25 DOI : 10.3831/KPI.2008.11.4.025 A Study on Subchronic Toxicity Test and Method of Increasing Output of Scolopendrid Pharmacopuncture Kim Sung-chul *Dept. of Acupuncture & Moxibustion, Gwang-Ju Oriental Medical, Hospital in WonKwang University ABSTRACT Purpose Methods Results Conclusions The purpose of this study was to investigate sub-chronic toxicity of scolopendrid pharmacopuncture in mouse and method of increasing output of scolopendrid pharmacopuncture. In order to prove the clinical safety of scolopendrid pharmacopuncture during 90 days, We have observed the physical reaction(side effect) and clinical pathology test after scolopendrid pharmacopuncture treatment and investigated method of increasing Output of scolopendrid pharmacopuncture for 90%, 80%, 70% ethanol. In subchronic toxicity test, there was no significant sign in clinical sign, opthalmological values, body weights, hematological values and urinalysis values. And we could see that food consumptions and water consumptions increased significantly, albumin, triglycerides, GPT in blood chemical values and Liver, Testis(right) in organ weights changed significantly in some groups, compared with those in the S1 group. But these changes were observed within the scope of physiology. So there was no sign of toxication in subchronic toxicity test, and we can tell that NOAEL(No Observed Adverse Effect Level) is above 0.286mg/kg/day. And 70% ethanol solution of scolopendrid was yielded the most amount of substance. This study demonstrates that scolopendrid pharmacopuncture is to treatment of safety for a long time and we can obtain much amount from 70% ethanol solution of scolopendrid. Key words LD50, sub-chronic toxic test, scolopendrid pharmacopuncture I Scolopendra subspinipes mutilans L. Koch Correspondence : Kim Sung-chul, Dept. of Acupuncture & Moxibustion, Gwang-Ju Oriental Medical, Hospital in WonKwang University Tel. 062-670-6442 E-mail : kscndl@hanmail.net This research was supported by KPI(Korea Pharmacopuncture Institute) in 2007. Received - 08.11-25 Revised - 08.11-28 Accepted - 08.11-30
26 11 4 2008 12 4800mg/kg histamine L5 CT MRI LD50 30mg/kg II 1. 40g ICR mouse 2 2. Alcohol 128.6 g 1000 Electronic magnetic stirrer Tost MS 300, KOREA 3 Electronic magnetic stirrer 3 105 ~107, 5-6 2 8 Rotary Vacuum evaporator EYELA, JAPAN 70 3 90% 1 2 8 Rotary Vacuum evaporator 80% 1 2 8 Rotary Vacuum evaporator 70% 1 2 8 Rotary Vacuum evaporator Rotary Vacuum evaporator 200 300 2 0.45, 0.1 Freezing dryer KOREA 200 9.88g 7.68% 128.6g 1000 Electronic magnetic stirrer 3
27 3. 1 0.1mg/kg 0.5mg/kg, 1mg/kg SP- R4 8 8.75 /g 13 2 30 1 mouse 2 30 8 1 1 Ether HEMAVET 950, U.S.A. DRI-CHEM 4000i, Fuji 2-1. Electronic magnetic stirrer 2 105 ~107, 5-6 90% 80% 70% 8 Rotary Vacuum evaporator 200-300 8 2 0.45 0.1 1 Freezing dryer 200 9.88g 7.68% Scheme 1. Manufacturing process of Scolopendrid pharmacopuncture 2-2. 1 2-3. 1 2-4. HEMAVET 950, U.S.A. Total erythrocyte count, RBC, Hemoglobin, Hb, Hematocrit, HCT, Mean cell volume, MCV, Mean cell hemoglobin, MCH, Mean cell hemoglobin concentration, MCHC, Total leucocyte count, WBC Platelet, PLT 2-5. vacutainer tube vacutainer, BECTON DICKINSON, U.S.A. 3000rpm 5 Total protein, Albumin, BUN, Creatinine, Triglyceride, Total cholesterol, GOT, GPT, A/G ratio DRI-CHEM 4000i, Fuji 2-6. Bayer Diagnostics Multistix 10SG Not. 5J06C, U.S.A REF 2300 03536597 Clinitek STA- TUS, U.S.A specific gravity, ph, leukocyte, nitrite, protein, glucose, ketone, urobilinogen, bilirubin, blood 2-7.
28 11 4 2008 12 2-8. ether 2-9. 3 Levene s test one way ANOVA test Scheffe SPSS Ver 10.1 III 0.1mg/kg 0.5mg/kg, 1mg/kg 8.75 /g 13 2 1. Table 1 Table 1. Mortality and Clinical signs of mice with injected SP for 13 weeks Group Dose Level Mortality % (dead/total N) Signs 0 0(0/8) NAD 100 0(0/8) NAD 500 0(0/8) NAD 1000 0(0/8) NAD N : Animal Numbers NAD : No Abnormality Detected 2. 13 R4 7 Table 2 Table 2. Body weight of mice with injected SP for 13 weeks (g) Weeks of treatments 0 1 2 3 4 5 6 7 8 9 10 11 12 13 28.1 31.6 35.0 39.0 39.9 41.6 40.5 41.8 42.0 43.8 44.9 46.1 46.1 45.8 0.65 0.42 0.88 1.14 1.49 1.74 1.25 1.52 1.43 1.69 2.13 2.08 2.19 2.83 8 8 8 8 27.9 31.8 35.6 38.4 40.1 40.5 41.6 42.0 42.5 43.1 44.5 45.6 44.8 44.9 0.65 0.35 0.60 0.86 0.98 0.90 0.81 0.90 1.14 0.96 1.29 1.12 1.16 1.92 8 8 8 8 28.1 32.1 36.0 39.4 40.6 41.5 42.0 42.8 43.8 44.4 44.9 45.8 45.6 45.6 0.62 0.49 0.83 1.19 1.35 1.73 1.77 1.70 2.15 1.84 1.70 1.78 1.75 2.56 8 8 8 8 27.6 31.9 36.1 38.4 39.5 40.9 41.3 40.1 41.6 43.3 44.1 44.5 45.6 45.6 0.83 0.32 0.43 0.67 0.86 0.74 0.63 0.91 1.09 0.75 1.10 1.03 1.89 2.06 8 8 8 8
29 3. R4 5 R1 R3 4 R4 6 7 13 Table 3, 4 Table 3. Food consumptions of mice with injected SP for 13 weeks(g) Mean Food Consumption(g/mice/day) during Week 1 2 3 4 5 6 7 8 9 10 11 12 13 5.8 5.9 6.0 7.1 6.3 5.1 5.2 7.1 6.4 5.5 5.8 5.7 5.7 0.56 0.41 0.26 0.00 0.10 0.29 0.17 0.98 0.71 0.29 0.41 0.41 0.43 8 8 8 5.9 5.9 5.8 6.3 6.0 5.7 5.4 5.7 6.3 5.4 5.8 5.9 6.0 0.59 0.39 0.20 0.39 0.21 0.51 0.13 0.38 0.69 0.32 0.45 0.50 0.54 8 8 8 6.1 6.0 6.0 6.5 6.4 5.8 5.3 5.9 6.9 5.6 5.9 6.0 6.1 0.59 0.43 0.27 0.45 0.20 0.47 0.02 0.39 0.76 0.33 0.35 0.43 0.47 6 6 6 6 6 6 6 6 6 6 6 6 6 6.5 6.1 5.8 6.5 6.7* 5.9 5.3 7.2 6.8 6.0 6.3 6.2 6.4 0.70 0.43 0.16 0.47 0.13 0.50 0.11 1.01 0.76 0.45 0.62 0.62 0.69 8 8 8 *, significantly different from values of group at P 0.05. Table 4. Water consumptions of mice with injected SP for 13 weeks(ml) Mean Water Consumption (ml/mice/day) during Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14.5 13.7 13.0 9.9 9.0 9.3 5.2 8.2 5.4 11.5 6.5 8.3 8.6 0.85 0.98 1.11 0.12 0.61 1.61 0.36 1.42 0.02 2.85 0.75 0.48 0.46 8 8 8 13.5 12.6 11.7 8.9 7.5 12.0 5.3 6.6 4.3 9.1 6.3 6.8 8.2 0.48 0.47 0.46 0.69 0.88 0.63 0.27 0.66 0.13 1.96 0.94 0.82 0.14 8 8 8 13.8 11.8 9.8 12.3* 6.8 11.8 5.3 7.0 4.9 8.5 6.6 8.8 8.5 0.49 0.95 1.40 1.05 1.30 0.85 0.17 0.52 0.35 1.28 0.79 0.07 0.13 8 8 8 14.5 12.2 9.8 11.8 9.0 12.9* 7.5* 8.3 8.8 8.9 10.5 14.1 15.0** 0.36 0.90 1.44 1.88 1.10 0.42 0.88 1.33 1.37 1.59 2.35 3.73 1.89 8 8 8 *, **, significantly different from values of group at P 0.05, P 0.01.
30 11 4 2008 12 4. Table 5 Table 5. Hematological values of mice with injected SP for 13 weeks WBC (K/ul) RBC (M/ul) Hb (g/dl) HCT (%) MCV (fl) MCH (pg) MCHC (g/dl) PLT (K/ul) 3.2 9.4 12.0 46.5 49.2 12.8 25.6 924.0 0.58 0.47 1.34 3.47 2.33 1.26 1.52 134.3 8 8 8 8 8 8 8 8 3.8 10.9 12.8 51.0 50.4 12.9 25.6 1286.5 0.77 1.42 1.29 4.31 2.41 1.36 2.15 360.2 8 8 8 8 8 8 8 8 3.6 9.5 12.7 46.6 49.6 13.6 27.2 1059.5 0.81 0.42 0.68 1.66 2.50 1.05 0.78 130.5 8 8 8 8 8 8 8 8 4.1 9.8 13.2 49.0 50.7 13.7 26.7 870.1 1.02 0.40 0.78 1.69 2.42 1.09 0.95 166.8 8 8 8 8 8 8 8 8 5. Albumin R2 R1 Triglycerides R2 R3 GPT R4 Table 6 Table 6. Blood Chemical values of mice with injected SP for 13 weeks Total protein Albumin A/G ratio Creatinine BUN Total cholesterol Triglyceri des (g/dl) (g/dl) (g/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (U/L) (U/L) GOT GPT 4.8 2.3 1.0 0.4 27.1 115.0 101.1 159.5 51.3 0.20 0.08 0.09 0.09 2.05 21.27 7.78 17.00 3.79 8 8 8 8 8 8 8 8 8 4.6 2.1* 0.8 0.4 31.2 94.0 143.8** 236.4 79.0 0.11 0.07 0.05 0.07 1.18 10.52 11.04 36.46 21.87 8 8 8 8 8 8 8 8 8 4.9 2.2 0.9 0.2 27.0 118.8 144.6* 196.9 72.3 0.20 0.06 0.06 0.05 2.71 11.84 15.85 25.31 14.90 8 8 8 8 8 8 8 8 8 5.3 2.4 0.8 0.3 30.8 103.9 93.8 152.1 28.3** 0.15 0.21 0.10 0.07 3.76 2.94 10.05 14.77 3.57 8 8 8 8 8 8 8 8 8 *, **, significantly different from values of group at P 0.05, P 0.01.
31 6. Table 7 Table 7. Urinalysis values of mice with injected SP for 13 weeks GLU (Glucose) BIL (Bilirubin) KET (Ketone) SG (Specific Gravity) BLO (Blood) ph PRO (Protein) (mg/dl) URO (Urobilinogen) (E.U./dL) Animal No. (0) (100) (500) (1000) 8 8 8 8-3 2 4 4 -+ 100 5 6 4 4 + 250 negative 3 4 4 4 + s 2 1 2 ++ m 1 4 3 2 +++ l 2 negative 1 2 -+ t 2 2 1 + 15 3 5 3 4 ++ 40 2 3 3 1 1.010 4 2 2 4 1.015 2 2 1.020 2 2 2 1.025 4 2 2 >=1.030 2 negative 8 8 8 8 -+ intact -+ lysed + s ++ m +++ l 5.5 6 6.5 2 7 3 4 7.5 1 2 2 8 8.5 1 1 2 =9 4 3 3 4 - -+ t + 30 4 2 4 4 ++ 100 4 6 4 4 +++ =300 0.2 NIT 1 2-5 5 6 5 (Nitrite) + 3 3 2 3-7 8 7 5 LEU (Leukocytes) -+ t + s 1 1 3 ++ m +++ l 7. Table 8
32 11 4 2008 12 Table 8. Opthalmological values of mice with injected SP for 13 weeks Group Dose NO pthalmological examination 0 8 NAD 100 8 NAD 500 8 NAD 1000 8 NAD N : Animal Numbers NAD : No Abnormality Detected 8. Liver R2 R1 Testis Right R4 Table 9,10 9. Absolute organ weights of mice with injected SP for 13 weeks Body weight Liver Kidney Lt. Rt. Spleen Testis Lt. Rt. Brain Lung Heart (g) (mg) (mg) (mg) (mg) (mg) (mg) (mg) (mg) (mg) 45.8 1906.6 337.1 322.7 118.2 139.5 134.3 503.8 203.3 184.4 2.83 52.71 36.45 39.51 11.95 6.71 5.82 13.77 17.61 21.12 44.9 2264.2** 322.3 302.6 115.0 137.3 127.3 508.5 208.2 192.5 1.92 83.49 26.23 27.69 10.76 9.66 8.80 16.93 19.20 21.18 45.6 1996.6 321.5 298.8 123.5 135.0 129.2 507.1 211.2 191.1 2.56 70.86 30.64 24.42 11.33 8.69 4.56 7.27 17.51 23.18 45.6 1854.0 323.5 317.0 131.0 125.1 119.8* 498.3 203.1 185.0 2.06 136.82 32.22 33.23 15.23 3.46 4.22 16.31 13.79 16.79 *, **, significantly different from values of group at P 0.05, P 0.01. Table 10. Relative organ weights of mice with injected SP for 13 weeks Body weight Liver Kidney Lt. Rt. Spleen Testis Lt. Rt. Brain Lung Heart (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) 45.8 4.26 0.73 0.69 0.26 0.31 0.30 1.12 0.44 0.40 2.83 0.27 0.04 0.05 0.02 0.02 0.02 0.05 0.02 0.02 44.9 5.09* 0.71 0.67 0.25 0.31 0.28 1.14 0.46 0.42 1.92 0.25 0.04 0.04 0.02 0.02 0.01 0.05 0.03 0.03 45.6 4.45 0.70 0.65 0.27 0.30 0.29 1.13 0.46 0.41 2.56 0.28 0.03 0.02 0.01 0.02 0.01 0.06 0.02 0.03 45.6 4.13 0.70 0.69 0.29 0.28 0.27 1.10 0.44 0.40 2.06 0.35 0.05 0.05 0.03 0.01 0.01 0.03 0.02 0.03 *, significantly different from values of group at P 0.05,
33 9. 90% 80% 70% Lowry Assay 10g/500ml 2 1 80% 6.20mg/g 70% 6.05mg/g 90% 5.85mg/g 2 5ml 70% 0.207mg/ml 80% 0.141mg/ml 90% 0.102mg/ml 70% 70%, 80% 2 5ml 1 3 3 70% 3 1 70% 70% 57 105-107 5-6 50 Figure 1. Scolopendrid solution for 90%, 80%, 70% ethanol before experiment Figure 2. Scolopendrid solution for 90%, 80%, 70% ethanol after 1 month Figure 3. 90% ethanol solution of scolopendrid after 1 month Figure 4. 80% ethanol solution of scolopendrid after 1 month Figure 5. 70% ethanol solution of scolopendrid after 1 month 80% 70% 90% 80% 70% 80% 70% Figure 1-5 1000ml -70 EYELA FDU-2100, Japan 105, 10 90% 80%, 70% 70% 80% 40-50 57
34 11 4 2008 12 3 70% Figure 6 Figure 6. Scolopendrid extract for 90%, 80%, 70% ethanol after freezing dryness IV 24 Organization for Economic Cooperation and Development; OECD National Academy of Science; NAS 6 OECD 10% 10 4800mg/kg 0.03g 10cc
35 LD50 30mg/kg Hep G2 L929 Peritoneal macrophage nitric oxide Table 1-10 LD50 50% LD50 30mg/kg R4 5 R3 4 R4 6 7 13 Albumin R2 Triglycerides R2 R3 GPT R4 Liver R2 Testis Right R4 13 70 mouse 18 90 mouse 12% 2,970 0.1mg/kg, 0.5mg/kg, 1mg/kg 13 2 2mg/kg/week NOAEL; No Observed Adverse Effect Level 0.286mg/kg/day Alcohol 105 ~107, 5-6 57 105-107 5-6 50 90% 90% 80% 70% Lowry Assay 10g/500ml 1 2 2 5ml 2 5ml 70% 0.207mg/ml 80%
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