Food Chemistry 114 (2009) 1321–1327

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Food Chemistry

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In vitro antioxidant analysis and characterisation of antler velvet extract

Ran Zhou, Shufen Li *

Key Laboratory for Green Chemical Technology of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

a r t i c l e i n f o

Article history:Received 15 May 2008Received in revised form 4 November 2008Accepted 5 November 2008

Keywords:Antler velvetChemical compositionSupercritical fluid extractionCo-solventAntioxidant activity

0308-8146/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.foodchem.2008.11.010

* Corresponding author. Tel./fax: +86 22 27402720E-mail address: shfli@tju.edu.cn (S. Li).

a b s t r a c t

The chemical composition and antioxidant properties of antler velvet extract prepared by supercriticalCO2 extraction with co-solvent are presented in this study. The composition in different extracts wasdetermined by radioimmunoassay analysis (RIA), high performance liquid chromatography (HPLC) andthin layer chromatographic (TLC), separately. The antioxidant properties including hydroxyl radical-scav-enging by phenanthroline-Fe(II) oxidation, inhibition of lipid peroxidation from lipoprotein induced byiron and the ability of the extract to protect 2-deoxy-D-ribose against hydroxyl radical-mediated degra-dation were assessed. The extract mainly contained three biological bases, two sex hormones, five phos-pholipids and p-hydroxybenzaldehyde, which may contribute greatly to antioxidant activity. The antlervelvet extracts demonstrated activity in the three antioxidant assays, and did so in a concentration-dependent fashion. The supercritical extract technology showed obvious advantage, and the inhibitoryactivity of SFE extract was obviously higher than that of traditional refluxing extracts.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, there has been an increasing awareness of thebenefits of functional foods and the interest in the discovery of nat-ural antioxidants has risen exponentially. Several compounds,which may be attributed to the high antioxidant activity of extractsfrom many plants, have been identified, including some phenoliccompounds, carotenoids, phycobiliproteins, chlorophyll and somechlorophyll degradation products (Bhat & Madyastha, 2001; Jaimeet al., 2005; Mendiola et al., 2005; Pinero Estrada, Bermejo Bescos,& Villar del Fresno, 2001). Many reports about phytochemistry andantioxidant properties of herbs, spices and plant-derived medicinalpreparations have been published (Hinneburg, Dorman, & Hiltun-en, 2006; Kumar, Ganesan, & Bhaskar, 2008; Tadhani, Patel, & Sub-hash, 2007; Yi, Yu, Liang, & Zeng, 2008).

Antler velvets are bony cranial appendages that develop on topof permanent frontal protuberances (the pedicles) in male deer(and females in the reindeer, Rangifer tarandus), and undergo peri-odic replacement (Kim et al., 2005). Antler velvet, as a kind of tra-ditional Chinese medicine for over two thousand years, has beenconsidered as one of the most effective and powerful invigorantsfor strengthening the kidney, sexual-reinforcing, prolonging lifeand anti-aging (Kim et al., 2005; Sunwoo, Nakano, & Sim, 1997;Takikawa, Kokuba, Kajihara, Dohi, & Tahara, 1997; Wang, Zhao, &Qi, et al., 1988; Wang et al., 1988a; Wang et al., 1988b). Traditionalmedical reports and clinical observations convincingly show thatantler velvet contains many active components which may be

ll rights reserved.

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the reasons of anti-aging and curing various diseases (Bhavnani,2000; Ditchkoff, Spicer, Masters, & Lochmiller, 2001; Gómez, Gar-cía, Landete-Castillejos, & Gallego, 2006; Li, Jiang, Jiang, & Fang,2001; Li et al., 2007; Sunwoo, Nakano, & Sim, 1998). Among thesecomponents, estradiol, progesterone, uracil, hypoxanthine, uridineand p-hydroxybenzaldehyde were reported to have the inhibitoryeffect on monoamine oxidase (MAO) (Chen, Wang, Zhang, & Zhang,1992; Wang & Chen, 1989; Wang et al., 1988a; Wang et al., 1988b;Yang, 1995; Zhang, Liu, Chen, & Bai, 1992). Moreover, phospholip-ids as the important ingredient in antler velvet are connected withthe metabolism of life. All of them can obviously increase the activ-ity of superoxide dismutase (SOD) in vivo, inhibit the activity ofmonoamine oxidase B (MAO-B) and reduce the content of lipofus-cin (LF) in organism (Chen, Wang, & Wu, 1990). They have the bio-logical effects of decreasing blood lipid, promoting the recovery offatty liver and delaying serum and lipid peroxidation. All of abovestudies are strong evidences of the anti-aging influence of deervelvet.

For the important properties of these active components in ant-ler velvet, each of them is responsible for not only antler growthand development, but also biomedical and nutraceutical uses ofantlers. However, little information is available on chemical andantioxidant properties of antler products due to the incompleteunderstanding of their applications and pharmacological proper-ties. To make antler products acceptable as nutraceuticals andfunctional foods, the chemical and antioxidant properties of antlervelvet have to be thoroughly investigated.

The advantages of supercritical fluid extraction (compared withliquid extraction) are that it is relatively rapid because of the lowviscosities and high diffusivities associated with supercritical

1322 R. Zhou, S. Li / Food Chemistry 114 (2009) 1321–1327

fluids. The extraction can be selective to some extent by controllingthe density of the medium and the extracted material is easilyrecovered by simply depressurizing, allowing the supercriticalfluid to return to gas phase and evaporate leaving no or little sol-vent residues.

But to our knowledge, there are still no reports about the chem-ical components and anti-oxidation activity of antler velvet extractextracted by supercritical fluid. Thus, the objective of this workwas to study the chemistry and antioxidant properties of antlervelvet active components extracted by supercritical fluid.

2. Materials and methods

2.1. Materials

Antler velvet (Cervus Nippon Temminck var. mantchurieus Sain-hoe) was provided by The Xiuzheng Pharmaceutical Group (Jilin,China). It was sectioned at a thickness of 2–3 mm, and then waspowdered into the size of 160–180 mesh after freeze-drying(Christ Alpha1-2, Marin Christ, Germany).

SFE grade carbon dioxide stored in a cylinder with a dip tubewith a purity of 99.9% was purchased from Liu Fang Gas Co.(Tianjin, China); The standard samples, including estradiol, pro-gesterone, uracil, hypoxanthine, uridine and p-hydroxybenzalde-hyde, all with purity greater than 99.0% were obtained fromSigma Chemical Co. (USA). Sphingomyelin (SPH) with purity of98.0% was acquired from Sigma Chemical Co. (USA). Phosphati-dylcholine (PC), phosphatidylethanolamine (PE), lysophosphati-dylcholine (LPC) and lysophosphatidylethanolamine (LPE) allwere bought from Fluka Co. (Germany). All the other agentswere of analytical grade (99%). The ethanol solvent used as co-solvent was filtered through a 0.45 lm nylon membrane priorto utilization.

2.2. Extraction procedures

Supercritical fluid extraction was performed on a speed SFEinstrument (Applied Separations Inc., Allenton, PA, USA) equippedwith a HPLC pump. Four grams of antler velvet powder or its mix-ture with co-solvent are weighed and packed into the extractioncolumn (32 cm3 capacity). Liquid CO2 was pressurised with ahigh-pressure pump and then charged into the extraction columnto desired pressure. The pressure was controlled to an accuracyof about 2% over the measuring range. The extraction columnwas heated through an oven and its temperature was indicatedand controlled by the thermocouple within ±1 �C. After the columnhold equilibration at desired constant pressure and temperature,the dynamic extraction process started, that is, liquid co-solventfrom the tank was pressurized with co-solvent pump and chargedinto the extraction column together with carbon dioxide in a cer-tain ratio. The supercritical CO2 and co-solvent with dissolvedcompounds passed through a heated micrometre valve, and weresubsequently expanded to ambient pressure at room temperature.The extracts were collected in a trapping vial, and the total amountof CO2 was measured by a calibrated wet-test metre at known tem-perature and pressure. Finally, the collected extracts were driedunder vacuum equipment (ZK-30 (BS), Huabei Apparatus Co.,Ltd., Tianjin, China) for further analysis.

The conventional extraction processes are consisting of 75%ethanol refluxing extraction and methanol refluxing extraction.Four grams of material were extracted by refluxing with 120 mlsolvent at 90 �C for 1 h. The extraction was carried out repeatedlyfor three times. Finally, the extract was condensed under vacuumwith a rotary evaporator to remove the ethanol and methanol sol-vent for further analysis.

2.3. Sample preparation and analysis methods

2.3.1. Determination of estradiol and progesterone in extracts byradioimmunoassay analysis (RIA)

Fifty milligrams of extracts obtained by supercritical carbondioxide and co-solvent were dissolved with 10 ml methanol. Thecontents were vortexed vigorously for 1 min and then centrifugedat 5000g for 10 min (TGL-16M, Xiangyi Centrifuge Instrument Co.,Ltd., Hunan, China). The supernatant was collected and calibratedto 10 ml by methanol. One millilitre of the supernatant solutionwas dried under vacuum equipment (ZK-30 (BS), Huabei ApparatusCo., Ltd., Tianjin, China) and the final solid were redissolved in a so-dium phosphate buffer (PBS) of pH 6.8 containing 0.2% bovine ser-um albumin (BSA). The sample solution was then stored at 4 �C forRIA.

The concentrations of estradiol (E2) and progesterone (P) weredetermined in conditioned media by radioimmunoassay using acommercially available estradiol kit (Union Medical and Pharma-ceutical Technology Tianjin Ltd., China) and a progesterone kit(Chemclin Biotech Co., Ltd., Beijing, China), respectively. The incu-bation mixture consisted of 0.2 ml of 125I-E2 or 125I-P, correspond-ing to 0.2 ml of E2 (or 0.1 ml of P) standard solution containing 0, 5,25, 100, 300, 750, or 2000 pg/ml (or 0, 0.1, 0.5, 2.0, 10, 30, or150 ng/ml), 0.2 ml of diluted antiserum. Each calibration point oran unknown sample was assayed in duplicate; tubes correspond-ing to total activity (T), binding capacity ((B/T)0) and nonspecificcounts (N/T) were also prepared. After 1.5 h incubation at 37 �C,1.0 ml of precipitating reagent was added to precipitate immunecomplexes, and then the RIA tubes were refrigerated in an ice-bath.After 10 min, the tubes were centrifuged at 24 �C at 3000g for20 min, and then each deposition was transferred to a vial for c-counting.

2.3.2. Determination of three biological base components and p-hydroxybenzaldehyde in extracts by HPLC

In a typical experiment, about 10 mg of the extracts by super-critical carbon dioxide and co-solvent extraction were dissolvedwith 5 ml of 3% methanol solution, and then one milliliter of result-ing solution was filtered for HPLC analysis. The analysis was per-formed on a liquid chromatography system equipped with anisocratic pump (LabAlliance, Model Series III) and an ultraviolet–visible detector (LabAlliance, model 500). The column used wasan Agilent TC C18 separation column (5 lm, 250 � 4.6 mm i.d.,Agilent Technologies, USA). The mobile phase was 0.07% of aceticacid methanol-water (3:97, v/v) pH 3.5 at a flow-rate of 1.0 ml/min. Detection was at a wavelength of 254 nm for the biologicalbase components including uracil, hypoxanthine and uridine;while for the determination of p-hydroxybenzaldehyde, the mobilephase used was methanol-water (50:50, v/v) at a flow-rate of 1 ml/min with wavelength of 280 nm.

A series of mixture standards of uracil, hypoxanthine and uri-dine in the range of 0.0017–0.0333 mg/ml and p-hydroxybenzalde-hyde standard in the range of 0.001–0.01 mg/ml were prepared inthe mobile phase. Quantification was carried out by integration ofthe peak areas using external standard calibration. A linear re-sponse with a correlation coefficient of 0.999 (n = 6) was obtainedfor the standards. For all experiments, the standards and extractswere filtered through a 0.45 lm cellulose esters membrane beforeinjected into the HPLC system. The injection volume was 20 lL.The chromatograms of the standards and samples extracted bysupercritical carbon dioxide are presented in Fig. 1.

2.3.3. Identification of phospholipids by thin layer chromatography(TLC)

Phospholipids were reported to have the capability of anti-oxi-dation and anti-senescence. In this paper, the phospholipids pres-

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R. Zhou, S. Li / Food Chemistry 114 (2009) 1321–1327 1323

ent in supercritical carbon dioxide (SC-CO2) extracts were evalu-ated with TLC. The TLC plates (pre-coated with a 0.25 mm layerof silica gel 60) were obtained from Qingdao Chemical Co. (China).One microlitre mixture standard solution (5 lg/ll) and 4 lL SC-CO2 extract solutions (50 lg/ll) were applied on a plate. The sol-vent system was a mixture of chloroform: methanol: acetic acid:ethanol: water = 25: 4: 6: 3: 0.5(v/v). For the visual evaluation ofthe different lipids, the plate was charred with iodine vapour andput into an oven at 70 �C for 20 min. The standard mixture of lipidswas run in parallel with the samples for identification of spots.

2.4. Antioxidant properties assays of extract

2.4.1. Ascorbate-iron(III)/EDTA-H2O2-catalysed hydroxyl radical-mediated 2-deoxy-D-ribose degradation

The ability of the extract to inhibit hydroxyl radical-mediated 2-deoxy-D-ribose degradation was carried out essentially as de-scribed by (Halliwell, Gutteridge, & Aruoma, 1987; Jin, Cai, Li, &Zhao, 1996). The reaction mixture contained 100 lL of extract dis-solved in 50 mmol/L phosphate buffer (pH 7.4), 100 lL of 60 mmol/L 2-deoxy-D-ribose in phosphate buffer, 200 lL of a premixed100 mmol/L FeCl3, and 1.04 mmol/L EDTA (1:1, v/v) solution,100 lL of 10 mmol/L H2O2 and 100 lL of 2.0 mmol/L aqueousascorbic acid. Tubes were vortexed and incubated at 37 �C for60 min. After incubation, the colour was developed by addition of1 ml of 20% trichloroacetic acid (TCA) and 1 ml of 0.8% thiobarbitu-ric acid (TBA) and heated in a boiling water-bath (95–100 �C) for20 min. The reaction was stopped by a 5 min ice H2O bath. To eachtube 2 ml of n-butanol was added, and the mixture was vigorouslyvortexed. After centrifugation, the extent of oxidation wasestimated from the absorbance of the organic layer at 532 nm by

ultraviolet spectrophotometry (UV 1600, Ruili Analysis InstrumentFactory Co., Beijing, China). The percentage inhibition was calcu-lated using Eq. (1) and the IC50 values (the half maximal inhibitoryconcentration, represents the concentration of an inhibitor that isrequired for 50% inhibition of its target) were estimated using anon-linear regression algorithm with the use of Origin 7.5 for Win-dows, USA.

Percentage inhibitionð%Þ ¼ 1� A532 nmin the presence of sampleA532 nmin the absence of sample

� �

� 100 ð1Þ

2.4.2. Hydroxyl radical-scavenging by phenanthroline-Fe (II) oxidationassay

Fenton reaction is key reaction in organism which produces hy-droxyl radicals. The principle can be described as follow:

Fe2þ þH2O2 ! Fe3þ þ OHþ OH�

Hydroxyl radicals can attack aromatic compound (salicylic acid)hydroxylation into hydroxylation products (2,3-dihydroxyl ben-zoic acid), which can be detected by colorimetry. The adding ofantioxidants can reduce hydroxyl radicals and hydroxylation sothat the efficiency of scavenging hydroxyl radical can bedetermined.

The ability of the hydroxyl radical-scavenging was carried outas described by (Jin et al., 1996; Özyürek et al., 2007). Two hundredmicrolitre of (3.75 mmo1/L) phenanthroline, 200 lL (3.75 mmo1/L) FeSO4 and 400 lL (0.05%) H2O2 were added into 400 lL of ex-tract dissolved in 0.76 mol/L phosphate buffer (pH 7.4). After 1 hof incubation at 37 �C, the absorbance at 532 nm was recorded.

1324 R. Zhou, S. Li / Food Chemistry 114 (2009) 1321–1327

The percentage scavenging activity was calculated using Eq. (1)and IC50 values were calculated by a non-linear regression algo-rithm with the use of Origin 7.5 for Windows, USA.

2.4.3. Inhibition of lipid peroxidation of lipoprotein induced by ironLipid peroxidation was estimated by modified the method of

(Tauseef, Sharma, & Fahim, 2007; Zhang, Yu, Zhou, & Xiao, 1996).The lipid peroxidation (LPO reaction) system contained 0.2% leci-thin in 0.1 mol/L PBS (pH 7.4), 100 lL of extract dissolved in0.1 mol/L PBS (pH 7.4) and 200 lL 25 mmol/L FeSO4 � 7H2O, the to-tal volume was 2.0 ml. Reaction mixture was incubated at 37 �C ina water-bath for 1 h. Reaction was stopped by adding 1.0 ml 20%TCA. Following by adding 1.0 ml of 0.8% thiobarbituric acid, allthe tubes were placed in boiling water-bath for 20 min and thenshifted to crushed ice-bath before centrifuged at 2500g for10 min. the rate of inhibition in each of the samples was assessedby measuring optical density of the supernatant at 532 nm usingspectrophotometer (UV 1600, Ruili Analysis Instrument FactoryCo., Beijing, China) against blank.

3. Results and discussion

3.1. Supercritical CO2 with co-solvent extraction and componentsanalysis of extract

The total extract yield (defined as extract mass in per unit massantler velvet material) by supercritical CO2 with co-solvent extrac-tion was 72.0 mg/g (dry wt.) antler velvet material under the opti-mal conditions (75% ethanol as co-solvent, extraction pressure was30 MPa, extraction temperature was 35 �C, extraction time was30 min).

3.1.1. HPLC–RIA analysis for the three biological base components, p-hydroxybenzaldehyde and two sex hormones

As uracil (Ur), hypoxanthine (HX), uridine (U) and p-hydroxy-benzaldehyde (PHBD) have inhibitory activities on senescence,contents of the three biological base components and PHBD inthe extracts were determined by HPLC shown in Fig. 1. While thecontents of two sex hormones was identified by RIA. It is indicatedfrom Table 1 that under optimum conditions of using 75% ethanolas co-solvent in this experiment, the yield of U, HX, Ur and PHBDare 0.2785 mg/g, 0.2611 mg/g, 0.2682 mg/g and 0.0013 mg/g,respectively. The yield of E2 and Pro was 0.08767 ng/g and25.66 ng/g.

3.1.2. Identification for phospholipids in the extract by TLCThe phospholipids constituents in extracts were identified by

thin layer chromatography (TLC), shown in Fig. 2. Lane 1 was theTLC of phospholipids standards, Lane 2–6 were the TLC of antler

Table 1HPLC and RIA analysis results of SFE and refluxing extracts of antler velvet.

Extraction methods Extract yield % Consumption of solvent (ml/g) S

1a 7.2 15 YC

2b 11 90 YC

3c 6.9 90 YC

a SC-CO2 extraction with 75% ethanol as co-solvent at 35 �C.b 75% Ethanol refluxing extraction.C Methanol refluxing extraction.d U, uracil; HX, hypoxanthine; Ur, uridine; PHBD, p-hydroxybenzaldehyde; E2, estradie Yield, extract yield expressed as mg/g dry wt. antler velvet material for U, HX, Ur, Pf Content, expressed as mg/g extract for U, HX, Ur, PHBD; ng/g extract for E2 and Pro

velvet extracts. The result indicated that PC, PE, SPH, LPC, LPEand an unknown lipid were found in the extracts obtained withSC-CO2 in the presence of both static and dynamic co-solventand refluxing extraction, however, there was only one unknown li-pid in the extracts obtained with pure supercritical CO2.

3.1.3. Comparison of active components by different extractionmethods

It must be noticed from extraction data (Table 1) that the yieldand content of active components obtained by supercritical CO2

and 75% ethanol extraction were higher than that obtained by75% ethanol refluxing extraction. Even more interesting was thelarge increase of active components purity except for PHBD inthe supercritical CO2 and co-solvent extract and the recovery of ac-tive components was almost up to 100%. Moreover, the consump-tion of organic solvent by supercritical CO2 and co-solventextraction was only 15 ml/g, which was 1/6 times of that with tra-ditional refluxing extraction methods. Therefore, the technology ofsupercritical CO2 and co-solvent extraction shows obvious advan-tage over traditional refluxing extraction methods.

3.2. Antioxidant properties of antler velvet extracts

3.2.1. Ascorbate-iron(III)/EDTA-H2O2-catalysed hydroxyl radical-mediated 2-deoxy-D-ribose degradation

As highly reactive oxygen species, hydroxyl radicals are capableof attacking most biological substrates, e.g. polyunsaturated fattyacids, carbohydrates, DNA and proteins. The prevention of suchdeleterious reactions is highly significant in terms of both humanhealth and the shelf-life of foodstuffs, cosmetics and pharmaceuti-cals. Therefore, it was considered important to assess the protec-tive ability of the antler velvet extract against hydroxyl radicals.The system of hydroxyl radicals-mediated 2-deoxy-D-ribose degra-dation is used to estimate the capability of anti-oxidative injury ofextract. In this assay, hydroxyl radicals are generated by Fentonreaction using EDTA, iron(III) ions, ascorbic acid and hydrogen per-oxide. The hydroxyl radicals fragment the 2-deoxy-D-ribose sub-strate into 2-thiobarbituric acid reactive substances (TBARS),which can be quantified spectrophotometrically at 532 nm. A sam-ple able to inhibit the formation of TBARS in this assay may be de-scribed as hydroxyl radical scavenger, capable of protectingcarbohydrates from oxidative degradation and injury.

The Fig. 3A(1)–(4) showed that the aqueous ascorbic acid,supercritical CO2 with co-solvent extract of antler velvet (SFE ex-tract), 75% ethanol extract (ethanol extract) and methanol extract(methanol extract) were both capable of protecting 2-deoxy-D-ribosefrom oxidative degradation by scavenging hydroxyl radicals andpresented a concentration-dependent fashion. The IC50 of SFEextract was about 10 mg/ml which was much higher than that of

ample Compositionsd

U HX Ur PHBD E2 Pro

ielde 0.28 0.26 0.27 0.0013 0.088 25.7ontentf 3.9 3.6 3.7 0.018 1.2 356.4ield 0.23 0.24 0.27 0.017 0.057 13.8ontent 2.1 2.2 2.5 0.15 0.52 125.2ield 0.25 0.27 0.33 0.014 0.059 12.4ontent 3.7 3.9 4.8 0.20 0.85 179.2

ol; Pro, progesterone.HBD; ng/g dry wt. antler velvet material for E2 and Pro..

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1: the standard mixtures of phospholipids 2: the extract obtained by SC-CO2 with static co-solvent 3: the extract obtained with SC-CO2 in the presence of dynamic

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4: the extracts obtained with pure supercritical CO2

5, 6: the extract obtained with 75 % ethanol and methanol refluxing

extraction

Fig. 2. Thin layer chromatography (TLC) of phospholipids in the extracts.

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Fig. 3. Antioxidant activity of antler velvet extracts and Vc by 2-deoxy-D-ribose degradation method (A(1)–A(4)), phenanthroline-Fe(II) oxidation method (B(1)–B(4)) andiron induced method (C(1)–C(4)).

R. Zhou, S. Li / Food Chemistry 114 (2009) 1321–1327 1325

aqueous ascorbic acid of 30 mg/ml. Moreover, the IC50 of ethanolextract and methanol extract were 40 mg/ml and 30 mg/ml,

respectively, which were obviously lower than that of SFE extract.Therefore, it is concluded that SFE extract of antler velvet can

1326 R. Zhou, S. Li / Food Chemistry 114 (2009) 1321–1327

effectively protect proteins and nucleic acid from injury, whichmay be the key to the anti-aging activity of antler velvet.

3.2.2. Hydroxyl radical-scavenging by phenanthroline-Fe(II) oxidationassay

Phenanthroline-Fe(II) is one of oxidation–reduction indicator incommon use, where the change of colour can be used to indicatethe alteration of the state of oxidation–reduction reaction. So theinhibitory effect on hydroxyl radical is an important index reflect-ing the anti-oxygenation of compounds. In the phenanthroline-Fe(II) assay, hydroxyl radicals (�OH) produced in H2O2/Fe2+ systemcan oxidise phenanthrolene-Fe2+ into phenanthrolene-Fe3+, andthe absorption peak at 532 nm(A532) was reduced significantly.When the drugs were added into the system, the change of A532

can be alleviated, so the extent of alleviation could be the criterionfor estimating the relative strength of anti-oxidation of extract.

The extracts were capable of inhibiting the hydroxyl radicalgreatly in a concentration-dependent fashion. As can be seen inFig. 3B(1)–(4), with the increasing of concentration of aqueousascorbic acid and antler velvet extract, the inhibitory effect was en-hanced dramatically. According to the simulation, the IC50 of SFEextract was 0.9858 mg/ml, which was relatively lower than thatof aqueous ascorbic acid. The IC50 of 75% ethanol and methanol ex-tract were 1.53 mg/ml and 1.16 mg/ml, respectively, which havelower inhibitory effect than SFE extract.

3.2.3. Inhibition of lipid peroxidation of lipoprotein induced by ironLipid peroxide damage tightly concerned with the generation

and development of many diseases, such as tumor, senescence,cardio cerebrovascular diseases, autoimmune disease, shock,inflammation and radiation injury. Investigation to the lipid perox-ide (LPO) injury will give a new clue to the therapy of diseases.Moreover, the therapeutic effect of several drugs has a close rela-tionship with their anti-oxidative property in pharmaceutical re-search, so it has significance in establishing a reasonable model.

In the function of radicals, peroxidation of polyunsaturatedfatty acid (PUFA) occurred. It can be seen from Fig. 3C(1)–(4) thatthe antler velvet extracts were capable of inhibiting the LPO. Withthe increasing of concentration of antler velvet extract, the inhibi-tory effect increased. However, further increase of the concentra-tion of antler velvet extract results in a slightly decrease of theinhibitory effect. The inhibitory effects of 75% ethanol and metha-nol extract were above 50% at the concentration from 0.01 mg/mlto 0.5 mg/ml, which were higher than that of SFE extract and aque-ous ascorbic acid. When the concentration of antler velvet SFE ex-tract was 10 mg/ml, the inhibitory effect could reach 51.82%.

The reason for obtaining these results is that the compositionsin antler velvet extract were complex. Both the anti-decrepitudecompositions and other components may generate antagonistic ac-tion, which has effect on inhibition of extract. The mechanism ofthis change needs further research.

4. Conclusions

It is possible to use antler velvet as a new source of natural anti-oxidants. From the compositional data of antler velvet extract, itcan be seen that there are many active compounds within this ex-tract including three biological bases, two sex hormones and fivephospholipids have been tentatively analyzed. In terms of activity,the extract demonstrated a range of antioxidant-related activitieswhich was concentration-dependent, viz., the ability to protectcarbohydrates from hydroxyl radical-mediated degradation, theability to scavenge hydroxyl radical and inhibit lipid peroxidation.Compared with the data of refluxing extraction, the results showthat the technology of supercritical CO2 with co-solvent extraction

has obvious advantage of lower consumptions of organic solventand time and high recovery of active components. The inhibitoryactivity of SFE extract was obviously higher than that of othertwo traditional refluxing extracts. The chemical components in ex-tracts and their investigation of antioxidant activity from animalmaterials were firstly reported. Additional work is therefore neces-sary to fractionate and identify the extracts further to elicit a betterunderstanding of how each chemical fraction contributes to theoverall antioxidant activity and whether the mixture of extractscontributes to a synergistic antioxidant activity.

Acknowledgments

The authors would like to acknowledgement the assistance ofDr. Junhua Ma, Tianjin Gong’ an Hospital, for work with RIA, andbe grateful for the financial support received from Tianjin NaturalScience Fund project 06YFJMJC10500, which made this studypossible.

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