高效液相色谱法测定辣椒干中辣椒素的含量_英文_

高效液相色谱法测定辣椒干中辣椒素的含量_英文_ ( Determination of Ca psa icin in Chill i Ca psic um a n n u um L . ) by HPL C 3 GAO Yi ,ZHONG Shi2an ,ZHOU Chun2shan ,CHEN Long2sheng ( )College of Chemistry and Chemical Engineering , Central South University , Changsha 410083 ,China ( ) Abstract :A simple ,rapid HPLC method was adapted to determine capsaicin in chilli Capsicum annuum L . with Burospher2 μ( ) () 100 Ccolumn I. D. 250 mm ×4. 6 mm ,5m,using MeOH2HO 70?30 ,V/ Vas mobile phase with an eluting rate of 0. 6 18 2 mL/ min and detected at 280 nm. There is a good linear relationship in the range of 1. 01,121. 2 mg/ L for capsaicin ,and the correlative coefficient is 019994. When the method was applied to analyse chilli extract ,and the relative standard deviation is 1. ( ) 38 % n = 6and the average recovery is 99. 88 %. Key words :HPLC ; Capsicum annuum ;capsaicin ; quantitative analyse 高效液相色谱法测定辣椒干中辣椒素的含量 3 艺 ,钟世安 ,周春山,陈龙 胜 高 ()中南大学化学化工学院 ,长沙 410083 摘 要 :本文建立了简便 、快速的分离测定辣椒干中辣椒素含量的高效液相色谱法 。HPLC 的条件 :Burospher2100 C柱 ( I. D. 250 mm ×4. 6 mm ,5μm) ;流动相为甲醇2水 (70?30 ,V/ V) ;流速为 0. 6 mL/ min ;波长为 280 nm。在 18 γ上述条件下 ,辣椒素在 1. 01,121. 2 mg/ L 的范围内线性关系良好 ,线性相关系数= 0. 9994 ;实验结果表明 ,该方 ( ) 法的相对偏差在 1. 38 % n = 6以内 ,平均回收率为 99. 88 % 。 关键词 :高效液相色谱 ;辣椒 ;辣椒素 ;定量 中图分类号 :O658 ;Q946. 91 ) ( , reCap saicin trans282methyl2N2vanillyl262nonenamide 2 Introduction 2 ferred as the bioactive component in Capsicum fruits ,is ( ) The fruits of the genus Capsicum Solanaceae,commonly principally used in clinical settings for treating neuropath2 known as red pepper , is used world2wide as a natural 3 ic pain such as diabetic neuropathy ,postherpetic neu2 condiment in food as well as raw material for pharmaceuti2 ralgia and so on. In addition , cap saicin is available in a cals. non2prescription cream to relieve pain associated with ( ) Pungency hot flavouris the most important quality at2 rheumatic diseases , osteoarthritis , and cluster headache . tribute of red pepper . The substances responsible for pun2 The pharmacological effects of cap saicin have been be2 gency is a group of alkaloids called cap saicinoids. More lieved to be highly selective for sensory afferent neurons than 20 cap saicinoids were characterised in Capsicum and derive from its specific interaction with the receptor . 1 ( ) genus. The most representatives are cap saicin Cand Due to its selectiveaction on a subset of primary afferent () dihydrocap saicin DC, constituting 90 % or more of the 4 neurones ,cap saicin is also a neuropharmacolo2gical tool total cap saicinoids. The other 10 % are nordihydrocap2 for the study of thin afferent fibres for almost half a centu2 () ( ) saicin NDC,homocap saicin HCand homodihydrocap2 ry. ( ) saicin HDC. Because of its various value , various methods have been 5 29 reported for quantifing cap saicin. However , most of Received Febrary 22 ,2005 ;Accepted May 27 ,2005 these methods ,though attaining sometimes better results , Fundation Item : Project supported by the Natural Science Foundation of are very laborious ,long2lasting or expensive . ()Hunan Province No . 04JJ 3080 The objective of this work is to establish an accurate and 3 Corresponding author Tel : 86273128830833 ; E2mail :zhongshian @yahoo . rapid method for the analyse of cap saicin by HPLC. com. cn 2005 Vol117 No16 天然产物研究与开发 798 into two group s : one from solvents and impurities with Experimental stronger polarity and less retention time ; the other from Materials and reagents cap saicinoids with similar structure of cap saicin and lower Capsaicin standard samples were obtained from Fluka Bio2 polarity. The above two group s could be easily separated chemika Co . . A chilli food2grade preparation ,purchased at because of the great difference of structure and chemical a local market ,was ground and sieved to give a fine pow2 and physical properties. However , cap saicin was difficult der . Solvents used for extraction were of analytical grade to separate from its analogues , it was important to select and those used chromatography were of HPLC grade . suitable chromatographic system. Instrumentation As the low solubility in water and high solubility in MeOH All chromatographic separations were carried out on a for cap saicin , different volume fractions of MeOH in mo2 Biotronik BT28100 liquid chromatography system equipped bile phase were experimented. It was observed that with with BT28200 variable2wavelength UV2Vis detector and a the increase of MeOH volume fraction ,the scouring ability Dynamic mixer . A C2R6A integrator recording retention was enhanced and the retention time of cap saicin became time and chromatograms was employed to evaluate peak shorter. However ,under this condition ,the extract that in2 ( areas. Reversed2phase column Burospher2100 col2 Ccluded many other compounds could not be separated 18 μ) properly because of the interaction of these components umn , I. D. 250 mm ×4 . 6 mm ,5mwas used at ambient temperature . and resulting in the overlapness of the apices in the chro2 matogram. On the contrary ,if the volume fraction of MeOH Standard solution was too low , the cap saicin peak was significantly broad2 Stock solution of cap saicin was prepared by dissolving ened and the analysis time became longer . staneard substance cap saicin into methanol to a concentra2 tion of 202 mg/ L and stored at 0,5 ?. The stock solu2 tion was further diluted with methanol to provide working standard solutions. Extraction of chilli sa mple 5 . 000 g chilli powder was extracted three times under ul2 trasonic wave with 50 mL 70 % ethanol for 20 min. The combined ethanol solution was evaporated to give a thick syrup . The syrup was dissolved in mobile phase and trans2 ferred into 25 mL standard flask ,which was stored at 5 ? for determination. HPL C determination Fig. 1 Chromatogra ms of standard sa mple Standard calibration graph was prepared for cap saicin by plotting peak area versus concentration. Linearity , repro2 ducibility and recovery were determined routinely. Sample solution was filtered through a micro2bore membrane with μμ( ) aperture of 0 . 45 m. This solution 6 L was injected into column. Analyse conditions : detection wavelength ,280 () nm ;mobile phase ,MeOH2HO 70?30 ,V/ V;flow2rate , 2 0 . 6 mL/ min. The column was regenerated by washing with methanol after analysis and equilibrated with the mo2 bile phase . Fig. 2 Chromatogra ms of extraction sa mple Results and Discussion Choose of detection wavelength After several experiments , good resolution was obtained () with mobile phase MeOH?HO = 70?30 ,V/ Vand flow 2From the scan of UV spectrophotometer ,it is obvious there rate of 0 . 6 mL/ min. From the HPLC , chromatogram of are two maximal absorption peaks at 230 nm and 280 nm. standard sample shown in Fig. 1 ,the peak of cap saicin in We choose 280 nm as the detection wavelength of cap2 Fig. 2 was determined. saicin. Quantitative analysis Chromatogra phic conditions In the selected chromatographic conditions ,the linearity of In the crude solution ,the absorption peaks can be divided 2005 Vol117 No16 高 艺等 :高效液相色谱法测定辣椒干中辣椒素的含量 799 method was evaluated in the range of 1 . 01,161 . 6 mg/ Lto be analyzed and by further processing as described 2 above . The average recovery of cap saicin from chilli exfor cap saicin. The reproducibility of analysis was estimated ( ) tract reach up 99 . 88 % Table 2. by the coefficient of variation of standard solutions of cap2 saicin in six successive assays performed on the same day Ta ble 1 Reproducibil ity of retention time and pea k area ( ) Table 1. ()No . Peak area Retention time min There is a good linear relationship in the range of 1 . 01, 1 97727 161090 121 . 2 mg/ L for cap saicin. The linear regression was ob2 2 94539 161133 tained of the peak area against concentration of standard 161233 3 95515 samples. It is as follows : Y = 1233 . 00708 + 814 . 3333 161052 4 96327 X is concen2 Y is peak area ,and X , R = 0 . 9994 . Where 161098 5 95791 () tration mg/ L. 161648 6 96834 Recovery experiment Average 96122 1613695 Recovery experiments were carried out in duplicate by the ( )RSD % 1115 1138 addition of standard solutions of cap saicin to the samples Ta ble 2 Recovery of ca psa icin in a chilli sa mple ρ()ρ()ρ()Originalmg/ L Addedmg/ L Total foundmg/ L ( )( )No . O A F Recovery R % Average recovery R % 1 20 54 . 74 53. . 93 53 . 85 99 . 95 2 30 64 . 21 63 . 89 64 . 07 99 . 60 34 . 18 99 . 88 3 40 74 . 27 74 . 36 74 . 01 100 . 1 Ta ble 3 Ca psa icin content in different kinds of chilli References ( )Type w % 1 Bosland PW , Votava EJ . Pepper : Vegetable and spice cap2 sicum. London : CAB Publishing ,2000 01283 Guizhou 2 Cordell GA , Araujo OE. Capsaicin : identification , nomencla2 01497 Hunan ture ,and pharmacotherapy. Annals of Pharmacotherapy , 1993 , 27 :3302336 01356 Sichun Alper BS ,Lewis PR. Treatment of postherpetic neuralgia :a sys2 3 Fujian 01148 tematic review of the literature . J ournal of Family Practice , 2002 ,51 :1212128 Sa mple determination Luo H ,Wan Y , Han J S. Capsaicin and its receptor2vanilloid re2 4 ceptor. Prog Physio Sci ,2003 ,34 :11215 The content of cap saicin in four kinds of chilli was deter2 Pankar DS , Magar NG. New method for the determination of 5 ( ) mined by above method Table 3. The contents of cap2 capsaicin by using multi2band thin2layer chromatography. J our2 saicin in chilli from Hunan and Sichun Provinces are rela2 nal of Chromatography ,1977 ,144 :1492152 tive higher . Zhu XL ,Liu BZ , Zong RW , et al . Analysis of chemical con2 6 stituents of capsicol by GC2MS. J Instru Anal ,2003 ,22 :67270 Concl usions Wu YY , Chen KX , Shao J S. The preparation technologies and 7 analysis methods of capsaicinoids. Chem World ,2004 ,2182221 HPLC analysis of cap saicin was carried out . There is a O Yang HX , Hu MB , Wang LZ. Determination of capsaicin in 8 good linear relationship in the range of 1 . 01,121 . 2 mg/ capsicum by RP2HPLC. Chin J Anal Chem , 1998 ,26 :1521 L for cap saicin ,the correlative coefficient is 0 . 9994 , the Laskaridou2Monnerville A. Determination of capsaicin and di2 9 ( ) relative standard deviation is 1 . 38 %n = 6and the aver2 hydrocapsaicin by micellar electrokinetic capillary chromatogra2 age recovery is 99 . 88 % ; phy and its application to various species of Capsicum , There is rich cap saicin in chilli from Hunan Province ; Solanaceae . J ournal of Chromatography A ,1999 ,838 :2932302 The method can be applied to the determination of cap2 Contreras10 2Padilla MC , Yahia EM. Changes in capsaicinoids during development , maturation and senescence of chilli pep2 saicin present in red pepper fruits and preparations made pers and relation with peroxidase activity. J ournal of Agricultur2 () from them chilli and oleoresin. al and Food Chemistry , 1998 ,46 :207522079