A fingerprint characteristic map quality detection method based on stagnation of spleen and stomach theory

Abstract

The present application belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a quality detection method of a fingerprint characteristic spectrum based on the theory of stagnation and injury of the spleen and stomach. The present application directly correlates the traditional Chinese medicine theory of "stagnation and injury of the spleen and stomach" with chemical fingerprints, links quality evaluation with preparation efficacy, and solves the problem of "recognition of medicine but not of authentication". The present application selects 5 characteristic components representing main medicinal ingredients and core efficacy categories in a representative prescription, so that the spectrum is more representative in efficacy, and is superior to single index or full component spectrum. The present application ensures effective separation and stable detection of multiple types of characteristic components through targeted pretreatment and chromatographic conditions, and the method validation is sufficient and specific. The digital control characteristic spectrum and relative retention time standard established by the present application are operable, can more sensitively identify abnormal key components, and are convenient for production and supervision.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, and specifically relates to a fingerprint feature spectrum quality detection method based on the theory of spleen and stomach stagnation. Background Technology

[0002] Fingerprint technology is a key means of quality control for traditional Chinese medicine, but most of the fingerprints currently established are "chemical fingerprints" that are weakly related to the specific TCM pathogenesis theories targeted by the medicines, resulting in the limitation of "recognizing the medicine but not the certificate".

[0003] In damp-heat constipation, "stagnation damaging the spleen and stomach" manifests as dampness obstructing qi and spleen deficiency leading to impaired function. Existing quality control methods for this preparation (such as single-index component determination or universal fingerprinting) are insufficient to effectively reflect the overall material basis for its correction of the "stagnation damaging the spleen and stomach" pathogenesis, resulting in a disconnect between chemical quality control and traditional Chinese medicine efficacy. Therefore, there is an urgent need to establish a characteristic fingerprinting detection method guided by the theory of "stagnation damaging the spleen and stomach" and capable of linking the active ingredients of the preparation, in order to achieve a more scientific and accurate evaluation of its quality.

[0004] In the fingerprint feature spectrum established in this invention, all five feature components are closely related to the pathogenesis theory of "stagnation in the spleen and stomach" and the indications for the preparation. Moreover, the components are not simply superimposed, but work together to exert a comprehensive effect of strengthening the spleen and resolving dampness, promoting qi circulation and eliminating stagnation, and moistening the intestines and relieving constipation: the tangeretin derived from Citrus aurantium can regulate glucose and lipid metabolism, break up qi stagnation and eliminate accumulation, directly targeting the core pathogenesis of dampness and constipation; the atractylodes ketone derived from Atractylodes macrocephala strengthens the spleen and dries dampness, improving the root cause of spleen deficiency and dampness; the paeoniflorin derived from Paeonia lactiflora soothes the liver and relieves urgency, alleviating abdominal pain and tenesmus; the isorhodoglycoside derived from Cistanche deserticola warms the kidneys and moistens the intestines, embodying the treatment principle of moistening without harming the body's vital energy; and the arecoline derived from Areca catechu promotes qi circulation and eliminates stagnation, targeting the symptoms of dampness stagnation. The five components mentioned above are derived from the main medicinal ingredients in the formula, encompassing various compounds such as polymethoxyflavonoids, sesquiterpenes, monoterpenoid glycosides, phenylethanol glycosides, and alkaloids. Their pharmacological effects complement and synergistically enhance each other—promoting qi circulation and relieving stagnation to address the symptoms, strengthening the spleen and drying dampness to consolidate the foundation, and moistening the intestines to promote bowel movements and protect yin, collectively reflecting the overall material basis for correcting the pathogenesis of "stagnation damaging the spleen and stomach." Therefore, the fingerprint spectrum established based on this characteristic component group can more scientifically and comprehensively reflect the consistency of the preparation's quality and efficacy.

[0005] The difficulty in determining the quality of fingerprint feature spectrum analysis methods for dehumidifying and laxative compound lies in: 1. The disconnect between chemical fingerprinting and traditional Chinese medicine theory: The chromatograms established by existing methods are only the "overall picture" or "indication" of chemical components. The characteristic peaks in the chromatograms lack a clear biological connection and logical correspondence with the improvement of the specific TCM pathogenesis theory of "stagnation and damage to the spleen and stomach". The chromatograms cannot reflect the efficacy orientation of the preparation. 2. Difficulty in screening and identifying key pharmacodynamic components: The preparation is composed of multiple medicinal materials with complex chemical compositions. The core challenge of this method is to scientifically screen and identify a group of characteristic components that can collectively embody the overall effects of "strengthening the spleen and resolving dampness, promoting qi circulation and relieving stagnation" from a massive number of compounds, and that are directly related to the improvement of the pathogenesis of "stagnation damaging the spleen and stomach," rather than randomly or empirically selecting a few components. 3. Balance between specificity and universality of analytical methods: A dedicated chromatographic method needs to be established that can effectively separate and detect the aforementioned diverse characteristic components (such as polymethoxyflavones, sesquiterpenes, monoterpenoid glycosides, phenylethanol glycosides, alkaloids, etc.) while eliminating interference from complex matrices. Simultaneously, the method must possess good stability and reproducibility to facilitate standardized application under different instrument, personnel, and laboratory conditions. Summary of the Invention

[0006] The purpose of this invention is to provide a fingerprint feature spectrum quality detection method based on the theory of spleen and stomach stagnation, and to improve the efficacy and safety of traditional Chinese medicine preparations in clinical applications by establishing quality control standards.

[0007] This invention relates to a quality control method for a traditional Chinese medicine preparation for treating damp-heat constipation using the principle of resolving dampness and promoting bowel movement. The preparation is composed of the following ingredients: raw immature bitter orange, raw atractylodes macrocephala, raw white peony root, trichosanthes fruit, cistanche deserticola, areca nut, lindera root, and hemp seed. Its main functions are to invigorate the spleen and resolve dampness, promote qi circulation and relieve stagnation, and moisten the intestines to relieve constipation. It is used for functional constipation (damp-heat constipation) of the damp-heat obstruction and qi stagnation type, characterized by incomplete bowel movements, sticky stools, rectal prolapse, and incomplete defecation, accompanied by abdominal distension, fatigue, heaviness in the body, bad breath, enlarged tongue with teeth marks, and regulation of glucose, lipid, and uric acid metabolism. It shows good efficacy for damp-heat constipation in patients with metabolic diseases.

[0008] One of the applicable preparations is a detoxifying and laxative compound, with the following formula: 100g of Citrus aurantium, 120g of Atractylodes macrocephala, 100g of Paeonia lactiflora, 150g of Trichosanthes kirilowii, 100g of Cistanche deserticola, 100g of Areca catechu, 100g of Lindera strychnifolia, and 100g of Cannabis sativa seed, to be prepared into 1000mL. The preparation method involves decocting the above eight ingredients twice with water, the first time for 2 hours and the second time for 1.5 hours. The decoctions are combined, the filtrate is concentrated to an appropriate volume, and 0.5-1.0g of steviol glycosides and 0.5g of ethylparaben (ethylparaben needs to be dissolved in 2-3ml of ethanol first) are added. Water is added to make 1000mL, which is then filled, sealed, and sterilized.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Based on the theory of spleen and stomach stagnation, the core active ingredients in the prescription are identified: (I) The core logic of the pathogenesis of spleen and stomach stagnation and its chemical composition Stagnant injury of the spleen and stomach is the core pathogenesis of digestive system syndromes such as food retention, fullness and distension in the epigastrium and abdomen, loss of appetite, and acid regurgitation. Its essence is the pathological chain of damp obstruction and qi stagnation → spleen deficiency with dampness exuberance → intestinal dryness and fluid depletion. This method breaks through the limitation of the traditional "chemical fingerprint without theoretical support", screens efficacy components guided by the pathogenesis of traditional Chinese medicine, and enables each characteristic peak of the fingerprint to correspond to the improvement effect of the pathological link of stagnant injury of the spleen and stomach, achieving "recognizing drugs more by syndrome and quality control in line with pathogenesis".

[0010] (2) Theoretical basis of stagnant injury of the spleen and stomach and corresponding pharmacodynamic effects of 5 characteristic components The 5 quality indexes of nobiletin, atractylon, paeoniflorin, isomorocetin, and arecoline determined by this fingerprint are not randomly selected. Instead, they are the core component group that closely adheres to the pathogenesis of stagnant injury of the spleen and stomach, covers the monarch, minister, assistant, and envoy herbs of the whole formula, and corresponds to the efficacy of treating both the principal and secondary aspects of the disease. The specific theory and chemical correspondence are as follows: 1. Nobiletin (derived from Fructus Aurantii Immaturus) - targeting the core pathogenesis: damp obstruction and qi stagnation, metabolic disorder Traditional Chinese medicine theory: The core pathogenesis of damp constipation is damp obstruction and qi stagnation, and the spleen and stomach fail to perform their transportation and transformation functions. Fructus Aurantii Immaturus is bitter and can descend and move downward, pungent and can disperse and move. It is sharp in nature and strong in power, and enters the spleen, stomach, and large intestine meridians. It is a key herb for breaking qi and eliminating accumulation, and is commonly used for symptoms such as food retention and qi stagnation, epigastric and abdominal pain, and constipation due to heat accumulation in the gastrointestinal tract. As a representative polymethoxyflavonoid component in Fructus Aurantii Immaturus, the high lipid solubility of nobiletin makes it easier to penetrate the intestinal mucosa to exert its pharmacodynamic effects, accurately representing the drastic nature of Fructus Aurantii Immaturus in "breaking qi and eliminating accumulation", and directly hitting the core link of "qi stagnation" in damp constipation.

[0011] Chemical pharmacodynamic effects: Nobiletin has a wide range of pharmacological activities such as lowering blood pressure, blood sugar, and blood lipids, and can improve various metabolic diseases such as tumors, diabetes, and hypertension; at the same time, it has significant anti-inflammatory and antioxidant stress effects; it can prevent liver steatosis and dyslipidemia by inhibiting hepatic fatty acid biosynthesis and increasing fatty acid oxidation, and has the effects of preventing obesity and assisting in reducing blood lipids. For the abnormal metabolism of sugar, lipid, and uric acid commonly associated with damp constipation patients, nobiletin can achieve multi-target intervention at the metabolic level, and is a key innovative index corresponding to "breaking qi and promoting qi movement, regulating metabolism" in the fingerprint.

[0012] 2. Atractylon (derived from Rhizoma Atractylodis Macrocephalae) - targeting the root cause: spleen deficiency with dampness exuberance Traditional Chinese medicine theory: The root cause of damp constipation lies in spleen deficiency. When the spleen fails to perform its transportation and transformation function, damp turbidity is generated internally and water and dampness accumulate. Rhizoma Atractylodis Macrocephalae is a key herb for strengthening the spleen, with the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, and is mainly used for symptoms such as poor appetite due to spleen deficiency, abdominal distension and diarrhea. Atractylon is a characteristic component in the volatile oil of Rhizoma Atractylodis Macrocephalae and is also one of the material bases for the "drying property" of Rhizoma Atractylodis Macrocephalae, representing the core efficacy dimension of Rhizoma Atractylodis Macrocephalae in "drying dampness and strengthening the spleen". The selection of atractylon instead of the conventional atractylenolides reflects the quality control idea of "comprehensively representing the efficacy of Rhizoma Atractylodis Macrocephalae from the dimension of volatile components",弥补了常规质控中对白术挥发油类活性成分关注不足的缺陷。 It should be noted that there is an incomplete sentence in the original text for item 17. The translation is based on the existing content as accurately as possible. If there are specific requirements for improvement or correction, please let me know.

[0013] Chemical Efficacy: As the main effective component of Atractylodes macrocephala, its volatile oil possesses a wide range of pharmacological activities, including regulating gastrointestinal motility, improving intestinal microbiota, anti-gastrointestinal inflammation, anti-tumor activity, and antioxidant activity. Modern research shows that components such as atractylone in Atractylodes macrocephala can promote the secretion of digestive juices and improve gastrointestinal motility; atractylone is not only the main component of Atractylodes macrocephala volatile oil but also a major antioxidant. Through multiple mechanisms such as promoting digestive juice secretion, improving gastrointestinal motility, and anti-oxidation, atractylone repairs damaged spleen and stomach function, fundamentally improving the pathological state of spleen deficiency and dampness accumulation. It is a unique and innovative indicator in fingerprinting corresponding to "strengthening the spleen and consolidating the foundation."

[0014] 3. Paeoniflorin (derived from white peony root) – for concurrent symptoms: abdominal pain, tenesmus. Traditional Chinese medicine theory states that patients with dampness-induced constipation often experience abdominal pain, tenesmus, and other discomforts, which are caused by dampness stagnation in the intestines and impaired Qi circulation. White peony root is bitter, sour, and slightly cold in nature, entering the liver and spleen meridians. It nourishes blood, softens the liver, and relieves pain, especially effective for pain caused by muscle spasms or visceral smooth muscle contractions. Paeoniflorin is the most abundant active ingredient in white peony root and is also the legally mandated indicator for its content determination in the Chinese Pharmacopoeia. It represents the core function of white peony root in "softening the liver and relieving pain" and is a key chemical characteristic in the formula for harmonizing Qi and blood and alleviating abdominal discomfort.

[0015] Chemical efficacy: Paeoniflorin is the main natural active ingredient extracted from the dried root of peony. It has broad anti-inflammatory and immunomodulatory effects. It exerts its pharmacological effects through multiple mechanisms, including anti-inflammatory, antioxidant, apoptosis-inhibiting, gut-brain axis regulation, gut microbiota regulation, and downregulation of mitochondrial apoptosis. It can inhibit gastrointestinal smooth muscle spasms and relieve stomach pain, making it suitable for stomach pain caused by improper diet or emotional stress. Paeoniflorin effectively relieves symptoms such as abdominal pain and tenesmus in patients with damp-heat constipation through its dual effects of inhibiting gastrointestinal smooth muscle spasms and anti-inflammation. It is a core indicator in fingerprint spectroscopy corresponding to "soothing the liver and relieving urgency".

[0016] 4. Verbena glycoside (derived from Cistanche deserticola) – For the main symptom: constipation due to intestinal dryness. Traditional Chinese medicine theory states that while dampness-induced constipation is caused by dampness obstructing qi, prolonged use of laxatives or a constitution lacking fluids can easily lead to intestinal dryness and depletion. Cistanche deserticola is sweet, salty, warm, and moistening, nourishing without being drying, and tonifying without being greasy. It enters the kidney and large intestine meridians, possessing the effects of tonifying kidney yang, replenishing essence and blood, and moistening the intestines to relieve constipation, especially effective for senile constipation and postpartum constipation. Vernacular glycoside, one of the main active components of phenylethanoid glycosides in Cistanche deserticola, belongs to the same class as echinacoside and verbascoside, but has received relatively little research attention. Choosing vernacular glycoside as a quality control indicator reflects the innovative approach of "abandoning common practices and selecting distinctive ones."

[0017] Chemical Efficacy: Vernaculosin possesses a wide range of biological functions, including anti-inflammatory, antioxidant, and anti-tumor effects. Studies have shown that its antioxidant activity is the most prominent among similar phenylethyl glycosides. Furthermore, vernaculosin also exhibits α-glucosidase inhibitory activity and improves insulin resistance. The polysaccharides and dietary fiber in Cistanche deserticola can increase intestinal mucus secretion and soften stool. Its laxative effect is mild and does not cause diarrhea, making it suitable for patients with long-term functional constipation. Vernaculosin, through its antioxidant and anti-inflammatory activities combined with the overall laxative effect of Cistanche deserticola, protects the intestinal mucosa while promoting bowel movements, embodying the therapeutic concept of "moistening without harming the body's vital energy." It is an innovative and distinctive indicator in fingerprinting corresponding to "warming the kidneys and moistening the intestines."

[0018] 5. Arecoline (derived from areca nut) – For symptoms of food stagnation, qi stagnation, and dampness in the intestines. Traditional Chinese medicine theory states that the root cause of damp-heat constipation lies in dampness stagnation in the intestines and the obstruction of Qi. Areca nut is bitter and pungent in taste, warm in nature, and enters the stomach and large intestine meridians. It has the effects of eliminating food stagnation, promoting Qi circulation, and promoting diuresis, and is often used for symptoms such as food stagnation and Qi stagnation, abdominal distension and constipation, and tenesmus after diarrhea. Arecoline is the core active ingredient of areca nut, stable in nature, and easy to standardize and control, making it the main form of pharmaceutical and laboratory analytical control standard. Arecoline represents the core efficacy of areca nut in "eliminating food stagnation, promoting Qi circulation, and promoting diuresis," directly addressing the key pathogenesis of damp-heat constipation as "dampness stagnation and excess."

[0019] Chemical effects: Arecoline can significantly accelerate the contraction frequency of gastric fundus muscle strips, induce contraction of colonic smooth muscle cells through M-cholinergic receptors, and its contractile effect is mediated through extracellular Ca2+. 2+ Arecoline mediates intestinal flow; it excites M-cholinergic receptors, increasing gastrointestinal smooth muscle tone, peristalsis, and digestive juice secretion; furthermore, arecoline can counteract gastric mucosal damage in rats and promote its repair, possibly related to its inhibition of gastric acid secretion and reduction of pepsin activity. Through multiple mechanisms, including stimulating cholinergic receptors, enhancing gastrointestinal motility, and promoting digestive juice secretion, arecoline directly targets the symptoms of "dampness stagnation in the intestines and stomach, food accumulation and qi stagnation," making it a core indicator in fingerprinting corresponding to "qi circulation and stagnation relief."

[0020] (III) Theoretical innovation of fingerprint spectrum quality indicators (different from traditional quality control) Theoretical binding, rather than purely chemical characterization Traditional fingerprinting only reflects the overall chemical composition and is disconnected from the pathogenesis in traditional Chinese medicine. This method uses the theory of spleen and stomach stagnation as the sole screening criterion. The five characteristic peaks correspond to "promoting qi circulation and relieving stagnation, strengthening the spleen and drying dampness, soothing the liver and relieving urgency, warming the kidney and moistening the intestines, and promoting diuresis and eliminating stagnation". The fingerprint is a chemical visualization of the improvement of the pathogenesis.

[0021] Synergistic effects, rather than the superposition of individual components. The five ingredients cover five major categories of chemical substances: polymethoxyflavonoids (hesperidin), sesquiterpenes (atractylone), monoterpenoid glycosides (paeoniflorin), phenylethanol glycosides (isosaurolol), and alkaloids (arecoline). They address both the symptoms and the root cause: hesperidin and arecoline work together to treat the symptoms (promoting qi circulation and diuresis), isaurolol moistens and protects yin (warming the kidneys and moistening the intestines), paeoniflorin relieves concurrent symptoms (soothing the liver, relieving spasms and pain), and atractylone strengthens the body (strengthening the spleen and drying dampness). This fully matches the entire pathogenesis of damp constipation: "dampness obstructing qi stagnation → spleen deficiency and dampness accumulation → intestinal dryness and fluid depletion".

[0022] Precise quality control, aligned with the clinical efficacy of traditional Chinese medicine Fingerprint spectroscopy uses relative retention time as a quantitative standard to directly correlate with the overall efficacy of the preparation in correcting stagnation in the spleen and stomach, solving the industry pain point of "qualified ingredients but poor efficacy", and achieving the unification of traditional Chinese medicine theory, chemical indicators and clinical efficacy.

[0023] Therefore, the fingerprint feature spectrum established by this method has all quality indicators derived from the TCM theory of spleen and stomach stagnation. It screens components from the pathogenesis, characterizes efficacy with components, and quantifies quality control with the spectrum, thus completely realizing the leap from "recognizing medicine" to "certifying" the quality control of Chinese medicine. It is an innovative method that conforms to the TCM syndrome differentiation and treatment, and scientifically evaluates the quality of preparations that strengthen the spleen, resolve dampness, and promote qi circulation.

[0024] A method for detecting the quality of fingerprint feature maps based on the theory of spleen and stomach stagnation includes the following steps: Step 1: Preparation of reference solution: Accurately weigh appropriate amounts of arecoline, paeoniflorin, isorhodoside, atractylone, and nobiletin reference standards, and place them in the same 10mL brown volumetric flask. Add an appropriate amount of acetonitrile-water mixed solvent (1:1 volume ratio), sonicate at 250W and 40kHz for 30 minutes to ensure complete dissolution, cool, dilute to the mark with the same mixed solvent, shake well, and filter through a 0.22µm microporous membrane to prepare a mixed reference solution containing 80µg of arecoline, 100µg of paeoniflorin, 100µg of isorhodoside, 120µg of atractylone, and 60µg of nobiletin per mL. Step Two: Preparation of test solution: Accurately measure 5.0 mL of this product and place it in a 50 mL stoppered conical flask. Accurately add 25 mL of methanol, weigh the sample, and sonicate (500 W power, 40 kHz frequency) for 30 minutes. After cooling, weigh the sample again and replenish the lost weight with methanol. Shake well and centrifuge at 3000 rpm for 10 minutes. Accurately measure the supernatant and filter it through a 0.22 µm microporous membrane. Collect the filtrate to obtain the test solution. Step 3: Determination method: Accurately pipette 10 μL each of the reference solution and the test solution and inject them into the liquid chromatograph, and record the chromatogram.

[0025] Chromatographic conditions: Column: Topsil C18 (4.6 x 250 mm, 5 μm); Mobile phase: 5 mmol / L ammonium formate solution (adjusted to pH 3.8 with formic acid) as mobile phase A, and acetonitrile-isopropanol mixed solution with a volume ratio of 95:5 as mobile phase B, eluted according to the specified gradient elution program; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 215 nm; The theoretical plate number calculated based on the atractylone peak should not be less than 5000.

[0026] The gradient elution procedure is as follows: 0~5min, mobile phase A: phase B, volume ratio 92:8; 5~25min, the volume ratio of mobile phase A: phase B changes from 92:8 to 80:20; 25~45min, the volume ratio of mobile phase A: phase B changes from 80:20 to 70:30; 45~55 min, the volume ratio of mobile phase A: phase B changes from 70:30 to 50:50; 55~60min, the volume ratio of mobile phase A: phase B changes from 50:50 to 10:90; 60~65min, mobile phase A: phase B, volume ratio 10:90; At 65-70 min, the volume ratio of mobile phase A to phase B changed from 10:90 to 92:8. 70~75 min, mobile phase A: phase B, volume ratio 92:8; Step 4: Generate a reference characteristic chromatogram: Select the chromatographic peaks that are present in the chromatograms of different batches of the Huashi Tongbian Mixture as common peaks, and use the average value calculation method to generate a reference characteristic chromatogram of the Huashi Tongbian Mixture.

[0027] The reference characteristic chromatogram generated in the further step four shows five chromatographic peaks, of which peaks 1, 2, 3, 4 and 5 correspond to the reference peaks of arecoline, paeoniflorin, isorhamnetin, atractylone and nobiletin, respectively.

[0028] The reference characteristic spectrum generated in step four uses the atractylone reference peak as the S peak. The relative retention times of each characteristic peak and the S peak are calculated. The relative retention times are all within ±10% of the specified values. The specified values ​​are: 0.32 (peak 1), 0.57 (peak 2), 0.72 (peak 3), and 1.09 (peak 5).

[0029] Compared with the prior art, the advantages of the present invention are as follows: 1. Strong theoretical relevance: It directly links the traditional Chinese medicine theory of "stagnation damaging the spleen and stomach" with chemical fingerprints, thus linking quality evaluation with the efficacy of the preparation and solving the problem of "recognizing the drug but not the certification"; 2. Scientific indicator selection: Five characteristic components of each major medicinal ingredient and core efficacy category in the representative formula are carefully selected to make the spectrum more representative of efficacy, which is superior to single indicator or full component spectrum. 3. Method-specific and stable: Targeted pretreatment and chromatographic conditions ensure effective separation and stable detection of multiple types of characteristic components, with thorough method validation and good specificity; 4. Precise and practical quality control: The established digital comparative feature spectrum and relative retention time standard are highly operable and can more sensitively identify abnormalities in key components, facilitating production and supervision. Attached Figure Description

[0030] The invention will now be further described with reference to the accompanying drawings; Figure 1 This is a repeatable chromatogram overlay of the present invention; S1-S6 are chromatograms obtained from 6 test solutions prepared according to the test solution preparation method; Figure 2 This is a chromatogram of the stability of the present invention; S1 is the chromatogram obtained by injecting the same test solution at 0 hours, S2 is the chromatogram obtained by injecting the same test solution at 2 hours, S3 is the chromatogram obtained by injecting the same test solution at 4 hours, S4 is the chromatogram obtained by injecting the same test solution at 8 hours, S5 is the chromatogram obtained by injecting the same test solution at 12 hours, and S6 is the chromatogram obtained by injecting the same test solution at 24 hours. Figure 3 This is a solution chromatogram of the reference material for the characteristic chromatogram of the present invention; wherein, S1 is the test sample and S2 is the reference material; Figure 4 The chromatogram of the test sample solution is a characteristic chromatogram of the present invention. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings. The instruments and equipment used in the embodiments are as follows: I. Instruments and Reagents 1.1 Instruments and Equipment .

[0032] 1.2 Test Materials .

[0033] 1.3 Reference Standard Information .

[0034] 1.4 Sample Information .

[0035] The formula for a dampness-resolving and laxative mixture consists of 100g of Citrus aurantium, 120g of Atractylodes macrocephala, 100g of Paeonia lactiflora, 150g of Trichosanthes kirilowii, 100g of Cistanche deserticola, 100g of Areca catechu, 100g of Lindera strychnifolia, and 100g of Cannabis sativa seed, prepared to a volume of 1000mL. The preparation method involves decocting the above eight ingredients twice with water, the first time for 2 hours and the second time for 1.5 hours. The decoctions are combined, and the filtrate is concentrated to a relative density of 1.05–1.15 (measured at 60℃). Then, 0.5–1.0g of steviol glycosides and 0.5g of ethylparaben (ethylparaben needs to be dissolved in 2–3mL of ethanol beforehand) are added. Water is added to make 1000mL, which is then filled, sealed, and sterilized.

[0036] Example 1: Study on the characteristic spectral method of a dampness-resolving and laxative compound Preparation of reference solution: Accurately weigh appropriate amounts of arecoline, paeoniflorin, isorhodoside, atractylone, and nobiletin reference standards, and place them in the same 10mL brown volumetric flask. Add an appropriate amount of acetonitrile-water mixed solvent (1:1 volume ratio), sonicate at 250W and 40kHz for 30 minutes to ensure complete dissolution, cool, dilute to the mark with the same mixed solvent, shake well, and filter through a 0.22µm microporous membrane to prepare a mixed reference solution containing 80µg of arecoline, 100µg of paeoniflorin, 100µg of isorhodoside, 120µg of atractylone, and 60µg of nobiletin per mL. Preparation of test solution: Accurately measure 5.0 mL of this product and place it in a 50 mL stoppered conical flask. Accurately add 25 mL of methanol, weigh the sample, and sonicate (500 W power, 40 kHz frequency) for 30 minutes. After cooling, weigh the sample again and replenish the lost weight with methanol. Shake well and centrifuge at 3000 rpm for 10 minutes. Accurately measure the supernatant and filter it through a 0.22 µm microporous membrane. Collect the filtrate to obtain the test solution. Determination method: Accurately pipette 10 μL each of the reference solution and the test solution and inject them into the liquid chromatograph, and record the chromatogram.

[0037] Chromatographic conditions: Column: Topsil C18 (4.6 x 250 mm, 5 μm); Mobile phase: 5 mmol / L ammonium formate solution (adjusted to pH 3.8 with formic acid) as mobile phase A, and acetonitrile-isopropanol mixed solution with a volume ratio of 95:5 as mobile phase B, with gradient elution as specified in the table below; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 215 nm; The theoretical plate number calculated based on the atractylone peak should not be less than 5000.

[0038] .

[0039] (1) System applicability Accurately pipette 10 μL of the reference solution and repeat the injection 6 times. Calculate the RSD values ​​of the retention times of arecoline, paeoniflorin, isorhodoside, atractylone, and nobiletin. The theoretical plate number and retention time are shown in the table below.

[0040] Table 1 System Applicability Results .

[0041] Table 2 System applicability retention time (min) results .

[0042] The results show that the theoretical plate number of atractylone is greater than 5000 and the RSD of retention time is less than 2.0%, indicating good system suitability.

[0043] (2) Exclusivity Accurately pipette 10 μL each of the negative solution, reference solution, and test solution, and inject them into the liquid chromatograph for determination.

[0044] Summary: The results show that arecoline, paeoniflorin, isorhodoside, atractylone, and tangeretin in the test solution and the reference solution have the same chromatographic peaks at the corresponding retention times. The resolution of arecoline, paeoniflorin, isorhodoside, atractylone, and tangeretin from adjacent peaks is greater than 1.50, and there is no interference from the negative sample solution, indicating good method specificity.

[0045] (3) Repeatability Accurately measure 5 mL of the dehumidifying and laxative mixture, and prepare 6 test solutions according to the preparation method for the test solution. Inject the solutions, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the relative standard deviation. The results are as follows (see below). Figure 1 .

[0046] Table 3. Repeatability Relative Retention Time Results .

[0047] Table 4. Repeatability of relative peak area results .

[0048] The results show that the relative retention time RSD of each chromatographic peak is less than 2.0%, and the relative peak area RSD is less than 2.0%, indicating good repeatability.

[0049] (4) Intermediate precision At different times, another analyst conducted the following experiments using different chromatographs.

[0050] Accurately measure 5 mL of the dehumidifying and laxative mixture, take 6 portions, and prepare 6 test solutions according to the test solution preparation method. Inject the samples, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the relative standard deviation.

[0051] Calculate the relative retention time and relative peak area of ​​each chromatographic peak for 12 samples for repeatability and intermediate precision, and calculate the relative standard deviation.

[0052] Table 5 Results of intermediate precision relative retention time .

[0053] Table 6 Results of intermediate precision relative peak area .

[0054] The results show that the relative retention time RSD and relative peak area RSD of each characteristic peak are less than 2.0%; when n=12, the relative retention time RSD and relative peak area RSD of each characteristic peak are less than 2.0%, indicating good intermediate precision.

[0055] (5) Solution stability Accurately measure 5 mL of the dehumidifying and laxative mixture and prepare one test solution according to the test solution preparation method. Inject the solution at 0, 2, 4, 8, 12, and 24 hours, respectively, and determine the relative retention time and relative peak area of ​​each chromatographic peak. Calculate the relative standard deviation. See [link to relevant documentation]. Figure 2 .

[0056] Table 7 Results of relative retention time for stability .

[0057] Table 8. Results of relative peak area for stability .

[0058] The results show that the relative retention time RSD of each characteristic peak is less than 2.0%, the relative peak area RSD is less than 3.0%, and the solution has good stability.

[0059] (6) Characteristic spectrum determination Chromatographic conditions: Column: Topsil C18 (4.6 x 250 mm, 5 μm); Mobile phase: 5 mmol / L ammonium formate solution (adjusted to pH 3.8 with formic acid) as mobile phase A, and acetonitrile-isopropanol mixed solution with a volume ratio of 95:5 as mobile phase B, with gradient elution as specified in the table below; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 215 nm; The theoretical plate number calculated based on the atractylone peak should not be less than 5000.

[0060] .

[0061] Preparation of reference solution: Accurately weigh appropriate amounts of arecoline, paeoniflorin, isorhodoside, atractylone, and nobiletin reference standards, and place them in the same 10mL brown volumetric flask. Add an appropriate amount of acetonitrile-water mixed solvent (1:1 volume ratio), sonicate at 250W and 40kHz for 30 minutes to ensure complete dissolution, cool, dilute to the mark with the same mixed solvent, shake well, and filter through a 0.22µm microporous membrane to prepare a mixed reference solution containing 80µg of arecoline, 100µg of paeoniflorin, 100µg of isorhodoside, 120µg of atractylone, and 60µg of nobiletin per mL. Preparation of test solution: Accurately measure 5.0 mL of this product and place it in a 50 mL stoppered conical flask. Accurately add 25 mL of methanol, weigh the sample, and sonicate (500 W power, 40 kHz frequency) for 30 minutes. After cooling, weigh the sample again and replenish the lost weight with methanol. Shake well and centrifuge at 3000 rpm for 10 minutes. Accurately measure the supernatant and filter it through a 0.22 µm microporous membrane. Take the filtrate to obtain the test solution. Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and record the chromatograms. The characteristic chromatogram of the reference solution is shown below. Figure 3 The characteristic chromatogram of the test solution is shown in the figure. Figure 4 .

[0062] The chromatogram of the test sample showed five chromatographic peaks. Peaks 1, 2, 3, 4, and 5 corresponded to the reference peaks of arecoline, paeoniflorin, isorhamnetin, atractylone, and nobiletin, respectively. Using the atractylone reference peak as the S peak, the relative retention times of each characteristic peak and the S peak were calculated. All relative retention times were within ±10% of the specified values. The specified values ​​were: 0.32 (peak 1), 0.57 (peak 2), 0.72 (peak 3), and 1.09 (peak 5).

Claims

1. A method for detecting the quality of fingerprint feature maps based on the theory of spleen and stomach stagnation, characterized in that, Includes the following steps: Step 1: Preparation of reference solutions: Weigh arecoline, paeoniflorin, isorhoside, atractylone, and tangeretin reference standards and place them in the same brown volumetric flask. Add acetonitrile-water mixed solvent, sonicate, cool, dilute with the same mixed solvent (acetonitrile-water), shake well, and filter through a 0.22µm microporous membrane to prepare a mixed reference solution containing 80µg arecoline, 100µg paeoniflorin, 100µg isorhoside, 120µg atractylone, and 60µg tangeretin per mL. Step Two: Preparation of test solution: Measure the dehumidifying and laxative mixture, add methanol, weigh, sonicate, cool and weigh again, make up the weight loss with methanol, shake well, centrifuge, accurately measure the supernatant and filter it through a 0.22µm microporous membrane, take the filtrate to obtain the test solution. Step 3: Determination method: Accurately pipette 10 μL each of the reference solution and the test solution and inject them into the liquid chromatograph, and record the chromatograms; Chromatographic conditions: Topsil C18 column, 4.6 x 250 mm, 5 μm; mobile phase: 5 mmol / L ammonium formate solution (pH 3.8) as mobile phase A, and acetonitrile-isopropanol mixture (95:5 v / v) as mobile phase B, gradient elution according to the specified gradient elution program; flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 215 nm; theoretical plate number calculated based on the atractylone peak should be no less than 5000. The gradient elution procedure is as follows: 0~5min, mobile phase A: phase B, volume ratio 92:8; 5~25min, the volume ratio of mobile phase A: phase B changes from 92:8 to 80:20; 25~45min, the volume ratio of mobile phase A: phase B changes from 80:20 to 70:30; 45~55 min, the volume ratio of mobile phase A: phase B changes from 70:30 to 50:50; 55~60min, the volume ratio of mobile phase A: phase B changes from 50:50 to 10:90; 60~65min, mobile phase A: phase B, volume ratio 10:90; At 65-70 min, the volume ratio of mobile phase A to phase B changed from 10:90 to 92:

8. 70~75 min, mobile phase A: phase B, volume ratio 92:8; Step 4: Generate a comparative feature map Chromatographic peaks present in all batches of the Huashi Tongbian Mixture were selected as common peaks, and a reference characteristic chromatogram of the Huashi Tongbian Mixture was generated using the average value calculation method. The formula for a dampness-resolving and laxative mixture consists of 100g of Citrus aurantium, 120g of Atractylodes macrocephala, 100g of Paeonia lactiflora, 150g of Trichosanthes kirilowii, 100g of Cistanche deserticola, 100g of Areca catechu, 100g of Lindera strychnifolia, and 100g of Cannabis sativa seed, prepared to a volume of 1000mL. The preparation method involves decocting the above eight ingredients twice with water, the first time for 2 hours and the second time for 1.5 hours. The decoctions are combined and filtered to obtain a filtrate. The filtrate is concentrated to a relative density of 1.05–1.15 and measured at 60℃ to obtain a concentrated solution. Then, 0.5–1.0g of steviol glycosides and 0.5g of ethylparaben are added to the concentrated solution to obtain a mixed solution. Ethylparaben needs to be dissolved in 2–3mL of ethanol first. Finally, the mixed solution is diluted with water to 1000mL, filled, and sterilized.

2. The fingerprint feature map quality detection method based on the theory of spleen and stomach stagnation as described in claim 1, characterized in that, In step one, the volume ratio of the acetonitrile-water mixed solvent is 1:

1.

3. The fingerprint feature map quality detection method based on the theory of spleen and stomach stagnation as described in claim 1, characterized in that, In step one, the reference solution was prepared by ultrasonic treatment at 250W and 40kHz for 30 minutes.

4. The fingerprint feature map quality detection method based on the theory of spleen and stomach stagnation as described in claim 1, characterized in that, In step one, the ultrasonic treatment conditions for preparing the test solution are 500W power and 40kHz frequency for 30 minutes.

5. The fingerprint feature map quality detection method based on the theory of spleen and stomach stagnation as described in claim 1, characterized in that, In step one, the centrifugation conditions for preparing the test solution are: 3000 rpm for 10 minutes.

6. The fingerprint feature map quality detection method based on the theory of spleen and stomach stagnation as described in claim 1, characterized in that, The reference characteristic chromatogram generated in step four shows five chromatographic peaks, among which peaks 1, 2, 3, 4, and 5 correspond to the reference peaks of arecoline, paeoniflorin, isorhamnetin, atractylone, and nobiletin, respectively.

7. The fingerprint feature map quality detection method based on the theory of spleen and stomach stagnation as described in claim 1, characterized in that, The control characteristic spectrum generated in step four uses the atractylone reference peak as the S peak. The relative retention times of each characteristic peak and the S peak are calculated. The relative retention times are all within ±10% of the specified values. The specified values ​​are: 0.32 for peak 1, 0.57 for peak 2, 0.72 for peak 3, and 1.09 for peak 5.

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