تاثير حلال هاي مختلف بر ميزان تركيبات فنلي و فعاليت آنتي اكسيداني ميوه عناب (Ziziphus jujube Miller)

Effect of Different Solvents on Total Phenolic Contents and Antioxidant Activity of Zizyphus jujube Miller Fruits

اين تحقيق به منظور بررسي اثر تيمار حلال هاي متانول، اتانول، استون (۵۰، ۹۰ و ۱۰۰ درصد) و آب مقطر بر روي ميزان تركيبات آنتي اكسيداني (فنل كل، تانن ها، فلاونوئيدها، آنتوسيانين ها) ميوه عناب (Ziziphus jujube Miller)، انجام شد. فعاليت آنتي اكسيداني هر يك از عصاره ها نيز به روش هاي DPPH و FRAP مورد ارزيابي قرار گرفتند. نتايج نشان داد كه استون ۵۰ درصد در استخراج تركيبات فنل و فلاونوئيد كل به ترتيب با مقادير (۷۹۸/۲۰ ميلي گرم گاليك اسيد در ۱۰۰ گرم ماده خشك) و (۱۸۷۱/۷۳ ميلي گرم كوئرستين در ۱۰۰ گرم ماده خشك)، بهترين عملكرد را در ميان ساير حلال ها داشته است. استخراج آنتوسيانين، با متانول ۵۰ درصد (۶/۳۱ ميلي گرم سيانيدين ۳- گلوكوزيد در ۱۰۰ گرم ماده خشك) و استخراج تانن با متانول ۱۰۰ درصد (۶۶۹/۴ ميلي گرم كاتكين در ۱۰۰ گرم ماده خشك)، داراي بالاترين بازده استخراج بوده است. در تمامي موارد آب از حداقل قابليت استخراج متابوليت هاي ثانويه در بين ساير حلال ها برخوردار بود. در بررسي خاصيت آنتي اكسيداني ميوه عناب به دو روش DPPH و FRAP مشخص گرديد كه خاصيت آنتي اكسيداني آن ها مستقيما تحت تاثير مقادير تركيبات فنلي قرار گرفته است. در ميان حلال هاي مورد استفاده، استون ۵۰ درصد بهترين بازده را در روش DPPH (۲۹/۰۲=IC۵۰) و روش FRAP (۵/۱۱ ميلي مول آهن II در ۱۰۰ گرم ماده خشك) نسبت به ساير حلال ها داشت. بر اساس نتايج مي توان گفت درصد مهار كنندگي DPPH و FRAP با كاهش غلظت حلال هاي متانول و استون رابطه مستقيم دارد. نتايج اين تحقيق نشان داد كه تفاوت در نوع، خلوص و قطبيت حلال ها و تفاوت در جزئيات روش استخراج عصاره (مانند زمان استخراج، سرعت اختلاط، نسبت ميزان پودر به حلال و اندازه ذرات پودر) بر ميزان استخراج متابوليت هاي ثانويه تاثير مي گذارند.

Introduction: Phenolic compounds have an ability to scavenge free radicals and cause the balance of reactive oxygen species (ROS) in our body. This balance prevents atherosclerosis, coronary heart and cancer diseases. Butylated hydroxyl toluene (BHT) is a well-known synthetic antioxidant, which is restricted to be used due to its probable toxic effects. Therefore, replacement of synthetic antioxidants with plant materials with high amounts of antioxidant activity, which protect the body from free radicals and many diseases caused by lipid peroxidation, is an appropriate option. Ziziphus jujuba Miller is one of the forty species belonging to Rhamnaceae family, which produces a great deal of industrial raw materials for horticultural, ornamental, food, and pharmaceutical industries. Antioxidants can be extracted by various solvents and extraction methods. Solvent extraction is the most common method used for separating natural antioxidants. Solvent properties undoubtedly play a key role in the extraction of antioxidative compounds. The type and yield of antioxidant extracted have been found to vary as affected by the solvent properties such as polarity, viscosity and vapor pressure. Therefore, it is difficult to develop a unified standard method for the extraction of antioxidants from all plant materials. Materials and Methods: Plant materials Fresh fruits were collected from Birjand, Iran, in late summer 2014. The samples were air dried under the shade at room temperature. Dried fruits were ground by using a mortar and pestle and were separately extracted by distilled water and organic solvents such as methanol, ethanol and acetone (50%, 90% and100% (v/v)). After filtering through the Whatman paper #3 and removing the solvents (using a rotary evaporator (BUCHI V-850)) and water (using a freeze dryer, (OPERON, FDB-5503, Korea)), the dried extracts were stored in refrigerator for further analysis. Determination of Total Phenolic Content (TPC) Samples were measured for TPCs colorimetrically using the Folin-Ciocalteu method with modification. Absorbance was read at 725 nm against blank using UV-Visible spectrophotometer (Cecil. UK.). A calibration curve was prepared using a standard solution of Gallic acid (0.2- 1mg/ml). Results were expressed as mg Gallic acid/g dry extract (mg GA/g DE). Determination of Total Flavonoid Content (TFC). TFC was determined using the method of Huang et al. (13) with minor modifications. Absorption was measured at 430 nm using UV-VIS spectrophotometer (Cecil. UK.). TFC was determined using a standard curve with quercetin as the standard, and expressed as mg of quercetin equivalents (CE)/g dry extract (mg QE/g DE). Determination of Total Anthocyanin Content (TAC). TAC was measured using a spectrophotometric differential pH method. Its absorbance was read at 510 and 700nm. Results were expressed as milligrams of cyanidin-3-glucoside (CY.) equivalents per g of dry extract. Determination of Total Tannin Content. For determination of tannins in the sample extracts, vanillin–HCl method was used. The absorbance was read at 500 nm using UV vis hydrogen donating ability or radical scavenging activity. DPPH assay expressed as IC50 and percentage inhibition. Lower IC50 value indicates higher antioxidant activity. Results and Discussion: Efficiency of different solvent extractions depends on the matrix of plant materials as well as the type of extractable compounds. The correct selection of solvent can improve the extraction yield of antioxidants from plants matrices considerably. For this reason, in the present study, some selected types of solvent showed different results. For extraction of total phenol and flavonoid compound, acetone 50% was the best yield. In methanolic extract (50, 100%), the highest amounts of anthocyanin and total tannin were reported. In all extracts, water had the least efficiency in comparison with other solvents. High correlation was observed in total phenolic content and antioxidant activity which was determined by DPPH and FRAP assay. Acetone 50% was the most potent for scavenging free radicals and reducing a ferric-tripyridyltriazine, Fe (III)-TPTZ, complex to ferrous, Fe (II) in all extracts. Conclusions: The results of the present study indicated that polarity, selectivity, viscosity, and vapor pressure are important physicochemical properties that should be considered when selecting a suitable solvent for the extraction of bioactive compounds from plant materials.