Total phenolic, total flavonoids, antioxidant and antimicrobial activities of scrophularia striata boiss extracts.

BACKGROUND
Scrophularia striata (Scrophulariaceae family) is an herbaceous plant that is traditionally used for treatment of microbial infections.


OBJECTIVES
Antimicrobial and antioxidant activity of different extracts (methanolic, ethanolic, aqueous and ethyl acetate) from S. striata aerial parts was evaluated.


MATERIALS AND METHODS
The antimicrobial activity of different extracts from S. striata was evaluated against a large number of bacteria and fungi by micro broth dilution. Total phenolic and flavonoid contents were measured and their antioxidant activities evaluated by DPPH assay and beta carotene linoleic acid test.


RESULTS
Antimicrobial screening exhibited the positive relation between the total phenolic content and its antimicrobial activity but their antioxidant activity had a negative relation.


CONCLUSIONS
Further studies are recommended against clinical isolate of sensitive bacteria and deep investigation on flavonoid and phenolic compounds of S. striata and detecting the antioxidant portion in aqueous extract.


Background
Scrophularia striata, an herbaceous flowering plant from Scrophulariaceae family, is commonly known as figwort. It has square stems, opposite leaves and open two-lipped flowers forming clusters at the end of their stems. The name Scrophularia comes from scrofula, a form of tuberculosis, since several species have been used to treat tuberculosis. It has been traditionally used for treatment of allergies, rheumatics and chronic inflammatory diseases. Some pharmacological activities of S. striata have been confirmed. Scrophularia extracts decrease edema, cell infiltration and proliferation of activated T-lymphocytes in joint tissues (1). Additionally, it inhibits a number of inflammatory factors such as PGE-2, leukotriene B4, NO, IL-1β, IL-4, INF-γ, but did not have any effect on the production of IL-10 (2).
Ethanolic extract of S. striata aerial parts acts as a nitric oxide inhibitor in a dose dependent manner in peritoneal macrophages and exhibited an anti-inflammatory effect (3). The antimicrobial activity of S. striata ethanolic extract leaves against Staphylococcus aureus and Escherichia coli (4), Enterococcus faecalis and Newcastle diseases (5) were confirmed. Additionally, this extract exhibited synergism activity with deoxycycline and of loxacin with FIC index 0.43 and 0.314, respectively (4). Ethanolic extract of S. striata had inhibitory effect against matrix metalloproteinase (MMP) (6). The leaves and seeds of S. striata contain both anticancer and cell growth enhancing activities (7).

Objectives
The purpose of this research is to evaluate the antimicrobial activity of different extracts from S. striata aerial parts against Gram positive, Gram negative bacteria and fungi. We also evaluated the total phenolic compounds in each extract and their antioxidant activity.

Plant Material and Extraction
The dried aerial parts of Scrophularia striata was collected from Kolm Valley (Ilam Province, Iran) in April 2010 and identified by Agriculture Department of Barij Essence Center, Kashan, Iran and authenticated under number 193-1. The aerial sample was ground and subjected to extraction by percolation method with water, methanol, ethyl acetate and ethanol-water (70:30, volume/volume). The powdered S. striata aerial parts were mixed with solvent at the ratio of 1:10 (w/v) in a percolator for 24 h at ambient temperature. Then, the mixture was filtered through Whatman filter paper No. 2, the residue rinsed with the same solvent and the extract dried under vacuum.

Total Phenolic Content (TPC)
Total phenolic contents of crude extracts were determined by a spectrophotometer using the Folin-Ciocalteu's reagent (Merck) (8). A concentration 1 mg/mL of each extract was prepared. 0.1 mL of extract and 0.5 mL of Folin-Ciocalteu's reagent (10%) was added. After 3-8 minute, 0.4 mL of 7.5% (w/v) sodium carbonate solution was added and mixed. Following 1 hour at ambient temperature, the absorbance of reaction mixture was measured at 765 nm. TPC was calculated using the following equation: W = ((Abs-0.0089)/0.0647) × 100, where Abs is absorbance and w is the weight (µg). All tests were conducted in triplicate and averaged. The results were expressed as mg of TPC per gram of dry extract as Gallic acid.

Total Flavonoid Content (TFC)
The modified aluminum chloride colorimetric method was used (9). 0.5 mL of diluted standard solution and each extract were separately mixed with 1.5 mL of ethanol (95%), 0.1 mL of aluminum chloride (10%), 0.1 mL potassium acetate (1 M) and 2.8 mL distilled water. Following incubation at room temperature for 30 minutes, the absorbance of the reaction mixture was measured at 415 nm with a spectrophotometer. The results were expressed as mg of TFC per gram of dry extract as Quercetin (QE).

Free Radical Scavenging Activity by DPPH Method
Radical scavenging activity of the extracts was determined using DPPH radical scavenging activity assay (10) with a slight modification. A stock solution of each extract and BHT as the reference antioxidant (63 mg/1.6 mL) was prepared in its own solvent and diluted with methanol in the range of concentrations. 0.1 mL of diluted extract (7.8 to 2000 μg/mL) were added to 2 mL of freshly prepared DPPH methanol solution and mixed. After 30 minutes, the absorbance of reaction mixture was measured at 490 nm by spectrophotometer. Inhibition of free radical DPPH in inhibition percent (I%) was calculated and a sample concentration providing 50% inhibition (IC50) was calculated by plotting inhibition percentages against concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as means. BHT was used as positive control.

β-carotene/ Linoleic Acid Bleaching Test (BCBT)
A diluted β-carotene in chloroform was transferred from pipette into a flask. Chloroform was removed using a rotary evaporator under vacuum at 50 °C for 10 minutes, and then 25 μL of linoleic acid, 200 mg of Tween 80 and 100 mL aerated distilled water were added to the flask with vigorous shaking. The emulsion (2.5 mL) was added to a tube containing 0.35 mL of the extract (2 mg/mL) and the absorbance immediately measured at 490 nm against a blank as zero time. Blank sample, devoid of β-carotene, was prepared for background subtraction. The tubes were placed in a water bath at 50 °C and the oxidation of the emulsion was monitored by measuring absorbance at 490 nm over a 120 minutes period. The antioxidant property (inhibition percentage, I%) of the samples was determined. BHT was used as positive control (11). Bacterial suspensions were prepared in Brain Heart Infusion (BHI) (Merck) to a concentration of approximately 10 8 CFU/mL using standard routine spectrophometric methods. Suspensions of fungi were made in sabouraud dextrose broth (10 6 CFU/mL). Subsequent dilutions were made from the above suspensions, which were then used in the tests.

Microbial Strains and Antimicrobial Evaluation by Micro Broth Dilution Assay
The minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) values of extracts were determined by micro broth dilution assay. The extracts were twofold serially diluted with 10% DMSO which contains 25.6-0.2 mg/mL of each extract. Antibiotics were used as positive control. These dilutions were prepared in a 96-well microtitre plate. MOPS-buffered RPMI 1640 (fungi) (12), Cation adjusted Muller Hinton broth (non fastidious bacteria) (13) and Todd Hewitt broth (fastidious bacteria) (14) were used as broth media. After shaking, 100 µl of extract dilutions was added to each well. The above microbial suspensions was diluted (1 × 10 6 CFU/mL for bacteria; 10 4 CFU/mL for fungi) and then 100 µl was added to each well and incubated at 35 ºC. MICs were defined as the lowest concentration of extract dilutions that inhibits bacteria and fungi after 24, 48 hours, respectively. MLC values were the first well that showed no growth on solid media.

Results
The antimicrobial activity of S. striata extracts against different microorganisms was calculated by micro broth dilution assay ( S. striata methanolic extract on different gram negative ones excluding P. aeruginosa, filamentous fungi and yeast was inhibitory effect. Antimicrobial effect of S. striata extracts on filamentous fungi is weak and only its ethanolic extract was effective against C. albicans, C. glabrata and A. parasiticus. Among gram negative ones, E. coli and P. aeruginosa showed more sensitivity to ethanolic extract and P. aeruginosa to methanolic extract. Total phenolic and flavonoid content of S. striata extracts were evaluated. The results have been revealed in Table 2. Total phenolic content of ethanolic extract (79.7 mg GAC/g), ethyl acetate (65.5 mg GAC/g) was higher than that of methanolic extract (49.1 mg GAC/g) and aqueous extract (36.6 mg GAC/g). The total flavonoid content of ethyl acetate extract (27.5 mg QE/g) was higher than ethanolic extract (9.8 mg (QE/g), methanolic extract (8.2 mg QE/g) and aqueous extract (5.1 mg QE/g). Ethyl acetate extract had higher total flavonoid content and ethanolic extract had higher total phenolic content. S. striata water extract had the lower phenolic and flavonoid content than the other extracts. The amount of phenolic content in methanolic extract was lower than ethanolic or ethyl acetate extract however its flavonoid content was a small lower than ethanolic extract.
Antioxidant activity of S. striata extracts was evaluated by DPPH and BCBT assays ( Table 2). In DPPH assay, IC50 of extracts revealed that S. striata aqueous extract with IC50 195 µg/mL had higher antioxidant activity. The IC50 of methanolic and ethanolic extracts was the same. Ethyl acetate extract had higher IC50. The antioxidant activity of S. striata was weaker than BHT ( Table 2). In BCBT assay, evaluation of inhibition percent (I%) showed that aqueous, methanolic, ethanolic and ethyl acetate extracts had I% 70.2%, 68%, 64.8% and 58.3%, respectively.

Discussion
Oxygen and nitrogen reactive species are essential for energy storage, toxification, chemical signaling and immunological functions also they control endogenous enzymes like superoxide dismutase, glutathione peroxidase and catalase. Failure in defense mechanisms is the agent of producing oxygen's free radicals that can disturb essential molecules such as DNA, lipids and proteins. These injuries are associated with cardiovascular diseases, cancer and other chronic diseases.
Natural antioxidants can protect body against oxidative injuries and decrease the risk of different chronic diseases (15). This hypothesis has been reached by the researcher who evaluated the relation between natural antioxidant in medicinal plants and the decrease in degenerative diseases. Medicinal plant's antioxidants such as phenolic represent a large group of antioxidant compounds. Phenolic compounds are able to control enzymatic activity and different clusters of herbal phenolic such as polyphenols are able to attach to proteins and this attachment causes the further attachment to cellular transporters and receptors. Flavonoids are a common cluster of phenolic compounds that can synthesize from phenyl alanine amino acid.
From the above explanation, we expected that ethanolic or ethyl acetate extracts with higher level of total phenolic content would exhibit higher antioxidant activities. Although many studies exhibit that there are a positive relation between total phenolic content and their antioxidant activities (16), our research demonstrates that there is no positive relation between total phenolic content of S. striata extracts and its antioxidant activity. The S. striata aqueous, methanolic and ethanolic extracts had higher antioxidant activity than ethyl acetate extract. The result showed a higher flavonoid content of ethyl acetate extract than of others, and that this amount is the same in ethanolic and methanolic extracts. It was expected that ethyl acetate extract would have the higher antioxidant activity than that of other extracts but surprisingly the aqueous extract has the higher antioxidant activity. Since the natural antioxidant include many different compounds such as phenol, nitrous compounds, caroteonids and many unidentified compounds (17), compounds other than the phenolic compounds may be responsible for antioxidant activity in water, methanolic or ethanolic extract or sometimes the structure of flavonoids in ethyl acetate extract may decrease its antioxidant activity. Rice-Evans et al (18) showed that hydroxyl groups, the amount of conjugation and its kind, are two important factors in antioxidant potential of phenolic compounds. Stronger antioxidants usually are more conjugated and have more hydroxyl groups that make the antioxidant strong enough to scavenge the free radicals. Further study is needed for demonstration of this issue.
S. striata is traditionally used for treatment of infectious diseases. The use of S. striata in treatment of infectious diseases has important role in reducing of chemical's side effect and breaking the antimicrobial resistance. The result of this study has been shown that the antimicrobial activity of S. striata is depended on the type of microorganism. The result of antimicrobial activity of S. striata ethanolic extract against S. aureus is coincidental to other studies (4). P. aeruginosa is resistant gram negative bacteria to many antibiotics, so its sensitivity to methanolic and ethanolic extract of S. striata is an important issue for finding the effective fraction against this resistant bacterium. Ethanolic extract from aerial parts of S. striata have higher antimicrobial activity than that of aqueous extract, although its flavonoid content is lower than methanolic or ethyl acetate extract. However, its phenolic content is higher than that of others in that it exhibits the positive relation between the total phenolic content and its antimicrobial activity. Further study is recommended against clinical isolate of sensitive bacteria and deep investigation on flavonoid and phenolic compounds of S. striata and identifying the antioxidant portion in aqueous extract.