Hederasaponin composition, hedera leaf extract comprising composition, and use thereof

Abstract

A hederasaponin composition and a hedera leaf extract. The hederasaponin composition comprises hederasaponin C, α-hederasaponin, hederasaponin B, and hederasaponin D in a mass ratio of 12-25:5-10:1:1-2. The hederasaponin composition and the hedera leaf extract can be used for preparing drugs for treating or preventing acute bronchitis, expectorants, and antitussives.

Description

Ivy saponin compositions, ivy leaf extracts including the compositions, and their applications Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to an ivy saponin composition, an ivy leaf extract comprising the composition, and their applications. Background Technology

[0002] Ivy ethanol extract possesses various effects, including antispasmodic, mucolytic, anti-inflammatory, antibacterial, and antiviral properties. Ivy preparations are currently marketed in many countries worldwide and widely used for respiratory-related diseases, such as respiratory mucositis, chronic bronchitis, and acute respiratory inflammation with cough. Currently, ivy ethanol extract is used as an expectorant in various dosage forms, including tablets, capsules, effervescent tablets, and oral liquids, and has been approved for marketing in several European countries. As a herbal medicine, ivy preparations have good safety and tolerability in children. The currently marketed Prospan brand ivy leaf extract syrup contains 7 mg / mL of 30% ethanol-extracted ivy leaf extract, 1.34 mg of potassium sorbate, 500 μg of anhydrous citric acid, 550 mg of sorbitol, 1.5 mg of xanthan gum, 3 mg of flavoring, and purified water to a final volume of 1 mL. The ivy leaf extract is a crude 30% ethanol extract, with lower levels of saponins and other active ingredients.

[0003] The main medicinal substances in ivy are saponins, including triterpenoid saponin disaccharide glycosides such as ivy saponin B, ivy saponin C, and ivy saponin D, and monosaccharide glycoside α-ivy saponin. Other components include flavonoids, coumarins, polyacetylenes, phenolic acids, alkaloids, and volatile oils. However, Prospan brand ivy leaf extract is a 30% ethanol crude extract, and its medicinal components are not clearly defined; the effective components are not sufficiently concentrated, resulting in large dosages; and the unclear composition is also detrimental to quality control. Moreover, the ivy leaf extract obtained by the current preparation method is highly dependent on the germplasm resources of ivy leaves, and the production and quality of the extract are easily affected by the medicinal material resources.

[0004] The prior art discloses a method for preparing extracts containing ivy saponins.

[0005] CN1856317A discloses a method for preparing ivy leaf extract and the extract prepared by this method (application date: 2004.09.10; publication date: 2006.11.01). This technology employs the following methods: a) converting pulverized ivy leaves into α-hedendrine by fermenting them with water to provide a first extract rich in α-hedendrine, containing at least 3% α-hedendrine; b) treating pulverized ivy leaves with superheated steam to inhibit the conversion of phadermoid C into α-hedendrine, providing a second extract rich in phadermoid C, containing at least 5% phadermoid C; c) mixing the first and second extracts to obtain an extract containing adjusted phadermoid C and adjusted α-hedendrine content. This technical solution solves the problem of enrichment of ivy saponin C and α-hedendrine, but does not disclose other pharmacologically active ingredients or anti-inflammatory activities.

[0006] The preparation method of ivy leaf extract was disclosed in the paper "Determination of Four Major Saponins in Ivy and Its Extract by One Test and Multiple Evaluation Method" (Journal of Pharmaceutical Analysis 2018, 38(06):986-996): Weigh an appropriate amount of dried ivy leaves, crush them into coarse powder, and extract them twice by reflux with 80% ethanol-water at 85-90℃. The first time, add 9 times the amount of water and reflux for 1 h, and the second time, add 5 times the amount of water and reflux for 0.5 h. Filter, combine the filtrates, recover the ethanol until there is no alcohol odor, dissolve in 6 times the amount of water, let stand overnight at 4℃, filter, pass through a DM130 macroporous adsorption resin column, wash with water until colorless, and then elute with 3 times the amount of 30% ethanol and 3 times the amount of 70% ethanol-water. Collect the 70% ethanol-water eluent, concentrate the filtrate, dry, and crush to obtain the extract. The extract of English ivy leaf contained the highest content of hedyotis diffusa saponin C (66%), the highest content of hedyotis diffusa saponin D (7%), the highest content of hedyotis diffusa saponin B (3%), and the highest content of α-hedyotis diffusa saponin (22%), with the total content of the four saponins being at most 80%. However, this technical solution aimed at enriching saponins and did not disclose anti-inflammatory effects or other pharmacologically active components.

[0007] The method of simultaneous determination of four saponins in ivy oral liquid using a single test and multiple evaluation method (Chinese Traditional and Pharmaceutical Preparations, Vol. 40, No. 8, 2018) was used. The extract was obtained by extraction with 80% ethanol, treatment with alkali solution, standing overnight at 4°C, and filtration through a DM130 macroporous resin column with gradient elution of 30% ethanol and 70% ethanol. The 70% ethanol eluent was collected and concentrated to obtain the extract, but the composition, content and anti-inflammatory effect of the extract were not disclosed.

[0008] The abstract of the optimized preparation process of ivy extract (Yang Ziwen, 2018) describes the following: extraction with 80% ethanol, followed by treatment with 1% alkali (based on the amount of the medicinal material), cold precipitation at 4°C for 8 hours, and filtrate through an HPD-100 macroporous resin column with gradient elution of 30% and 80% ethanol. The 80% ethanol eluent was collected, concentrated, and the extract was obtained. The average content of ivy saponin C in the extract was 40.44%, and the average content of α-ivy saponin was 17.09%, with a recovery rate of 65.24%. The combined content of ivy saponin C and α-ivy saponin reached 57.52%. However, the other components and their contents, as well as the anti-inflammatory effects, of the extract were not disclosed.

[0009] CN104306416A discloses a drug for treating bronchitis, pneumonia, and asthma, and its preparation method (publication date: 2015.01.28). It describes a method for preparing ivy saponins: ivy stems and leaves are weighed, and extracted in an extraction vessel with 10-90% ethanol solution at a mass-to-volume ratio of ivy stems / leaves to ethanol solution of 1:3-10. After reflux extraction, the mixture is filtered, centrifuged, concentrated under reduced pressure, and rotary evaporated to a paste-like consistency. The paste is dissolved in deionized water, passed through a macroporous resin column, and eluted sequentially with water and ethanol solutions. The ethanol eluent is concentrated under reduced pressure, vacuum dried at 60°C, and weighed to obtain ivy saponins. The specification discloses the effects of an oral ivy solution made from a mixture of ivy saponins and ivy volatile oil on SOD activity in the serum, lung tissue, and BALF of rats with chronic bronchitis, but does not disclose the anti-inflammatory effects of the saponins.

[0010] CN102188465A discloses a method for preparing an extract of ivy plant and its uses (publication date: 2011.09.21). The method describes extracting the ivy plant extract using an extraction solvent, passing the extract over a macroporous adsorption resin, and drying the resulting alcohol eluent to obtain the extract. The extract contains a total saponin content of not less than 30%. This extract can be used alone or in combination with other extracts for the preparation of drugs for treating rheumatoid arthritis, respiratory diseases, and hepatitis. However, the specification only uses the enrichment of total saponin content as the optimization target, without disclosing the relationship between total saponins, saponin C, and α-saponins and the efficacy.

[0011] Existing technologies for extracting ivy primarily focus on increasing the content and recovery rate of saponins. For example, gradient elution with 30% ethanol + 70% ethanol is used to remove highly polar impurities such as polysaccharides and pigments, aiming to enrich the saponins in the ivy extract and increase their content. However, existing technologies do not investigate whether the anti-inflammatory efficacy of the extract is related to the saponin content or specific component ratios. Common sense dictates that higher saponin content should lead to better efficacy, or that a higher content of a component with strong anti-inflammatory effects is preferable. However, the applicant's research has revealed phenomena that may contradict common sense. Existing technologies have not studied the content and ratio of saponins in the extract, nor have they established a relationship between these ratios and anti-inflammatory efficacy. It is necessary to further optimize the saponin content and ratio in the extract from the perspective of improving its anti-inflammatory efficacy. Summary of the Invention

[0012] The purpose of this invention is to solve the technical problem that the components of ivy leaf extract are unclear and its anti-inflammatory effect is poor in the prior art.

[0013] This invention provides an ivy saponin composition with a defined composition and better anti-inflammatory effect. By screening and optimizing the proportion of saponin components, the composition exhibits synergistic effects among its components, thereby improving its anti-inflammatory efficacy. Moreover, it is not limited by the source of medicinal materials or extraction process. Extracts with different component contents obtained from different sources of medicinal materials and processes can be adjusted by increasing or decreasing the component content to obtain the saponin composition of this invention, which can also achieve a good anti-inflammatory effect.

[0014] The technical solution adopted in this invention is:

[0015] An ivy saponin composition comprising ivy saponin C, α-ivy saponin, ivy saponin B and ivy saponin D, wherein the mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B and ivy saponin D is (12-25):(5-10):1:(1-2).

[0016] Preferably, the mass ratio of ivy saponin C to ivy saponin D is (8-16):1.

[0017] More preferably, the mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B, and ivy saponin D is (16-25):(5-7):1:(1-2).

[0018] Alternatively, the preferred mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B, and ivy saponin D is (12-16):(7-10):1:1.

[0019] The present invention also provides an ivy leaf extract, the extract comprising ivy saponin C, α-ivy saponin, ivy saponin B and ivy saponin D, wherein the mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B and ivy saponin D is (12-25):(5-10):1:(1-2), wherein the content of ivy saponin C in the extract is not less than 25%, the content of total flavonoids is not less than 4%, the content of total polysaccharides is not less than 2%, and the content of total saponins in the ivy leaf extract is not less than 70%.

[0020] Preferably, the mass ratio of hedyoside C, α-hedyoside, hedyoside B, and hedyoside D in the extract is (12-21):(5-9):1:(1-1.6).

[0021] More preferably, the mass ratio of hedyotis diffusin C, α-hedyotis diffusin, hedyotis diffusin B, and hedyotis diffusin D in the extract is (12-21):(5-6):1:(1-1.6).

[0022] More preferably, the mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B, and ivy saponin D in the extract is (12-17):(6-9):1:(1-1.4).

[0023] More preferably, in the extract, the mass ratio of hedyotis diffusin C, α-hedyotis diffusin, hedyotis diffusin B, and hedyotis diffusin D is (17-20):(5-9):1:(1.4-1.6).

[0024] Furthermore, the extract contains at least 10% α-hederonine.

[0025] Furthermore, the extract contains at least 30% hederaside C, at least 2% hederaside D, and at least 2% hederaside B.

[0026] Preferably, the ivy leaf extract includes organic acid compounds, flavonoid compounds, saponin compounds, glycoside compounds and other compounds, wherein the organic acid compounds include one or more of quinic acid, neochlorogenic acid, cryptochlorogenic acid, asiatic acid, hydroxyasiatic acid, 3-O-sulfuric acid, and 3-O-sulfuric acid.

[0027] The flavonoid compounds include one or both of isoquercitrin and flavoside;

[0028] The glycoside compound includes one or more of the following: ethyl 3-O-β-D-glucopyranoside butyrate, (E)-4-O-β-D-glucopyranoside p-coumaric acid, 3-methylbut-2-enyl[β-D-furanopyranosyl-(1→6)]-β-D-glucopyranoside, icariin F2, myresculoside, cincaxiol B19-O-β-D-glucopyranoside, and nomiline 17-β-D-glucopyranoside.

[0029] The saponin compound includes one or two of β-hederin and pyrolysis saponin A;

[0030] The other compounds include one or more of 1-O-galloylpropanetriol, rhodioloside D, 3-methoxy-4-hydroxyphenol 1-O-[β-D-furanotropic-(1→6)]-β-D-glucopyranoside, icariin B5, camphorate H, and cimicifuga epoxide glycoside.

[0031] Preferably, the ivy leaf extract comprises α-hederone, hederone B, hederone C, and hederone D, and one or more of the following compounds: quinic acid, neochlorogenic acid, cryptochlorogenic acid, asiatic acid, hydroxyasiatic acid, 3-O-sulfuric acid, 3-O-sulfuric acid, oleanolic acid, ethyl 3-O-β-D-glucopyranoside butyrate, (E)-4-O-β-D-glucopyranoside-p-coumaric acid, myresculoside, β-hederone, pyrolytic acid A, rhodioloside D, 3-methoxy-4-hydroxyphenol 1-O-[β-D-furanoside-(1→6)]-β-D-glucopyranoside, anodic acid H, and cimicifuga epoxide glycoside.

[0032] More preferably, the ivy leaf extract includes chlorogenic acid, shikimic acid, 3-O-p-coumarylquinic acid, sennain, aescin, α-hederone, hederone B, hederone C and hederone D.

[0033] The present invention also provides the use of the ivy saponin composition or ivy leaf extract in the preparation of medicaments for the treatment or prevention of acute bronchitis.

[0034] The present invention also provides the use of the ivy saponin composition or ivy leaf extract in the preparation of expectorant and antitussive drugs.

[0035] The present invention also provides a medicament for treating or preventing acute bronchitis, comprising the ivy saponin composition or ivy leaf extract.

[0036] The present invention also provides an expectorant and antitussive drug, comprising the ivy saponin composition or ivy leaf extract.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention screened and optimized the content ratio of four components in ivy saponins, obtaining an ivy saponin composition with a defined composition and better anti-inflammatory effect, thus solving the problem of limited medicinal material sources. The four components in the composition of this invention have a synergistic effect, achieving a better anti-inflammatory effect than a single component. Conventional technology suggests that if a single component has a good anti-inflammatory effect, increasing its content will correspondingly increase the anti-inflammatory effect of the composition. For example, in this invention, the anti-inflammatory activity of the four components in the composition, from highest to lowest, is ivy saponin C > α-ivy saponin > ivy saponin B > ivy saponin D. Therefore, according to conventional understanding, increasing the content of ivy saponin C and α-ivy saponin should result in a better anti-inflammatory effect than increasing the content of ivy saponin B and ivy saponin D. However, the experimental results of this invention demonstrate that, under the same weight gain, the anti-inflammatory effect of the composition obtained by increasing the content of ivy saponin B and ivy saponin D is superior to that of the composition obtained by increasing the content of ivy saponin C and α-ivy saponin. This contradicts conventional understanding. This proves that there is a synergistic effect among the components of the composition of this invention. At a specific content ratio, the four components have a more superior anti-inflammatory effect, overcoming the technical bias of improving the overall efficacy of the composition by simply increasing the content of the component with the highest anti-inflammatory effect, and producing unexpected technical effects.

[0039] This invention also provides an ivy leaf extract with a defined composition and better anti-inflammatory effect, which is more conducive to reducing patient dosage and improving patient compliance. The extract of this invention is not limited by the source of the medicinal materials or the extraction process. Different sources of medicinal materials or different extraction processes yield extracts with different component contents. The component contents can be adjusted according to the scheme of this invention to obtain the saponin composition of this invention, and such extract can also achieve good anti-inflammatory effects. The composition of this invention has a wide range of applications and has great economic value and industrial benefits. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0041] Example 1: Screening of anti-inflammatory activity of ivy saponins

[0042] 1. Experimental process

[0043] 1.1 Medicines and Reagents

[0044] COX-2 Inhibitor Screening Kit (Beyotime Biotechnology), α-Ivy Saponin (China National Institute for Food and Drug Control), Ivy Saponin B / D (Chengdu Professional Biotechnology Co., Ltd.), Ivy Saponin C (self-made by Zhejiang Conba Pharmaceutical R&D Center), DMSO (Diamond).

[0045] 1.2 Test Methods

[0046] According to the kit's testing requirements, the drug administration group was set at a concentration of 100 μg / mL, the positive control group (celecoxib), and the DMSO group. Following the kit instructions, a 100% enzyme activity control group, a blank control group, a positive inhibitor group, and a sample group were set up. For the sample group, the test sample, ivy saponins, was added to each well, mixed, and incubated at 37°C for 10 min. 5 μL of COX-2 Probe was added to each well in each group, followed by 5 μL of COX-2 Substrate working solution, mixed, and incubated at 37°C in the dark for 5 min before fluorescence measurement. Detection was performed using a Tecan microplate reader with excitation wavelength of 560 nm and emission wavelength of 590 nm.

[0047] Inhibition rate (%) = (RFU) 100％酶活性对照 -RFU 样品 ) / (RFU 100％酶活性对照 -RFU 空白对照 )×100%.

[0048] 2. Test Results

[0049] Table 1. Inhibitory activity of hederine monomers against COX-2 enzyme

[0050] The experimental results showed that ivy saponins exhibited different inhibitory activities against COX-2 enzyme, thus possessing different anti-inflammatory effects. The order of anti-inflammatory activity of the four ivy saponins was ivy saponin C > α-ivy saponin > ivy saponin B > ivy saponin D.

[0051] Example 2: Screening of anti-inflammatory activity of ivy saponin compositions

[0052] 1. Experimental process

[0053] 1.1 Sample preparation

[0054] The ivy saponins from Example 1 were dissolved in methanol to prepare stock solutions of different concentrations, with ivy saponin C at a concentration of 0.3967 mg / mL, α-ivy saponin at 0.4168 mg / mL, ivy saponin B at 0.4812 mg / mL, and ivy saponin D at 0.5182 mg / mL. Methanol was then added to each solution to prepare test samples with a concentration of 100 μg / mL, as shown in Table 2 below.

[0055] Table 2. Preparation of Ivy Saponin Compositions

[0056] 1.2 Test Methods

[0057] The COX-2 enzyme inhibition activity test was performed according to the method in Example 1, and the inhibition rate of different test samples was statistically analyzed.

[0058] 2. Test Results

[0059] The experimental results are shown in Table 3. Different compositions consist of four types of ivy saponins, each with different inhibition rates. Combining Tables 2 and 3, composition 2, compared to composition 1, increases the content of ivy saponin C by one part and α-ivy saponin by one part, resulting in a decrease in ivy saponin C content from 54.5% to 54.2%, and an increase in α-ivy saponin content from 45.5% to 45.8%. However, the anti-inflammatory activities of ivy saponin C and α-ivy saponin are similar, resulting in little difference in anti-inflammatory activity between composition 1 and composition 2. Composition 3, based on composition 1, increases the content of ivy saponin B by one part and ivy saponin D by one part, with the total number of parts being the same as composition 2. The content of ivy saponin C decreases from 54.2% in composition 2 to 50%; the content of α-ivy saponin decreases from 45.8% to 41.6%; while the contents of ivy saponin B and ivy saponin D increase. The inhibition rate comparison showed that the inhibition rate of composition 3 was 58.26%, which was significantly higher than the inhibition rate of composition 2 (41.15%).

[0060] Similarly, in compositions 4-6, composition 5 increased the amount of hedyotis diffusin C by 2 parts and α-hedyotis diffusin by 1 part compared to composition 4. Correspondingly, the content of hedyotis diffusin C decreased and the content of α-hedyotis diffusin increased in the monomer component ratio of the mixture, but the inhibition rate did not change significantly compared to composition 4. Composition 6 increased the amount of hedyotis diffusin B by 1 part and hedyotis diffusin D by 2 parts compared to composition 4. The total number of parts in the composition was the same as in composition 5, but the content of hedyotis diffusin C and α-hedyotis diffusin decreased in composition 6. The inhibition rate comparison showed that the inhibition rate of composition 6 was 60.73%, higher than the inhibition rate of composition 5 (55.62%).

[0061] In compositions 7-9, composition 8 increases the amount of ivy saponin C by 2 parts compared to composition 7, thus increasing the proportion of saponin C in the monomer mixture. At the same dosage concentration, composition 8 showed an inhibition rate of 57.96%, which was superior to the inhibition rate of 49.30% of composition 7. Composition 9 increased the amount of ivy saponin B and ivy saponin D by 1 part each compared to composition 7, while keeping the amount of ivy saponin C unchanged. This is equivalent to replacing the 2 parts of ivy saponin C in composition 8 with 1 part of ivy saponin B and 1 part of ivy saponin D. However, the corresponding inhibition rate of composition 9 was 61.19%, which was higher than that of composition 8.

[0062] Based on the inhibition rates of the monomeric compounds in Table 1, it can be seen that the inhibition rates of ivy saponin C and α-ivy saponin are higher than those of ivy saponin B and ivy saponin D. Compositions 3, 6 and 9 improved the efficacy of the compositions by adding ivy saponin B and ivy saponin D, which have lower inhibition rates, respectively. They replaced the efficacy contributions of ivy C and α-ivy saponin, and even achieved better efficacy, producing unexpected technical effects.

[0063] Compositions 10-11 were compared with a composition containing hederone C and α-hederone at a ratio of 1:1:1:1 to four hederones in a 3:1 ratio. The results showed that the inhibition rates of compositions 10 and 11 were 41.40% and 49.01%, respectively, both lower than those of compositions 3, 6, and 9 at the same dosage. Furthermore, replacing an equal amount of hederone C with hederone B and hederone D in composition 10 did not increase the inhibition rate.

[0064] Therefore, when ivy saponin C and α-ivy saponin are used in combination with ivy saponins B and D, they may not necessarily increase the anti-inflammatory effect of the composition. The four saponins in the composition need to be in the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D = (12-25): (5-10): 1: (1-2) to achieve the technical effect of synergistic effect.

[0065] Table 3. Inhibitory activity of ivy saponin compositions against COX-2 enzyme

[0066] The applicant further investigated the components of the ivy leaf extract and found that ivy saponin C, α-ivy saponin, ivy saponin B, and ivy saponin D are important pharmacologically active components of the ivy leaf extract. Simultaneously, comparing the component ratios in the ivy leaf extract with those in the composition of Example 2 revealed that when the saponin ratio in the extract is within the range of the aforementioned composition, the ivy leaf extract also exhibits good anti-inflammatory effects. The following describes the preparation of different extracts using *Ivy japonica* as raw material. The production batch numbers for *Ivy japonica* medicinal materials are Z226b-210602, Z226b-210502, LX220501, and LX220502.

[0067] Example 3: Preparation of Extract 1

[0068] Weigh 200g of dried ivy leaves (production batch number Z226b-210602), crush them into coarse powder, and extract twice with 12 times the volume of the medicinal material using 80% ethanol under reflux at 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add 60mL (0.3 times the feed amount) of 5% sodium hydroxide solution, add water to a total volume of 12 times the feed amount, adjust the pH to 9.5-11, refrigerate (0-4℃) and let stand overnight, filter, pass through HPD100 macroporous adsorption resin, wash with water until colorless, then elute with 3 times the column volume of 70% ethanol, collect the 70% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0069] Example 4: Preparation of Extract 2

[0070] Weigh 200g of dried ivy leaves (production batch number Z226b-210502), crush them into coarse powder, and extract twice with 9 times the volume of the medicinal material using 85% ethanol under reflux at 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add 0.4 times the feed volume of 5% sodium hydroxide solution, add water to a total volume of 9 times the feed volume, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, and adsorb the filtrate through HPD100 macroporous resin. Wash with water until colorless, then elute with 5 times the column volume of 60% ethanol. Collect the 60% ethanol eluent, concentrate, dry, and crush to obtain the final product.

[0071] Example 5: Preparation of Extract 3

[0072] Weigh 200g of dried ivy leaves (production batch number LX220501), crush them into coarse powder, and extract twice with 12 times the volume of 80% ethanol under reflux at 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add water to potassium hydroxide solution to a total volume of 12 times the feed amount, adjust the pH to 9.5-11, refrigerate and let stand overnight, filter, adjust the pH of the filtrate to neutral, pass the filtrate through a polyamide column (30-60 mesh), wash with water until colorless, then elute with 3 times the column volume of 70% ethanol, collect the 70% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0073] Example 6: Preparation of extract from control group 1

[0074] Based on Example 3, the alkaline solution was adjusted to 1% of the amount of medicinal material, and a DM130 chromatographic column was used, with 30% ethanol added for elution, as detailed below:

[0075] Weigh 200g of dried ivy leaves (production batch number Z226b-210602), crush them into coarse powder, and extract twice with 12 times the volume of the medicinal material in 80% ethanol under reflux at 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add 0.2 times the feed volume of 5% NaOH solution, add water to a total volume of 12 times the feed volume, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, pass through DM130 macroporous adsorption resin, wash with water until colorless, then elute with 3 times the column volume of 30% ethanol, and then elute with 3 times the column volume of 70% ethanol. Collect the 70% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0076] Example 7: Preparation of extract from control group 2

[0077] The control group 2 extract was prepared according to the preparation method described in "Determination of Four Major Saponins in Ivy and Its Extracts by One Test and Multiple Evaluation Method" (Journal of Pharmaceutical Analysis, 2018, 38(06), 986-996).

[0078] Weigh 200g of dried ivy leaves (production batch number LX220502), pulverize into coarse powder, and extract twice by reflux with 12 times the volume of 80% ethanol-water at 85-90℃. For the first extraction, reflux with 9 times the volume of ethanol-water for 1 hour, and for the second extraction, reflux with 5 times the volume of ethanol-water for 0.5 hours. Filter, combine the filtrates, recover the ethanol until no alcohol odor remains, dissolve in 6 times the volume of ethanol-water, let stand overnight at 4℃, filter, pass through a DM130 macroporous adsorption resin column, wash with water until colorless, and then elute with 3 times the volume of 30% ethanol-water and 3 times the volume of 70% ethanol-water. Collect the 70% ethanol-water eluent, concentrate the filtrate, dry, and pulverize to obtain the final product.

[0079] Example 8: Preparation of extract from control group 3

[0080] Weigh 200g of dried ivy leaves (production batch number LX220501), crush them into coarse powder, and extract twice with 12 times the volume of the medicinal material using 80% ethanol under reflux at 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add 0.3 times the feed volume of 5% sodium hydroxide solution, add water to a total volume of 12 times the feed volume, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, adsorb with 5 times the feed volume of alkaline alumina, wash with 3 times the column volume of water, collect the washing liquid, concentrate, dry, add an appropriate amount of methanol for ultrasonic extraction, filter, concentrate and dry the filtrate to obtain the final product.

[0081] Example 9: Component Detection

[0082] 1. Test Methods

[0083] 1.1 Determination methods for the content of four types of ivy saponins

[0084] Preparation of reference solutions: Accurately weigh appropriate amounts of ivy saponin C and α-ivy saponin reference standards, and dissolve them in methanol to prepare a mixed solution containing 0.5 mg of ivy saponin C and 0.2 mg of α-ivy saponin per 1 mL. Separately, take appropriate amounts of ivy saponin B and ivy saponin D reference standards, and dissolve them in methanol to prepare solutions containing 0.5 mg of ivy saponin B and 0.5 mg of ivy saponin D per 1 mL, respectively.

[0085] Preparation of the test solution: Weigh 50 mg of ivy leaf extract accurately, place it in a 50 mL volumetric flask, add 80% methanol, sonicate to dissolve and dilute to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0086] Chromatographic conditions and system suitability test: ZORBAX Eclipse XDB-C18 Analytical column (4.6*250mm 5-Micron); acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 4; column temperature 25℃; detection wavelength 205nm; injection volume 10μL, flow rate 1.0mL / min.

[0087] Table 4 Liquid phase elution gradient

[0088] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0089] 1.2 Determination of total saponin content

[0090] Preparation of reference solution: Take an appropriate amount of ivy saponin C, accurately weigh it, and add methanol to prepare a solution containing 0.4 mg per 1 mL.

[0091] Preparation of standard curve: Accurately measure 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of the reference solution into stoppered test tubes, evaporate to dryness in a water bath, accurately add 5 mL of perchloric acid to each, shake well, react in a 70°C water bath for 15 minutes, remove, cool in an ice bath for 5 minutes, remove, shake well, use the corresponding reagent as a blank, and detect at wavelengths of 322 nm and 406 nm using ultraviolet-visible spectrophotometry (General Rule 0401). Plot the standard curve with absorbance as the ordinate and concentration as the abscissa.

[0092] Preparation of the test solution: Weigh 50 mg of ivy leaf extract accurately, place it in a stoppered conical flask, add 50 mL of water, sonicate to dissolve, remove, extract twice with 100 mL of water-saturated n-butanol solution each time, combine the n-butanol layers, recover to dryness under reduced pressure, dissolve the residue in methanol and dilute to 50 mL in a volumetric flask, shake well to obtain the test solution.

[0093] Assay: Accurately measure 0.1 mL of the test solution and place it in a stoppered test tube. Dry the tube in a water bath. Following the method under the preparation of the standard curve, starting from "accurately add 5 mL of perchloric acid", determine the absorbance according to the method. Read the weight of ivy saponin C in the test solution and calculate it from the standard curve.

[0094] 1.3 Determination of total flavonoid content

[0095] Preparation of test solution: Accurately weigh 0.5g of ivy leaf extract into a 100mL volumetric flask, add 70% ethanol, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0096] Preparation of the standard curve: Accurately measure 0.2 mL, 0.6 mL, and 1.0 mL of rutin control solution and place them in 10 mL volumetric flasks respectively. Add water to each flask to a final volume of 3 mL. Accurately add 2 mL each of acetate-sodium acetate buffer (pH 4.5) and 0.1 mol / L aluminum trichloride solution, shake well, and then add 70% ethanol to the mark. Shake well again. Using the corresponding reagents as blanks, scan the entire wavelength range of 200–600 nm using ultraviolet-visible spectrophotometry (Appendix VA) to obtain the spectrum. Select the wavelength of maximum absorption for detection and plot the standard curve with absorbance as the ordinate and concentration as the abscissa.

[0097] Assay: Accurately measure 0.5 mL of the test solution and place it in a 10 mL volumetric flask. Following the method under the preparation of the standard curve, starting from "add water to make up to 3 mL", measure the absorbance according to the method and read the amount of rutin in the test solution. Calculate the rutin content from the standard curve.

[0098] 1.4 Determination of total polysaccharide content

[0099] Preparation of test solution: Take 0.5g of ivy leaf extract, add 5mL of 25% methanol and sonicate to dissolve. Pass the solution through an ODS column (2cm*25cm), elute with 25% methanol, collect 150mL (twice the column volume of eluent), and discard the rest.

[0100] Preparation of standard curve: Accurately measure 0.0 mL, 0.2 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose control solution and place them into 20 mL stoppered test tubes. Add water to each tube to a final volume of 1 mL. Accurately add 1 mL of 5% phenol solution, shake well, then add 5 mL of sulfuric acid, shake well, heat in a 100°C water bath for 20 min, remove and cool in an ice bath for 5 min, let stand for 20 min, and measure the absorbance at a wavelength of 488 nm. Plot the standard curve using absorbance and concentration.

[0101] Assay: Accurately measure 0.5 mL of the test solution and place it in a 20 mL volumetric flask. Following the method under the preparation of the standard curve, starting from "add water to make up to 1 mL", measure the absorbance according to the method. Read the amount of glucose in the test solution from the standard curve and calculate the result.

[0102] 2. Test Results

[0103] The compositional analysis of ivy leaf extracts 1-3 and control groups 1-3 showed that the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D in extracts 1-3 was (12-21): (5-9): 1: (1-1.6), which is consistent with the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D = (12-25): (5-10): 1: (1-2). However, the saponin content ratios in control groups 1-3 were not within the above range. Specifically, the D / B ratio of control group 1 (0.78), the α / B ratio of control group 2 (3.21), and the C / B ratio of control group 3 (11.00) were significantly lower than the range of the compositions.

[0104] Table 5 Parameters of different ivy leaf extracts

[0105] Example 10: Pharmacological test of extract

[0106] 1. Experimental Procedure

[0107] 1.1 Sample preparation

[0108] (1) Preparation of test samples: Ivy leaf extract 1-3 and control group 1-3 were prepared with methanol to prepare test samples with a final concentration of 50 μg / mL.

[0109] (2) Preparation of spiked reference standard: Based on the extraction content determination results of Example 9, different amounts of saponin were added to control groups 1 and 3, and the content was determined by the method of Example 9, so that the obtained extract has the corresponding saponin ratio of the composition.

[0110] Table 6 Preparation of spiked control group

[0111] Table 7 Parameters of extracts from the spiked control group

[0112] 1.2 Test Methods

[0113] The COX-2 enzyme inhibition activity test was performed according to the method in Example 1, and the inhibition rate of different test samples was statistically analyzed.

[0114] 2. Test Results

[0115] The experimental results are shown in Table 8. The saponin content ratios of extracts 1-3 and spiked control 1 are all within the range of the composition of Example 2, that is, they meet the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D = (12-25): (5-10): 1: (1-2), and the corresponding average inhibition rates are all higher than 77%. The saponin content ratio of spiked control 3 is also within the range of the composition of Example 2, but the average inhibition rate is only 61.57%. The saponin content of control 1, spiked control 2, control 2, and control 3 does not meet the range of the composition ratio, and the corresponding inhibition rates are all less than 67%.

[0116] Spiked control 1, with the addition of hederaside D, increased the D / B ratio from 0.78 to 1.55, decreased the C / D ratio from 25.17 to 12.74, and showed no significant change in total polysaccharide and total flavonoid content. However, the inhibition rate increased from 63.17% to 78.11%, a 15% increase. Spiked control 2, with the addition of hederaside C and hederaside D, increased the D / B ratio from 0.78 to 3.27, increased the C / B ratio from 19.70 to 23.37, and decreased the C / D ratio from 25.17 to 7.15. The total polysaccharide and total flavonoid content remained largely unchanged, and its inhibition rate was 66.64%, similar to that of control 1.

[0117] A comparison of saponin contents between spiked control 1 and spiked control 2 revealed that the content of ivy saponin C in spiked control 2 (47.08%) increased by 2.56% compared to spiked control 1 (44.52%), the content of α-ivy saponin (10.51%) decreased by 1.28% compared to spiked control 1 (11.79%), the content of ivy saponin B (2.01%) decreased by 0.25% compared to spiked control 1 (2.26%), and the content of ivy saponin D (6.58%) increased by 3.09% compared to spiked control 1 (3.49%). Combined with the anti-inflammatory activity results of saponin monomers in Table 1, the order of anti-inflammatory activity was ivy saponin C > α-ivy saponin > ivy saponin B > ivy saponin D, with inhibition rates at a dosage concentration of 100 μg / mL of 36.87%, 33.16%, 27.06%, and 22.23%, respectively. In spiked control 2, the increase in hedyotis diffusin C was replaced by the decrease in α-hedyotis diffusin at a 1:1 ratio, resulting in a 1.28% increase in hedyotis diffusin C. Similarly, the decrease in hedyotis diffusin B was replaced by the increase in hedyotis diffusin D at a 1:2 ratio, resulting in a 2.59% increase in hedyotis diffusin D. Total flavonoids and total polysaccharides remained largely unchanged. However, with a 4% increase in saponin content, the inhibition rate of spiked control 2 actually decreased from 78.11% in spiked control 1 to 66.64%, indicating that the proportions of the four saponins in ivy leaf extract achieve better anti-inflammatory effects within the specific range of spiked control 1. Combining the saponin ratios and inhibition rates of extracts 1-3 and spiked control 1, it was found that all four saponins met the requirements of the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D = (12-25): (5-10): 1: (1-2), with inhibition rates greater than 77% and good anti-inflammatory activity. Preferably, the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D = (12-21): (5-9): 1: (1-1.6). Furthermore, adding external standards to ensure the component content of ivy leaf extracts meets the above conditions can also achieve good anti-inflammatory effects. Therefore, the source of ivy leaf extracts and extraction methods are not limited. Even if the content of specific saponins in the ivy leaf raw material is limited, or different extraction methods result in different saponin content ratios, the content of saponins in the extract can be made to meet the requirements of this invention by adding external standards, thereby obtaining better anti-inflammatory activity. Therefore, this invention can increase the source of raw materials for ivy leaf extract; at the same time, it reduces the requirements for saponin content in ivy leaf medicinal materials, expands the base of medicinal material sources, and reduces production costs.

[0118] The prior art discloses a method for preparing Control 2, which differs from Control 1 in that it does not undergo alkali treatment. The obtained Control 2 extract has a higher content of saponin C, but the crude extract, without alkali treatment, suffers from greater loss of flavonoids and polysaccharides during column chromatography elution. The resulting hedera saponin C content is 61.3%, the total flavonoid content is 1.4%, and the total polysaccharide content is 1.6%, corresponding to an inhibition rate of 66.73%, similar to the 63.17% inhibition rate of Control 1 extract but significantly lower than that of extracts 1-3 and spiked Control 1. Component analysis revealed that even though the total saponin content of Control 2 is 79.6%, slightly higher than that of extracts 1-3 and spiked Control 1, the total flavonoid and total polysaccharide contents are only one-third of those of extracts 1-3 and spiked Control 1. Anti-inflammatory results indicate that saponins, total flavonoids, and total polysaccharides are all effective components of hedera leaf extract. If the total flavonoid and total polysaccharide content is low, the anti-inflammatory effect will also be poor. Therefore, the total flavonoid and total polysaccharide content also needs to meet the requirements of the scope of this invention.

[0119] Spiked control 3 was supplemented with ivy saponin C, increasing the C / B ratio from 11.0 in control 3 to 14.97, thus satisfying the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D = (12-25): (5-10): 1: (1-2). However, the inhibition rate of spiked control 3 was 61.57%, similar to the inhibition rate of control 3 (62.78%). A comprehensive comparison revealed that the total flavonoid and total polysaccharide contents of both control 3 and spiked control 3 were less than 1%, and the total saponin content was 56.34%, significantly lower than that of extracts 1-3 or spiked control 1. This indicates that total flavonoid, total polysaccharide, and total saponin content are the pharmacodynamic indicators of ivy leaf extract. Combining the component contents of extracts 1-3 and spiked control 1, it was found that ivy leaf extract exhibited better anti-inflammatory effects when the total flavonoid content was greater than 4%, the total polysaccharide content was greater than 2%, and the total saponin content was greater than 70%.

[0120] Table 8. Inhibitory activity of different ivy leaf extracts on COX-2 enzyme

[0121] Example 12: Identification of Extract Components

[0122] The chemical composition of extracts 1-3 and spiked control extracts 1-3 was determined, and component analysis was performed in conjunction with the medicinal materials of Hedera helix and Hedera sinensis.

[0123] 1. Test Methods

[0124] 1.1 Preparation of test sample

[0125] Take 0.01g of each of the extracts 1-3 and spiked control samples 1-3 into a 1.5mL ep tube, add 1mL of 80% methanol, sonicate for 1 hour, centrifuge at 10000rpm for 10 minutes, and transfer the supernatant to an Agilent sample vial for later use.

[0126] Weigh 1g each of English ivy powder and Chinese ivy powder, make two parallel extracts. Add 20mL of pure water to one extract and 20mL of 80% methanol to the other, and extract by sonication for 1 hour. Filter the extract through a corresponding 0.22μm microporous membrane, collect the filtrate, and concentrate it to 2mL by centrifugation. Pass the concentrate through an SPE column, elute with methanol-water (9:1, v / v), collect the eluent, and concentrate to dryness by centrifugation. Redissolve in 1mL of methanol-water (9:1, v / v), centrifuge at 10000rpm for 10 minutes, and transfer the supernatant to an Agilent sample vial for later use.

[0127] 1.2 Detection Method

[0128] Liquid chromatography conditions: Agilent ZORBAX Eclipase XDB-C18 column (Analytical 4.6×250mm, 5-micron); column temperature 30℃; flow rate 1mL / min; UV detection wavelength 205nm; mobile phase, (A) 0.05% formic acid-water, (B) acetonitrile; gradient: 0min, 5% B; 30min, 30% B; 60min, 85% B; 65min, 95% B; 70min, 95% B.

[0129] Mass spectrometry conditions: negative ion mode, dry gas temperature 320℃; dry gas flow rate 8L / min; nebulizer gas pressure 45psig; sheath gas temperature 350℃; sheath gas flow rate 11L / min; source voltage 3500V; fragmentation voltage 175V; skimmer voltage 65V; primary ion range 100-1700m / z; collision energies 10V, 20V, 40V; automatic secondary acquisition, dynamic exclusion enabled.

[0130] Positive ion mode: Drying gas temperature 320℃; Drying gas flow rate 8L / min; Nebulizing gas pressure 45psig; Sheath gas temperature 350℃; Sheath gas flow rate 11L / min; Source voltage 4000V; Fragmentation voltage 175V; Skimmer voltage 65V; Primary ion range 100-1700m / z; Collision energy 10V, 20V, 40V; Automatic secondary acquisition, dynamic exclusion enabled.

[0131] 2. Test Results

[0132] The component analysis results of extracts 1-3 and controls 1-3 are shown in Table 9. The components included organic acids, flavonoids, glycosides, saponins, and others, totaling 32 compounds. Among them, 10 were classified as organic acids, 2 as flavonoids, 8 as glycosides, 6 as saponins, and 6 as other compounds. The detection and classification of these compounds further clarified the effective components of the ivy leaf extract.

[0133] A comparison of the components of the six extracts revealed that nine components were commonly detected in extracts 1-3 and controls 1-3; 23 components, different from controls 2 and 3, were detected in extracts 1-3 and control 1, as shown in Table 10. The difference between spiked controls 1 and 2 and control 1 was the increased amounts of hedyotis diffusin C and hedyotis diffusin D; the difference between spiked control 3 and control 3 was the increased amount of hedyotis diffusin C. Spiking had no effect on the types of components.

[0134] Controls 2, 3, and spiked control 3 did not contain any common distinguishing compounds, and their saponin ratio, total flavonoid content, total polysaccharide content, total saponin content, and the ratio of total saponins to extracts 1-3 and spiked control 1 were different, corresponding to poorer anti-inflammatory effects.

[0135] According to the component detection results in Table 10, the anti-inflammatory effects of Control 1, Spiked Control 1 and Spiked Control 2 in Table 8 show that even though the above three extracts all contain common distinguishing compounds, the anti-inflammatory effect of Spiked Control 1 is significantly better than that of Control 1 and Spiked Control 2. It can be seen that the proportion of saponins, total flavonoid content, total polysaccharide content and total saponin content also directly affect the anti-inflammatory results.

[0136] Therefore, extracts 1-3 and spiked control 1 all contain the common distinguishing compounds listed in Table 10. The corresponding saponins conform to the ratio of ivy saponin C: α-ivy saponin: ivy saponin B: ivy saponin D = (12-25): (5-10): 1: (1-2). When the total flavonoid content is greater than 4%, the total polysaccharide content is greater than 2%, and the total saponin content is greater than 70%, the ivy leaf extract exhibits better anti-inflammatory effects. This technical solution provides an ivy leaf extract with better anti-inflammatory effects and more clearly defined components. Furthermore, by detecting the above-mentioned components in the ivy leaf extract, it can also be confirmed whether the obtained ivy leaf extract meets the component requirements.

[0137] Table 9. Component Analysis of Ivy Leaf Extract

[0138] In Table 9, * indicates that 1 represents the presence of the component in both the raw material ivy and the extract, and 2 represents that the component was not detected in ivy but was detected in the extract (this may be due to the complexity of the raw material's composition or a transformation that occurred during extraction).

[0139] In the # column, 1 indicates that the component is shared by both English ivy and Chinese ivy; 2 indicates that the component is unique to English ivy or that its content is higher than that of Chinese ivy.

[0140] Table 10 Results of component analysis of different ivy leaf extracts

[0141] Example 13: Pharmacodynamic effects of ivy leaf extract on a lipopolysaccharide (LPS)-induced acute bronchitis model in mice.

[0142] 1. Experimental reagents and materials

[0143] LPS (Sigma-Aldrich (Shanghai) Trading Co., Ltd.); Prospan oral solution (manufactured by Engelhard Arzneimittel, batch number: 22G080A); Ambroxol hydrochloride oral solution (manufactured by China Resources Sanjiu (Nanchang) Pharmaceutical Co., Ltd., batch number: 2112020J); Honey-processed Sichuan fritillary bulb and loquat syrup (manufactured by Kyoto Nianci'an General Factory Co., Ltd., batch number: Y12102104); Extract 3 samples prepared in Example 5 (self-made by Zhejiang Kang Enbei Traditional Chinese Medicine Co., Ltd.).

[0144] 2. Animal grouping

[0145] After three days of acclimatization feeding, SPF-grade Balb / c mice were divided into 6 groups of 10 mice each, based on their average body weight.

[0146] Table 11 Animal Grouping

[0147] 3. Route of administration and timing of administration

[0148] Administer orally via gavage for 3 consecutive days at a dose of 0.2 mL / 10 g body weight.

[0149] 4. Model Replication

[0150] Healthy mice were treated with a pipette, and 10 mg / kg of LPS saline solution (6 mg / mL) was administered intranasally for 3 consecutive days. The mice were then sacrificed after 5 days. The medication was administered once daily, starting two days before model establishment and continuing until model establishment.

[0151] 5. Indicator Testing

[0152] Serum was collected from animals one hour after the last administration to detect changes in the inflammatory factor TNF-α.

[0153] 6. Statistical methods

[0154] All data are expressed as Mean ± SD. The significance analysis was performed using the t-test to determine the differences between each group and the model group. P < 0.05 was considered statistically significant.

[0155] 7. Experimental Results

[0156] As shown in Table 12, the serum TNF-α levels in the model group mice were significantly higher at the end of drug administration compared to the control group (P<0.01). Compared to the model group, all drug groups reduced serum TNF-α levels at the end of drug administration. 2 mg / kg dexamethasone acetate significantly reduced serum TNF-α levels compared to the model group (P<0.01). 5.8 mL / kg Nin Jiom Pei Pa Koa showed no difference compared to the model group, while 3.2 mL / kg Prospan significantly reduced serum TNF-α levels compared to the model group (P<0.05). 3.11 mg / kg extract 3 showed a significant difference compared to the model group (P<0.05). The experimental results indicate that extract 3 can effectively inhibit the growth of inflammatory factors in LPS-induced acute bronchitis in mice, demonstrating a therapeutic effect, and its anti-inflammatory effect is superior to that of the Prospan group.

[0157] Table 12 Effects of ivy leaf extract on serum TNF-α in mice with acute bronchitis (n=10, Mean±SD) Note: **P<0.01 vs model group; *P<0.05 vs model group

[0158] Example 14: Expectorant effect of ivy leaf extract on a mouse expectorant model (phenol red method)

[0159] 1. Experimental reagents and materials

[0160] Phenol red (GENERAL-REAGENT); ELISA reader (Thermo); Drug source as shown in Example 13.

[0161] 2. Animal grouping

[0162] Sixty SPF-grade ICR mice were randomly divided into 6 groups of 10 mice each after a three-day acclimatization period. The groups are shown in Table 13.

[0163] Table 13 Animal Grouping

[0164] 3. Route of administration and timing of administration

[0165] The medication was administered orally once daily for three consecutive days, with a dosage of 0.2 mL / 10 g body weight.

[0166] 4. Model Replication

[0167] Thirty minutes after the last administration of the drug to mice, 0.1 mL / 10 g of 5% phenol red solution was injected intraperitoneally. The animals were sacrificed 30 minutes later, and the trachea was dissected. A flattened No. 7 needle was inserted 0.3 cm into the trachea and secured with suture. 0.5 mL of 5% sodium bicarbonate solution was drawn into a 1 mL syringe and used to irrigate the respiratory tract three times through the needle. The irrigating fluid was withdrawn on the last attempt and injected into a test tube. This procedure was repeated three times, yielding approximately 1 mL of irrigating fluid, which was then centrifuged at 1500 rpm for 5 minutes. 150 μL of the supernatant was then transferred to a 96-well plate.

[0168] 5. Indicator Testing

[0169] The OD value at 546 nm was measured using an ELISA reader, and the instrument was zeroed using 5% sodium bicarbonate solution.

[0170] 6. Statistical methods

[0171] All data are expressed as Mean±SD. The t-test was used to analyze the significance between groups, and the difference between each group and the model group was tested. P<0.05 was considered statistically significant.

[0172] 7. Experimental Results

[0173] As shown in Table 14, 3.2 mL / kg Prospan significantly increased tracheal phenol red secretion in mice compared to the model group (P<0.05); 2.57 mL / kg ambroxol hydrochloride oral solution promoted tracheal phenol red secretion in mice, but showed no difference compared to the model group (P>0.05); 3.11 mg / kg extract 3 significantly increased tracheal phenol red secretion in mice compared to the model group (P<0.05); 5.8 mL / kg honey-processed fritillaria and loquat paste showed no significant promoting effect. Therefore, extract 3 can increase tracheal phenol red secretion in female mice and has a certain expectorant effect.

[0174] Table 14 Results of the expectorant effect of ivy leaf extract Note: *P<0.05 vs model group

[0175] Example 15: Study on the antitussive effect of ivy leaf extract on a mouse antitussive model (ammonia method)

[0176] 1. Experimental reagents and materials

[0177] Ammonia water (Sinopharm Chemical Reagent Co., Ltd.); Dextromethorphan hydrobromide tablets (Guangzhou Baiyunshan Guanghua Pharmaceutical Co., Ltd.); the sources of the drugs are as shown in Example 13.

[0178] 2. Animal grouping

[0179] Fifty SPF-grade ICR female mice were acclimatized for three days and then divided into 5 groups of 10 mice each, based on their average body weight. The group assignments are shown in Table 15.

[0180] 3. Route of administration and timing of administration

[0181] Administer orally via gavage for 3 consecutive days at a dose of 0.2 mL / 10 g body weight.

[0182] 4. Model Replication

[0183] One hour after the last administration, the mice were placed in the YLS-8A multifunctional cough and asthma induction device, and 12% ammonia water was nebulized for 10 seconds. The cough latency period and the number of coughs within 2 minutes were observed and recorded.

[0184] 5. Indicator Testing

[0185] The incubation period of a cough is the total number of coughs within 2 minutes.

[0186] 6. Statistical methods

[0187] All data are expressed as Mean ± SD. The significance analysis was performed using the t-test to determine the differences between each group and the model group. P < 0.05 was considered statistically significant.

[0188] 7. Experimental Results

[0189] As shown in Table 16, after 3 consecutive days of administration to mice, 3.2 mL / kg Prospan, 1.03 tablets / kg dextromethorphan hydrobromide, 5.8 mL / kg honey-processed fritillaria cirrhosa and loquat syrup, and 3.11 mg / kg extract 3 all prolonged the cough latency period in mice, showing a significant difference compared to the model group (P<0.01). According to the Prospan oral solution instructions, the ratio of dried ivy extract to crude drug is 5–7.5:1, with an average of 1 mg of dried ivy extract containing 6.25 mg of crude drug. Since each milliliter of the oral solution contains 7 mg of dried ivy extract, it can be inferred that 1 mL of the oral solution contains 43.75 mg of ivy. Therefore, the corresponding crude drug dose (calculated per mouse) for a Prospan oral solution dosage of 3.2 mL / kg is 3.2 * 43.75 = 140 mg / kg. The yield of extract 3 was 3.9%, corresponding to a crude drug content of 3.11 / 3.9% = 79.74 mg / kg. The experimental results indicate that extract 3 can prolong the cough latency period in mice, exhibiting a certain antitussive effect, and the corresponding crude drug content is lower than that in the Prospan group.

[0190] Table 16 Effect of ivy leaf extract on cough latency in mice (n=10, Mean±SD) Note: **P<0.05 vs model group

[0191] As shown in Table 17, after three consecutive days of administration to mice, 3.2 mL / kg Prospan, 1.03 tablets / kg Dextromethorphan hydrobromide, 5.8 mL / kg honey-processed fritillaria cirrhosa and loquat syrup, and 3.11 mg / kg Extract 3 all reduced the number of coughs in mice, showing significant differences compared to the model group (P<0.01). The experimental results indicate that Extract 3 can reduce the number of coughs in mice, exhibiting a certain antitussive effect, and the corresponding amount of raw herb is less than that in the Prospan group.

[0192] Table 17 Effect of ivy leaf extract on the total number of coughs in mice within 2 minutes (n=10, Mean±SD) Note: **P<0.05 vs model group

Claims

1. An ivy saponin composition, characterized in that... The ivy saponin composition includes ivy saponin C, α-ivy saponin, ivy saponin B and ivy saponin D, wherein the mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B and ivy saponin D is 12-25:5-10:1:1-2.

2. The ivy saponin composition according to claim 1, characterized in that... The mass ratio of ivy saponin C to ivy saponin D is 8–16:

1.

3. The ivy saponin composition according to claim 1, characterized in that... The mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B, and ivy saponin D is 16–25: 5–7: 1: 1–2.

4. The ivy saponin composition according to claim 1, characterized in that... The mass ratio of hedyotis diffusin C, α-hedyotis diffusin, hedyotis diffusin B, and hedyotis diffusin D is 12–16:7–10:1:

1.

5. An ivy leaf extract, characterized in that, The ivy leaf extract comprises ivy saponin C, α-ivy saponin, ivy saponin B, and ivy saponin D, wherein the mass ratio of ivy saponin C, α-ivy saponin, ivy saponin B, and ivy saponin D is 12-25:5-10:1:1-2. The extract contains not less than 25% ivy saponin C, not less than 4% total flavonoids, not less than 2% total polysaccharides, and not less than 70% total saponins.

6. The ivy leaf extract as described in claim 5, characterized in that... The mass ratio of hedyotis diffusin C, α-hedyotis diffusin, hedyotis diffusin B, and hedyotis diffusin D is 12–21:5–9:1:1–1.

6.

7. The ivy leaf extract as described in claim 5, characterized in that... The extract contains no less than 10% α-hederonine.

8. The ivy leaf extract as described in claim 5, characterized in that... The extract contains at least 30% hedyotis diffusin C, at least 2% hedyotis diffusin D, and at least 2% hedyotis diffusin B.

9. The ivy leaf extract as described in claim 5, characterized in that... The ivy leaf extract further includes organic acid compounds, flavonoid compounds, saponin compounds, glycoside compounds and other compounds, wherein the organic acid compounds include one or more of quinic acid, neochlorogenic acid, cryptochlorogenic acid, asiatic acid, hydroxyasiatic acid, 3-O-sulfuric acid, and 3-O-sulfuric acid. The flavonoid compounds include one or both of isoquercitrin and flavoside; The glycoside compound includes one or more of the following: ethyl 3-O-β-D-glucopyranoside butyrate, (E)-4-O-β-D-glucopyranoside p-coumaric acid, 3-methylbut-2-enyl[β-D-furanopyranosyl-(1→6)]-β-D-glucopyranoside, icariin F2, myresculoside, cincaxiol B19-O-β-D-glucopyranoside, and nomiline 17-β-D-glucopyranoside. The saponin compound includes one or two of β-hederin and pyrolysis saponin A; The other compounds include one or more of 1-O-galloylpropanetriol, rhodioloside D, 3-methoxy-4-hydroxyphenol 1-O-[β-D-furanotropic-(1→6)]-β-D-glucopyranoside, icariin B5, camphorate H, and cimicifuga epoxide glycoside.

10. The ivy leaf extract as described in claim 9, characterized in that... The ivy leaf extract comprises α-hederone, hederone B, hederone C, and hederone D, and one or more of the following compounds: quinic acid, neochlorogenic acid, cryptochlorogenic acid, asiatic acid, hydroxyasiatic acid, 3-O-sulfuric acid, 3-O-sulfuric acid, oleanolic acid, ethyl 3-O-β-D-glucopyranoside butyrate, (E)-4-O-β-D-glucopyranoside-p-coumaric acid, myresculoside, β-hederone, genistein A, rhodioloside D, 3-methoxy-4-hydroxyphenol 1-O-[β-D-furanoside-(1→6)]-β-D-glucopyranoside, anodic acid H, and cimicifuga epoxide glycoside.

11. The ivy leaf extract as described in claim 10, characterized in that... The ivy leaf extract includes chlorogenic acid, shikimic acid, 3-O-p-coumarylquinic acid, sennain, aescin, α-hederone, hederone B, hederone C and hederone D.

12. The use of the ivy saponin composition according to any one of claims 1 to 4 or the ivy leaf extract according to any one of claims 5 to 11 in the preparation of a medicament for the treatment or prevention of acute bronchitis.

13. The use of the ivy saponin composition according to any one of claims 1 to 4 or the ivy leaf extract according to any one of claims 5 to 11 in the preparation of expectorant and antitussive drugs.

14. A drug for treating or preventing acute bronchitis, characterized in that... The drug comprises the ivy saponin composition as described in any one of claims 1 to 4 or the ivy leaf extract as described in any one of claims 5 to 11.

15. An expectorant and antitussive drug, characterized in that... The drug is the ivy saponin composition as described in any one of claims 1 to 4 or the ivy leaf extract as described in any one of claims 5 to 11.

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