LIU Hai-qing (劉海青)

Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, Haikou 570228, China

ABSTRACT Objective:To set up a database of three-dimensional fluorescence spectra of traditional Chinese medicines, Screening of medicinal materials by three-dimensional fluorescence spectroscopy,Methods:The paper Summarizes the three-dimensional fluorescence fingerprints, screens medicinal materials on basis of verifying the three-dimensional fluorescence spectra of the reference crude herbs of the national standard Chinese medicines, and combines with visual basic6.0 and microsoft access 2003 technology system to construct a three-dimensional fluorescence fingerprint database of TCMs.Results:the identification and quality control of traditional Chinese medicines (TCMs) was accuately and swiftly achieved.Conclusion:The relevant analysis on the operating system of database and the three-dimensional fluorescence spectrum database of TCMs indicates that this operating system can provide convenient channels for the inspection, verification and quality control of TCMs.

KEYWORDS Traditional Chinese medicines (TCM); Three-dimensional fluorescence; Fingerprinting;

INTRODUCTION

As a cultural treasure that has been handed down to China for thousands of years, traditional Chinese medicine (TCM) still occupies a decisive position in the current society that is featured with a developed science and technology culture and plays a role that could not be replaced by Western medicines especially in chronic cases.However,even in the context of advanced information technology, the complicated ingredients and medicinal properties of TMC cannot be fully and clearly recognized.ZHANG Jucheng, TAO Yuji and others established solid three-dimensional fluorescence fingerprints of pseudo-ginseng and its roots, obtained the corresponding solid threedimensional fluorescence spectrum chart through experiments, and select two or more fluorescence peaks as characteristic fluorescence peaks in the detection so as to identify pseudo-ginseng and its roots quickly and efficiently[1].XU Yongqun, Li Xingwang and others apply the statistical pattern recognition method to design the identification program of the three-dimensional fluorescence fingerprints of TCM injections and identify the program of four injections: acanthopanax,Carthamus tinctorius L., panax notoginseng saponins and saliae miltiorrhizae composita.Studies have shown that this program has high resolution,is easy to be automated, and can be used for authenticity identification and quality control of TCM injections[2].YAO Jie, WEI Yongju and others establish a new method for authenticating Mangnolia officinalis based on three-dimensional fluorescence fingerprinting, and the final results show that the three-dimensional fluorescence fingerprints of real and fake Magnolia officinalis are remarkably different in terms of the number of fluorescence peaks, the positions of peaks and peak intensity[3].WANG Lin, PANG Qichang and others use liquid crystal spectrographs to detect the images of fluorescence spectra of golden cypress and ginseng by means of spectral imaging, and their final experimental results also indicate that this image detection can provide effective pixels for the construction of TCM fingerprints[4].ZHANG Jucheng, YAN Heping and other scholars measure the absorption spectra and the three-dimensional fluorescence spectra of camellia pitardii in different regions and find out that camellia pitardiis with different absorption spectra is significantly different[5].

With the continuous enhancement of China's international influence, TCM have gradually been recognized by experts around the world and related research has been carried out.However, the complicated varieties of TCMs and the diversity of its ingredients have increased the difficulty of discrimination[6].Taking bletilla striata as an example, its flowers and leaves can be used to separate more than 50 kinds of Chinese herbal ingredients including stilbenes, phenanthrenes,phenanthrenes and pyrimidines, whose structures are also required to be identified.At the same time,most Chinese herbal medicines contain fluorescent properties.Under this condition, the fluorescent effects provide multiple paths for deep research on TCM[7].In this regard, the study on Chinese herbal medicine can generally be conducted in both microscopic and macroscopic aspects.On the microscopic level, the effect of drug properties and pharmacology is studied from the perspective of molecules of TMC ingredients.On the macroscopic side, TMC can be taken as an entirety, and accurate identification and quality control are conducted on the TMC ingredients by means of fingerprinting[8].Based on computer technology, this article applies the three-dimensional fluorescence technology to construct a unique standard fingerprint map for all types of TMCs and ingredients, further achieves accurate comparison, search and verification of TCM ingredients, and provides a realistic basis for TCM authentication.

OVERVIEW OF THREE-DIMENSIONAL FLUORESCENCE FINGERPRINT

Three-dimensional fluorescence spectra can reflect the changes in light intensity as the emission wavelength varies, which can more clearly reflect the complete spectrum information than the conventional or synchronized fluorescence spectra[9].Specifically,its two representations are isohypse spectrum chart and equiangular three-dimensional projection drawing.In general, three-dimensional fluorescence spectra can more vividly display information such as relative fluorescence intensity and maximum emission or excitation.At the present stage, threedimensional fluorescence spectra have begun to play its unique role in mapping the fingerprints in the study and determination of TCM extracts.

Under normal circumstances, after TCMs absorb ultraviolet and other energy, electrons and other minor components in the TCM molecules return from the excited state to the ground state and release the absorbed energy——fluorescence.The material fluorescence information reflected by the three-dimensional fluorescence spectra enables the spectra to better display the abundant TCM data on account of the features of information such as breadth, depth and high sensitivity[10].

MAPPING AND ACQUISITION OF THREEDIMENSIONAL FLUORESCENCE SPECTRUM OF TCM

Generally speaking, the accuracy and reproducibility of the spectra should be ensured in the drawing of standard TCM search maps.Fluorescence is used as a high-grade sensitive analytical measurement method but with low reproducibility and accuracy.For this reason, this paper applies the Standard Chinese Medicine sold by National Institutes for the control of pharmaceutical and biological products in the mapping and obtains the standard mapping by scanning the TCM placed under the same instrument parameters.

Pre-treatment Plan for TCMs

First, 0.04 g standard reference medicine was collected and placed in a test tube with 8 ml of distilled water.Afterwards, the tube was placed and heated in boiling water for 10 minutes and then placed in cold water until room temperature.Again,liquid medicine that was cooled to room temperature was separated.The supernatant liquid was partially placed in a 25 ml container and diluted to the mark.After shaking it uniformly, a 0.1 mg/ml liquid was obtained.In the end, scans are performed according to the specified parameters of the three-dimensional fluorescence mapping in the instrument.

Introduction of Development Tools for the Retrieval System for TCM Databases

The article employs Visual Basic 6.0 and Microsoft Access 2003 as the development tools for the retrieval system for TCM databases.These tools are featured with advantages of simple and easy operation and powerful functions, which provides a workable channel for its wide availability.Therefore, the application of these tools can ensure the universality of the retrieval system for TCM database.In the meantime, this system uses Visual Basic 6.0 as a link, which, compared with other connection tools, holds the advantages of high flexibility and easy preservation.

Construction of the Retrieval System for Three-Dimensional Fluorescence Fingerpint Database of TCMS

The TCM database system studied in the article mainly includes two parts: the database operating system and the three-dimensional fluorescence spectrum database of TCM.Table 1 demonstrates the database structure of the system.

Table 1.The Structure of Retrieval System for Three-Dimensional Fluorescence Fingerprint Database of TCMS

According to the database structure in Table 1, operators can delete and add spectral data and search or find medicine names in the TCM database anytime and anywhere.Figure 1 illustrates the details.

Figure 1.Schematic Diagram of Retrieval System for Three-Dimensional Fluorescence Fingerprint Database of TCMS

The final function of this retrieval system is the procedure of retrieval and search for TCMs.The most complicated algorithm involved is simplified in this paper.We directly derived the spectral files from the F-4500 fluorescence spectrophotometer, processed the related documents of the spectral files to form a matrix and facilitate data processing, and excluded the influence of environment on the scattered light.Thus, in the range of Ex: (200 nm-400 nm) and Em:(250 nm-600 nm), first-order, second-order and thirdorder scattering occur.As shown in Table 2, i and j represent the horizontal and vertical coordinates respectively; Ex and Em indicate the horizontal and vertical coordinates of the three-dimensional fluorescence fingerprints of TCM.

Table 2.Mapping Relation between Coordinates and Arrays of Three-Dimensional Fluorescence Fingerpints

In light of Table 2, the following set of functions can be derived:

where the first-order scattering condition is Ex = Em, so 250 + 5i = 195 + 5j, j - i = 11.

Exclude the similar parts: 5nm: 8 ＜ j - i ＜ 14

Similarly, the second-order and third-order scattering conditions are:

Em = 2Ex, so 250 + 5i = 2(195 + 5j), j - 0.5i = -14

Exclude the similar parts: 5nm: -17 ＜ j - 0.5i ＜ -11

Em = 3Ex, so 250 + 5i = 3(195 + 5j), j - 0.33i = -22

Exclude the similar parts: 5nm: -19 ＜ j - 0.33i ＜ -25

In light of the retrieval system for the threedimensional fluorescence fingerprint database of TCMs, most common TCMs can be accurately identified.Furthermore, TCMs are affected by natural climate and environmental origin due to their natural, complex and different medicinal properties.This operating system can conduct the further retrieval of TCMs through different scattering conditions to enhance the accuracy of the search results.

CONCLUSION

In general, the retrieval system for the threedimensional fluorescence fingerprint database of TCMs constructed in this article can achieve the strict identification of ingredients, categories, and quality of most TCMs.However, on account of influential factors such as the place of production,climatic environment and natural factors, the actual application value of TCMs is also affected.In addition, the highly sensitive nature of fluorescence in TCMs can also cause reproducibility in the fluorescence spectroscopic analysis.In view of this,the future improvement of the three-dimensional fluorescence fingerprint database of TCMs can adopt a more scientific and reasonable statistical method in the study to correct the human errors.What's more, in consideration of the differences in the growth environment of the same species of herbs,in the study of three-dimensional fluorescence, it is suggested to expand the fluorescence spectra as much as possible, conduct more comprehensive experiments on medicinal herbs with different production habits in order to reduce the uncertainty of herbs, and enhance the reproducibility of the spectra, thereby bringing more authority to the threedimensional fluorescence fingerprint database of TCMs.

ACKNOWLEDGEMENT

Key research project of hainan province，A pilot trial on the development of the health-care and anti-inflammatory products of Clinacanthus nutans ferment，S100007043008