Preparation method and application of coptis soup based on reflux extraction optimization

Abstract

The method comprises the following steps: weighing 40 g of coptis chinensis decoction pieces, 30 g of dried ginger, 30 g of licorice roots, 30 g of cassia twigs, 25 g of Chinese dates, 25 g of pinellia ternate and 20 g of ginseng, crushing the materials into coarse powder, sieving the coarse powder, adding 70% ethanol, and soaking the coarse powder for a period of time; heating to a boiling state, keeping a slight boiling state, refluxing for 1 hour, stirring once every 15 minutes, filtering while the solution is hot, and reserving residues; adding 70% ethanol again, timing after boiling, refluxing for 1 hour, filtering and combining the two filtrates; and concentrating the filtrate into thick extract without ethanol smell, and preparing freeze-dried powder by adopting a freeze-drying process. The preparation method is high in powder yield. The freeze-dried powder prepared by the invention can obviously reduce serum inflammatory factors (IL-1beta and IL-6), improve gastric mucosa atrophy (GAST, PGA / PGC indexes) and inhibit gastric tissue pathological damage, and can be used for reversing gastric precancerous lesions.

Description

Technical Field

[0001] The present invention relates to the field of medicine and is a medicine for treating gastric precancerous lesions, and in particular to a preparation method of Huanglian decoction based on reflux extraction optimization and an application thereof. Background Art

[0002] According to WHO data, gastric cancer ranks fifth in incidence and fourth in mortality among malignant tumors worldwide. In 2020, China had 479,000 new cases of gastric cancer, accounting for approximately 44.0% of the global total, and 375,000 deaths, accounting for approximately 48.6% of the global total. Precancerous lesions of the stomach (PLGC) are an independent risk factor for gastric cancer. Timely and effective treatment and reversal of PLGC are particularly important for preventing gastric cancer. [1] Principle of existing technology: Currently, Western medicine treatment of PLGC mainly relies on eradication of Helicobacter pylori (Hp) and gastric mucosal protective agents. The former reduces inflammatory stimulation through quadruple therapy (proton pump inhibitor (PPI) + a bismuth agent + two antibiotics), while the latter protects the mucosa through physical coverage and mucus secretion (such as sucralfate, teprenone). [2] Technical disadvantages: Even after successful H. pylori eradication, the persistent chronic inflammatory microenvironment (such as high expression of IL-6 and TNF-α) still drives the progression of dysplasia. [3] The reversal rate of H. pylori eradication for PLGC is limited, only 35%-40%, and there is no significant improvement in intestinal metaplasia and dysplasia; long-term use of PPIs increases the risk of fractures and leads to hypomagnesemia. [4] Problems such as poor patient compliance caused by antibiotic resistance and drug side effects cannot be ignored [5] The global clarithromycin resistance rate is >30% (up to 45% in some parts of China), and the metronidazole resistance rate is >60%, resulting in the eradication rate of the quadruple regimen dropping to 70%-80%. [6] Existing drugs have a single target, targeting only Hp infection or mucosal protection, and lack the coordinated regulation of multiple pathways (such as Wnt / β-catenin, HIF-1α / VEGF). [7] .

[0003] Theoretical basis of TCM treatment of PLGC: According to clinical observations, PLGC belongs to the category of "fullness", "stomach distension" and "epigastric pain" in the theoretical system of traditional Chinese medicine. The nature of the disease is mostly "root deficiency and superficial excess", root deficiency is mainly spleen and stomach qi deficiency and / or yin deficiency, and superficial excess includes blood stasis, heat toxicity, etc. Huanglian Decoction comes from Article 173 of "Treatise on Febrile Diseases", "For patients with typhoid fever who have heat in the chest, evil qi in the stomach, abdominal pain, and vomiting, Huanglian Decoction is the main treatment". It is composed of 7 herbs: coptis slices, dried ginger, cinnamon twigs, pinellia tuber, ginseng slices, licorice slices, and jujube. The whole prescription "uses both cold and heat", opens with pungent and descends with bitter, and tonifies and purges at the same time. It has the functions of clearing the upper part and warming the lower part, harmonizing the stomach and relieving adverse reactions. It is one of the representative prescriptions of the harmonizing method and is widely used in the clinical treatment of chronic digestive system diseases with remarkable efficacy. [8] .

[0004] The above content involves the following documents:

[0005] [1]SUGANO K, MOSS SF, KUIPERS E J. Gastric Intestinal Metaplasia: RealCulprit or Innocent Bystander as a Precancerous Condition for Gastric Cancer? [J].Gastroenterology,2023,165(6):1352-66.e1.

[0006] [2] Analysis of methods of combining traditional Chinese and western medicine to cure drug-resistant Helicobacter pylori[J]. Chinese Journal of Clinical Physicians (9(15):716-20.

[0007] [3]SHARAFUTDINOV I,TEGTMEYER N,LINZ B,et al.A single-nucleotide polymorphismin Helicobacter pylori promotes gastric cancer development[J].Cell Host Microbe,2023,31(8):1345-58.e6.

[0008] [4]LESPESSAILLES E,TOUMI H.Proton Pump Inhibitors and Bone Health: AnUpdate Narrative Review[J].Int J Mol Sci, 2022, 23(18).

[0009] [5]UHE I, HAGEN ME, RIS F, et al. Cell-free DNA liquid biopsy for early detection of gastrointestinal cancers: A systematic review [J]. World JGastrointest Oncol, 2021, 13(11): 1799-812.

[0010] [6]WANG L, LI Z,TAY CY,et al.

[0011] [7]WU J,

[0012] [6] Liu Lin, Fan Xiaohui, Tang Xudong. Transformation of gastric mucosal inflammation to carcinoma.pdf, Chinese Journal of Integrated Traditional and Western Medicine 44(2024). Summary of the Invention

[0013] The present invention provides a method for preparing Huanglian decoction based on reflux extraction optimization with a high powder yield, and the HLD freeze-dried powder prepared by the present invention can treat gastric precancerous lesions, further verifying the mechanism of action of HLD freeze-dried powder in treating gastric precancerous lesions.

[0014] In view of this, the technical solution adopted by the present invention is: a preparation method of Huanglian Decoction based on reflux extraction optimization, comprising the following steps:

[0015] Weigh 40g of Coptis chinensis slices, 30g of dried ginger, 30g of liquorice, 30g of cinnamon twig, 25g of jujube, 25g of pinellia, and 20g of ginseng, grind them into coarse powder, sieve them, add 70% ethanol, and soak them for a while;

[0016] Heat to boiling, start counting, and reflux for 1 hour while slightly boiling. Stir every 15 minutes, filter while hot, and retain the residue. Add 70% ethanol again, start counting after boiling, reflux for 1 hour, and filter and combine the two filtrates.

[0017] The above filtrate is concentrated to a thick extract without ethanol taste, and freeze-dried powder is prepared by freeze-drying process.

[0018] Furthermore, the sieving adopts No. 2 sieve, and the particle size is 2-4 mm.

[0019] Furthermore, the soaking is performed at room temperature for 30 minutes.

[0020] Furthermore, the concentration is carried out under reduced pressure at 40° C. until there is no ethanol smell.

[0021] Furthermore, the freeze-drying process is to pre-freeze at -20°C and then dry under reduced pressure in a freeze dryer (<50pa, <-50°C).

[0022] The present invention also provides a HLD freeze-dried powder prepared by the method.

[0023] Furthermore, the invention provides the use of the HLD freeze-dried powder in preparing a product for reversing gastric precancerous lesions.

[0024] Specifically, HLD inhibited the PI3K / Akt signaling pathway, downregulated the expression of Cyclin D1 and IL-1β proteins, and activated apoptosis-related proteins Cleaved-Caspase3 and Bax.

[0025] Furthermore, the dosage range of HLD lyophilized powder is 1.12-2.24 g / kg / d, and the administration method is oral gavage suspension.

[0026] The HLD freeze-dried powder is used in the preparation of supplies for reducing serum inflammatory factors IL-1β and IL-6.

[0027] The HLD freeze-dried powder is used in the preparation of a medicine for improving gastric mucosal atrophy.

[0028] The present invention has the following beneficial technical effects:

[0029] 1. Extraction efficiency and active ingredients are significantly improved

[0030] Reflux extraction process parameters: Use 70% ethanol as the solvent, reflux twice (2000ml for the first time, 1600ml for the second time), total extraction time 2h, and maintain the stability of the active ingredient in a slightly boiling state. The crushing particle size is controlled to pass through a No. 2 sieve, the particle size is 2-4mm, and the soaking time is optimized to 30min to improve the dissolution efficiency. Concentration and freeze-drying process: Concentrate under reduced pressure at 40℃ until there is no ethanol smell, pre-freeze at -20℃ and then freeze-dry (<50Pa, <-50℃) to ensure the retention of active ingredients and the stability of the preparation. Using the above process parameters, the powder yield is increased to 24.30%, and the content of key active ingredients (such as berberine hydrochloride and epiberberine) is significantly higher than that of the percolation method and boiling method, and the process parameters are stable and controllable to ensure the consistency of the preparation quality.

[0031] UPLC-Q-TOF-MS was used to quantitatively analyze seven key components, including 6-gingerol, caffeic acid, and magnolamine, ensuring the standardization and controllability of active ingredients after process optimization. The reflux method extract freeze-dried powder contained significantly higher levels of anti-inflammatory and anti-tumor components, such as palmatine hydrochloride (0.256 μg / mL) and berberine hydrochloride (12.08 μg / mL), than those obtained using other methods.

[0032] 2. Clarify the mechanism of efficacy of drugs for reversing gastric precancerous lesions

[0033] Animal experiments using an MNNG-induced PLGC rat model have confirmed that high-dose HLD lyophilized powder can significantly reduce serum inflammatory factors (IL-1β, IL-6), improve gastric mucosal atrophy (GAST, PGA / PGC indicators), and inhibit the PI3K / Akt pathway to reduce cell proliferation and promote apoptosis. This is the first time that its molecular mechanism of action in reversing gastric precancerous lesions has been elucidated: HLD inhibits the PI3K / Akt signaling pathway, downregulating Cyclin D1 and IL-1β protein expression, and simultaneously activating apoptosis-related proteins (Cleaved-Caspase 3, Bax), thereby blocking the progression of gastric precancerous lesions.

[0034] 3. Preparation stability and clinical application advantages

[0035] The freeze-dried powder preparation process (pre-freezing at -20°C, followed by vacuum freeze-drying) effectively preserves heat-sensitive components, ensuring the stability of active ingredients during long-term storage and avoiding the perishable nature of traditional decoctions. The suspension formulation (containing 0.5% CMC-Na) facilitates precise dosing and, combined with methodological validation (recovery rate >97%, RSD <2%), provides reliable technical support for standardized production and clinical translation.

[0036] Methodological validation: The sample recovery rate is 97.49% to 101.76%, and the precision (RSD < 2%) meets the requirements of the 2020 edition of the "Chinese Pharmacopoeia", ensuring the reliability of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1LC-MS results of the active ingredients of Huanglian Decoction under different extraction processes; A. Reference substance; B. Percolation extraction; C. Reflux extraction; D. Decoction extraction; 1. 6-gingerol; 2. Caffeic acid; 3. Magnolia pine; 4. Jatrorrhizine; 5. Epiberberine; 6. Palmatine hydrochloride; 7. Berberine hydrochloride;

[0039] Figure 2 Flow chart of animal experiments for HLD treatment of PLGC;

[0040] Figure 3 The stomach tissue morphology of animals in each group is shown;

[0041] Figure 4 H&E pathological staining images of gastric tissues of rats in each group;

[0042] Figure 5 The TUNEL staining analysis of gastric tissues of rats in each group; Note: Compared with the Vehicle group, **P<0.01; Compared with the PLGC group, ## P < 0.01;

[0043] Figure 6 Effects of HLD lyophilized powder on gastric tissue protein in PLGC model rats; Note: Compared with the Vehicle group, * P<0.05, ** P<0.01; compared with the PLGC group, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0044] 1. Reflux extraction method

[0045] 1.1 Weigh 40g of Coptis chinensis slices, 30g of dried ginger, 30g of liquorice root, 30g of cinnamon twig, 25g of jujube, 25g of pinellia tuber, and 20g of ginseng. Grind: Grind 200g of the above herbs into a coarse powder (pass through a No. 2 sieve, particle size approximately 2-4mm). Place in a round-bottom flask, add 2000ml of 70% ethanol, and soak at room temperature for 30 minutes.

[0046] 1.2 After connecting the apparatus, heat to boiling, time the boil, and reflux for 1 hour, stirring every 15 minutes. Filter through four layers of gauze while still hot, retaining the residue. Add another 1600 ml of 70% ethanol, time the boil, and reflux for 1 hour. Filter and combine the two filtrates.

[0047] 1.3 Transfer the above decoction to a rotary evaporator (40°C, -0.3, -0.1Mpa) and concentrate it to a thick extract without ethanol taste. Freeze it at -20°C and transfer it to a tray. Place it in a freeze dryer (<50pa, <-50°C) and dry it under reduced pressure. Then grind it into a clean 50mL centrifuge tube, seal it tightly with a sealing strip, and store it in a desiccator. The freeze-dried powder is obtained. The powder yield is calculated using the formula "Powder yield (%) = dry paste powder weight / decoction piece weight × 100%". HLD freeze-dried powder powder yield: 48.59g / 200g*100% = 24.30%.

[0048] 2. Percolation extraction

[0049] 2.1 Weigh 40g of Coptis chinensis slices, 30g of dried ginger, 30g of liquorice root, 30g of cinnamon twig, 25g of jujube, 25g of pinellia tuber, and 20g of ginseng. Grind: Grind 200g of the above herbs into a coarse powder (pass through a No. 2 sieve, particle size approximately 2-4mm). Add 240ml of 70% ethanol to submerge the herbs, stir well, and soak in a sealed container at room temperature for 4 hours.

[0050] 2.2 Load the moistened medicinal materials into the percolation cylinder in portions, gently compacting them. Cover the top with filter paper or gauze to prevent the materials from dispersing when adding solvent. Slowly add 70% ethanol from the top of the percolation cylinder until the medicinal materials are submerged. Close the stopcock and allow to soak for 12 hours. Open the stopcock and control the percolation rate to 2ml / min. Collect the percolation liquid to 2000ml. When the liquid level approaches the medicinal powder, add solvent promptly to maintain a stable liquid level.

[0051] 2.3 Transfer the percolate to a rotary evaporator and follow the procedure in 1.3 to calculate the powder yield. HLD freeze-dried powder yield: 42.24 g / 200 g * 100% = 21.12%.

[0052] 3. Boiling extraction method

[0053] 3.1 Weigh 40g of Coptis chinensis slices, 30g of dried ginger, 30g of liquorice root, 30g of cinnamon twig, 25g of jujube, 25g of pinellia tuber, and 20g of ginseng. Grind: Grind 200g of the above herbs into a coarse powder (pass through a No. 2 sieve, particle size approximately 2-4mm). Place in a stainless steel pot, add 2000ml of pure water, and soak at room temperature for 1 hour.

[0054] 3.2 Bring to a boil over high heat, then reduce heat and simmer for 30 minutes, stirring three times to prevent the bottom from becoming sticky. Filter through four layers of gauze while still hot. Add 1600ml of purified water to the residue and boil again for 30 minutes. Filter and combine the two decoctions.

[0055] 3.3 Transfer the decoction to a rotary evaporator and follow the procedure in 1.3 to calculate the powder yield. HLD freeze-dried powder yield: 40.63 g / 200 g * 100% = 20.32%.

[0056] 4. Preparation of Standard Working Solution

[0057] Take 2.5 mg each of 6-gingerol, caffeic acid, magnolamine, jatrorrhizine, epiberberine, palmatine hydrochloride, and berberine hydrochloride and place them in a 25 ml volumetric flask. Add methanol to the scale to obtain a 0.1 mg / mL mixed reference solution, marked as A.

[0058] 5. Preparation of Test Sample Working Solution

[0059] Accurately weigh 0.5 g of freeze-dried powder of Huanglian Decoction extracted by the percolation, reflux, and decoction methods and label them B, C, and D, respectively. Extract at a solid-liquid ratio of 1:25 g / mL for 30 min at 180 W. Centrifuge and concentrate the extract to a concentration of 0.1 g of the original herbal medicine per mL. Filter through a 2.5 μm microporous membrane to obtain the extract.

[0060] 6. UPLC-Q-TOF-MS method for the detection of the main active ingredients in HLD

[0061] 6.1 Instruments and reagents

[0062] Acquity UPLC I-class ultra-high performance liquid chromatograph, Xevo G2-XS Q-Tof time-of-flight mass spectrometer; all chromatographic reagents were imported from FISHER;

[0063] Chromatographic column: Waters Acquity BEH C18, 2.1*100 mm, 1.7 μm; mobile phase: A-ACN, B-H2O (0.1% HCOOH); column temperature: 35°C; injection volume: 1 μl; gradient elution program see Table 1.

[0064] Table 1. Gradient elution program

[0065] Time(min) A％ B％ Curve Flow rate (ml / min) 0.00 0.5 99.5 Initial 0.4 2 0.5 99.5 6 0.4 4 8 92 6 0.4 6 14 86 6 0.4 15 30 70 6 0.4 24 45 55 6 0.4 27 70 30 6 0.4 31 90 10 6 0.4 33 90 10 6 0.4 33.5 0.5 99.5 6 0.4 36 0.5 99.5 6 0.4

[0066] 6.2 Mass spectrometry conditions

[0067] Ionization mode: electrospray ionization, positive ion mode / negative ion mode; capillary voltage: 3 kV; cone voltage: 40 V; ion source temperature: 100°C; desolvation temperature: 250°C; desolvation gas flow rate: 400 L / hr (pos) / 600 L / hr (neg); scan mode: MSe; low energy channel collision voltage: 6 V; high energy channel collision voltage: 10-30 V; scan range: 100-1400 DaLockMas uses leucine-enkephalin LE, whose m / z is [M+H]+=556.2771, [MH]-=554.2615.

[0068] 7. Test results

[0069] The contents of 6-gingerol, caffeic acid, magnolamine, jatrorrhizine, epiberberine, palmatine hydrochloride and berberine hydrochloride in the freeze-dried powder obtained by the percolation method, reflux extraction method and boiling method were determined. See Table 2 for details. Figure 1 .

[0070] Table 2 Results of target component content determination in freeze-dried powder under different extraction methods (n=6)

[0071] Target ingredients Percolation Reflux extraction method Decoction method 6-Gingerol 17.14 ng / mL 21.22 ng / mL 18.820 ng / mL Caffeic acid 11.03 ng / mL 13.22 ng / mL 10.53 ng / mL Magnolia alkaloids 20.18 ng / mL 18.720 ng / mL 15.49 ng / mL Jatrorrhizine 25.33 ng / mL 30.17 ng / mL 26.55 ng / mL Epiberberine 39.71 ng / mL 43.97 ng / mL 41.56 ng / mL Bamatin hydrochloride 0.137 μg / mL 0.256 μg / mL 0.097 μg / mL Berberine hydrochloride 10.94 μg / mL 12.08 μg / mL 13.16 μg / mL

[0072] Combine Figure 1 The results in Table 2 show that through the optimized reflux extraction process (70% ethanol, two micro-boiling refluxes), the powder yield was increased to 24.30%, and the content of key active ingredients (such as berberine hydrochloride and epiberberine) was significantly higher than that of the percolation method and boiling method. In addition, the process parameters were stable and controllable, ensuring the consistency of the preparation quality.

[0073] 8. Methodological Performance Evaluation

[0074] The analytical method was systematically validated through sample recovery, precision, and stability tests. The RSD% was <2%, meeting the chromatographic methodology validation requirements of the 2020 edition of the Chinese Pharmacopoeia (precision RSD ≤ 2%, recovery RSD ≤ 5%). The results are shown in Table 3.

[0075] Table 3. Methodology performance evaluation results

[0076]

[0077] 9. Selection of extraction process for Huanglian Decoction

[0078] Based on the results of the contents of 6-gingerol, caffeic acid, magnolamine, jatrorrhizine, epiberberine, bamipine hydrochloride and berberine hydrochloride in the freeze-dried powders of the percolation, reflux extraction and boiling methods, we selected the freeze-dried powder of the reflux extraction method with higher overall extraction quality for subsequent pharmacodynamic and molecular mechanism studies on the treatment of gastric precancerous lesions with Huanglian Decoction.

[0079] 10. Study on the therapeutic effect of HLD freeze-dried powder suspension on PLGC model rats

[0080] 10.1 Reproduction of PLGC Pathological Model in Rats

[0081] Male Sprague-Dawley rats, weighing 180 ± 20 g, were purchased from Beijing Sibeifu, license number: 110324220101552336. The experimental animal care and animal welfare committee reviewed and approved this experiment with the license number: XKY-20240820. Throughout the experiment, rats had free access to water and free movement, and the animal experimental center was naturally lit. The room temperature was 20–25°C (±0.5°C), with a 12-hour light / dark cycle, relative humidity of 55% ± 5%, and a maximum temperature difference of ≤4°C. Rats were admitted to the Animal Experimental Center of Chongqing Academy of Animal Science and were fed adaptively for 7 days before being weighed and randomly divided into five groups of eight rats each: a control group (Vehicle), a PLGC model group, a positive drug morodan (MLD) group, a low-dose HLD-L group, and a high-dose HLD-H group.

[0082] A PLGC rat model was established by oral gavage and ad libitum drinking of MNNG solution, combined with irregular feeding for 20 weeks. Specifically, the control group rats were given free access to a 170 μg / mL MNNG solution prepared in pre-cooled (4°C) purified water. They were fasted for three days at an unspecified time each week and allowed to eat freely the rest of the time. Two model rats were randomly sacrificed at weeks 15 and 18, and gastric tissue was obtained for H&E staining to determine the extent of gastric tissue damage.

[0083] 10.2 Preparation of HLD Suspension and Animal Administration

[0084] After the PLGC rat model was successfully established, each treatment group was gavaged with the corresponding treatment regimen once a day for 6 weeks. The control group was gavaged with the same volume of purified water every day, and the model group was gavaged with the same volume of purified water containing 0.5% sodium carboxymethylcellulose (CMC-Na) (see Figure 2 According to the formula: dry powder dosage = raw material amount × yield, the HLD lyophilized powder yield in this study was 24.30%. Therefore, the low and high doses of HLD lyophilized powder were: 1.12 g / kg / d lyophilized powder (equivalent to 5.3 g / kg / d of raw material weight) and 2.24 g / kg / d lyophilized powder (equivalent to 10.6 g / kg / d of raw material weight), respectively. The drug solution was prepared according to the formulation using purified water containing 0.5% CMC-Na at a volume of 5 mL / kg for gavage.

[0085] 10.3 Collection of experimental samples from model rats

[0086] 24 hours after the last administration, the rats were anesthetized by intraperitoneal injection of 20% urethane solution at a standard rate of 1 mL / 100 g according to their body weight. The rat limbs were fixed, the abdominal aorta was exposed, and the whole blood of the rats was collected into a negative pressure blood collection tube containing a coagulant and an inert separation gel (yellow cover, 10 mL). After gently inverting the tube to mix, it was placed in a 4°C refrigerator for 1 hour, centrifuged at 3000 rpm / min in a pre-cooled centrifuge for 10 minutes, and the upper serum sample was aspirated. To avoid repeated freezing and thawing of the samples, the serum samples of each animal were evenly divided into 6 1.5 mL EP tubes and frozen in a -80°C refrigerator for later use. The abdominal cavity was opened to remove the gastric tissue, and the overall morphology of the gastric tissue was observed and photographed for archiving. The gastric body was cut open along the greater curvature of the stomach, and the stomach was turned inside out ( Figure 3 ) and rinsed with saline. The stomach was cut open along the greater curvature, photographed, and preserved. The stomach was then cut into four portions. One portion was fixed in 10% neutral formalin for subsequent pathological staining. To avoid repeated freezing and thawing, the remaining three portions of gastric tissue were aliquoted into three cryovials, stored in liquid nitrogen, and then transferred to a -80°C freezer for later use.

[0087] 10.4 Serum biochemical index level detection and results of rats in each group

[0088] To investigate the therapeutic effect of HLD on PLGC rats, double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure serum levels of inflammatory factors, gastric function-related factors, and gastric acid secretion-related factors. Serum GAST and PGA / PGC expression levels reflect the degree and specific location of gastric mucosal atrophy in rats. IL-1β and IL-6 are important factors in the degree of gastric mucosal inflammation in rats. IL-1β is the most potent inhibitor of gastric acid secretion and indirectly reflects gastric function. Results showed that compared with the vehicle group, serum levels of GAST, IL-1β, and IL-6 in the PLGC group were significantly increased (P < 0.01), while PGA / PGC expression was significantly decreased (P < 0.01). This suggests that MNNG treatment significantly damages gastric mucosal cells in rats. After HLD treatment, serum levels of GAST, IL-1β, and IL-6 were significantly decreased in the HLD-H group compared with the PLGC model group (P < 0.05). The above results showed that the inflammatory indicators and gastric tissue atrophy-related indicators in the serum of rats in the high-dose HLD group were significantly improved (P < 0.05), indicating that HLD has a significant therapeutic effect on MNNG-induced PLGC in rats (Table 4).

[0089] Table 4. Comparison of serum GAST, PGA / PGC, IL-1β, and IL-6 in rats of each group (xˉ±s, n=8)

[0090] Group GAST (ng·L-1) PGA / PGC IL-1β (ng·L-1) IL-6 (pg·L-1) Vehicle group 13.24±1.26 1.46±0.23 74.13±1.62 40.62±4.65 PLGC group <![CDATA[15.32±2.43 ** ]]> <![CDATA[1.03±0.21 ** ]]> <![CDATA[79.67±2.27 ** ]]> <![CDATA[46.32±9.51 ** ]]> HLD-H group <![CDATA[13.73±2.19 # ]]> <![CDATA[1.41±0.37 ## ]]> <![CDATA[72.38±3.35 ## ]]> <![CDATA[43.73±10.39 # ]]> HLD-L group <![CDATA[13.62±3.11 # ]]> <![CDATA[1.39±0.52 ## ]]> <![CDATA[74.38±2.59 ## ]]> 44.67±11.16 MLD group <![CDATA[13.17±2.73 ## ]]> <![CDATA[1.37±0.66 ## ]]> <![CDATA[76.49±3.02 # ]]> <![CDATA[42.66±6.75 # ]]>

[0091] Note: Compared with the Vehicle group,* P<0.05, ** P<0.01; compared with the PLGC group, # P<0.05, ## P < 0.01;

[0092] 10.5 Pathological examination and results of gastric tissues of rats in each group

[0093] H&E staining was used to examine the pathological changes in the stomach tissues of rats in each group before and after HLD treatment. The results showed that the stomach tissues of rats in the Vehicle group had good morphology, rosy color, deep folds and good elasticity; the stomach tissues of rats in the PLGC model group had poor morphology, poor elasticity of the gastric body, grayish-white color, and shallow folds; after HLD treatment, the stomach tissue morphology improved, the color returned to rosy color, and the depth of the gastric body folds improved ( Figure 3 ). After H&E staining, the gastric tissue section showed ( Figure 4 ). In the PLGC model group, rats showed desquamation of gastric mucosal epithelial cells, vacuolation of some mucosal cells, atrophy of the intrinsic glands, and marked congestion and dilation of capillaries, as well as inflammatory cell infiltration and fibrosis. After HLD treatment, the loss of intrinsic glands in the gastric mucosa was alleviated, as were the degree of gastric gland atrophy and inflammatory infiltration. This suggests that HLD treatment significantly improves the pathological changes in gastric tissue of PLGC rats.

[0094] 10.6 Observation of gastric epithelial cell apoptosis by Tunel staining

[0095] In order to observe the apoptosis of gastric mucosal cells in MNNG-induced PLGC rats treated with HLD, TUNEL staining was used to detect gastric tissue. The results are shown in Figure 5 Image J software was used to analyze the Tunel+ fluorescence intensity. The results showed that the apoptosis of cells in the model group was significantly enhanced compared with that in the control group. After HLD treatment, the Tunel+ intensity of gastric mucosal cell apoptosis was significantly reduced (P < 0.01). This result suggests that HLD treatment can improve the apoptosis state of gastric tissue.

[0096] Effects of 10.7HLD freeze-dried powder on gastric tissue protein in PLGC model rats

[0097] Western blotting was used to examine the expression of related proteins in gastric tissue of rats in each group. Results showed that compared with the vehicle group, the expression of PI3K and p-Akt proteins in gastric tissue was significantly increased after MNNG modeling (P < 0.01), while HLD treatment significantly decreased these expressions (P < 0.05). Furthermore, HLD treatment significantly inhibited the expression of cyclin D1 and inflammatory cytokine IL-1β (P < 0.05 or P < 0.01), particularly in the HLD-H group. Furthermore, HLD intervention significantly increased the expression of Cleaved-Caspase 3 and Bax, downstream apoptosis-related signaling factors in the PI3K / Akt signaling pathway (P < 0.05 or P < 0.01), promoting apoptosis. These results suggest that HLD treatment significantly ameliorates mucosal damage in MNNG-induced PLGC rat models, possibly through inhibition of key proteins in the PI3K / Akt pathway.

Claims

1. A method for preparing Huanglian decoction based on reflux extraction optimization, characterized in that: The following steps are involved: Weigh 40g of Coptis chinensis slices, 30g of dried ginger, 30g of liquorice, 30g of cinnamon twig, 25g of jujube, 25g of pinellia tuber, and 20g of ginseng, grind them into coarse powder, sieve them, add 70% ethanol, and soak them for a while; Heat to boiling, keep it at a slight boiling state and reflux for 1 hour, stirring every 15 minutes, filter it while hot, and keep the residue; Add 70% ethanol again, time after boiling, reflux for 1 hour, filter and combine the two filtrates; The above filtrate is concentrated to a thick extract without ethanol taste, and freeze-dried powder is prepared by freeze-drying process.

2. The method for preparing Huanglian Decoction based on reflux extraction optimization according to claim 1, characterized in that: The sieving adopts No. 2 sieve, and the particle size is 2-4 mm.

3. The method for preparing Huanglian Decoction based on reflux extraction optimization according to claim 1, characterized in that: The soaking is performed at room temperature for 30 minutes.

4. The method for preparing Huanglian Decoction based on reflux extraction optimization according to claim 1, characterized in that: The concentration was carried out under reduced pressure at 40° C. until no ethanol smell was found.

5. The method for preparing Huanglian Decoction based on reflux extraction optimization according to claim 4, characterized in that: The freeze-drying process comprises pre-freezing at -20°C and then drying under reduced pressure in a freeze dryer (<50 Pa, <-50°C). 6.HLD freeze-dried powder, characterized by: The product is prepared by the Huanglian Decoction preparation method based on reflux extraction optimization as described in any one of claims 1 to 5.

7. Use of the HLD lyophilized powder according to claim 6 in the preparation of a product for reversing gastric precancerous lesions.

8. The application according to claim 7, characterized in that: HLD inhibits the PI3K / Akt signaling pathway, downregulates the expression of Cyclin D1 and IL-1β proteins, and activates apoptosis-related proteins Cleaved-Caspase3 and Bax.

9. Use of the HLD freeze-dried powder according to claim 6 in the preparation of a product for reducing serum inflammatory factors IL-1β and IL-6.

10. Use of the HLD freeze-dried powder according to claim 6 in the preparation of a drug for improving gastric mucosal atrophy.

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