Materials
Tripterygium wilfordii, is a plant of the genus Tripterygium of the family Celastraceae, is a root of a perennial liana. In this study, the crude material of Tripterygium wilfordii was collected in the early spring and at the end of summer from Ren-ai Township, Nantou County and Shih-ding Township, Taipei County.
After identify the origin of Tripterygium wilfordii, used methanol to extract.
Animals and treatment regimens
It was separated to two parts. The first part was acute toxicity test (LD50) and the second part was 28-day treatment to demonstrate the relationship between dosage and toxicity. Crude material of Tripterygium wilfordii was collected in different seasons for the acute toxicity test. Methanol extracts from different part of Tripterygium wilfordii, such as root cortex, stem etc, were given orally to mice for the acute toxicity test and 28-day-treatment in rats for the evaluation of dosage and toxicity.
In the acute toxicity test was used male ICR-mice to survey the sublethal dose.
The animals were randomly divided into nine groups. The groups and dosages were described in tab. 2 .
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In the 28-day-treatment in rats for the evaluation of dosage and toxicity, the animals were randomly divided into ten groups. Group 1 is blank, treated with vehicle. Groups 2 to 4 were treated with extraction of stem in three different dosages, stem low dose (SL), stem middle dose (SM) and stem high dose (SH).
Groups 5 to 7 were treated with extraction of root in three different dosages, root low dose (RL), root middle dose (RM) and root high dose (RH). The final three groups were treated with root cortex low dose (CL), root cortex middle dose (CM) and root cortex high dose (CH).
Male Sprague–Dawley rats, weight 250 to 300g, purchased from BioLASCO Taiwan Co., Ltd were used in this study. Rats were housed 2–3 per cage with food and water available ad libitum, on a 12:12 h light:dark schedule (lights on 0800 h). Room/cage temperature was maintained at 23±1 ℃. The experiment was started after two-week adaptation of the environment for all rats.
I. Before the experiment, the rats were fasted for 12 to 16 hours. The treatment regimens were parallel test, the animals were randomly assigned to ten groups, each group consisted of eight rats.
i. Control group: treated with saline, PO. 0.1 ml/kg.
ii. Tripterygium wilfordii stem extraction groups: treated with three different dosages, PO.
iii. Tripterygium wilfordii root extraction groups: treated with three different dosages, PO.
iv. Tripterygium wilfordii root cortex extraction groups: treated with three different dosages, PO.
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The dosages treated rats in 28-day treatment were chose from a fifth sublethal dose.
The groups and dosages were described in the table below.
Group Appellation No. Dose (mg/kg)
Group 1 Blank B1~B8 Saline
Group 2 Stem low dose SL1~SL8 12
Group 3 Stem middle dose SM1~SM8 60
Group 4 Stem high dose SH1~SH8 300
Group 5 Root low dose RL1~RL8 16
Group 6 Root middle dose RM1~RM8 80
Group 7 Root high dose RH1~RH8 400
Group 8 Root cortex low dose CL1~CL8 12 Group 9 Root cortex middle dose CM1~CM8 30 Group 10 Root cortex high dose CH1~CH8 150
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II. The rats were orally treated with crude extracts of the Tripterygium wilfordii in different dosage for 28 days as previously described. The toxic responses of rats in different groups were observed and recorded daily, if any rat died during the experiment period will be anatomized.
Animals were observed daily and recognized the change in its skin, hair, eyes and mucous membrane. And recorded of the time when the respiratory system, circulatory system, nervous system, activity of limbs, behavior changed, and recorded the degree and the duration of the change. The consumption of water and food was recorded every day, and the body weights of rats were weighted weekly. If the rats died during the experiment period, rats were sent to be anatomized right away or refrigerated then be anatomized.
After 28 days of treatment with crude extracts of the Tripterygium wilfordii, the animals were sacrificed by over dose of Zoltil®,
i. Blood and urine test
1. Blood routine test: After experiment period, blood collected from all the animals and tested the A/G, RBC and WBC etc.
2. Blood biochemistry test: After experiment period, blood collected from all the animals and tested the ALT, AST, BUN, Cr, Alb and TP.
3. Urine test: Before sacrifice, collected the urine from all the animals.
The test including pH volume, urine protein, urine sugar and blood cell in urine.
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ii. Gross anatomy and Pathologic histology
1. Gross anatomy: All the animals were going gross anatomy, examined the skull, thorax, abdominal cavity and the tissue inside. Weighed the organs such as liver, lung and kidney.
2. Pathologic histology: The animals in each group had quickly
dissected the organs such as liver, lung, heart, brain etc. These tissues and ever organ had to do pathological sections and stained. Then recognized the pathological change in rats, especially in liver and kidney, to evaluate the toxicity of Tripterygium wilfordii.
If organ or tissue had observed pathological changes in high dose, we observed the corresponding organ and tissue in middle and low dose group.
3. Pathologic test: After weighing, the liver and kidney were fixed in 10% formaldehyde, and cut a piece in the place then took into a tissue embedding cassette. After dehydrated over night, used paraffin wax to embed, stored at -20 and to take ℃ pathological sections. In order to fix and dry the paraffin wax the sections were put into oven, then stained by H/E. The sections were observed by microscope.
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Results
I. The collections of Tripterygium wilfordii:
After collected the fresh Tripterygium wilfordii, we compared the
configuration and characteristic marks of the fresh Tripterygium wilfordii with the describption in Flora of Taiwan. After it had been confirmed, then took the crude drug organized sections to confirm the origin. The description about Tripterygium wilfordii in Flora of Taiwan is below.
Fig. 1. The drawing paint of Tripterygium wilfordii in Flora of Taiwan III.
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II. Prepartion of Tripterygium wilfordii and acute toxicity test i. Preparation for Tripterygium wilfordii
The crude material of Tripterygium wilfordii, including stem, leaf, root, cortex, and commercial products, were soaked in methanol separately.
The methanol had to cover up Tripterygium wilfordii. After infiltration for three days, the methanol extract was filtered and then repeated the protocol twice, three times in all. The methanol extracts were collected and evaporated under vacuum. The extracts were prepared for acute toxicity test.
Yields and extractions rate of Tripterygium wilfordii were described in the following table (Tab. 1).
Tab. 1. The extractions rate of Tripterygium wilfordii.
Material collect time and material part for extraction
Weight before
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ii. Acute toxicity test
According to the results from acute toxicity test, the toxicity was
different as the collection of Tripterygium wilfordii in different seasons.
The crude extracts from collection of Tripterygium wilfordii in the early spring were almost nontoxic, as the LD50s of extraction of stem and leaf were over 10g/kg. The collection of Tripterygium wilfordii at the end of summer revealed different LD50s. The LD50s of stem and leaf are 3.53 g/kg and 5.06 g/kg, respectively. The LD50s of root without cortex and cortex are 3.95 g/kg and 1.67 g/kg, respectively. The commercial
products we bought in China, the LD50 of the extraction was over 10 g/kg.
Results of acute toxicity test are showed below.
Material source and
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iii. Evaluation of the toxicity
The sublethal doses of the different parts of Tripterygium wilfordii were showed below.
High Dose Middle dose Low Dose
Stem 300 mg/kg 60 mg/kg 12 mg/kg
Root 400 mg/kg 80 mg/kg 16 mg/kg
Cortex 150 mg/kg 30 mg/kg 6 mg/kg
To evaluate the hepatoxicity/renal toxicity in rats, we didn’t find the obvious pathological change in the liver and kidney.
The results of histopathological finding of the kidney and liver were shown in table 2 to table 4 below.
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Tab. 2. The samples of mice’s pathological organization and serial numbers
Group Sample code Submitted samples
1 Stem 1.25 g/kg Liver, kidney
2 Leaf 7.5 g/kg
3 Root 1.25 g/kg
4 Cortex 1.25 g/kg
5 Cortex 0.625 g/kg
6 Root 2.5 g/kg
7 Stem 2.5 g/kg
8 Leaf 2.5 g/kg
9 Blank
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Tab. 3. The standard of pathological assay Observation fate:
No significant lesions (NSL)
Modification: Degeneration, Necrosis, …
Distribution: Focal, Multifocal, Local Extensive and Diffuse
Degree: Minimal, slight, Moderate, Moderate/Severe and Severe/High Duration: Acute, Subacute, and Chronic
Exudate: Serous, Fibrinous, and Purulent
Severity of lesions was graded according to the methods described by Shackelford et al. (2002) with modification (Toxicologic Pathology 30; 93-96, 2002). Degree of lesions was graded from one to four depending on severity: 1 =minimal (<1%); 2 =slight (1-25%); 3 =moderate
(26-50%); 4 =moderate/severe (51-75%); 5 =severe/high (76-100%).
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Tab. 4. Summary of pathological incidence in mice
Organ Histopathology Group
1 2 3 4 5 6 7 8 9 Kidney
Degeneration/necrosis, dilation, distal tubule, focal, slight1
- - - - 1/1# - - - -
Regeneration, tubule, focal, minimal
- - 1/1 - - -
Liver - - - - - - - - -
-: No significant lesions.
1: Degree of lesions was graded from one to five depending on severity: 1 =minimal (<1%); 2
=slight (1-25%); 3 =moderate (26-50%); 4 =moderate/severe (51-75%); 5 =severe/high (76-100%).
#Incidence: Affected mice/Total examined mice (n=1).
Comments:
Lesions of kidney were graded minimal to slight and were considered as non-specific lesion in mice.
According to the result of histopathological finding of the kidney and liver, treated with the subleathal dose of the extraction of Tripterygium wilfordii, including stem, leaf, root and cortex, would not make lesions in the liver or kidney.
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Appendix 1. Pathology – individual micro findings of mice.
Organ Histopathological finding Animal code 1 2 3 4 5 6 7 8 9
Kidney - - - - - - -
Degeneration/necrosis, dilation, distal tubule, focal
2 Regeneration, tubule, focal 1
Liver - - - - - - - - -
-: No significant lesions, NSL. N: no section.
1: Degree of lesions was graded from one to five depending on severity: 1 =minimal (<1%); 2
=slight (1-25%); 3 =moderate (26-50%); 4 =moderate/severe (51-75%); 5 =severe/high (76-100%).
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Fig.2. Histopathological finding of the kidney and liver in groups 1 (stem 1.25g/kg) and 2 (leaf 7.5g/kg) treated mice. No significant lesions was noted in the kidney (A. 40x, B. 100x, C. 400x) and liver (D. 40x, E, 100x, F. 400x). H&E stain.
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Fig. 3. Histopathological finding of the kidney and liver in groups 3 (root 1.25g/kg) and 4 (cortex 1.25g/kg) treated mice. Focal, minimal tubular regeneration was found in the Group 3.
No significant lesions was noted in the kidney (A. 40x, B. 100x, C. 400x) and liver (D. 40x, E, 100x, F. 400x) in group 4. H&E stain.
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Fig. 4. Histopathological finding of the kidney and liver in groups 5 (cortex 0.625g/kg) and 6 (root 2.5g/kg) treated mice. Focal, slight tubular dilation and degeneration were found in the Group 5. No significant lesions was noted in the kidney (A. 40x, B. 100x, C. 400x) and liver (D.
40x, E, 100x, F. 400x) in group 6. H&E stain.
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Fig. 5. Histopathological finding of the kidney and liver in groups 7 (stem 2.5g/kg) and 8 (leaf 2.5g/kg) treated mice. No significant lesions was noted in the kidney (A. 40x, B. 100x, C. 400x) and liver (D. 40x, E, 100x, F. 400x) in group 7 and 8. H&E stain.
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Fig.6. Histopathological finding of the kidney and liver in group 9 (control group) mice. No significant lesions was noted in the kidney (A. 40x, B. 100x, C. 400x) and liver (D. 40x, E, 100x, F. 400x) in group 9. H&E stain.
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iv. The effect of blood, urine and the relationship between dosage and
hepatoxicity/renal toxicity in rats, after PO. Tripterygium wilfordii for 28 days.
The groups of rats treated with the extractions of the stem, root and cortex of Tripterygium wilfordii in three different dosages were summarized below.
Group Appellation No. Dose (mg/kg)
Group 1 Blank B1~B8 Saline
Group 2 Stem low dose SL1~SL8 12
Group 3 Stem middle dose SM1~SM8 60
Group 4 Stem high dose SH1~SH8 300
Group 5 Root low dose RL1~RL8 16
Group 6 Root middle dose RM1~RM8 80
Group 7 Root high dose RH1~RH8 400
Group 8 Root cortex low dose CL1~CL8 12
Group 9 Root cortex middle dose CM1~CM8 30
Group 10 Root cortex high dose CH1~CH8 150
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The rats treated with the extractions of the stem, root and cortex of
Tripterygium wilfordii in three different dosages in the morning continuously for 28 days. Observed the effect on the animals in different dosages, and recorded the consumption of water and food every day, and weighed weekly. The consumption of water and food, and weight gain were showed in figures 7 to 9 and tables 5 to 7.
There were no significant differences observed.
The urine tests before the animals treated with Tripterygium wilfordii were summarized in table 8. Treated with Tripterygium wilfordii continuously for 28 days, the skin, hair, eyes, mucous membrane respiratory system, circulatory system, nervous system, activity of limbs were no abnormal change in the experiment period, and the consumption of water and food and the rats body weight were changed slightly. There were no differences in the organ weights between every group (Tab. 9). The results of urine assay in every group were not significantly different, either (Tab. 10 and Tab. 11). The results of the blood biochemistry assay are shown on tables 12 to 13.
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Fig.7. The consumption of food when treated with Tripterygium wilfordii continuously for 28 days.
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Tab. 5. The weekly change of consumptions of food when treated with extractions of different parts of Tripterygium wilfordii continuously for 28 days.
Dose (mg/kg)
Mean of food consumption per day in different week (g) 1st week 2nd week 3rd week 4th week Blank 23.89±2.22 28.28±0.75 27.30±0.41 28.51±1.09 Stem
12 27.09±0.44 27.09±0.71 26.79±0.71 24.71±1.62 60 26.29±0.61 26.75±0.31 26.06±0.51 25.49±0.49 300 24.12±1.49 25.06±0.42 23.56±0.74 24.13±0.37 Root
16 26.18±0.48 28.26±0.73 28.79±0.54 26.81±0.36 80 25.42±0.77 27.04±0.50 27.62±0.63 26.38±0.64 400 23.29±0.74 25.74±0.74 27.02±0.47 23.83±0.73 Root cortex
6 27.07±0.62 27.99±0.55 28.33±0.39 25.93±0.41 30 27.11±0.40 26.88±0.32 26.93±0.46 25.87±0.48 150 24.43±0.78 26.20±0.46 27.61±0.46 23.33±1.26 Data were expressed as mean±SE
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Fig. 8. The consumption of water when treated with Tripterygium wilfordii continuously for 28 days.
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Tab. 6. The weekly change of consumptions of water when treated with extractions of different parts of Tripterygium wilfordii continuously for 28 days.
Dose (mg/kg)
Mean of water consumption per day in different week (ml) 1st week 2nd week 3rd week 4th week Blank 38.39±5.65 46.87±2.24 45.97±0.74 47.78±3.38 Stem
12 43.30±0.98 44.33±3.34 44.91±0.93 42.13±5.16 60 44.03±1.30 46.05±0.98 42.76±3.45 42.95±1.59 300 40.75±2.13 41.77±1.23 40.86±1.19 41.87±2.02 Root
16 42.62±1.19 49.98±2.41 52.96±1.83 47.93±2.17 80 41.92±2.34 50.80±1.43 49.18±1.87 48.69±2.68 400 38.77±1.14 44.62±1.28 46.36±2.96 37.88±1.88 Root cortex
6 43.55±1.07 47.08±1.35 46.54±0.89 44.15±2.50 30 41.79±0.79 42.99±1.39 43.39±0.80 40.91±1.19 150 41.97±1.36 47.67±1.20 49.55±1.80 41.55±2.44 Data were expressed as mean±SE
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Tab. 7. Summary of the weekly change of body weights when treated with extractions of different parts of Tripterygium wilfordii continuously for 28 days.
Body weight (g) 0 1st week 2nd week 3rd week 4th week Blank 299.38±2.25 331.75±4.63 361.13±5.95 386.38±6.14 404.63±7.10 Stem 12 mg/kg 302.00±3.05 349.38±2.67 384.25±6.47 400.88±7.01 418.75±6.93 Stem 60 mg/kg 290.88±6.48 342.75±3.48 366.13±6.13 385.25±6.01 391.63±7.81 Stem 300 mg/kg 293.63±3.59 332.25±8.44 356.00±10.58 367.00±11.37 390.00±10.44
Root 16 mg/kg 293.13±1.62 337.00±2.97 366.75±5.51 383.88±10.30 399.75±8.12 Root 80 mg/kg 304.50±2.10 340.75±3.76 365.63±6.46 387.88±6.15 395.88±19.19 Root 400 mg/kg 293.50±3.05 325.29±7.62 356.86±5.53 381.71±6.37 393.50±6.79
Root Cortex 6 mg/kg
302.25±2.13 349.25±4.54 377.50±7.06 397.25±8.48 414.13±17.20
Root Cortex 30 mg/kg
302.25±3.28 350.00±3.82 374.63±4.35 393.50±6.32 411.13±10.99
Root Cortex 150 mg/kg
300.63±3.44 336.75±7.03 359.13±8.16 387.86±9.71 393.43±17.64
Data were expressed as mean±SE
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Fig. 9. The weekly change of body weights when treated with extractions of different parts of Tripterygium wilfordii continuously for 28 days.
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Tab. 8. The results of urine tests before treated with Tripterygium wilfordii.
Dose
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Tab.9. Organ-weight changes of rats after 28-day treatment with crude extracts of different parts of Tripterygium wilfordii.
Dose (mg/kg) Brain (g) Heart (g) Lung (g) Spleen (g) Liver (g) Kidney (g) Adrenal gland (g)
Testis (g) Epididymis (g)
Blank 2.05±0.05 1.29±0.04 2.00±0.27 0.59±0.01 9.14±0.30 2.57±0.08 0.05±0.00 2.95±0.09 1.07±0.04 Stem
12 2.13±0.05 1.29±0.05 1.67±0.12 0.71±0.03 9.23±0.24 2.68±0.11 0.05±0.00 3.02±0.09 1.22±0.08 60 2.15±0.07 1.24±0.05 1.72±0.06 0.66±0.04 8.23±0.18 2.46±0.10 0.05±0.00 3.22±0.09 1.31±0.21 300 2.07±0.04 1.18±0.04 1.85±0.11 0.61±0.03 9.00±0.30 2.53±0.08 0.11±0.06 3.26±0.11 0.85±0.03 Root
16 2.06±0.05 1.29±0.05 1.66±0.08 0.63±0.04 9.61±0.47 2.71±0.10 0.05±0.00 2.95±0.09 1.11±0.11 80 2.02±0.04 1.24±0.04 1.62±0.05 0.57±0.03 8.63±0.29 2.58±0.09 0.05±0.01 2.98±0.10 1.25±0.10 400 2.07±0.02 1.24±0.04 1.94±0.11 0.64±0.02 9.06±0.46 2.60±0.07 0.06±0.00 2.79±0.13 0.85±0.04 Root cortex
6 2.18±0.07 1.29±0.05 1.65±0.08 0.68±0.03 9.50±0.39 2.66±0.07 0.05±0.01 3.29±0.11 1.28±0.13 30 2.04±0.01 1.31±0.07 1.67±0.13 0.58±0.02 8.65±0.22 2.54±0.03 0.05±0.00 2.86±0.31 1.09±0.09 150 2.06±0.02 1.33±0.05 2.01±0.23 064±0.02 9.29±0.26 2.69±0.12 0.06±0.01 3.07±0.25 0.93±0.06 Data were expressed as mean±SE
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Tab. 10. Urine routine test of rats after 28-day treatment with crude extracts of different parts of Tripterygium wilfordii.
Dose (mg/kg)
Volume ml
Appearance pH S. Gravity Protein mg/dl Blank 10.98±1.66 Y 7.00±0.00 1.01±0.00 31.25±17.68 0.13±0.35 0.50±0.71 0.00±0.00 Stem
12 10.41±2.56 Y 7.06±0.82 1.02±0.00 25.00±0.00 1.38±3.11 2.50±1.08 0.00±0.00 60 9.06±1.67 Y 6.88±0.23 1.02±0.00 25.00±0.00 0.25±0.71 2.63±1.85 0.00±0.00 300 11.48±3.72 Y 6.94±0.50 1.02±0.00 18.75±11.57 1.63±1.19 2.13±1.46 0.00±0.00 Root
16 7.56±2.96 Y 7.00±0.65 1.01±0.01 50.00±26.73 0.86±0.69 1.86±1.46 0.00±0.00 80 11.26±3.27 Y 7.38±0.52 1.01±0.00 31.25±17.68 0.25±0.46 2.75±1.91 0.13±0.35 400 10.98±3.89 Y-B 7.08±0.49 1.01±0.00 25.00±0.00 1.00±0.63 3.17±1.94 0.00±0.00 Root cortex
6 9.39±2.97 Y 7.44±0.82 1.01±0.00 43.75±25.88 1.25±1.12 1.88±1.13 0.00±0.00 30 9.48±1.98 Y 7.13±0.35 1.01±0.00 37.50±23.15 1.38±0.92 1.88±1.89 0.00±0.00 150 10.92±3.43 Y 7.33±0.52 1.01±0.00 25.00±0.00 1.17±1.47 2.83±1.17 0.00±0.00 Data were expressed as mean±SE
Y － yellow
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Tab. 11. Urine routine of rats after 28-day treatment with crude extracts of different parts of Tripterygium wilfordii.
Dose (mg/kg)
BUN (mg/dl) Creatinine(U) (ml/min)
Ccr (ml/min) Urine volume (ml/min)
BW (g) Ccr/BW (%) Blank 113.8±38.67 92.79±6.38 2.43±0.29 0.008±0.000 392.88±6.86 0.62±0.08 Stem
12 70.16±7.68 104.94±9.68 2.06±0.23 0.008±0.001 418.75±6.93 0.49±0.05 60 51.91±8.34 117.88±5.78 1.95±0.07 0.006±0.001 399.50±6.69 0.49±0.02 300 159.28±52.57 94.99±12.24 3.45±0.73 0.008±0.001 390.00±10.44 0.88±0.18 Root
16 27.40±0.88 107.76±21.19 1.98±0.11 0.005±0.001 399.75±8.12 0.50±0.03 80 71.98±10.08 101.36±14.38 2.16±0.10 0.008±0.001 405.00±7.56 0.53±0.03 400 67.50±9.05 117.18±13.43 2.60±0.18 0.008±0.001 393.50±17.20 0.66±0.04 Root cortex
6 53.93±12.52 129.40±10.89 2.57±0.11 0.007±0.001 414.13±10.99 0.62±0.03 30 51.59±3.39 114.44±6.27 1.74±0.09 0.007±0.000 411.13±5.69 0.43±0.02 150 49.12±12.34 94.47±6.33 2.14±0.38 0.008±0.001 407.67±12.31 0.53±0.09 Data were expressed as mean±SE
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Tab. 12. Blood routine of rats after 28-day treatment with crude extracts of different parts of Tripterygium wilfordii.
Dose Blank 3.15±0.23 7.81±0.16 15.10±0.21 45.64±1.23 58.44±0.88 19.38±0.25 33.16±0.52 962.25±33.42 Stem
12 3.08±0.25 7.69±0.19 14.64±0.33 43.26±1.1 56.30±0.44 19.05±0.17 33.86±0.19 902.75±38.73 60 3.64±0.17 7.51±0.19 14.36±0.17 43.58±0.67 58.19±1.13 19.18±0.32 32.99±0.34 967.63±51.09 300 3.48±0.37 7.33±0.22 14.40±0.27 42.25±0.86 57.77±1.28 19.70±0.53 33.89±0.37 855.75±82.13 Root
16 6.00±0.50* 7.48±0.17 14.44±0.18 43.14±0.66 57.76±0.99 19.33±0.26 33.50±0.20 937.86±54.14 80 6.66±0.46** 7.75±0.12 15.00±0.26 43.66±0.59 56.39±0.55 19.38±0.22 34.36±0.35 1024.88±9.43 400 8.48±1.37** 8.19±0.35 14.36±0.34 48.82±1.71 59.74±0.9 17.72±1.01 32.62±1.44 1197.40±127.75 Root cortex
6 5.84±0.35* 7.66±0.13 14.4±0.20 44.04±0.95 57.53±1.29 18.93±0.15 32.99±0.71 945.86±33.27 30 5.95±0.20* 8.61±0.28 15.03±0.23 52.34±2.62 60.76±2.03 17.59±0.58 29.18±1.34 1062.75±55.27 150 5.62±0.91* 8.00±0.31 14.30±0.21 46.90±2.12 58.57±0.74 18.00±0.74 30.78±1.40 1057.33±107.68 Data were expressed as mean±SE
*p<0.05 vs blank; **p<0.01 vs blank; ***p<0.001 vs blank
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Tab. 13. Blood chemistry of rats after 28-day treatment with crude extracts of different parts of Tripterygium wilfordii.
Dose Blank 1.70±0.07 3.93±0.29 2.48±0.20 39.78±3.84 19.18±2.02 89.13±9.45 8.31±0.70 0.31±0.04 Stem
12 1.81±0.04 4.58±0.24 2.95±0.16 40.29±3.70 18.99±1.99 93.88±5.54 10.58±0.63 0.39±0.03 60 1.87±0.08 4.74±0.15 3.02±0.13 44.66±6.48 24.28±1.43 116.00±5.03 11.69±0.76 0.38±0.02 300 1.59±0.08 3.37±0.36 3.09±0.24 39.53±4.69 19.96±1.92 81.00±10.35 10.58±1.20 0.25±0.03 Root
16 1.75±0.04 4.24±0.26 2.96±0.18 52.44±3.48 20.91±1.85 114.38±13.20 10.15±1.12 0.39±0.01 80 1.67±0.05 4.66±0.15 3.06±0.11 55.96±3.10 23.19±1.55 117.38±6.22 11.29±0.49 0.34±0.02 400 1.57±0.11 4.70±0.27 2.85±0.17 53.17±5.39 20.32±1.59 110.33±6.59 9.80±0.35 0.33±0.02 Root cortex
6 1.80±0.07 4.70±0.11 3.01±0.05 53.60±2.27 19.80±0.81 121.00±8.32 12.80±0.69 0.33±0.02 30 1.64±0.05 4.69±0.22 3.05±0.12 50.91±2.58 18.01±0.73 121.00±6.45 12.88±0.68* 0.43±0.02 150 1.61±0.06 4.83±0.23 3.01±0.13 70.25±8.63*** 24.57±2.59 116.29±11.55 14.49±2.99** 0.40±0.03 Data were expressed as mean±SE
*p<0.05 vs blank; **p<0.01 vs blank; ***p<0.001 vs blank
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Tab. 14. The changes of the result of blood coagulation test after 28-day treatment with crude extracts of different parts of Tripterygium wilfordii.
Dose (mg/kg) Prothrombin time (sec)
APTT (sec) Fibrinogen (mg/dl) Blank 11.49±0.43 26.96±1.21 200.84±5.65 Stem
12 12.46±0.24 24.76±1.35 207.38±5.00
60 11.35±0.33 22.79±0.59 206.91±7.25
300 12.88±0.80 27.28±2.13 207.10±16.35 Root
16 12.84±0.47 21.59±2.23 202.64±12.83
80 11.66±0.34 23.46±1.33 204.55±3.07
400 12.12±0.76 29.12±2.50 238.76±10.10 Root cortex
6 13.13±0.46 40.94±2.15*** 195.64±12.61 30 12.34±0.44 39.00±0.86*** 198.20±5.72 150 13.20±1.06 40.03±1.12*** 225.60±7.10 Data were expressed as mean±SE
*p<0.05 vs blank; **p<0.01 vs blank; ***p<0.001 vs blank
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Tab. 15. The assay of pathological histopathology after 28-day treatment with crude extracts of different parts of Tripterygium wilfordii.
Organ Histopathology Group
B SH RH CH
Kidney
Nephritis, pelvis, focal - - - 1/8 Postmortem, diffuse - - - 1/8
Liver - - - -
-: No significant lesions.
1: Degree of lesions was graded from one to five depending on severity: 1 =minimal (<1%); 2 =slight (1-25%); 3 =moderate (26-50%); 4 =moderate/severe (51-75%); 5
=severe/high (76-100%).
2Incidence: Affected rats/Total examined rats (n=8).
.
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Appendix2. Pathology – individual micro findings of rats.
Organ Histopathological finding Animal code Blank (B)
1 2 3 4 5 6 7 8
Kidney - - - -
Liver - - -
Organ Histopathological finding Animal code Stem high (SH)
1 2 3 4 5 6 7 8
Kidney - - - -
Liver - - -
Organ Histopathological finding Animal code Root high (RH)
1 2 3 4 5 6 7 8
Kidney - - N - - - - -
Liver - - N - - -
Organ Histopathological finding Animal code
Root cortex high (CH)
1 2 3 4 5 6 7 8
Kidney - - - - - -
Nephritis, pelvis, focal (artificial gavage error) 3
Postmortem, diffuse 5
Liver - - - -
-: No significant lesions. N: no tissue
1: Degree of lesions was graded from one to five depending on severity: 1 =minimal (<1%); 2 =slight (1-25%); 3 =moderate (26-50%); 4 =moderate/severe (51-75%); 5
=severe/high (76-100%).
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Fig. 10. Histopathological finding of the kidney and liver in groups B, SH and RH treated rats. No significant lesions were noted in the kidney (A) and liver (B), 400x. H&E stain.
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Fig. 11. Histopathological finding of the kidney and liver in group CH treated rats. No significant lesions were noted in the kidney (A) and liver (B), 400x. H&E stain.
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Discussion
The aim of this study is to demonstrate the relationship between different parts of Tripterygium wilfordii, such as stem, root and root cortex, and the different dosages with the toxicity in the liver and kidney.
Tripterygium wilfordii is a plant of the genus Tripterygium of the family Celastraceae ,which is a root of a perennial liana (Jia, 1985). In
Supplements to Compendim of Materia Medica, it was used to be a insecticide (Ma et al., 2006). Effect of Tripterygium wilfordii were well known as expel heat, detoxify, detumescence, insecticide and stanch
bleeding. It has been widely used in clinical practices such as rheumatism, kidney diseases, skin diseases and immunodificent syndrome (Qin et al., 1981; Tao et al., 1989; Liao and Li, 1994; Jiang, 1994). Owing to the narrow therapeutic window, the active ingredient maybe the poison composition (Ma et al., 2006). The common adverse drug reactions of Tripterygium wilfordii such as gastrointestinal disorders, the blood system reaction, cardiovascular system disorders and lesion of the liver and kidney (Jia, 2006). How to increase the safety in use of Tripterygium wilfordii is important.
Tripterygium wilfordii is a woody vine native to Eastern and Southern China, Korea, Japan, and Taiwan (Ma et al., 1999); and can find it in Jin-Qua-Shi, Keelung City, Shih-ding Township, Taipei County and Neihu District, Taipei City. Tripterygium wilfordii can be collected in Jin-Qua-Shi, Keelung City, Shih-ding Township, Taipei County in autumn.
After collected in autumn, the crude medicines were washed out the soils,
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sun-drying, and sliced. According to the traditional use, we collected Tripterygium wilfordii in autumn, and authenticated.
The poison composition of Tripterygium wilfordii was centralized in the root cortex. The common reason of intoxication is overdose or use by mistake. The adverse drug reactions of Tripterygium wilfordii were made direct lesion in gastrointestinal, cardiovascular system, nerve system and urinary system. The syndromes were progressive, such as nausea,
vomiting, abdominal pain, diarrhea and the pulse was thin and rapid. In blood system, the blood pressure decreased and lead to headache, dizzy, weak, palpitation, anxious and even lead to twitch. In urinary system, the ADRs were lumbago, oliguria, hematuria and proteinuria. The causes of death are acute renal failure, lesion of nerve cell in the central nerve system, critical myelosuppression and single or multiple organ failures (Jia, 2006).
There is general agreement that the active components responsible for the therapeutic effect of extracts of Tripterygium wilfordii are a group of diterpenoids (Zheng et al., 1994). Although many diterpenoid compounds have been isolated from Tripterygium wilfordii, triptolide and tripdiolide have been documented to be responsible for most of the biologic activity of the EA extract (Tao et al., 2000).
Triptolide has been reported to be able to suppress the production of a wide range of proinflammatory cytokines, including interleukin-2 (IL-2),
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interferon-γ (IFNγ), IL-6, and tumor necrosis factor (TNF), as well as inhibit the up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) (Tao et al., 1996). Previous studies have shown that triptolideexerts its antiinflammatory and immunosuppressive actions by directly suppressing the transcription of thegenes that encode
interferon-γ (IFNγ), IL-6, and tumor necrosis factor (TNF), as well as inhibit the up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) (Tao et al., 1996). Previous studies have shown that triptolideexerts its antiinflammatory and immunosuppressive actions by directly suppressing the transcription of thegenes that encode