Food Chemistry 117 (2009) 681–686

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Food Chemistry

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In vitro and in vivo antioxidant activity of Pinus koraiensis seed extractcontaining phenolic compounds

Xiao-Yu Su a, Zhen-Yu Wang a,*, Jia-Ren Liu b

a School of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, PR Chinab Harvard Medical School, 300 Longwood AVE, Boston 02115-5737, United States

a r t i c l e i n f o

Article history:Received 11 September 2008Received in revised form 20 April 2009Accepted 21 April 2009

Keywords:Pinus koraiensis seedAntioxidant activityD-GalactoseRadiationPhenolic compounds

0308-8146/$ - see front matter � 2009 Published bydoi:10.1016/j.foodchem.2009.04.076

* Corresponding author. Address: School of Food SciInstitute of Technology, 202 HaiHe Road, NanGangChina. Tel./fax: +86 45186282909.

E-mail address: tougao08212007@yahoo.cn (Z.-Y.

a b s t r a c t

A polyphenolics extract from Pinus koraiensis seed (PKS) has been investigated for its in vivo and in vitroantioxidant activity. The total phenolic content of PKS extract was 264 mg of gallic acid equivalents/g drymaterial. The extract was found to show remarkable scavenging activity on 2,2-diphenyl-picrylhydrazyl(DPPH) (EC50 0.023 ± 0.004 mg/ml), OH� (EC50 0.391 ± 0.055 mg/ml), O�2 (EC50 4.37 ± 0.19 mg/ml), as wellas a high and dose-dependent reducing power. The extract also showed strong suppressive effect on ratliver lipid peroxidation and erythrocyte haemolysis. Ageing and radiation models were used to evaluatethe antioxidant effect in vivo. PKS extract highly inhibited oxidative damage induced by D-galactose andc-ray. The results have indicated that intragastric administration of the extract can increase levels ofsuperoxide dismutase (SOD) and glutathione (GSH), and decrease malondialdehyde (MDA) content.

� 2009 Published by Elsevier Ltd.

1. Introduction

It has been suggested that natural antioxidants are more safeand healthy than synthetic antioxidants which used in food (Mah-fuz, Omer, Ibrahim, & Nuri, 2007; Velioglu, Mazza, Gao, & Oomah,1998). Pinus koraiensis was one of the most important sources usedfor timber and traditional medicinal plant (Yang, Ding, Sun, &Zhang, 2005). It has been reported that the Pinus plants have effecton releasing fatigue, anti-ageing and anti-inflammatory (Watana-be, Momose, & Handa, 1995). There are nutritional compounds inPinus seeds (Isidorov, Vinogorova, & Rafaowski, 2003). P. koraiensisis distributed in Changbai Mountain, Northeast China. P. koraiensisseeds are a kind of nutritional and delicious food. The seed wasappreciated for abundant nutrition and excellent production asan edible plant seed. In preliminary study, we confirmed that theputamina of P. koraiensis seed (abbreviated as PKS) was rich in phe-nolic compounds. The antioxidant ability of phenolic compoundshas been widely accepted by the scientific community and ex-pected to prevent relative disease (Chewa, Lima, Omara, & Khoob,2008). The antioxidant capacity of PKS extract can be explored tohealth food which prevents ageing and rancidity. The purpose ofthe present study was to evaluate the in vivo and in vitro antioxi-dant activity of PKS extract.

Elsevier Ltd.

ence and Engineering, HarbinDistrict, Harbin 150090, PR

Wang).

Phenolic compounds are widely distributed as secondarymetabolites of medicinal plants, as well as in some edible plants(Hagerman et al., 1998; Soong & Barlow, 2004). Several studieshave shown medicinal plants and foods to have good antioxidantproperties and high phenolic compound content. Phenolic com-pounds were found to have effect on oxidation damage (Ribeiro,Barbosa, Queiroz, Knodler, & Schieber, 2008), for instance applepolyphenols has been confirmed to do resistance on immune in-jury and cancerisation induced by radiation.

An antioxidant can prevent organism from oxidative damage inmany ways (Goli, Barzegar, & Sahari, 2005), in order to evaluate theantioxidant systematically, we did different assays to determinethe true antioxidant potential of PKS extract.

2. Materials and methods

2.1. Chemicals and reagents

The superoxide dismutase (SOD), malondialdehyde (MDA), glu-tathione (GSH) and glutathione peroxidase (GSH-px) commercialkits were purchased from Jiancheng Institute of Biotechnology,China. D-Galactose was purchased from Amresco Inc., USA. The D-galactose was dissolved in physiological saline (0.9% saline and dis-tilled water) at concentration of 10 mg/ml. 2,2-Diphenyl-pic-rylhydrazyl (DPPH), ascorbic acid, tris (hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetic acid diso-dium salt (EDTA-2Na) were purchased from Sigma Chemical Co.,USA. Folin–Ciocalteu phenol reagent, gallic acid, 30% hydrogen

682 X.-Y. Su et al. / Food Chemistry 117 (2009) 681–686

peroxide, sodium salicylate, pyrogallol, 2-thiobarbituric acid andother chemicals in the studies were of highest quality commer-cially available from local suppliers (Shanghai, China).

2.2. Sample extraction

The P. koraiensis seeds were collected from the Yichun ForestryStore (Heilongjiang Province, China). The voucher specimen wasidentified by Dr. Zhenyu Wang, College of Food Science and Engi-neering, Harbin Institute of Technology, China. The dried putaminaof PKS (1500 g) was grinded by pulveriser and extracted with pro-portional ethanol (40%) by ultrasonic apparatus (KX-1740QT), atultrasonic procedure of 80 �C, 200 W and 2 h. The extract was cen-trifuged at 3000 rpm for 5 min and the residue was extracted onceagain with new solvent under the same conditions. The superna-tant was evaporated and the extract powder was obtained byfreeze drying. The PKS extract powder (50 g) was stored at 4 �C un-til required. Before the assays, the extract was dissolved at re-quired concentration.

2.3. Determination of total phenolic content

Total phenolic content was performed according to the Folin–Ciocalteau method (Slinkard & Singleton, 1977) with some modifi-cations. Briefly, 0.1 ml of sample (0–5 mg/ml), 1.9 ml distilledwater and 1.0 ml of Folin–Ciocalteau’s reagent were seeded in atube, and then 1.0 ml of 100 g/l Na2CO3 was added. The reactionmixture was incubated at 25 �C for 2 h and the absorbance of themixture was read at 765 nm. The sample was tested in triplicateand a calibration curve with six data points for gallic acid was ob-tained. The results were compared to a gallic acid calibration curveand the total phenolic content of PKS extract was expressed as mgof gallic acid equivalents per gram of extract.

2.4. OH� scavenging assay

The scavenging capacity of PKS extract on OH� was evaluatedaccording to the reaction of sodium salicylate and residual hydroxylradical. OH� scavenging assay was performed according to a litera-ture procedure (Wang, Gao, Zhou, Cai, & Yao, 2008) with a few mod-ifications. Hydroxyl radicals were generated by Fenton reaction inthe system of FeSO4 and H2O2. The reaction mixture was consistedof 0.5 ml FeSO4 (8 mM), 0.8 ml H2O2 (6 mM), 0.5 ml distilled water,1.0 ml of various concentrations PKS extract and 0.2 ml sodiumsalicylate (20 mM). The total mixture (3.0 ml) was incubated at37 �C for 1 h and then the absorbance of the mixture was recordedat 562 nm. Ascorbic acid was used as the positive control. The scav-enging activity was calculated using the following Eq. (1):

Scavenging ðor inhibitionÞ rate ð%Þ ¼ ½1� ðA1 � A2Þ=A0� � 100

where A0 is the absorbance of the control (without extract), A1 is theabsorbance of the extract addition and A2 is the absorbance withoutsodium salicylate. Matlab 7.0 analysis software was applied to ana-lyse the regression of concentration and scavenging rate.

2.5. DPPH radical scavenging activity assay

The capacity of PKS extract to reduce the 2,2-diphenyl-pic-rylhydrazyl (DPPH) radical was assessed using the method in a lit-erature (Blois, 2002). Two millilitres of a solution of DPPH inethanol (8.62 � 10�2 mM) were mixed with 2 ml of various con-centrations extract during 30 min at room temperature and theabsorbance was recorded at 517 nm (Hsu, Coupar, & Ng, 2006).The normal purple colour of DPPH will turn into yellow when itssingle electron is paired with a hydrogen atom coming from a po-tential antioxidant. Ascorbic acid was used as the positive control.

In this method, the percentage of DPPH scavenging activity wascalculated according to absorbance difference, as has been de-picted in Eq. (1), where A0 is the absorbance of DPPH alone, A1 isthe absorbance of DPPH + extract and A2 is the absorbance of theextract only.

2.6. O�2 scavenging assay

In brief, 0.2 ml of different concentrations extract was added to5.7 ml of 50 mM Tris–HCl buffer (pH 8.2). The mixture was incu-bated at 25 �C for 10 min and 0.1 ml of 6 mM pyrogallol (25 �C)was added. The absorbance of the reaction mixture was measuredat 320 nm every 30 s until the reaction proceeded to 5 min (thesame concentration extract was used as the blank to eliminateinterference). O�2 scavenging activity was expressed by the oxida-tion degree of a test group in comparison to that of the control.The percentage of inhibition effect was calculated according toEq. (1), where A0 is the absorbance of the Tris–HCl buffer withpyrogallol, A1 is the absorbance of the extract addition and A2

was the absorbance of extract blank.

2.7. Inhibition of lipid peroxidation in rat liver homogenate

The inhibition effect of PKS extract on lipid peroxidation wasdetermined according to the thiobarbituric acid method. FeCl2–H2O2 was used to induce the liver homogenate peroxidation (Yen& Hsieh, 1998). In this method, 0.2 ml of PKS extract (0–10 mg/ml) was mixed with 1.0 ml of 1% liver homogenate (each 100 mlhomogenate solution contains 1.0 g rat liver), then 50 ll of FeCl2

(0.5 mM) and H2O2 (0.5 mM) was added. The mixture was incu-bated at 37 �C for 60 min, then 1.0 ml of trichloroacetic acid(15%) and thiobarbituric acid (0.67%) was added and the mixturewas heated up in boiled water for 15 min. The absorbance was re-corded at 532 nm (Buege & Aust, 1978). Ascorbic acid was used asthe positive control. The percentage of inhibition effect was calcu-lated according to Eq. (1), where A0 is the absorbance of the control(without extract), A1 is the absorbance of the extract addition andA2 is the absorbance without liver homogenate.

2.8. Inhibition of erythrocyte haemolysis in rat blood

The blood sample was obtained from mice eyepit and made to0.5% erythrocyte suspension for the assay. The reaction mixturewas consisted of 1.0 ml of erythrocyte suspension (0.5%), 1.0 ml ex-tract (0.02–0.2 mg/ml) and 0.1 ml H2O2 (100 mM). The mixturewas incubated at 37 �C for 60 min, then four times volume distilledwater was added to the mixture and centrifuged at 1000 rpm for10 min. The absorbance of the supernatant was read at 415 nm.The percentage of erythrocyte haemolysis inhibition effect was cal-culated according to Eq. (1), where A0 is the absorbance of thesupernatant without extract, A1 is the absorbance of the extractaddition and A2 was the absorbance of extract solution.

2.9. Reducing ability assay

The reducing ability of PKS extract was assayed as described in areference (Tsai, Huang, & Mau, 2006). Various concentrations ex-tract (0–1.0 mg/ml) was added to 2.5 ml of phosphate buffer(200 mM, pH 6.6) and 2.5 ml of potassium ferrocyanate[K3Fe(CN)6] (1%). After incubation at 50 �C for 20 min, 2.5 ml of tri-chloroacetic acid (10%) was added to the mixture before centrifu-gation at 1000 rpm for 10 min. The supernatant (5 ml) wasgathered and mixed with 5 ml of distilled water and 1 ml of ferricchloride (0.1%). The absorbance was measured at 700 nm. Thereducing ability was weighed by the absorbance value of the reac-tion solution and a higher absorbance indicated increased reducing

X.-Y. Su et al. / Food Chemistry 117 (2009) 681–686 683

power (Jayaprakasha & Bhimanagouda, 2007). Ascorbic acid wasused as positive control.

2.10. Ageing model

Kunming male mice, weighing about 18–22 g were obtainedfrom the Experimental Animal Center of Heilongjiang Tumour Hos-pital. Mice were kept in our animal house at an ambient tempera-ture of 25 �C and 45–55% relative humidity with a 12 h each of darkand light cycle. Animals had free access to food and water. After1 week of acclimatisation, the mice were randomly divided intofive groups (12 mice per group) and i.p. injected with 0.3 ml of D-galactose (150 mg/kg) once daily for 60 days. Simultaneously, theageing control group (group AC) mice were p.o. administered with0.3 ml of physiological saline each; the PKS treatment group (groupPKS) mice were p.o. administered with different dose of extract(250, 500 and 1000 mg/kg). The normal control group (group NC)mice were i.p. injected and p.o. administered with 0.3 ml of phys-iological saline.

2.11. Radiation model

c-Ray was used to induce radiation oxidative damage (radiationdose is 5 grey, dose-rate 0.95 grey/min; radiation time 6.15 min,radiation radius 145 cm). The mice were divided into five groups(12 mice per group). The PKS groups were p.o. administered with250, 500 and 1000 mg/kg of extract once a day. The radiation con-trol (group RC) and normal group (group NC) were p.o. adminis-tered with 0.3 ml of physiological saline. After 21 days, the micewere irradiated by c-ray according to foregoing dose expect thenormal group. The determination was carried out at the fifth dayafter c-ray irradiation.

2.12. Measurement of superoxide dismutase (SOD) activity andmalondialdehyde (MDA) level

Mice were sacrificed by dislocating; the brains and livers wereremoved rapidly and homogenised in physiological saline (ice-bath). Brain and liver were used for ageing model and radiationmodel assays, respectively. The homogenate was centrifuged at4000 rpm (4 �C) for 15 min and the supernatant was gathered forassays. Test of total SOD activity was carried out by using commer-cial kits. SOD activity was expressed as units per microgram ofbrain or liver protein. MDA content was determined by thiobarbi-turic acid reaction (TBAR) method and expressed as nanomoles permilligram of brain or liver protein.

2.13. Determination of glutathione peroxidase (GSH-px) activity andglutathione (GSH) content

Mice were sacrificed by dislocating; the brains and livers wereremoved rapidly and homogenised in cold physiological saline(ice-bath). Brain and liver were used for ageing model and radia-tion model assays, respectively. GSH content and GSH-px activitywere measured by commercial kits.

2.14. Protein assay

Protein concentration was measured by Bradford method (Lu,Li, Huang, & Zhang, 2007). Bovine serum albumin was used asthe standard.

2.15. Statistical analysis

Data was expressed as mean ± SD. The Duncan test and a one-way analysis of variance were used for multiple comparisons by

analysis of SPSS 14.0. The results were considered statistically sig-nificant if the P-values were 0.05 or less.

3. Results and discussion

3.1. Total phenolic content

Proportional ethanol (40%, v/v) was used for extraction and theextract yield was 3.5 g/100 g. This solvent system destroys thecell membranes, simultaneously dissolving the phenolic com-pounds. Phenolic content of PKS extract was measured by Fo-lin–Ciocalteau method and the total phenolic content in theextract was 264 ± 10.52 mg/g. The Folin–Ciocalteau method issimple and can be used in characterising botanical samples, forexample sorghum (Awika, Rooney, & Waniska, 2004), soybean(Takahashi et al., 2005), white and black sesame seed (Shahidi,Liyana-Pathirana, & Wall, 2006). The result showed PKS extractcontained high phenolic content compared with some otherplants, such as black soybean seed (Astadi, Astuti, Santoso, & Nug-raheni, 2009), bayberry (Zhou et al., 2009) and Vietnam bitter tea(Thuong, Nguyen, Ngo, et al., 2009). PKS was a nice potentialantioxidant plant.

3.2. OH� scavenging activity

As the positive control, ascorbic acid showed an excellent scav-enging activity on OH�, the half inhibition concentration(EC50) = 0.103 ± 0.031 mg/ml. PKS extract was also observed tohave strong scavenging activity, with an EC50 value of0.391 ± 0.055 mg/ml (Fig. 1a).

3.3. DPPH radical scavenging activity

Fig. 1b showed the scavenging capacity of PKS extract on DPPH(ascorbic acid as the positive control). A dose–response relation-ship is found in DPPH scavenging activity of PKS extract and anincrease in concentration is synonymous of an increase in scav-enging capacity. Ascorbic acid showed high scavenging activity(EC50 = 0.002 ± 0.0001 mg/ml). The PKS extract also had strongDPPH scavenging effect, with an EC50 value of 0.023 ± 0.004 mg/ml. Among the levels used in the experiment, 0.09 mg/ml varietywas the strongest one with a scavenging rate of 91.64%, ascorbicacid as standard was 96.46% at the same concentration. At0.09 mg/ml, the scavenging ability of PKS extract on DPPH wasnot significantly different from ascorbic acid. The strong scaveng-ing capacity of PKS extract on DPPH was possibly due to the phe-nolic compounds which could act as a hydrogen donorantioxidant.

3.4. O�2 inhibition activity

Spontaneity oxidation of pyrogallol will proceed in slightlyalkaline condition and the O�2 was produced with absorption at320 nm. Antioxidant can inhibit the spontaneity oxidation of pyro-gallol but it has been found that the reaction is instable in 1–3 min.Repetitious tests showed the reaction was stable in 4–5 min andconsequently the data at 5 min was adopted to evaluate the inhibi-tion activity. Matlab 7.0 analysis software was applied to analysethe regression of concentration and inhibition rate. PKS extractwas found to have inhibition activity and the EC50 was4.37 ± 0.19 mg/ml (Fig. 2a), as the positive control, EC50 of ascorbicacid was 0.069 ± 0.013 mg/ml. The result showed PKS extract had acertain extent inhibition on O�2 but was not as strong as ascorbicacid. PKS extract effectively inhibited O�2 at concentrations of 4–8 mg/ml. The inhibition rate was above 80% at 8 mg/ml.

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Fig. 1. (a) The OH� scavenging activity of PKS extract. (b) The DPPH radical scavenging activity of PKS extract.

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Fig. 2. (a) The O�2 inhibition activity of PKS extract. (b) The inhibition of PKS extract for erythrocyte haemolysis in rat blood.

684 X.-Y. Su et al. / Food Chemistry 117 (2009) 681–686

3.5. Inhibition of lipid peroxidation in rat liver homogenate

Lipid peroxidation was an oxidative deterioration process ofpolyunsaturated fatty acids which induced by radical. Lipid struc-ture of cell velum was damaged by radical and accordingly lipidperoxidation of viscera homogenate was increased. MDA contentcan indicate the degree of lipid peroxidation. FeCl2–H2O2 systemwas used to induce lipid peroxidation in rat liver homogenate. Inthe assay, PKS extract was confirmed to have strong inhibitionfor lipid peroxidation. 88.47% of the peroxidation was inhibitedby PKS extract at concentration of 4 mg/ml (Fig. 3a). At 7 mg/ml,PKS extract showed an inhibition rate of 90.21%. Ascorbic acid, asthe positive control, at concentrations of 4 and 7 mg/ml, 51.93%and 83.13% of the peroxidation was inhibited, respectively. SoPKS extract has highly strong resistance on peroxidation. The phe-nolic compounds in PKS extract can provide phenolic hydroxyl toaccept electrons and scavenge OH� which induced by FeCl2–H2O2.

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Fig. 3. (a) The lipid peroxidation inhibition activity of PKS

The results are considered to be noteworthy when compared tothe findings of other studies concerning medicinal plants (Sultana,Anwar, & Przybylski, 2007; Tepe, Sokmen, Akpulat, Yumrutas, &Sokmen, 2006).

3.6. Inhibition of erythrocyte haemolysis in rat blood

Oxidant damage of cell film which induced by H2O2 can result inerythrocyte haemolysis. The result showed erythrocyte haemolysiswas effectively inhibited by PKS extract at concentrations of 0.02–0.2 mg/ml. At concentration of 0.04 mg/ml, the inhibition rate wasnearly 50%. At concentrations of 0.06, 0.08 and 0.2 mg/ml, the inhi-bition rate was 55.3%, 57.9% and 59%, respectively. Inhibition rateof ascorbic acid (the positive control) was 60% at 0.04 mg/ml. At0.06, 0.08 and 0.2 mg/ml, the inhibition rate was 65.1%, 69.4%and 75.8%, respectively (Fig. 2b). In this assay, PKS extract wasfound to have inhibition activity for erythrocyte haemolysis; this

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extract. (b) The reducing ability assay of PKS extract.

Table 1Effect of PKS extract on SOD, GSH-px activity and MDA, GSH content for ageing mice brain.

Group SOD (U/mg protein) GSH-px (U/mg protein) MDA (nmol/mg protein) GSH (mg/g protein)

NC 391 ± 32.8 688 ± 47.3 2.88 ± 0.77 3.85 ± 0.84AC 316 ± 21.9b 540 ± 43.7b 3.64 ± 0.61a 3.09 ± 0.60a

PKS (250 mg/kg) 321 ± 36.0 550 ± 34.6 3.40 ± 0.54 3.35 ± 0.55PKS (500 mg/kg) 345 ± 15.9c 559 ± 39.7 2.98 ± 0.48c 3.71 ± 0.58c

PKS (1000 mg/kg) 374 ± 36.3d 561 ± 30.0 2.87 ± 0.53c 3.86 ± 0.54c

N = 12.a P < 0.05 vs. NC group.b P < 0.01 vs. NC group.c P < 0.05 vs. AC group.d P < 0.01 vs. AC group.

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effect can prevent cell films from lipid oxidation and protecterythrocyte.

3.7. Reducing ability assay

The reducing power of PKS extract was presented in Fig. 3b. Asdescribed in Fig. 3b, PKS extract was found to have strong and in-creased reducing ability at concentrations of 0.12–0.2 mg/ml. Inthis assay, the yellow colour of the test solution changes to variousshades of green and blue (Barros, Baptista, & Ferreira, 2007). Thepresence of reducers causes the conversion of the Fe3+/ferricyanidecomplex to the ferrous form. The reducing power of PKS extract in-creased with a higher concentration. All spectrophotometric mea-surements were repeated twice with at least three replicates. Atconcentrations of 0.02, 0.04 and 0.08 mg/ml, the reducing powerof PKS extract was around 0.17, 0.23 and 0.38, respectively, whilea solution of ascorbic acid at the same concentration, the positivecontrol used in this test, had a reducing power value of 0.358,0.712 and 1.713, respectively.

3.8. Antioxidative effects for ageing mice

Subacute mice ageing were induced by injection of D-galactose.The biological redox substance in mice can be disturbed by long-term injection of D-galactose, such as decreasing of GSH level,SOD and GSH-px activity. The antioxidant effect of PKS extractfor ageing mice was presented in Table 1. The results indicated thatPKS extract could strongly inhibit mice ageing. Mice were p.o.administered with PKS extract and the SOD activity was enhanced9% and 18% at dose of 500 and 1000 mg/kg, respectively. The MDAcontent was decreased 18% and 21% at 500 and 1000 mg/kg,respectively, the GSH content was also effectively increased at doseof 500 and 1000 mg/kg. At dose of 1000 mg/kg, the PKS extractshowed the greatest antioxidant effect for ageing mice. The resultsalso indicated that PKS extract might be used as natural antioxi-dants and alternatives to synthetic anti-ageing reagent. The potentanti-ageing effect of PKS extract in vivo maybe owe to its antioxi-dative activity. The antioxidant principle was to reduce organism

Table 2Effect of PKS extract on SOD, GSH-px activity and MDA, GSH content for radiation mice li

Group SOD (U/mg protein) GSH-px (U/mg pr

NC 501 ± 38.6 890 ± 48.2RC 417 ± 22.2b 740 ± 43.7b

PKS (250 mg/kg) 423 ± 36.5 750 ± 34.6PKS (500 mg/kg) 434 ± 23.4 759 ± 39.7PKS (1000 mg/kg) 475 ± 34.4d 761 ± 27.6

N = 12.a P < 0.05 vs. NC group.b P < 0.01 vs. NC group.c P < 0.05 vs. RC group.d P < 0.01 vs. RC group.

harm and prevent radical formation and histiocytic period wasprolonged.

3.9. Antioxidative effects for radiation mice

Radiation can result in oxidant damage and disturb the balanceof enzyme activity in mice livers. The important enzyme activityand substance content related to radiation injury were detectedin this assay. The results of radiation model test were showed inTable 2. It was found that PKS extract could potently inhibit oxi-dant damage induced by c-ray. PKS extract was ingested by p.o.administration and the radiation damage on mice was highly de-creased. The results showed ingestion of PKS extract in vivo couldincrease SOD and GSH level of radiation mice to normal level.Ingestion of PKS extract can maintain the balance of enzyme actionand release the radiation damage. At dose of 1000 mg/kg, PKS ex-tract can remarkably enhance SOD activity. Also the MDA contentwas highly decreased at the same dose (decreasing of 11% and 16%,respectively). At dose of 500 and 1000 mg/kg, the GSH content inmice liver was much higher than the RC group.

PKS (homologous from Pinus plant) was reported to have manybioactivities, such as anti-cancer, antioxidant, anti-virus and anti-bacterial. Free radicals, oxidant-related enzyme and lipid peroxida-tion are important factors which highly impact oxidation andageing. In this study, antioxidant properties of PKS extract (40%ethanol) were demonstrated by various assay systems. The resultssuggested that PKS extract had potent antioxidant activities inmultiple mechanisms. Especially, the scavenging activity on OH�

was very strong and nearly as effective as ascorbic acid (Fig. 1a).It was worth mentioning that inhibition effect of PKS extract for li-pid peroxidation was even higher than ascorbic acid (Fig. 3a). Invivo assays, the PKS extract was found to increase the levels of anti-oxidant enzymes (SOD, GSH) and decrease MDA content in brainand liver. It was confirmed that PKS extract could protect the tis-sues against oxidative damage. Enhancing of SOD activity andGSH content in mice viscera indicated the in vivo antioxidant activ-ity of PKS extract and the related mechanism. The present studycan be considered a forward step in this direction.

ver.

otein) MDA (nmol/mg protein) GSH (mg/g protein)

3.98 ± 0.48 4.42 ± 0.654.62 ± 0.48a 3.26 ± 0.78b

4.40 ± 0.63 3.44 ± 0.514.08 ± 0.42 3.83 ± 0.56d

3.86 ± 0.52c 4.20 ± 0.49d

686 X.-Y. Su et al. / Food Chemistry 117 (2009) 681–686

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