| BACKGROUNDUncaria macrophylla, belonging to Rubiaceae Uncaria, "Chinese Pharmacopoeia"2010 edition gathers, as a kind of Uncaria genuine traditional Chinese medicine. Uncaria macrophylla mainly grow in Guangdong, Guangxi, Yunnan and other provinces. Uncaria macrophylla is Chinese herbal drugs used for epileptic seizures and endogenous liver wind in traditional Chinese medical treatment.As a traditional Chinese medicine, the stems and branches with hook of Uncaria plants are always used. Recent studies have found that the chemical composition of leaves with Uncaria with similar stems. So the leaves have great potential to use as medicinal resource. However, due to the current absence of a systematic study of medicine and pharmacology and toxicology, Uncaria leaf has not been the development and utilization, resources has been in a lot of abandoned state. In Uncaria genus and related species of medicinal plants, Uncaria macrophylla leaves have the greatest weight and maximum yield. Uncaria macrophylla leaves present on the pharmacological impact have not been reported.OBJECTIVEResource survey of Uncaria macrophylla, pharmacognosy and determination of chemical composition, on this basis, the pharmacological impact of Uncaria macrophylla preliminary study, the main effect were observed on the CNS and the role of anti-drug dependence, the further development and utilization of drugs and provide experimental basis.METHODS1. Uncaria macrophylla mainly grown in southern and southwestern regions of China. We used plant taxonomy and plant geography methods, the nearest choice of site, large areas in western Guangdong Uncaria abundance, utilization and ecological environment, and investigation, the production of wax leaf samples collected leaves.2. Collected specimens of Uncaria macrophylla herbs, and leaves the character identification. The microscopic identification was used to observe the stoma and cell, the paraffin section used to the histological structure of main vein and diachyma, permeabilizing with chloral hydrate used to the microstructure of the powder, the reaction of alkaloid precipitation used as the physical and chemical identification and TLC used for the control.3. Determination of rhynchophylline and isorhynchophylline by HPLC, chromatographic column Hypersil ODS (4.6mm×200mm,5um), the mobile phase was methanol-water (containing 0.5% triethylamine, glacial acetic acid adjusted PH6.334)(67:33), and the flow rate was 0.8ml/min. The injection volume was 5.0μl Detection wavelength was 245nm. Column temperature was room temperature.4. The impact of Uncaria macrophylla on the CNS4.1 Locomotor activity test in mice:after fasting for 12h without limiting the water intake,60 Kunming mice with equal numbers in male and female were randomly divided into 6 groups, namely, control group (saline group with the same capacity), positive drug group (diazepam 1mg/kg), Uncaria macrophylla hooked stems group(20mg/kg), low dose of Uncaria macrophylla leaves group (5mg/kg), medium dose of Uncaria macrophylla leaves group(10mg/kg), and high dose of Uncaria macrophylla leaves group(20mg/kg). Each group of the mice was put into the YLS-1A multi-functional locomotor activity determinate 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 60min,120min the numbers of themice with locomotor activity were recorded. Each time lasted 5 min.4.2 Subthreshold dose of peniobarbital sodium inducing sleep test in mice:55 Kunming mice with equal numbers in male and female were randomly divided into 5groups, control group (saline group with the same capacity), positive drug group (diazepam lmg/kg), Uncaria macrophylla hooked stems group (20mg/kg), low dose of Uncaria macrophylla leaves group (5mg/kg), and high dose of Uncaria macrophylla leaves group (20mg/kg). Each group had 10 animals. They were administered medicine with different doses by intragastric administration. After 60 min, each mouse was administered pentobarbital sodium (30mg/kg) by intraperitoneal injection and the disappearance of righting reflex in mice for more than 1 min was regarded as the standard of sleeping. The number of the sleeping mice in each group was recorded with in 15min.4.3 Subthreshold dose of pentobarbital sodium inducing sleeping time of mice test:40 Kunming mice were equally divided between male and female. They were randomly divided into 5 groups, grouping is as the above. Each group had 8 animals. They were administered medicine with different doses by intragastric administration. After 60 min, each mouse was administered pentobarbital sodium (50mg/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)4.4 Experiment on convulsions of mice induced by Strychnine sulfate:75 Kunming mice with equal numbers in male and female were divided into 5 groups randomly, with 15 in each group. Each group of the mice was administered drugs by ig administration. After 60 min, 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 with in 2h, as well as the deaths of the mice. Convulsions indicator reaction was tonic clonic limbs in mice.4.5 Experiment on convulsions of mice induced by Strychnine sulfate:75 SPF mice which passed the determination of nature place preference were randomly divided into①normal control group,②model group of amphetamine,③model+Uncaria macrophylla hooked stems group(20mg/kg),④model+high dose of Uncaria macrophylla leaves group (20mg/kg)⑤model+low dose of Uncaria macrophylla leaves group (5mg/kg),⑥Uncaria macrophylla hooked stems group(20mg/kg).②～⑤mice were injected with methamphetamine(2 mg/kg) in the odd number day (Am 8:00), and equal volume of normal saline were injected to mice in the even number day (Am 8:00).③～⑤mice were given (ig) corresponding dose Uncaria macrophylla every evening (Pm 20:00).①mice were given equal volume normal saline instead of amphetamine, other process just same as②group.⑥mice were given (ig) corresponding dose of rhynchophylline in the odd number day (Am 8:00), and equal volume of normal saline were injected to mice in the even number day (Am 8:00). In the day 1 morning(8:00), after given with normal saline, amphetamine or Uncaria macrophylla, the mice were put in the white box; in the day 2 morning(8:00), after given with normal saline, mice were put in the black box. The training in CPP shuttle box was kept for 8 consecutive days (dl-d8). The animal ethnology analytical system was used to record and analyze rat staying-time in drug-paired compartment for 15 min. After examination of CPP, all mice in each group were decapitated quickly. Rapidly stripping the whole brain, frozen in liquid nitrogen is set after 1 minute, put under test in a -80℃save. Test parts of the brain of mice, weighing, homogenized, processed by centrifugation into the sample solution, measured by fluorescence spectrophotometry in the brain NE, DA,5-HT content.6. Statistical Methods:Statistical software SPSS 13.0 was used to analyze the experimental data. The experimental data were indicated by mean±standard deviation (±s). Paired t test, repeated measures analysis of variance, ANOVA,.LSD method was used in multiple comparisons between groups when variance is homogeneous. Dunnett's T3 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. Luoding City, Guangdong Province, an excellent ecological environment and forest plant communities preserved relatively intact, is suitable for the growth of south region herbal medicine. And there is a profound cultural background of folk medicine; the resources should be actively protected, further rational development and utilization. The large leaves of Uncaria findings of pharmacognosy, Uncaria macrophylla leaves large, including many single-cell non-glandular hairs,2-3 palisade cells, thin leaf cells containing clusters of calcium oxalate crystal jade, plain shaft type stomatal. By the method of TLC, we could find that there were at least two kinds of the same alkaloids.2. HPLC results showed that rhynchophylline and isorhynchophylline in 0.02μg～5μg good linear relationship, r rhynchophylline=0.9999, r isorhynchophylline= 1.0000, average recoveries were 98.52% and 99.12%, RSD rhynchophylline= 2.045%, RSD isorhynchophylline=2.171%. Uncaria macrophylla leaves alkali content of 0.016%, hook 0.015%, isorhynchophylline of Uncaria macrophylla leaves alkali content is 0.006%, and Uncaria macrophylla hook 0.004%.3. The impact of Uncaria macrophylla on the CNS3.1. The impact of drugs on locomotor activity of mice:between treatment groups using repeated measures analysis of variance, repeated factor of four levels of data does not satisfy Sphericity text (P= 0.009), taking Greenhouse-Geisser correction of the results. Difference between treatment groups was statistically significant (F= 4.187, P=0.003), four time points were statistically significant (F=71.010, P <0.001), time factor and there is no interaction between groups (F=1.605, P=0.091). At. each time point differences between different groups using One-Way ANOVA, 30min after drug administration before and two points in time homogeneity of variance (P=0.238,0.215), the six treatment groups no significant difference between (F drug before=2.209, P drug before=0.067; F after drug 30min=0.987, P drug after 30min=0.434); drug 60min after the homogeneity of variance (P=0.102), the differences between the six treatment groups was statistically significant (F=3.346, P =0.009), control group were higher than the other five groups (P=0.009,0.001, 0.001,0.002,0.008), while the remaining five were no significant differences between groups (P values> 0.05); 120min after drug homogeneity of variance (P= 0.199), the differences between the six treatment groups was statistically significant (F120min=3.538, P120min=0.008), higher than the hook stem control group, positive medicine group, low dose leaves group, leaves the high dose group (P<0.001, P=0.004,0.017,0.005). Each group activities at different time points between the number of repeated measures analysis of variance with the blank group at each time point the data does not satisfy Sphericity text (P=0.022), Greenhouse-Geisser correction of the results obtained, differences between the four time points was statistically significance (F=4.010, P=0.018); to 60 minutes after drug administration before and the difference was statistically significant; positive drug data set to meet the Sphericity text (P=0.077), four time points were statistically significant (F=9.261, P<0.001); to 60 minutes after drug administration before and the difference was statistically significant. Hook stem group, leaves the low dose group, middle dose group leaves, leaves to meet the high dose group data are assumed (P=0.179,0.865,0.085,0.294), four time points were statistically significant (F= 11.901, P<0.001; F=17.515, P<0.001; F=21.998, P<0.001; F=14.332, P<0.001), both to 30 minutes after drug administration before and the difference was significant. Overall, the number of activities in mice before and after drug administration there are differences between different times, six groups were administered prior to the maximum, except the blank group, and after administration of the other five the number of activities decreased with time, to 120min minimum. Hook group and leaves each dose administered 30 minutes before with the difference statistically significant, positive to the drug group and 60 minutes after drug administration before the difference was statistically significant, leaves rapid onset.3.2 Control group injected subthreshold dose of sodium pentobarbital sleep (30mg/kg) after the loss of righting reflex in mice was 0. Positive drug group had 6 mice to sleep, hook group of 7 mice to sleep, leaves the low dose group,5 mice to sleep, high-dose group,4 mice sleep. Test results in Table 3, because 50% of the lattice theory of frequency less than 5, take the results of Fisher exact test, p=0.015, significant difference may be that the 5 differences between experimental groups was significant. Sleep time in mice in each group of data by one-way ANOVA analysis showed significant group differences (F=5.676, P=0.001), need further pairwise comparisons (using LSD method). The control group and the low dose group was no significant difference (P=0.959), and the hook stem no significant difference (P= 0.274), and positive drug group were significantly different (P=0.004), High-dose group and leaves were significantly different (P=0.001). The results show that leaves the high dose group the threshold dose of pentobarbital sodium can result the duration of sleep in mice, and compared with the positive drug not significantly different (P=0.569).3.3 Mice by intraperitoneal injection of strychnine (2mg/g), the mice occurred within 5min tonic convulsions and death. The number of mice in each group survived by X2 test,50% of the grid is less than 5, take the results of Fisher exact test, P=0.133, the difference was not statistically significant, can be considered among the five experimental groups of mice the number of seizures. There was no significant difference. Convulsion time of mice by one-way ANOVA analysis, levene test Heterogeneity of variance, Welch method with approximate F test, P= 0.003, significant group differences, the pairwise comparison, the positive drug group and control group, hook stem high-dose group were significantly different, the other four were no significant differences in leaf low dose group and the positive drug group was not significantly different (P=0.142), but the other 3 groups there was no significant difference, still can not believe that similar efficacy between them.4. The impact of Uncaria macrophylla in amphetamine dependent mice Before administration of the variance in each group of data is Heterogeneity of variance (P=0.022), the results obtained Welch P=0.309, the difference was statistically significant. Control group of mice before and after administration of the residence time in the medicine chest with no significant change (P=0.162). In comparison with the pre-administration, the model after administration in mice with medicine cabinet in the residence time was significantly longer (P=0.002). White boxes indicate that the model generated mice CPP, CPP mouse model of amphetamine success. Compared with the previous administration, the low dose group and high dose group, with leaf stems the hook stem group, pure hook in mice after administration of the residence time in the medicine chest with no significant difference. Compared with the control group, model group mice after administration of the medicine cabinet with the length of stay was significantly longer in (P=0.001); and rhynchophylline low dose group and high dose group of mice after administration of the medicine cabinet with associated length of stay was not significantly different. The low dose group, the hook stem group, pure hook group compared with the model group were significantly (P<0.001, P=0.006, P<0.001). With One-Way ANOVA on the assay results of each group were analyzed, each group NE, DA,5-HT data homogeneity of variance (P=0.738, P=0.120, P=0.072), the difference between the groups was statistically significant (P<0.001, P<0.001, P= 0.033). Multiple comparisons with LSD, compared with the control group, model group, NE, DA levels were higher (P<0.001),5-HT content decreased (P=0.033); model+NE content increased hook group (P=0.044) pure hook, model+hook, model+high and low dose group NE were lower than model group (P<0.001); pure hook, model+hook, model+high and low dose group were lower than model group DA (P=0.002, P<0.001, P=0.001, P<0.001); pure hook group, model group of 5-HT+hook was higher than the model group (P=0.002, P=0.01); model+high dose group, Model+low-dose group,5-HT content compared with the model group increased, but the difference was not significant (P=0.169, P=0.069).CONCLUSION1. Character identification and micro-features of leaves of Uncaria macrophylla. Have morphological rules, and that can be used as experimental evidence of identification of pharmacognosy. Results of alkaloid precipitation and TLC analysis show that Uncaria macrophylla leaves and Uncaria control medicines have the same chemical compositions, especially in the alkaloids are at least two aspects of the same ingredients.2. Uncaria macrophylla with a high content in rhynchophylline and isorhynchophylline. This method is accurate, simple, rapid, reproducible, high precision, can be used to determine the contents of total alkaloid and monomer composition rhynchophylline, isorhynchophylline for different types and parts of Uncaria genus.3. Uncaria macrophylla can significantly reduce locomotor activity, increase the number of the hypnotized mice and extend the mice's sleeping time. However, the effect of decreasing the number of deaths induced by strychnine sulfate and the convulsive latent time in mice induced by strychnine sulfate was not obvious. Mice could produce conditioned place preference due to amphetamine, prolonging the time-staying in drug-paired compartment. The time-staying in drug-paired compartment could be reduced to some extent by various parts of Uncaria macrophylla, and eliminate CPP effect significantly. As the same time, we found that Uncaria macrophylla did not cause CPP effect in normal mice. The results showed that the determination of neurotransmitters, the brain area model group DA, NE,5 HT content were different from those of a control group, suggesting that these three monoamine neurotransmitters is closely related with the amphetamine dependence. Uncaria macrophylla can to some extent in each group to improve the changes in neurotransmitter levels, so that tends to normal levels. |
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