Laboratory Study On The Pharmacological Effects Of Uncaria Tomentosa

BackgroundUncaria tomentosa is the dry stem of Rubiaceae Uncaria plants, which is commonly known as Cat's Claw. The plant originated in Peru, Ecuador and other tropical rainforest areas in Central America. It has the name of Cat's Claw because of the claw-shaped hooks on its leaf base. It is reported that Uncaria tomentosa has the effects of enhancing immunity, increasing DNA repair, inhibiting cancer cell proliferation, restraining the decrease of leucocyte caused by chemotherapy, anti-forgotten and anti-inflammatory. The herb is often used to treat ulcers, rheumatoid arthritis, tumor, as well as prevention and treatment of leukemia, AIDS and so on. Since 1970s, overseas scholars have carried out researches on chemical composition, pharmacological effects and clinical application of Uncaria tomentosa and as a result, they have confirmed a lot of traditional efficacy of the herb. In addition, they have found that the herb can help resist many incurable diseases troubling a lot of people, so that it has gradually become one of the best-selling herbal medicines in the international market.Uncaria tomentosa and Ramulus et uncus uncariae both belong to Rubiaceae Uncaria plant. Their main chemical composition is similar, which both are alkaloids and indole-based alkaloids. The similarity in chemical composition of these two drugs sure determines that their pharmacological effects also have similarities. Ramulus et uncus uncariae, a Chinese traditional medicine, is used as a drug for calming the liver and checking wind whose main effect is to inhibit the central nervous system. Does Uncaria tomentosa have the same effect? The effect of Uncaria tomentosa on the central nervous system has not been reported yet.The article is going to study preliminarily the pharmacological effects of Uncaria tomentosa on the central nervous system, observe the effect of Uncaria tomentosa on rats' blood pressure through hypertensive rat models and probe for the mechanism for the purpose of providing more experimental bases to develop the resources of Uncaria plants.OBJECTIVE1. To study and compare the sedative and hypnic effect, as well as the anticonvulsant effect of Uncaria tomentosa from Peru and ramulus et uncus uncariae from China.2. To study and compare the antihypertensive effect and the mechanism of Uncaria tomentosa produced in Peru and ramulus et uncus uncariae produced in China.METHODS1. Locomotor activity test in mice: After fasting for 12h without limiting the water intake, 50 Kunming mice with equal numbers in male and female were randomly divided into 5 groups, namely, control group (saline group with the same capacity), Ramulus et uncus uncariae group (320mg/kg), low dose of Uncaria tomentosa group (80mg/kg), medium dose of Uncaria tomentosa group (160mg/kg), and high dose of Uncaria tomentosa group (320mg/kg). Each group of the mice was put into the YLS-1A multi-functional locomotor activity determinator for little animals and the mice were adapted to the environment for 5 min. Then the number of the mice with locomotor activity was determined before administering any medicine for 5 min. Keep the lab quiet while recording. Each group was administered different medicine according to different doses by intragastric administration (ig) respectively. After 30 min and 60 min, the numbers of the mice with locomotor activity were recorded. Each time lasted 5min.2. Subthreshold dose of pentobarbital sodium inducing sleep test in mice: 50 Kunming mice were equally divided between male and female, grouping is as the above. Each group had 10 animals. They were administered medicine with different doses by intragastric administration. After 60 min, each mouse was administered pentobarbital sodium (30 mg / kg) by intraperitoneal injection and the disappearance of righting reflex in mice for more than 1min was regarded as the standard of sleeping. The number of the sleeping mice in each group was recorded within 15 min.3. Subthreshold dose of pentobarbital sodium inducing sleeping time of mice test: 50 Kunming mice were equally divided between male and female. They were randomly divided into 5 groups, grouping is as the above. Each group had 10 animals. They were administered medicine with different doses by intragastric administration. After 60 min, each mouse was administered pentobarbital sodium (50 mg / kg) by intraperitoneal injection. The sleeping time of the mice in each group was recorded. (The disappearance of the righting reflex in mice after administration was regarded as the time of falling asleep, and the time from the disappearance of righting reflex to recovery was sleeping time.)4. Experiment on convulsions of mice induced by Strychnine sulfate: 50 Kunming mice with equal numbers in male and female were divided into 5 groups randomly, with 10 in each group. Each group of the mice was administered drugs by ig administration. After 60min, each group was administered strychnine sulfate by intraperitoneal injection (2mg / kg), and the number of the convulsive mice was recorded after the injection of strychnine sulfate within 2h, as well as the deaths of the mice. Convulsive reaction has been based on mice limbs or tonic-clonic seizures for the target.5. Reproduction of hypertensive rat model: Low frequency and low-voltage alternating current electric was used to shock the feet of the rats, combining noise to stimulate the rats. They were stimulated repeatedly by the combination of low frequency and low-voltage alternating current electric, as well as noise for 28d to establish a stable chronic hypertensive animal model induced by stress. The rat tail artery manometer was used to measure repeatedly the blood pressure of the rats 3 times, taking the average. The rats with systolic blood pressure greater than 18.7Kpa (140mmHg) and diastolic blood pressure greater than 12.0Kpa (90mmHg) were regarded as successful models, removing the animals which didn't comply with the above requirements.6. Experimental groups of hypertensive rat model: the rats with blood pressure in line with the requirements (other than the normal control group) were randomly divided into 3 groups. Each group had 10 animals. They were model control group (with the same amount of distilled water), Uncaria tomentosa group (120 mg / kg) and Ramulus et uncus uncariae group (120 mg / kg). Another 10 rats belong to the normal control group (with the same amount of distilled water).7. Detection indicator of hypertensive rat model: the rats were measured blood pressure by the rat tail artery manometer before the establishment of the model, after the establishment of the model and after the experiment. The average value was recorded. After the experiment, each rat was drawn 2ml blood from its abdominal aorta, which was mingled with EDTA-Na2 (100 g / L) and trasylol in a test tube. Plasma was centrifuged form the complex in 4℃and preserved in -20℃for future use. It was solubilized at room temperature and centrifuged again to get supernatant. Radioimmunoassay (RIA) was used to measure the content of Ang II, ET and CGRP content in the plasma.8. Statistical Methods: Statistical software SPSS13.0 was used to analyze the experimental data. The experimental data were indicated by mean±standarddeviation (x|-±s). Repeated measures analysis variance and the single-factor analysis of variance (ANOVA) were used to analyze the data. LSD method was used in multiple comparisons between groups when variance is homogeneous. Games-Howell method was used when variance is not homogeneous. Chi-square test was used to deal with count data. P <0.05 indicates that the data have statistical significance.RESULTS1. The impact of the drugs: the result shows that there is interaction between the time point and the groups (F=4.065, P=0.000), main effects between groups (F=8.201, P=0.000) and main effects between the time points (F=41.712, P=0.000). The control group had no changes in frequency on the mouse locomotor activity before and after administration (P>0.05). After 30min of administration, the number of the mice with the locomotor activity in Ramulus et uncus uncariae group was reduced. Compared with the condition before administration, the difference was significant (P<0.01). Compared with the control group, there was also a significant difference (P<0.01); But the locomotor activity of mice in the low dose of Uncaria tomentosa group, medium dose of Uncaria tomentosa group, and high dose of Uncaria tomentosa group had no changes. Compared with the condition before administration and with the control group, there was no significant difference (P> 0.05). Compared with Ramulus et uncus uncariae group, the locomotor activity of mice after 30min of administration in the low dose of Uncaria tomentosa group, medium dose of Uncaria tomentosa group, and high dose of Uncaria tomentosa group had significant difference (P<0.01). After 60min of administration, the locomortor activity of mice in both Ramulus et uncus uncariae group and the low dose of Uncaria tomentosa group, medium dose of Uncaria tomentosa group, high dose of Uncaria tomentosa group were significantly reduced. Compared with the previous administration, the difference was significant (P <0.05 or P<0.01). Compared with the blank control group, there was also a very significant difference (P<0.01). Comparing the low dose of Uncaria tomentosa group, medium dose of Uncaria tomentosa group, high dose of Uncaria tomentosa group with Ramulus et uncus uncariae group, there was no significant difference (P> 0.05).2. The impact of the drugs on sleeping mice induced by the threshold dose of pentobarbital sodium: There were only 3 mice disappearing righting reflex in control group after being administered pentobarbital sodium (30 mg / kg) by intraperitoneal injection. There were 8 mice which fell asleep in both Ramulus et uncus uncariae group and the low dose of Uncaria tomentosa group. There were 9 sleeping mice respectly in the medium dose of Uncaria tomentosa group and the high dose of Uncaria tomentosa group. The numbers of the sleeping mice were analyzed by Chi-square test, and the result showed that x~2 = 13.098, v = 4, P = 0.011 (bilateral), and there was significant difference. That is to say, differences among the five groups were significant. The numbers of the sleeping mice in the medium dose of Uncaria tomentosa group and the high dose of Uncaria tomentosa group were the most.3. The effect of the drugs on mouse sleeping time induced by pentobarbital sodium: the data of mouse sleeping time in each group were analyzed by one-way ANOVA. It showed that there was significant difference among groups (F=24.304, P=0.000). LSD method was used for further analyse. There was no significant difference between control group and the low dose of Uncaria tomentosa group (P=0.379). But compared with other groups, control group was significantly different (P<0.01). There was no difference between Ramulus et uncus uncariae group and the high dose of Uncaria tomentosa group (P=0.343). Compared with other groups, they were significantly different (P<0.05). The result shows that both Ramulus et uncus uncariae and Uncaria tomentosa can obviously prolong the sleeping time of the mice induced by subthreshold dose of pentobarbital sodium. The effect of Uncaria tomentosa strengthened with the increased dose.4. The effect of the drugs on convulsive mice induced by strychnine sulfate: the mice were administered strychnine sulfate (2mg/kg) by intraperitoneal injection. Most mice would have tonic convulsions and die within 10min. The number of mice with convulsions in each group was analyzed by Chi-square test. The result shows that, x~2 - 2.500, v = 4, P = 0.645 (bilateral), the difference was not significant. That is to say, the numbers of the convulsive mice among the five experimental groups had no significant difference. 8 mice in control group died and 2 mice in Ramulus et uncus uncariae group died. The numbers of the deaths in low dose of Uncaria tomentosa group, medium dose of Uncaria tomentosa group and high dose of Uncaria tomentosa group were 6, 4 and 2 respectly. The numbers of the deaths were analyzed by Chi-square test, whose result shows that, x~2 = 11.303, v = 4, P = 0.023 (bilateral), the difference was significant. That is to say, differences among the 5 experimental groups were significant. The number of the deaths in Ramulus et uncus uncariae group and in high dose of Uncaria tomentosa group was the least.5. The effect of the drugs on blood pressure in hypertensive rat models:The impact of the drugs on systolic blood pressure: there is interaction between the time point and the groups (F=16.240, P=0.000), main effects between groups (F=9.951, P=0.000) and main effects between the time points (F=98.796, P=0.000). One-way ANOVA was used to analyze the points of time, and the repeated measures analysis of variance was used to analyze the main effects of the total time among groups. Before the establishment of the hypertensive rat models, the systolic blood pressure of all rats was not statistically significant (P> 0.05). After the establishment of the model, compared with the normal control group, the systolic blood pressure of the rats in model control group, Uncaria tomentosa group and Ramulus et uncus uncariae group were significantly different (P = 0.000), but there was no significant difference among the three groups (P> 0.05). After the treatment, the systolic blood pressure of the rats in Uncaria tomentosa group, Ramulus et uncus uncariae group and normal control group were significantly different, compared with control group (P=0.000), but they had no significant difference among the three groups (P> 0.05).The impact of the drugs on diastolic blood pressure: there is interaction between the time point and the groups (F=3.388, P=0.009), no effect between groups (F=1.634, P=0.199) and main effects between the time points (F=17.661, P=0.000). One-way ANOVA was used to analyze the points of time, and the repeated measures analysis of variance was used to analyze the main effects of the total time among groups. Before the establishment of the rat model, the diastolic pressure of the rats in each group had no significant difference (P> 0.05). After the establishment of the rat model, compared with normal control group, the diastolic blood pressure of rats in model control group was significantly different (P = 0.028), the diastolic blood pressure of rats in Uncaria tomentosa group was also different (P=0.027), the diastolic blood pressure of rats in Ramulus et uncus uncariae group was different as well (P = 0.014). but there were no significant difference among the three groups (P>0.05). After the treatment, compared with the model control group, the diastolic blood pressure of the rats in the normal control group was signifieantly different (P=0.014), the diastolic blood pressure of the rats in Uncaria tomentosa group was also different (P = 0.013), the diastolic blood pressure of the rats in Ramulus et uncus uncariae group was different as well (P=0.008). But there were no significant difference among the three groups ( P> 0.05).In short, before the establishment of model animals, the blood pressure of all the rats was not statistically significant (P>0.05). After the establishment of model animals, all the rats have increased blood pressure (P <0.05). After 7-day treatment, the model group of animals had no significant changes in blood pressure (P> 0.05). Blood pressure of rats in both Uncaria tomentosa group and Ramulus et uncus uncariae group decreased significantly (P <0.01). In inter-group comparison, the two drug-treated groups compared with the model group were significantly different (P <0.05), but the two drug-treated groups themselves had no significant difference (P> 0.05).6. The effect of the drugs on Ang II, ET and CGRP of the hypertensive rats: Ang II and ET levels of the model group were significantly increased. Compared with the normal control group, Ramulus et uncus uncariae group, and Uncaria tomentosa group, there was significant difference (P<0.01). CGRP levels in model group decreased significantly. Compared with Ramulus et uncus uncariae group, Uncaria tomentosa group and normal control group, the difference was significant (P <0.01).CONCLUSION1. Uncaria tomentosa can significantly reduce locomotor activity, increase the number of the hypnotized mice and extend the mice's sleeping time. Uncaria tomentosa has effects of sedation and hypnosis, which is similar to ramulus et uncus uncariae2. Uncaria tomentosa has the effect of decreasing the number of deaths induced by strychnine sulfate. However, the effect on the number of convulsive mice was not obvious. The anticonvulsant effect of Uncaria tomentosa is similar to Ramulus et uncus uncariae.3. Uncaria tomentosa has an obvious hypotensive effect. Its effect of lowering blood pressure may be related to the regulation of renin - angiotensin - aldosterone system. Ramulus et uncus uncariae has the same effect.