Composition capable of inhibiting neuraminidase activity and application thereof

Abstract

The invention discloses a composition capable of inhibiting neuraminidase activity and application of the composition. The composition is prepared from pericarpium citri reticulatae powder and platycodon grandiflorum powder. Wherein the mass ratio of the pericarpium citri reticulatae powder to the platycodon grandiflorum powder is 5: (2-4). The dried orange peel powder and the platycodon grandiflorum powder are used as main functional components, the dried orange peel powder and the platycodon grandiflorum powder are combined according to the ratio of 5: 3, and in-vitro tests prove that the prepared food has the properties of inhibiting neuraminidase activity and improving respiratory tract health.

Description

Technical Field

[0001] The invention belongs to the technical field of functional compositions, and in particular relates to a composition capable of inhibiting neuraminidase activity and application thereof. Background Art

[0002] Influenza (flu) is a respiratory infectious disease caused by influenza virus. The normal invasion and replication cycle of the virus is divided into six stages, including: adsorption, invasion, uncoating, biosynthesis, assembly and release. After the virus enters the host, the hemagglutinin on its surface binds to the hemagglutinin receptor on the surface of the epithelial cell, and then the virus enters the cell. Its RNA uses the substrate provided by the host cell to replicate and express, and finally completes the assembly and protrudes from the host cell in the form of budding. After protrusion, the mature influenza virus is not completely separated from the host cell, and still relies on the sialic acid residue at the end of the hemagglutinin molecule to connect with the sugar group on the surface of the hemagglutinin receptor molecule by glycosidic bond. At this time, neuraminidase is needed to catalyze the hydrolysis of the glycosidic bond so that the virus can detach from the host cell and infect other cells for spread. Inhibiting the activity of neuraminidase in this process can significantly block the spread and transmission of the virus in the body, so it is a target for the treatment of influenza.

[0003] Currently, the anti-influenza drugs such as oseltamivir and zanamivir used in clinical practice are all neuraminidase inhibitors. However, such drugs can cause gastrointestinal discomfort such as nausea, vomiting, diarrhea, abdominal pain, and adverse reactions such as dizziness, headache, insomnia, and fatigue. Therefore, this proposal proposes a composition of plant extracts that can inhibit neuraminidase activity.

[0004] Platycodon grandiflorum (Jacq.) A.DC. is a perennial herbaceous plant of the Campanulaceae family. Its main medicinal part is the dried rhizome. It tastes bitter and pungent, is neutral in nature, and mainly enters the lung meridian. It has the effects of relieving cough and expectoration, clearing the lungs and relieving throat pain. It has a good therapeutic effect on cough with phlegm, sore throat, etc. The chemical components of Platycodon grandiflorum include triterpenoid saponins, flavonoids, and phenols. Recent studies have shown that Platycodon grandiflorum can inhibit the activity of cathepsin and TMPRSS2 protease (transmembrane serine protease 2), thereby blocking the new coronavirus from entering human cells to achieve an antiviral effect.

[0005] Tangerine peel is the dried mature peel of the Rutaceae plant Citrus reticulata Blanco and its cultivated varieties. After picking the ripe fruits, peel the peel, sun-dry or low-temperature dry to obtain tangerine peel, which tastes bitter and spicy, is warm in nature, and belongs to the lung and spleen meridians. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm. It is mainly used for abdominal distension, poor appetite, vomiting and diarrhea, and cough with phlegm. Modern research shows that the main components of tangerine peel include flavonoids such as hesperidin, alkaloids, and volatile oils. Its volatile oil components can block the contraction and spasm of bronchial smooth muscles caused by acetylcholine chloride and histamine phosphate, thereby achieving the effect of relieving cough and asthma; limonene in its volatile oils also has antibacterial and stimulates the respiratory mucosa to secrete mucus and promote sputum; flavonoid components such as hesperidin have antiviral functions and can prevent throat discomfort caused by viral infections.

[0006] At present, there is no research report that shows that tangerine peel powder, platycodon powder and their combination can inhibit the activity of neuraminidase. Summary of the invention

[0007] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0008] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0009] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a composition having the ability to inhibit neuraminidase activity.

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions: a composition capable of inhibiting neuraminidase activity, comprising tangerine peel powder and platycodon powder;

[0011] Wherein, in terms of mass ratio, the ratio of the tangerine peel powder to the platycodon powder is 5:(1-5).

[0012] As a preferred embodiment of the composition of the present invention, the mass ratio of the tangerine peel powder to the platycodon powder is 5:(2-4).

[0013] As a preferred embodiment of the composition of the present invention, the mass ratio of the tangerine peel powder to the platycodon powder is 5:(2-3).

[0014] As a preferred embodiment of the composition of the present invention, the hesperidin content in the tangerine peel powder is ≥ 0.1%.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a composition capable of inhibiting neuraminidase activity, comprising uniformly mixing tangerine peel powder and platycodon powder to prepare the composition.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide a composition having the ability to inhibit neuraminidase activity for use in the preparation of food or health products.

[0017] As a preferred embodiment of the application of the present invention, the food includes powders, soft / hard capsules, compressed candies, soft candies, drinks, jellies, etc.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide a composition for inhibiting neuraminidase activity for use in the preparation of medicines.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention combines tangerine peel powder and platycodon powder to synergistically inhibit neuraminidase activity and improve the function of throat discomfort symptoms.

[0021] (2) The present invention uses tangerine peel powder and platycodon powder as main effective ingredients, wherein the tangerine peel powder and platycodon powder are combined in a certain ratio. The prepared food is verified by in vitro tests to have the properties of inhibiting neuraminidase activity and improving respiratory health. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0023] Figure 1 This is a graph showing the effect of the tangerine peel and platycodon grandiflorum composition on the level of LPS-induced cellular inflammatory factors in an embodiment of the present invention.

[0024] Figure 2 This is a scoring chart of the immediate effect of the tangerine peel and platycodon grandiflorum composition on throat discomfort in the implementation of the present invention.

[0025] Figure 3 This is a scoring chart of the long-term effect of the tangerine peel and platycodon grandiflorum composition on throat discomfort in the implementation of the present invention. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0029] The raw materials used in the embodiments of the present invention are all common commercially available products.

[0030] Example 1

[0031] This embodiment provides a method for preparing a composition, the main steps of which are:

[0032] (1) Preparation of tangerine peel powder: Add 10 times the weight of drinking water to the tangerine peel raw material, boil and extract for 1.5 hours, and filter to obtain the first extract;

[0033] Add 8 times drinking water to the residue, boil and extract for 1.5 hours, and filter to obtain the second extract;

[0034] The first extract and the second extract are combined, concentrated at a temperature of 50-100°C and a vacuum degree of -0.05 to -0.09 MPa, and then spray-dried, crushed and sieved through a 100-mesh sieve at an inlet air temperature of 175-205°C and an outlet air temperature of 75-105°C. The obtained tangerine peel powder has a hesperidin content of ≥0.1% as determined by the hesperidin content test under the tangerine peel item in the Chinese Pharmacopoeia-2020.

[0035] (2) Preparation of Platycodon grandiflorum powder: extract the original Platycodon grandiflorum medicinal material with water at 60°C for 2 hours, concentrate the obtained water extract to a relative density of 1.12-1.17, sieve, spray dry under the conditions of inlet air temperature of 175-205°C and outlet air temperature of 75-105°C, and pass through an 80-mesh sieve to obtain Platycodon grandiflorum powder; wherein the ratio between the amount of the original medicinal material and the amount of the extracted Platycodon grandiflorum powder is 5:1.

[0036] (3) Preparation of a composition: 50 g of dried tangerine peel powder and 10 g of platycodon grandiflorum powder were mixed uniformly to prepare a composition having the ability to inhibit neuraminidase activity.

[0037] Example 2

[0038] This embodiment provides a method for preparing a composition, wherein the preparation method of tangerine peel powder and platycodon grandiflorum powder is consistent with that of embodiment 1, except that:

[0039] (3) Preparation of a composition: 50 g of dried tangerine peel powder and 20 g of platycodon grandiflorum powder were mixed uniformly to prepare a composition having the ability to inhibit neuraminidase activity.

[0040] Example 3

[0041] This embodiment provides a method for preparing a composition, wherein the preparation method of tangerine peel powder and platycodon grandiflorum powder is consistent with that of embodiment 1, except that:

[0042] (3) Preparation of a composition: 50 g of dried tangerine peel powder and 30 g of platycodon grandiflorum powder were mixed uniformly to prepare a composition having the ability to inhibit neuraminidase activity.

[0043] Example 4

[0044] This embodiment provides a method for preparing a composition, wherein the preparation method of tangerine peel powder and platycodon grandiflorum powder is consistent with that of embodiment 1, except that:

[0045] (3) Preparation of a composition: 50 g of dried tangerine peel powder and 40 g of platycodon grandiflorum powder were mixed uniformly to prepare a composition having the ability to inhibit neuraminidase activity.

[0046] Comparative Example 1

[0047] This comparative example provides a method for preparing a composition, wherein the preparation method of tangerine peel powder and platycodon grandiflorum powder is consistent with that of Example 1, except that:

[0048] (3) Preparation of a composition: Take 50 g of dried tangerine peel powder and 50 g of platycodon grandiflorum powder, mix them evenly, and prepare a composition having the ability to inhibit neuraminidase activity.

[0049] Comparative Example 2

[0050] Preparation of tangerine peel powder: The tangerine peel powder is prepared through water extraction, concentration, drying and other steps, and the hesperidin content is ≥ 0.1%. The specific preparation process is the same as that in Example 1.

[0051] Comparative Example 3

[0052] Preparation of Platycodon grandiflorum powder: It is prepared through water extraction, concentration, drying and other processes, wherein the extraction ratio of Platycodon grandiflorum powder is 4:1, and the specific preparation process is the same as Example 1.

[0053] In vitro test protocol:

[0054] 1. Preparation of standard curve detection:

[0055] a. Add 70 μl of neuraminidase detection buffer (provided by the neuraminidase inhibitor screening kit (PD309) purchased from Beyotime Biotech) to each well of a 96-well fluorescent ELISA plate.

[0056] b. Add 0, 1, 2, 5, 7.5, and 10 μl of neuraminidase to each well, respectively.

[0057] c. Add 0-20 μl of Milli-Q water to each well to make the total volume of each well 90 μl.

[0058] 2. Preparation of sample testing (refer to Table 1):

[0059] a. Add 70 μl of neuraminidase assay buffer to each well of a 96-well fluorescence ELISA plate.

[0060] b. Add 10 μl of neuraminidase to each well.

[0061] c. Add 10 μl of the neuraminidase inhibitor sample to be screened to each well (refer to Table 1 for grouping: each group of samples was dissolved in pure water, and the sample concentrations were 200, 400, 800, 1600 and 3200 μg / ml, respectively).

[0062] 3. Detection:

[0063] a. Mix by shaking for about 1 minute.

[0064] b. Incubate at 37°C for 2 minutes to allow the inhibitor and neuraminidase to fully interact with each other. The standard curve wells are also incubated together.

[0065] c. Add 10 μl of neuraminidase fluorescent substrate (provided by the neuraminidase inhibitor screening kit (PD309) purchased from Beyotime Biotechnology Co., Ltd.) to each well.

[0066] d. Vibrate to mix for about 1 minute.

[0067] e. After incubation at 37°C for 30 minutes, fluorescence was measured with an excitation wavelength of 322 nm and an emission wavelength of 450 nm.

[0068] 4. Calculation:

[0069] The inhibition percentage of the sample on neuraminidase can be calculated based on the standard curve.

[0070] For the detection of effective inhibitors, the OD value and concentration data obtained by the final fluorescence measurement of samples of different concentrations were input into the Graphpad prism software, and the IC50 of the inhibitor was calculated using the Log (inhibitor) vs. response—Variable slope (four parameters) under the Dose-response-Inhibition option.

[0071] 5. See Table 1 for test results.

[0072] Table 1

[0073] Grouping IC50(ug / ml) Tangerine peel powder 309.2 Platycodon grandiflorum powder 367.8 Tangerine peel powder: Platycodon grandiflorum powder = 5:1 621.8 Tangerine peel powder: Platycodon grandiflorum powder = 5:2 301.0 Tangerine peel powder: Platycodon grandiflorum powder = 5:3 167.6 Tangerine peel powder: Platycodon grandiflorum powder = 5:4 316.7 Tangerine peel powder: Platycodon grandiflorum powder = 5:5 857.0

[0074] It can be seen that the effect of inhibiting neuraminidase activity is best under the condition of tangerine peel powder: platycodon powder = 5:3.

[0075] Cell assay protocol:

[0076] 1. Macrophages (RAW264.7) were revived and plated;

[0077] 2. LPS (1 ug / ml) and sample (100 ug / ml) (5 mg of the sample in Example 3 was dissolved in 50 ml of water) were co-cultured for 24 h;

[0078] 3. Collect the cell supernatant, centrifuge at 4°C, 5000 rpm for 5-10 min, and collect the supernatant;

[0079] 4. Perform ELISA to detect the concentration of IL-6, TNF-α and IL-1β. All reagents are provided by the purchased ELISA kit. The specific method is as follows:

[0080] 4.1) Move all reagents to room temperature (18-25°C) to equilibrate for at least 30 minutes, and prepare reagents according to the above method for use.

[0081] 4.2) Sample addition: Set up standard wells and sample wells. Add standard or sample to each well, shake gently to mix, cover with plate sticker, and incubate at 37℃ for 2 hours.

[0082] 4.3) Discard the liquid, spin dry, and do not wash.

[0083] 4.4) Add labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0084] 4.5) Discard the liquid in the wells, spin dry, and wash the plate 3 times. Soak for 2 minutes each time, 200 μl / well, and spin dry.

[0085] 4.6) Add enzyme-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0086] 4.7) Discard the liquid in the wells, spin dry, and wash the plate 5 times. Soak for 2 minutes each time, 200 μl / well, spin dry.

[0087] 4.8) Add substrate solution to each well in sequence and incubate at 37°C in the dark for 15-30 minutes.

[0088] 4.9) Add stop solution to each well in sequence to terminate the reaction.

[0089] 4.10) Within 5 minutes after the reaction is terminated, the optical density (OD value) of each well is measured in sequence at a wavelength of 450 nm using a microplate reader.

[0090] 5. Experimental results see Figure 1 .

[0091] Statistical analysis is the difference between the treated and LPS-induced models, and the horizontal line is the difference between the tangerine peel and platycodon composition and the control composition. *** / ###, P≤0.001; ** / ##, P≤0.01; * / #, P≤0.05; ns, P>0.05.

[0092] The experimental results showed that at the cellular level, after treatment with the tangerine peel and platycodon grandiflorum combination, the levels of IL-6, TNF-α and IL-1β were significantly reduced by 18.2%, 19.0% and 25.9% respectively compared with the LPS-induced group, and the degree of reduction in the levels of TNF-α and IL-1β was significantly higher than that of the control combination (P≤0.05).

[0093] Example 5

[0094] Somatosensory testing program:

[0095] 1. Subjects: 40 people

[0096] 2. Grouping:

[0097] Group A: The tangerine peel and platycodon grandiflorum composition of Example 3 (n=17) was taken directly, 1 bag / time, 3 times / day;

[0098] Group B: control composition (n=12), directly consumed, 1 bag / time, 6 times / day; wherein the control composition is a common throat-clearing and throat-soothing product on the market, which is a solid beverage with platycodon, polygala, licorice and almond as main ingredients;

[0099] Group C: positive control (n=11), taken orally, 1 bag / time, 2 times / day; wherein the positive composition is an OTC non-prescription drug, which is a granule OTC non-prescription drug mainly composed of Scutellaria baicalensis, Oleander, Platycodon grandiflorum, Rhizoma Anemarrhenae, Sterculia lychnophora, Prunus armeniaca, Citrus aurantium, Fructus aurantii and the like.

[0100] 3. Inclusion criteria:

[0101] The comprehensive score of the Throat Sensation Assessment Questionnaire was ≥14;

[0102] Exclusion criteria:

[0103] Caused by lesions of the nasopharynx, pharynx, larynx, nose, larynx, esophagus, neck, tuberculosis, and metastatic lung cancer.

[0104] People under 18 years old / over 65 years old, pregnant and lactating women and those who are allergic to the test samples.

[0105] Patients with serious diseases such as cardiovascular, cerebrovascular, liver, kidney, hematopoietic system, bronchial and lung diseases, as well as mental illnesses and sleep disorders.

[0106] Having taken items related to the purpose of the sample test in a short period of time, which may affect the judgment of the results.

[0107] 4. Phase 1: Instant physical sensation test. All subjects filled out a throat physical sensation assessment questionnaire before taking the sample, immediately after taking it, 5 minutes after taking it, and 30 minutes after taking it. The staff collected the questionnaire data and conducted statistical analysis.

[0108] 5. The second stage: long-term somatosensory testing. All subjects filled out the throat somatosensory assessment questionnaire before taking the sample, 3 days after taking it, 7 days after taking it, and 14 days after taking it. The staff collected the questionnaire data and conducted statistical analysis.

[0109] Questionnaire questions:

[0110]

[0111] There are 14 questions in total, and each question is scored according to severity: 0 points for no symptoms at all; 1 point for mild symptoms; 3 points for moderate symptoms; 5 points for severe symptoms; 7 points for unbearable symptoms, and the total score is finally added up.

[0112] The test results can be found in Figure 2 , Figure 3 The test results showed that the scores of the throat sensation questionnaire immediately, 5 minutes and 30 minutes after taking the tangerine peel and platycodon grandiflorum combination were significantly lower than the initial values ​​(P≤0.001);

[0113] The throat sensation questionnaire scores at 3, 7 and 14 days after taking the tangerine peel and platycodon grandiflorum combination were significantly lower than the initial values ​​(P≤0.01).

[0114] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A composition for inhibiting neuraminidase activity, characterized in that: include, Tangerine peel powder and platycodon powder; wherein, by mass ratio, the ratio of the tangerine peel powder to the platycodon powder is 5:(1-5).

2. The composition according to claim 1, characterized in that: The mass ratio of the tangerine peel powder to the platycodon powder is 5:(2-4).

3. The composition according to claim 1 or 2, characterized in that: The mass ratio of the tangerine peel powder to the platycodon powder is 5:(2-3).

4. The composition according to claim 3, characterized in that: The hesperidin content in the tangerine peel powder is ≥0.1%.

5. The method for preparing the composition according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing tangerine peel powder and platycodon grandiflorum powder evenly to prepare the composition.

6. Use of the composition according to any one of claims 1 to 4 in the preparation of food or health products.

7. The use according to claim 6, characterized in that: The food includes powder, soft / hard capsule, compressed candy, soft candy, beverage, and jelly.

8. Use of the composition according to any one of claims 1 to 4 in the preparation of medicines.

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