Analysis And Identification Of Twelve Seeds Traditional Chinese Medicine By Multilevel Infrared Spectra

Objective The Fourier transform infrared spectra(FTIR), second derivative infrared spectra(SD-IR), and two-dimensional correlation infrared spectra(2D-IR) was applied to analyze and identify the twelve seeds traditional Chinese medicine(TCM). The shape, the position and the intensity of their characteristic absorption peaks had a lot of differences in infrared spectra(IR) of TCM. By summing up the information of specific functional group frequencies from twelve kinds of seeds TCM, to build a set of Infrared Spectra method suitable for rapid discrimination of seeds TCM, and provide the research mentality and the scientific basis for the development of these seeds TCM.Methods The specific methods for the analysis of IR spectra of mixtures included “macrointerpretation”, “tri-step identification”, and “intelligent analysis”. The three methods are defined as spectral “’multilevel infrared spectral macro-fingerprints analysis”. According to the spectral macro-fingerprints feature of mixtures, to analyze the overlapped peaks in infrared spectra of twelve seeds TCM and obtain the relational information concerning mixture samples from their infrared spectra. At the same time, the statistical techniques were used to analysis the specific components of seeds qualitatively and quantitatively.Results According to the differences of components in infrared spectra of twelve seeds, seeds can be divided into high fats, high proteins, carbohydrates and others. The infrared spectra of Sinapis alba L.(SA) and Raphanus sativus L.(RS) can be observed the characteristics absorption peaks assigned to the fats, and the relative intensity of the peaks were higher. So SA and RS can be divided into seeds contained high fats. The infrared spectra of Glycine max(L.) Merr.(GM), Pharbitis nil(L.) Choisy(PN) and Holarrhena antidysenteriaca Wall. ex A.DC.(HA) had the absorption peaks assigned to proteins, and the relative intensity of the peaks was highest. The three seeds can be divided into seeds contained high proteins. According to the obvious absorption peaks of Abrus precatorius L.(AP), Strychnos nux-vomica L.(SN), Nelumbo nucifera Gaertn.(NN), Vaccaria segetalis(Neck.) Garcke(VS), and Ginkgo biloba L.(GB) in the range of 1200-950cm-1, were similar to significant peaks of the carbohydrates, the major components of five seeds were classified as the carbohydrates. The infrared spectra of Prunus persica(L.) Batsch(PP) and Areca catechu L.(AC) was different from the others. The obvious absorption peaks of PP and AC were assigned to the organic acids and the carbohydrates, respectively. The shape of the peaks of the carbohydrates in AC, was different from other seeds contained the carbohydrates, and the infrared spectra of AC had the absorption peaks assigned to organic acids. Therefore, the two samples were classified as others.1. Correlation coefficients of the IR spectra of SA and RS were 0.9783. The two kinds of seeds belonged to the same Cruciferae family, and they both contained large amounts of fats and proteins. The relative intensity of characteristic absorption peaks reflected the relative content of fats and proteins. According to the absorption peak at 1711cm-1 assigned to the organic acid in RS, displayed the differences of two kinds of TCM in the SD-IR spectra. The differences of two TCM were more significant in the 2D-IR spectra. With the change of temperature, the fats in SA and the proteins in RS were changed obviously. The Tri-step infrared spectroscopy can be used to fast analyze and distinguish the two kinds of herbs containing the same major component.2. According to the relative intensity of absorption peaks of fats and proteins, to distinguish the GM, PN, and HA. The mass ratio of fat to protein in HA is the least, and the mass ratio of fat to protein in GM is the highest. PN and HA both had a peak at 1711cm-1 assigned to the organic acid in the SD-IR spectra. And the relative contents of the organic acid were less than the relative contents of the fats in PN. The 2D-IR spectra showed the more differences of TCM. The number of stronger auto peaks in GM, PN, and HA was 2, 3, and 5 respectively.3. The major components of AP, SN, NN, VS, and GB were the carbohydrates. Obviously, the spectra of GB and VS were very similar to the spectra of starch in the IR spectra. The five TCM both had a certain amount of proteins. The relative intensity of the absorption peaks of components reflected the relative contents of the components. The relative contents of starch were highest in GB, and the relative contents of proteins were highest in AP. The SD-IR spectra reflected more subtle differences between several samples. In two-dimensional correlation infrared spectra, the five samples can be visually distinguished due to their significant differences in auto-peak position and intensity. The number of stronger auto peaks of five TCM in the region of 800-1300cm-1, was 5,7,4,4, and 3 respectively, and the position of the strongest auto-peaks occurred at about 1196cm-1、1207cm-1、1220cm-1、1222cm-1 and 884cm-1.4. PP and AC contained different major components. The IR spectra of the two samples were almost different. The major components of PP were the fats, and the major components of AC were the carbohydrates. SD-IR spectra can reveal minor spectral features presenting within strong bands in the original spectra, which is very helpful in interpreting the components with low concentrations and weak absorption peaks. The SD-IR spectra of PP had the absorption peaks assigned to the proteins, and the SD-IR spectra of AC occurred near 1603cm-1, 1517cm-1 and 1440cm-1 the absorption peaks assigned to the skeletal vibrations of aromatic rings. The 2D-IR spectra of two seeds had 8 and 11 auto peaks respectively, and the position of the strongest auto-peaks occurred at about 1136cm-1 and 855cm-1 respectively. AC had an auto peak at 1066cm-1, which corresponds to the negative cross peaks related to the auto peaks. In the region of 1600-1750cm-1, PP had two auto peaks which the cross peaks corresponding to each other was negative, and AC had two auto peaks which the cross peaks corresponding to each other was positive.Conclusions The spectra of the TCM can be interpreted and compared directly and qualitatively by “multilevel infrared spectral macro-fingerprints analysis”. Without separation procedures, the analysis of mixtures would be much easier and quicker and it is possible to obtain an overall evaluation of the whole sample. According to the major components of the twelve kinds of seeds, TCM can be grouped into four categories. The fingerprint characteristic of the samples can show the similarities and differences between seed samples, and provided the relevant information of chemical constituents. The relative intensity of absorption peaks can reflect the relative contents in the TCM. The IR spectra combined with computer analysis software provided a method to analysis and identify the seeds TCM.