Study on Coagulation Activity of Callicarpa nudiflora

Ziming YANG，Sheng HUANG，Xiaojie YAN，Li ZHANG，Zhixin GU，Dianpeng Ll*

1.Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization，Guangxi Institute of Botany，Chinese Academy of Sciences，Guilin 541006，China；

2.Hunan Jiuzhitang Co.，Ltd.，Changsha 410008，China；

3.Hainan Jiuzhitang Pharmaceutical Co.，Ltd.，Haikou 570311，China

Study on Coagulation Activity of Callicarpa nudiflora

Ziming YANG1，Sheng HUANG2，3，Xiaojie YAN1，Li ZHANG1，Zhixin GU2，Dianpeng Ll1*

1.Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization，Guangxi Institute of Botany，Chinese Academy of Sciences，Guilin 541006，China；

2.Hunan Jiuzhitang Co.，Ltd.，Changsha 410008，China；

3.Hainan Jiuzhitang Pharmaceutical Co.，Ltd.，Haikou 570311，China

［Objective］This study aimed to study the coagulation effect of Callicarpa nudiflora. ［Method］The effects of Callicarpa nudiflora on blood coagulation system of mice were investigated by measuring prothrombin time（PT），thrombin time（TT），activated partial thromboplastin time（APTT）and plasma fibrinogen（FIB）.［Result］Compared with the control group，the crude extracts and eluted parts of Callicarpa nudiflora all significantly shortened PT，APTT and TT，and increased the content of FIB in mice.［Conclusion］The crude extracts and eluted parts of Callicarpa nudiflora have a coagulation-promoting effect through affecting several links of the coagulation process in mice.

Callicarpa nudiflora；Coagulation；Mice；Prothrombin time

C allicarpa nudiflora Hook.et Arn. （Verbenaceae: Callicarpa）is mainly produced in Hainan Province of China，and it is a local medicinal material in Hainan. Callicarpa nudiflora is also distributed in Guangdong and Guangxi of China［1］，Vietnam，India and Malaysia.Callicarpa nudiflora is bitter and slightly acrid in taste，and is neutral in nature. The roots，stems and leaves of Callicarpa nudiflora can all be used as medicine，and they have detoxifying，anti-bacterial，anti-inflammatory，stopping bleeding and dissipating blood stasis，anti-swelling functions.Callicarpa nudiflora is mainly used to treat acute infectious hepatitis，suppurative inflammation，respiratory and gastrointestinal bleeding and thrombocytopenic purpura embolism.If Callicarpa nudiflora is used externally，it can treat burns，rheumatism swelling，bruises and swelling，traumatic bleeding，tuberculosis hemoptysis and gastrointestinal bleeding.Callicarpa nudiflora is one of the common herbs in the Li ethnic group in Hainan Province［2-5］. There have been currently rare researches on effective coagulation promoting parts of Callicarpa nudiflora and the relevant mechanisms.Studies have reported that flavonoids is the main active ingredient in Callicarpa nudiflora that promotes coagulation［6］. In order to explore effective coagulation-promoting parts of Callicarpa nudiflora and the relevant mechanism，the effects of main active ingredients from Callicarpa nudiflora on blood coagulation in mice were investigated，and the relevant coagulation mechanism was primarily studied.

Materials and Methods

Materials

Test animal The SPF Kunming mice，with weight of 18-22 g，were provided by the Hunan Slack King of Laboratory Animal Co.，Ltd（Produc-tion license number:SCXK （Xiang）2009-0004）.

Drugs and reagents The dry powder extract of Callicarpa nudiflora was provided by the Hainan Jiuzhitang Pharmaceutical Co.，Ltd.The Anluoxue was produced by the Changzhou Yabang PharmaceuticalCo.， Ltd（batch number:1103030）.The PT，TT，APTT and FIB assays were produced by the Beijing Shidi Scientific Instruments Co.，Ltd.The sodium citrate，methanol and other reagents were all of analytical grade.

Equipment and instruments The used equipment and instruments included AT-200 electronic balance（METTLER，Germany），DL-0601 electronic balance （Xiangyi，Hunan，China），LG-PABER coagulation factor analyzer（Shidi，Beijing，China），QD highspeed centrifuge （Heima，Zhuhai，China）， R-200 rotary evaporator（Buchi Labortechnik AG CH-9230）and Agilent 1100 series HPLC （Agilent，USA）.

Methods

Extraction and separation of chemical ingredients A certain amount（500 g）of dry powder extract of Callicarpa nudiflora was completely dissolved in a certain amount of deionized water （w/v，1/20）.The solution was first filtered through bag and then through cotton plug.The obtained filtrate was loaded on HP-20 macroporous resin，and then eluted to colorless with deionized water， 5% methanol， 40% methanol， 60% methanol and 80%methanol，respectively.The obtained deionized water elute，5% methanolelute，40% methanol elute，60%methanol elute and 80%methanol elute were dried in a rotary evaporator at 50℃.And then，the deionized waterextract，5% methanol extract，40%methanol extract，60%methanol extract and 80% methanol extract were obtained.The HPLC analysis showed that the components of non-adsorbed fraction by macroporous resin and of 5% methanol elute were all contained in the deionized water elute，so the three parts were collectively referred to as water elute.

Effects of Callicarpa nudiflora on blood coagulation system in mice

The Kunming mice were randomly divided into control group（equivalentvolume distilled water），crude dry extract group （1.0 g/kg），water elute group （1.0 g/kg），40%methanol elute group（1.0 g/kg），60%methanol elute group （1.0 g/kg），80%methanol elute extract（1.0 g/kg）and positive control group （Anluoxue，0.004 g/kg）.There were total 12 mice in each group.The water and drugs were given to mice by gavage one a day （0.2 ml/10 g body weight），which lasted for 7 continuous days.Blood was sampled from eyeballs of mice 60 min after the last administration.The blood samples were immediately mixed with certain amounts of 109 mmol/L sodium citrate（v/v，9/1）.They were centrifuged at 3 000 rpm for 10 min.Subsequently，the obtained plasmas were transferred to new tubes.For each plasma sample，the PT，TT，APTT and FIB were measured using LG-PABER coagulation factor analyzer.The decrement rates of PT，TT and APTT and increment rate of FIB were calculated according to the following formulas:

Decrement rate of PT（%）=（Average PT in the control group-Average PT in the administration groups）/ Average PT in the control group×100；

Decrement rate of TT （%）=（Average TT in the control group-Average TT in the administration groups）/ Average TT in the control group×100；

Decrement rate of APTT （%）=（Average APTT in the control group-Average APTT in the administration groups）/Average APTT in the control group×100；

Increment rate of FIB （%）=（Average FIB content in the administration groups-Average FIB content in the control group）/Average FIB content in the control group×100.

Data processing and statistics The data were analyzed using SPSS 11.5.The final data were expressed as±s.T-test was conducted for two independent samples，whileANOVA was conducted for pairwise comparisons among multiple samples.When P value was less than 0.05，the difference was considered to be statistically significant.

Results and Analysis

Effect of Callicarpa nudiflora on plasma PT of mice

As shown in Table 1，compared with that in the control group，the PTs in the crude dry extract group，water elute group，40% methanol elute group，60%methanol elute group and 80%methanol elute group were reduced by 34.2%，31.5%，31.5%，37.7%and 38.4%，respectively （P＜0.01）after a 7-d administration.However，compared with that in the crude dry extract group，the PTs in various elute groups were not changed significantly.

Effect of Callicarpa nudiflora on plasma TT of mice

As shown in Table 2，compared with that in the control group，the TTs in the crude dry extract group，water elute group，40% methanol elute group，60%methanol elute group and 80%methanol elute group were reduced by 19.8%，17.1%，12.3%，6.7% and 9.5%，respectively（P＜0.05 or P＜0.01）after a 7-d administration.There were no significant differences in TT between the crude dry extract group and elute groups.

Table 1 Effect of Callicarpa nudiflora on plasma PT of mice

Table 1 Effect of Callicarpa nudiflora on plasma PT of mice

*indicates significant difference at the 0.05 level compared with the control group；** indicates significant difference at the 0.01 level compared with the control group.

Group Dose∥g/kg PT∥s Decrement rate∥% Control - 14.6±1.8 -Crude dry extract 1.0 9.6±0.9**34.2 Water elute 1.0 10.0±0.9**31.5 40%methanol elute 1.0 10.0±0.6**31.5 60%methanol elute 1.0 9.1±0.8**37.7 80%methanol elute 1.0 9.0±0.7**38.4 Anluoxue 0.004 8.2±0.4**43.8

Effect of Callicarpa nudiflora on plasma APTT of mice

Table 3 showed that compared with that in the control group，the APTTs in the crude dry extract group，water elute group，40%methanol elutegroup，60%methanol elute group and 80%methanol elute group were reduced by 31.8%，33.4%，14.9% ，27.9%，40.3%and 35.3%，respectively（P＜0.01）after a 7-d administration. Compared with that in the crude dry extract group，the APTT in the 60% methanol elute group was significantly reduced（P＜0.01）.

Table 2 Effect of Callicarpa nudiflora on plasma TT of mice

Table 2 Effect of Callicarpa nudiflora on plasma TT of mice

*indicates significant difference at the 0.05 level compared with the control group；** indicates significant difference at the 0.01 level compared with the control group.

Group Dose∥g/kg TT∥s Decrement rate∥% Control - 25.2±1.7 -Crude dry extract 1.0 20.2±1.5**19.8 Water elute 1.0 20.9±1.5**17.1 40%methanol elute 1.0 22.1±1.5**12.3 60%methanol elute 1.0 23.5±1.6*6.7 80%methanol elute 1.0 22.8±2.3**9.5 Anluoxue 0.004 21.1±1.5**16.3

Table 3 Effect of Callicarpa nudiflora on plasma APTT of mice（±s，n=12）

Table 4 Effect of Callicarpa nudiflora on plasma FIB of mice

Table 4 Effect of Callicarpa nudiflora on plasma FIB of mice

*indicates significant difference at the 0.05 level compared with the control group；** indicates significant difference at the 0.01 level compared with the control group.

Group Dose∥g/kg FIB∥g/L Increment rate∥% Control - 1.15±0.13 -Crude dry extract 1.0 1.75±0.19**52.2 Water elute 1.0 1.64±0.18**42.6 40%methanol elute 1.0 1.40±0.10**21.7 60%methanol elute 1.0 1.47±0.21**27.8 80%methanol elute 1.0 1.36±0.15**18.3 Anluoxue 0.004 1.83±0.17**59.1

Effect of Callicarpa nudiflora on plasma FIB of mice

As shown in Table 4，after a 7-d administration，the FIBs in the crude dry extract group，water elute group，40% methanolelute group，60% methanolelute group and 80% methanol elute group were increased by 52.2%，42.6%，21.7%，27.8%and 18.3%，respectively （P＜0.01）compared with that in the control group.No significant differences were observed in FIB between the crude dry extract group and several elute groups.

Conclusions and Discussion

Currently，there have been some products made from extracts of Callicarpa nudiflora on the market，such as Luohuazizhu capsule，Luohuazizhu particle，Luohuazizhu tablet，anti-inflammatory suppository and compound casuarinas tablet［7-8］.These different types of preparations are commonly made from extracts or crude water extracts of Callicarpa nudiflora， which have dark colors and miscellaneous components.The coagulation promoting factions of Callicarpa nudiflora and their chemical composition are still unclear.The chemical base for coagulation effect of Callicarpa nudiflora is still unknown，greatly hindering the development and promotion of medicinal products of Callicarpa nudiflora.Prothrombin time（PT）can accurately and effectively reflect the performance of extrinsic coagulation system；thrombin time （TT）can effectively reflect the performance of endogenous coagulation system；activated partial thromboplastin time （APTT）reflects the common performance of extrinsic and endogenous coagulation systems；plasma fibrinogen（FIB）reflect coagulation effect indirectly through the effects of Callicarpa nudiflora on fibrinolytic system［9-10］.In this experiment，Kunming mice were treated as the research object，and the effects of Callicarpa nudiflora on blood coagulation system of experimental animal were investigated by measuring PT，TT，APTT and FIB.The results showed that the crude dry extract of Callicarpa nudiflora could significantly shorten the PT，APTT and TT，and increase the FIB content in mice.It suggests that Callicarpa nudiflora achieves the coagulation effect through affecting both extrinsic and intrinsic coagulation pathways.Compared with the control group，various elutes of Callicarpa nudiflora showed more obvious coagulation effects，indicating that various elutes of Callicarpa nudiflora all have coagulation-promoting effects.

In conclusion，the crude extract and various elutes of Callicarpa nudiflora all have better coagulation effects，and they achieve the coagulation-promoting effects mainly through endogenous thrombin， exogenous thrombin and both extrinsic and intrinsic coagulation pathways，as well as the effects on fibrinolytic system.

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Responsible editor：Tingting XU

Responsible proofreader：Xiaoyan WU

裸花紫珠凝血活性研究

杨子明1，黄胜2，3，颜小捷1，张利1，谷陟欣2，李典鹏1*（1.广西植物功能物质研究与利用重点实验室/广西植物研究所，广西桂林541006；2.九芝堂股份有限公司，湖南长沙410008；3.海南九芝堂药业有限公司，海南海口 570311）

［目的］研究裸花紫珠凝血作用。［方法］以小鼠为研究对象，通过测定其凝血酶原时间（PT）、凝血酶时间（TT）、活化部分凝血活酶时间（APTT）、血浆纤维蛋白原（FIB）4项指标观察裸花紫珠对试验动物凝血系统的影响。［结果］与空白对照组比较，裸花紫珠粗提物组及各洗脱部位组均能能明显缩短PT、APTT、TT的时间，能明显提高FIB含量。［结论］裸花紫珠及各洗脱部位可以通过影响实验动物凝血系统的多个环节发挥其凝血作用。

裸花紫珠；凝血；小鼠；凝血酶原时间

国家科技支撑计划（SQ2010BAJY1411-07-05）；海南省中药现代化专项 （2015ZY20）；海南重大科技项目（ZDZX2013008-01-04）；广西植物功能物质研究与利用重点实验室主任基金项目（ZRJJ2014-7，ZRJJ2015-14）；广西壮族自治区八桂学者专项经费资助。

杨子明（1982-），男，广西兴安人，助理研究员，硕士，从事植物活性物开发与利用研究，E-mail：yangziming198310@126.com。*通讯作者，研究员，博士，从事植物活性物开发与利用研究。

2015-09-14

Supported by National Key Technology Research and Development Program（SQ2010BAJY1411-07-05）；Special Fund for Modernization of Traditional Chinese Medicine in Hainan Province （2015ZY20）；Major Science and Technology Project of Hainan Province （ZDZX2013008-01-04）；Director Fund of Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization （ZRJJ2014-7，ZRJJ2015-14）；Bagui Scholar Program of Guangxi.

*Corresponding author.E-mail:yangziming198310@126.com

Received:September 14，2015 Accepted:October 19，2015

修回日期 2015-10-19