Reactive oxygen species(ROS), kinds of active chemical materials which were generated by the biological oxidation, includes O₂^-, H₂O₂, ·OH and so on(Sies, 1986). With the process of oxidation in vivo, ROS will result in the damage to all kinds of biological macromolecules, such as protein, fat, and DNA(David et al., 1997; Shukla et al., 2009). Furthermore, this damage is also likely to contribute to the decline and death of cells, coronary heart diseases, atheromas, cancers, diabetes, arthritis, neurological disorders etc(Germanas et al., 2007; Devasagayam et al., 2004; Finkel et al., 2000).

Many synthetic antioxidants such as butylated hydroxyanisole(BHA) and butylated hydroxytoluene(BHT), which have been proved to possess potential health risk to human beings(Botterweck et al., 2000), are hoped to be replaced by some natural antioxidants. Recent studies suggest that blueberry and apple extracts could prolong the life of fruit flies(Peng et al., 2012; 2011), and daily consumption of blueberry juice would improve elderly people’s memory function(Krikorian et al., 2010). All of these functions result from the great antioxidant capacity of natural products. Furthermore, plants’ substances with great antioxidant capacities and health benefits have been extracted from persimmon skin, orange peel, tea, grape seeds and skins, even some of which are of great interest in the industry after they are put into practice(Wolfe et al., 2003; Bocco et al., 1998; Tang et al., 2002; Yusuf et al., 2004).

Medicine mulberry(Morus nigra)which is rarely seen in nature is the only black mulberry in China. Medicine mulberry is regarded as folk medicine by Uygur people, ethanol extracts of whose fruits have been drinking to keep fit for a long time, and its fruits are often used as anti-pharyngitis drugs. However, reports on the antioxidant activity of extracts from each part of it are still unknown. Therefore, this investigation is going to explore the antioxidant activity of the extracts of Medicine mulberry’s fruits, branches, leaves, and roots, in terms of total polyphenols content, total flavonoids content, free radical scavenging capacity and Fe²⁺-chelating activity, aiming to find a new natural green food antioxidant and lay a foundation for the future reasonable development of Medicine mulberry.

Ascorbic acid(Vc), gallic acid, rutin, 1,1-diphenyl-2-picrylhydrazyl(DPPH) and lecithin were purchased from Sigma(St. Louis, Mo, USA); potassium ferricyanide, ferrozine, thiobarbituric acid(TBA), phosphoric acid, ethanol, sodium hydroxide, trichloroacetic acid(TDA), sodium carbonate were purchased from Kelong(Chengdu, Sichuan, China); iMark microplate reader(Japan), FA2004A electronic balance(Shanghai, China), B.U Chi R-210 rotavapor(Flawil, Switzerl and ).

The samples of Medicine mulberry(Morus nigra)were collected from Xinjiang Hetian Sericulture Science Institute in November 2012. The leaves, branches and roots were dried at 50 ℃ and the fruits stored in -80 ℃ refrigerator.

Each sample(20 g)was powdered at low temperature and extracted with 200 mL 75%(V/V)ethanol, for 2 h, 3 times, and the filtrate was evaporated to dryness until the weight would never change. The dried extracts of each sample were named as EEML, EEMB, EEMF and EEMR(ethanolic extracts of mulberry leaves, branches, fruits and roots, respectively). The volume was adjusted with 75%(V/V)ethanol to 500 mL. Percentage yields of EEML, EEMB, EEMF and EEMR of dry weight were 20.16%, 7.7%, 86.76% and 17.65%(w/w), respectively.

Total polyphenols content was estimated by using the Folin-Ciocalteu colorimetric method described previously but with a slight modification(Bae et al., 2007). 0.1 mL extract was mixed with 0.1 mL Folin-Ciocalteu reagent(50%, V/V), shaken vigorously, and after 5 min reaction, 3 mL Na₂CO₃(0.2 g·mL^-1)was added into each mixture. The reaction mixture was incubated at 20 ℃ for 2 h. The absorbance was determined at 765 nm. Results were compared with a gallic acid calibration curve and expressed as milligram of gallic acid equivalent per one mL of extracts.

The total flavonoids content assay was carried out by the previously described way with a few modifications and used rutin as a st and ard flavonoid(Hairi et al., 1991). 0.5 mL(50 mg·mL^-1)NaNO₂ was added into 0.1 mL extract and shaken vigorously, leaving the mixture at room temperature for 6 min, and then, 0.5 mL Al(NO₃)₃ was added into reaction mixture. After 6 min st and ing, 2.5 mL NaOH was mixed together with mixture and left at room temperature for 15 min. The absorbance was determined at 510 nm. Results were compared with a rutin calibration curve and expressed as milligram of rutin equivalent per one mL of extracts.

The reducing power assay was in accordance with the previous reference(Yen et al., 1995). 80 μL extracts were added to 1 mL phosphate buffer(pH6.6), 1 mL potassium ferricyanide(10 mg·mL^-1)was mixed with the buffer and incubated at 50 ℃ for 20 min before having a ice-water bath. Soon afterwards, 1 mL trichloroacetic acid(100 mg·mL^-1) and 1 mL ferric chloride(1 mg·mL^-1)were added to the mixture in turn, shaken vigorously. The absorbance was determined at 700 nm after 10 min incubation and compared with an ascorbic acid calibration curve.

The assay was carried out following the previously described approach with a few modifications(Chu et al., 2000). Different volumes of extracts(20, 50, 80, 100, 200, 300, 400 μL)were reacted with 250 μL DPPH respectively, and then the total volume was adjusted with 70%(V/V)ethanol to 4 mL and shaken vigorously. After incubated at 25 ℃ for 10 min, the absorbance was determined with a Microplate reader at 517 nm. A lower level of absorbance indicated a stronger scavenging activity. The scavenging ability was calculated according to following formula: Scavenging effect(%)=[1-(A_s/A_c)] × 100. Where A_c is the absorbance of the control groups and A_s is the absorbance in the presence of extracts.

According to the previous reference with a few modifications(Jeong et al., 2009), 1.5 mL FeSO₄(1.5 mmol·L^-1) and 1 mL H₂O₂(6 mmol·L^-1)were mixed together, then water bath in 37 ℃. After 30 min, hydroxyl radical was generated from Fenton reaction. 0.5 mL salicylic acid(20 mmol·L^-1)as well as various volume extracts were added into the mixture and the total volume was adjusted with 70%(V/V)ethanol to 4 mL, allowed to react for 1 h at 37 ℃. The absorbance was determined at 562 nm. A lower level of absorbance indicated a stronger scavenging ability.

The percentage of hydroxyl radical was calculated by using the formula given below: Scavenging effect(%)=[1-(A_s/A_c)] × 100. Where A_c is the absorbance of the control and A_s is the absorbance in the presence of extracts or other scavenger.

According to a literature procedure, the assay was measured but with a few modifications(Chang et al., 2011). Fe²⁺-chelating ability of extracts was monitored by the absorbance of the ferrous iron-ferrozine complex at 562 nm. Different volumes of extracts(100-700 μL)were added into 1 mL FeCl₂ (0.2 mmol·L^-1). The reaction was initiated by the addition of 0.5 mmol·L^-1 ferrozine(0.1 mL)dissolved in filled water, then the total volume was adjusted with 70%(V/V)ethanol to 4 mL. The absorbance of the Fe²⁺-ferrozine complex was measured at 562 nm against a blank group. A lower level of absorbance indicated a stronger chelating activity.

According to the reference described previously with a few modifications(Tamura et al., 1991), lecithin(300 mg)was dissolved in 30 mL phosphate buffer(10 mmol·L^-1, pH7.4)by using an ultrasonic cleaner, which was carried out in ice-water. Different volumes of extracts(0.2-1.2 mL)were mixed with 1 mL sonicated solution(10 mg·mL^-1), 1 mL FeCl₃ (0.4 mmol·L^-1) and 1 mL ascorbic acid(0.4 mmol·L^-1), then the total volume was adjusted with 70%(V/V)ethanol to 4.5 mL and shaken vigorously. The mixture was added into 2 mL TCA-TBA-HCl after incubated at 37 ℃ for 60 min in a dark place, the centrifuge tubes were put into boiling water for 15 min, which was then in ice-cold water bath for 5 min and centrifuged at 5 000 r·min^-1 for 5 min. The absorbance of supernatant was measured at 532 nm. The blank group contained all reagents except lecithin. A lower level of absorbance indicated a stronger protective activity.

Inhibiting lipid oxidation capacity of each sample was calculated via the formula: Inhibition(%)=[1-(A_s/A_c)] × 100. Where A_c is the absorbance of the control and A_s is the absorbance in the presence of extracts.

Each experiment was carried out in triplicate. The data were analyzed by SPSS(version 18.0) and expressed as meanst and ard deviation(SD). Results were considered significantly at P < 0.05.

According to the regression equations of gallic y=1.212 3x + 0.033 5(R²=0.999 9), the concentration of the total polyphenols was calculated. As is shown in Fig. 1, EEMR has the highest concentration of polyphenols, reaching1.643 4 mg·mL^-1, and EEML contains the lowest polyphenols, only 0.247 9 mg·mL^-1. In other words, each gram of dry extract contains 30.74 mg(EEML), 222.32 mg(EEMB), 232.84 mg(EEMR) and 27.20 mg(EEMF)of polyphenols separately.

	Fig.1 Total polyphenols content and total flavonoids content of each sample

On the basis of the regression equations of rutin y=0.419 5x-0.009 3(R² =0.999 7), the concentration of the flavonoids content were calculated. Just as the content of total polyphenols, EEMR also has highest content of flavonoids. Fig. 1 shows EEMR contains the highest total flavonoids content, reaching 0.801 7 mg·mL^-1. The total flavonoids content in these extracts are in the order of EEMR>EEMB>EEMF>EEML. The high content of polyphenols and flavonoids is far more than those in other tested mulberry(Chang et al., 2011). It has been widely recognized that due to the free radical scavenging capacity and strong reducing power of polyphenols in vivo(Birt et al., 2001), it has the function of inhibiting atherosclerosis and tumor cell proliferation(Cheung et al., 2003). Therefore, the main reason why the mulberry branches and roots have been regarded as an herbal medicine is the abundant polyphenols in them. Besides, the difference of polyphenols content and flavonoids content indicated that the four samples of polyphenols not only exist as flavonoids.

There are linear regressions and a significant relationship between total flavonoids and reducing power in all extracts. It can be obviously found in Tab.1 that EEMF has strongest reducing power, which was about 1.17-fold, 2.82-fold, 7.37-fold, greater than that of EEMR, EEMB and EEML respectively. Except EEMF, the polyphenols and flavonoids content of EEMR, EEMB and EEML were significantly correlated with reducing power, the linear correlation coefficient of total polyphenols and reducing power is r=0.998 0, and the total flavonoids content correlated extraordinarily well(Fig. 2). Furthermore, as is shown in Tab.2, the reducing power of different concentration of EEMF, EEMR, EEMB and EEML were noticeably correlated with total flavonoids. In many vitro studies, polyphenols and flavonoids compounds were demonstrated the main material of antioxidant activities(Chew et al., 2009; Socha et al., 2009; Proestos et al., 2006). Our data are consistent with the previous studies.

	Fig.2 Linear regressions of reducing power and flavonoids concentration

Tab. 1 IC₅₀ in DPPH radical, ·OH radical(mg·mL^-1), maximum inhibition rate of lipid oxidation(%) and reducing ability^①

Tab.1IC50 in DPPH radical, ·OH radical(mg·mL-1), maximum inhibition rate of lipid oxidation(%) and reducing ability① Samples DPPH ·OH radical Lipid Reducing ability* EEMF 0.108±0.009c 0.561±0.016c 71.138±2.333a 2.144±0.115a EEMR 0.140±0.016c 1.841±0.077b 37.777±4.126b 1.839±0.020b EEMB 0.337±0.017b 3.004±0.136a 33.746±5.328bc 0.759±0.036c EEML 1.400±0.050a 1.760±0.104b 30.975±2.199c 0.291±0.018d Vc 0.017±0.003d 0.026±0.044d — — ①Column wise values with same letter indicate no significant difference (P>0.05). Results are displayed with means Standard deviations for n=3. * Expressed as ascorbic acid equivalent.

Tab. 2 The linear regressions of different concentration flavonoids and reducing power

Tab.2The linear regressions of different concentration flavonoids and reducing power Samples Linear regressions EEML Y=0.002 7 x + 0.021 0(R2=0.998 9) EEMB Y=0.005 9 x + 0.042 0(R2=0.999 2) EEMR Y=0.012 2 x + 0.139 1(R2=0.998 8) EEMF Y=0.017 1 x + 0.055 8(R2=0.998 9)

The capability of scavenging radical is regarded as one of the regular indexes of evaluating the ability of antioxidant activity. Among the ROS, hydroxyl radical is the most reactive and induces severe damage to bio-molecules(Sakanaka et al., 2005). Because of the hydroxyl radical’s high reactivity which enables itself to react with a wide range of molecules found in living cells such as amino acids, sugars, lipids and nucleotides, hydroxyl radical scavenging is an important antioxidant activity(Stohs et al., 1995). From the Fig. 3, it can be easily found EEMF has the strongest capacity of scavenging ·OH radical, which may as a result of the high content of anthocyanins, which had the functions of excellently resisting oxidation, getting rid of hydroxyl radicals(Kang et al., 2006).

	Fig.3 Scavenging hydroxyl radical activity of the samples

Besides, the four extracts have similar ability of scavenging radical. As is shown in Fig. 4, four extracts all possess the ability of scavenging DPPH radical. Although EEMR, EEMB and EEML have the similar trend in the reaction system, EEMF has the strongest scavenging capacity obviously, it can be demonstrated IC₅₀ in the Tab.1.

	Fig.4 Scavenging DPPH effects of the samples

Fig. 5 implies that the abilities of inhabiting lipid oxidation of the four extracts are remarkably different. With the increasing concentration, the capacities of inhabiting lipid oxidation were on the increasing trend, and EEMR and EEMF were stronger than EEML and EEMB at the same concentration. Moreover, anti-lipid oxidation capacity of EEMF was proportional to the concentration, and EEMR exhibited the strongest capability by 37.78%, while EEMF was 71.14%, revealing that EEMF is the most effective in preventing lipid oxidation in vitro. Lipid oxidation is a harmful process occurring in a cell’s physiological metabolism. Induced by ROS, lipid oxidation will lead to cellular damage and promote the pathological progression of carcinogenesis, atherosclerosis and diabetes(Wang et al., 2007). Medicine mulberry fruit wine is seen as high-end nutritional nourishment by Uygur. In the experiment, EEMF’s ability to inhibit the oxidation of lipid is stronger than that of the others, which will contribute to the cell membrane antioxidant, aging prevention and pigmentation.

	Fig.5 Liposome protection of the samples

Compared with the control group, it was easily found all of the extracts have Fe²⁺-chelating activity from Fig. 6. Obviously, EEMR and EEMB have chelating activities, meanwhile EEMF and EEML even have stronger chelating activities than theirs. Although as the essential metal element for the body, iron contains an unpaired electron, so that they are able to participate in the single-electron transfer reactions, which is harmful to human body(Lloyd et al., 1997). It can be figured out from this experiment that despite the same chelating Fe²⁺ function of the four extracts, chelating ability of EEMF is much stronger. So EEMF can replace deferoxamine which is the only iron chelator in clinical used for the treatment of iron overload disease.

	Fig.6 Fe2+-chelating activity of the samples

Although the content of total flavonoids in EEMF is lower than that in EEMR, EEMB and EEML, EEMF’s reducing power is more powerful than that of others. According to some reports, per 100 grams of fruits extract liquid have the content of 418 mg of V_C and 6.2 g of reducing sugar(Maimaiti et al., 2007). Apart from that, it was found that Medicine mulberry fruits possess higher content of antioxidants, such as β-carotene, V_B and V_E(Maimaiti et al., 2002). Besides, anthocyanins content in Medicine mulberry fruits reached 0.02%(w/w)(Jiang, 2010). The main antioxidant physiological function of anthocyanins is scavenging free radicals, studies have shown that the antioxidant capacity of anthocyanins is 20 times higher than the V_E, 50 times higher than V_C, and even stronger than that of rutin(Jiang, 2010; Tsuda et al., 2003; Wang et al., 2000). What's more, anthocyanins could protect cells from damage, and prevent lung cancer cell proliferation(Chen et al., 2006).

A large number of Medicine mulberry(Morus nigra)are planted in Xinjiang to fix s and s. However, because of the heavy drug smell of their leaves, the leaves can not be used to feed silkworms. A great quantity of branches were cut down every year, but they only to use as firewood, this is not only a waste lots of resources, but also produces environmental pollution. From this investigation, Medicine mulberry could be developed into a kind of antioxidant additives which are new and green.