CN108956843B - Rapid multi-information thin-layer identification method for pinellia ternate, bighead atractylodes rhizome and gastrodia elata decoction freeze-dried powder - Google Patents

Abstract

The invention relates to a rapid multi-information thin-layer identification method for pinellia ternate, bighead atractylodes rhizome and gastrodia elata decoction freeze-dried powder. The method is characterized in that: a test sample and a reference medicinal material solution are obtained by a simple and quick pretreatment method, the same test sample solution is adopted, four medicinal materials are simultaneously identified on two thin-layer plates, liquorice and bighead atractylodes rhizome are arranged on one thin-layer plate, and exocarpium citri rubrum and rhizoma gastrodiae are arranged on the other thin-layer plate. After the sheet is unfolded, directly detecting fluorescent spots of the liquorice and the red tangerine peel under an ultraviolet lamp of 365 nm; spraying 10% sulfuric acid ethanol solution for color development, and inspecting fluorescent spots of the Atractylodes macrocephala after fluorescence enhancement under an ultraviolet lamp of 365 nm; spraying a mixed solution of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution which are mixed in equal volumes for color development, and inspecting the brownish red spots of the color of the gastrodia elata through lamplight in a dark room, wherein the brownish red spots are overlapped with each other, but do not interfere with each other under respective inspection conditions. Provides a simple, convenient and quick thin-layer method for controlling the quality of the pinellia ternate, bighead atractylodes rhizome and gastrodia elata decoction freeze-dried powder.

Description

A rapid multi-information thin-layer identification method for freeze-dried powder of Banxia Baizhu Tianma Decoction

technical field

The invention relates to a rapid multi-information thin-layer identification method for freeze-dried powder of Banxia Baizhu Tianma Decoction.

Background technique

In traditional Chinese medicine compound preparations, especially those prepared by traditional decoction, according to the extraction principle of similarity and compatibility, it is difficult to extract part of the fat-soluble components in the preparation, while more water-soluble components are extracted, and the mutual interference is serious. After water extraction, the original medicinal materials are identified by the thin layer of fat-soluble components, some of which can no longer be used, and the identification feature points of water-soluble components must be found. Due to technical difficulties, the thin layer identification of the water-extracted preparations is low. , quantitative measurement indicators are few. Taking the 2015 edition of the Chinese Pharmacopoeia as an example, the water-decocted Yiganning granules are composed of thirteen traditional Chinese medicines, and only the thin-layer identification of four medicinal materials has been added, and the identification and renewal rate is 30%. Not only the identification update rate is low, but the identification method is also very cumbersome and complicated. The completion of the four identifications requires a sample of 85g or 15g (containing lactose), only a pretreatment solvent of 386ml, and a time of 10 to 12 hours. For 4 identifications, 4 kinds of test solutions need to be prepared, and 4 times of unfolding are completed on 4 thin-layer plates; the contained Erding granules are composed of 4 medicinal materials, only one thin-layer identification has been added, and the renewal rate is 25 %. The pretreatment requires 15g or 3g of samples (lactose-containing), 82ml of processing solvent, and 2 hours of time; the Erbao granules contained are composed of eleven medicinal materials, and the thin-layer identification of four medicinal materials has been added, with an update rate of 36%. And the identification method is also very cumbersome and complicated. For the 4 identifications, 3 kinds of test solutions should be prepared, and 4 times of expansion are completed on 4 thin-layer plates. A total of 80 g of sample, 295 ml of pretreatment solvent, and 8 hours of time are required. If the developing agent and the developing time are added, the organic solvent and time spent will be even more, etc. There are countless similar examples.

To sum up, the thin-layer identification is basically one variety. If there are N identifications, N kinds of test solutions, N thin-layer plates, and N times are prepared to identify the traditional identification mode of N medicinal materials. In order to eliminate interference, the pretreatment procedures of the samples are complicated and cumbersome, and a large number of organic reagents need to be repeatedly purified, which is labor-intensive, time-consuming, reagent-intensive, pollutes the environment, endangers health, and has a long detection cycle. In this way, it generally takes a week to complete a quality standard test containing 6 to 7 thin-layer identification and two content determinations. In case of retest, the time will be doubled, and the detection speed will seriously restrict the modern production speed of traditional Chinese medicine. Therefore, finding a simple and fast detection method for traditional water-extracted preparations, improving detection efficiency, and reducing detection costs is a difficulty that must be overcome in the quality control of traditional Chinese medicine.

Banxia Baizhu Tianma Decoction is a classic prescription announced by the State Administration of Traditional Chinese Medicine. , There are eight herbs in the freeze-dried powder. Its prescription ratio and preparation process are as follows:

Prescription: Pinellia 1.5 cents each, Tianma, Poria, Orange 1.00 each, Atractylodes 3.5 cents, and Licorice 5.0 cents.

Preparation method: Take the six herbs in the prescription, add one piece of ginger and two pieces of jujube, boil with water, concentrate the decocting liquid, freeze-dry, and obtain freeze-dried powder.

In order to ensure the quality of the preparation, a thin-layer identification study was carried out on the freeze-dried powder of Banxia Baizhu Tianma Decoction.

SUMMARY OF THE INVENTION

The present invention is aimed at the current one variety, and if there are N items of identification, N kinds of test solution solutions, N pieces of thin-layer plates, and N times are unfolded to identify the traditional identification mode of N medicinal materials. By studying the parameters of different combinations of developing agents and their proportions, color developing agents, thin-layer carriers, and detection conditions, the same test solution was created to identify 4 samples on two thin-layer plates under 4 different inspection conditions. A new mode of one-test, multiple-evaluation thin-layer identification of medicinal herbs. Licorice and Atractylodes are on another thin-layer board, and orange red and gastrodia elata are on a thin-layer board. The fluorescence enhancement detection of Atractylodes Rhizoma water extract preparation and the color development technology of Gastrodia elata are reported for the first time.

The scheme adopted by the present invention to solve its technical problem is:

A. Take 0.5 g of Banxia Baizhu Tianma Decoction freeze-dried powder, add 2 ml of methanol, sonicate for 10 minutes, centrifuge, and use the supernatant as the test solution; take another Atractylodes Rhizoma control medicinal material 0.2 g, add 2 ml of methanol, and sonicate for 20 minutes , the supernatant is used as Atractylodes Rhizoma reference medicinal material solution; then take 0.1 g of licorice reference medicinal material, add 30 ml of water, decocted for 20 minutes on low heat, filter, concentrate the filtrate to dryness, add 2 ml of methanol to the residue to dissolve, as licorice reference medicinal material solution; Licorice reference medicinal material solution 2-3 μl, Atractylodes Rhizoma reference medicinal material solution 6-8 μl, and test solution 8-10 μl, respectively, were spotted on the same silica gel GF ₂₅₄ thin-layer plate, and the volume ratio was 13:2:3:0.5 chloroform -Ethyl acetate-methanol-concentrated ammonia as the developing agent, unfolded, taken out, dried with hot air, and inspected under an ultraviolet lamp at 365 nm. In the chromatogram of the test product, the same color fluorescence was displayed at the position corresponding to the chromatogram of the licorice reference medicinal material. The main spot (Fig. 1); then sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spot color was clear, and inspected under an ultraviolet lamp at 365 nm. Show the same color fluorescent main spots (Figure 2);

B. Take 0.2g each of gastrodia elata reference medicinal material and orange red reference medicinal material, add 2 ml of methanol respectively, ultrasonically treat for 20 minutes, and use the supernatant as the respective reference medicinal material solution; take another appropriate amount of gastrodin and hesperidin reference substance, add methanol to make Each 1ml contains 1mg of solution as the respective reference solution; absorb 3-4μl of gastrodin reference solution, 5-6μl of hesperidin reference solution, 5-6μl of reference medicinal material solution, and the test solution under item A 6-8 μl, respectively, were placed on the same silica gel GF ₂₅₄ thin-layer plate, and ethyl acetate-butanone-methanol-formic acid-water with a volume ratio of 8:3:1:0.2:0.2 was used as the developing agent, and spread to about 3cm, Take it out, dry it with hot air, and then place it in the same developing agent to expand to 10cm, take it out, dry it with hot air, and inspect it under an ultraviolet lamp at 365 nm. Bright blue fluorescent main spot (Fig. 3); inspected under ultraviolet light at 254 nm, in the chromatogram of the test substance, the main spots of the same color were displayed at the positions corresponding to the chromatograms of the hesperidin reference substance and the orange-red reference medicinal material respectively ( Figure 4); then spray the mixed solution of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution mixed with a volume ratio of 1:1, heat at 105 ° C until the spots are clear, place in a dark room, inspect through light, the test sample In the chromatogram, at the positions corresponding to the chromatograms of gastrodin and gastrodia elata, there were main spots of the same color (Fig. 5).

The principle of the present invention is as follows:

According to the chemical structure and properties of the active ingredients of traditional Chinese medicine, following the extraction principle of similarity and compatibility, and using suitable extraction solvents, the test sample and the reference medicinal material solution were prepared simply and quickly. Then use different components, different proportions, and different polarities of developing agents to expand, and various chemical components will follow different developing agents, depending on the ability of adsorption, desorption, re-adsorption and re-desorption. The respective thin layer plates were well separated. Then, with the help of various active ingredients with similar polarities, on the same thin-layer board, under different inspection conditions, although overlapping, through different color rendering methods, they can be displayed at different levels without interfering with each other. Individually different colored spots to obtain informative thin-layer chromatograms. Realize the desire for simple, fast, low-cost, and high-efficiency thin layers.

The innovations and beneficial effects of the present invention are as follows:

1. Use chloroform-ethyl acetate-methanol-concentrated ammonia with a volume ratio of 13:2:3:0.5 as a developing agent to develop licorice and Atractylodes. There is no information spot; spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spot color is clear, and check the fluorescent main spot after Atractylodes fluorescens under the ultraviolet lamp at 365nm, the spot is clear and bright, and it is easy to inspect. Its innovation is that 1) Atractylodes has always detected the color spots of fat-soluble components and vanillin sulfuric acid solution, and its sensitivity is not high. Sensitive fluorescent spots; 2) The developing agent is alkaline, which can not only make a large part of the acidic components in the compound preparation salt, slow down its adsorption and desorption capacity, reduce the interference of the thin layer background, and make the Atractylodes Rhizoma covered by the acidic components. Uncovering the spots can also enhance the fluorescence intensity of the Atractylodes Rhizoma fluorescent spots after color development; 3) So far, after decocting in Atractylodes Rhizoma, the fat-soluble components are lost, and the thin-layer identification information spots cannot be found; 4) This What was identified and inspected was the most water-soluble fluorescent spot of Atractylodes macrocephala, and the developing agent just pushed some of the lipid-soluble fluorescent spots of the reference medicinal material to the front of the developing agent, which solved the problem that Atractylodes macrocephala was mainly water-soluble components in the preparation, while the control medicinal material of Atractylodes macrocephala was mainly water-soluble. The high content of fat-soluble components resulted in the apparent inconsistency of the two spots, making it impossible to describe the problem. 5) It provides an effective identification method for the detection of Atractylodes Rhizoma in the water-decocted compound preparation, which is innovative and practical.

2. Using ethyl acetate-butanone-methanol-formic acid-water with a volume ratio of 8:3:1:0.2:0.2 as the developing agent, expand Gastrodia elata and orange-red, first inspect it under an ultraviolet light at 365 nm, and the orange-red 3 fluorescent main Spots, and then inspected under UV light at 254 nm for tan spots of orange red and hesperidin, two chemical constituents next to each other (Figure 6). Then spray the mixed solution of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution in equal volume, bake at 105°C until the spots are clear, put it in a dark room, and check the brown-red color of gastrodin and gastrodia elata control medicinal materials through light. The main spot; comprehensive analysis of this identification, it is considered that: under the bright blue fluorescent spot is the tan spot of hesperidin, and the bright blue fluorescent spot overlaps with the colorized gastrodin spot, although they overlap. However, one observes the fluorescent spots, the other observes the tan spots, and the other observes the colored spots, which do not interfere with each other, which is the first report.

3. So far, the color-developing agents for the identification of Gastrodia elata thin layers are 10% phosphomolybdic acid ethanol solution, which is a wide-range color-developing agent and reacts with most of the components, showing blue spots, Difficulty identifying characteristic spots from color. Not only that, but the 10% phosphomolybdic acid ethanol solution is a blue solution, which interferes with the color background of the sprayed thin-layer board, and the color development speed is very slow. It must be heated for a long time, and it is easy to appear color development. If it is not in place, the accuracy of the identification will be affected. And what this application adopts is the mixed solution of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution mixed in equal volume, which not only obviously improves the color development speed of Gastrodia elata, but also makes the color of the spots of gastrodin, an active ingredient of Gastrodia elata. From blue to reddish brown, it is obviously different from the blue spots of hesperidin, which helps to improve the ability of inspectors to judge thin-layer spots.

4. The mixed solution composed of equal volume of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution, as a thin-layer identification color developer, not only has a fast color development speed, but also can make different effective components of traditional Chinese medicine produce different effects. The color, which has not been reported yet, provides a new color developer for the field of thin layer identification, which is innovative and has a prospect of popularization and application.

5. Compared with the traditional thin-layer identification exemplified under the background technology item, the identification is completed, and only 0.5g of the sample, 0.2g of the reference medicinal materials of Atractylodes japonica, 0.2 g of the reference medicinal material of Gastrodia elata, 0.1 g of the reference medicinal material of licorice, 11 ml of organic treatment solvent, and Dosage 30ml, time 1 hour. The method is simple, fast, efficient, and has a lot of detection information, which are not available in the reported methods so far. It provides an effective quality control method for the quality supervision of the freeze-dried powder of Banxia Baizhu Tianma Decoction. It reflects the necessity of detection technology innovation and the significant beneficial effects it brings.

Description of drawings

Fig. 1 is the TLC diagram of licorice of the lyophilized powder of Banxia Baizhu Tianma Decoction under UV lamp 365nm.

Figure 2 is the TLC image of Atractylodes Rhizoma Atractylodes Rhizoma viewed under an ultraviolet lamp at 365 nm after color development of the lyophilized powder of Banxia Baizhu Tianma Decoction in 10% sulfuric acid ethanol solution.

Figure 3 is the orange-red TLC image of the freeze-dried powder of Banxia Baizhu Tianma Decoction under UV lamp 365nm

Figure 4 shows the TLC images of tangerine red and hesperidin of the freeze-dried powder of Banxia Baizhu Tianma Decoction under UV light at 254 nm

Fig. 5 shows the coloration of Gastrodia elata and gastrodin after the freeze-dried powder of Banxia Baizhu Tianma Decoction was sprayed with a mixed solution of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution in a volume ratio of 1:1 TLC diagram

Figure 6 shows the TLC of the freeze-dried powder of Banxia Baizhu Tianma Decoction under dual-light source UV lamps at 365 nm and 254 nm

Figures 1 and 2 are TLC chromatograms of the same TLC plate under different inspection conditions, among which, 1. Atractylodes Rhizoma blank 2. Atractylodes Rhizoma reference material 3.4.5 Sample 6. Licorice blank 7. Licorice reference material

Figures 3 and 4 are the TLC chromatograms of the same thin-layer plate under different inspection conditions, in which 1. gastrodin 2. gastrodia elata reference material 3.4.5 sample 6. orange red reference material 7. hesperidin reference substance

1.2.3 Sample in Figure 6 4. Orange-red reference medicinal material 5. Hesperidin

The specific embodiments of the present invention are as follows:

A. Take 0.5 g of Banxia Baizhu Tianma Decoction freeze-dried powder, add 2 ml of methanol, sonicate for 10 minutes, centrifuge, and use the supernatant as the test solution; take another Atractylodes Rhizoma control medicinal material 0.2 g, add 2 ml of methanol, and sonicate for 20 minutes , the supernatant is used as Atractylodes Rhizoma reference medicinal material solution; then take 0.1 g of licorice reference medicinal material, add 30 ml of water, decocted for 20 minutes on low heat, filter, concentrate the filtrate to dryness, add 2 ml of methanol to the residue to dissolve, as licorice reference medicinal material solution; Licorice reference medicinal material solution 2-3 μl, Atractylodes Rhizoma reference medicinal material solution 6-8 μl, and test solution 8-10 μl, respectively, were spotted on the same silica gel GF ₂₅₄ thin-layer plate, and the volume ratio was 13:2:3:0.5 chloroform -Ethyl acetate-methanol-concentrated ammonia as the developing agent, unfolded, taken out, dried with hot air, and inspected under an ultraviolet lamp at 365 nm. In the chromatogram of the test product, the same color fluorescence was displayed at the position corresponding to the chromatogram of the licorice reference medicinal material. The main spot; then sprayed with 10% sulfuric acid ethanol solution, heated at 105 ℃ until the spot color is clear, and inspected under the ultraviolet lamp at 365 nm. main spot;

B. Take 0.2g each of gastrodia elata reference medicinal material and orange red reference medicinal material, add 2 ml of methanol respectively, ultrasonically treat for 20 minutes, and use the supernatant as the respective reference medicinal material solution; take another appropriate amount of gastrodin and hesperidin reference substance, add methanol to make Each 1ml contains 1mg of solution as the respective reference solution; absorb 3-4μl of gastrodin reference solution, 5-6μl of hesperidin reference solution, 5-6μl of reference medicinal material solution, and the test solution under item A 6-8 μl, respectively, were placed on the same silica gel GF ₂₅₄ thin-layer plate, and ethyl acetate-butanone-methanol-formic acid-water with a volume ratio of 8:3:1:0.2:0.2 was used as the developing agent, and spread to about 3cm, Take it out, dry it with hot air, and then place it in the same developing agent to expand to 10cm, take it out, dry it with hot air, and inspect it under an ultraviolet lamp at 365 nm. Bright blue fluorescent main spot; inspected under ultraviolet light at 254nm, in the chromatogram of the test substance, the main spots of the same color appear respectively at the positions corresponding to the chromatograms of the hesperidin reference substance and the orange-red reference medicinal material; then spray with volume The mixed solution of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution mixed in a ratio of 1:1, heated at 105 ° C until the spots are clear, placed in a dark room, and inspected through light. On the corresponding positions of the gastrodia elata control material chromatogram, the main spots of the same color were respectively displayed.

Claims (2)

1. a fast multi-information thin-layer identification method for the freeze-dried powder of Banxia Baizhu Tianma Decoction, is characterized in that: A. Take 0.5 g of Banxia Baizhu Tianma Decoction freeze-dried powder, add 2 ml of methanol, sonicate for 10 minutes, centrifuge, and use the supernatant as the test solution; take another Atractylodes Rhizoma control medicinal material 0.2 g, add 2 ml of methanol, and sonicate for 20 minutes , the supernatant is used as Atractylodes Rhizoma reference medicinal material solution; then take 0.1 g of licorice reference medicinal material, add 30 ml of water, decocted for 20 minutes on low heat, filter, concentrate the filtrate to dryness, add 2 ml of methanol to the residue to dissolve, as licorice reference medicinal material solution; Licorice reference medicinal material solution 2-3 μl, Atractylodes Rhizoma reference medicinal material solution 6-8 μl, and test solution 8-10 μl, respectively, were spotted on the same silica gel GF ₂₅₄ thin-layer plate, and the volume ratio was 13:2:3:0.5 chloroform -Ethyl acetate-methanol-concentrated ammonia as the developing agent, unfolded, taken out, dried with hot air, and inspected under an ultraviolet lamp at 365 nm. In the chromatogram of the test product, the same color fluorescence was displayed at the position corresponding to the chromatogram of the licorice reference medicinal material. The main spot; then sprayed with 10% sulfuric acid ethanol solution, heated at 105 ℃ until the spot color is clear, and inspected under the ultraviolet lamp at 365 nm. main spot; B. Take 0.2g each of gastrodia elata reference medicinal material and orange red reference medicinal material, add 2 ml of methanol respectively, ultrasonically treat for 20 minutes, and use the supernatant as the respective reference medicinal material solution; take another appropriate amount of gastrodin and hesperidin reference substance, add methanol to make Each 1ml contains 1mg of solution as the respective reference solution; absorb 3-4μl of gastrodin reference solution, 5-6μl of hesperidin reference solution, 5-6μl of reference medicinal material solution, and the test solution under item A 6-8 μl, respectively, were placed on the same silica gel GF ₂₅₄ thin-layer plate, and ethyl acetate-butanone-methanol-formic acid-water with a volume ratio of 8:3:1:0.2:0.2 was used as the developing agent, and spread to about 3cm, Take it out, dry it with hot air, and then place it in the same developing agent to expand to 10cm, take it out, dry it with hot air, and inspect it under an ultraviolet lamp at 365 nm. Bright blue fluorescent main spot; inspected under ultraviolet light at 254nm, in the chromatogram of the test substance, the main spots of the same color appear respectively at the positions corresponding to the chromatograms of the hesperidin reference substance and the orange-red reference medicinal material; then spray with volume The mixed solution of 3% phosphomolybdic acid ethanol solution and 10% sulfuric acid ethanol solution mixed in a ratio of 1:1, heated at 105 ° C until the spots are clear, placed in a dark room, and inspected through light. On the corresponding positions of the gastrodia elata control material chromatogram, the main spots of the same color were respectively displayed. 2. A kind of fast multi-information thin-layer identification method for freeze-dried powder of Banxia Baizhu Tianma Decoction according to claim 1, characterized in that every 1 g of said freeze-dried powder is equivalent to 2.4-2.8g of native medicinal materials.

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