CN105136966A - Quality detection method for Liangfu pill-like preparations - Google Patents

Abstract

The invention discloses a quality detection method for Liangfu (of which the main compositions are rhizoma alpiniae officinarum and rhizoma cyperi) pill-like preparations. The method is a thin-layer chromatography method, and detection is performed by taking alpha-cyperone, cyperotundone, nootkatone, galangin and kaempferol as index components. The expansion conditions of the thin-layer chromatography comprise petroleum ether and ethyl acetate with the volume ratio of 5:1, and the thin-layer plate is silica gel GF254 plate. The quality detection method is capable of simultaneously detecting five index components in the Liangfu pill-like preparations, the environment adaptability is high, the method is simple, separation of the index components is good, and effective guarantee is provided for comprehensive detection on the quality of the Liangfu pill-like preparations.

Description

A kind of quality detection method of Liangfu pill preparation

technical field

The invention relates to the technical field of drug quality control methods, in particular to a quality detection method for Liangfu pill preparations.

Background technique

Liangfu pills were first recorded in "Liangfang Jixiong" written by Xie Yuanqing in the Qing Dynasty. The representative preparation of TCM has a history of nearly a thousand years in clinical application of traditional Chinese medicine. As a typical traditional Chinese medicine compound preparation, Liangfu Pills has a fixed medicinal composition, simple preparation process, remarkable clinical curative effect and great potential for development.

As a compound preparation of traditional Chinese medicine, Liangfu pill has the characteristics of multi-component, multi-target, multi-level, and overall effect. However, in the first part of the Chinese Pharmacopoeia 2010 edition, only α-cyperone is used as the index component of Liangfu pill, so it is difficult to accurately , comprehensively reflect the quality of the preparation, let alone its function and indications.

Therefore, there is a need for a detection method that can fully reflect the quality of Liangfu pill preparations.

Contents of the invention

The inventors have found through research that α-cyperone, cyperenone, and narone can be used as index components of cyperone, and galangin and kaempferol can be used as index components of galangal. Therefore, the present invention uses α-cyperone, cyperenone, citron, galangin, and kaempferin as index components for quality detection.

Therefore, in order to solve the above problems, the invention provides a kind of quality detection method of Liangfu pill preparation, and this method is thin-layer chromatography method, and it comprises the following steps:

(1) get α-cyperone, cyperenone, citron, galangin, kaempferin reference substance, mix, add methanol and be mixed with reference substance solution;

(2) Spot the reference substance solution on the thin-layer plate, after the developer is developed, take it out for inspection, and obtain the Rf of α-cyperone, cyperenone, citron, galangin and kaempferol Value; The developer is petroleum ether-ethyl acetate with a volume ratio of 5:1; the thin-layer plate is a silica gel GF ₂₅₄ plate;

(3) Take Liangfu pill preparation or its powder, add methanol to extract, and filter;

(4) evaporate the filtrate gained in step (3) to dryness, add methanol to dissolve the residue, as need testing solution;

(5) detect according to the thin-layer chromatography method of step (2), the Rf value of each spot on the thin-layer plate is compared with α-cyperone, cyperenone, citron, galangin, kaempferol By comparing the Rf value of the product, we can know the quality of Liangfu pill preparations.

Further preferably, in step (2), the unfolding temperature is 4-30°C.

Further preferably, in step (2), the unfolding humidity is 32%-88%.

Further preferably, in step (2), the applied sample volume is 2-10 μL, preferably 4 μL.

Further preferably, in step (2), the inspection method is: inspect the thin-layer board under a 254nm ultraviolet lamp; or, spray sulfuric acid ethanol solution on the thin-layer board to develop color, heat until the spot color is obvious, and put it under ultraviolet light View under lamp 365nm.

Further preferably, the concentration of the sulfuric acid ethanol solution is 10%.

Further preferably, the concentration of the reference substance solution is 1 mg/mL.

Further preferably, the extraction in step (3) is ultrasonic extraction.

Further preferably, the ultrasonic extraction time is 30 minutes.

Further preferably, in step (3), the volume-to-weight ratio of the methanol and Liangfu pill preparation or its powder is 25:1mL/g; in step (4), the methanol and Liangfu pill preparation The volume-to-weight ratio of its powder is 1:1 mL/g.

Further preferably, the Liangfu pill preparations include Liangfu pills, Liangfu dropping pills or Liangfu soft capsules.

The quality detection method of the present invention can simultaneously detect 5 kinds of index components (α-cyperone, cyperenone, citron, galangin and kaempferol) in Liangfu pill preparations, which are far greater than In the Chinese Pharmacopoeia 2010 edition, only α-cyperone is used as the detection method of the indicator component of Liangfu pills. Simultaneously, the method of the invention is simple and convenient, has high environmental adaptability and good separation of each index component, and provides an effective guarantee for comprehensively testing the quality of Liangfu pill preparations.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

In the thin-layer chromatogram of the reference substance in the accompanying drawings, the spots from top to bottom are α-cyperone, cyperenone, citron, galangin, and kaempferol in turn.

Fig. 1 is the thin-layer chromatogram of the inventive method in embodiment 1 under ultraviolet lamp 254nm: 1 is reference substance, and 2-5 is Liangfu pill sample.

Fig. 2 is the thin-layer chromatogram of the inventive method in embodiment 1 under the ultraviolet lamp 365nm: 1 is reference substance, and 2-5 is Liangfu pill sample.

Fig. 3 is the thin-layer chromatogram of comparative example 1 method in the embodiment 1 under the ultraviolet lamp 254nm: 1 is the reference substance, 2-4 is the Liangfu pill sample.

Fig. 4 is the thin-layer chromatogram of comparative example 1 method in embodiment 1 under the ultraviolet lamp 365nm: 1 is reference substance, 2-4 is Liangfu pill sample.

Fig. 5 is the thin-layer chromatogram of the comparative example 2 method in the embodiment 1 under the ultraviolet lamp 254nm: 1 is the reference substance, 2-4 is the Liangfu pill sample.

Fig. 6 is the thin-layer chromatogram of the comparative example 2 method in the embodiment 1 under the ultraviolet lamp 365nm: 1 is the reference substance, 2-4 is the Liangfu pill sample.

Fig. 7 is the thin-layer chromatogram of the comparative example 2 method in the embodiment 1 under the ultraviolet lamp 254nm: 1 is the reference substance, 2-4 is the Liangfu pill sample.

Fig. 8 is the thin-layer chromatogram of the comparative example 2 method in the embodiment 1 under the ultraviolet lamp 365nm: 1 is the reference substance, 2-4 is the Liangfu pill sample.

Fig. 9 is the thin-layer chromatogram of the extraction method investigation test in Example 2 under the ultraviolet lamp 254nm: 1 is the reference substance, 2 is the ultrasonic extraction method, and 3 is the reflux extraction method.

Fig. 10 is the thin-layer chromatogram of the extraction method investigation test in Example 2 under the ultraviolet lamp 365nm: 1 is the reference substance, 2 is the ultrasonic extraction method, and 3 is the reflux extraction method.

Fig. 11 is the thin-layer chromatogram of the sample volume investigation test in Example 3 under the ultraviolet lamp 254nm: the sample volumes of 1-6 are 1, 2, 4, 6, 8, and 10 μL in sequence.

Fig. 12 is the thin-layer chromatogram of the sample volume investigation test in Example 3 under the ultraviolet lamp 365nm: the sample volumes of 1-6 are 1, 2, 4, 6, 8, and 10 μL in sequence.

Figure 13-16 The thin layer chromatograms of the TLC investigation test in Example 4 under the ultraviolet lamp 365nm: the TLC plates in Figure 9-12 are Qingdao Haiyang, Qingdao Hailang, Qingdao Yuminyuan, Tianjin Silida 1 is the reference substance, and 2-5 are samples of Liangfu pills.

Figures 17 and 18 are respectively the thin-layer chromatograms of the development temperature investigation test in Example 4 under the ultraviolet lamp at 365nm: Figure 13 is at 4°C, Figure 14 is at 30°C, 1 is the reference substance, and 2-5 are Liangfu pills sample.

Fig. 19 is the thin-layer chromatogram of each preparation under the embodiment 4 under the ultraviolet lamp 254nm: 1 is the reference substance, 2 is the reference medicinal material of galanga, 3 is the reference medicinal material of Cyperus cyperi, 4-9 is the sample of Liangfu pills, 10 -12 is Liangfu dropping pill, and 13-15 is Liangfu soft capsule.

Fig. 20 is the thin-layer chromatogram of each preparation under the ultraviolet lamp 365nm under embodiment 4: 1 is reference substance, 2 is galanga reference medicinal material, 3 is Cyperus cyperi reference medicinal material, 4-9 is Liangfu pill sample, 10 -12 is Liangfu dropping pill, and 13-15 is Liangfu soft capsule.

Detailed ways

The source of raw material, instrument and reagent in the specific embodiment of the present invention is as follows:

KQ5200DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); silica gel G thin-layer board (Qingdao Ocean Chemical Factory, self-made silica gel G board).

Liangfu Pills (Beijing Tongrentang Pharmaceutical Co., Ltd.), Liangfu Soft Capsules (Tianjin Pacific Pharmaceutical Co., Ltd.), Liangfu Dropping Pills (Guizhou Huangguo Lishuang Pharmaceutical Co., Ltd.); Galangin (China Institute for the Control of Pharmaceutical and Biological Products, 111699 -200501), kaempferone (Chengdu Mansite Biotechnology Co., Ltd., must-12020812), narone (US Sigma Company, 10112423), α-cyperone (Jiangxi Bencao Tiangong Technology Co., Ltd., 1443- 080529), cyperenone (self-made), Cyperus reference medicinal materials (National Institute for the Control of Pharmaceutical and Biological Products, 120918-200809), galangal reference medicinal materials (National Institute for the Control of Pharmaceutical and Biological Products, 1263-0301).

Petroleum ether (60-90°C), ethyl acetate, cyclohexane, acetone, and methanol were all analytically pure, provided by Chengdu Kelong Chemical Reagent Factory.

Example 1

1. Preparation of reference substance solution: Take an appropriate amount of reference substance of galangin, kaempferone, citronone, α-cyperone, and cyperenone, and add methanol to make a solution containing 1 mg per 1 mL, as the reference substance solution .

2. Preparation of the test solution: Take 1 g of Liangfu pill powder, add 25 mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test solution.

3. TLC conditions:

Developing agent: petroleum ether (60-90°C)-ethyl acetate (5:1);

Thin layer board: silica gel GF ₂₅₄ board;

Sample volume: 4 μL each for the test solution and the reference solution;

The temperature was 22°C and the relative humidity was 58%.

4. Inspection: inspect under the ultraviolet lamp at 254nm, the results are shown in Figure 1, the spots of each index component are obvious and well separated; then spray with 10% sulfuric acid ethanol for color development, heat at 105°C until the spot color is obvious, and place the ultraviolet lamp at 365nm Under the inspection, the results are shown in Figure 2. The spots of each index component are obvious and well separated.

Comparative example 1

1. Preparation of the reference substance solution: take an appropriate amount of reference substances of galangin, kaempferone, citronone, α-cyperone, and cyperenone, and add methanol to make a solution containing 1 mg per 1 mL as the reference substance solution .

2. Preparation of the test solution: Take 1 g of Liangfu pill powder, add 25 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test solution.

3. TLC conditions:

Developing agent: cyclohexane-acetone (3:1);

Thin layer board: silica gel GF ₂₅₄ board;

Sample volume: 4 μl each of the test solution and the reference solution;

The temperature was 22°C and the relative humidity was 58%.

4. Inspection: put it under the ultraviolet lamp (254nm) to inspect, the results are shown in Figure 3, the spots of each index component are not obvious, and the separation is poor; then spray with 10% sulfuric acid ethanol for color development, and heat at 105°C until the spots are clearly colored. Put under the ultraviolet lamp (365nm) to examine, the result is shown in Figure 4, each index component, the spot is not obvious, and separation is poor.

Comparative example 2

According to the method of Example 1, the difference is that the developer is petroleum ether-ethyl acetate=3:1, and other conditions remain unchanged. The results are shown in Figures 5 and 6.

It can be seen that under this condition, α-cyperone, cyperenone, and narone were not successfully separated.

Comparative example 3

According to the method of Example 1, the difference is that the developer is petroleum ether-ethyl acetate=7:1, and other conditions remain unchanged, the results are shown in Figure 7 and Figure 8.

It can be seen that under this condition, the span of galangin and kaempferol is too small to be completely separated.

It can be seen from the comparison that, compared with the method of the comparative example, the method of the present invention has moderate Rf values of the five components to be detected, clear main color spots and better separation, high sensitivity, stable method and good reproducibility.

Embodiment 2 extraction method investigation

Take 1g of Liangfu pill powder, add 25mL of methanol, investigate the two extraction methods of reflux extraction and ultrasonic extraction respectively, the extraction time is 30min, filter, the filtrate, evaporate to dryness, add 1mL of methanol to the residue to dissolve, as the test solution, TLC Chromatographic conditions and inspection methods are the same as in Example 1, and the results are shown in Figures 9 and 10.

The results show that the main spots are clearer in both extraction methods. Because the operation of the ultrasonic extraction method is simpler and more convenient, the preferred ultrasonic extraction method is used as the preparation method of the test sample in the present invention.

Example 3 point sample volume investigation

According to the method of Example 1, the same silica gel GF254 thin-layer plate was taken, and the sample volumes of 1, 2, 4, 6, 8, and 10 μL were investigated respectively. The results are shown in Figure 11 and Figure 12.

The results showed that when the spotting volume of the test solution was 2-10 μL, the clarity and separation of the spots were better, and the optimum spotting volume was 4 μL.

Embodiment 4 durability investigation

1. Inspection of thin-layer boards from different manufacturers

According to the method of Example 1, Qingdao Hailang, Qingdao Haiyang, Qingdao Yuminyuan, and Tianjin Sleda thin-layer panels were selected, and the results are shown in Figures 13-16.

The results show that the method of the present invention has good adaptability to different brands of thin-layer boards, and can meet the identification requirements.

2. Investigation of different temperatures

According to the method of Example 1, the thin-layer plate after sampling was taken, and the relative humidity (RH) was 54%, and the temperature environment was respectively developed at 4°C and 30°C. The results are shown in Figures 17 and 18. .

The results show that the method of the present invention has good adaptability to different temperatures and can meet the identification requirements.

3 Investigation of different humidity

According to the method of Example 1, the thin-layer plate after sampling was taken, and developed under the condition of 25° C. and humidity environments of 32% and 88%, respectively.

The results show that the method of the present invention has good adaptability to different humidity and can meet the identification requirements.

4. Investigation of different batches of Liangfu pills and their preparations

Twelve batches of samples were taken, and experiments were carried out according to the method of Example 1, and the results are shown in Figures 19 and 20.

The results showed that in the chromatogram of the test product, there were fluorescent spots of the same color at the positions corresponding to the chromatograms of the reference medicinal material and the reference product. It shows that the method of the present invention has high stability and good reproducibility, and can effectively detect Liangfu pills and various reagents thereof.

In summary, the quality detection method of the present invention can simultaneously detect 5 kinds of index components (α-cyperone, cyperenone, citron, galangin and kaempferone) in Liangfu pill preparations element), which is far greater than the detection method in the Chinese Pharmacopoeia 2010 edition, which only uses α-cyperone as the index component of Liangfu pills. Simultaneously, the method of the invention is simple and convenient, has high environmental adaptability and good separation of each index component, and provides an effective guarantee for comprehensively testing the quality of Liangfu pill preparations.

Claims (10)

1. a quality detection method of Liangfu pill preparations, characterized in that: the method is a thin-layer chromatography method, and it may further comprise the steps: (1) get α-cyperone, cyperenone, citron, galangin, kaempferin reference substance, mix, add methanol and be mixed with reference substance solution; (2) Spot the reference substance solution on the thin-layer plate, after the developer is developed, take it out for inspection, and obtain the Rf of α-cyperone, cyperenone, citron, galangin and kaempferol Value; The developer is petroleum ether-ethyl acetate with a volume ratio of 5:1; the thin-layer plate is a silica gel GF ₂₅₄ plate; (3) Take Liangfu pill preparation or its powder, add methanol to extract, and filter; (4) evaporate the filtrate gained in step (3) to dryness, add methanol to dissolve the residue, as need testing solution; (5) detect according to the thin-layer chromatography method of step (2), the Rf value of each spot on the thin-layer plate is compared with α-cyperone, cyperenone, citron, galangin, kaempferol By comparing the Rf value of the product, we can know the quality of Liangfu pill preparations. 2. The quality detection method according to claim 1, characterized in that: in step (2), the unfolding temperature is 4-30°C. 3. The quality detection method according to claim 1, characterized in that: in the step (2), the unfolded humidity is 32%-88%. 4 . The quality detection method according to claim 1 , characterized in that: in step (2), the applied sample volume is 2-10 μL, preferably 4 μL. 5. The quality detection method according to claim 1, characterized in that: in step (2), the inspection method is: inspecting the thin-layer plate under a 254nm ultraviolet lamp; Or, spray sulfuric acid ethanol solution on the thin-layer plate to develop color, heat until the spot color is obvious, and inspect it under a 365nm ultraviolet lamp. 6. The quality detection method according to claim 5, characterized in that: the concentration of the sulfuric acid ethanol solution is 10%. 7. The quality detection method according to claim 1, characterized in that: the concentration of the reference substance solution is 1 mg/mL. 8. The quality detection method according to claim 1, characterized in that: the extraction in the step (3) is ultrasonic extraction. 9. according to the quality detection method described in any one of claim 1-8, it is characterized in that: in step (3), the volume-to-weight ratio of described methanol and Liangfu pill preparation or its powder is 25:1mL /g; In step (4), the volume-to-weight ratio of the methanol and the Liangfu pill preparation or its powder is 1:1mL/g. 10. The quality detection method according to claim 1, characterized in that: the Liangfu pill preparations include Liangfu pills, Liangfu dripping pills or Liangfu soft capsules.