| Irkutsk Anemone Rhizome belongs to genus Ranunculaceae plants of Anemone altaica Fisch.exe C.A.Mey. It is warm-natured and pungent, and it distributes mainly in Shannxi, Shanxi, Henan, Neimenggu, and Gansu Province of China. It can dispel sputum and soothe the nerves in Pharmacopoeia. Irkutsk Anemone Rhizome is used for the treatment for fever, faint, epilepsy, neural function disorder, tinnitus deafness, bosom frowsty abdominal distension, loss of appetite, and the treatment for the ulcer sores tinea. Irkutsk Anemone Rhizome contains a variety of active ingredients such as anemonin, beta-sitosterol and succinic acid, it can obviously inhibit the gastrointestinal myoelectric and central nervous activity. Except for that, there is no other modern pharmacological effects reported. Therefore, it is necessary to study furtherly for pharmacological effects, therapeutic efficacy and effective component analysis in Irkutsk Anemone Rhizome. In this study, we had a preliminary study for antioxidant, antimicrobial and analgesic biological activity of Irkutsk Anemone Rhizome, and we also analyzed succinic acid which is one of the active ingredients and fat-soluble chemical constituents. All of these provided a reference for in-depth research and use of Irkutsk Anemone Rhizome. The main findings were as follows:(1) The in vitro antioxidant activities of the extracts with different polarity solvents of Irkutsk Anemone Rhizome were evaluated. The powdered plant material was sequentially extracted two times with95%ethanol and75%ethanol by the method of reflux extraction, then petroleum ether, chloroform, ethyl acetate, n-butanol and water extractive fractions were obtained respectively by systematically separating the extracts. The antioxidant activities of the five extractive fractions for diphenyl bitter acyl radicals (DPPH-) scavenging, antioxidant properties of beta-carotene/linoleic acid system and the total reducing power were determined by spectrophotometric method. The results showed that in DPPH-radical-scavenging assay, the ethyl acetate extract exhibited the highest scavenging capacitiy towards DPPH. At a final concentration for2.0mg/mL, scavenging rate towards DPPH-of the ethyl acetate extract was95.41%, and the chloroform extract was89.93%which was slightly lower than the ethyl acetate extract, followed by water extract and the inhibition rate of87.54%. The IC50of chloroform extract was0.22mg/mL, ethyl acetate extract was0.21mg/mL, water extract was0.52mg/mL. In β-carotene/linoleic acid bleaching system, the chloroform extract showed the highest inhibitory activity, and it decreased from77.93%to37.95%, followed by ethyl acetate extract, decreased from72.41%to19.55%, again, the inhibitory rate of water extract decreased from74.14%to11.14%. In ferric ion reducing power system, reducing power of ethyl acetate and chloroform extracts with increasing concentrations increased faster than others. The reducing power of the ethyl acetate extract was highest when the concentration was between2.0mg/mL to5.0mg/mL, the second was chloroform extract, the third was water extract, then the fourth was petroleum ether extract, and the last was butanol extract. All of the five extractive fractions from Irkutsk Anemone Rhizome had antioxidant activity, and the antioxidant capacity of the ethyl acetate extractive fractions were stronger than others.(2)The in vitro antimicrobial activities of the extracts with different polarity solvents of Irkutsk Anemone Rhizome were studied. The antimicrobial activities of the above five extractive fractions were evaluated using the Oxford cup and96-well plates methods. The chloroform and ethyl acetate extractive fractions had antibacterial effects on all tested bacterial species while the petroleum ether, n-butanol and water extractive fractions had no antibacterial activity; The antibacterial activity of chloroform fraction was higher than the ethyl acetate fraction. The ethyl acetate extract had inhibitory effect on all tested bacterial species at6.25mg/mL while chloroform extrac at0.78mg/mL. The chloroform and ethyl acetate extracts had bactericidal effects on all tested bacterial species at50mg/mL.(3) The analgesic activity of Irkutsk Anemone Rhizome was studied. We use the mouse hot plate method and acetic acid writhing test to study analgesic activity of Irkutsk Anemone Rhizome. The extendation effects of chloroform and ethyl acetate extracts for the hot-plate pain threshold were better than others, but compared with the control group, no significant differences (P>0.05), there was no significant effects on mitigation central pain caused by thermal stimulation. The chloroform and ethyl acetate extracts at the dose of500mg/kg exhibited significant reduction of the number of writhes at the rate of65.72%and25.38%, respectively. Chloroform and ethyl acetate extracts could inhibit significantly narcotic pain in abdominal cavity mucosa caused by acetic acid. The inhibition caused by Chloroform extracts was significant different from the control group (P<0.01). The petroleum ether, n-butanol and water extractive fractions had no analgesic activity.(4) An HPLC method for the determination of succinic acid in Irkutsk Anemone Rhizome was established. Kromasil C18(150mm×4.6mm,5μm) column was used and methyl alcohol-0.02mol/L KH2PO4buffer solution (7:93) as the mobile phase, with the detection wavelength of214nm, column temperature of30℃and rate of flow0.5mL/min. The results showed that the linearity relationship of succinic acid was good in the range of4～48μg, and the average recovery rate of succinic acid in samples was99.15%with RSD of2.04%. The content of succinic acid in Irkutsk Anemone Rhizome was0.200%～0.318%. The method was reliable, repeatable and precise, which could be used for the rational use and quality control of Irkutsk Anemone Rhizome. (5) The fat-soluble components of Irkutsk Anemone Rhizome from five different places were extracted by Soxlet extraction, esterified and then analyzed by gas chromatography-mass spectrometry.18,22,20,20and20fat-soluble compounds were identified from Irkutsk Anemone Rhizome in Ningxia, Shanxi, Henan, Sichuan and Shaanxi, respectively. Their relative content were88.14%,96.17%,71.37%,89.67%,77.08%, respectively.There were eight common components in the five kinds of Irkutsk Anemone Rhizome, in which hexadecanoic acid and9,12-octadecadienoic acid were the chief components. The main fat-soluble components of Irkutsk Anemone Rhizome from Ningxia and Shaanxi province were linoleic acid (Ningxia46.5%, Shaanxi30.88%), linolenic acid (Ningxia17.2%, the Shaanxi18.02%) and palmitic acid (Ningxia15.8%,16.33%in Shaanxi). The main fat-soluble components of Irkutsk Anemone Rhizome from Shanxi and Sichuan province were linoleic acid (Shanxi36.81%, Sichuan,40.09%), palmitic acid (Shanxi25.72%, Sichuan,13.7%) and rock celery acid (Shanxi22.97percent, Sichuan24.3percent). The main fat-soluble components of Irkutsk Anemone Rhizome from Henan province were linoleic acid (37.3%) and palmitic acid (19.3%). There were significant differences of the fat-soluble constituents and their contents were found in the five kinds of Irkutsk Anemone Rhizome. These results provide references for the identification and clinical application of Irkutsk Anemone Rhizome. |
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