Anthocyanin are known as large group of red-blue natural pigments giving different color in plants and their products.1 They belong to flavonoids, a large group of phenolic compounds, divided to aglycons and glycosylated form.1 More than 400 anthocyanins have been recognized in nature. Anthocyanin play different role in plants such as antioxidants, phytoalexins or as antibacterial agents.2 Nowadays anthocyanin consider as health benefit compounds.3 They shows different pharmacological properties comprise improving cognitive decline and neural dysfunction4 reducing capillary permeability and fragility, inhibitory effect on NO· production and effective in inflammatory diseases which are associated with NO·.2
Evidence of many researchers suggests that anthocyanin poses confirmed potential potency in cancer prevention and decrease risk of human breast cancer, human melanoma cancer and human ovarian cancer.1 Anti-cancer properties of these compounds related to several mechanisms, they are powerful free radial scavenger and have antioxidant effects because of phenolic structure. They are anti-proliferator agents towards several cancer cell lines and can be induced apoptosis. Moreover, they able to inhibit angiogenesis and invasiveness in cancer cells.5
Berberis sp. Fruits are rich sources of many bioactive compounds such as anthocyanin.6 Berberis integerrima belongs to Berberidaceae. Synthesis of different secondary metabolites in this plant make it as therapeutic agent in different disease such as gastrointestinal diseases, bleeding, sore throat, fever, malaria and hepatitis.7
The aim of this study was to separation of anthoyanin fraction of Berberis integerrima Bunge fruits (AFBI), evaluation of anthocyanin content in this fraction and antioxidant activity assessment of this fraction by different methods: DPPH, ABTS radial scavenging assay, inhibition of lipid peroxidation and nitric oxide.
MATERIALS AND METHODS
Berberis integerrima Bunge fruits were manually harvested during August and September 2104, from Kohmar (Fars Province, Iran), and were identified by Ms. S. Khademian in the museum of medicinal plants, Shiraz University of Medical Sciences, Shiraz, Iran (voucher no. P. M. 396).
325 g of freeze-dried fruits powder was percolated with ethanol for 48h and the extract concentrated in rotary evaporator under vacuum followed by speed vacuum to get 140 g gummy material.
30 g of the crude extract was then suspended in 0.3% TFA then filtered and shake well with ethyl acetate in decanter. The aqueous phase was collected, this extraction repeated three times. This phase was loaded an amberlite column (2.5×45 cm), then rinsed with distilled water containing 0.3% TFA to remove polysaccharides .The anthocyanin were eluted with 0.3% TFA in methanol then concentrated by rotary evaporator. Yield of extraction and anthocyanin fractionation were 20% and 2.57%, respectively.
Anthocyanin content measurement
The pH-differential method was used to determine the anthocyanin content in the fraction.8
0.4 ml of sample was mixed with 3.6 mL of corresponding buffers. Buffers were 0.4 M sodium acetate solution and 0.025 M potassium chloride solution adjusted to pH 4.5 and 1.0 with HCl, respectively. The absorbance of each dilution was measured at 519 nm and distilled water used as blank. Anthocyanin content was calculated by the following equation:
DPPH free radical scavenging activity
Fresh DPPH radical solution (100 mM) was mixed with different concentrations anthocyanin fraction. The reaction mixture was incubated at room temperature for 30 min in the dark. The DPPH radical inhibition was measured at 490 nm by using a microplate reader. All measurements were performed in triplicates. The antioxidant activity was calculated as follows:
“A”: the absorbance
Control: contains DPPH without sample; blank: methanol.
Nitric Oxide radical assay
Nitric oxide (NO) radical scavenging method was performed with slight modification.9 Sodium nitroprusside in phosphate buffer (0.2 M, pH 7.4) was mixed with each sample and incubated (27 °C for 150 min). After incubation, 100 μL of o Griess reagent was added to each sample and then incubated 5 min at room temperature. Finally, absorbance was measured at 542 nm.
Nitric oxide radical scavenging was determined as follow:
Blank: Absorbance of sample without any reagent
Control: Absorbance of control without sample
The base of this assay is the ability of different substances to scavenge 2,2’ azinobis (3-ethylbenzthi-azoline-6-acid) (ABTS•+) radical. Colorful ABTS radical change into colorless neutral form. These radicals were freshly prepared by adding ammonium persulfate (2.45 Mm) solution to ABTS solution (7 Mm) and kept in dark for 16 h. This ABTS•+ solution was diluted with ethanol to gain an absorbance of 0.70 ± 0.02 at 734 nm. 200 μl ABTS•+ Solution and 20 μl of tested samples (6.25-3200 μg/mL) and the absorbance was recorded at 734 nm. The antioxidant activity of was calculated by the following equation:11
FRAP (Ferric-Reducing Antioxidant Power) assay
In the presence of antioxidants, ferric and tripyridyltriazine (TPTZ) reduce and change its color complex. The FRAP reagent included TPTZ solution (10 mM) in HCl (40 mM), FeCl3 (20 mM) and acetate buffer (0.3 M , pH 3.6). The fresh mixture was prepared and incubated at 37 °C.
Then 20 μL of each sample and 180 μL of FRAP reagent were mixed in a 96-well microplate reader then the absorbance of the reaction mixture was measured at 593 nm after incubation at 37 °C for 10 min. Results reported as IC50.9
The thiobarbituric acid (TBA) assay was carried out by the method of Rael et al. with slight modifications 9. Initially mixture prepared in a screw-cap vial contain 4.0 mg of a sample in 4 mL of 99.5% ethanol, 4.1 mL of 2.5% linoleic acid in 99.5% ethanol, 8.0 mL of 0.02 M phosphate buffer (pH 7.0) and 3.9 mL of water then this mixture was placed at 40 °C in the dark. To 0.1 mL of this mixture, 9.7 mL of 75% (v/v) ethanol and 0.1 mL of 30% ammonium thiocyanate was added. Then 0.1 mL of 0.02 M ferrous chloride in 3.5% hydrochloric acid was added to the reaction mixture, the absorbance was measured at 500 nm. The absorbance was measured at this wavelength every 24 h until the day after that the absorbance of the control reached its maximum value.
Percentage inhibition was calculated by the following expression:
Percentage of inhibition = [(Ao- A1) / Ao] × 100
where Ao is absorbance of control and A1 is absorbance of sample is absorbance of sample.
RESULT AND DISCUSSION
Results of phytochemical studies of Berberis species showed that various parts of these plants used for different purpose. Berberis sp. fruits have promising nutraceutical compounds with potential health advantages.12 The former studies demonstrated anthocyanins are effective biological agents in wide range of disease such as cancer, neuralage-related disease.13
In this study, anthocyanin of AFBI measurement by the pH-differential method showed that anthocyanin content of fruits before use of amberlite column as a stationary phase is 14.36 ± 0.33 mg/g and anthocyanin content is 34.51 ± 0.42 mg/g after separation of fraction by column.
These results showed the effective anthocyanin separation using amberlite. it seems that the value of anthocyanin content of our study is comparable to previous reported including anthocyanin content of orange Nasturtium flowers (Tropaeolum majus) was 72 ± 10 mg/100 g,14 fresh Rubus glaucus cv Hull (75 mg/100 g), a hybrid of strawberries (Fragaria anannassa ) fruit is 71.8 mg/100 g.15 Anthocyanin content can be affected by seasonal variations.16 This variation in anthocyanin contents may be due to stability of anthocyanin related to multifactorial such as temperature, pH, presence of phenols and metals complex and structures.17
In DPPH assay radical react with hydrogen donor antioxidant and DPPH color convert from purple to yellow. Antioxidant activity related to change of color and measure by reduction of DPPH maximum absorption.18-19 The IC50 of this assay reduce to 66 ± 0.04 μg/mL after loading on column while IC50 of Quercetin is 26.51 ± 0.06 μg/mL (Table 1).
FRAP assay engaged to assessment radical scavenging of AFBI. Reaction of TPTZ-Fe (III) complex by antioxidant leads to form TPTZ-Fe (II) complex. The absorbance of this complex at 593 nm shows the reducing power of samples.14 The reduction of IC50 value after loading on column show reducing power of sample increased by using this type of column.
The results of ABTS assay have the same trend as DPPH and FRAP assays and antioxidant activity increased by loading on column (Table 1) In nitric oxide radical scavenging assay, nitric oxide radicals (NO•) were produced by sodium nitroprusside, capable to interacts with oxygen and produce nitrite ions (NO-).16 The results of Nitric oxide scavenging ability of 200 μg/mL concentration of AFBI was 97.04 ± 0.69 after using amberlit column for anthocyanin purification, AFBI exhibited high nitric oxide scavenging ability, as reactive nitrogen species play critical role in cardiovascular disease, antioxidant be effective in prophylaxis of cardiovascular diseases.20 According to Table.1, decreased IC50 of DPPH and FRAP assay and increased nitric oxide scavenging ability in loaded sample exhibit the efficiency of amberlite column to purify phenolic compound as mentioned in previous study.21 Furthermore, strong antioxidant activity of this fraction due to present of anthocyanin.22 Previous study revealed there is a linear relationship between the values of the antioxidant activity and the anthocyanin quantity in blackberries, red raspberries, black raspberries and strawberries.23 Anthocyanins able to scavenge free radicals by donate of phenolic hydrogen atoms.24 Besides hydrogen donation, other mechanisms engaged by anthocyanin are metal chelation, and protein binding.25 Anthocyanins as powerful antioxidant prevent important biological macromolecule against oxidative stress. In vivo study confirm that dietary consumption of blueberry Black raspberry anthocyanin protect blood cells from free radicals and oxidative stress significantly.26,27 So, it reveals the critical role of anthocyanin against reactive oxygen species cellular events such as cellular aging, mutagenesis, carcinogenesis, and coronary heart disease which may be mediated by destabilization of membranes, DNA damage and oxidation of low-density lipoprotein (LDL).28
The protective effect of AFBI on lipid peroxidation was determined by the ferric thiocyanate method (FTC). peroxides is a product of linoleic acid peroxidation, which able to oxidized Fe2+ to Fe3+.The complex of Fe3+ ion with SCN had a maximum absorbance at 500 nm.20
As shown in figure 1. The percentages of inhibition are about 39% in first day and 95% after 10 days. The inhibition percentage of lipid peroxidation was observed 66.863% for BHT and 64.06% for Vit C in the tenth day. According to these, it seems that AFBI is more effective than BHT and Vit C to protect lipid from peroxidation.
Lipid peroxidation assay exhibited the strong protective effect of AFBI on lipid peroxidation after ten days and there is significant difference with BHT and Vit C and AFBI.
Lipid peroxidation is results of free-radicals attack and oxidation of polyunsaturated fatty acids (PUFAs) which participate in membrane composition.20-29 It is assumed that lipid oxidative damages related to change genes involved in developmental processes such as differentiation and aging and different disease.30 So the ability of AFBI to prevent lipid peroxidation is promising to modify age related complications.
Our research shows the high antioxidant activity of Berberis integerrima Bunge fruits. The high level of anthocyanins content suggested that these fruits considered as appropriate source of functional antioxidant and improve human health. Further studies help to reveal more biological effects.
Berberis integerrima belongs to Berberidaceae comprise various secondary metabolites which candidate it as therapeutic herb in different disease. This study provides isolation of anthocyanin fraction of Berberis integerrima Bunge fruit by column chromatography. Anthocyanin content was measured by PH-differential method and antioxidant activities of this fraction quantified by various methods (DPPH, FRAP, ABTS) before and after loading on column as well as lipid peroxidation assay. Determination of antioxidant activities using different methods is worthwhile to realize the mechanism of antioxidant activities.