A quality control method for an anti-inflammatory and analgesic composition

Abstract

The application discloses a quality control method for a swelling and pain relieving composition, which comprises the following steps: A, preparing a receiving side solution; B, using a YUS chamber to prepare a test sample solution, fixing rat skin in a 0.5 cm2 diffusion chamber, with the outer epidermis side as a drug administration side, adding a swelling and pain relieving extract to the drug administration side container, with the inner epidermis side as a receiving side, adding a receiving liquid to the receiving side container to two-thirds of the container, finally covering the top of the two containers with a fresh-keeping film, opening a constant temperature water bath to maintain a constant temperature of 37.5 DEG C, waiting for 36-48 hours, passing through a 0.22 mu m microporous filter membrane, taking the filtrate, and obtaining the test sample solution; C, using a liquid chromatography program to separate and then detecting by a UV detector. The method relies on a high performance liquid chromatograph, extracts transdermal components through a YUS chamber through an isolated rat skin, and forms a new type of quality control method for a swelling and pain relieving composition.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical quality control, specifically to a method for quality control of an anti-inflammatory and analgesic composition. Background Technology

[0002] The swelling-reducing and analgesic tincture, a representative example, is composed of the following medicinal herbs: Costus root, Saposhnikovia root, Schizonepeta root, Asarum root, Acanthopanax bark, Cinnamon twig, Achyranthes root, Ligusticum root, Cynanchum root, Angelica root, Curcuma rhizome, Eucommia bark, Ulmus pumila root, Ulmus pumila root, Zanthoxylum nitidum root, Pterocarya rhizome, Gardenia fruit, Sparganium rhizome, Agarwood, Camphor, and Menthol. This swelling-reducing and analgesic tincture is a traditional Chinese medicine product manufactured by Guangxi Zhuang Autonomous Region Huahong Pharmaceutical Group Co., Ltd. It is for external use and has the effects of relaxing muscles and promoting blood circulation, reducing swelling and relieving pain. It is mainly used to treat traumatic injuries, rheumatic bone pain, boils of unknown origin, and swelling and pain from mumps.

[0003] Current research on anti-inflammatory and analgesic tinctures mainly focuses on the determination of aristolochic acid A using liquid chromatography-mass spectrometry, the determination of palmatine hydrochloride using high-performance liquid chromatography, and the determination of camphor and menthol content using capillary gas chromatography. Anti-inflammatory and analgesic tinctures can be applied topically to the affected area or taken orally; however, research on their transdermal absorption is very limited. Therefore, this experiment aims to study the anti-inflammatory and analgesic tincture through in vitro transdermal experiments, providing a new method for quality control. Summary of the Invention

[0004] The purpose of this invention is to provide a quality control method for anti-inflammatory and analgesic compositions. This method utilizes high-performance liquid chromatography (HPLC) to extract components transdermally from isolated rat skin via a Uss chamber, thus forming a novel quality control method for anti-inflammatory and analgesic compositions.

[0005] The technical solution of the present invention is as follows:

[0006] The quality control method for the aforementioned anti-inflammatory and analgesic composition includes the following steps:

[0007] A. Prepare a 0.9% physiological saline solution by weight. Add PEG400 to the 0.9% physiological saline solution and dissolve it completely to obtain the receiver-side solution for later use. The weight ratio of physiological saline solution to PEG400 is 4-6:1.

[0008] B. The test solution was prepared using a Uss chamber, with rat skin fixed at 0.5 cm. 2 In the diffusion chamber, the outer epidermal side is the drug delivery side, and an anti-inflammatory and analgesic extract is added to the drug delivery side chamber; the inner epidermal side is the receiving side, and receiving liquid is added to the receiving side chamber until it is two-thirds full. Finally, plastic wrap is placed over the top of both chambers, and a constant temperature water bath is turned on to maintain a constant temperature of 37.5°C. After 36-48 hours, the solution is filtered through a 0.22μm microporous membrane, and the filtrate is collected to obtain the test solution.

[0009] C. Separate using liquid chromatography with programmed temperature increase, and then detect using an ultraviolet detector;

[0010] The liquid chromatography conditions are as follows:

[0011] The chromatographic column is a C18 column;

[0012] The mobile phase was acetonitrile-0.1% acetic acid water, with gradient elution: 0–2 min, 5% acetonitrile; 2–4 min, 5%–10% acetonitrile; 4–8 min, 10%–30% acetonitrile; 8–26 min, 30%–95% acetonitrile; 26–30 min, 95%–95% acetonitrile.

[0013] Volumetric flow rate: 0.1-0.5 mL / min;

[0014] Column temperature 35-45℃;

[0015] The detection wavelength is 250-270nm.

[0016] Preferably, the liquid chromatography conditions are as follows:

[0017] The chromatographic column is a C18 column;

[0018] The mobile phase was acetonitrile-0.1% acetic acid water, with gradient elution: 0–2 min, 5% acetonitrile; 2–4 min, 5%–10% acetonitrile; 4–8 min, 10%–30% acetonitrile; 8–26 min, 30%–95% acetonitrile; 26–30 min, 95%–95% acetonitrile.

[0019] Volumetric flow rate: 0.3 mL / min;

[0020] Column temperature 40.0℃;

[0021] Detection wavelength: 262nm.

[0022] Preferably, in the liquid chromatography conditions, the C18 column has the following specifications: 150 mm × 3.0 mm, 3 μm.

[0023] The swelling-reducing and pain-relieving composition is made from the following medicinal materials: costus root, saposhnikovia root, schizonepeta, asarum root, eleutherococcus bark, cinnamon twig, achyranthes root, chuanxiong rhizome, cynanchum paniculatum, angelica root, turmeric root, red eucommia bark, large umbellatus, small umbellatus, zephyranthes root, scutellaria barbata, scutellaria barbata, sparganium rhizome, gardenia fruit, sparganium rhizome, agarwood, camphor, and menthol.

[0024] The method for preparing the swelling-reducing and analgesic extract includes the following steps:

[0025] Take all the medicinal materials except camphor and menthol, extract them with alcohol, then add camphor and menthol and mix well to obtain the final product.

[0026] When the composition is prepared into an anti-inflammatory and analgesic tincture, in step B, 5 ml of the anti-inflammatory and analgesic tincture is added to the administration chamber.

[0027] The chromatogram detected in step C should contain chromatographic peaks of the following components:

[0028] Gamma-aminobutyric acid, psoralen, propyl gallate, methyl furoate, DL-mannitol, phytohexene, 3,4-dimethoxyphenol, schizonepetoside E_qt, 4-hydroxybenzoic acid, genipin, terbinafine, styrene, vanillic acid, baicalin, o-hydroxycinnamic acid, allocinnamic acid, kaempferol, oleanolic acid, azelaic acid, isopsoralen), MKN-004C, α-estradiol, estriol, eugenol, dibutyl phthalate, xanthocyanin, 1-palmitoyl monoglyceride, 4,5-Di-O-caffeoylquinic acid, R-(+)-1-phenyl-1-propanol, 2-hydroxy-4-methylbenzaldehyde, 2-methoxy-4-vinylphenol, phenylpropynic acid, p-methoxybenzoic acid, asarone, genipin, 3,4-dihydroxyphenyl-2-propanone, 2,6-dimethoxy-1,4-benzoquinone, maleic acid, 5-hydroxymethylfurfural.

[0029] The chromatogram detected in step C should contain chromatographic peaks of the following components:

[0030]

[0031]

[0032]

[0033] The beneficial effects of this invention are as follows:

[0034] This invention establishes a novel method for detecting anti-inflammatory and analgesic compositions. This method is beneficial for further monitoring the stability of the production process and ensuring the stability, uniformity, and controllability of its quality.

[0035] This invention improves the quality control standards for anti-inflammatory and analgesic compositions, especially anti-inflammatory and analgesic tinctures, effectively preventing product counterfeiting and ensuring the normal production and distribution order of this product.

[0036] The method of the present invention has good feasibility, stability and reproducibility. Attached Figure Description

[0037] Figure 1 High-performance liquid chromatography (HPLC) chromatogram of the anti-inflammatory and analgesic tincture 1;

[0038] Figure 2 High-performance liquid chromatography (HPLC) chromatogram of the anti-inflammatory and analgesic tincture 2. Detailed Implementation

[0039] The present invention will be further illustrated below by way of examples. It should be understood that the examples of the present invention are for illustrative purposes and not for limiting the present invention.

[0040] Example 1

[0041] A method for quality control of an anti-inflammatory and analgesic composition includes the following steps:

[0042] A. Prepare a 0.9% physiological saline solution by weight. Add PEG400 to the 0.9% physiological saline solution and dissolve it completely to obtain the receiver-side solution for later use. The physiological saline solution and PEG400 are mixed in a weight ratio of 4:1.

[0043] B. The test solution was prepared using a Uss chamber, with rat skin fixed at 0.5 cm. 2 In the diffusion chamber, the outer epidermal side is the drug delivery side, and an anti-inflammatory and analgesic extract is added to the drug delivery side chamber; the inner epidermal side is the receiving side, and receiving liquid is added to the receiving side chamber until it is two-thirds full. Finally, plastic wrap is placed over the top of both chambers, and a constant temperature water bath is turned on to maintain a constant temperature of 37.5°C. After 40 hours, the solution is filtered through a 0.22μm microporous membrane, and the filtrate is collected to obtain the test solution.

[0044] C. Separate using liquid chromatography with programmed temperature increase, and then detect using an ultraviolet detector;

[0045] The liquid chromatography conditions are as follows:

[0046] The chromatographic column was a C18 column, 150 mm × 3.0 mm, 3 μm;

[0047] The mobile phase was acetonitrile-0.1% acetic acid water, with gradient elution: 0–2 min, 5% acetonitrile; 2–4 min, 5%–10% acetonitrile; 4–8 min, 10%–30% acetonitrile; 8–26 min, 30%–95% acetonitrile; 26–30 min, 95%–95% acetonitrile.

[0048] Volumetric flow rate: 0.1 mL / min;

[0049] Column temperature 35℃;

[0050] The detection wavelength is 250nm.

[0051] The swelling-reducing and pain-relieving composition is made from the following medicinal materials: costus root, saposhnikovia root, schizonepeta, asarum root, eleutherococcus bark, cinnamon twig, achyranthes root, chuanxiong rhizome, cynanchum paniculatum, angelica root, turmeric root, red eucommia bark, large umbellatus, small umbellatus, zephyranthes root, scutellaria barbata, scutellaria barbata, sparganium rhizome, gardenia fruit, sparganium rhizome, agarwood, camphor, and menthol.

[0052] The method for preparing the swelling-reducing and analgesic extract includes the following steps:

[0053] Take all the medicinal materials except camphor and menthol, extract them with alcohol, then add camphor and menthol and mix well to obtain the final product.

[0054] Example 2

[0055] A method for quality control of an anti-inflammatory and analgesic composition includes the following steps:

[0056] A. Prepare a 0.9% physiological saline solution by weight. Add PEG400 to the 0.9% physiological saline solution and dissolve it completely to obtain the receiver-side solution for later use. The physiological saline solution and PEG400 are mixed in a weight ratio of 6:1.

[0057] B. The test solution was prepared using a Uss chamber, with rat skin fixed at 0.5 cm. 2 In the diffusion chamber, the outer epidermal side is the drug delivery side, and an anti-inflammatory and analgesic extract is added to the drug delivery side chamber; the inner epidermal side is the receiving side, and receiving liquid is added to the receiving side chamber until it is two-thirds full. Finally, plastic wrap is placed over the top of both chambers, and a constant temperature water bath is turned on to maintain a constant temperature of 37.5°C. After 48 hours, the solution is filtered through a 0.22μm microporous membrane, and the filtrate is collected to obtain the test solution.

[0058] C. Separation was performed using liquid chromatography with a temperature ramping program, followed by detection using a UV detector. The chromatogram of the anti-inflammatory and analgesic tincture sample showed high absorption at 3.25 min, 8.91 min, 9.11 min, 10.99 min, 11.32 min, 11.19 min, and 17.83 min. This indicates that among the components of the anti-inflammatory and analgesic tincture, highly polar components are slightly more abundant, while less polar components are fewer, but some individual components have higher concentrations, as follows. Figure 1 As shown.

[0059] The liquid chromatography conditions are as follows:

[0060] The chromatographic column was a C18 column, 150 mm × 3.0 mm, 3 μm;

[0061] The mobile phase was acetonitrile-0.1% acetic acid water, with gradient elution: 0–2 min, 5% acetonitrile; 2–4 min, 5%–10% acetonitrile; 4–8 min, 10%–30% acetonitrile; 8–26 min, 30%–95% acetonitrile; 26–30 min, 95%–95% acetonitrile.

[0062] Volumetric flow rate: 0.5 mL / min;

[0063] Column temperature 45℃;

[0064] The detection wavelength is 270nm.

[0065] The swelling-reducing and pain-relieving composition is made from the following medicinal materials: costus root, saposhnikovia root, schizonepeta, asarum root, eleutherococcus bark, cinnamon twig, achyranthes root, chuanxiong rhizome, cynanchum paniculatum, angelica root, turmeric root, red eucommia bark, large umbellatus, small umbellatus, zephyranthes root, scutellaria barbata, scutellaria barbata, sparganium rhizome, gardenia fruit, sparganium rhizome, agarwood, camphor, and menthol.

[0066] The composition is prepared into an anti-inflammatory and analgesic tincture. In step B, 5 ml of the anti-inflammatory and analgesic tincture is added to the administration chamber.

[0067] High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect the separated components. The mass spectrometry conditions were: electrospray ionization (ESI) source, positive and negative ion modes, scan range m / z 100–1000; collision energy 35 e V; ion transfer tube temperature 320 °C; vaporizer temperature 350 °C; positive and negative ion spray voltages 3500 V and 3000 V, respectively.

[0068] After mass spectrometry analysis, the detected chromatogram contained the following components:

[0069]

[0070]

[0071] Example 3

[0072] A method for quality control of an anti-inflammatory and analgesic composition includes the following steps:

[0073] A. Prepare a 0.9% physiological saline solution by weight. Add PEG400 to the 0.9% physiological saline solution and dissolve it completely to obtain the receiver-side solution for later use. The physiological saline solution and PEG400 are mixed in a weight ratio of 5:1.

[0074] B. The test solution was prepared using a Uss chamber. Rat skin was fixed in a 0.5 cm² diffusion chamber, with the outer epidermal side as the administration side. An anti-inflammatory and analgesic extract was added to the administration side chamber. The inner epidermal side was the receiving side. A receiving solution was added to the receiving side chamber until it was two-thirds full. Finally, plastic wrap was placed over the top of both chambers, and a constant temperature water bath was turned on to maintain the temperature at 37.5°C. After 36 hours, the solution was filtered through a 0.22 μm microporous membrane, and the filtrate was collected to obtain the test solution.

[0075] C. Separation was performed using liquid chromatography with a temperature ramping program, followed by detection using a UV detector. The chromatogram of the anti-inflammatory and analgesic tincture sample showed high absorption at 3.25 min, 8.91 min, 9.11 min, 10.99 min, 11.32 min, 11.19 min, and 17.83 min. This indicates that among the components of the anti-inflammatory and analgesic tincture, highly polar components are slightly more abundant, while less polar components are fewer, but some individual components have higher concentrations, as follows. Figure 2 As shown.

[0076] The liquid chromatography conditions are as follows:

[0077] The chromatographic column was a C18 column, 150 mm × 3.0 mm, 3 μm;

[0078] The mobile phase was acetonitrile-0.1% acetic acid water, with gradient elution: 0–2 min, 5% acetonitrile; 2–4 min, 5%–10% acetonitrile; 4–8 min, 10%–30% acetonitrile; 8–26 min, 30%–95% acetonitrile; 26–30 min, 95%–95% acetonitrile.

[0079] Volumetric flow rate: 0.3 mL / min;

[0080] Column temperature 40.0℃;

[0081] Detection wavelength: 262nm.

[0082] The swelling-reducing and pain-relieving composition is made from the following medicinal materials: costus root, saposhnikovia root, schizonepeta, asarum root, eleutherococcus bark, cinnamon twig, achyranthes root, chuanxiong rhizome, cynanchum paniculatum, angelica root, turmeric root, red eucommia bark, large umbellatus, small umbellatus, zephyranthes root, scutellaria barbata, scutellaria barbata, sparganium rhizome, gardenia fruit, sparganium rhizome, agarwood, camphor, and menthol.

[0083] The composition is prepared into an anti-inflammatory and analgesic tincture. In step B, 5 ml of the anti-inflammatory and analgesic tincture is added to the administration chamber.

[0084] High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect the separated components. The mass spectrometry conditions were: electrospray ionization (ESI) source, positive and negative ion modes, scan range m / z 100–1000; collision energy 35 eV; ion transfer tube temperature 320 °C; vaporizer temperature 350 °C; positive and negative ion spray voltages 3500 V and 3000 V, respectively.

[0085] After mass spectrometry analysis, the detected chromatogram contained the following components:

[0086]

[0087]

[0088] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting an anti-inflammatory analgesic composition, characterized by, It comprises the following steps: A. Prepare 0.9% physiological saline by weight, add PEG400 to 0.9% physiological saline, dissolve thoroughly, prepare the receiving side solution, and reserve; wherein the weight ratio of physiological saline and PEG400 is 4-6:1; B, Yuse chamber was used to prepare the test solution, the rat skin was fixed in 0.5cm 2 In the diffusion chamber, the outer epidermis side was the administration side, and the anti-inflammatory extract was added to the administration side chamber. The inner epidermis side was the receiving side, and the receiving liquid was added to the receiving side chamber to two-thirds of the chamber. Finally, the fresh-keeping film was covered on the top of the chambers, the constant temperature water bath was opened to maintain 37.5℃, and 36-48 hours were waited. The 0.22μm micropore filter membrane was used to filter, the filtrate was taken, and the test solution was obtained. C. Separate by liquid chromatography program temperature, and then detect by ultraviolet detector; The liquid chromatography conditions are: The chromatographic column is C18 chromatographic column; The mobile phase is acetonitrile-0.1% acetic acid water, gradient elution: 0-2 min, 5% acetonitrile; 2-4 min, 5%-10% acetonitrile; 4-8 min, 10%-30% acetonitrile; 8-26 min, 30%-95% acetonitrile; 26-30 min, 95%-95% acetonitrile; The volume flow rate is 0.1-0.5 m L / min; The column temperature is 35-45℃; The detection wavelength is 250-270 nm; The swelling and pain relieving composition is made of the following medicinal materials: costus root, siler, schizonepeta, asarum, acanthopanax, cassia twig, radix anemarrhenae, chuanxiong, xuchangqing, baizhi, zedoary turmeric, red eucommia, large umbrella, small umbrella, two-edged needle, yellow bamboo, gardenia, sparganium, agarwood, camphor and menthol; The swelling and pain relieving extract preparation method comprises the following steps: Take all the medicinal materials except camphor and menthol, alcohol extract, then add camphor and menthol, mix evenly, and get it; The chromatogram detected in step C should contain the following components: Gamma-aminobutyric acid, psoralen, propyl gallate, 2-furoic acid methyl ester, DL-mannitol, folikalin, 3,4-dimethoxyphenol, schizonepetoside E_qt, 4-hydroxybenzoic acid, geniposidic acid, terrein, styrene, vanillic acid, baicalin, o-hydroxycinnamic acid, another cinnamic acid, kaempferol, oleanolic acid, azelaic acid, isopsoralen, MKN-004C, alpha-estradiol, estriol, eugenol, dibutyl phthalate, zanthoxylum toxicol, 1-palmitic acid monoglyceride, 4,5-Di-O-caffeoylquinic acid, R-(+)-1-phenyl-1-propanol, 2-hydroxy-4-methylbenzaldehyde, 2-methoxy-4-vinylphenol, phenylpropynic acid, p-methoxybenzoic acid, asarone, geniposide, 3,4-dihydroxyphenyl-2-propanone, 2,6-dimethoxy-1,4-benzoquinone, maleic acid and 5-hydroxymethylfurfural.

2. The swelling and pain relieving composition detection method of claim 1, wherein: The liquid chromatography conditions are: The chromatographic column is C18 chromatographic column; The mobile phase is acetonitrile-0.1% acetic acid water, gradient elution: 0-2 min, 5% acetonitrile; 2-4 min, 5%-10% acetonitrile; 4-8 min, 10%-30% acetonitrile; 8-26 min, 30%-95% acetonitrile; 26-30 min, 95%-95% acetonitrile; The volume flow rate is 0.3 m L / min; The column temperature is 40.0℃; The detection wavelength is 262 nm.

3. The method for detecting the anti-inflammatory and analgesic composition as described in claim 1, characterized in that: The liquid chromatography condition is: C18 chromatographic column specification is 150 mm*3.0 mm, 3 μm.

4. The detection method of the anti-inflammatory and analgesic composition according to claim 1, wherein the composition is prepared into an anti-inflammatory and analgesic tincture. In step B, 5 ml of the anti-inflammatory and analgesic tincture is added to the administration side chamber.

5. The detection method of the anti-inflammatory and analgesic composition according to claim 1, wherein the composition is prepared into an anti-inflammatory and analgesic tincture. In step C, the chromatogram detected should contain the following components: 。

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