TOTAL PHENOLIC AND FLAVONOID CONTENTS AND BIOLOGICAL ACTIVITIES OF CACHRYS CRISTATA DC. EXTRACTS

The total phenolic/flavonoid contents and antioxidant potential of the methanol, ethyl-acetate, acetone and water extracts obtained from the aerial parts and fruits of Cachrys cristata DC. (Apiaceae) were compared. The total phenolic contents of the tested extracts were determined using Folin-Ciocalteu’s reagent. The amounts per g of dry plant extract of gallic acid (GA) and quercetin (Qu) ranged between 22.60-166.97 mg, and 8.91-46.02 mg, respectively. The antioxidant activity, expressed as IC50, ranged from 1.784-17.621 mg/mL and from 1.01-3.42 mg L(+)-ascorbic acid (Vitamin C)/g when tested with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ABTS, respectively. The antimicrobial activity of the extracts was investigated by the microwell dilution assay, for the most common human gastrointestinal pathogenic bacterial strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella enteritidis ATCC 13076, Bacillus cereus ATCC 10876, Listeria monocytogenes ATCC15313, Staphylococcus aureus ATCC 25923 and yeast Candida albicans ATCC 10231. The results indicate that C. cristata can be regarded as a potential source of antioxidant and antimicrobial agents.


INTRODUCTION
Many studies have been examined the chemical composition and biological activities of various extracts of plants from the Apiaceae family (Wang et al., 2012).Species from this family are characterized by the presence of furanocoumarins, flavones and flavonols.The most common flavonols are quercetin and kaempferol (Miean and Mohamed, 2001).
In various extracts of the Apiaceae species, coumarins are often detected as the most abundant group of secondary metabolites.Also known as benzopyrones, they represent a large group of phenolic compounds occurring in green plants, as well as in fungi and bacteria (Murray et al., 1982).They have antimicrobial, antitumoral and anti-aggregate activities and have been reported to be potent antioxidants with anti-inflammatory properties (Hoult et al., 1994, Paya et al., 1994, Ng et al., 1996, Hoult and Paya, 1996, Cai et al., 1997, Su et al., 1998, Kang et al., 1999).Furanocoumarins, alternatively known as furocoumarins, are a category of coumarins that occur as plant constituents predominantly in two families, the Apiaceae and the Rutaceae (Fujioka et al., 1999).
Essential oil isolated by hydrodistillation from the aerial parts and fruits of this species was analyzed using combined GC and GC/MS (Matejić et al., 2012).The aerial parts of C. cristata were rich in sesquiterpenes (45.7%) and oxygenated sesquiterpenes (32.9%), while the fruit oil consisted of a higher percentage of sesquiterpenes (48.3%) and oxygenated sesquiterpenes (36.7%).In the oil from aerial parts, dominant constituents were phytol (13.1%) and germacrene D (12.9%), while in the fruit oil of C. cristata most abundant were suberosin (19.7%) and germacrene D (12.3%) (Matejić et al., 2012).
In this work, we present a first report on the antioxidant and antimicrobial activities of C. cristata methanol, ethyl-acetate, acetone and water extracts.

Plant material
Aerial plant parts and fruits of Cachrys cristata were collected in July 2010 from Rujan Mountain, southeast Serbia.A voucher specimens for C. cristata (BEOU 16434), has been deposited at the Herbarium of the Institute of Botany and Botanical Garden "Jevremovac", Faculty of Biology, University of Belgrade, Serbia.

Preparation of plant extract
Plant material was air dried in the dark and ground to a powder.The aerial plant parts and fruits (10 g) were powdered and extracted with 100 ml water, methanol, acetone and ethyl acetate.The mixture was exposed to ultrasound for 30 min and after 24 h standing in the dark was filtered.The methanol, ethyl-acetate and acetone solvents were removed by evaporation under reduced pressure, at maximum temperature of 40ºC.Water extract was frozen and later dried by freeze-drying.After evaporation of the solvent, the crude extract was subjected to subsequent analysis.

Determination of total phenolic content
The total phenolic content of extracts was determined spectrophotometrically by the Folin-Ciocalteu method as described by Singleton et al. (1999), with some modifications.Briefly, 300 µl of different extract solutions and 1 500 µl of 1:10 Folin-Ciocalteu reagent were mixed.After 6 min in the dark, 1 200 µl of 7.5% Na 2 CO 3 were added.After incubation for 2 h in the dark at room temperature, the absorbance at 740 nm was measured (Shimadzu, UV-Visible PC 1650 spectrophotometer).The total phenolic concentration was calculated from the GA calibration curve (10-100 mg/l).Data were expressed as GA equivalents/g extract averaged from three measurements.

Determination of flavonoid content
The total flavonoid content was evaluated using aluminum nitrate nonahydrate according to the procedure reported by Woisky and Salatino (1998) with some modifications.The sample for determination was prepared by mixing 600 µl of extract solution and 2 580 µl of a mixture containing 80% C 2 H 5 OH, 10% Al(NO 3 ) 3 x9H 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 (Shimadzu, UV-Visible PC 1650 spectrophotometer).The total flavonoid concentration in the extract was calculated from the quercetin hydrate (Qu) calibration curve (10-100 mg/l) and expressed as Qu equivalents/g dry extract.Measurements were performed in triplicates.

Evaluation of DPPH-scavenging activity
The antioxidant activity of extract was evaluated by the radical scavenging method, using the stable radical DPPH as reagent (Blois, 1958).The effect of antioxidant compounds on DPPH radical scavenging is due to their hydrogen-donating ability or radical-scavenging activity.When a solution of DPPH is mixed with a substance that can donate a hydrogen atom, this gives rise to the reduced form diphenylpicrylhydrazine with the loss of its violet color (Molyneux, 2004).The water, methanol, ethyl-acetate and acetone solution of the investigated extract (300 μl) (the extract concentrations between 1 000 and 7 500 μg/ml) was added to 2 700 μl methanolic solution of DPPH (concentration 0.04 mg/ml), and after shaking, the reaction mixture was left to react in the dark for 30 min at room temperature.Absorbance of the remaining DPPH radical was measured Percentage of absorption decrease (on 517 nm) = (A0-A1)x100/A0 Concentrations that decrease absorption of DPPH solution by 50% (IC 50 ) were obtained from the absorption curve of DPPH solution at 517 nm from the concentrations for each compound and standard antioxidant.For calculation of these values, Origin 7.0 software was used.Tests were carried out in triplicate.

ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid) radical-scavenging activity was determined by the method of Miller and Rice-Evans (1997) with some modifications.The ABTS + solution was prepared by mixing 19.2 mg of ABTS with 5 ml of potassium persulfate (2.46 mM).The solution was kept at room temperature in the dark for 12-16 h before use.The ABTS + solution (1 ml) was diluted with 100-110 ml water in order to obtain an absorbance 0.7±0.02 at 734 nm.Fresh ABTS + solution was prepared for each analysis.Seventy five μl of antioxidant or standard solution was mixed with 3 ml of diluted ABTS + solution and incubated at 30ºC for 30 s.The absorbance at 734 nm was measured (Shimadzu, UV-Visible PC 1650 spectrophotometer).Water was used as a blank.ABTS radical-scavenging activity in the different extracts was calculated from a vitamin C calibration curve (0-2 mg/l) and expressed as vitamin C/g dry extract.All experimental measurements were carried out in triplicate and were expressed as the average of three analyses ± standard deviation.

Microbial cultures
The antimicrobial activity of all tested samples was evaluated using laboratory control strains obtained from the American Type Culture Collection: Gramnegative bacteria: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella enteritidis ATCC 13076; Gram-positive bacteria: Bacillus cereus ATCC 10876, Listeria monocytogenes ATCC15313, Staphylococcus aureus ATCC 25923 and yeast Candida albicans ATCC 10231.

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 the genera) (NCCLS 2003).Serial dilutions of the tested samples (methanol, ethyl acetate and acetone extracts of C. cristata (100 mg/ml in a 30% ethanol and water extract; 100 mg/ml in 5% DMSO) were prepared in a 96-well microtiter plate over the range of 50.0-0.1 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 plates were incubated for 24 h at 37ºC.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 (Sartoratto et al., 2004).The 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 minimal bactericidal/fungicidal concentrations (MBC/MFC), broth was taken from each well without visible growth and inoculated in Mueller-Hinton agar (MHA) for 24 h at 37ºC.MBC/ MFC were defined as the lowest sample concentration killing 99.9% of bacterial/fungal cells.

total phenolic content
The total phenolic contents of the examined plant extracts (Table 1), expressed as gallic acid equivalents (GA) per gram of dry extract, ranged between 22.60 and 166.97 mg GA/g.The highest phenolic content was found in the acetone extract of aerial plant parts (3 mg/ml extract).

Flavonoid content
A summary of the quantities of flavonoids detected in the tested extract is shown in Table 1.The concentrations of flavonoids in plant extracts ranged from 8.91 to 46.02 mg Qu/g.The highest flavonoid content was measured in the ethyl-acetate extract of aerial parts (with 5 mg/ml extract concentration).Because of their presence in plants, flavonoids are important components of human and animal diet.Due to the different biological activities of plant secondary metabolites, their regular consumption may have serious consequences for health, both positive and negative (Stobiecki and Kachlicki, 2006).

DPPH-scavenging activity
The free radical-scavenging capacities of the tested extract were measured by the DPPH assay and the results are shown in Table 2.A lower IC 50 value indicates higher antioxidant activity.The water extract of C. cristata fruits possessed the strongest antioxidant activity compared to the other extracts.

ABtS-scavenging activity
The results from the ABTS assay are shown in Table 1.The amount ranged from 1.01 to 3.42 mg vitamin C/g of C. cristata species.The highest content was identified in the acetone extract of C. cristata aerial parts (3 mg/ml extract) and the lowest in the ethylacetate fruits extract (5 mg/ml extract).
Various solvents were used to achieve the extraction of active substances with diversity in their polarity.The acetone extract of C. cristata aerial parts showed relatively high antioxidant activity that is in accordance with the high concentration of its total phenols and flavonoids.Based on the results of this study, the extract with the highest antioxidant activity had the highest concentration of phenols.Phenolic compounds are very important plant constituents because of their scavenging ability on free radicals due to their hydroxyl groups.Therefore, the phenolics in plants directly contribute to their antioxidant action.

Antimicrobial activity
All tested extracts of C. cristata (except the water extract) showed inhibitory antimicrobial activity against all tested strains at concentrations of 0.78-12.5 mg/ml, but most of them did not show bactericidal effect at concentrations of 50.0 mg/ml (Table 3).As reference antibiotics, streptomycin and chloramphenicol, and antimycotic nystatin were used.
The ethyl-acetate extract of aerial plant parts and fruits showed the best activity against Gram-positive bacteria with an inhibitory and bactericidal concentration range of 0.78-6.25 mg/ml.The same extract had inhibitory effect on the P. aeruginosa strain at concentrations of 3.125-6.250mg/ml, but the highest concentration of this extract did not show bactericidal effect on this treated bacterial strain.Similar results were observed for the C. albicans strain.Methanol extracts of aerial parts and fruits showed similar inhibitory effects to the ethyl-acetate extract, but the acetone extract had lower effectiveness compared to the others (except on Bacillus cereus).B. cereus was the most sensitive strain to extracts of Cachrys cristata.This is very important because C. cristata is a spice plant while Bacillus cereus is a bacterium that often causes food spoilage.

CONCLUSIONS
Methanol, ethyl-acetate, acetone and water extracts from Cachrys cristata are potential protective agents against oxidative stress provoked by DPPH and hydroxyl radicals.They expressed good antioxidant activity in comparison with the synthetic antioxidants BHA and Vitamin C. Ethyl-acetate and methanol extracts showed an inhibitory effect at low concentrations against a selected group of microorganisms that cause food spoilage.Our results point to their potential use as natural preservatives and in the production of food that could prevent gastrointestinal disorders.

Table 1 .
Total phenolic/flavonoid contents and antioxidant capacities by ABTS assay of aerial plant parts and fruits C. cristata extracts.Each value in the table was obtained by calculating the average of three analyses ± standard deviation.

Table 2 .
Antioxidant activities of C. cristata extracts using DPPH scavenging method.

Table 3 .
Antimicrobial activity of aerial parts and fruits C. cristata extracts against pathogenic microbial strains using Micro-well Dilution Assay.