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Separation and Purification Technology 65 (2009) 275–281

Contents lists available at ScienceDirect

Separation and Purification Technology

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upercritical fluid extraction of monoamine oxidase inhibitor fromntler velvet

an Zhou, Jinyu Wang, Shufen Li ∗, Ying Liuey Laboratory for Green Chemical Technology of State Education Ministry, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

r t i c l e i n f o

rticle history:eceived 23 June 2008eceived in revised form 24 October 2008ccepted 25 October 2008

a b s t r a c t

Supercritical fluid extraction (SFE) of the monoamine oxidase (MAO) inhibitor from antler velvet with CO2

was explored. The effect of different parameters, such as particle size, the kind of co-solvent, extractiontemperature and pressure, on the extraction yield (Y) and the total inhibitory activity (TI) on MAO-B wereinvestigated using three-level orthogonal array design. The experimental results show that when the

eywords:onoamine oxidase inhibitor

ntler velvetupercritical fluid extractionupercritical carbon dioxide

absolute ethanol as co-solvent was used and the particle size was 120 �m, the extraction temperaturewas 70 ◦C, the extraction pressure was 30 MPa, the extract yield reached 3.58% and the TI of the extractwas 3319.13 U/g. Evaluation of the inhibitory activity of extract on MAO indicated that the extracts hadstrong inhibitory effects on MAO-B, but had slight effects on MAO-A. The MAO-B activity was inhibited by93.77% at the concentration of 278.15 mg/L, which was much higher than that of water extract and ethanol

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. Introduction

Antler velvet is unossified horn cut from male deer, whichelongs to the Cervidae. It is generally termed “Lu rong” in China,nd “Nokyong” in Korea, “Tokujo” in Japan or “Pantui” in Russia [1].ntler velvet has been used in traditional Chinese medicines forver 2000 years. It has long been recognized as one of the mostffective and powerful invigorants for strengthening the kidney,exual-reinforcing, and prolonging life [2,3]. Recently, there wereany studies on the components and pharmacological effects of

ntler velvet. Many researches made it evident that antler velvetnd its extracts have some inhibitory activities on arthritis [1,4–6],nti-inflammatory effects [7], anti-stress activities [8], inhibitoryction on monoamine oxidase B [9–11], and anti-aging activities12–14], etc. The compositions of estradiol, uracil, hypoxanthine,-hydroxybenzaldehyde and phospholipids in antler velvet wereeported to have the inhibitory effect on monoamine oxidaseMAO).

MAO is an important enzyme in the metabolism of a wideange of monoamine neurotransmitters such as noradrenaline,

opamine, and serotonin (5-HT). Two different forms, MAO-A andAO-B, have been identified, which play key roles in monoamine

eurotransmitter metabolism and involve in many neuropsychi-try disorders [15,16]. Inhibition of MAO can effectively prevent

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

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383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.seppur.2008.10.036

tradiol, uracil, hypoxanthine, p-hydroxybenzaldehyde and phospholipidsed, which were reported to have the inhibitory effect on MAO.

© 2008 Elsevier B.V. All rights reserved.

epression and various oxidative stresses in the brain [17]. Theompositions which have the function of inhibiting the activity onAO were called MAO inhibitors in this paper. Some MAO inhibitors

ppeared to be effective to prevent and treat neural disease, suchs Parkinson’s disease or Alzheimer’s disease (AD) [18]. However,ome MAO inhibitors were reported to have severe adverse effects19]. Thus, it caused great interest to find new MAO inhibitors with-ut severe side-effects from natural product [15,19,20].

Previous studies on pharmacological effects of antler velvetere generally performed by using its water extract or organic

olvent extract. These extracts were obtained commonly by sol-ent extraction or boiling water [4,8–13]. These processes canpend long extraction times and consume large quantities of sol-ents, especially they can result in the loss or degradation of activeomponents. These disadvantages can be avoided by using theupercritical fluid extraction (SFE). Compared with liquid extrac-ion, SFE is relatively rapid because of the low viscosities andigh diffusivities associated with supercritical fluids. The extrac-ion can be selective to some extent by controlling the density ofhe medium and the extracted material is easily recovered by sim-ly depressurizing, allowing the supercritical fluid to return to gashase and evaporate leaving no or little solvent residues. However,one of investigations has been reports about the extraction of the

onoamine oxidase inhibitor by supercritical CO2.In this paper, supercritical fluid extraction (SFE) of the

onoamine oxidase (MAO) inhibitor from antler velvet with CO2as explored and optimized. Evaluation of the inhibitory activ-

ty of the SFE extract on MAO was made, and the compositions

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f estradiol, uracil, hypoxanthine, p-hydroxybenzaldehyde andhospholipids in the SFE extracts were also identified which areonsidered to have the inhibitory effect on MAO.

. Experimental methods

.1. Chemicals and raw materials

Antler velvet (Cervus nippon TEMMINCK var. mantchuricuswinhoe) was provided and identified by Tianjin Shencao pharma-eutical zoo breeding Co. Ltd. Kunming rats (30 ± 5 g), 7 weeks old,ere supplied by Beijing Experimental Zoo Centre.

Carbon dioxide (99.99% pure) supplied in a cylinder was pur-hased from Liu Fang Gas Co. (Tianjin, China). Serotonin creatinineulfate complex (5-HT, 99.0%), Benzylamine hydrochloride (BA,9.0%), Sphingomyelin (SPH, 98.0%) were obtained from Sigmahemical Co. (USA). The standard samples of estradiol (E2), uracilU), hypoxanthine (HX) and p-Hydroxybenzaldehyde (PHBD), allith purity more than 99.0% were obtained from Sigma Chemicalo. (USA). Sphingomyelin (SPH) with purity of 98.0% was acquired

rom Sigma Chemical Co. (USA). Phosphatidylcholine (PC), phos-hatidylethanolamine (PE), lysophosphatidylcholine (LPC) and

ysophosphatidylethanolamine (LPE) all were bought from Flukao. (Germany). Butyl acetate and cyclohexane were HPLC grades99.0%) obtained from Guangfu chemical reagent Co. (Tianjin,hina).

.2. Supercritical fluid extraction procedure

Supercritical CO2 extraction was experimentally performedith Spe-ed SFE instrument (Applied Separations Inc., Allenton, PA,SA), shown schematically in Fig. 1. Antler velvet powder or its mix-

ure with co-solvent were weighed and packed into the extractionolumn (32 cm3 capacity) (6). The ratio of co-solvent to antler velvetowder was 1:1 (v/m). Liquid CO2 was pressurized with a high-ressure pump (3) and then charged into the extraction column

6) to desired pressure. The pressure was controlled to an accu-acy of about 2% over the measuring range. The extraction columnas heated through an oven (7) and its temperature was indicated

nd controlled by the thermocouple (12) within ±1 ◦C. The extrac-ion process started after the column hold equilibration for 20 min

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Fig. 1. Schematic diagram of the

n Technology 65 (2009) 275–281

t working conditions. The supercritical CO2 with dissolved com-ounds passed through a heated micrometer valve (9), and wasubsequently expanded to ambient pressure at room temperature.he flow-rate of CO2 was controlled at 1.0 L/min (ambient temper-ture and pressure) in this study. A calibrated wet-test meter (11)t known temperature and pressure measured the total amountsf CO2.

The SFE extracts were precipitated in a 20 mL collecting vial10). Collected extracts were dried under vacuum equipment (ZK-0 (BS), Huabei Apparatus Co., Ltd., Tianjin, China). Finally, the totalxtract yield (defined as extract mass in per unit mass antler vel-et material) was calculated. The MAO activity and the chemicalompositions in extract were also analyzed.

.3. MAO activity assays

An in vitro assay was designed and reported to measure thenhibitory activity of the extract on MAO-A and MAO-B [21].

.3.1. Preparation of the source of MAO activityRat brain mitochondrial fraction as a source of MAO activity

as prepared. Male Kunming rats were killed by stunning andecapitation. The head was dissected and the brain tissue wasemoved. Then the brain tissue was homogenized on ice in 10 vol. of00 mmol/L sodium phosphate buffer (PBS, pH 7.4) with 0.32 mol/Lucrose. The mixture was centrifuged (TGL-16M, Xiangyi Centrifugenstrument Co., Ltd., Hunan, China, 600 × g, 10 min, 4 ◦C) immedi-tely. The precipitation fraction was removed, and the supernatantiquid was continued to centrifuge (15,000 × g, 10 min, 4 ◦C). Aban-oning the supernatant this time, the solids were suspended in theame buffer and centrifuged (15,000 × g, 10 min, 4 ◦C) again. Thenal solids were suspended in the above PBS solution. The proteinoncentration of brain mitochondrial extract was diluted with theame PBS to 1 mg/mL. Protein concentration was estimated by theowry method which uses bovine serum albumin as the standard22]. The brain mitochondrial extract was stored at −20 ◦C.

.3.2. MAO activity assays with UV spectrophotometer methodMAO activity was assessed by UV spectrophotometer method.

erotonin creatinine sulfate complex (5-HT) and Benzylamineydrochloride (BA) were diluted as specific substrates for MAO-

experimental apparatus.

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and B in distilled water deoxygenated by N2 bubbling. The 5-HTnd BA with the concentration of 0.02 and 0.01 mol/L, respectivelyere stored under nitrogen at −20 ◦C.

The SFE extract were dissolved in PBS (0.1 M, pH 7.4) to formifferent concentration of extract solution. Take a test tube, adding00 �L extract solution, 200 �L rats brain mitochondrial extract and00 �L PBS in sequence. Then the mixture was pre-incubated for0 min at 37 ◦C. The substrate 5-HT for MAO-A or BA for MAO-B washen added, and the tubes were incubated at 37 ◦C for 20 min. Thessay was terminated by adding 2 mL of 2 mol/L HCl. Blank assayas prepared by adding 2 mL 2 mol/L HCl to inhibit all MAO activ-

ty prior to the reaction, and performed subsequently in the samerocedure. After the addition of 3 mL of butyl acetate for MAO-product or cyclohexane for MAO-B product, the end productsere extracted to organic phase (for 30 min, at 4 ◦C) and cen-

rifuged (600 × g, 10 min) at room temperature. The absorbencyf the organic phase was determined by a UV Spectrometer (UV-600 Spectrometer, Beijing Rayleigh Analytical Instrument Corp.) at42 nm for MAO-B activity and 280 nm for MAO-A activity. An activ-ty unit was defined as 0.01 absorbance produced by 1 mg proteineaction in 1 h. So the enzyme activity was expressed as follows:

nzyme activity = the absorbance of testing tube × 3content of proteins in 200 �L × 0.01

.4. Identification methods of components having inhibitoryctivity on MAO

.4.1. Determination of estradiol in extracts byadioimmunoassay analysis (RIA)

About fifty milligrams of SFE extracts were dissolved with 10 mLethanol. The contents were vortexed vigorously for 1 min and

hen centrifuged at 5000 × g for 10 min (TGL-16M, Xiangyi Cen-rifuge Instrument Co., Ltd., Hunan, China). The supernatant wasollected and calibrated to 10 mL by methanol. One milliliter of theupernatant solution was dried under vacuum equipment (ZK-30BS), Huabei Apparatus Co., Ltd., Tianjin, China) and the final solidere redissolved in a sodium phosphate buffer (PBS) of pH 6.8 con-

aining 0.2% bovine serum albumin (BSA). The sample solution washen stored at 4 ◦C for RIA analysis.

The concentration of estradiol (E2) was determined in condi-ioned media by radioimmunoassay using a commercially availablestradiol kit (Union Medical and Pharmaceutical Technology Tian-in Ltd., China). The incubation mixture consisted of 0.2 mL of25I-E2, corresponding to 0.2 mL of E2 standard solution containing, 5, 25, 100, 300, 750, or 2000 pg/mL, 0.2 mL of diluted antiserum.ach calibration point or an unknown sample was assayed in dupli-ate; tubes corresponding to total activity (T), binding capacityB/T)0 and nonspecific counts (N/T) were also prepared. After 1.5 hncubation at 37 ◦C, 1.0 mL of precipitating reagent was added torecipitate immune complexes, and then the RIA tubes were refrig-rated in an ice bath. After 10 min, the tubes were centrifuged at4 ◦C at 3000 × g for 20 min, and then each deposition was trans-erred to a vial for �-counting.

.4.2. Identification of biological base components and-hydroxybenzaldehyde (PHBD) in extracts by HPLC

About 10 mg of SFE extracts were dissolved in 5 mL of% methanol aqueous solution, and the resulting solution wasltered for HPLC analyses. The analysis was performed on

high-performance liquid chromatography system equippedith an isocratic pump (LabAlliance, Model Series III) and anltraviolet–visible detector (LabAlliance, model 500) was used.he column used for separation was an Agilent TC C18 separationolumn (5 �m, 250 × 4.6 mm i.d., Agilent Technologies, USA). The

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n Technology 65 (2009) 275–281 277

obile phase was 0.07% of acetic acid methanol–water (3:97, v/v)t a flow-rate of 1 mL/min. Detection was made at a wavelengthf 254 nm for uracil and hypoxanthine; while for the determi-ation of p-hydroxybenzaldehyde, the mobile phase used wasethanol–water (50:50, v/v) at a flow-rate of 1 mL/min with wave-

ength of 280 nm.A series of standards of uracil, hypoxanthine, p-

ydroxybenzaldehyde in the range of 0.001–0.1 mg/mL wererepared in the mobile phase. Quantification was carried out by

ntegration of the peak areas using external standard calibration.linear response with a correlation coefficient of 0.999 (n = 6) was

btained for the standards. For all experiments, the standards andxtracts were filtered through a 0.45 �m membrane filter beforenjected into the HPLC system. The injection volume was 20 �L.he chromatograms of the standards and extracts obtained by SFEre presented in Fig. 2.

.4.3. Identification of phospholipids by thin layerhromatography (TLC)

Phospholipids were reported to have the capability of antiox-dation and anti-senescence. In this paper, the phospholipidsresent in supercritical carbon dioxide (SC-CO2) extracts werevaluated with TLC. The TLC plates (pre-coated with a 0.25-mmayer of silica gel 60) were obtained from Qingdao Chemical Co.China). One microlitre mixture standard solution (5 �g/�L) and�L SC-CO2 extract solutions (50 �g/�L) were applied on a plate.he solvent system was a mixture of chloroform:methanol:aceticcid:ethanol:water = 25:4:6:3:0.5 (v/v). For the visual evaluation ofhe different lipids, the plate was charred with iodine vapor and putnto an oven at 70 ◦C for 20 min. The standard mixture of lipids wasun in parallel with the samples for identification of spots.

. Results and discussion

.1. Optimization of SFE parameters for extracting MAO inhibitorrom antler velvet

An L9 (34) matrix orthogonal array design (OAD) was utilized toptimize the supercritical CO2 extraction of MAO inhibitor fromntler velvet (Table 1). The effect of particle size (440, 215 and65 �m), the kind of co-solvent (1# (pure CO2), 2# (50% ethanol)nd 3# (absolute ethanol)), extraction temperature (60, 70 and0 ◦C) and extraction pressure (20, 30 and 40 MPa) on the extractionield (Y) and the total inhibitory activity of the extracts on MAO-BTI) was studied.

The experimental results were listed in Table 1, where the meanalues of each Y and TI for the corresponding factors at each levelere calculated according to the assignment of the experiment.

or example, the Y and TI of the three trials with the particle size of65 �m were evaluated as mean values of the corresponding threeuns. The mean values of the three levels of each factor (e.g. particleize) reveal how the extraction yield will change when changing theevel of that factor. Fig. 3 shows Y and TI as a function of levels of thetudied factors. In all cases it should be noted that each reported Ynd TI is the average of three measurements, in each of which thearameter of interest was kept constant, and the other parametersere changed (Fig. 3). The ANOVA results of OAD (Table 1) show

hat the variable B, namely the kind of co-solvent, had the highestnalysis of variable (ANOVA) value of 0.99 and exerted the largestffect on Y. The variable A, namely particle size had the largest effect

n TI.

.1.1. Effect of particle sizeThe particle size of antler velvet material plays important roles

n the SFE of monoamine oxidase inhibitor, which has the second-

278 R. Zhou et al. / Separation and Purification Technology 65 (2009) 275–281

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ig. 2. HPLC chromatograms of reference substances of uracil, hypoxanthine (1), p).

ighest effect on Y. As can be seen from Fig. 3(A), with particleize decreasing the specific surface area of particle would increase,he mass transfer distance would shorten. These all would resultn promoting the extraction yield. As for another indicator of TI,article size has the largest effect on the TI. The TI significantly

ncreases with decreasing the particle size. These results suggesthat particle size is the most important factor, and the objectiveompounds possessing inhibitory activity on MAO-B maybe mainlyistribute inside the cell of antler velvet tissue. The diffusion speedf the components from tissue to the bulk phase of SC-CO2 is the

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able 1he orthogonal array design and experimental results (x̄ ± S.D.).

o. Particle size (�m) Co-solventa Temperature (◦C)

1(440) 1(1#) 1(60)1(440) 2(2#) 2(70)1(440) 3(3#) 3(80)2(215) 1(1#) 2(70)2(215) 2(2#) 1(60)2(215) 3(3#) 3(80)3(165) 1(1#) 3(80)3(165) 2(2#) 1(60)3(165) 3(3#) 2(70)

ield (Y)Ij/kjb 1.61 1.9IIj/kjb 1.95 1.5IIIj/kjb 2.51 2.5ANOVAc 0.90 0.9

otal inhibitory effects on MAO-B (TI)Ij/kjb 751.55 1230.3IIj/kjb 1086.70 959.7IIIj/kjb 1712.85 1360.9ANOVAc 961.30 401.2

a The kind of co-solvent: 1# (pure CO2), 2# (50% ethanol) and 3# (absolute ethanol).b The average value of each index at every level for certain factor.c The abbreviation of analysis of variable, that is, the extreme difference equals the max

xybenzaldehyde (2) and samples extracted by supercritical carbon dioxide (3 and

ain limited steps. The smaller particle size could result in thearger specific surface area of the particle and more touching chanceetween antler velvet powder grounds with SC-CO2. Therefore theI is promoted significantly.

.1.2. Effect of the kind of co-solventThe effect of the kind of co-solvent on Y and TI of SFE extract

as investigated and results are shown in Fig. 3(B). As can beeen, it has the highest impact on Y and the lowest influencen TI. The extraction yield is 1.90% when not using co-solvent,

Pressure (MPa) Y (%) TI (U/g)

1(20) 1.28 ± 0.113 540.66 ± 23.12(30) 1.29 ± 0.102 775.22 ± 34.33(40) 2.26 ± 0.098 938.78 ± 27.73(40) 2.11 ± 0.100 1463.5 ± 28.51(20) 1.04 ± 0.124 407.72 ± 37.92(30) 2.71 ± 0.203 1388.9 ± 56.52(30) 2.31 ± 0.147 1686.8 ± 42.33(40) 2.45 ± 0.085 1696.4 ± 53.31(20) 2.78 ± 0.098 1755.3 ± 31.8

0 1.59 1.709 2.06 2.108 2.43 2.279 0.84 0.57

3 881.60 901.239 1331.34 1283.649 1338.16 1366.240 456.56 465.01

imum minus the minimum for a certain factors.

ification Technology 65 (2009) 275–281 279

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hich is better than the result of 1.59% obtained by adding0% ethanol aqueous solution (v/v) as co-solvent. When absolutethanol is used as co-solvent, the extraction yield is the highest2.58%), which enhances 35.78% being compared with the resultbtained by pure supercritical CO2. The addition of ethanol pro-otes the polarity of supercritical CO2 and its solubility for the

olute with high polarity, but when the polarity of co-solvent isoo large, the capability of extracting liposoluble components willecrease.

Though the TI increases with the addition of absolute ethanol,he variation has the least impact on TI among four factors. Finally,he absolute ethanol was selected as co-solvent.

.1.3. Effect of extraction temperature and pressureThe variation of temperature during SFE affects the satu-

ated vapor pressure of solutes, the volatility of the analytesnd desorption of the analytes. At higher temperatures, analytesecome more volatile and the saturated vapor pressure of solutes

ncreases. The results in Fig. 3(C) indicate that the extractionield increased with the increasing of extraction temperature.oreover, the TI increased by 51.01% from 60 to 70 ◦C. But with

he further increasing of extraction temperature, the extractionield of active components which has inhibitory effect on MAO-

showed little change and the active components which areot resistant to high temperature denaturized and deactivated sohat TI has slight change from 70 to 80 ◦C. Therefore, the appro-riate extraction temperature of 70 ◦C was determined to obtainelatively high extraction yield and the highest biological activ-ty.

Analytical solubility of a solute in a supercritical fluid (SCF)epends on the balance of SCF density and solute vapor pressure,here both of them are controlled by pressure of the SCF. As can be

nferred from the results, extraction recovery is usually enhanceds the pressure rises. An increase in pressure causes an increase inhe fluid density, and thus it has an effect of increasing in the sol-ating power of the supercritical fluid, responsible for quantitativextraction yield and total inhibition activity on MAO-B. As shown

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Fig. 3. Effect of particle size (A), concentration of ethanol (B), extracti

ig. 4. Effects of particle size on the extraction yield and total inhibitory activity onAO-B.

n Fig. 3(D) the optimized pressure was 30 MPa for the extractionf monoamine oxidase inhibitor from antler velvet.

Taking into account of the importance of particle size for yieldnd TI, further study on the influence rules of particle size wasbserved. Six levels of particle size were examined (Fig. 4). It isound that the extraction yield increases with the particle sizeecreasing. But the TI reaches a maximum at 120 �m and then lev-ls off after further decreasing the particle size. The results revealhat the active substance is almost extracted completely when thearticle size is 120 �m, and with further decreasing the particleize, much impurity without inhibition activity on MAO-B is furtherbtained.

Finally, the optimal operation parameters were obtained. Whenhe co-solvent was absolute ethanol, the particle size was 120 �m,

he extraction pressure was 30 MPa, the extraction temperatureas 70 ◦C, the mean extraction yield and total inhibition activityn MAO-B were 3.58% and 3319.13 U/g, respectively, and the RSD%or two factors were 8.99% and 1.74%, respectively.

on temperature (C) and extraction pressure (D) on the Y and TI.

2 ification Technology 65 (2009) 275–281

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.2. Evaluation of the inhibitory activity of SFE extract on MAO

Different concentration of SFE extract in PBS was used to furthernvestigate the does-dependent manner. The experimental resultsllustrated in Fig. 5(A) exhibit that the MAO-B inhibition increasesignificantly with the increasing concentration of the extract solu-ion. The MAO-B inhibition reaches 93.77% when the concentrationf extract solution was 278.15 mg/L. However, the extract has slightnhibitory effect on MAO-A. The MAO-A inhibition was 24.18% athe same concentration of 278.15 mg/L. The literature reportedalues were plotted in Fig. 5(B) for comparison [10,23]. It can beeen from Fig. 5(B) that the water extract has 85% inhibition activityt the concentration of 96,000 mg/L. Therefore, the inhibition ofFE extract was much higher than that of water extract. It also cane seen from Fig. 5 that the MAO-B inhibition of SFE extract was.47–5.52 times higher than that of ethanol extracts. There may beore active components in SFE extract than those in water extract

nd ethanol extracts. The structure of active components may beimilar to the structure of MAO substrate and coenzyme, whichan prevent the combination between substrate and MAO and hashe influence on MAO membrane morphology in mitochondria.

ig. 5. Inhibitory effects on MAO of the SFE extract (A), water extract (the data fromhen [10]) and ethanol extracts (the data from Yang [23]) (B) from antler velvet.

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.3. Identification of components having inhibitory activity onAO in SFE extracts

It is concluded from above experimental results that the antlerelvet SFE extract had strong inhibitory function on MAO-B. Itas been reported that estradiol, uracil, hypoxanthine and p-ydroxybenzaldehyde all have the function of inhibiting MAOctivity [11,21,23–25]. Phospholipids as the important ingredient inntler velvet are connected with the metabolism of life, which alsoan obviously inhibit on the activity of MAO-B [26]. These composi-ions might be the key for reducing signs normally associated withenility. Therefore, the content of estradiol, uracil, hypoxanthine,-hydroxybenzaldehyde and five phospholipids were measured inFE extracts.

.3.1. RIA-HPLC analysis for estradiol, two biological baseomponents and p-hydroxybenzaldehyde

The content of estradiol was identified by RIA, and the contentf the two biological base components and PHBD was deter-ined by HPLC shown in Fig. 2. It is showed that under optimum

onditions of using absolute ethanol as co-solvent in this exper-ment, the contents of E2, U, HX and PHBD in the SFE extracts

ere 9.335 ng/g, 172.8 �g/g, 12.82 �g/g and 25.14 �g/g, respec-ively.

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

hin layer chromatography (TLC), shown in Fig. 6. Lane 1 was theLC of phospholipids standards, Lanes 2–3 were the TLC of antlerelvet extracts. The results indicated that PC, PE, SPH, LPC, LPE andn unknown lipid were found in the extracts obtained with SC-O2 in the presence of co-solvent. However, there was only onenknown lipid in the extracts obtained with pure supercritical CO2.he phospholipids constituents existing in the SFE extract perhapsere another factor for producing strong inhibitory MAO activi-

ies.

. Conclusion

In this study, the extraction conditions of extracting monoaminexidase inhibitors by SC-CO2 from antler velvet and the inhibitoryctivity of extract on MAO were investigated. The extraction yield

ould reach 3.58% and the TI is 3319.13 U/g when co-solvent is

bsolute ethanol, the particle size is 120 �m, the extraction tem-erature is 70 ◦C and the extraction pressure is 30 MPa. The SFExtract has significant inhibitory activity on MAO-B which is higherhan that of water extract and ethanol extract, and did so in a

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oncentration-dependent fashion. The activity of MAO-B is inhib-ted by 93.77% at the SFE extract concentration of 278.15 mg/L. Thective components are identified by suitable analytical methodsuch as RIA, HPLC and TLC. The identified results indicate thathere is a diversity of compounds in the SFE extracts, which per-aps play an important role in inhibiting activity on MAO. Theseesults indicate that antler velvet can be used as a new and naturalesource of MAO inhibitors that may be used in the food and phar-aceutical products for preventing senescence. Further studies are

till required in order to fractionate and identify the extracts fur-her to elicit a better understanding of how each chemical fractionontributes to the overall inhibitory activity on MAO.

cknowledgements

The authors are grateful for the financial support received fromianjin Natural Science Fund project 06YFJMJC10500, which madehis study possible.

eferences

[1] Y.K. Kim, K.S. Kim, K.H. Chung, J.G. Kim, K.S. Kim, Y.C. Lee, Y.C. Chang, C.H.Kim, Inhibitory effects of deer antler aqua-acupuncture, the pilose antlerof Cervus korean TEMMINCK var. mantchuricus Swinhoe, on type II collagen-induced arthritis in rats, International Immunopharmacology 3 (2003)1001–1010.

[2] Z.A. Pan, The Pharmacopeia of the People’s Republic of China (I), first, ChemicalIndustry Press, Beijing, 2000, pp. 264–265.

[3] H.Z. Zheng, Z.H. Dong, J. She, Modern Study of Traditional Chinese Medicine, 6,Xue Yuan Press, Beijing, 1999, pp. 5048–5077.

[4] S.K. Kang, K.S. Kim, S.I. Kim, K.H. Chung, I.S. Lee, C.H. Kim, Immunosupperssiveactivity of deer antler extracts of Cervus korean TEMMINCK var. mantchuricusSwinhoe, on type II collagen-induced arthritis, In Vitro Cellular & Developmen-tal Biology-Animal 42 (2006) 100–107.

[5] K.H. Kim, K.S. Kim, B.J. Choi, K.H. Chung, Y.C. Chang, S.D. Lee, K.K. Park, H.M. Kim,C.H. Kim, Anti-bone resorption activity of deer antler aqua-acupuncture, thepilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe (Nokyong)in adjuvant-induced arthritic rats, Journal of Ethnopharmacology 96 (2005)497–506.

[6] K.S. Kim, Y.H. Choi, K.H. Kim, Y.C. Lee, C.H. Kim, S.H. Moon, S.G. Kang, Y.G.Park, Protective and antiarthritic effects of deer antler aqua-acupuncture(DAA), inhibiting dihydroorotate dehydrogenase on phosphate ions-mediatedchondrocyte apoptosis, and rat collagen-induced arthritis, InternationalImmunopharmacology 4 (2004) 963–973.

[7] Z.Q. Zhang, B.X. Wang, H.O. Zhou, Y. Wang, H. Zhang, Purification and par-tial characterization of anti-inflammatory peptide from pilose antler of Cervusnippon Temminck, Acta Pharmaceutica Sinica 27 (1992) 321–324.

[8] H.H. Sunwoo, T. Nakano, J.S. Sim, Effect of water-soluble extract from antler ofwapiti (Cervus elaphus) on the growth of fibroblasts, Canadian Journal of AnimalScience 77 (1997) 343–345.

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n Technology 65 (2009) 275–281 281

[9] X.G. Chen, D.Y. Chang, Z.Y. Cui, B.X. Wang, Effects of the water extract of piloseantler on some biochemical indicators related to aging in old mice, Pharmacol-ogy and Clinics of Chinese Materia Medica 8 (1992) 17–20.

10] X.G. Chen, Y.G. Jia, B.X. Wang, Study on inhibitory effects of the water extractof pilose antler on monoamine oxidase in old mice, China Journal of ChineseMateria Medica 17 (1992) 107–110.

11] B.X. Wang, X.H. Zhao, X.W. Yang, S. Kaneko, M. Hattori, T. Namba, Y. Nomura,Identification of the inhibitor for monoamine oxidase B in the extract from deerantler (Rokujo), Journal of Medical and Pharmaceutical Society for WAKAN-YAKU 5 (1988) 116–122.

12] B.X. Wang, X.H. Zhao, S.B. Qi, S. Kaneko, M. Hattori, T. Namba, Y. Nomura, Effectsof repeated administration of deer antler (Rokujo) extract on biochemicalchanges related to aging in senescence-accelerated mice, Chemical & Pharma-ceutical Bulletin (Tokyo) 36 (1988) 2587–2592.

13] B.X. Wang, X.H. Zhao, X.W. Yang, S. Kaneko, M. Hattori, T. Namba, Y. Nomura,Inhibition of lipid peroxidation of deer antler (Rokujo) extract in vivo and invitro, Journal of Medical and Pharmaceutical Society for WAKAN-YAKU 5 (1988)123–128.

14] H.B. Xu, Y.Z. Li, X.G. Chen, X.B. Sun, B.X. Wang, Study on the anti-aging actionof phosphate of pilose antler, Pharmacology and Clinics of Chinese MateriaMedica 8 (1992) 29–31.

15] H. Haraguchi, Y. Tanaka, A. Kabbash, T. Fujioka, T. Ishizu, A. Yagi,Monoamine oxidase inhibitors from Gentiana lutea, Phytochemistry 65 (2004)2255–2260.

16] M. Strolin Benedetti, P. Dostert, Monoamine oxidase, brain aging and degener-ative diseases, Biochemical Pharmacology 38 (1989) 555–561.

17] A.S. Kalgutkar, N. Castagnoli Jr., B. Testa, Selective inhibitors of monoamineoxidase (MAO-A and MAO-B) as probes of its catalytic site and mechanism,Medicinal Research Reviews 15 (1995) 325–388.

18] M. Weinstock, E. Gorodetsky, T. Poltyrev, A. Gross, Y. Sagi, M. Youdim, A novelcholinesterase and brain-selective monoamine oxidase inhibitor for the treat-ment of dementia comorbid with depression and Parkinson’s disease, Progressin Neuro-Psychopharmacology and Biological Psychiatry 27 (2003) 555–561.

19] L.D. Kong, C.H.K. Cheng, R.X. Tan, Inhibition of MAO A and B by some plant-derived alkaloids, phenols and anthraquinones, Journal of Ethnopharmacology91 (2004) 351–355.

20] L.D. Kong, R.X. Tan, A.Y.H. Woo, C.H.K. Cheng, Inhibition of rat brain monoamineoxidase activities by psoralen and isopsoralen: implications for the treatmentof affective disorders, Pharmacology and Toxicology 88 (2000) 75–80.

21] B.X. Wang, X.G. Chen, Inhibitory effect of hypoxanthine on monoamine oxidaseactivity, Acta Pharmaceutica Sinica 24 (1989) 573–577.

22] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurementwith the Folin phenol reagant, Journal of Biological Chemistry 193 (1951)265–275.

23] X.W. Yang, HPLC analysis and inhibitory effect of base components in piloseantler of sika deer and red deer on monoamine oxidase activity, Chinese Tra-ditional Herbal Drugs 26 (1995) 17–19.

24] X.G. Chen, B.X. Wang, J. Zhang, H. Zhang, Inhibitory effect of uracilon monoamine oxidase activity, Chinese Biochemical Journal 8 (1992)81–85.

25] D.P. Holschneider, T. Kumazaw, K. Chen, J.C. Shih, Tissue-specific effects ofestrogen on monoamine oxidase A and B in the rat, Life Sciences 63 (1998)155–160.

26] X.G. Chen, B.X. Wang, Y.D. Wu, Inhibitory effect of phospholipids of pilose antleron monoamine oxidase activity, Pharmacology and Clinics of Chinese MateriaMedica 6 (1990) 14–17.