Honeysuckle and lu jujube root composition

Abstract

This invention provides a honeysuckle and codonopsis composition. The composition, which includes the determination of the main components of codonopsis, scientifically combines honeysuckle (containing iridoids and phenolic acids) with codonopsis (containing alkynes, polysaccharides, and newly discovered compounds) to form a synergistic compound system. An integrated quality control method of "multi-index content determination - dual-wavelength characteristic spectroscopy" ensures stable quality. The invention elucidates that this composition exerts its anti-inflammatory and antioxidant effects through a multi-pathway synergistic mechanism: the honeysuckle component targets and inhibits inflammatory signaling pathways such as NF-κB and MAPK, while the codonopsis component achieves systemic protection by regulating intestinal flora, enhancing the mucosal barrier, and modulating immunity. This composition is suitable for the prevention and treatment of inflammatory diseases such as enteritis and chemotherapy-induced intestinal injury, reflecting a deep integration of the traditional Chinese medicine principle of "clearing heat and strengthening the body, treating both the root cause and symptoms" with modern pharmacological mechanisms.

Description

Technical Field

[0001] This invention relates to the field of medicinal material composition technology, specifically a honeysuckle and codonopsis composition. Background Technology

[0002] Honeysuckle oral liquid is a traditional Chinese medicine, mainly used for clearing heat and detoxifying, and dispersing wind-heat. It is suitable for symptoms such as wind-heat cold, sore throat, prickly heat, and boils. Its core ingredient, honeysuckle, has antiviral, antibacterial, anti-inflammatory, and immunomodulatory effects. Its main functions include: clearing heat and detoxifying: relieving symptoms of heat toxicity such as fever, sore throat, prickly heat, and boils; antiviral and antibacterial: inhibiting influenza A virus, Staphylococcus aureus, etc.; anti-inflammatory and swelling-reducing: reducing congestion and swelling of the throat mucosa; and immunomodulatory: enhancing resistance and preventing respiratory infections.

[0003] Codonopsis pilosula enters the spleen and lung meridians. It contains Codonopsis pilosula polysaccharide (16.8%), Codonopsis pilosula glycoside (10.62%), and Atractylodes lancea lactone III (more than 0.01%). It has the effects of tonifying the spleen and lungs, nourishing blood and promoting body fluids. It is mainly used to treat symptoms such as spleen and lung qi deficiency and qi and blood deficiency. It should not be used with Veratrum nigrum. The traditional processing method uses the "three rubbing and three kneading" technique, and it can be made into oral liquid, honey-fried slices and other dosage forms.

[0004] In the existing technology, the two medicinal materials mentioned above are usually used as separate medicines, but their effects are singular and cannot form a combination of diverse effects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a honeysuckle and codonopsis composition to solve the problems mentioned in the background. This invention features a novel structure and ensures stable quality through an integrated quality control method of "multi-index content determination - dual-wavelength characteristic spectroscopy." It elucidates that this composition exerts its anti-inflammatory and antioxidant effects through a multi-pathway synergistic mechanism: the honeysuckle component targets and inhibits inflammatory signaling pathways such as NF-κB and MAPK, while the codonopsis component provides systemic protection by regulating intestinal flora, enhancing the mucosal barrier, and modulating immunity. This composition is suitable for the prevention and treatment of inflammatory diseases such as enteritis and chemotherapy-induced intestinal injury, embodying a deep integration of the traditional Chinese medicine principle of "clearing heat and strengthening the body, treating both the symptoms and the root cause" with modern pharmacological mechanisms.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: a honeysuckle and codonopsis composition, wherein the preparation method of the composition includes:

[0007] 1) To determine the content of the main components of Codonopsis pilosula, an HPLC method was established for the determination of the content of syringophenol glycoside, L-tryptophan, codonopsis glycoside, codonopsis glycoside alkaloid, codonopsis glycoside I, and atractylodes lactone III in Codonopsis pilosula oral liquid;

[0008] 2) Toxicity analysis of Codonopsis pilosula oral liquid, prevention and relief of intestinal damage caused by chemotherapy drugs, intestinal protection through anti-inflammation and improvement of stem cell function, and protection of intestinal mucosa by increasing intestinal probiotics;

[0009] 3) Determination of relevant pharmacopoeia indicators of honeysuckle medicinal materials, and network pharmacological detection of in vivo exposed components of honeysuckle oral liquid;

[0010] 4) Evaluation of the heat-clearing efficacy and anti-tumor activity of honeysuckle oral liquid and its key components;

[0011] 5) Deepen the scientific understanding of single Chinese herbal medicines (honeysuckle / codonopsis) or their preparations (oral liquid) through modern scientific and technological means, and ultimately serve product quality control and value enhancement.

[0012] Furthermore, according to step 1), the main chemical components of Codonopsis pilosula are glycosides, lignins, alkaloids, coumarins, organic acids, sugars, etc., such as glycosides including codonopsis glycoside, codonopsis glycoside I, syringoglycoside, etc., and terpenoids including atractylodes lactone II, III, etc.

[0013] Furthermore, according to step 2), the toxicity analysis of the Codonopsis pilosula oral liquid was conducted as follows: using healthy C57BL / 6 mice as a model, after 30 days of oral administration of Codonopsis pilosula oral liquid, the weight and survival rate of the mice were not significantly affected, and the liver and kidney function indicators were normal, indicating that Codonopsis pilosula oral liquid has no obvious toxic side effects.

[0014] Furthermore, according to step 2), the Codonopsis pilosula oral liquid prevents and alleviates intestinal damage caused by chemotherapy drugs: an intestinal damage model is constructed using irinotecan, and Codonopsis pilosula oral liquid at 1.5g / kg is administered before chemotherapy (LDS / CPT11), during chemotherapy (LDS+CPT11), and after chemotherapy (CPT11 / LDS).

[0015] Furthermore, according to step 2), the Codonopsis pilosula oral liquid exerts an intestinal protective effect through anti-inflammatory and stem cell function enhancement: a mouse ulcerative colitis model is constructed using dextran sulfate sodium (DSS). By downregulating intestinal inflammatory factors (such as IL-6 and IL-1β) and increasing the expression of stem cell markers (such as Lgr5 and Olfm4), and reducing the infiltration of neutrophils and macrophages, it alleviates DSS-induced intestinal mucosal damage and restores intestinal barrier function.

[0016] Furthermore, according to step 2), the Codonopsis pilosula oral liquid exerts a protective effect by influencing intestinal immunity and metabolic mechanisms: RNA-seq sequencing analysis of the Codonopsis pilosula oral liquid treatment group and the untreated group of ulcerative colitis revealed that compared with the wild control group, the DSS model group had a total of 1346 differentially expressed genes; the Codonopsis pilosula oral liquid treatment group had 2108 differentially expressed genes.

[0017] Furthermore, according to step 3), the determination of relevant pharmacopoeia indicators of honeysuckle medicinal materials: based on the method under the honeysuckle testing item in the 2020 edition of the pharmacopoeia, the indicators such as total phenolic acid, total ash, acid ash, moisture, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C of 12 batches of honeysuckle medicinal materials from different places of origin and years were tested.

[0018] Furthermore, according to step 3), the network pharmacology includes: Network pharmacology method: using the pharmmapper database to predict possible targets of the eight blood-entering components of the above-mentioned key in vivo exposed components, and collecting all heat-cleaning-related proteins from the GeneCards database. Using the PEAC seq dataset, a 3D integrated analysis is performed on the metabolomics results and the results from pharmmapper and GeneCards. Network pharmacology results: the pharmmapper database output a total of 400 possible targets, the GeneCards database output a total of 496 heat-cleaning-related proteins, and in the metabolomics results, there are a total of 375 proteins related to key metabolic pathways.

[0019] Furthermore, according to step 4), the evaluation of the heat-clearing efficacy of the honeysuckle oral liquid and its key components: The heat-clearing effect of the honeysuckle oral liquid was evaluated using an LPS-induced rat fever model. Sixty male SD rats (200g ± 20g) were randomly divided into 6 groups. The specific administration regimens are shown in Table 3.1. The content of the corresponding key heat-clearing components in the Zhenao honeysuckle oral liquid was used as the control for the total phenolic acid and iridoid ethers. Chlorogenic acid was selected as the representative phenolic acid component, and swertiamarin was used as the substitute component for iridoid ethers to examine the correctness of the predicted results of the above metabolomics and network pharmacology.

[0020] Furthermore, according to step 4), the antitumor activity evaluation of the honeysuckle oral liquid and key components was carried out by administering honeysuckle oral liquid to rats by gavage for different time periods. The drug-containing serum was diluted to concentrations of 20%, 15%, 10% and 5% in DMEM complete medium, and the inhibitory effect of the drug-containing serum on A549 cells was detected by CCK-8 assay.

[0021] The beneficial effects of this invention are:

[0022] 1. Both of these inventions follow a systematic research chain: "basic analysis of chemical substances → establishment and improvement of quality standards → elucidation of efficacy mechanisms." Secondly, in terms of quality control, both have broken through the traditional model of qualitative analysis based on a single component, and have shifted to establishing a multi-component, holistic evaluation system. Both have established HPLC multi-index content determination and holistic fingerprinting methods to more comprehensively reflect the chemical characteristics of medicinal materials and preparations. Finally, in terms of efficacy verification, both have adopted modern animal models and molecular biology techniques to explore and confirm the specific pharmacological mechanisms of action of the products, combining traditional efficacy with modern scientific evidence. This reflects the common trend of modern Chinese medicine research moving towards standardization, scientification, and internationalization.

[0023] 2. This invention integrates in vivo process analysis (serum pharmacochemistry), metabolomics, and network pharmacology. It not only clarifies the "components entering the bloodstream," but also screens and verifies 12 phenolic acids and iridoids directly related to the core efficacy (clearing heat) as key quality markers through systems biology methods. For the first time, it applies the Quality Assay Multiple Selection (QAMS) method for efficient and low-cost multi-component quantification. It significantly supplements the existing pharmacopoeia standards, adding modern quality control items such as multi-component content determination, characteristic spectroscopy, heavy metals, and pesticide residues, and introduces new identification technologies such as carbon spectroscopy / mass spectrometry imaging. Its efficacy research takes a unique approach, focusing on the new direction of "intestinal protection," and deeply reveals the novel mechanism by which the product exerts its effects through a comprehensive network of regulating intestinal flora and repairing the mucosal barrier.

[0024] 3. This invention analyzes all the chemical characteristics of medicinal materials (including the discovery of new compounds), and then verifies their overall efficacy (immune regulation, intestinal protection) and explores new mechanisms (microbial regulation) through animal models. The screening of its quality markers is based more on the comprehensiveness of chemical analysis (characteristic spectrum, polysaccharides, various small molecules) and its correlation with the authenticity of the place of origin. Finally, the quality control is achieved by the combination of the overall characteristic spectrum and multiple indicator components.

[0025] 4. In terms of quality standard construction, this invention has established multi-component content determination and characteristic chromatograms, significantly expanded the safety control items (such as heavy metals, pesticide residues, and ash content), and extended the standards to both raw medicinal materials (Codonopsis pilosula) and finished preparations, forming a more complete quality standard system.

[0026] 5. Compared with existing technologies, this invention scientifically combines honeysuckle (containing iridoids and phenolic acids) with Codonopsis pilosula (containing alkynes, polysaccharides, and newly discovered compounds) to form a synergistic compound system. A comprehensive quality control method of "multi-index content determination - dual-wavelength characteristic spectroscopy" ensures stable quality. Furthermore, this invention elucidates that the composition exerts its anti-inflammatory and antioxidant effects through a multi-pathway synergistic mechanism: the honeysuckle component targets and inhibits inflammatory signaling pathways such as NF-κB and MAPK, while the Codonopsis pilosula component provides systemic protection by regulating intestinal flora, enhancing the mucosal barrier, and modulating immunity. This composition is suitable for the prevention and treatment of inflammatory diseases such as enteritis and chemotherapy-induced intestinal injury, embodying a deep integration of the traditional Chinese medicine principle of "clearing heat and strengthening the body, treating both the symptoms and the root cause" with modern pharmacological mechanisms. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the liquid chromatography (LC) spectra of the reference standard (A) and sample (B) of the honeysuckle and codonopsis composition of the present invention;

[0028] Figure 2 This is a schematic HPLC chromatogram of the reference standard ST and the oral liquid sample of the honeysuckle and codonopsis composition of the present invention;

[0029] Figure 3 This is a schematic diagram illustrating the effect of the honeysuckle and codonopsis composition of the present invention, specifically the codonopsis oral liquid, on mouse body weight and survival.

[0030] Figure 4 This is a schematic diagram illustrating how the honeysuckle and codonopsis composition of the present invention, specifically the codonopsis oral liquid, significantly alleviates intestinal mucosal damage caused by irinotecan.

[0031] Figure 5 This is a schematic diagram illustrating how the honeysuckle and codonopsis composition of the present invention, specifically the codonopsis oral liquid, significantly alleviates intestinal damage caused by DSS.

[0032] Figure 6 This is a schematic diagram illustrating how the honeysuckle and codonopsis composition of the present invention, specifically the codonopsis oral liquid, downregulates intestinal inflammatory factors, increases the expression of stem cell markers, and reduces the infiltration of neutrophils and macrophages.

[0033] Figure 7 This is a schematic diagram illustrating how the honeysuckle and codonopsis composition of the present invention, in the form of codonopsis oral liquid, exerts its protective effect by influencing intestinal immunity and metabolism.

[0034] Figure 8 This is a schematic diagram illustrating how the honeysuckle and codonopsis composition of the present invention, specifically the codonopsis oral liquid, exerts its protective effect on the intestinal mucosa by increasing the intestinal probiotic population.

[0035] Figure 9 A schematic diagram illustrating the integrated analysis results of the heat-clearing effect of the honeysuckle and codonopsis composition of the present invention, specifically the genuine honeysuckle oral liquid.

[0036] Figure 10 This is a schematic diagram comparing the heat-clearing effects of the honeysuckle oral liquid and its key components in the honeysuckle and codonopsis composition of the present invention.

[0037] Figure 11 This is a schematic diagram illustrating the effect of chlorogenic acid and swertiamarin in the honeysuckle and codonopsis composition of the present invention on pro-inflammatory factors.

[0038] Figure 12 This is a schematic diagram of the preparation process of the honeysuckle and codonopsis composition of the present invention;

[0039] Figure 13 This is a schematic diagram of the standard curve of syringophenol glycosides in the honeysuckle and codonopsis composition of the present invention.

[0040] Figure 14 This is a schematic diagram of the L-tryptophan standard curve of the honeysuckle and codonopsis composition of the present invention;

[0041] Figure 15 This is a schematic diagram of the standard curve of codonopin I in the honeysuckle and codonopsis composition of the present invention;

[0042] Figure 16 This is a schematic diagram of the standard curve of codonopsis glycoside glycoside in the honeysuckle and codonopsis composition of the present invention;

[0043] Figure 17 This is a schematic diagram of the standard curve of codonopsis glycosides in the honeysuckle and codonopsis composition of the present invention;

[0044] Figure 18 This is a schematic diagram of the standard curve of atractylodes lactone III in the honeysuckle and codonopsis composition of the present invention;

[0045] Figure 19 This is a schematic diagram of the chromatographic analysis of the iridoid ethers in the honeysuckle and codonopsis composition of the present invention.

[0046] Figure 20 This is a schematic diagram of the chromatographic analysis of the phenolic acid components of the honeysuckle and codonopsis composition of the present invention in the Zhenao honeysuckle oral liquid;

[0047] Figure 21 This is a schematic diagram illustrating the anti-inflammatory activities of the six phenolic acids and seven iridoids in the honeysuckle and codonopsis composition of the present invention.

[0048] Figure 22 This is a schematic diagram showing the inhibitory effect of the honeysuckle and codonopsis composition of the present invention on A549 cells in rat serum containing the drug at different administration times;

[0049] Figure 23 This is a schematic diagram illustrating the inhibitory effect of cisplatin (A) and honeysuckle oral liquid-containing serum (B) on A549 cells in the honeysuckle and codonopsis composition of the present invention.

[0050] Figure 24 This is a schematic diagram illustrating the determination of the synergistic index between cisplatin and honeysuckle oral liquid in the serum containing the honeysuckle and codonopsis composition of the present invention.

[0051] Figure 25 This diagram illustrates the inhibitory effects of chlorogenic acid (A) and swertiamarin (C) in the honeysuckle and codonopsis composition of the present invention, as well as the inhibitory effects of cisplatin combined with chlorogenic acid (B) and swertiamarin (D) on A549 cells.

[0052] Figure 26 This is a schematic diagram illustrating the inhibitory effect of cisplatin on A549-DDP cells in the honeysuckle and codonopsis composition of the present invention.

[0053] Figure 27 This is a schematic diagram illustrating the inhibitory effect of the honeysuckle oral liquid of the honeysuckle and codonopsis composition of the present invention on A459-DDP cells;

[0054] Figure 28 This is a schematic diagram illustrating the inhibitory effect of the honeysuckle oral liquid of the honeysuckle and codonopsis composition of the present invention on A459-DDP after alcohol precipitation.

[0055] Figure 29 This is a schematic diagram illustrating the inhibitory effects of chlorogenic acid and swertiamarin in the honeysuckle and codonopsis composition of the present invention on A459-DDP cells. Detailed Implementation

[0056] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0057] Please see Figures 1 to 29 The present invention provides a technical solution: the method for preparing the composition includes:

[0058] 1) To determine the content of the main components of Codonopsis pilosula, an HPLC method was established for the determination of the content of syringophenol glycoside, L-tryptophan, codonopsis glycoside, codonopsis glycoside alkaloid, codonopsis glycoside I, and atractylodes lactone III in Codonopsis pilosula oral liquid;

[0059] 2) Toxicity analysis of Codonopsis pilosula oral liquid, prevention and relief of intestinal damage caused by chemotherapy drugs, intestinal protection through anti-inflammation and improvement of stem cell function, and protection of intestinal mucosa by increasing intestinal probiotics;

[0060] 3) Determination of relevant pharmacopoeia indicators of honeysuckle medicinal materials, and network pharmacological detection of in vivo exposed components of honeysuckle oral liquid;

[0061] 4) Evaluation of the heat-clearing efficacy and anti-tumor activity of honeysuckle oral liquid and its key components;

[0062] 5) Deepen the scientific understanding of single Chinese herbal medicines (honeysuckle / codonopsis) or their preparations (oral liquid) through modern scientific and technological means, and ultimately serve product quality control and value enhancement.

[0063] Determination of the main components of Codonopsis pilosula:

[0064] The main chemical components of Codonopsis pilosula include glycosides, lignins, alkaloids, coumarins, organic acids, and sugars. Glycosides include codonopsis glycoside, codonopsis glycoside I, and syringoside, while terpenoids include atractylodes lactone II and III. Codonopsis glycoside, codonopsis glycoside alkaloid, codonopsis glycoside I, and atractylodes lactone III are important small-molecule components in Codonopsis pilosula. Studies have shown that these components are directly related to the anti-inflammatory, antioxidant, and anti-tumor effects of Codonopsis pilosula. To reflect and control the intrinsic quality of Codonopsis pilosula oral liquid, an HPLC method was established to determine the content of syringoside, L-tryptophan, codonopsis glycoside, codonopsis glycoside alkaloid, codonopsis glycoside I, and atractylodes lactone III in Codonopsis pilosula oral liquid.

[0065] (1) Experimental materials

[0066] Instruments: Agilent 1260 high performance liquid chromatograph, Mettler AG135 0.0001 g balance, KQ5200DE CNC ultrasonic cleaner.

[0067] Reagents: Syringophenol glycoside, L-tryptophan, Codonopsis pilosula glycoside I, Codonopsis pilosula glycoside, Codonopsis pilosula glycoside, and Atractylodes macrocephala lactone III reference standards were purchased from Nantong Feiyu Biotechnology Co., Ltd., all with a purity of 98% HPLC grade. Acetonitrile and methanol were purchased from Thermo Fisher Scientific, Inc., USA. Water was purified water purchased from Hangzhou Wahaha Group Co., Ltd., and other reagents were of analytical grade.

[0068] (2) Methods and Results

[0069] Chromatographic conditions: Column: V-0203052-5250 Svea (4.6 mm x 250 mm, 5 μm); Mobile phase: 0.1% phosphoric acid (A) – acetonitrile (B); Flow rate: 0.8 mL / min; Gradient elution program: 0–10 min, 95%–90% (A); 10–30 min, 90%–85% (A); 30–50 min, 85%–80% (A); 50–65 min, 80%–70% (A); 65–75 min, 70%–50% (A); 75–80 min, 50%–30% (A); 80–85 min, 30% (A); 85–86 min, 30%–10% (A); 86–93 min, 10% (A); 93–95 min, 10%–95% (A); 95–110 min… min; 95% (A), detection wavelength 210 nm, column temperature 25 ℃, injection volume 25 μL.

[0070] Preparation of reference solutions: Accurately weigh appropriate amounts of syringoglycoside, L-tryptophan, codonopsis glycoside I, codonopsis glycoside, codonopsis glycoside, and atractylodes lactone III standards, dilute to volume with methanol, and prepare a 1 mg / mL reference stock solution. Take appropriate amounts of each reference stock solution and mix them, then dilute with methanol to obtain mixed reference stock solutions with concentrations of 25, 50, 50, 25, 50, and 50 µg / mL for syringoglycoside, L-tryptophan, codonopsis glycoside I, codonopsis glycoside, codonopsis glycoside, and atractylodes lactone III, respectively.

[0071] Preparation of the test solution: Dry and pulverize the Codonopsis pilosula herb, accurately weigh 2 g of Codonopsis pilosula powder into a 25 mL stoppered conical flask, add 10 mL of 90% methanol, seal, shake, weigh, in a 75℃ water bath for 1 h, cool, weigh, make up the weight loss, transfer to a 50 mL centrifuge tube, centrifuge, collect the supernatant, and filter through a 0.22 µm filter membrane to obtain the test solution.

[0072] Methodological examination:

[0073] 1) Chromatographic Condition Determination: Accurately pipette 25 μL of each of the above solutions and inject them into the liquid chromatograph (Agilent 1100 HPLC). Use a V-0203052-5250 Svea (4.6 mm 250 mm, 5 μm) and perform the tests under the aforementioned chromatographic conditions. See [link to chromatographic conditions]. Figure 1 Furthermore, the peak shape and separation effect were quite ideal (1: syringoside; 2: L-tryptophan; 3: codonopsis glycoside I; 4: codonopsis glycoside; 5: codonopsis glycoside; 6: atractylodes lactone III).

[0074] 2) Linearity test: Take the mixed reference stock solution prepared above, dilute it with methanol to obtain mixed reference solutions of different concentrations, centrifuge and collect the supernatant, inject and determine the sample. Plot a standard curve with the peak area of ​​each reference as the ordinate and the concentration as the abscissa, and record the limit of detection (LOD) and limit of quantitation (LOQ). See Tables 6-10 to 6-16 for details.

[0075] Table 6-10 Results of linear relationship investigation of syringoglycosides

[0076] Concentration (µg / mL) 12.5 6.25 3.125 1.5625 0.78125 0.390625 area 6610.9 3372.9 1708.9 854.9 433.2 213.5

[0077] Table 6-11 Results of the linear relationship investigation of L-tryptophan

[0078] Concentration (µg / mL) 50 25 12.5 6.25 3.125 1.5625 0.78125 area 60140.8 33471.4 17282.4 8769.9 4390.9 2206.5 1109.3

[0079] Table 6-12 Results of linear relationship investigation of codonopin I

[0080] Concentration (µg / mL) 25 12.5 6.25 3.125 1.5625 0.78125 area 7891.1 4065.8 2038.9 1024.2 524.5 251.6

[0081] Table 6-13 Results of linear relationship investigation of Codonopsis pilosula glycosides

[0082] Concentration (µg / mL) 12.5 6.25 3.125 1.5625 0.78125 0.390625 area 9521.2 4801.6 2408.2 1209.6 610.3 308.7

[0083] Table 6-14 Results of linear relationship investigation of Codonopsis pilosula glycosides

[0084] Concentration (µg / mL) 25 12.5 6.25 3.125 1.5625 0.78125 area 25369.3 13284 6745 3392.6 1710.8 865.1

[0085] Table 6-15 Results of linearity study of atractylodes lactone III

[0086] Concentration (µg / mL) 25 12.5 6.25 3.125 1.5625 0.78125 area 11104.5 5630 2831.7 1419.8 714.6 355.9

[0087] Table 6-16 Results of Linearity Study

[0088] Element Regression equation <![CDATA[r 2 ]]> Linear range (µg / mL) Syringophenol glycosides y = 528.18x + 32.696 0.9999 0.39~12.50 L-Tryptophan y = 1337.2x + 235.98 0.9997 0.78~50.00 Codonopinion I y = 315.53x + 44.362 0.9998 0.78~25.00 Codonopsis pilosula glycoside y = 760.95x + 22.2 1 0.39~12.50 Codonopsis glycosides y = 1013.1x + 250.28 0.9994 0.78~25.00 Atractylodes lactone III y = 443.86x + 35.059 0.9999 0.78~25.00

[0089] The experimental results showed that syringophenol glycoside, L-tryptophan, codonopin I, codonopinyl glycoside, codonopinyl glycoside, and atractylodes lactone III exhibited good linearity in concentration ranges of 0.39–12.50, 0.78–50.00, 0.78–25.00, 0.39–12.50, 0.78–25.00, and 0.78–25.00 µg / mL, respectively, as detailed in Table 6-16.

[0090] Content determination of samples: The content of 10 batches of Codonopsis pilosula oral liquid samples was determined. The results are shown in Table 6-17 (1: syringoside; 2: L-tryptophan; 3: codonopsis pilosula glycoside I; 4: codonopsis pilosula glycoside; 5: codonopsis pilosula glycoside; 6: atractylodes macrocephala lactone).

[0091] Table 6-17 Determination of the content of each component in Codonopsis pilosula oral liquid (mg / mL)

[0092] serial number L-Tryptophan Codonopinion I Codonopsis pilosula and Gan Ning Codonopsis glycosides Atractylodes lactone III DSKFY-0302 NA NA NA 0.057 0.024 DSKFY-0303 NA NA NA 0.065 0.023 DSKFY-0304 NA NA NA 0.063 0.022 DSKFY-0305 NA NA NA 0.062 0.022 DSKFY-0306 NA NA NA 0.057 0.023 DSKFY-0307 NA NA NA 0.055 0.023 DSKFY-0308 NA NA NA 0.052 0.021 DSKFY-0309 NA NA NA 0.056 0.022 DSKFY-0310 NA NA NA 0.056 0.021 DSKFY-0311 NA NA NA 0.058 0.021

[0093] Results: The content of codonopsis glycoside in 10 batches of Codonopsis pilosula oral liquid ranged from 0.052 to 0.065 mg / mL, with an average of 0.058 mg / mL. The content of atractylodes lactone III ranged from 0.021 to 0.024 mg / mL, with an average of 0.022 mg / mL. The total amount of codonopsis glycoside and atractylodes lactone III per 1 mL of this product shall not be less than 0.08 mg.

[0094] Example 1, Toxicity analysis of Codonopsis pilosula oral liquid: Using healthy C57BL / 6 mice as a model, after 30 days of oral administration of Codonopsis pilosula oral liquid, there was no significant effect on mouse body weight or survival rate, and liver and kidney function indicators were normal, indicating that Codonopsis pilosula oral liquid has no obvious toxic side effects. See details below. Figure 3 .

[0095] Example 2: Prevention and Alleviation of Chemotherapy-Induced Intestinal Injury by Codonopsis pilosula Oral Solution: Irinotecan is a chemotherapy drug that easily causes severe gastrointestinal reactions. This study used irinotecan to construct an intestinal injury model and administered Codonopsis pilosula oral solution at 1.5 g / kg before chemotherapy (LDS / CPT11), during chemotherapy (LDS+CPT11), and after chemotherapy (CPT11 / LDS). The results showed that taking the oral solution before chemotherapy significantly alleviated irinotecan-induced intestinal mucosal damage, maintained intestinal barrier function, and reduced intestinal bleeding in mice. Therefore, Codonopsis pilosula oral solution can effectively prevent and alleviate chemotherapy-induced intestinal toxicity and has a good protective effect. (See details...) Figure 4 .

[0096] Example 3: Codonopsis pilosula oral liquid exerts intestinal protective effects through anti-inflammatory and stem cell function enhancement: A mouse ulcerative colitis model was constructed using dextran sulfate sodium (DSS). It was found that Codonopsis pilosula oral liquid alleviated DSS-induced intestinal mucosal damage and restored intestinal barrier function by downregulating intestinal inflammatory factors (such as IL-6 and IL-1β), increasing the expression of stem cell markers (such as Lgr5 and Olfm4), and reducing neutrophil and macrophage infiltration. (See details...) Figure 5 and Figure 6 .

[0097] Example 4: Codonopsis pilosula oral liquid exerts a protective effect by influencing intestinal immunity and metabolic mechanisms: RNA-seq sequencing analysis of ulcerative colitis patients treated with and untreated with Codonopsis pilosula oral liquid revealed 1346 differentially expressed genes in the DSS model group compared to the wild-type control group; the Codonopsis pilosula oral liquid treatment group had 2108 differentially expressed genes. GO and KEGG analyses of these differentially expressed genes showed that the differentially expressed genes after drug treatment were mainly enriched in immunoglobulin A (IgA)-related signaling pathways such as intestinal immunity, necrosis, extracellular interactions, protein digestion and absorption, and inflammatory lipid metabolism. The experimental results also confirmed that Codonopsis pilosula oral liquid can exert a protective effect by activating intestinal immune function, reducing inflammatory lipids and cell cycle, and promoting nutrient absorption. See details... Figure 7 .

[0098] Example 5: Codonopsis pilosula oral liquid enhances the protective effect of intestinal mucosa by increasing intestinal probiotics: Intestinal bacterial dysbiosis is crucial for the occurrence of enteritis. Analysis of intestinal microbiota data showed that the increased abundance ratio (F / B) of Firmicutes to Bacteroidetes in inflammatory mice indicated that DSS caused dysbiosis. However, Codonopsis pilosula oral liquid significantly reduced the abundance ratio of Firmicutes to Bacteroidetes and increased the relative abundance of beneficial bacteria, such as Alistipes, Muribacacea, and Alloprevotella. Therefore, the oral liquid can reshape the intestinal microenvironment and promote intestinal mucosal repair. (See details...) Figure 8 .

[0099] The above results confirm that Codonopsis pilosula oral liquid significantly alleviates intestinal mucosal damage caused by chemotherapy drugs irinotecan and DSS, and restores intestinal barrier function. It exerts its protective effect through a comprehensive regulatory mechanism. The drug can increase the relative abundance of beneficial bacteria, reshape the intestinal microenvironment, significantly reduce the inflammatory microenvironment, and has immunomodulatory effects. At the same time, it promotes intestinal mucosal repair and nutrient metabolism. This study is the first to explore the application potential and mechanism of action of Codonopsis pilosula oral liquid in intestinal damage caused by chemotherapy drugs and DSS. It has found its important role in regulating the intestinal microenvironment and immune response, and confirmed that the preventive and protective effect of the oral liquid has no significant adverse reactions.

[0100] Determination of relevant pharmacopoeia indicators of honeysuckle medicinal materials: According to the methods under the honeysuckle testing item in the 2020 edition of the pharmacopoeia, 12 batches of honeysuckle medicinal materials from different origins and years were tested for indicators such as total phenolic acid, total ash, acid ash, moisture, chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C. As shown in Table 4.1, the total phenolic acid content of honeysuckle medicinal materials from older years was lower, the total phenolic acid content of second-harvest flowers and flowers from the previous year was lower, and the chlorogenic acid content of second-harvest flowers was lower. The results suggest that the first harvest of the current year should be used as the raw material for preparing honeysuckle oral liquid.

[0101] Table 4.1 Pharmacopoeia-related indicator test results of 12 batches of honeysuckle medicinal materials

[0102] Serial Number batch number Origin Total phenolic acids (%) (converted to water content) Total Ash % acid ash% Moisture% Chlorogenic acid % (converted to water) Isochlorogenic acid A% (converted to water) Isochlorogenic acid C% (converted to water) 1 190301 Pingyi, Shandong 3.90 5.54 0.54 10.0 2.50 1.13 0.27 2 190501 Pingyi, Shandong 3.77 6.41 1.12 9.5 2.38 1.13 0.27 3 190502 Pingyi, Shandong 4.16 5.67 0.44 9.0 2.61 1.26 0.29 4 20181202 Pingyi, Shandong 3.62 5.71 0.74 8.5 2.41 0.98 0.23 5 20181202 Pingyi, Shandong 3.68 5.57 0.58 9.5 2.47 0.97 0.24 6 2022.05 Shandong first batch 4.46 6.12 1.02 8.5 2.96 1.28 0.22 7 2022.5 Shandong Borun Henan first batch 3.95 9.42 3.93 9.5 2.34 1.37 0.23 8 2022.5 Shandong Borun Hebei first batch 4.33 6.41 1.11 9.0 3.00 1.14 0.19 9 Second crop of flowers in 2021 Pingyi, Shandong 1.88 7.26 1.90 9.0 1.18 0.58 0.11 10 2021 biennial flowers Pingyi, Shandong 3.63 6.35 0.80 8.5 2.30 1.10 0.23 11 2021 Henan 3.83 8.16 2.45 8.5 2.26 1.34 0.22 12 2021 Hebei 2.85 7.35 1.52 8.5 1.83 0.81 0.20 Pharmacopoeia content limits >3.8% <10.0% <3.0% <12.0% >1.5%

[0103] Chromatographic conditions for analysis of key quality markers: Instrument: Shimadzu LC-20AT high performance liquid chromatograph; Column: Luna® C18(2) 100Å (250 mm × 4.6 mm, 5 μm); Mobile phase: 0.4% phosphoric acid water (A) - acetonitrile (B); Flow rate: 1 mL / min; Column temperature: 28 ℃; Injection volume: 5 μL; Detection wavelengths: 240 nm and 327 nm; Gradient elution program is shown in Table 4.2.

[0104] Table 4.2 Gradient elution conditions

[0105] Time (min) A(%) B(%) 0 90 10 15 88 12 32 75 25 43 50 50 47 0 100 55 0 100 57 90 10 66 90 10

[0106] Validation of analytical methods for key quality markers:

[0107] Preparation of iridoid reference solutions: Accurately weigh appropriate amounts of iridoid reference standards loganic acid (I1), strychnosine (I2), strychnosine (I3), strychnosine (I5), strychnosine (I6), and strychnosine (I7), and add 75% methanol to prepare mixed solutions containing 0.2083, 0.3750, 1.4583, 0.1250, 0.2500, and 0.2917 mg per mL, respectively, as iridoid reference solutions.

[0108] like Figure 19 As shown, the analytical method for iridoid components is as follows: Take an appropriate amount of the iridoid reference solution prepared under section 4.3.1 and dilute it as sample 1; take an appropriate amount of the test solution prepared under section 4.3.3 and dilute it as sample 2; mix the iridoid reference solution and the test solution and dilute them appropriately as sample 3. Determine the iridoid components at a detection wavelength of 240 nm according to the chromatographic conditions under section 4.2. The chromatogram is shown in Table 4.1. The six iridoid components showed good separation and moderate retention time, and can be used for subsequent analysis. In the figure, Line 1: (Sample 1; Line 2: Sample 2; Line 3: Sample 3).

[0109] Preparation of phenolic acid reference solutions: Accurately weigh appropriate amounts of phenolic acid reference standards neochlorogenic acid (P1), chlorogenic acid (P2), cryptochlorogenic acid (P3), isochlorogenic acid B (P4), isochlorogenic acid A (P5), and isochlorogenic acid C (P6), and add 75% methanol to prepare mixed solutions containing 0.30, 1.10, 0.35, 0.16, 0.27, and 0.24 mg per mL, respectively, as phenolic acid reference solutions.

[0110] like Figure 20 As shown, the analytical method for phenolic acid components is as follows: Take an appropriate amount of the phenolic acid reference solution prepared under section 4.3.1 and dilute it as sample 4; take an appropriate amount of the test solution prepared under section 4.3.3 and dilute it as sample 5; mix the phenolic acid reference solution and the test solution and dilute them appropriately as sample 6. Measure the phenolic acid components at a detection wavelength of 327 nm according to the chromatographic conditions under section 4.2. The chromatogram is shown in Table 4.2. The six phenolic acid components show good separation and moderate retention time, and can be used for subsequent analysis. In the figure (Line 4: Sample 4; Line 5: Sample 5; Line 6: Sample 6).

[0111] Network pharmacology of in vivo exposed components of honeysuckle oral liquid:

[0112] Network pharmacology approach: The pharmmapper database was used to predict the possible targets of the eight blood-entering components of the above key in vivo exposed components. The GeneCards database was used to collect all proteins related to heat-cleaning. The PEACseq dataset was used to perform a 3D integrated analysis of the metabolomics, pharmmapper, and GeneCards results.

[0113] Network pharmacology results: The pharmmapper database output 400 possible targets, the GeneCards database output 496 proteins related to heat-cleaning, and the metabolomics results showed that there were 375 proteins related to key metabolic pathways.

[0114] The key components for clearing heat in honeysuckle oral liquid were integrated and identified: Using the PEAC seq dataset, a three-dimensional map was constructed based on the correlation score of each protein. The metabolomics analysis results and network pharmacology results were successfully integrated. The results showed that the two top-ranked targets were MAPK3 (erk1) and AKT1. These two targets are regulated by representative phenolic acids and iridoids such as chlorogenic acid and loganic acid, respectively. Therefore, they were listed as key components for clearing heat.

[0115] Example 1: Evaluation of the heat-clearing efficacy of honeysuckle oral liquid and its key components:

[0116] Efficacy evaluation of heat-clearing properties: A rat fever model induced by LPS lipopolysaccharide was used to evaluate the heat-clearing effect of honeysuckle oral liquid. Sixty male SD rats (200 g ± 20 g) were randomly divided into 6 groups. The specific administration regimens are shown in Table 3.1. The contents of the corresponding key heat-clearing components in Zhenao honeysuckle oral liquid were used as controls for the key pharmacological components of total phenolic acids and iridoids. Chlorogenic acid was selected as the representative phenolic acid component, and swertiamarin was used as the substitute component for iridoids to examine the correctness of the predicted results of the above metabolomics and network pharmacology. The rats were injected with LPS (1 mg / kg) intraperitoneally. The rectal temperature of the rats was measured using a live in vivo scanning system (Chengdu Taimeng) at 0 h, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 and 3.0 h after treatment, and the rectal temperature curve was plotted.

[0117] Table 3.1 LPS-induced fever model in rats

[0118] Grouping Dosing regimen LPS 1 mg / kg LPS intraperitoneal injection Zhenao Honeysuckle Oral Liquid (HEP) 1 mg / kg LPS administered intraperitoneally, followed by approximately 9 ml / kg of 800 mg / kg (crude drug weight) administered by gavage. Chlorogenic acid combined with swertiamarin (CA+SWE) 1 mg / kg LPS intraperitoneal injection followed by 20 + 10 mg / kg CA + SWE via gavage Aspirin 1 mg / kg LPS intraperitoneal injection followed by 20+10 mg / kg aspirin intraperitoneal injection Chlorogenic acid (CA) 1 mg / kg LPS intraperitoneally followed by 20 mg / kg CA via gavage Swertia triterpenoids (SWE) 1 mg / kg LPS intraperitoneally followed by 10 mg / kg SWE by gavage

[0119] After intraperitoneal injection of lipopolysaccharide in rats, the anal temperature increased significantly at 0.5 h and 2.25 h. Compared with the LPS group, the positive control drug aspirin group significantly reduced the area under the curve of anal temperature (###, p < 0.001), indicating that the model was successfully established.

[0120] The antipyretic effects at 0.5 h and 2.25 h were examined. Compared with the rectal temperature of the model group, the HEP, CA+SWE, CA, and SWE groups all showed significant differences (**, p < 0.01; ***, p < 0.001). However, there was no significant difference in efficacy between the CA+SWE group and the HEP group (ns). This indicates that the combined use of total phenolic acids and total iridoids (calculated as chlorogenic acid and swertiamarin) in the preparation is as effective as using Zhenao Honeysuckle Oral Liquid. This proves that the integrated analysis results of metabolomics and network pharmacology are correct, and phenolic acids and iridoids can be used as quality markers in Zhenao Honeysuckle Oral Liquid.

[0121] Chlorogenic acid showed a significant advantage in clearing heat in the first 0.5 h (^^^, p < 0.001); while in the later 2.25 h, swertiamarin showed a greater advantage in clearing heat than chlorogenic acid (^, p < 0.05).

[0122] Detection of relevant inflammatory factors: To further evaluate the accuracy of the predicted pathways in this experiment, we assessed the effects of Zhenao Honeysuckle Oral Liquid, chlorogenic acid, and swertiamarin on the expression of PGE2, IL-1, and IL-6, the main fever-related inflammatory mediators involved in the downstream of the MAPK and PI3K-AKT pathways.

[0123] Compared with the LPS model group, HEP, CA, SWE, and CA+SWE groups all showed good inhibitory effects at 0.5 h, with significant differences compared with the LPS model group (**, p < 0.01; ***, p < 0.001). The combined treatment of chlorogenic acid and swertiamarin and the effects of Zhenao honeysuckle oral liquid on PGE2, IL-1, and IL-6 were not significantly different (ns); while the therapeutic effect of chlorogenic acid alone was better than that of swertiamarin (^, ​​p < 0.05; ^^^, p < 0.001).

[0124] Furthermore, at 2.25 h, there was no significant difference (ns) between the combined treatment of chlorogenic acid and swertiamarin and the effects of Zhenao honeysuckle oral liquid. However, the therapeutic effects of chlorogenic acid and swertiamarin were reversed, with swertiamarin showing a significant advantage (^, p < 0.05; ^^, p < 0.01). It is speculated that this may be related to the differences in the absorption and metabolism rates of the two small molecules in vivo and the forms in which they exert their effects. This result is basically consistent with the heat-clearing effect, indicating that phenolic acids and iridoids can serve as quality markers for heat-clearing in oral liquids.

[0125] Anti-inflammatory activity assessment: HEK-293T cells with good growth and confluence of 80%–90% were collected, digested with trypsin to obtain cell suspension, and an appropriate amount was evenly spread in 96-well plates and incubated statically at 37 °C in a 5% CO2 incubator. When the cell confluence in the wells reached 50%–70%, PGL4.32 plasmid (100 ng / well), Renilla plasmid (8.3 ng / well), and liposome 2000 (PEI, 1 mg / mL) were evenly mixed in blank medium and incubated for 15 min. The mixture was then diluted with an appropriate amount of blank medium and added to the cell 96-well plates. The plates were incubated at 37 °C in a 5% CO2 incubator for 24 h for transfection.

[0126] like Figure 21 As shown, the experiment was divided into a blank group (Con), a model group (Mod, TNF-α concentration of 5 ng / mL), a positive control group (Dex, 10⁻⁵ mol / L dexamethasone), and various drug administration groups (10⁻⁵ mol / L). The positive control group and all drug administration groups used culture medium containing 5 ng / mL TNF-α as a solvent and were administered for 6 days. After h, the anti-inflammatory activities of six iridoids and swertiamarin, as well as six phenolic acids, in honeysuckle oral liquid were detected using the NF-κB dual-luciferase reporter gene system. Seven iridoids, including loganic acid, strychnosine, strychnosine, strychnosine, strychnosine, strychnosine, and swertiamarin, and six phenolic acids, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C, all showed significant NF-κB inhibitory activity. The anti-inflammatory activities of strychnosine and swertiamarin were essentially equivalent, with no significant statistical difference. (Comparison with Group Con:) * P < 0.05 ** P < 0.01, *** P < 0.001, ### P<0.001; ns, no significant difference; n=5).

[0127] Example 2: Evaluation of the antitumor activity of honeysuckle oral liquid and its key components:

[0128] Preparation of drug-containing serum in rats: 24 male SD rats (200 g ± 20 g) were randomly divided into a Con group and drug-treated groups (30 min group, 1 h group, and 2 h group), with 6 rats in each group. After weighing, blood was collected from the abdominal aorta of the Con group after anesthesia. The drug-treated groups were administered honeysuckle oral liquid by gavage at a dose of 17.5 mL / kg. Blood was collected from the abdominal aorta at 30 min, 1 h, and 2 h after anesthesia. Whole blood was collected into vacuum blood collection tubes and left at room temperature for 2 h. After centrifugation at 3500 rpm for 10 min at 4 °C, blank serum and drug-containing serum were obtained. Three times the amount of methanol was added, and the mixture was vortexed for 3 min to precipitate the protein. After centrifugation at 12000 rpm for 10 min at 4 °C, the supernatant was dried under nitrogen and used for later use.

[0129] like Figure 22 As shown, the inhibitory effect on A549 cells was investigated: Rat serum containing honeysuckle oral liquid was administered by gavage at different time points and diluted to 20%, 15%, 10%, and 5% concentrations in DMEM complete medium. The inhibitory effect of the drug-containing serum on A549 cells was detected by CCK-8 assay. The IC50 values ​​for the 30 min, 1 h, and 2 h administration groups were 1.868%, 7.5%, and 2.454%, respectively. The diagram shows the inhibitory effect of drug-containing serum on A549 cells in rats after 30 min (A), 1 h (B), and 2 h (C) administration (n=5).

[0130] like Figure 23 As shown, cisplatin concentrations were set at 2.5, 5, 10, 20, 40, and 80 μM, and honeysuckle-containing serum concentrations were set at 6.25%, 12.5%, and 25%. The inhibitory effects of cisplatin and honeysuckle-containing serum on A549 cells were investigated separately. The IC50 of cisplatin was 31.37 µM, and the IC50 of the honeysuckle-containing serum was 7.438% (n=6 in the figure).

[0131] like Figure 24 As shown, the inhibitory effect of the above concentrations of cisplatin combined with honeysuckle-containing serum on A549 cells was investigated by CCK-8 assay. The synergy index (CI value) was analyzed using CompuSyn software. <1 indicates synergistic effect, =1 indicates additive effect, and >1 indicates antagonistic effect. The results showed that the combination of different concentrations of cisplatin and honeysuckle oral liquid-containing serum exhibited synergistic inhibitory effects on A549 cells at some concentrations. Preferably, the ratio of cisplatin to drug-containing serum was 40 μM cisplatin and 12.5% ​​drug-containing serum, as shown in the figure (n=6).

[0132] like Figure 25As shown in the figure, the concentration of cisplatin was set at 40 µM, and the concentrations of chlorogenic acid and swertiamarin were 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM. The inhibitory effects of chlorogenic acid and swertiamarin, as well as cisplatin in combination with the two, on A549 cells were investigated by CCK-8 assay. The use of chlorogenic acid and swertiamarin alone, or the combination of the two with cisplatin, did not show a dose-dependent inhibitory effect on A549 cells (n=6 in the figure).

[0133] like Figure 26 As shown, the inhibitory effect of cisplatin on A549 cisplatin-resistant (A549-DDP) cells was investigated by setting cisplatin concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM, and the IC50 value of cisplatin was 53.43 μM (n=6 in the figure).

[0134] like Figure 27 As shown in the figure, the concentrations of honeysuckle oral liquid were set at 0.03175, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg / mL. The inhibitory effect of honeysuckle oral liquid on A549-DDP cells was investigated by CCK-8 assay. Honeysuckle oral liquid did not show a dose-dependent inhibitory effect on A549-DDP cells (n=6 in the figure).

[0135] like Figure 28 As shown, after precipitating the sugars in the honeysuckle oral liquid with 70% ethanol, the mixture was centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was then dried under reduced pressure. The concentrations of the honeysuckle oral liquid were set at 0.040, 0.079, 0.159, 0.317, 0.625, 1.25, 2.5, 5, and 10 mg / mL. The inhibitory effect of the ethanol-precipitated honeysuckle oral liquid on A549-DDP cells was investigated using CCK-8 assay. The ethanol-precipitated honeysuckle oral liquid did not show a dose-dependent inhibitory effect on A549-DDP cells (n=6 in the figure).

[0136] like Figure 29 As shown, the concentrations of chlorogenic acid and swertiamarin were set at 31.25, 62.5, 125, 250, 500, and 1000 μM. The inhibitory effects of chlorogenic acid and swertiamarin on A549-DDP cells were investigated using CCK-8 assay. Neither chlorogenic acid nor swertiamarin showed a dose-dependent inhibitory effect on A549-DDP cells.

[0137] By combining network pharmacological analysis of the main components exposed in vivo in honeysuckle oral liquid, integrating target proteins from the GeneCards heat-clearing database, and metabolomics analysis results, it was revealed that phenolic acids and iridoids mainly exert their antipyretic effects by regulating the MAPK and PI3k-AKT pathways through the two targets MAPK3(erk1) and AKT1, which are the main key components for heat-clearing.

[0138] By comparing the effects of phenolic acids and iridoids on LPS-induced fever and the expression of major fever-related inflammatory mediators PGE2, IL-1, and IL-6, it was found that the combined treatment with these compounds did not differ significantly from the heat-clearing efficacy of the oral liquid. However, chlorogenic acid showed a significant advantage in heat-clearing effect in the early stage, while swertiamarin showed a slight advantage in heat-clearing effect in the later stage. Furthermore, all six phenolic acids and six iridoids in the honeysuckle oral liquid showed significant NF-κB inhibitory activity, indicating that phenolic acids and iridoids can serve as quality markers for heat-clearing in oral liquids.

[0139] The inhibitory activity of honeysuckle oral liquid, representative phenolic acids, and iridoids on tumor cells A549 and A549-DDP was evaluated using the CCK-8 assay. The results showed no dose-dependent significant inhibitory effect on lung cancer A549 cells.

[0140] The foregoing has shown and described the basic principles and main features of the present invention and its advantages. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention.

[0141] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A honeysuckle and codonopsis composition, characterized in that: The method for preparing the composition includes: 1) To determine the content of the main components of Codonopsis pilosula, an HPLC method was established for the determination of the content of syringophenol glycoside, L-tryptophan, codonopsis glycoside, codonopsis glycoside alkaloid, codonopsis glycoside I, and atractylodes lactone III in Codonopsis pilosula oral liquid; 2) Toxicity analysis of Codonopsis pilosula oral liquid, prevention and relief of intestinal damage caused by chemotherapy drugs, intestinal protection through anti-inflammation and improvement of stem cell function, and protection of intestinal mucosa by increasing intestinal probiotics; 3) Determination of relevant pharmacopoeia indicators of honeysuckle medicinal materials, and network pharmacological detection of in vivo exposed components of honeysuckle oral liquid; 4) Evaluation of the heat-clearing efficacy and anti-tumor activity of honeysuckle oral liquid and its key components; 5) Deepen the scientific understanding of single Chinese medicines or their preparations through modern scientific and technological means, and ultimately serve product quality control and value enhancement.

2. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 1), the main chemical components of Codonopsis pilosula are glycosides, lignins, alkaloids, coumarins, organic acids, sugars, etc. For example, glycosides include codonopsis glycoside, codonopsis glycoside I, syringoglycoside, etc., and terpenoids include atractylodes lactone II, III, etc.

3. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 2), the toxicity analysis of the Codonopsis pilosula oral liquid was conducted as follows: using healthy C57BL / 6 mice as a model, after 30 days of oral administration of Codonopsis pilosula oral liquid, the weight and survival rate of the mice were not significantly affected, and the liver and kidney function indicators were normal, indicating that Codonopsis pilosula oral liquid has no obvious toxic side effects.

4. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 2), the Codonopsis pilosula oral liquid prevents and alleviates intestinal damage caused by chemotherapy drugs: an intestinal damage model is constructed using irinotecan, and Codonopsis pilosula oral liquid 1.5g / kg is administered before chemotherapy (LDS / CPT11), during chemotherapy (LDS+CPT11), and after chemotherapy (CPT11 / LDS).

5. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 2), the Codonopsis pilosula oral liquid exerts an intestinal protective effect through anti-inflammatory and stem cell function enhancement: a mouse ulcerative colitis model is constructed using dextran sulfate sodium (DSS). By downregulating intestinal inflammatory factors and increasing the expression of stem cell markers, as well as reducing the infiltration of neutrophils and macrophages, the intestinal mucosal damage caused by DSS is alleviated and the intestinal barrier function is restored.

6. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 2), the Codonopsis pilosula oral liquid exerts a protective effect by influencing intestinal immunity and metabolic mechanisms: RNA-seq sequencing analysis of the Codonopsis pilosula oral liquid treatment group and the untreated group of ulcerative colitis revealed that compared with the wild control group, the DSS model group had a total of 1346 differentially expressed genes; the Codonopsis pilosula oral liquid treatment group had 2108 differentially expressed genes.

7. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 3), the determination of relevant pharmacopoeia indicators of honeysuckle medicinal materials: based on the method under the honeysuckle testing item in the 2020 edition of the pharmacopoeia, the indicators such as total phenolic acid, total ash, acid ash, moisture, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C of 12 batches of honeysuckle medicinal materials from different places of origin and years were tested.

8. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 3), the network pharmacology includes: Network pharmacology approach: The pharmmapper database was used to predict the possible targets of the eight blood-entering components of the above key in vivo exposed components. The GeneCards database was used to collect all proteins related to heat-cleaning. The PEAC seq dataset was used to perform a 3D integrated analysis of the metabolomics, pharmmapper, and GeneCards results. Network pharmacology results: The pharmmapper database output 400 possible targets, the GeneCards database output 496 proteins related to heat-cleaning, and the metabolomics results showed that there were 375 proteins related to key metabolic pathways.

9. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 4), the evaluation of the heat-clearing efficacy of honeysuckle oral liquid and its key components: The heat-clearing effect of honeysuckle oral liquid was evaluated using an LPS-induced rat fever model. Sixty male SD rats (200 g ± 20 g) were randomly divided into 6 groups. The specific administration regimen is shown in Table 3.

1. The content of the corresponding key heat-clearing components in the Zhenao honeysuckle oral liquid was used as the control of total phenolic acids and iridoids. Chlorogenic acid was selected as the representative component of phenolic acids, and swertiamarin was used as the substitute component of iridoids to examine the correctness of the predicted results of the above metabolomics and network pharmacology.

10. The honeysuckle and codonopsis composition according to claim 1, characterized in that: According to step 4), the antitumor activity evaluation of the honeysuckle oral liquid and key components is as follows: the drug-containing serum of rats after being administered honeysuckle oral liquid by gavage for different time periods is diluted with DMEM complete medium to concentrations of 20%, 15%, 10% and 5%, and the inhibitory effect of the drug-containing serum on A549 cells is detected by CCK-8 assay.

Images