壮药滇桂艾纳香止血效应的实验研究
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摘要
目的：
1.研究滇桂艾纳香止血作用的药效学及急性毒性。
2.从凝血功能调节、子宫平滑肌收缩、血管收缩以及相关药效进行综合考察,探讨滇桂艾纳香发挥止血作用的作用机制。
方法：
第一部分滇桂艾纳香不同总提取物药效学研究
通过测定滇桂艾纳香水提取物半数致死量(LD50)和50%乙醇提取物、70%乙醇提取物的最大耐受量(MTD),考察滇桂艾纳香的急性毒性。通过研究滇桂艾纳香不同总提取物对小鼠出血时间的影响、对小鼠凝血时间的影响、对家兔血浆复钙时间的影响、对小鼠离体子宫平滑肌的影响以及对家兔离体动脉条平滑肌的影响,明确滇桂艾纳香不同总提取物的止血作用。
第二部分滇桂艾纳香有效部位的实验研究
通过对滇桂艾纳香不同部位提取物凝血作用研究,包括石油醚部位、氯仿部位、乙酸乙酯部位、正丁醇部位及70%乙醇部位,分为高、中、低剂量三个剂量组,研究其对小鼠出血时间、凝血时间、家兔血浆复钙时间、小鼠离体子宫平滑肌、家兔离体动脉条的影响,初步筛选出滇桂艾纳香的有效活性部位。
第三部分滇桂艾纳香止血作用机制研究
(1)研究滇桂艾纳香水提液、含药血清对小鼠离体子宫平滑肌、对大鼠在体子宫平滑肌及家兔血管平滑肌的影响；(2)研究滇桂艾纳香对早孕大鼠流产子宫出血量及对子宫内膜形态的影响；(3)研究滇桂艾纳香大鼠静脉血中血栓素(TXA2)、前列环素(PGI2)与TXA2/PGI2含量,以及对大鼠血清肿瘤坏死因子(TNF-α)的影响；(4)研究滇桂艾纳香水提液对凝血酶原时间(PT),活化部分凝血活酶时间(APTT),纤维蛋白原(FIB)的影响；(5)研究滇桂艾纳香对药物流产后大鼠子宫组织中一氧化氮(NO)、一氧化氮合酶(NOS)及血管紧张物质内皮素(ET)等的影响；(6)研究滇桂艾纳香对药物流产后大鼠子宫蜕膜中层粘连蛋白(LN)和纤维粘连蛋白(FN)的影响；(7)研究滇桂艾纳香的抗炎作用,采用小鼠耳肿胀、大鼠足肿胀法及小鼠腹腔毛细血管通透性法进行研究。通过以上指标考察滇桂艾纳香止血作用及其凝血机制。
结果：
第一部分滇桂艾纳香不同总提取物药效学研究
1.实验结果表明,滇桂艾纳香有较好的止血作用。滇桂艾纳香水提物、50%乙醇提取物、70%乙醇提取物均可缩短小鼠出血时间、凝血时间,以及缩短体外给药家兔血浆复钙时间。并能在一定程度上增强小鼠离体子宫平滑肌的收缩频率及收缩力及家兔离体动脉条平滑肌的收缩力,其中70%乙醇提取物作用最为显著。
2.急性毒性实验结果显示,滇桂艾纳香水提取物对昆明种小鼠灌胃给药(ig)的LD50为143.10g生药/kg体重；滇桂艾纳香50%乙醇提取物对昆明种小鼠的MTD为259.60g生药/kg体重,70%乙醇提取物对昆明种小鼠灌胃给药的MTD为309.68g生药/kg体重。分别相当于成人日用量的519倍及619倍,表明毒性低。
第二部分滇桂艾纳香有效部位的实验研究
滇桂艾纳香乙酸乙酯部位、正丁醇部位及70%乙醇部位三个部位均可使小鼠出血时间、凝血时间及体外给药家兔血浆复钙时间缩短,并能在一定程度上增强小鼠离体子宫平滑肌的收缩频率及收缩力及家兔离体动脉条平滑肌的收缩力(P<0.05)。
第三部分滇桂艾纳香止血作用机制研究
(1)滇桂艾纳香水煎液及含药血清均能加强小鼠离体子宫平滑肌、大鼠在体子宫平滑肌及家兔血管平滑肌的收缩能力,减少早孕大鼠流产子宫出血量,与空白组比较有显著性差异(P<0.05)；(2)改善子宫内膜组织形态,促进子宫内膜组织的修复,减少子宫出血(P<0.05)；(3)滇桂艾纳香可升高大鼠血浆中血栓素(TXA2)的含量显著降低大鼠血浆中前列环素(PGI2)的含量,同时升高TXA2/PGI2值,促进血小板粘附、聚集并能收缩血管,从而达到止血作用(P<0.05)；(4)滇桂艾纳香可缩短凝血酶原时间(PT)、活化部分凝血活酶时间(APTT),显著提高大鼠静脉血中纤维蛋白原(FIB)含量,促使血液凝固,而止血(P<0.05)。对肿瘤坏死因子(TNF-α)也有一定的抑制作用,但与空白组比较无显著性差异(P>0.05)；(5)可降低子宫局部一氧化氮合酶(NOS)和一氧化氮(NO)含量(P＜0.01),升高内皮素(ET)含量(P<0.05)；(6)可降低LN、FN在蜕膜组织中的表达,利于蜕膜排出体外(P<0.01)；(7)可明显抑制二甲苯致小鼠耳廓肿胀(P＜0.05),抑制角叉菜胶诱发的大鼠足跖肿胀(P＜0.01),以及降低小鼠腹腔毛细血管通透性(P<0.05),提示具有较好的抗炎作用。
结论：
1.滇桂艾纳香有较好的止血作用。其中70%乙醇提取物作用最为显著。
2.滇桂艾纳香可升高大鼠血浆中TXA2的含量,显著降低大鼠血浆中PGI2的含量,同时升高TXA2/PGI2值,促进血小板粘附、聚集,表明具有较好的止血作用。
3.可促进子宫内膜修复,降低子宫局部NO和NOS含量,升高ET含量,收缩血管,提示具有较好的止血作用。
4.滇桂艾纳香可降低LN、FN在蜕膜组织中的表达,利于蜕膜排出体外,减少出血时间。
5.抑制二甲苯致小鼠耳廓肿胀度和角叉菜胶诱发的大鼠足跖肿胀度,提示具有较好的抗炎作用。
6.滇桂艾纳香急性毒性结果显示,毒性低。
Objective
1.Research on the pharmacodynamics of hemostatic effect and toxicology of the Blumea riparia (BI.) DC
2. Research on the mechanism of the hemostatic effect of the Blumea riparia (BI.) DC from uterine contraction、coagulation function regulation、vasoconstriction and other factors.
Methods
The first part Research on the efficacy of different total extracts in the blumea riparia (BI.) DC
To Study the acute toxicity of water extract of Blumea riparia (BI.) DC by LD50 and 50%,70% ethanol extract of Blumea riparia (BI.) DC by MTD. The hemostatic effect of extracts of Blumea riparia (BI.) DC was observed by the bleeding and coagulation time of small rats、the plasma recalcifieation time of rabbits and the effects of uterine smooth muscle of small rats and artery of rabbits.
The second part Research on the active sites in the blumea riparia (BI.) DC
The hemostasis effect of different extracts of Blumea riparia (BI.) DC was observed by the bleeding and coagulation time of small rats、the plasma recalcifieation time of rabbits and the effects of uterine smooth muscle from small rats and artery of rabbits of high、middle and low dose extracts of petroleum ether、chloroform、ethyl acetate、n-butanol and 70% ethanol, and tried to find the active sites of Blumea riparia (BI.) DC preliminarily.
The third part Research on the mechanism of hemostatic effect of the Blumea riparia (BI.) DC
(1)Study of the effect of uterine contraction on isolated uterine smooth muscle from small rats and artery of rabbits by water extract and medicated serum of Blumea riparia (BI.) DC; (2) Study of the effect of Blumea riparia (BI.) DC to endometrial morphology and amount of bleeding;(3) Study of the content of TXA2、PGI2、TXA2/PGI2 in venous blood of rats and TNF-αin serum of rats; (4)Study of the effect of coagulation function by uterine bleeding quantity of early pregnant rabbits、PT、APTT、FIB and endometrial morphology; (5)Study of the effect of Blumea riparia (BI.) DC oncontent of NO、NOS and ET in uterine tissue of rats which were drug aborted; (6) Study of the effect on LN and FN in uterine decidua of rats which were drug aborted; (7) Study of the anti-inflammatory effect by experiments of ears swelling、foot swelling、penetrability of abdominal capillaryvessel. Through the indicators above research of the hemostasis mechanism of Blumea riparia (BI.) DC.
Results
The first part Research on the efficacy of total extracts in the blumea riparia (BI.) DC
1. Results showed the Hemostatic effect of Blumea riparia (BI.) DC was good. The water extract、50% ethanol extract and 70% ethanol extract all could shorten bleeding time and bleeding time of small rats both and shorten PRT of rabbits'isolated uterine smooth muscle by treated in vitro. In the same time, enhanced contraction frequency and contractility of isolated uterine smooth muscle of small rats and rabbits, arid the effects of 70% ethanol extrac was the most remarkable.
2. The result of acute toxicity showed that LD50 of water extract was 143.1034g herb/kg by intragastric administration; the most tolerance dose of 50% ethanol extract and 70% ethanol extract were 259.60g herb/kg、309.68g herb/kg by intragastric administration to small rats. Equivalent to 519 and 619 times of adult dosage respectively, it showed that the toxicity of Blumea riparia (BI.) DC was low.
The second part Research on the active sites in the Blumea riparia (BI.) DC
The extracts of ethyl acetate、n-butanol and 70% ethanol could shorten bleeding time and coagulation time of small rats both and shorten PRT of rabbits'isolated uterine smooth muscle by treated in vitro, in the same time, enhanced contraction frequency and contractility of isolated uterine smooth muscle from small rats and artery from rabbits in certain (P＜0.05)
The third part Research on the mechanism of hemostatic effect of the Blumea riparia (BI.) DC
(1) The contraction ability of isolated uterine smooth muscle from small rats、uterine smooth muscle from rabbits and vascular smooth muscle from rabbits was strengthened by water extract and medicated serum of Blumea riparia (BI.) DC, and uterine bleeding was reduced, compared with the normal group, the different was significant (P＜0.05).
(2) Improved endometrial morphology, promoted the repair of uterine endometrium, reduced uterine bleeding, compared with the normal group, the different was significant(P＜0.05).(3) Blumea riparia(BI.)DC could increased TXA2 in plasma of rats, decreased PGI2 and increased TXA2/PGI2, promoted platelet adhesion、aggregation, and contracted the blood vessels to hemostasis, compared with the model group, the different was significant (P＜0.05). (4) Effected the endogenesis coagulation system and exogenous coagulation system to shorten PT、APTT to hemostasis, besides, the content of FIB obvioudly increased, promoted blood coagulation and inhibition fibrinolysis, compared with the model group, the different was significant (P＜0.01).TNF-αwas restrained in certain, the expression had no significant (P＞0.05).(5) Decreased the content of NO(P＞0.05)、NOS(P＜0.01)in part of uterine and increased the content of ET (P＜0.05). (6) Decreased the expression of LN and FN, helped the decidua excrete, the different was significant (P＜0.01). (7) Blumea riparia (BI.) DC could inhibit ears swelling induced by dimethylbenzene (P＜0.05)、foot swelling induced by carrageenan (P＜0.01)、decreased of penetrability of abdominal capillaryvessel induced by acetic acid (P＜0.05),the results showed that the anti-inflammatory effects of Blumea riparia (BI.) DC. was good.
Conclution
1. The hemostatic effect of Blumea riparia (BI.) DC was good, and the effect of 70% ethanol extracts was the most remarkable.
2. Blumea riparia (BI.) DC could increase TXA2 in plasma of rats, decreased PGI2 distinguished and increased TXA2/PGI2, promoted platelet adhesion、aggregation, and contracted the blood vessels to hemostasis to stanch bleeding.
3. Promoted the repair of endometrium, decreased the content of NO、NOS and increased the content of ET shrink vascularto stop bleeding.
4. Blumea riparia (BI.) DC could decrease the expression of LN and FN, helpe the decidua excrete and reduce uterine bleeding.
5. Inhibited ears swelling induced by dimethylbenzene、foot swelling induced by carrageenan、decreased of penetrability of abdominal capillaryvessel induced by acetic acid to inhibit inflammation.
6. The result of acute toxicity showed that the toxicity in the blumea riparia (BI.) DC was low.
1.研究滇桂艾纳香止血作用的药效学及急性毒性。
2.从凝血功能调节、子宫平滑肌收缩、血管收缩以及相关药效进行综合考察,探讨滇桂艾纳香发挥止血作用的作用机制。
方法：
第一部分滇桂艾纳香不同总提取物药效学研究
通过测定滇桂艾纳香水提取物半数致死量(LD50)和50%乙醇提取物、70%乙醇提取物的最大耐受量(MTD),考察滇桂艾纳香的急性毒性。通过研究滇桂艾纳香不同总提取物对小鼠出血时间的影响、对小鼠凝血时间的影响、对家兔血浆复钙时间的影响、对小鼠离体子宫平滑肌的影响以及对家兔离体动脉条平滑肌的影响,明确滇桂艾纳香不同总提取物的止血作用。
第二部分滇桂艾纳香有效部位的实验研究
通过对滇桂艾纳香不同部位提取物凝血作用研究,包括石油醚部位、氯仿部位、乙酸乙酯部位、正丁醇部位及70%乙醇部位,分为高、中、低剂量三个剂量组,研究其对小鼠出血时间、凝血时间、家兔血浆复钙时间、小鼠离体子宫平滑肌、家兔离体动脉条的影响,初步筛选出滇桂艾纳香的有效活性部位。
第三部分滇桂艾纳香止血作用机制研究
(1)研究滇桂艾纳香水提液、含药血清对小鼠离体子宫平滑肌、对大鼠在体子宫平滑肌及家兔血管平滑肌的影响；(2)研究滇桂艾纳香对早孕大鼠流产子宫出血量及对子宫内膜形态的影响；(3)研究滇桂艾纳香大鼠静脉血中血栓素(TXA2)、前列环素(PGI2)与TXA2/PGI2含量,以及对大鼠血清肿瘤坏死因子(TNF-α)的影响；(4)研究滇桂艾纳香水提液对凝血酶原时间(PT),活化部分凝血活酶时间(APTT),纤维蛋白原(FIB)的影响；(5)研究滇桂艾纳香对药物流产后大鼠子宫组织中一氧化氮(NO)、一氧化氮合酶(NOS)及血管紧张物质内皮素(ET)等的影响；(6)研究滇桂艾纳香对药物流产后大鼠子宫蜕膜中层粘连蛋白(LN)和纤维粘连蛋白(FN)的影响；(7)研究滇桂艾纳香的抗炎作用,采用小鼠耳肿胀、大鼠足肿胀法及小鼠腹腔毛细血管通透性法进行研究。通过以上指标考察滇桂艾纳香止血作用及其凝血机制。
结果：
第一部分滇桂艾纳香不同总提取物药效学研究
1.实验结果表明,滇桂艾纳香有较好的止血作用。滇桂艾纳香水提物、50%乙醇提取物、70%乙醇提取物均可缩短小鼠出血时间、凝血时间,以及缩短体外给药家兔血浆复钙时间。并能在一定程度上增强小鼠离体子宫平滑肌的收缩频率及收缩力及家兔离体动脉条平滑肌的收缩力,其中70%乙醇提取物作用最为显著。
2.急性毒性实验结果显示,滇桂艾纳香水提取物对昆明种小鼠灌胃给药(ig)的LD50为143.10g生药/kg体重；滇桂艾纳香50%乙醇提取物对昆明种小鼠的MTD为259.60g生药/kg体重,70%乙醇提取物对昆明种小鼠灌胃给药的MTD为309.68g生药/kg体重。分别相当于成人日用量的519倍及619倍,表明毒性低。
第二部分滇桂艾纳香有效部位的实验研究
滇桂艾纳香乙酸乙酯部位、正丁醇部位及70%乙醇部位三个部位均可使小鼠出血时间、凝血时间及体外给药家兔血浆复钙时间缩短,并能在一定程度上增强小鼠离体子宫平滑肌的收缩频率及收缩力及家兔离体动脉条平滑肌的收缩力(P<0.05)。
第三部分滇桂艾纳香止血作用机制研究
(1)滇桂艾纳香水煎液及含药血清均能加强小鼠离体子宫平滑肌、大鼠在体子宫平滑肌及家兔血管平滑肌的收缩能力,减少早孕大鼠流产子宫出血量,与空白组比较有显著性差异(P<0.05)；(2)改善子宫内膜组织形态,促进子宫内膜组织的修复,减少子宫出血(P<0.05)；(3)滇桂艾纳香可升高大鼠血浆中血栓素(TXA2)的含量显著降低大鼠血浆中前列环素(PGI2)的含量,同时升高TXA2/PGI2值,促进血小板粘附、聚集并能收缩血管,从而达到止血作用(P<0.05)；(4)滇桂艾纳香可缩短凝血酶原时间(PT)、活化部分凝血活酶时间(APTT),显著提高大鼠静脉血中纤维蛋白原(FIB)含量,促使血液凝固,而止血(P<0.05)。对肿瘤坏死因子(TNF-α)也有一定的抑制作用,但与空白组比较无显著性差异(P>0.05)；(5)可降低子宫局部一氧化氮合酶(NOS)和一氧化氮(NO)含量(P＜0.01),升高内皮素(ET)含量(P<0.05)；(6)可降低LN、FN在蜕膜组织中的表达,利于蜕膜排出体外(P<0.01)；(7)可明显抑制二甲苯致小鼠耳廓肿胀(P＜0.05),抑制角叉菜胶诱发的大鼠足跖肿胀(P＜0.01),以及降低小鼠腹腔毛细血管通透性(P<0.05),提示具有较好的抗炎作用。
结论：
1.滇桂艾纳香有较好的止血作用。其中70%乙醇提取物作用最为显著。
2.滇桂艾纳香可升高大鼠血浆中TXA2的含量,显著降低大鼠血浆中PGI2的含量,同时升高TXA2/PGI2值,促进血小板粘附、聚集,表明具有较好的止血作用。
3.可促进子宫内膜修复,降低子宫局部NO和NOS含量,升高ET含量,收缩血管,提示具有较好的止血作用。
4.滇桂艾纳香可降低LN、FN在蜕膜组织中的表达,利于蜕膜排出体外,减少出血时间。
5.抑制二甲苯致小鼠耳廓肿胀度和角叉菜胶诱发的大鼠足跖肿胀度,提示具有较好的抗炎作用。
6.滇桂艾纳香急性毒性结果显示,毒性低。
Objective
1.Research on the pharmacodynamics of hemostatic effect and toxicology of the Blumea riparia (BI.) DC
2. Research on the mechanism of the hemostatic effect of the Blumea riparia (BI.) DC from uterine contraction、coagulation function regulation、vasoconstriction and other factors.
Methods
The first part Research on the efficacy of different total extracts in the blumea riparia (BI.) DC
To Study the acute toxicity of water extract of Blumea riparia (BI.) DC by LD50 and 50%,70% ethanol extract of Blumea riparia (BI.) DC by MTD. The hemostatic effect of extracts of Blumea riparia (BI.) DC was observed by the bleeding and coagulation time of small rats、the plasma recalcifieation time of rabbits and the effects of uterine smooth muscle of small rats and artery of rabbits.
The second part Research on the active sites in the blumea riparia (BI.) DC
The hemostasis effect of different extracts of Blumea riparia (BI.) DC was observed by the bleeding and coagulation time of small rats、the plasma recalcifieation time of rabbits and the effects of uterine smooth muscle from small rats and artery of rabbits of high、middle and low dose extracts of petroleum ether、chloroform、ethyl acetate、n-butanol and 70% ethanol, and tried to find the active sites of Blumea riparia (BI.) DC preliminarily.
The third part Research on the mechanism of hemostatic effect of the Blumea riparia (BI.) DC
(1)Study of the effect of uterine contraction on isolated uterine smooth muscle from small rats and artery of rabbits by water extract and medicated serum of Blumea riparia (BI.) DC; (2) Study of the effect of Blumea riparia (BI.) DC to endometrial morphology and amount of bleeding;(3) Study of the content of TXA2、PGI2、TXA2/PGI2 in venous blood of rats and TNF-αin serum of rats; (4)Study of the effect of coagulation function by uterine bleeding quantity of early pregnant rabbits、PT、APTT、FIB and endometrial morphology; (5)Study of the effect of Blumea riparia (BI.) DC oncontent of NO、NOS and ET in uterine tissue of rats which were drug aborted; (6) Study of the effect on LN and FN in uterine decidua of rats which were drug aborted; (7) Study of the anti-inflammatory effect by experiments of ears swelling、foot swelling、penetrability of abdominal capillaryvessel. Through the indicators above research of the hemostasis mechanism of Blumea riparia (BI.) DC.
Results
The first part Research on the efficacy of total extracts in the blumea riparia (BI.) DC
1. Results showed the Hemostatic effect of Blumea riparia (BI.) DC was good. The water extract、50% ethanol extract and 70% ethanol extract all could shorten bleeding time and bleeding time of small rats both and shorten PRT of rabbits'isolated uterine smooth muscle by treated in vitro. In the same time, enhanced contraction frequency and contractility of isolated uterine smooth muscle of small rats and rabbits, arid the effects of 70% ethanol extrac was the most remarkable.
2. The result of acute toxicity showed that LD50 of water extract was 143.1034g herb/kg by intragastric administration; the most tolerance dose of 50% ethanol extract and 70% ethanol extract were 259.60g herb/kg、309.68g herb/kg by intragastric administration to small rats. Equivalent to 519 and 619 times of adult dosage respectively, it showed that the toxicity of Blumea riparia (BI.) DC was low.
The second part Research on the active sites in the Blumea riparia (BI.) DC
The extracts of ethyl acetate、n-butanol and 70% ethanol could shorten bleeding time and coagulation time of small rats both and shorten PRT of rabbits'isolated uterine smooth muscle by treated in vitro, in the same time, enhanced contraction frequency and contractility of isolated uterine smooth muscle from small rats and artery from rabbits in certain (P＜0.05)
The third part Research on the mechanism of hemostatic effect of the Blumea riparia (BI.) DC
(1) The contraction ability of isolated uterine smooth muscle from small rats、uterine smooth muscle from rabbits and vascular smooth muscle from rabbits was strengthened by water extract and medicated serum of Blumea riparia (BI.) DC, and uterine bleeding was reduced, compared with the normal group, the different was significant (P＜0.05).
(2) Improved endometrial morphology, promoted the repair of uterine endometrium, reduced uterine bleeding, compared with the normal group, the different was significant(P＜0.05).(3) Blumea riparia(BI.)DC could increased TXA2 in plasma of rats, decreased PGI2 and increased TXA2/PGI2, promoted platelet adhesion、aggregation, and contracted the blood vessels to hemostasis, compared with the model group, the different was significant (P＜0.05). (4) Effected the endogenesis coagulation system and exogenous coagulation system to shorten PT、APTT to hemostasis, besides, the content of FIB obvioudly increased, promoted blood coagulation and inhibition fibrinolysis, compared with the model group, the different was significant (P＜0.01).TNF-αwas restrained in certain, the expression had no significant (P＞0.05).(5) Decreased the content of NO(P＞0.05)、NOS(P＜0.01)in part of uterine and increased the content of ET (P＜0.05). (6) Decreased the expression of LN and FN, helped the decidua excrete, the different was significant (P＜0.01). (7) Blumea riparia (BI.) DC could inhibit ears swelling induced by dimethylbenzene (P＜0.05)、foot swelling induced by carrageenan (P＜0.01)、decreased of penetrability of abdominal capillaryvessel induced by acetic acid (P＜0.05),the results showed that the anti-inflammatory effects of Blumea riparia (BI.) DC. was good.
Conclution
1. The hemostatic effect of Blumea riparia (BI.) DC was good, and the effect of 70% ethanol extracts was the most remarkable.
2. Blumea riparia (BI.) DC could increase TXA2 in plasma of rats, decreased PGI2 distinguished and increased TXA2/PGI2, promoted platelet adhesion、aggregation, and contracted the blood vessels to hemostasis to stanch bleeding.
3. Promoted the repair of endometrium, decreased the content of NO、NOS and increased the content of ET shrink vascularto stop bleeding.
4. Blumea riparia (BI.) DC could decrease the expression of LN and FN, helpe the decidua excrete and reduce uterine bleeding.
5. Inhibited ears swelling induced by dimethylbenzene、foot swelling induced by carrageenan、decreased of penetrability of abdominal capillaryvessel induced by acetic acid to inhibit inflammation.
6. The result of acute toxicity showed that the toxicity in the blumea riparia (BI.) DC was low.
引文
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