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11 velvet sponge,,, sponge. CP sponge CRW (p < 0.001)(Fig. 4), CRW velvet sponge (p < 0.001). CP CRW (p < 0.05). HS-II, CP CRW.,, CP CRW,. HS-II CP sponge (p < 0.05). (cholesterol ester), (triglyceride), (free fatty acid), (cholesterol) (monoglyceride). phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), sphingomyelin (SM), phosphatidyl serine (PS)

phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC),

12 (U.K). cholesterol ester, phosphatidyl choline., velvet sponge. CLA.(p<0.05) velvet sponge velvet HS, HS-II, CP, CRW MC.(p < 0.05) spongehs, HS-II, CRW. CPsponge.. PA. velvet sponge. PC, SM. HS-II.

14 I. Title The study on the production of domestic brand velvet antler and its active constituents and pharmaceutical efficacy II. Purpose and Significance of Research Despite its several ill conditions in domestic and abroad, Korean deer farming is maintaining its continuous development and it shows contrasting appearance from decrease in other stock breeding business. History of Korean deer farming is comparatively short but has showed rapid growth. About 160,000 deer are raised as of end of 2002 in 12,000 farms and these are 299% and 209% increases each respectively compared to past decade that is evaluated that it shows potentiality in development of deer farming. It is evaluated that it is because of special features of deer farming that are low cost, low labor, environmental friendly stock breeding, high benefits in that industry and if only a few problems are solved additionally, deer farming will solidify its position as the best farm household earning source. The biggest problem of deer

decrease in other stock breeding business. History of Korean deer farming is comparatively short but has showed rapid growth.

15 farming is that even if Korea has largest velvet market in the world, its market possession rate is below 30% which is derived from the fact that its cost is more expensive compared to foreign products and has low productivity due to lack of related research. This study was conducted to establish the high valued(bland) velvet antler production using medicinal herbs and special feed sources and its quality and pharmaceutical efficacy for intensifying competitiveness of domestic deer farming industry through differentiation to imported velvet antlers. III. Contents of Research 1) Brand velvet antler production This research was conducted to development of feed source for brand velvet antler production and to establishment of velvet antler productivity by feed sources. Also this research established proper condition for brand velvet antler production by feed sources. 2) Assesment of quality for brand velvet antler This research assessed basic quality of brand velvet antler produced by different feed sources. Assessment was conducted to analyse crude protein, crude fiber, ether extract, and crude ash and amino acid and collagen contents. Biochemical composition of blood serum was also analysed with condition of different feed sources. 3) Analysing of active constituents in brand velvet antler

This study was conducted to establish the high valued(bland) velvet antler production using medicinal herbs and special feed sources and its quality and pharmaceutical efficacy for intensifying

16 Active constituents were analysed with GAGs, sialic acid, and uronic acid for carbohydrates and phospolipid and neutral lipid for lipid. 4) Pharmaceutical efficacy of brand velvet antler This research was conducted to determine pharmaceutical efficacy of brand velvet antler. Intake and growth rate was assessed to evaluate value of brand velvet antler as a growth factor in experimental animal. Also the efficacy of antidioxin was determined for brand velvet antler in rats exposed by dioxin. IV. Results of Research 1) Brand velvet antler production All feed sources were not significantly differ in production of velvet antler. 2) Assesment of quality for brand velvet antler a. Feeding of Dangyi and medicinal herbs mixture The content of crude protein was higher in section of velvet and sponge of antler produced by feeding of Dangyi and medicinal herbs mixture than in antler produced by feeding of ordinary feed. The content of crude ash was lower in section of velvet and sponge of antler produced by feeding of Dangyi and medicinal herbs mixture than in

17 antler produced by feeding of ordinary feed. Biochemical composition of blood serum was different in some constituents by feeding condition. b. Feeding of mealed bark of lacquer tree Chemical composition was not significant in comparison of velvet antlers produced by feeding of mealed bark of lacquer tree and ordinary feed. Amino acid was also not significant between control and treatment with mealed bark of lacquer tree. Biochemical composition of blood serum differed significantly in some constituents between control and treatment. c. Feeding of Chungung and CLA Chemical composition was not significant in comparison of velvet antlers produced by feeding of Chungung, CLA and ordinary feed. Amino acid was also not significant between treatments. Biochemical composition of blood serum was not significant between treatments. d. Feeding of mulberry, Lycii Fructus, and by-product silage of oriental medicine Chemical composition was not significant in comparison of velvet antlers produced by feeding of mulberry, Lycii Fructus, and by-product silage of oriental medicine. Amino acid was also not significant between treatments. Biochemical composition of blood serum differed significantly in some constituents between treatments.

Biochemical composition of blood serum differed significantly in some constituents between control and treatment. c. Feeding of Chungung and CLA Chemical composition was not significant in comparison of velvet antlers produced by feeding of Chungung, CLA and ordinary feed.

18 3) Analysing of active constituents in brand velvet antler The contents of glycosaminoglycans, uronic acid, sialic acid increased in the top section and sponge part and those was higher in brand velvet antler than in ordinary velvet antler. Total lipids content increased in the top section and velvet part of antlers of all groups. The concentrate of inorganic ions decreased in treatments. Phospholipid was higher in brand velvet antler than in ordinary velvet antler. 4) Pharmaceutical efficacy of brand velvet antler We investigated anti-toxic effect in dioxin(tcdd) medicated rat under the purpose of clinical effect of velvet produced from diverse medication fed deer for brand velvet. In this study, from the examination animals exposed to environmental hormone, dioxin, improved result in medical plants fed deer velvet group is shown compared to ordinary velvet treated group and untreated group in forage ingestion amount, weigh loss, viscera weigh.

20 목 차 제 1 장 연구과제의 개요 제1절 연구개발의 목적 및 필요성 제 2 장 국내외 기술개발 현황 제1절 국내외 관련기술의 현황과 문제점 제2절 앞으로의 전망 제 3 장 연구개발수행 내용 및 결과 제1절 제1세부과제: 브랜드녹용의 생산 제2절 제2세부과제: 브랜드녹용의 품질평가 제3절 제3세부과제: 브랜드녹용의 활성성분분석- 79 제4절 제4세부과제: 브랜드녹요의 약리효과규명- 100 제 4 장 목표달성도 및 관련분야에의 기여도 제 5 장 연구개발 결과의 활용계획 제 6 장 연구개발과정에서 수집한 해외과학기술 정보 제 7 장 참고문헌

------- 40 제2절 제2세부과제: 브랜드녹용의 품질평가 --- 49 제3절 제3세부과제: 브랜드녹용의 활성성분분석- 79 제4절 제4세부과제: 브랜드녹요의 약리효과규명- 100 제 4 장 목표달성도 및 관련분야에의 기여도

22 사육호수 사육두수 호당사육두수 호수 및 두수 두수 년도 총소비량 국산녹용소비량 소비량(kg) 년도

23 년도 생 녹 용 건 녹 용 국산 수입 계 국산 수입 계 합계 (건용) ,516 (39,167) 22,560 (8,048) 143,076 (47,215) - 67,388 67, , ,126 (36,112) 80,458 (26,551) 191,584 (63,223) - 112, , ,503 증감 - 9,390 (7.8%) 57,898 (357%) 48,508 (33.9%) - 44,892 44,892 60,900 (53.2%)

25 혼합사료 육림부산물 g,mg/dl AST ALT T-protein Albumin Cholesterol Glucose Ca P 분석항목

45 Control Treatment Elk weight (kg) Antler weight (g)

48 구기자 뽕잎 한약부산물 사 일리지 사슴번호 좌 우 평균 2487± ± 평균 3370± ± 평균 2201± ±229

65 Item AST ALT ALP TP ALB CHOL T-BIL GLU BUN CRE GGT D-BIL UA LDH CA IP TG HDL CPK A/G Before casting Control treatment 30.25± ± ± ± b ± b ± ± ± a ± ± b ± ±18.32 n.d 0.03± ± ± ± ± a ± a ± a ± ±25.12 n.d 0.003± b ± b ± b ± ± a ± ± ± a ± b ± ± ± ± ± ± a ± ±0.32 Cutting time control treatment 38.00± ± ± ± a ± a ± ± ± b ± ± a ± ± ± ± ± ± ± ± b ± b ± b ± ±9.31 n.d 0.08± a ± a ± a ± ± b ± ± ± b ± a ± ± ± ± ± ± ± ±0.10

71 g/dl 10 Total protein IU/ L 45 Alanine amino trans feras e IU /L A lk a lin e p h o s p h a t a s e C o n t r o l C L A 천 궁

72 g/dl 9 Total protein IU/L 40 Alanine aminotransferase

73 IU/L Alkaline phosphatase Control CLA 천 궁

75 velvet velvet velvet 22.74±3.37 Bab ±0.75 b ±0.81 A ±3.94 b ±0.06 Bb 17.65±4.25 b 28.99±4.43 ab 28.16±1.50 a 30.54± ±3.47 a 26.44±2.89 a 26.63±4.38 a 31.70±3.31 a 31.76±2.77 a 31.72± ±5.84 a 28.93±3.09 a 28.62±2.27 a

76 Item 구기자 뽕 잎 한약부산물 AST 57.0 b ± b ± a ±17.8 ALT 20.6 b ± b ± a ±2.5 ALP 685.0± ± ±31.0 TP 6.9± ± ±0.17 ALB 3.2± ± ±0.20 AHOL 80.3 ab ± b ± a ±9.4 TBIL 0.367± ± ±0.100 GLU ± ± ±4.583 BUN b ± a ± ab ±1.418 CRE 1.500± ± ±0.058 GGT b ± a ± a ±1.732 DBIL 0.167± ± ±0.058 UA 0.800± ± ±0.000 LDH b ± b ± a ± CA c ± b ± a ±0.115 IP 7.167± ± ±0.100 AMY b ± a ± a ±3.512 TG ± ± ±2.517 HDL b ± b ± a ±8.000 LDL a ± b ± a ±2.082 AG 0.900± ± ±0.173

81 1) Folch method(jordi Floch, M. Less et al, 1957) 1g chloroform : methanol(2 : 1) 30ml 5 (sonication) 10ml water organic layer -20. dry material percentage. 2) 10mg / 100 µl chloroformsolid phase extraction. column aminopropyl column (Alltech). Hexane 4 ml column 100 µl(10 mg) column loading chloroform : 2-propanol (15 : 1) 6 ml elution, ether : acetic acid (100 : 2) 5 ml elution free fatty acid. methanol, 4 ml hexane : 2-propanol : ethanol : 0.1M ammonium acetate :formic acid (420:350:100:50:0.5) containing 5% phosphoric acid, 4 ml elution ) chloroform HPLC,. HITACHI pump (model L-6200) ELS detector (SEDEX 75). Column Lichrosphere DIOL-100 column, column colmun heater (FIAtron)50. flow rate 0.8ml/min elution A (Hex :acetic acid, 99:1)

82 elution B (Hex : 2-propanol : acetic acid, 84:15:1) 0 min-12%b, 25 min-100%b, 26 min-100%b, 29 min-12%b gradient program. flow rate 1ml/min elution A(Hex : 2-propanol : Acetic acid, 82:17:1) elution B( 2-propanol : Water : Acetic acid, 85:14:1) 0 min-5%b, 25 min-40%b, 35min-100%B, 46 min-5%b gradient program. retention timepeak. 10 mg. < 3-5> Aminopropyl column elk Student's t-test.

83 ,,,.,,,,.,,,. velvet sponge,,, sponge ( 3-23). CP spongecrw (p < 0.001)( 3-9), CRW velvet sponge (p < 0.001) ( 3-10). CP CRW (p < 0.05)( 3-11). HS-II, CP CRW.,, CP CRW,. (sonication) Forch method. velvet sponge, velvet, sponge (Table. 3, 4, 5). velvet sponge. HS-II CP sponge (p < 0.05).

84 solid phase extraction method. ELS detector(sedere, model SEDEX75, France) HPLC (HITACHI, model L-6200, Japan),. (cholesterol ester), (triglyceride), (free fatty acid), (cholesterol) (monoglyceride) (Fig. 7). phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), sphingomyelin (SM), phosphatidyl serine (PS) (U.K) (Fig. 7). cholesterol ester, phosphatidyl choline., velvet sponge (Fig. 9). CLA.(p<0.05) velvet sponge velvet HS, HS-II, CP, CRW MC.(p < 0.05) spongehs, HS-II, CRW. CPsponge.. PA. velvet sponge.

85 PC, SM. HS-II. 3-24, 25,26,., ( 3-27).., fatty acid Lyso-form,, (Quinn MT et al, 1988) (Parthasarathy s et al, 1989) (Mandel H et al, 1998) (Anliker B et al, 2004)..

86 Carbohydate analysis < 3-23> Weights and Chemical Analysis of Antler Group GAG a Uronic Acid a Sialic Acid a V-T 0.53± ± ±0.03 V-M 0.42± ± ±0.02 Control V-B 0.40± ± ±0.01 S-T 5.29± ± ±0.02 S-M 0.82± ± ±0.02 S-B 0.83± ± ±0.02 V-T 0.54± ± ±0.03 V-M 0.47± ± ±0.01 HS-II V-B 0.53± ± ±0.01 S-T 5.55± ± ±0.08 S-M 0.98± ± ±0.01 S-B 0.59± ± ±0.02 V-T 0.67± ± ±0.04 V-M 0.48± ± ±0.01 CP V-B 0.45± ± ±0.02 S-T 6.65± ± ±0.05 S-M 1.17± ± ±0.06 S-B 0.85± ± ±0.11 V-T 0.89± ± ±0.03 V-M 0.83± ± ±0.19 CRW V-B 0.85± ± ±0.03 S-T 7.47± ± ±0.18 S-M 1.49± ± ±0.03 S-B 1.27± ± ±0.05 V-T : velvet layer top section, V-M : velvet layer middle section, V-B : velvet layer bottom section,

87 S-T : sponge layer top section, S-M : sponge layer middle section, S-B : sponge layer bottom section a percentage of dry material CE TG FFA CHOL mv 400 DG 200 MG retention time < 3-6> chromatogram CE : cholestrol ester, TG : triglyceride, FFA : free fatty acid, DG : diglyceride, CHOL : cholesterol, MG : monoglyceride

88 PC 400 PE PS mv 300 SM LPC PA retention time chromatogram PA : phosphatidic acid, PE : phosphatidyl ethanolamine, PC : phosphatidyl choline SM : sphingomyelin, PS : phosphatidyl serine, LPC :lyso-phosphatidyl choline

89 DG TG SM PC mv x X sec Sec CE LPC FFA PS CHOL PE PA MG Pneutral h osph olipids s ( ( ug u g ) ) < 3-8> percentage of dry material V-T V-M V-B S-T S-M S-B 0 control HS-II CP CRW Group < 3-9> Comparison of glycosaminoglycan from different fodders-treated antlers.

90 *p < 0.05, **p < 0.01, ***p < percentage of dry material V-T V-M V-B S-T S-M S-B 0 control HS-II CP CRW Group < 3-10> Comparison of uronic acid from different fodders-treated antlers. *p < 0.05, **p < 0.01, ***p < 0.001

91 1.0 percentage of dry material V-T V-M V-B S-T S-M S-B 0.0 control HS-II CP CRW Group < 3-11> Comparison of sialic acid from different fodders-treated antlers. *p < 0.05, **p < 0.01, ***p < 0.001

92 ) lipid analysis U.K PC mv PE SM PS PA Retention Tim e CE FFA 800 mv 600 C H O L 400 TG Retention tim e < 3-12> HPLC chromatography of neutral lipids and phospholipids from antler

93 <표3-24> Phospholipid composition of antler-i group TL / DM a PL / TL b PA c U.K c PE c PC c SM c PS c V-T 81.4± ± ± ± ± ± ± ±4.7 V-M 108.7± ± ± ± ± ± ±15.0 n.d control V-B 106.1± ± ± ± ± ± ±11.6 n.d S-T 41.0± ± ± ± ± ± ± ±3.4 S-M 29.8± ± ± ± ± ± ± ±6.9 S-B 24.2± ± ± ± ± ± ± ±1.3 V-T 81.5± ± ±4.5* 177.8± ± ± ± ±5.7* V-M 105.9± ± ±14.4* 139.9± ± ± ± ±23.3* JW V-B 90.6± ± ± ± ± ± ± ±9.6* S-T 38.6± ± ±18.8* 213.4±15.4* 231.9± ± ± ±13.2* S-M 28.5± ± ±4.5* 192.9±10.2* 143.6±8.0* 354.0±13.9* 236.3± ±14.3* S-B 24.4± ± ±3.1* 154.5±9.7* 129.2±8.6* 311.7± ± ±4.5* V-T 84.7± ±0.09* n.d 258.6±37.6* 341.5±51.6* 590.6± ±15.0 n.d V-M 117.1± ±0.09 n.d 163.6± ±26.3* 342.2±26.6* 156.5±17.5 n.d HS V-B 107.9± ±0.05 n.d 160.7± ± ± ±16.1 n.d S-T 39.8± ±0.10* 117.9± ±43.0* 371.7±41.1* 777.4±65.2* 315.6± ±8.0* S-M 28.8± ±0.09* 117.7± ± ±28.2* 672.3±42.9* 290.1± ±0.9* S-B 22.4± ±0.10* 238.6±1.9* 234.9± ± ±91.1* 221.2± ±4.2* V-T 87.7± ±0.14* n.d 172.2± ± ±52.2* 242.4± ±0.9* V-M 112.2± ±0.06* n.d 109.3±8.8* 114.4±5.6* 181.0±40.1* 172.4± ±4.5* MC V-B 119.3± ±0.03* n.d 129.8± ±7.7* 151.8±28.2* 147.9±1.8* n.d S-T 40.2± ±0.02* n.d 242.6± ±9.7* 661.1± ± ±9.7* S-M 32.5± ±0.15 n.d 253.9± ±18.8* 514.1± ± ±4.8 S-B 25.9± ± ±8.5* 251.2± ± ±34.0* 242.3±24.0 n.d a total lipids in dry material (mg / g), b phospholipids in total lipids (mg / 10mg),

94 c each phospholipid in total lipids (ug / 10mg), * p < 0.05 <표 3-25> Phospholipid composition of antler-ii group TL / DM a PL / TL b PA c U.K c PE c PC c SM c PS c V-T 81.4± ± ± ± ± ± ± ±4.7 V-M 108.7± ± ± ± ± ± ±15.0 n.d control V-B 106.1± ± ± ± ± ± ±11.6 n.d S-T 41.0± ± ± ± ± ± ± ±3.4 S-M 29.8± ± ± ± ± ± ± ±6.9 S-B 24.2± ± ± ± ± ± ± ±1.3 V-T 75.5± ±0.04* n.d 257.6±19.4* 272.3± ±29.4* 280.0±20.1* 104.8±1.3* V-M 117.2± ±0.04 n.d 176.1± ± ± ±25.1 n.d HS-II V-B 97.1± ±0.01 n.d 159.2± ± ± ±21.3 n.d S-T 31.2±2.4* 2.14±0.09* n.d 415.5±24.5* 405.8±38.4* 811.3±15.6* 400.4±18.3* 110.8±0.9* S-M 19.1±1.2* 1.73±0.03* 79.1±9.2* 275.2± ±19.3* 690.3±2.4* 319.6±17.9* 104.2±14.7* S-B 14.4±1.7* 1.48±0.07* 239.3±2.7* 216.6± ± ±15.4* 236.6± ±5.8* V-T 74.2± ±0.05* n.d 226.7± ± ± ±40.0* 110.5±9.4* V-M 105.4± ±0.07 n.d 148.0± ± ± ±21.9* n.d CRW V-B 116.3± ±0.06* n.d 133.4± ±8.2* 181.3± ±15.0* n.d S-T 37.6± ±0.03* 259.8±11.5* 206.0±16.3* 207.3± ±26.9* 245.2± ±13.6 S-M 24.8± ±0.11* 293.9±20.2* 194.8±58.2* 151.0±33.6* 586.9±33.9* 371.7±66.2* 65.1±12.1* S-B 22.0± ±0.18* 314.8±6.9* 225.6± ± ± ±52.1* n.d V-T 60.4± ±0.04* n.d 214.2± ± ±26.3* 269.9±24.1* 104.3±2.2* V-M 93.0± ±0.03 n.d 158.2± ±17.5* 253.7± ±21.9 n.d CP V-B 92.7± ± ± ± ± ± ±15.7 n.d S-T 33.8± ±0.09* 245.2±2.6* 354.1±25.6* 403.5±21.2* 744.1±67.5* 363.0±9.9* 116.3±2.1* S-M 22.9±4.3* 1.73±0.08* 264.6±6.1* 258.3±3.9* 201.9± ±54.6* 299.2± ±7.8* a S-B 20.0± ±0.05* 263.4±6.1* 260.1± ± ±45.4* 257.0±16.1* 102.9±2.2* total lipids in dry material (mg / g), b phospholipids in total lipids (mg / 10mg),

95 c each phospholipid in total lipids (ug / 10mg), * p < 0.05 group TL / DM a PL / TL b PA c U.K c PE c PC c SM c PS c T 65.9± ± ± ± ± ± ± ±11.4 control M 53.6± ± ± ± ± ± ± ±8.6 B 42.7± ± ± ± ± ± ± ±10.9 T 73.2± ±0.06* 80.6± ±25.5* 412.8±47.3* 554.3± ±21.5* 146.7±10.8* CLA M 49.7± ±0.03* 81.9± ± ± ± ± ±8.0* B 38.3± ±0.04* 153.0± ±16.3* 207.3±26.7* 466.1± ± ±14.7 T 68.9± ± ±21.0* 220.1± ± ± ± ±5.5 Coumarin M 50.3± ± ±21.1* 210.9± ± ± ± ±8.5 B 47.6± ± ± ± ± ± ± ±17.2 a total lipids in dry material (mg / g), b phospholipids in total lipids (mg / 10mg), c each phospholipid in total lipids (ug / 10mg), * p < 0.05

97 < 3-27> Neutral lipids composition of antler-iii group TL / DM NL / TL CE TG FFA CHOL T 65.9± ± ± ±51 438±11 683±78 control M 53.6± ± ± ±67 336±25 609±106 B 42.7± ± ± ±26 355±45 630±89 T 73.2± ± ± ±9 283±26 568±41 CLA M 49.7± ± ± ±7 248±65 525±85 B 38.3± ± ± ±28 290±47 659±36 T 68.9± ± ± ±46 428±18 767±75 Coumarin M 50.3± ± ±86 243±23 258±42 627±32 B 47.6± ± ± ±28 239±25 542±80 a total lipids in dry material (mg / g), b phospholipids in total lipids (mg/10mg), c each phospholipid in total lipids (ug / 10mg), * p < 0.05

98 2 PL in TL ( mg / 10mg ) 1 Top Middle Bottom 0 control CLA coumarin group < 3-13> Phospholipids in total lipids-i *p < 0.05, **p < 0.01, ***p < 0.001

99 3 PL in TL ( mg / 10mg ) 2 1 V-Top V-Middle V-Bottom S-Top S-Middle S-Bottom 0 control JW HS MC HS-II CRW CP group < 3-14> Phospholipids in total lipids-ii *p < 0.05, **p < 0.01, ***p < 0.001

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107 Weight G1(N C) G2(T T) G3(일반) G4(중원) G5(호산) G6(청풍) G7(청록원) W eek 30.0 Intake G1(N C) G2(T T) G3(일반) G4(중원) G5(호산) G6(청풍) G7(청록원) W eek

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136 The study on the production of domestic brand velvet antler and its active constituents and pharmaceutical efficacy

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical