| Aconiti kusnezoffii Radix is the dried roots of Aconitum kusnezoffii Reichb.,which has been used in folk medicine to treat rheumatism,paralysis due to cold, herniaand pain.Diterpenoid alkaloids are the main bioactive constituents in Aconiti kusnezoffiiRadix,especially aconitine,hypaconitine and mesaconitine.These aconitine-typealkaloids are the main active constituents in Aconitum,but they have high toxicity.Unreasonable use of these drugs can lead to poisoning,which has seriously restrictedtheir clinical application.So,it is essential to strictly control the contents of thesealkaloids to ensure safe use of this medicine.In this study,eight alkaloids were isolated from the ethyl acetate crude extract ofAconiti kusnezoffii Radix.Besides aconitine(cw-6),hypaconitine(cw-7) andmesaconitine(cw-8),the others’ structures were identified by NMR,MS and IR spectraanalysis.They were songorine(cw-1),songoramine(cw-2),talatizamine(cw-3),karakoline(cw-4),3-deoxyaconitine(cw-5).Among the eight compounds,songorine,songoramineand karakoline were obtained from Aconiti kusnezoffii Radix for the first time.The paper also established a HPLC-ELSD method to determine the content ofsongorine in Aconiti kusnezoffii Radix.1Extraction,isolation and structure identification of the chemicalconstituents of alkaloids1.1Extraction10kg dried Aconiti kusnezoffii Radix were powdered and then percolated withethyl acetate-NH3·H2O(20:1).The extract was evaporated and acidulated by0.5%HClto pH=1~2,and then extracted by ethyl acetate for four times.The lower extract was combined,evaporated,and basified by0.5%NaOH to pH=11~12,and then extracted byethyl acetate for four times.The upper extract was combined,evaporated to afford thecrude extract (310g).1.2Isolation and purificationThe crude extract(310g) was subjected to silica gel column chromatography withgradient elution of petroleum ether-ethyl acetate-diethylamine to give five fractions.Fraction1(40g) was separated by petroleum ether-acetone-diethylamine to givesubfraction A(10g) and subfraction B(5g).Separation of the subfraction A by columnchromatography provided compound cw-1(2.0g) and cw-6(860mg).Subfraction B wasfurther separated by column chromatography,and then recrystallizated to yieldcompound cw-3(1.0g).Fraction2(35g) was separated by petroleum ether-acetone-diethylamine to give subfraction C(9.4g) and subfraction D(3.0g).The subfraction Cwas isolated by column chromatography,and compound cw-7(560mg) and C-1(5.2g)were obtained.C-1was separated by column chromatography,and thenrecrystallizated to yield compound cw-5(200mg).Subfraction D was separated bycolumn chromatography,and then recrystallizated to yield compound cw-2(550mg).Fraction4(42g) was separated by column chromatography and eluted by petroleumether-acetone-diethylamine to give subfraction E(17g).Subfraction E was furtherseparated by column chromatography to yield compound cw-8(4.0g) and E-1(5.0g).E-1was separated by column chromatography,and then recrystallizated to yieldcw-4(70mg).1.3Structure identificationCompounds cw-6,7and8were identified as aconitine,hypaconitine,andmesaconitine by comparison of TLC behavior with those of authentic samples.The structures of others were established as songorine(cw-1),songoramine(cw-2),talatizamine(cw-3),karakoline(cw-4) and3-deoxyaconitine(cw-5) on basis of NMR,MS and IR spectra analysis.Songorine,songoramine and karakoline were obtained fromAconiti kusnezoffii Radix for the first time.2Content determination of songorine(cw-1) by HPLC-ELSD 2.1Instruments and chromatographic conditionsAgilent1200HPLC and Alltech2000ELSD were applied for the determination.An Agilent Extend-C18(4.6×250mm,5μm) was used as the chromatographic column.The mobile phase was acetonitrile-0.1%triethylamine(40:60),the column temperaturewas25℃,the flow rate was1.0mL·min-1,and the injection volume was20μL.For the ELSD,the drift tube temperature was set at96℃,the gas(air) pressure was2.50bar,and the impactor was closed.2.2Methodology investigation2.2.1Linear relationWithin the range of1.5μg~30μg,the lgS(S:peak area) of compound cw-1and thelgm(m:sample mass) were in a good linear relationship.Regression equation waslgS=1.6311lgm+0.1061,R2=0.9991.2.2.2Precision testThe intra-day and inter-day RSD of the peak area of compound cw-1was0.26%and1.54%,respectively.It indicated that the method had a good precision.2.2.3Repeatability testSix samples were extracted in parallel and the results showed that the RSD of thecontent of compound cw-1was1.45%,which indicated that the method had a goodrepeatability.2.2.4Stability testThe same sample solution was analyzed every2hours in10hours,and the resultsshowed that the RSD of the peak area of compound cw-1was1.68%,which indicatedthat the sample solution was stable within10hours.2.2.5Recovery testThe average recovery of compound cw-1was98.69%,and the RSD was1.59%,which indicated that the method had a good recovery.2.3Content determinationThe average content of songorine in Aconiti kusnezoffii Radix from Anguo inHebei province was0.9238mg·g-1.The average content of songorine in Aconiti kusnezoffii Radix from Panshi in Jilin province was1.319mg·g-1.The average contentof songorine in Aconiti kusnezoffii Radix from Dali in Yunnan province was1.768mg·g-1.The average content of songorine in Aconiti kusnezoffii Radix fromBozhou in Anhui province was1.635mg·g-1.The results showed that the content of songorine in Aconiti kusnezoffii Radixvaried slightly from different sources,but the most of them were over0.1%.Thecontent of songorine is high in Aconiti kusnezoffii Radix,so it is necessary to includethe content determination of songorine into the quality criteria of this drug.3SummaryOn the basis of the domestic and foreign studies, the chemical constituents ofalkaloids from Aconiti kusnezoffii Radix was deeply investigated in this paper. Eightalkaloids were isolated and identified, and three of them were obtained from this plantfor the first time. In this study, a HPLC-ELSD method was also developed for thedetermination of songrine. The results of this paper provided support to complete thequality criteria and to promote safer and better use of this herb. |
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