when compared with ethyl-acetate extracts. In addition, methanol and ethyl-acetate extract of O. hispidus were found to contain noticeable amount of phenol and flavonoids that could have great importance as a therapeutic agent in preventing or slowing oxidative stress related degenerative diseases. Both extracts of different parts of this plant species showed good antimicrobial activity. Therefore, O. hispidus can be further harnessed for novel antioxidant/antimicrobial compounds, which is well evidenced by the present study.