Radical scavenging and antioxidant activities of methanolic extracts from Hypericum species growing in Bulgaria

Thirteen Hypericum species growing in Bulgaria were investigated for free radical-scavenging activity, antioxidant activity, total tannins and total flavonoids contents. Methanolic extracts from the Hypericum species were analyzed for radical scavenging and antioxidant activities using DPPH-, ABTS- free radicals, total antioxidant activity and inhibition of lipid peroxidation by ferric thiocyanate (FTC) method. Butylated hydroxytoluene and ascorbic acid were used as positive controls. Methanolic extracts from H. cerastoides , H. perforatum and H. maculatum demonstrate the highest antioxidant activities and are potential sources of natural antioxidant compounds. The quantification of tannins and flavanoids were determined in Hypericum species using Folin-Chiocalteu reagent and AlCl3, respectively. The amounts of the tannins ranged from 1.30 ± 0.01 mg/100 g dw in H. elegans to 8.67 ± 0.02 g/100 g dw in H. perforatum . The highest concentration of flavonoids was found in H. cerastoides (1.22 ± 0.02 g/100g dw), and the lowest amount was established in H. olympicum (0.20 ± 0.03 g/100g dw).


INTRODUCTION
Free radical production and lipid peroxidation are involved in the pathogenesis of some chronic diseases, including atherosclerosis, cardiac and cerebral ischemia, neurodegenerative disorders, carcinogenesis, diabetes, and rheumatic disorders. [1][2][3][4] In recent years, there has been an increased interest in phenolic compounds derived for their possible health benefits. The anticarcinogenic, antimutagenic, and cardioprotective effects of phenolic compounds are reported to be generally associated with their antioxidant properties of eliminating free radicals and alleviating lipid peroxidation. [5] The genus Hypericum L. is a large group of herbs or shrubs consisting of approximately 450 species in 36 sections. [6] Hypericum species have been known for their antidepressant, analgesic, spasmolytic, antiviral and wound healing effects for many years. [7,8] Phytochemical investigations have shown the presence of naphthodianthrones hypericin and pseudohypericin, [9] tannins, flavonoids, xanthones, benzophenones. [10][11][12] Numerous substances have been suggested to act as antioxidants. Various phenolic antioxidants such as flavonoids, tannins, coumarins and more recently procyanidins have been shown to scavenge radicals in a dose dependent manner and therefore are viewed as promising therapeutic drugs for free radical pathologies. [13] Flavonoids and tannins are the major plant compounds with antioxidant activity. [14] The aim of the current study is to evaluate the antioxidant property of methanolic extracts from Hypericum species, including scavenging activities against DPPH and ABTS radicals, total antioxidant activity and inhibition of lipid peroxidation. Moreover, the total flavonoids and tannins were quantified.

Plant material and extraction
Aerial parts of Hypericum species used for this study were collected during the flowering season from wild habitats. Voucher specimens from all plants samples were deposited in the Herbarium of the Institute of Botany-Bulgarian Academy of Sciences (SOM) [ Table 1].
Air-dried aerial parts were sequentially extracted with CH 2 Cl 2 and then with MeOH. The MeOH extracts (0.01 mg dw/ml) were assayed to determine antioxidant activities.

Quantification of tannins
The quantification of tannins in the aerial parts was performed according to the European Pharmacopoeia [15] involving Folin-Chiocalteu reagent and pyrogallol as standard. The analyses were carried out at 760 nm. All determinations were performed in triplicate (n = 3).

Quantification of flavonoids
The content of the flavonoids in the aerial parts was established spectrophotometrically at 430 nm by creating a complex with AlCl 3 according to the European Pharmacopoeia. [16] The content of flavonoids was calculated as hyperoside. The measurements were carried out using a Shimadzu UV-1203 spectrophotometer (Japan). All determinations were performed in triplicate (n = 3).

Determination of antioxidant activity DPPH radical-scavenging activity
Scavenging activity of Hypericum extracts against DPPH radical was assessed according to the method of Blois [17] with some modifications. Briefly, 1 ml of Hypericum extracts (0.01 mg dw/ml) was mixed with 4 ml of 0.005 mg/ml DPPH methanol solution. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured at 517 nm. Ascorbic acid and BHT were used as references. The ability to scavenge DPPH radical was calculated by the following equation: DPPH radical scavenging activity (%)= Abs control − Abs sample Abs control 100 , where Abs control is the absorbance of DPPH radical in methanol; Abs sample is the absorbance of DPPH radical solution mixed with sample extract /standard. All determinations were performed in triplicate (n = 3).

ABTS radical scavenging assay
For ABTS assay, the procedure followed the method of Arnao et al. [18] with some modifications. The stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 14 h at room temperature in the dark. The solution was then diluted by mixing 1 ml ABTS solution with 60 ml methanol to obtain an absorbance of 0.706 ± 0.01 units at 734 nm using a spectrophotometer. Fresh ABTS solution was prepared for each assay. Plant extracts (1 ml) were allowed to react with 1 ml of the ABTS solution and the absorbance was taken at 734 nm after 7 min using a spectrophotometer. The ABTS scavenging capacity of the extract was compared with that of BHT and ascorbic acid and percentage inhibition calculated as ABTS radical scavenging activity Abs control − Abs sample Abs control 100 where Abs control is the absorbance of ABTS radical in methanol; Abs sample is the absorbance of ABTS radical solution mixed with sample extract/standard. All determinations were performed in triplicate (n = 3).

Total antioxidant activity (ferric reducing antioxidant power, FRAP)
The FRAP assay was done according to the method of Benzie and Strain [19] with some modifications. The stock

Determination of antioxidant activity in linoleic acid system by the FTC method
The antioxidant activity of Hypericum extracts against lipid peroxidation was measured through ammonium thiocyanate assay, as described by Takao et al., [20] with some modifications. The reaction solution, containing 0.

Tannins and flavonoids content
The amount of tannins, measured by Folin-Ciocalteu method, was expressed as pyrogallol equivalent (PE) and ranged from 1.30 to 8.67 g/100 g dry weight (dw) [ Table 2]. The total flavonoids content in the extracts was expressed as g hyperoside equivalent (HE) and varied from 0.20 to 1.22 g/100 g dw. The highest level of tannins was found in H. perforatum (8.67 ± 0.02 g PE/100 g dw), followed by H. maculatum 7.06 ± 0.01 g PE/100 g dw). H. elegans has the lowest content of tannins (1.30 ± 0.01 g PE/100 g dw) and significantly low level of flavonoids (0.43 ± 0.03 g HE/100 g dw). H. cerastoides demonstrated the highest amount of flavonoids (1.22 ± 0.02 g HE/100 g dw) and moderate quantity of tannins (5.75 ± 0.01 g PE/100 g dw). Other species with high content of flavonoids were H. tetrapterum (1.13 ± 0.02 g HE/100 g dw) and H. perforatum (1.12 ± 0.01 g QE/100 g dw). The least quantity of flavanoids was found in H. olympicum (0.20 ± 0.03 g QE/100 g dw).

DPPH, ABTS radical-scavenging and total antioxidant activity
The radical scavenging activity of Hypericum extracts (0.01 mg dw/ml) was compared with those of BHT and ascorbic acid at the same concentration and expressed as % of inhibition against DPPH and ABTS, respectively [   Although, H. olympicum has low content of tannins and flavonoids, the species showed high total antioxidant activity probably due to the presence of other compounds. This fact unambiguous proves that antioxidant potential is affected by many factors. In the present study, the antioxidant activity of Hypericum extracts was determined by peroxidation of linoleic acid using the ferric thiocyanate method (FTC) [ Figure 1]. During linoleic acid peroxidation, peroxides were formed and these compounds oxidized Fe 2+ to Fe 3+ . The Fe 3+ ion formed a complex with SCN -, which had a maximum absorbance at 500 nm. [19] Thus, a high absorbance value was an indication of high peroxide formation during the emulsion incubation. As shown in Figure 1, the absorbance of the control at 500 nm increased to a maximal value of 2 after 96 h, while H. barbatum was the only Hypericum sample, the absorption of which increased to 2. The highest significant diminution was demonstrated by H. cerastoides (0.7) followed by H. richeri (0.75) and H. elegans (0.78). However, the antioxidant activity of these Hypericum species was slightly less effective than that of BHT, a widely used commercial antioxidant. These results indicate that polar extracts from Hypericum species can significantly inhibit the peroxidation of linoleic acid and reduce the formation of hydroperoxide, thus implying that these plants are powerful natural antioxidants.

CONCLUSION
The obtained results revealed that all tested Hypericum species exhibited radical scavenging. Furthermore, antioxidant activities of methanolic extracts from H. cerastoides, H. perforatum and H. maculatum were found to be the most potent. These species could be evaluated as rich sources of antioxidants.