| OBJECTIVE1. Using different extraction methods to extract volatile oil from different parts of Amomum, and identified the extracted essential oil from different parts of the Amomum by GC-MS methods.2. With the Trichophyton rubrum (CMCC (F) Tla), the Trichonphyton mentagrophyton (CMCC (F) T5a), and the Microsporum gypseum (CMCC (F) M2a), as the research objects, studied the antibacterial activity of volatile oil for fungi and bacteria.3. With hydroxypropyl-β-cyclodextrin as the inclusion material, optimization of preparation technology of Amomum villous oil-HP-β-CDinclusion complex by orthogonal experiment, and identified the incusion complex with UV, GC-MS, DSC et al.4. Determined the content of CP and BA in the inclusion complex by high performance liquid chromatography, and studied the stability of inclusion complex and the solubilization of hydroxypropyl-β-cyclodextrinof on the volatile oil.5. Bioavailability study With camphor and bornyl acetate for the determination of indicators, inspected the bioavailability of Amomum volatile oil-hydroxypropyl-β-cyclodextrin inclusion complex and volatile oil CMC-Na mixture in SD rats.Research Methods1. Extraction of Amomi Essential Oil and Identification1.1 Hydrodistillation method Taken the Amomum villosum, peeled, crushed seed and shell separately, than 24 mesh screen. One hundred grams of plant material and 1000 mLwater were placed in a round bottom flask, soaked over night and install extraction equipment of essential oil, Set in boiling water for 6 h, standing 3 h, so that oil and water can be separated, collected essential oil and rewashed extractor with anhydrous ether repeatedly, recoveried ether vacuum, dried essential oil with anhydrous sodium sulfate and then obtain the clear essential oil. stored in 4℃refrigerator for spare。Bligh-Dyer method One hundred grams of plant material and five times the amount of methanol:chloroform:water= 1:1:0.5 mixture of solvent, soaked for three days, ultrasonic treatment (40℃,40 KHZ,45 min), filtration, extraction repeated three times, combined filtrate, centrifugal 4000 r/min for 5 min, get lower, removed solvent by vacuμm rotary evaporator, and then obtained essential oil. stored in 4℃refrigerator for spare。The analyses of the essential oil were run on a Hewlett Packard GC-MS system (GC 6890 series II; MSD 5973, Hewlett Packard, Guangzhou, China).The column was J & W DB-5MS quartz capillary column (30 mm×250μm×0.25μm). Oven temperature was programmed as following:Initial column temperature was 50℃, with 5℃/min rise to 125℃(to keep 15 min), then 5℃/min rise to 250℃. Sample was dissolved with ether; injection port for the streaming mode; gasification temperature was 250℃; carrier gas He as the constant current mode, flow rate of 1.0 mL/min, the average line speed 37 cm/sec, split ratio was 10:1, autosampler of 1.0μL; MS conditions:mass spectrometry ionization energy of 70eV, electron multiplier tube voltage of 1905V, the ion source temperature of 230℃, quadrupole temperature of 150℃, mass scan range 30 amu-550amu. The compounds in volatile oils were identified by GC-MS by mass fragmentation pattern and spectral comparison with standards in the Wiley275 and Nist05 library.2. Antibacterial activity of Amomum essential oil2.1 Determination of antibacterial and antifungal activityIn vitro antibacterial activity of the essential oils were evaluated by disc diffusion method using Sabouraud agar with determination of inhibition zones (IZ). The incubation conditions used were 96 h at 28℃. The essential oil (10μL) and the positive control were applied on the paper discs (the disc diameter is 6 mm). Then disc papers were placed in the inoculated plates. Each group repeated three times. used Ketoconazole as positive control. After specified culture conditions, the diameter of growth inhibition zones were measured. The scale of measurement as the following:>15 mm zone of inhibition was strongly inhibitory; 10mm-15mm zone of inhibition was moderately inhibitory and<10 mm was weakly inhibitory.3. Preparation and identification of the inclusion complex of Amomum volatile oilTaken HP-β-CD amount, set in 100 mL flask with stopper, added appropriate amount of water, and made it into 40% dubbed HP-β-CD aqueous solution, added the ethanol solution of volatile oil (essential oil:ethanol=1:2 V:V) in a ultrasonic cleaner in 40℃slowly, ultrasound inclusion to the provisions of time, removed, washed with petroleμm ether three times (each 5 mL), removed the volatile oil which is not included, frozen ice, dried in freeze-drying machine, removed out and set the dryer to save backup. With composite score of the utilization of volatile oil, content of oil and the inclusion yield inclusion as the evaluation index, preferred the best method of inclusion, optimizated the best inclusion process by orthogonal test of L9 (34) method, and identificated the inclusion complex by Microscopic identification method, UV spectrophotometry, Differential Scanning Calorimetry analysis, and Gas Chromatography.4. Stability of inclusion and effects of solubilization on Amomum volatile oil.With Bornyl Acetate and Camphor as index, determined the content of BA and CP in inclusion complex and physical mixture by HPLC, respectively. Weighed inclusion complex and physical mixture, filled in the capsule, and set the capsule in strong light (light of 4000 lx), high temperature (temperature of 60℃), high humidity (relative humidity of 90%) of the environment, respectively. Taken samples at 0 d,2 d,4 d,6 d, 8 d,10 d, according to determination conditions, determined the contents of BA and CP in inclusion complex and physical mixture. Determined solubilization of hydroxypropyl-β-cyclodextrin on the volatile oil by phase solubility method.5. Bioavailability of inclusion complex in rat12 SD rats, fasting but water before administration 12 h, were divided into 2 groups randomly, each group for 6, the volatile oil group and volatile oil-hydroxypropyl-β-cyclodextrin inclusion complex group. A group was given inclusion complex of volatile oil (water as solvent, content n of bornyl acetate and camphor in inclusio was 5.89%,2.70% respectively), B group was given essential oil mixed suspension (0.15% sodium carboxymethyl cellulose solution as solvent, content of bornyl acetate and camphor in the cellulose solution was 38.55%,17.49% respectively). Drug dose of bornyl acetate and camphor was 500 mg-Kg-1 and 230 mg-Kg-1 for rats respectively. Inclusion complex of volatile oil was dissolved in water directly to fed the same dose for the two treated groups, respectively. Taken 1.0 mL of blood in the tail vein at 0.167 h,0.5 h,1.0 h,2.0 h,3.0 h,4.0 h,6.0 h,8.0 h and 12.0 h, transferred the blood into the EP tube, centrifugalled for 10 min at 8000r-min-1, the supernatant transferred to another EP tube and stored at-20℃refrigerator for spare.6. Statistical analysisAll data are described by mean±standard deviation, using SPSS 13.0 statistical software to analysis. Statistical methods are used as following:1. dates of antibacterial activity were analyzed by One-Way ANOVA, considering the main effects only, significant level for the a=0.05.2. datas of inclusion process were analyzed by analysis of variance orthogonal design information, according to preliminary test basis, considering the main effects only, significant level for the a= 0.05.3. dates of inclusion and physical stability of the main components and pharmacokinetics in rats were analyzed by Independent-Samples T Test in different groups at same time, dates of inclusion and physical stability of the main components were analyzed by analysis of variance of repeated measures in same group at different time. Significance level for the a=0.05.Results1.By GC-MS analysis,138 chemical constituents were identified from the dry mature seeds and shell of four volatile oil, volatile oils include bornyl acetate (5%-47%), camphor (4%-17%), borneol (1.5%-6%), camphene (0.2%-3%),α-pinene (0.2%-3%),β-pinene (0.2%-5%) and a-copalene (0.1%-2%), and, in which the relative content of vinyl material total composition of total volatile oil-40% to 10%. Eucalyptus alcohol, big root geranium ene Ring, mint enolase, tricosane, and other new 35 kinds of compositions were identified from this plants for the first time. The volatile oil yield of seeds was 3.57% (g/g) by HD method, and volatile oil yield of husk was 0.97% (g/g); the volatile oil yield of seeds was 7.01% (g/g) by B-D method, and the yield of husk was 5.37% (g/g). The results of antibacterial showed that the volatile oil had shown significant inhibitory activity for Trichophyton rubrum, Trichonphyton mentagrophyton and Microsporμm gypseum. 2. According to the results of orthogonal test method, the best conditions for inclusion process was:HP-β-CD and volatile oil ratio was 10:1 (g/mL), HP-β-CD concentration of 40%, inclusion time of 80 min, inclusion temperature of 45℃. The results of various factors on the degree of influence:A> C> B> D. Analysis of variance showed that factor A and factor C on the inclusion process had a significant differences (P<0.05), factor B and factor D of the inclusion process had no significant differences (P> 0.05). A variety of identification methods showed that the preparation of inclusion complexs was successful, and the component of inclusion did not change after inclusion.3. Determinated the content of camphor and bornyl acetate by HPLC method.The best chromatographic conditions were column:Hypersil ODS C18 column (4.0 mm×250 mm,5μm); mobile phase (methanol-water=75:25); flow rate of 1 mL·min-1; detection wavelength of 210 nm; column temperature was 30℃, and the injection volume of 10μL. The results of stability showed that the content of bornyl acetate and camphor were significantly higher than the physical mixture under the high temperature, humidity stability and high light conditions, solubility test showed that hydroxypropyl-β-cyclodextrin concentration of 50% could increase the solubility of essential oil of about 35 times.4. Results of BioavailabilityFed the rats with inclusion complex and the mixture in both groups respectively, determinated the concentrations of BA and CP at different time points, got curves of the average plasma concentration-time, calculated by DAS2.1.1 software, pharmacokinetic parameters of both groups were analysed by One-WayANOVA, the results showed that the bioavailability of inclusion group were significantly higher than Mixture group in the AUCo-t value, AUC0-∞values and Cmax values. ConclusionsExtraction efficiency of BD method is higher than that of HD method. The volatile oil showed significant inhibitory effects for selected fungi, opened up a new application for the essential oil in the pharmaceutical industry and food industry development and utilization of scientific theory and experimental evidence. Inclusion complex showed more greater stability in high temperature, high humidity, strong light than the physical mixture, and hydroxypropyl-β-cyclodextrin could increase the solubility of Amomum volatile oil significantly. AUC values obtained from Cmax values of inclusion complex were significantly higher than that of Amomum volatile oil suspension, which showed the inclusion complex has higher bioavailability. |
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