The Preliminary Study On The Spectrum-effect Relationship Of Volatile Oil From Acorus Tatarinowii Schott

Objective1. To make clear of the present quality situation of volatile oil from Acorus tatarinowiiSchott, and provide the reference for the quality evaluation of A. tatarinowii.2. To explicit the pharmacological activities of volatile oil from A. tatarinowii through the pharmacodynamic tests, and screen the effective components.3. To explore spectrum-effect relationship of volatile oil from A. Tatarinowii preliminarily, and construct the pharmacodynamic finger-print, thus providing comprehensive and reasonable quality evaluation for A. tatarinowii.Methods1. Acorus tatarinowii Schott samples were collected from different habitat and batches, the volatile oil was extracted from A. tatarinowii by using steam distillation method, and analysed and identified by GC-MS. Peak area normalization method was used for calculating the relative contents of the characteristic components that were methyleugenol, cis-methylisoeugenol, trans-methylisoeugenol, γ-asarone, β-asarone and α-asarone, hierarchical cluster analysis was used for classifying the 50 batches of samples by their relative contents of the characteristic components, and the test samples for pharmacodynamic tests were selected from each cluster category.2. To compare pharmacological activities of volatile oil from different batches of A. Tatarinowii through blood coagulation experi- ments, hypoxia tolerance tests, pentobarbital-sodium for sleep experiments, square water maze experiments and step through tests. Mice were administered by methyleugenol, methylisoeugenol,β-asarone and α-asarone respectively to screen the effective components.3. The GC technique was used to record the gas chromatographic fingerprints of 50 batchs volatile oil of A. Tatarinowii, and identified the β-asarone、 α-asarone. Combined with the results of pharmacodynamic tests and hierarchical cluster analysis, the reference fingerprints, namely the pharmacodynamic fingerprints, were generated by gas chromatographic fingerprints of the samples that have significant pharmacological activity imported into "similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine 2004A edition" software.Results1. GC-MS results showed that:The characteristic components of 50 batches volatile oil of Acorus tatarinowii Schott were methyleugenol, cis-methylisoeugenol, trans-methylisoeugenol, γ-asarone, β-asarone and a-asarone. Peak area normali-zation method was used for calculating the relative contents of the each characteristic components (methyleugenol for 0～40.653%, cis-methylisoeugenol for 0.057～23.583%, trans-methylisoeugenol for 0～2.859%, γ～asarone for 0～24.322%, β～asarone for 1.301～91.804%,α ～asarone for 0.727～ 44.151%). The relative contents of 6 characteristic components of 50 batchs samples totalled 3.677～98.681%, the mean of its total relative contents was 86.356%. By hierarchical cluster analysis, the 50 batches of volatile oil of A. Tatarinowii could be clustered into 10 categories in the first layer, the second layer was clustered into 8 categories, Ⅰ,Ⅱ was clustered into a class, this 2 categories totalled 31 batches samples, the main component of the 31 batches samples was β-asarone, followed by a-asarone in common, the 31 batches of samples should be the mainstream of A. Tatarinowii. Eventually 7 batches of representative samples were selected for pharmacodynamic experiment(No.34 sample was not included because amount is not enough).2. Pharmacodynamic experiments results showed that:In the blood coagulation experiments, A, D, E, F, G group of volatile oil of A. Tatarinowii with high relative contents of asarone had significant anticoagulant activit, anticoagulant activit of B, C group with low relative contents of asarone was not significant. In the hypoxia tolerance experiments, D, E, F, G group of volatile oil rich in β-asarone could significantly prolong survival time in mice, while A, B, C group poor in β-asarone had no statistical significance. In the pentobarbital-sodium for sleep experiments,7 batches of volatile oil of A. Tatarinowii (A, B, C, D, E, F, G group) was significantly shortened sleep time of NIH male mice, and showed analeptic action. In the square water maze experiments, D, E, F, G group had significant difference, enhancing intelligence effect of A, B, C group poor inβ-asarone was not obvious. In the step through tests,7 batches of volatile oil of A. Tatarinowii all prolonged the latency, showed the enhancing intelligence activity3. Effective components screening results showed that:In the blood coagulation experiments, only β-asarone and E group showed significant activity, effect of methyleugenol, while methylisoeugenol, a-asarone and B group was not obvious.In the hypoxia tolerance experiments, β-asarone and E group could significantly prolong survival time in mice, while methyleugenol, methylisoeugenol, a-asarone and B group had no significant difference. In the pentobarbital-sodium for sleep experiments, methyl-eugenol, methyl isoeugenol, β-asarone, B and E group all showed analeptic action, a-asarone effect was not obvious. In the square water maze experiments and the step through tests, 6 treatment group (methyleugenol, methylisoeugenol,β-asarone, a-asarone, B and E group) all had statistical significance, and all significantly improved memory impairment induced by scopolamine in mice.4. GC results showed that:This experiment established Gas chromato-graphic fingerprints of 50 batches of volatile oil of A. Tatarinowii, and analyzed by "similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine 2004A edition" software for similarity evaluation, similarity were below 0.05, it indicated the great quality difference between the samples of volatile oil from A. Tatarinowii. Combined with the results of pharmacodynamic experiments, pharmacodynamic fingerprint of volatile oil from A. Tatarinowii was preliminary established.Conclusion1. The GC-MS analysis methods of volatile oil from A. Tatarinowii were established, results of peak area normalization method and hierarchical cluster analysis showed great quality difference between the 50 batches of volatile oil of A. Tatarinowii. The quality difference would cause effect difference, so the exploration of the relationship between spectrum and effect was very necessary.2. Different batches of volatile oil of A. Tatarinowii were different in anticoagulation, antihypoxia, analeptic action and enhancing intelligence, it implied that the difference of quality indeed can cause differences in efficacy. D, E, F, G group samples all had statistical significance in 5 pharmacodynamic experiments, the main component of the four samples was β-asarone, so infered β-asarone is main active constituents of volatile oil.3. Different chemical constituents from volatile oil of A. Tatarinowii produced different influence in its pharmacological function, Some activities was mainly result of a component, and some activities was the interaction result of many components, it indicated the material basis is different in different pharmacological function from the same kind of medicine. The results of B、E group were consistent with the results of pharmacodynamic experiments, which showed that the results of the two rounds pharmacodynamic experiments were accurate and reliable. β-asarone in five efficacy trials showed significant efficacy, it indicated that β-asarone was the main active constituents of volatile oil of A. Tatarinowii combined with the results of pharmacodynamic experiments.4. This experiment established gas chromatographic fingerprints of 50 batches of volatile oil of A. Tatarinowii, the precision, repeatability and stability of all measurement were up to standard, which could provide reference for quality evaluation of A. Tatarinowii herbs. It was the first time to carry out the exploration of spectrum-effect relationship of volatile oil from A. Tatarinowii, and provided new ideas and methods for the quality evaluation model of traditional Chinese medicine.