Hedera helix extract, preparation method therefor, and use thereof

Abstract

Provided are a Hedera helix extract, a preparation method therefor, and use thereof. The preparation method comprises extracting Hedera helix leaves with 75%-85% ethanol, concentrating, adjusting the pH value to 9.5-11, standing, filtering to obtain a crude extract, passing the crude extract through a chromatographic column, and eluting with 60%-80% ethanol to prepare the Hedera helix extract. The chromatographic column is an HPD100 macroporous resin column, a polyamide column or a C18 column. The Hedera helix extract can be used for preparing drugs with anti-inflammatory, expectorant, and antitussive effects, as well as for treating acute bronchitis.

Description

An Ivy Extract, Its Preparation Method and Application Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, specifically to an ivy extract, its preparation method, and its application. 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 English ivy are saponins, including triterpenoid saponin disaccharide glycosides such as ivy saponin B, ivy saponin C, and ivy saponin D, and monosaccharide glycosides such as α-ivy saponin. Other components include flavonoids, coumarins, polyacetylenes, phenolic acids, alkaloids, and volatile oils.

[0004] Various methods for preparing ivy extracts have been disclosed in the prior art.

[0005] 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) involved 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 anti-inflammatory effect, extract yield, and saponin content of the extract were not disclosed.

[0006] The abstract of the paper "Optimization of Preparation Process of Hedera helix extract" (Yang Ziwen, 2018) describes an extraction process using 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 passing through an HPD-100 macroporous resin column with gradient elution of 30% and 80% ethanol. The 80% ethanol eluent was collected, concentrated, and then the extract was obtained. This method mainly optimized the extraction content and recovery rate of hederaside C and α-hederaside, but did not cover other saponin components such as hederaside B and hederaside D. Furthermore, it did not disclose other components of the extract, nor did it disclose the extract yield or the anti-inflammatory effect of the extract.

[0007] 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 using an ethanol solution with a purity of 10-90% 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 ivy saponins used alone.

[0008] 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, loading the extract onto a macroporous adsorption resin, and drying the resulting alcohol eluent to obtain the extract. The total saponin content of the extract is 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.

[0009] Existing technologies for extracting ivy primarily focus on increasing the content and recovery rate of saponins. For example, elution using a 30% ethanol + 70% ethanol gradient is employed 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 saponins is related to their content or recovery rate. Common sense dictates that higher saponin content should lead to better efficacy, but the applicant's research has revealed a potential contradiction with this common sense. Existing technologies have not established a relationship between the amount of alkali used in the extraction process, the elution gradient, and the anti-inflammatory efficacy, nor have they conducted corresponding research. It is necessary to further optimize the extraction process from the perspective of improving the anti-inflammatory efficacy of ivy extracts. Summary of the Invention

[0010] 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.

[0011] This invention provides an ivy extract with a simpler preparation process, more defined components, and better anti-inflammatory effects. By optimizing the extraction process, this invention increases the yield of soluble active ingredients, thereby enhancing the anti-inflammatory efficacy of the extract while maintaining a lower saponin content, shortening the production cycle, and saving production costs.

[0012] The technical solution adopted in this invention is:

[0013] A method for preparing an ivy extract, comprising: extracting ivy leaves with 75%–85% ethanol, concentrating the extract, adjusting the pH to 9.5–11, allowing it to stand, filtering the crude extract, passing it through a chromatography column, and eluting with 60%–80% ethanol to obtain the ivy extract; wherein the chromatography column is one of an HPD100 macroporous resin column, a polyamide column, or a C18 column.

[0014] Furthermore, when the chromatography column is a polyamide column, it is preferable to adjust the pH of the crude extract to neutral before passing it through the chromatography column.

[0015] Furthermore, when the chromatography column is a C18 column, the crude extract can be eluted with 30% ethanol first, and then with 60% to 80% ethanol, and the eluent eluted with 60% to 80% ethanol can be collected.

[0016] Furthermore, the chromatography column is preferably an HPD100 macroporous resin column.

[0017] Furthermore, the method includes the following steps:

[0018] (1) Take ivy leaves, crush them, extract them by reflux with 75% to 85% ethanol, filter and combine the filtrates;

[0019] (2) Concentrate the filtrate until there is no alcohol odor, adjust the pH to 9.5-11, refrigerate and stand for 8-15 hours, and filter to obtain crude extract;

[0020] (3) The crude extract is passed through a chromatography column, washed with water until colorless, and then eluted with 3 to 5 column volumes of 60% to 80% ethanol. The ethanol eluent is collected, concentrated and dried to obtain the ivy extract. The chromatography column is one of HPD100 macroporous resin column, polyamide column or C18 column.

[0021] In step (1), the reflux extraction can be performed multiple times, such as 2 to 4 times, preferably 2 to 3 times.

[0022] In step (1), the volume of 75% to 85% ethanol is generally 5-12 mL / g based on the weight of ivy leaves used.

[0023] In step (1), the reflux extraction temperature is preferably 80-90℃.

[0024] In step (3), when the chromatography column is a polyamide column, it is preferable to adjust the pH of the crude extract to neutral before passing it through the chromatography column.

[0025] When the chromatography column is a C18 column, after the crude extract passes through the column, it can first be eluted with 30% ethanol, and then eluted with 3 to 5 column volumes of 60% to 80% ethanol. The eluent obtained from the 60% to 80% ethanol eluent should be collected, concentrated, and dried. The elution volume of 30% ethanol is 2 to 3 column volumes, and this portion of the eluent should be discarded.

[0026] Most preferably, the chromatography column is an HPD100 macroporous resin column.

[0027] In step (2), an alkaline solution is preferably used to adjust the pH value, including sodium hydroxide solution or potassium hydroxide solution. After adjusting the pH value with the alkaline solution, water can be added to a total volume of 5 to 12 times the amount of ivy leaves (volume-to-mass ratio).

[0028] The concentration of the sodium hydroxide solution or potassium hydroxide solution is preferably 3-8%, more preferably 5%.

[0029] The volume of the alkaline solution is preferably 0.2 to 0.4 times the amount of ivy leaves used, more preferably 0.2 to 0.3 times, and even more preferably 0.3 times.

[0030] More preferably, the mass of sodium hydroxide or potassium hydroxide in the alkaline solution is 1-2% of the amount of ivy leaves added.

[0031] The ivy mentioned includes various medicinal materials such as Western ivy (Hedera helix L.), Chinese ivy (Hedera nepalensis), Canary ivy (Hedera canariensis), leather-leaved ivy (Hedera colchica), and Japanese ivy (Hedera rhombea). Among them, Western ivy and Chinese ivy are more commonly used in drug treatment.

[0032] The present invention also provides an ivy extract prepared by the above preparation method.

[0033] Furthermore, the ivy extract includes α-hederone, hederone C, hederone B and hederone D, and the total mass fraction of α-hederone, hederone C, hederone B and hederone D is greater than 50%.

[0034] Furthermore, in the ivy extract, the mass content of α-hederone is greater than 10%, the mass content of hederone C is greater than 30%, the mass content of hederone B is greater than 2%, and the mass content of hederone D is greater than 1%.

[0035] The present invention also provides the use of the ivy extract in the preparation of medicaments for the treatment or prevention of acute bronchitis.

[0036] The present invention also provides the application of the ivy extract in the preparation of expectorant and antitussive drugs.

[0037] The present invention also provides a medicament for treating or preventing acute bronchitis, comprising the ivy extract.

[0038] The present invention also provides an expectorant and antitussive drug, comprising the ivy extract.

[0039] The beneficial effects of this invention are as follows:

[0040] This invention optimizes the preparation process, enriching the anti-inflammatory components in ivy raw materials and clarifying that ivy extracts with specific component contents have better efficacy. This invention increases the yield of the extract while reducing the saponin content, but the resulting extract exhibits better anti-inflammatory effects, achieving unexpected technical results. This invention overcomes the technical bias of prior art that simply increases saponin content, obtaining an ivy extract with better anti-inflammatory effects and higher yield by simplifying the elution process. This is more conducive to the development of oral solution formulations and reduces the use of solubilizers. This invention can effectively enrich the anti-inflammatory components in ivy leaf extract, increasing the anti-inflammatory activity of the extract. Therefore, it has a better anti-inflammatory effect at the same weight of raw material, which helps to reduce the dosage and improves the economic efficiency and utilization rate of the raw material. Detailed Implementation

[0041] 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.

[0042] Taking 200g of processed ivy leaves as an example, ivy leaf extract was prepared according to the following embodiment. The production batch number of the ivy medicinal material is LX220501.

[0043] Example 1: Preparation of Extract 1

[0044] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 1000mL (5 times the volume of the medicinal material) of 85% ethanol at 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add 80mL (0.4 times the amount of material) of 5% sodium hydroxide solution, add water to a total volume of 5 times the amount of material, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, 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.

[0045] Example 2: Preparation of Extract 2

[0046] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 times that of the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add 0.3 times the amount of 5% NaOH solution, add water to a total volume of 12 times the amount of the medicinal material, adjust the pH to 9.5-11, refrigerate and 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.

[0047] Example 3: Preparation of Extract 3

[0048] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 75% ethanol at a volume ratio of 9 times the medicinal material, at an extraction temperature of 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 9 times the feed volume, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, pass through HPD100 macroporous adsorption resin, wash with water until colorless, then elute with 2 times the column volume of 80% ethanol, collect the 80% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0049] Example 4: Preparation of Extract 4

[0050] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 85% ethanol at a volume ratio of 5 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add potassium hydroxide solution, add water to a total volume of 5 times the feed amount, adjust the pH to 9.5-11, refrigerate and 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 5 times the column volume of 60% ethanol, collect the 60% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0051] Example 5: Preparation of Extract 5

[0052] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add potassium hydroxide solution, add water to a total volume of 12 times the feed amount, adjust the pH to 9.5-11, refrigerate and 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.

[0053] Example 6: Preparation of Extract 6

[0054] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 75% ethanol at a volume ratio of 9 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add potassium hydroxide solution, add water to a total volume of 9 times the feed amount, adjust the pH to 9.5-11, refrigerate and 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 2 column volumes of 80% ethanol, collect the 80% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0055] Example 7: Preparation of Extract 7

[0056] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 85% ethanol at a volume ratio of 5 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add sodium hydroxide solution, add water to a total volume of 5 times the feed amount, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, concentrate to 100-200mL, and then pass through a C18 column (particle size 60μm). Elute with water until colorless, then elute with 5 times the column volume of 60% ethanol. Collect the 60% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0057] Example 8: Preparation of Extract 8

[0058] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 85% ethanol at a volume ratio of 5 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add water to sodium hydroxide solution to a total volume of 5 times the feed amount, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, concentrate to 100-200mL, and pass through a C18 column (particle size 60μm). Elute with water until colorless, then elute with 2 times the volume of 30% ethanol, and then elute with 5 times the column volume of 60% ethanol. Collect the 60% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0059] Example 9: Preparation of Extract 9

[0060] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add sodium hydroxide solution, add water to a total volume of 12 times the feed amount, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, concentrate to 100-200mL, and then pass through a C18 column (particle size 60μm). Elute 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.

[0061] Example 10: Preparation of Extract 10

[0062] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add sodium hydroxide solution, add water to a total volume of 12 times the feed amount, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, concentrate to 100-200mL, and pass through a C18 column (particle size 60μm). Elute with water until colorless, then elute with 2 column volumes of 30% ethanol, and then elute with 3 column volumes of 70% ethanol. Collect the 70% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0063] Example 11: Preparation of Extract 11

[0064] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 75% ethanol at a volume ratio of 9 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until there is no alcohol odor, add sodium hydroxide solution, add water to a total volume of 9 times the feed amount, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, concentrate to 100-200mL, and then pass through a C18 column (particle size 60μm). Elute with water until colorless, then elute with 2 column volumes of 80% ethanol. Collect the 80% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0065] Example 12: Preparation of Extract 12

[0066] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 75% ethanol at a volume ratio of 9 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add sodium hydroxide solution, add water to a total volume of 9 times the feed amount, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, concentrate to 100-200mL, and pass through a C18 column (particle size 60μm). Elute with water until colorless, then elute with 2 column volumes of 30% ethanol, and then elute with 2 column volumes of 80% ethanol. Collect the 80% ethanol eluent, concentrate the filtrate, dry, and crush to obtain the final product.

[0067] Example 13: Preparation of Control Group 1

[0068] Based on Example 2, 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:

[0069] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 (12 times the volume of the medicinal material) under reflux at 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add 0.2 times the amount of 5% NaOH solution, add water to a total volume of 12 times the amount of the medicinal material, 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.

[0070] Example 14: Preparation of Control Group 2

[0071] Based on control group 1, an HPD100 chromatographic column was used, as follows:

[0072] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 times the medicinal material, at an extraction temperature of 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, and add water to a total volume of 12 times the feed volume. Adjust the pH to 9.5-11, refrigerate and stand overnight, filter, and pass through HPD100 macroporous adsorption resin. Wash with water until colorless, then elute with 3 times the column volume of 30% ethanol, and then 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 15: Preparation of Control Group 3

[0074] Based on Example 2, elution with 30% ethanol was added, as follows:

[0075] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add 0.3 times the feed amount of 5% NaOH solution, add water to a total volume of 12 times the feed amount, adjust the pH to 9.5-11, refrigerate and stand overnight, filter, pass through HPD100 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 16: Preparation of Control Group 4

[0077] Weigh 200g of dried ivy leaves, crush them into coarse powder, and extract twice with 80% ethanol at a volume ratio of 12 times the medicinal material, at an extraction temperature of 80-90℃. Filter and combine the filtrates, recover the ethanol until no alcohol odor remains, add 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 and stand overnight, filter, adsorb with 5 times the feed amount 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 product.

[0078] The preparation processes of Examples 1 to 16 are shown in Table 1 below.

[0079] Table 1 Preparation process

[0080] Example 17: Component Detection

[0081] The test items include: the content of saponin C, α-saponin, saponin B, and saponin D in the extract.

[0082] Yield = (Weight of extract / Amount of medicinal materials used) * 100%.

[0083] Saponin transfer rate = weight of extract * saponin content in extract / (weight of corresponding medicinal slices * saponin content in medicinal slices) * 100%.

[0084] 1. Experimental instruments and materials:

[0085] The production batch number of the processed ivy leaf slices was LX220501 (Zhejiang Kang Enbei Pharmaceutical Co., Ltd.). Extracts CCT-1 to CCT-12 and control group extracts 1 to 4 were prepared according to Examples 1 to 16.

[0086] 2. Detection method:

[0087] Chromatographic conditions and system suitability test: ZORBAX Eclipse XDB-C18 Analytical 4.6*250mm 5-Micron column; acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 2; column temperature 25℃; detection wavelength 205nm; injection volume 10μL, flow rate 1.0mL / min.

[0088] Table 2 Gradient elution program

[0089] 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 ivy saponin D per 1 mL, respectively.

[0090] 3. Experimental Results

[0091] The yields, saponin active ingredient contents, and saponin transfer rates of extracts CCT-1–12 and controls 1–4 are shown in Table 3. The saponin transfer rates of extracts CCT-2 and controls 1–3 are shown in Table 4.

[0092] Table 3. Quality analysis results of ivy extracts prepared by different processes

[0093] Table 4 shows the transfer rates of some extracts.

[0094] Comparing extract 2 and control group 1, it was found that the contents of saponin C and α-saponin were not significantly different in the extracts obtained under different alkalization conditions, different macroporous resin column packing materials, and different elution conditions. However, the saponin transfer rate of extract 2 was significantly higher than that of control group 1. Further studies using control groups 2–4 were conducted to investigate the effects of different preparation conditions on the component content and transfer rate of ivy leaf extracts.

[0095] Control groups 1 and 2 were prepared under the same alkalinization conditions and eluents, using different macroporous resin column packings. The content of the four saponins in control group 2 was higher than that in control group 1. Extract 2 and control group 4 were prepared under the same elution conditions, but control group 4, using an alumina chromatography column, showed a significantly lower content of saponins. This indicates that the enrichment of active ingredients is related to the column chromatography packing; different columns yield different active ingredients with varying contents. Among extracts 1–12, the HPD100 macroporous resin column yielded the highest yield and saponin content.

[0096] Control groups 2 and 3 used the same chromatography column and eluent. Under different pH conditions, after increasing the amount of alkali solution, the content of the four saponins in control group 3 and the yield of the extract were both higher than those in control group 2. Furthermore, the transfer rates of saponin C and α-saponin were relatively increased in control group 3. This indicates that increasing the amount of alkali solution within a specific pH range improved the saponin transfer rate, yield, and content of the extract. This is because increasing the amount of alkali solution enhances the sedimentation of polysaccharides and proteins in the ivy leaf alcohol extract, reducing the precipitation of active ingredients, especially α-saponins. However, excessively high alkali concentrations can disrupt the ester bond at position 28 of ivy saponin C, leading to hydrolysis and the formation of α-ivy saponin. Using 0.3 times the amount of alkali solution and adjusting the pH to the range of 9.5–11 not only increased the water precipitation effect of the alcohol extract but also prevented ivy saponin C from reducing its transfer rate due to alkali hydrolysis and the formation of α-ivy saponin.

[0097] Extracts 1-12, obtained using HPD-100, polyamide, and C18 columns, had saponin contents of 50-60%. Except for extract 2, the total saponin content of the others was lower than that obtained by elution with DM130 + 30% + 70% ethanol in control group 1. In terms of yield, extracts 1-3 had higher yields than control group 1, but extracts 4-12 had lower yields. According to conventional standards for evaluating extract quality, higher saponin content and yield generally indicate better technical effects; therefore, control group 1 should be considered a better extraction method. However, this invention tested the anti-inflammatory effects of the extracts. The results showed that the content of saponins, as the main active ingredient in the extracts, is not linearly correlated with the anti-inflammatory effect. Even with a lower saponin content, the anti-inflammatory effect may be better, producing unexpected technical benefits. See Example 18 for details.

[0098] Example 18: Evaluation of the COX-2 inhibitory activity of ivy leaf extract

[0099] 1. Experimental reagents and materials

[0100] COX-2 inhibitor screening kit (Beyotime Biotechnology), ivy extracts (CCT-1~12, control group 1~4) (self-made by Zhejiang Kang Enbei Pharmaceutical Co., Ltd.), DMSO (Diamond).

[0101] 2. Experimental Methods

[0102] According to the kit's testing requirements, three concentrations were set for the drug administration groups: 12.5 μg / mL, 25 μg / mL, and 50 μg / mL; a positive control group (celecoxib); and a 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 also set up. For the sample group, the test sample (ivy leaf extract) 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. The mixture was mixed, incubated at 37°C in the dark for 5 min, and then fluorescence was measured. Detection was performed using a Tecan microplate reader with an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

[0103] 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%

[0104] 3. Experimental Results

[0105] Different extracts of ivy exhibited varying inhibitory activities against COX-2, with half-maximal inhibitory rates (IC50) of 1,000 and 1,000%. 50 See Table 5 below for details. Based on the yield, the concentration of the extract (IC50) can be calculated as follows: I1–12. 50 The values ​​ranged from 7.64 to 13.78 μg / mL, which translates to 113.52 to 328.10 μg / mL of processed medicinal slices. The IC50 values ​​for control groups 1–4 were... 50 The values ​​ranged from 35.92 to 249.9 μg / mL, which translates to a medicinal slice content of 520.58 to 6941.67 μg / mL.

[0106] Based on the saponin content results in Table 3, the content of the four saponins in control group 1 was 62.1%, which was slightly higher or similar to the saponin content of extracts 1-12, and the yield was also higher than that of extracts 4-12. However, the IC50 after converting the amount of medicinal slices was lower. 50 The saponin content was 3 to 7 times that of extracts 1-12, corresponding to an anti-inflammatory activity of 1 / 7 to 1 / 3 of that of extracts 1-12. It is evident that the saponin content of extracts 1-12 provided by this invention is similar to or lower than that of control group 1, but the anti-inflammatory effect is better. Saponins are the main active components of ivy extracts, but this technical solution achieves better anti-inflammatory effects with similar or lower saponin content, demonstrating unexpected technical benefits. Therefore, the quality evaluation of extracts should not be limited to yield and saponin content; high saponin content does not necessarily indicate good anti-inflammatory effects, and there is no linear correlation between saponin content and efficacy.

[0107] The anti-inflammatory results of control groups 1 and 2 showed that different chromatographic columns enriched different active pharmaceutical ingredients, with the HPD100 macroporous resin column enriching the active pharmaceutical ingredients better than the DM130 macroporous resin column. A comparison of extracts 2, 5, and 9 with control group 4 revealed that, under the same alkaline solution pH and eluent conditions, the anti-inflammatory activity of extracts obtained using the HPD100 macroporous resin column, polyamide column, and C18 column was 30–58 times higher than that obtained using the alumina column. Therefore, the HPD100, polyamide, and C18 columns can effectively enrich the anti-inflammatory components in ivy leaf extract, increasing its anti-inflammatory activity.

[0108] The anti-inflammatory effects of control groups 2 and 3 showed that only minor changes in the amount of alkaline solution had little impact on the enrichment of anti-inflammatory components. However, the selection of extraction solvent, alkaline solution, chromatographic column, and eluent is a crucial component of the purification process of crude ivy leaf extract, ultimately directly affecting its pharmacological activity.

[0109] Comparing extract 2 and control group 3, the total amount of the four saponins in control group 3 was 74%, corresponding to IC50. 50 The value (converted to the amount of processed medicinal slices) was 531.92 μg / mL; the total amount of the four saponins in extract 2 was 62.5%, corresponding to IC50 values. 50 The concentration (converted to the amount of processed medicinal slices) was 120.11 μg / mL, representing a 340% increase in activity compared to control group 3. This indicates that the anti-inflammatory components of the crude ivy leaf extract 2 were effectively enriched after separation using an HPD100 column and elution with 70% ethanol. Compared to control group 3, which yielded high-purity saponins through gradient elution with 30% and 70% ethanol, extract 2 exhibited better anti-inflammatory effects. This demonstrates that even using the same chromatographic column, the type and order of the eluent play a crucial role in the enrichment of anti-inflammatory components in ivy leaf extract. The choice of eluent directly affects the types and amounts of eluted components; different elution methods result in different enriched anti-inflammatory components, thus significantly influencing the efficacy of the extract.

[0110] Table 5. Inhibitory activity of various extracts of ivy against COX-2 enzyme.

[0111] Example 19: Pharmacodynamic effects of ivy leaf extract on a lipopolysaccharide (LPS)-induced acute bronchitis model in mice.

[0112] 1. Experimental reagents and materials

[0113] 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); CCT-2, CCT-5 and CCT-9 samples (self-made by Zhejiang Kang Enbei Traditional Chinese Medicine Co., Ltd.)

[0114] 2. Animal grouping

[0115] After three days of acclimatization feeding, SPF-grade Balb / c mice were divided into 8 groups of 10 mice each according to their body weight, as shown in Table 6 below.

[0116] Table 6 Animal Grouping

[0117] 3. Route of administration and timing of administration

[0118] Administer orally via gavage for 3 consecutive days at a dose of 0.2 mL / 10 g body weight.

[0119] 4. Model Replication

[0120] 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.

[0121] 5. Indicator Testing

[0122] Serum was collected from animals one hour after the last administration to detect changes in the inflammatory factor TNF-α.

[0123] 6. Statistical methods

[0124] 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, with p < 0.05 considered statistically significant.

[0125] 7. Experimental Results

[0126] As shown in Table 7, 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 CCT-2, CCT-5, and CCT-9 showed significant differences compared to the model group (P<0.05). The experimental results indicate that CCT-2, CCT-5, and CCT-9 effectively inhibited the growth of inflammatory factors in LPS-induced acute bronchitis in mice, demonstrating therapeutic effects, and their anti-inflammatory effects were superior to those of the Prospan group.

[0127] Table 7. 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

[0128] Example 20: Expectorant effect of ivy leaf extract on a mouse expectorant model (phenol red method)

[0129] 1. Experimental reagents and materials

[0130] Phenol red (GENERAL-REAGENT); ELISA reader (Thermo); Drug source as shown in Example 19.

[0131] 2. Animal grouping

[0132] Eighty SPF-grade ICR mice were randomly divided into eight groups of ten mice each after a three-day acclimatization period. The groups are shown in Table 8.

[0133] Table 8 Animal Grouping

[0134] 3. Route of administration and timing of administration

[0135] The medication was administered orally once daily for three consecutive days, with a dosage of 0.2 mL / 10 g body weight.

[0136] 4. Model Replication

[0137] 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.

[0138] 5. Indicator Testing

[0139] The OD value at 546 nm was measured using an ELISA reader, and the instrument was zeroed using 5% sodium bicarbonate solution.

[0140] 6. Statistical methods

[0141] 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.

[0142] 7. Experimental Results

[0143] As shown in Table 9, 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 CCT-2, CCT-5, and CCT-9 all 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 syrup showed no significant promoting effect. Therefore, CCT-2, CCT-5, and CCT-9 can increase tracheal phenol red secretion in mice and have a certain expectorant effect, with the expectorant effect being superior to that of the Prospan group.

[0144] Table 9. Expectorant effect of ivy leaf extract (n=10, Mean±SD) Note: *P<0.05 vs model group

[0145] Example 21: Study on the antitussive effect of ivy leaf extract on a mouse antitussive model (ammonia method)

[0146] 1. Experimental reagents and materials

[0147] 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 19.

[0148] 2. Animal grouping

[0149] Seventy SPF-grade ICR female mice were acclimatized for three days and then divided into seven groups of 10 mice each, based on their average body weight. The group assignments are shown in Table 10. Table 10: Animal Grouping

[0150] 3. Route of administration and timing of administration

[0151] Administer orally via gavage for 3 consecutive days at a dose of 0.2 mL / 10 g body weight.

[0152] 4. Model Replication

[0153] 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.

[0154] 5. Indicator Testing

[0155] The incubation period of a cough is the total number of coughs within 2 minutes.

[0156] 6. Statistical methods

[0157] 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.

[0158] 7. Experimental Results

[0159] As shown in Table 11, 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 CCT-2, CCT-5, and CCT-9 significantly prolonged the cough latency period 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, a Prospan oral solution dosage of 3.2 mL / kg corresponds to a crude drug dosage (calculated per mouse) of 3.2 * 43.75 = 140 mg / kg. The yields of CCT-2, CCT-5, and CCT-9 were 9.0%, 3.9%, and 4.3%, respectively. The corresponding crude drug amounts were calculated as dose / yield: CCT-2 had a crude drug amount of 3.11 / 9% = 34.44 mg / kg, CCT-5 had 3.11 / 3.9% = 79.74 mg / kg, and CCT-9 had 3.11 / 4.3% = 72.33 mg / kg. The experimental results indicate that CCT-2, CCT-5, and CCT-9 can prolong the cough latency period in mice, exhibiting some antitussive effect, and their corresponding crude drug amounts were lower than those in the Prospan group.

[0160] Table 11 Effect of ivy leaf extract on cough latency in mice (n=10, Mean±SD) Note: **P<0.05 vs model group

[0161] As shown in Table 12, 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 CCT-2, CCT-5, and CCT-9 all reduced the number of coughs in mice, showing significant differences compared to the model group (P<0.01). The experimental results indicate that CCT-2, CCT-5, and CCT-9 can reduce the number of coughs in mice, exhibiting a certain antitussive effect, and the corresponding amounts of raw herbs are less than those in the Prospan group.

[0162] Table 12 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. A method for preparing an ivy extract, characterized in that, The method is as follows: ivy leaves are extracted with 75% to 85% ethanol, concentrated, and the pH is adjusted to 9.5 to 11. After standing, the crude extract obtained by filtration is passed through a chromatography column and eluted with 60% to 80% ethanol to obtain the ivy extract; the chromatography column is one of HPD100 macroporous resin column, polyamide column or C18 column.

2. The method as described in claim 1, characterized in that, When the chromatography column is a polyamide column, the crude extract is adjusted to neutral pH and then passed through the chromatography column; When the chromatography column is a C18 column, the crude extract is passed through the chromatography column, first eluted with 30% ethanol, then eluted with 60% to 80% ethanol, and the eluent eluted with 60% to 80% ethanol is collected.

3. The method as described in claim 1, characterized in that, The method includes the following steps: (1) Take ivy leaves, crush them, extract them by reflux with 75% to 85% ethanol, filter and combine the filtrates; (2) Concentrate the filtrate until there is no alcohol odor, adjust the pH to 9.5-11, refrigerate and stand for 8-15 hours, and filter to obtain crude extract; (3) The crude extract is passed through a chromatography column, washed with water until colorless, and then eluted with 3 to 5 column volumes of 60% to 80% ethanol. The ethanol eluent is collected, concentrated and dried to obtain the ivy extract. The chromatography column is one of HPD100 macroporous resin column, polyamide column or C18 column.

4. The method as described in claim 3, characterized in that, The chromatography column is an HPD100 macroporous resin column.

5. The method as described in claim 3, characterized in that, In step (2), an alkaline solution is used to adjust the pH value. The alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution.

6. The method as described in claim 5, characterized in that, In step (2), the concentration of the sodium hydroxide solution or potassium hydroxide solution is 3-8%, and the volume of the alkaline solution is 0.2-0.4 times the amount of ivy leaves added.

7. The method as described in claim 5, characterized in that, In step (2), the mass of sodium hydroxide or potassium hydroxide in the alkaline solution is 1-2% of the amount of ivy leaves added.

8. The method as described in claim 5, characterized in that, In step (2), after adjusting the pH value of the alkaline solution, water is added until the total volume is 5 to 12 times the amount of ivy leaves added.

9. Ivy extract prepared by any one of claims 1 to 8.

10. The ivy extract as described in claim 9, characterized in that... The ivy extract includes α-hederone, hederone C, hederone B and hederone D, and the total mass fraction of α-hederone, hederone C, hederone B and hederone D is greater than 50%.

11. The ivy extract as described in claim 10, characterized in that... In the ivy extract, the mass content of α-hederone is greater than 10%, the mass content of hederone C is greater than 30%, the mass content of hederone B is greater than 2%, and the mass content of hederone D is greater than 1%.

12. The use of the ivy extract prepared by the method according to any one of claims 1 to 8 or the ivy extract according to any one of claims 10 to 11 in the preparation of a medicament for the treatment or prevention of acute bronchitis.

13. The use of the ivy extract prepared by the preparation method according to any one of claims 1 to 8 or the ivy extract according to any one of claims 10 to 11 in the preparation of expectorant and antitussive drugs.

14. A medicament for treating or preventing acute bronchitis, comprising an ivy extract prepared by any one of claims 1 to 8 or an ivy extract prepared by any one of claims 10 to 11.

15. An expectorant and antitussive drug, comprising an ivy extract prepared by any one of claims 1 to 8 or an ivy extract prepared by any one of claims 10 to 11.

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