METHOD FOR SCREENING ADULTERATION OF FIBRATE ANTI-HYPERLIPIDEMIA CHEMICALS IN TEA BY COMBINED METHOD OF HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY AND BIOLUMINESCENCE

Abstract

A method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography (HPTLC) and bioluminescence, which belongs to the field of food inspection. The method includes: firstly, formulating standard solutions of bezafibrate and ciprofibrate, and preparing a tea sample; pre-washing a thin-layer plate and then performing HPTLC spotting; performing HPTLC separation to move original mixed target objects mixed originally onto different positions of the thin-layer plate according to different molecular structures, so as to form a physical isolation; and subsequently, simultaneous detection of multiple targets in the tea sample could be realized conveniently through luminous bacteria coupled with the thin-layer plate by an immersed manner. The present disclosure establishes a method capable of detecting anti-hyperlipidemia chemicals in tea rapidly and quantitatively by the combined detection method of HPTLC and bioluminescence, which has the advantages of being economic, rapid, simple and convenient.

Claims

1. A method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence, comprising the steps of: formulating standard solutions of bezafibrate and ciprofibrate, preparing a tea sample; pre-washing a thin-layer plate and then performing HPTLC spotting; performing HPTLC separation to move original mixed target objects onto different positions of the thin-layer plate according to different molecular structures, so as to form a physical isolation; and subsequently, simultaneous detection of multiple targets in the tea sample could be realized conveniently through luminous bacteria coupled with the thin-layer plate by an immersed manner.

2. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 1, wherein the specific steps are as follows: (1) formulation of standard solutions: precisely weighing 10 mg of bezafibrate standard and 10 mg of ciprofibrate standard with an electronic balance, placed into a 10 mL volumetric flask respectively, and adjusting to a constant volume with methanol to obtain a standard stock solution with a concentration of 1 mg/mL; at normal times, storing the standard stock solution standing at 4° C. in dark, before the analysis working begin, taking 0.2 mL of the standard stock solution into a 10 mL volumetric flask, and diluted with methanol to a constant volume, so as to obtain a standard working solution with a concentration of 0.02 mg/mL; the standard working solution is ready-to-use; (2) preparation of tea sample solutions respectively: firstly, taking Folium nelumbinis, Apocynum venetum and Ginkgo biloba, uniformly crushed with an electric crusher respectively; then weighing 1.0 g of the crushed sample, and 10 mL of methanol is added, ultrasonic extracting in a water bath at 25° C. for 30 min, taken out and then centrifuged at 5000 r/min for 5 min; after the centrifugation, taking 5 mL of supernatant of the upper layer and charging it into a syringe, compressing plunger rod of the syringe to enable the supernatant to pass through a 0.45 μm nylon filter membrane, and the resulting sample supernatant could be directly used for HPTLC spotting; (3) HPTLC spotting and chromatographic conditions: firstly, manually pipetting the sample solution with a 100 μL spotting needle; spotting by a semi-automatic thin-layer spotting device, with the assistance of a 0.5 MPa nitrogen flow, sweeping the sample solution to a position 10 cm away from the bottom of the thin-layer plate with a liquid flow sweeping speed of 100 μL/s, a pre-discharge volume of 0.2 μL, a band width of 6 mm and a distance from both side edges of at least 15 mm; spotting each of the samples prepared by the steps (1) and (2), after finishing the spotting of one sample, manually taking out the spotting needle, washing it with methanol for three times, and then carrying out the spotting of a next sample; after finishing all the spotting, taking out the thin-layer plate and heated with an electric hair dryer for 1 min to volatilize the residual methanol attached at spotting positions; Carrying out chromatographic separation in a full-automatic thin layer chromatographic expander with a mobile phase ration: a volume ratio of ethyl acetate/methanol being 9/1 and an expanding distance being 60 mm; the chromatographic separation conditions: controlling relative humidity in an expanding tank by bubbling a saturated magnesium chloride solution for 3 min, so as to adjust the relative humidity to 35%, and pre-equilibrating the thin-layer plate for 10 min; when a front edge of the mobile phase reaching a predetermined height, the system automatically ending, taking out the thin-layer plate and baking it on a thin-layer heater at 80° C. for 5 min to obtain a bright and low-noise bioluminescence imaging background; (4) detection by bioluminescence imaging: immersing the expanded and dried thin-layer plate into a luminescent working suspension by using an automatic immersion device at an immersion speed of 1 mm/s and a retention time of 2 s, then putting the thin-layer plate immersed with the luminescent working suspension into a bioluminescence imager to detect imaging, with a imaging exposure time of 40 s, a imaging interval of 2 min, and taking 15 photographs continuously; and (5) analysis: saving the photographs taken by the bioluminescence imager, opening the photographs with a Videoscan software, digitizing the pixel gray scale in the photographs, and then setting integral parameters and conditions for quantitative analysis.

3. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 2, wherein the preparation process of the luminescent working suspension is as follows: (1) formulation of simulated seawater liquid medium: formulating a simulated seawater liquid medium according to the following formula: 30 g/L of NaCl, 5 g/L of Na.sub.2HPO.sub.4, 5 g/L of KH.sub.2PO.sub.4, 3 mL/L of glycerol, 5 g/L of peptone and 5 g/L of a yeast extract, adding 1 L of ultrapure water to dissolve under stirring; adjusting the pH value to 7.3-7.7 with 1 mol/L of sodium hydroxide solution, and carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot to obtain a simulated seawater liquid medium, then packaging the simulated seawater liquid medium and refrigerating it in a refrigerator for later use, and the simulated seawater liquid medium could be stored in an environment of 4° C. for 7 days when being idle; and (2) culture and preserve of luminous strains: inoculating luminous bacteria cryopreserved with glycerol into a triangular flask containing 100 mL of the liquid medium prepared in the step (1); wrapping the flask mouth with sterilized four-layer folded tin foil paper to ensure external oxygen could enter the flask during the culture process, and then culturing the bacteria in the flask under shaking at 100 r/min in an environment of 25° C. to obtain a bacterial mother liquor; then adding an equal volume of fresh liquid medium into the mature bacterial mother liquor to prepare a luminescent working suspension; and the luminescent working suspension can be stored in an environment of 4° C. for 3 days when being idle.

4. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 3, wherein the luminous strains of the step (2) are preserved by an agar plate method, specifically as follows: a. preparation of luminous bacteria medium: taking 10 g of agar, 30 g of NaCl, 5 g of Na.sub.2HPO.sub.4, 5 g of KH.sub.2PO.sub.4, 3 mL of glycerol, 5 g of peptone and 5 g of a yeast extract, and adding 1 L of ultrapure water to dissolve under stirring, then adjusting the pH value to 7.5±0.2 with a 1 mol/L sodium hydroxide aqueous solution, and then carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot; and when a sterilized culture medium cooled to 60° C., pouring it into a Petri dish with a diameter of 10 cm to form a plate while hot; and b. inoculation: firstly, immersing a sterilized inoculating loop into a mature luminous bacteria medium, and then shading with diagonal lines on the surface of the nutrient agar medium, repeating it for many times; culturing the inoculated nutrient agar medium in an environment of 25° C. in dark for 48 h, a bacterial mother liquor is obtained after obvious colonies being observed, and then transferring it into an environment of −4° C. in dark for preservation.

5. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 3, wherein in the step (2), the absorbance of the liquid medium to an incident light at 600 nm is taken as an index for determining a cell density; a clear simulated seawater liquid medium is taken as a reference, the absorbance OD.sub.600 of a medium to the incident light at 600 nm is monitored by a spectrophotometer during the culture process, and the medium with the value of OD.sub.600 reaching 0.7 is selected as the bacterial mother liquor.

6. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 2, wherein the thin-layer plate in the step (3) needs to be pre-washed, specifically as follows: firstly, pouring 10 mL of methanol into a clean expanding tank, then a blank thin-layer plate being put; expanding the thin-layer plate to the top end thereof, keeping for 5 min when the thin-layer plate expanded to the top end to wash off impurities as many as possible; then taking out the thin-layer plate and baking it on a thin-layer plate heater at 100° C. for 5 min to volatilize the residual organic solvent; and wrapping the dried thin-layer plate with aluminum foil paper for later use.

7. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 2, wherein the thin-layer material of the thin-layer plate is an ordinary silica gel.

8. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 2, wherein the specific process of the step (5) is as follows: saving the photographs taken by the bioluminescence imager in CPF, Black/white linear formats, opening the photographs with a Videoscan software, digitizing the pixel gray scale in the photographs, and then setting integral parameters and conditions for quantitative analysis.

9. The method for screening adulteration of fibrate anti-hyperlipidemia chemicals in tea by combined method of high performance thin layer chromatography and bioluminescence according to claim 1, wherein the luminous bacteria, a class of microorganisms, could be emitting visible light under normal physiological conditions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is an imaging diagram of separation results under the conditions of bacterial liquor immersed-bioluminescence inhibition (pseudo-color mode) in Examples 1-3.

[0032] Spotting tracks: 1. BZF, 2. CPF, 3. Folium nelumbinis, 4. Folium nelumbinis+standard, 5. Ginkgo biloba, 6. Ginkgo biloba+standard.

[0033] FIG. 2 is a standard curve of BZF.

[0034] FIG. 3 is a standard curve of CPF.

[0035] Table 1 is the evaluation of the quantitative detection abilities of the methods.

[0036] Table 2 is the evaluation of the quantitative accuracy of the methods.

DETAILED DESCRIPTION OF THE INVENTION

[0037] In the following examples, the analytical standards of ciprofibrate (≥99%, HPLC) and bezafibrate (≥96.0%, HPLC) are purchased from Aladdin (Shanghai, China). The analytically pure NaCl, Na.sub.2SO.sub.4, NaNO.sub.3 and other chemical reagents are purchased from Sigma-Aldrich. The high-efficiency thin-layer silica gel plate with a glass substrate (analytical type, size 10×20 cm, batch number 1.05729.0001) is purchased from Merck (Darmstadt, Germany). The thin-layer plate is washed once with methanol before use. Blank tea samples are purchased from local supermarkets.

[0038] The luminous bacterium used in the following examples is Aliivibrio fischeri (strain preservation number DSM 507), and the strain is purchased from the China General Microbiological Culture Collection Center, abbreviated as CGMCC.

Example 1

[0039] (1) formulation of simulated seawater liquid and solid medium: formulating a simulated seawater liquid medium according to the following formula: 30 g/L of NaCl, 5 g/L of Na.sub.2HPO.sub.4, 5 g/L of KH.sub.2PO.sub.4, 3 ml/L of glycerol, 5 g/L of peptone, and 5 g/L of a yeast extract, adding 1 L of ultrapure water to dissolve under stirring; adjusting the pH value to 7.5±0.2 with 1 mol/L of sodium hydroxide solution, and carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot to obtain a simulated seawater liquid medium, then packaging the simulated seawater liquid medium and refrigerating it in a refrigerator for later use, and the simulated seawater liquid medium could be stored in an environment of 4° C. for 7 days when being idle; and

[0040] (2) culture and preservation of luminous strains: inoculating luminous bacteria cryopreserved with glycerol into a triangular flask containing 100 mL of the liquid medium prepared in the step (1); wrapping the flask mouth with sterilized four-layer folded tin foil paper to ensure external oxygen could enter the flask during the culture process, and then culturing the bacteria in the flask under shaking at 100 r/min in an environment of 25° C. according to the method described by Chen et al. to obtain a bacterial mother liquor; then adding an equal volume of fresh liquid medium into the mature bacterial mother liquor to prepare a luminescent working suspension; and the luminescent working suspension can be stored in an environment of 4° C. for 3 days when being idle.

[0041] The luminous strains of bacteria were preserved by an agar plate method, and the method for preparing the nutrient agar plate was as follows: taking 10 g of agar, 30 g of NaCl, 5 g of Na.sub.2HPO.sub.4, 5 g of KH.sub.2PO.sub.4, 3 mL of glycerol, 5 g of peptone and 5 g of a yeast extract and adding 1 L of ultrapure water to dissolve under stirring, then adjusting the pH value to 7.5±0.2 with a 1 mol/L sodium hydroxide aqueous solution, and then carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot; and when a sterilized culture medium cooled to 60° C., pouring it into a Petri dish with a diameter of 10 cm to form a plate while hot; and inoculation: firstly, immersing a sterilized inoculating loop into a mature luminous bacteria medium, and then shading with diagonal lines on the surface of the nutrient agar medium, repeating it for many times; culturing the inoculated nutrient agar medium in dark in an environment of 25° C. for 48 h, obtaining a bacterial mother liquor when obvious colonies being observed, and then transferring it into an environment of −4° C. in dark for preservation.

[0042] (3) Formulation of standard solutions: precisely weighing 10±0.1 mg of bezafibrate standard and 10±0.1 mg of ciprofibrate standard with an electronic balance, respectively placed into a 10 mL volumetric flask, and adjusting to a constant volume with methanol to obtain a standard stock solution with a concentration of 1 mg/mL; at normal times, storing the standard stock solution standing at 4° C. in dark, immediately before the analysis working time, taking 0.2 mL of the standard stock solution into a 10 mL volumetric flask, and diluted with methanol to a constant volume, so as to obtain a standard working solution with a concentration of 0.02 mg/mL; the standard working solution is ready-to-use;

[0043] The standard curve of BZF was shown in FIG. 2, and the standard curve of CPF was shown in FIG. 3.

[0044] (4) Pre-washing of thin-layer plate: the small amount of organic residues in the stationary phase of the thin-layer plate would have a significant negative impact on the bioluminescence of the bacteria, so it was necessary to pre-wash the thin-layer plate with methanol before use. Firstly, pouring 10 mL of methanol into a clean expanding tank, then a blank thin-layer plate being put; expanding the thin-layer plate to the top end thereof, keeping for 5 min when the thin-layer plate expanded to the top end to wash off impurities as many as possible; then taking out the thin-layer plate and baking it on a thin-layer plate heater at 100° C. for 5 min to volatilize the residual organic solvent; and wrapping the dried thin-layer plate with aluminum foil paper for later use;

[0045] (5) HPTLC spotting and chromatographic conditions: firstly, manually pipetting the sample solution with a 100 μL spotting needle; spotting by a semi-automatic thin-layer spotting device, with the assistance of a 0.5 MPa nitrogen flow, sweeping the sample solution to a position 10 cm away from the bottom of the thin-layer plate with a liquid flow sweeping speed of 100 μL/s, a pre-discharge volume of 0.2 μL, a band width of 6 mm and a distance from both side edges of at least 15 mm; spotting each of the samples prepared by the step (3), after finishing the spotting of one sample, manually taking out the spotting needle, washing it with methanol for three times, and then carrying out the spotting of a next sample; after finishing all the spotting, taking out the thin-layer plate and heated with an electric hair dryer for 1 min to volatilize the residual methanol attached at spotting positions.

[0046] Chromatographic separation was carried out in a full-automatic thin layer chromatographic expander with a mobile phase ration: a volume ratio of ethyl acetate/methanol being 9/1 and an expanding distance being 60 mm. The chromatographic separation conditions: the relative humidity in an expanding tank was controlled by bubbling a saturated magnesium chloride solution for 3 min (the RH was adjusted to about 35% by bubbling the saturated magnesium chloride solution), the thin-layer plate was pre-equilibrated for 10 min, and it took about 25 min for the whole expanding process. When a front edge of the mobile phase reached a predetermined height, the system automatically ended, and the thin-layer plate was taken out and baked on a thin-layer heater at 80° C. for 5 min to volatilize the residual organic solvent to obtain a bright and low-noise bioluminescence imaging background.

[0047] (6) Detection by bioluminescence imaging: immersing the expanded and dried thin-layer plate into a luminescent working suspension by using an automatic immersion device at an immersion speed of 1 mm/s for a retention time of 2 s, and then putting the thin-layer plate immersed with the luminescent working suspension into a bioluminescence imager to imaging detect, with a imaging exposure time of 40 s, a imaging interval of 2 min, and 15 photographs taken continuously; and

[0048] (7) saving the photographs taken by the bioluminescence imager in CPF (Black/white linear) format, opening the photographs with a Videoscan software, digitizing the pixel gray scale in the photographs, and then setting integral parameters and conditions for quantitative analysis.

[0049] The imaging diagram of separation results under the conditions of bacterial liquor immersed-bioluminescence inhibition (pseudo-color mode) was shown in FIG. 1.

Example 2

[0050] (1) formulation of simulated seawater liquid and solid medium: formulating a simulated seawater liquid medium according to the following formula: 30 g/L of NaCl, 5 g/L of Na.sub.2HPO.sub.4, 5 g/L of KH.sub.2PO.sub.4, 3 ml/L of glycerol, 5 g/L of peptone, and 5 g/L of a yeast extract, adding 1 L of ultrapure water to dissolve under stirring; adjusting the pH value to 7.5±0.2 with 1 mol/L of sodium hydroxide solution, and carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot to obtain a simulated seawater liquid medium, then packaging the simulated seawater liquid medium and refrigerating it in a refrigerator for later use, and the simulated seawater liquid medium could be stored in an environment of 4° C. for 7 days when being idle; and

[0051] (2) culture and preservation of luminous strains: inoculating luminous bacteria cryopreserved with glycerol into a triangular flask containing 100 mL of the liquid medium prepared in the step (1); wrapping the flask mouth with sterilized four-layer folded tin foil paper to ensure external oxygen could enter the flask during the culture process, and then culturing the bacteria in the flask under shaking at 100 r/min in an environment of 25° C. according to the method described by Chen et al. to obtain a bacterial mother liquor; then adding an equal volume of fresh liquid medium into the mature bacterial mother liquor to prepare a luminescent working suspension; and the luminescent working suspension can be stored in an environment of 4° C. for 3 days when being idle.

[0052] The luminous strains of bacteria were preserved by an agar plate method, and the method for preparing the nutrient agar plate was as follows: taking 10 g of agar, 30 g of NaCl, 5 g of Na.sub.2HPO.sub.4, 5 g of KH.sub.2PO.sub.4, 3 mL of glycerol, 5 g of peptone and 5 g of a yeast extract and adding 1 L of ultrapure water to dissolve under stirring, then adjusting the pH value to 7.5±0.2 with a 1 mol/L sodium hydroxide aqueous solution, and then carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot; and when a sterilized culture medium cooled to 60° C., pouring it into a Petri dish with a diameter of 10 cm to form a plate while hot; and inoculation: firstly, immersing a sterilized inoculating loop into a mature luminous bacteria medium, and then shading with diagonal lines on the surface of the nutrient agar medium, repeating it for many times; culturing the inoculated nutrient agar medium in dark in an environment of 25° C. for 48 h, obtaining a bacterial mother liquor when obvious colonies being observed, and then transferring it into an environment of −4° C. in dark for preservation.

[0053] (3) Formulation of standard solutions: precisely weighing 10±0.1 mg of bezafibrate standard and 10±0.1 mg of ciprofibrate standard with an electronic balance, respectively placed into a 10 mL volumetric flask, and adjusting to a constant volume with methanol to obtain a standard stock solution with a concentration of 1 mg/mL; at normal times, storing the standard stock solution standing at 4° C. in dark, immediately before the analysis working time, taking 0.2 mL of the standard stock solution into a 10 mL volumetric flask, and diluted with methanol to a constant volume, so as to obtain a standard working solution with a concentration of 0.02 mg/mL; the standard working solution is ready-to-use;

[0054] The standard curve of BZF was shown in FIG. 2, and the standard curve of CPF was shown in FIG. 3.

[0055] (4) Preparation of a sample: firstly, taking and uniformly crushing a sample of Folium nelumbinis with an electric crusher; then weighing 1.0 g of the crushed sample and added it into 10 mL of methanol, ultrasonic extracting in a water bath at 25° C. for 30 min, taken out and then centrifuged at 5000 r/min for 5 min; after the centrifugation, taking 5 mL of supernatant of the upper layer and charging it into a syringe, compressing a plunger rod of the syringe to enable the supernatant to pass through a 0.45 μm nylon filter membrane, and the resulting sample supernatant could be directly used for HPTLC spotting;

[0056] (5) pre-washing of thin-layer plate: the small amount of organic residues in the stationary phase of the thin-layer plate would have a significant negative impact on the bioluminescence of the bacteria, so it was necessary to pre-wash the thin-layer plate with methanol before use. Firstly, pouring 10 mL of methanol into a clean expanding tank, then a blank thin-layer plate being put; expanding the thin-layer plate to the top end thereof, keeping for 5 min when the thin-layer plate expanded to the top end to wash off impurities as many as possible; then taking out the thin-layer plate and baking it on a thin-layer plate heater at 100° C. for 5 min to volatilize the residual organic solvent; and wrapping the dried thin-layer plate with aluminum foil paper for later use;

[0057] (6) HPTLC spotting and chromatographic conditions: firstly, manually pipetting the sample solution with a 100 μL spotting needle; spotting by a semi-automatic thin-layer spotting device, with the assistance of a 0.5 MPa nitrogen flow, sweeping the sample solution to a position 10 cm away from the bottom of the thin-layer plate with a liquid flow sweeping speed of 100 μL/s, a pre-discharge volume of 0.2 μL, a band width of 6 mm and a distance from both side edges of at least 15 mm; spotting each of the samples prepared by the step (3), after finishing the spotting of one sample, manually taking out the spotting needle, washing it with methanol for three times, and then carrying out the spotting of a next sample; after finishing all the spotting, taking out the thin-layer plate and heated with an electric hair dryer for 1 min to volatilize the residual methanol attached at spotting positions.

[0058] Chromatographic separation was carried out in a full-automatic thin layer chromatographic expander with a mobile phase ration: a volume ratio of ethyl acetate/methanol being 9/1 and an expanding distance being 60 mm. The chromatographic separation conditions: the relative humidity in an expanding tank was controlled by bubbling a saturated magnesium chloride solution for 3 min, adjusting the RH to about 35%, the thin-layer plate was pre-equilibrated for 10 min, and it took about 25 min for the whole expanding process. When a front edge of the mobile phase reached a predetermined height, the system automatically ended, and the thin-layer plate was taken out and baked on a thin-layer heater at 80° C. for 5 min to volatilize the residual organic solvent to obtain a bright and low-noise bioluminescence imaging background.

[0059] (7) Detection by bioluminescence imaging: immersing the expanded and dried thin-layer plate into a luminescent working suspension by using an automatic immersion device at an immersion speed of 1 mm/s for a retention time of 2 s, and then putting the thin-layer plate immersed with the luminescent working suspension into a bioluminescence imager to imaging detect, with a imaging exposure time of 40 s, a imaging interval of 2 min, and 15 photographs taken continuously; and

[0060] (8) saving the photographs taken by the bioluminescence imager in CPF (Black/white linear) format, opening the photographs with a Videoscan software, digitizing the pixel gray scale in the photographs, and then setting integral parameters and conditions for quantitative analysis.

[0061] The imaging diagram of separation results under the conditions of bacterial liquor immersed-bioluminescence inhibition (pseudo-color mode) was shown in FIG. 1.

Example 3

[0062] (1) formulation of simulated seawater liquid and solid medium: formulating a simulated seawater liquid medium according to the following formula: 30 g/L of NaCl, 5 g/L of Na.sub.2HPO.sub.4, 5 g/L of KH.sub.2PO.sub.4, 3 ml/L of glycerol, 5 g/L of peptone, and 5 g/L of a yeast extract, adding 1 L of ultrapure water to dissolve under stirring; adjusting the pH value to 7.5±0.2 with 1 mol/L of sodium hydroxide solution, and carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot to obtain a simulated seawater liquid medium, then packaging the simulated seawater liquid medium and refrigerating it in a refrigerator for later use, and the simulated seawater liquid medium could be stored in an environment of 4° C. for 7 days when being idle; and

[0063] (2) culture and preservation of luminous strains: inoculating luminous bacteria cryopreserved with glycerol into a triangular flask containing 100 mL of the liquid medium prepared in the step (1); wrapping the flask mouth with sterilized four-layer folded tin foil paper to ensure external oxygen could enter the flask during the culture process, and then culturing the bacteria in the flask under shaking at 100 r/min in an environment of 25° C. according to the method described by Chen et al. to obtain a bacterial mother liquor; then adding an equal volume of fresh liquid medium into the mature bacterial mother liquor to prepare a luminescent working suspension; and the luminescent working suspension can be stored in an environment of 4° C. for 3 days when being idle.

[0064] The luminous strains of bacteria were preserved by an agar plate method, and the method for preparing the nutrient agar plate was as follows: taking 10 g of agar, 30 g of NaCl, 5 g of Na.sub.2HPO.sub.4, 5 g of KH.sub.2PO.sub.4, 3 mL of glycerol, 5 g of peptone and 5 g of a yeast extract and adding 1 L of ultrapure water to dissolve under stirring, then adjusting the pH value to 7.5±0.2 with a 1 mol/L sodium hydroxide aqueous solution, and then carrying out sterilization treatment at 121° C. for 15 min within a high-pressure steam sterilization pot; and when a sterilized culture medium cooled to 60° C., pouring it into a Petri dish with a diameter of 10 cm to form a plate while hot; and inoculation: firstly, immersing a sterilized inoculating loop into a mature luminous bacteria medium, and then shading with diagonal lines on the surface of the nutrient agar medium, repeating it for many times; culturing the inoculated nutrient agar medium in dark in an environment of 25° C. for 48 h, obtaining a bacterial mother liquor when obvious colonies being observed, and then transferring it into an environment of −4° C. in dark for preservation.

[0065] (3) Formulation of standard solutions: precisely weighing 10±0.1 mg of bezafibrate standard and 10±0.1 mg of ciprofibrate standard with an electronic balance, respectively placed into a 10 mL volumetric flask, and adjusting to a constant volume with methanol to obtain a standard stock solution with a concentration of 1 mg/mL; at normal times, storing the standard stock solution standing at 4° C. in dark, immediately before the analysis working time, taking 0.2 mL of the standard stock solution into a 10 mL volumetric flask, and diluted with methanol to a constant volume, so as to obtain a standard working solution with a concentration of 0.02 mg/mL; the standard working solution is ready-to-use;

[0066] The standard curve of BZF was shown in FIG. 2, and the standard curve of CPF was shown in FIG. 3.

[0067] (4) Preparation of a sample: firstly, taking and uniformly crushing a sample of Ginkgo biloba with an electric crusher; then weighing 1.0 g of the crushed sample and added it into 10 mL of methanol, ultrasonic extracting in a water bath at 25° C. for 30 min, taken out and then centrifuged at 5000 r/min for 5 min; after the centrifugation, taking 5 mL of supernatant of the upper layer and charging it into a syringe, compressing a plunger rod of the syringe to enable the supernatant to pass through a 0.45 μm nylon filter membrane, and the resulting sample supernatant could be directly used for HPTLC spotting;

[0068] (5) pre-washing of thin-layer plate: the small amount of organic residues in the stationary phase of the thin-layer plate would have a significant negative impact on the bioluminescence of the bacteria, so it was necessary to pre-wash the thin-layer plate with methanol before use. Firstly, pouring 10 mL of methanol into a clean expanding tank, then a blank thin-layer plate being put; expanding the thin-layer plate to the top end thereof, keeping for 5 min when the thin-layer plate expanded to the top end to wash off impurities as many as possible; then taking out the thin-layer plate and baking it on a thin-layer plate heater at 100° C. for 5 min to volatilize the residual organic solvent; and wrapping the dried thin-layer plate with aluminum foil paper for later use;

[0069] (6) HPTLC spotting and chromatographic conditions: firstly, manually pipetting the sample solution with a 100 μL spotting needle; spotting by a semi-automatic thin-layer spotting device, with the assistance of a 0.5 MPa nitrogen flow, sweeping the sample solution to a position 10 cm away from the bottom of the thin-layer plate with a liquid flow sweeping speed of 100 μL/s, a pre-discharge volume of 0.2 μL, a band width of 6 mm and a distance from both side edges of at least 15 mm; spotting each of the samples prepared by the steps (3) and (4), after finishing the spotting of one sample, manually taking out the spotting needle, washing it with methanol for three times, and then carrying out the spotting of a next sample; after finishing all the spotting, taking out the thin-layer plate and heated with an electric hair dryer for 1 min to volatilize the residual methanol attached at spotting positions.

[0070] Chromatographic separation was carried out in a full-automatic thin layer chromatographic expander with a mobile phase ration: a volume ratio of ethyl acetate/methanol being 9/1 and an expanding distance being 60 mm. The chromatographic separation conditions: the relative humidity in an expanding tank was controlled by bubbling a saturated magnesium chloride solution for 3 min, (the RH was adjusted to about 35% by bubbling the saturated magnesium chloride solution), the thin-layer plate was pre-equilibrated for 10 min, and it took about 25 min for the whole expanding process. When a front edge of the mobile phase reached a predetermined height, the system automatically ended, and the thin-layer plate was taken out and baked on a thin-layer heater at 80° C. for 5 min to volatilize the residual organic solvent to obtain a bright and low-noise bioluminescence imaging background.

[0071] (7) Detection by bioluminescence imaging: immersing the expanded and dried thin-layer plate into a luminescent working suspension by using an automatic immersion device at an immersion speed of 1 mm/s for a retention time of 2 s, and then putting the thin-layer plate immersed with the luminescent working suspension into a bioluminescence imager to imaging detect, with a imaging exposure time of 40 s, a imaging interval of 2 min, and 15 photographs taken continuously; and

[0072] (8) saving the photographs taken by the bioluminescence imager in CPF (Black/white linear) format, opening the photographs with a Videoscan software, digitizing the pixel gray scale in the photographs, and then setting integral parameters and conditions for quantitative analysis.

[0073] The imaging diagram of separation results under the conditions of bacterial liquor immersed-bioluminescence inhibition (pseudo-color mode) was shown in FIG. 1.

Example 4

[0074] Apocynum venetum was selected as the sample in the step (4), and other steps were the same as those in Example 3.

Example 5

[0075] The detection abilities of the methods described in Examples 1-4 were evaluated. The specific results of quantitative detection ability evaluation were shown in Table 1, and the quantitative accuracy evaluation were shown in Table 2.

TABLE-US-00001 TABLE 1 Evaluation of quantitative detection abilities of methods Linear Quantifying Binomial Quantifying Quantitative Range Regression Regression Analyte [ng/zone] [mg/g] Equation R.sup.2 Equation R.sup.2 BZF 50-1000 0.05-1 y = 62.789x + 0.9279 y = −0.0621x2 + 0.9992 26312 126.59x + 16409 CPF 50-1000 0.05-1 y = 62.209x + 0.9837 y = 0.0242x2 + 0.9955 20305 37.32x + 24168

TABLE-US-00002 TABLE 2 Evaluation of the quantitative accuracy of methods Recovery rate % Additive amount Folium Apocynum Ginkgo Analyte [ng/zone] [μg/g] nelumbinis venetum biloba BZF 50 5 81 79 86 100 10 90 89 85 200 20 82 84 88 CPF 50 5 74 78 83 100 10 89 90 83 200 20 78 83 91