Contents Determination Of The Active Ingredient In Guangdong Uncaria Genus And Its Pharmacological Actions

BACKGROUNDRubiaceae of Uncaria genus plants all over the world there are about34species, distributed in Asia, Australia, Africa and Madagascar, and tropical America. There are eleven kinds and one variety in china, widely distributed in Guangdong, Guangxi, Fujian, Jiangxi, Hunan, Guizhou, Sichuan and other provinces. Here, the Guangdong Uncaria plants are mainly located in the Northwest area, rich resources and more varieties. There are about6species, such as Uncaria rhynchophylla (Miq) Miq. ex Havil., U. macrophylla Wall., U. hirsute Havil., U. sessilifructus Roxb., U. rhynchophylloides How and U.scandens (smith) Hutch.Rubiaceae Uncaria is a commonly used traditional Chinese medicine, and then "Chinese Pharmacopoeia"2010edition reproduce Uncaria rhynchophylla (Miq) Miq. ex Havil.,U. macrophylla Wall., U. hirsute Havil., U. sinensis (Oliv.) Havil.and U. sessilifructus Roxb..The plants of Uncaria genus are full of variety. They are belonging to multi-source Chinese herbal medicines.Modern clinical pharmacology studies have shown that Uncaria has the exact effect for high blood pressure, headaches, Vertigo, and so on. The genus has a wide range of chemical component, such as indole alkaloid, flavonoids and triterpene compounds, with effective part of indole alkaloids are the main. Currently, a comprehensive and systematic research of Uncaria resources in Guangdong Province was deficient. According to the survey of Uncaria resources in Guangdong Province is so rich that with some research value.In recent years, studies have shown that Uncaria medicine not only the traditional administration site of stems with hooks is a medicinal resource, the leaves and the stems also have potential as medicine resources. The resource is very rich, its Medicinal value or utilization of potential is huge. The study on the system of pharmacy, pharmacology and toxicology in different parts and different varieties of the Uncaria resources in Guangdong Province are less, so it has not been exploitation.OBJECTIVEContents determination of total alkaloid, hirsutine and rhynchophylline, isorhynchophylline in different varieties and different parts of the Uncaria genus of Guangdong, and central inhibition studies for those Uncaria genuses. For establishing methods for UV and HPLC determinations of alkaloids in Uncaria genus and compare their contents between different parts of Uncaria in Guangdong. Also for this new resource reasonable exploitation and utilization of medicinal plants provide Pharmacodynamics basis.METHODS1. Three ways to be selected, such as cold dipping method, thermal extraction method and ultrasonic extraction method, on this basis then compare a variety of extraction solvents, for example Petroleum Ether,80%Ethanol, Anhydrous Ethanol, Methanol, Ether and Chloroform.2. To establish methods for UV determinations of total alkaloid in U. rhynchophylla (Miq) Miq.ex Havil., U. macrophylla Wall. U. hirsute Havil., U. sinensis (Oliv.) Havil. and U. sessilifructus Roxb.. And compare their contents between different parts of them in Guangdong. Rhynchophylline and hirsutine as the reference substances, and then use them to calculate the total alkaloid content. The detect wavelengths are242nm and245nm respectively.3. To establish methods for HPLC determination of hirsutine, rhynchophylline and isorhynchophylline in Uncaria genus and compare their contents between different parts of these kinds Uncaria in Guangdong:①Determination of rhynchophylline and isorhynchophylline by HPLC, chromatographic column Hypersil ODS C18(4.6mm×200mm,5μm), the mobile phase was Methanol-water (containing0.50%triethylamine, glacial acetic acid adjusted pH6.33)(63:37), and the flow rate was0.80mL·min-1. The injection volume was5.00μL. Detection wavelength was245nm. Column temperature was room temperature.②Determination of hirsutine by HPLC, chromatographic column Hypersil ODS C18(4.6mm×200mm,5μm), the mobile phase was Methanol-water (containing0.50%triethylamine, glacial acetic acid adjusted pH5.35) with gradient elution, the injection volume was10.00μL and the column temperature was40℃. Detection wavelength was245nm.4. The research of central inhibition effect in different parts of four kinds of Uncaria genus①Locomotor activity test in mice:After fasting for12h without limiting the water intake,90Kunming mice with equal numbers in male and female were randomly divided into9groups, control group(saline group with the same capacity), U. macrophylla group(15g·kg-1), leaves of U. macrophylla group(15g·kg-1), U. rhynchophylla group(15g·kg-1), leaves of U. rhynchophylla group(15g·kg-1), U. rhynchophylloides group(15g·kg-1), leaves of U. rhynchophylloides group(15g·kg-1), U. hirsute group(15g·kg-1) and leaves of U. hirsute group(15g·kg-1). Each set of10 animals. 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 for5min.Then the number of the mice with locomotor activity was determined before administering any medicine for5min, keeping the lab quiet while recording. Each group was administered different medicine according to the same dose by intragastric administration (ig) respectively. After30min and60min the numbers of the mice with locomotor activity were recorded. Each time lasted5min.②Subthreshold dose of Sodium pentobarbital inducing sleep test in mice:180Kunming mice with equal numbers in male and female were randomly divided into9groups, control group(saline group with the same capacity), U. macrophylla group(15g·kg-1), leaves of U. macrophylla group(15g·kg-1), U. rhynchophylla group(15g·kg-1), leaves of U. rhynchophylla group(15g·kg-1), U. rhynchophylloides group(15g·kg-1), leaves of U. rhynchophylloides group(15g·kg-1), U. hirsute group(15g·kg-1) and leaves of U. hirsute group(15g·kg-1). Each group had20animals. They were administered medicine with the same dose by intragastric administration. After60min, each mouse was administered sodium pentobarbital (30mg·kg-1) by intraperitoneal injection and the disappearance of righting reflex in mice for more than1min was regarded as the standard of sleeping. The number of the sleeping mice in each group was recorded with in15min.③Threshold dose sodium pentobarbital-induced sleeping time in mice experiments: After fasting for12h without limiting the water intake,90Kunming mice were equally divided between male and female. They were randomly divided into9groups, grouping is as the above. Each group had10animals. They were administered medicine with the same dose by intragastric administration. After60min, each mouse was administered sodium pentobarbital (50mg·kg-1) 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).④Experiment on convulsions of mice induced by Strychnine Nitrate:After fasting for12h without limiting the water intake,90Kunming mice with equal numbers in male and female were divided into9groups randomly, with10in each group. Each group of the mice was drugs delivery by intragastric administration. After60min, each group was administered stryehnine Nitrate by intraperitoneal injection(1.5mg·kg-1), and the number of the convulsive and the death in mice were recorded after the injection of stryelinine Nitrate within2h.5. Statistical Methods:①97-2003software Excel was used to analyze the data processing of Content determination of main chemical components.②Statistical software SPSS13.0was used to analyze the experimental data. The experimental data were indicated by (x±s). Repeated measures analysis of repeated measures analysis of variance, One-Way ANOVA,.LSD method was used in multiple comparisons between groups when variance was homogeneous. Games-Howell method was used when variance was not homogeneous. Chi-square test was used to deal with count data. P<0.05indicates that the data have statistical significance.RESULTS1. Three ways to be selected, such as cold dipping method, thermal extraction method and ultrasonic extraction method, on this basis then compare a variety of extraction solvents, for example Petroleum Ether,80%Ethanol, Anhydrous Ethanol, Methanol, Ether and Chloroform. From timeliness and safety to consider, the experiment finalized with Anhydrous ethanol as the solvent, first infiltration of ammonia, then to add20times the volume of ethanol extraction of two times per40min, for the extraction of the sample solution.2. Rhynchophylline or hirsutine as the reference substances, and then to calculate the total alkaloid content. Results indicate that the total alkaloid content in various Uncaria species were different in different parts of Uncaria genus.①Calculation of the total alkaloid content by rhynchophylline, its linear range of6.00～14.00μg·ml-1r=0.9985. The total alkaloid content in the stems with hooks of U. macrophylla Wall is higher than other. The content of total alkaloid is0.478%.②Calculation of the total alkaloid content by hirsutine, its linear range of2.00～10.00μg·ml-1, r=0.998. The total alkaloid content in the Leaf of U. macrophylla Wall is highest in all. The content of total alkaloid is0.778%.3. The calibration curves of rhynchophylline and isorhynchophylline were in good linearity over the range of0.02～5.00μg(rl=0.9995, r2=0.9995). The average recovery rate were98.52%and99.12%, with RSD1=2.05%, RSD2=2.17%. The calibration curve of hirsutine was in good linearity over the range of0.02～0.4μg (r=0.9995). The average recovery rate was96.83%, with RSD=1.60%.4. The results of central inhibition effect①Mouse activity before and after the administration of a significant difference between the number sense (F=114.228, P<0.001), all of groups were administered prior to the maximum before the drug, in addition to the blank group, the remaining8groups to the number of activities with time after the drug was significantly decreased at60min of minimum. In the downward trend of U. macrophylla group is most clearly. Difference between the9groups was no significant (F=1.407, P=0.206) Administered before,30min after the drug was no significant difference between9groups (Pl=0.653,P2=0.150), but drug after60min, that there are significant differences between the all of groups(P=0.001). Drug after60min, control group was slightly on the rise and significantly higher than the other8groups. Point in time and interaction between groups(F=2.011, P=0.018). Through the6groups for each time point compared pairwise, all of drug delivery group30minutes after drug administration were significantly before differences.3time points by pairwise comparison of each group, before administration, there were no significant differences between the blank groups and the remaining8group.30min after the drug, Compared with the blank group, only U. macrophylla group was significant difference, other groups were not. After the drug60min, the blank group and U. rhynchophylloides group and leaves of U. rhynchophylloides group were not significantly different, and the remaining6groups were significantly differences.②Mouse righting reflex disappear (go to sleep) to positive, sleep number by the χ2test in mice showed that, χ2=27.596, v=8, P=0.001, there are significant differences. By the table, the blank group njected subthreshold dose of sodium pentobarbital sleep (30mg·kg-1) after the loss of righting reflex in mice was three. Number of mice fall asleep in the blank group was less than other groups. Among them, U. rhynchophylla mice fall asleep only number15. That is the most. And then there were14and12respectively in the groups of U. macrophylla and leaves of U. macrophylla.③Sleep time in mice in each group of data by One-Way ANOVA analysis showed significant group differences (F=4.765, P<0.001), need further pairwise comparisons (using Games-Howen method). Results show that, the blank group and the U. macrophylla group or leaves of U. macrophylla group was significant difference (Pl=0.001, P2=0.016).Also the blank group and the U. rhynchophylla group were significant difference (P<0.001). The results show that U. macrophylla group、leaves of U. macrophylla group and U. rhynchophylla group the threshold dose of pentobarbital sodium can result the duration of sleep in mice. and other groups compared with the blank group were not significantly different (P>0.05).④Mice by intraperitoneal injection of strychnine (1.5mg·kg-1), the mice occur within5min tonic convulsions and death. Convulsion in mice to positive, the number of mice in each group seizures by χ2test,χ2=10.827, v=8, P=0.212, there are no significant differences. Death in mice is positive, the number of mice in each group death by χ2test,χ2=27.569, v=8, P=0.001, there are significant differences. Number of death in mice in the blank group was more than other groups. Among them, the leaves of U. macrophylla group death in mice are the least.CONCLUSION1. Now Uncaria has isolated more than30kinds of compounds, mainly for the indole alkaloid, which rhynchophylline, isorhynchophylline, Isocorynoxeine and Corynoxeine accounted for more than50%of total alkaloids. Rhynchophylline and isorhynchophylline are isomer of each other, very often exists in the Uncaria plant by the relationship for1:1. So it is reasonable to evaluate Uncaria medicinal herbs by total alkaloid, rhynchophylline and isorhynchophylline. Chemically, rhynchophylline and isorhynchophylline can be dissolved in ethanol and chloroform. But the high toxicity and price more expensive of chloroform, so choose ethanol as solvent extraction is possible. Discovered through testing by ultrasonic extraction method has high efficiency, it need Less time and solvent than the specific heat method or the cold dipping method. So ultimately determine the ultrasonic extraction method for the experiment of extracting method.2. The calculation of the total alkaloid content by rhynchophylline in UV, Its size in order is U. macrophylla> leaves of U. macrophylla Wall.> U. hirsute> U. rhynchophylla> U. rhynchophylloides> stems of U. rhynchophylla (Miq.) Miq. ex Havil.>leaves of U. rhynchophylla (Miq.) Miq. ex Havil.>stems of U. macrophylla Wall.> stems of U. hirsuta Havil.> leaves of U. hirsute Havil.> stems of U. rhynchophylloides How.>leaves of U. rhynchophylloides How.. The calculation of the total alkaloid content by rhynchophylline in UV, its order is leaves of U. macrophylla Wall.>leaves of U. rhynchophylla (Miq.) Miq. ex Havil.>hooks and stems branch of U. macrophylla Wall.>hooks and stems branch of U. rhynchophylla (Miq.) Miq. ex Havil.>hooks and stems branch of U. hirsute Havil.>leaves of U. hirsute Havil.>hooks and stems branch of U. rhynchophylloides How.> leaves of U. rhynchophylloides How.3. Determinations by HPLC, leaves of U. macrophylla contained rhynchophylline and isorhynchophylline are the highest level in those Uncaria plant. The mass percentages are0.1632mg·g-1and0.0694mg·g-1. The content of hirsutine is also the highest level in the hooks and stems branch of Uncaria macrophylla Wall. It is202.5μg·g-1. Thiose methods are accurate, simple, rapid, reproducible, high precision, can be used to determine the contents of total alkaloid and monomer composition rhynchophylline, isorhynchophylline and hirsutine for different types and parts of Uncaria genus.4. These different kinds of Uncaria medicinal materials can significantly reduce locomotor activity, increase the number of the hypnotized mice and extend the mice’s sleeping time, and reducing strychnine nitrate convulsions caused by the number of death and the number of Convulsion in mice. Among them, U. macrophylla is most obvious.