CN116898885A

CN116898885A - Water leaching active matter for inhibiting infection of COVID-19XBB 1.5 variant strain and application thereof

Info

Publication number: CN116898885A
Application number: CN202310914618.8A
Authority: CN
Inventors: 江磊; 陈宇; 余朝霞
Original assignee: Qinghai Xueshang Yunchuan Biotechnology Co ltd; Northwest Institute of Plateau Biology of CAS
Current assignee: Qinghai Xueshang Yunchuan Biotechnology Co ltd; Northwest Institute of Plateau Biology of CAS
Priority date: 2023-07-25
Filing date: 2023-07-25
Publication date: 2023-10-20
Anticipated expiration: 2043-07-25
Also published as: CN116898885B

Abstract

A water leaching active matter for inhibiting the infection of a variant strain of COVID-19XBB 1.5 and application thereof relate to the technical field of medicines, and the water leaching active matter for inhibiting the infection of the variant strain of COVID-19XBB 1.5 is a kola seed water leaching active matter; the raw materials of the water extraction active matter of the kombucha are the kombucha fruits, roots and stems and leaves. The invention has the beneficial effects that: the kola seed water extraction active substance can be used for preventing and treating the infection of a variant strain of the COVID-19XBB 1.5; and the extract active matter of the kola vine fruit water can be used as an inhibitor for the infection of a variant strain of the COVID-19XBB 1.5, and can be applied to medicines.

Description

Water leaching active matter for inhibiting infection of COVID-19XBB 1.5 variant strain and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a water leaching active substance for inhibiting the infection of a variant strain of COVID-19XBB 1.5 and application thereof.

Background

The covd-19 virus constitutes a particular risk for vulnerable populations, especially the elderly or those with underlying health problems; the spike (S) protein of the COVID-19 virus plays a vital role in infiltrating and infecting human cells; the S protein aids the entry of viruses into these cells by interacting with the angiotensin converting enzyme 2 (ACE 2) receptor present on the surface of human cells; the S protein is glycoprotein and consists of two subunits S1 and S2, and plays a vital role in virus invasion and replication; in the S1 subunit, there is a Receptor Binding Domain (RBD) responsible for recognizing and binding to ACE2 receptor; on the other hand, the S2 subunit promotes fusion of the viral membrane with the host cell membrane, enabling the virus to invade and replicate within the host cell; ACE2 receptors are widely distributed in various tissues of the human body, particularly in the lungs, heart and kidneys; the interaction of the S protein of the virus with the ACE2 receptor causes structural changes, contributing to the invasion and infection of the virus; after the virus enters the cell, its genetic material (RNA) is released into the cytoplasm, and then the virus controls the machinery of the cell to begin to replicate, producing more viral particles; these particles are then released from the infected cells, enabling systemic transmission of the virus, which can damage the infected cells, leading to tissue damage and inflammation, particularly in the lungs; as the virus progresses, it can cause extensive inflammation, leading to severe symptoms such as fever, cough, and dyspnea; currently, researchers are exploring potential therapeutic approaches to the S protein that interfere with its interaction with ACE2 receptors, thereby preventing viral entry and infection; further studies are crucial to fully understand this interaction and to develop therapeutic methods and vaccines effective against covd-19.

Disclosure of Invention

In order to solve the problems, the invention provides a water leaching active substance for inhibiting the infection of a variant strain of COVID-19XBB 1.5 and application thereof.

The invention provides a water leaching active matter for inhibiting the infection of a variant strain of COVID-19XBB 1.5 and application thereof, wherein the water leaching active matter for inhibiting the infection of the variant strain of COVID-19XBB 1.5 is a kola seed water leaching active matter.

The water leaching method of the kola vine fruit water leaching active matter comprises the following steps:

step one: the fruits, roots and stems of the kola vine are dried in a drying room at room temperature for five days;

step two: cutting root, stem and leaf into segments with the length of 0.5 cm, and putting the segments and fruits into a nylon mesh bag;

step three: weighing, placing into an extractor, adding purified water 30 times the weight of the herbal medicine into the extractor for extraction, and extracting the herbal medicine for 2 hours once the water is boiled;

step four: drying the water extract at 60deg.C with an electrothermal drying oven to obtain dried water extract powder;

step five: re-dissolving the dried water extract powder in purified water, filtering to remove insoluble substances, and drying again with 60deg.C drying oven to obtain water extracted fructus Physalis active substance.

The kombucha water extract active substance is used as an inhibitor of the infection of a variant strain of COVID-19XBB 1.5 and is applied to medicines.

The application of the kola seed water extraction active substance in preventing and treating the infection of a variant strain of COVID-19XBB 1.5.

The invention has the beneficial effects that: the extract active substance of the kola seed water can be used for preventing and treating the infection of a variant strain of the COVID-19XBB 1.5; and the extract active matter of the kola vine fruit water can be used as an inhibitor for the infection of a variant strain of the COVID-19XBB 1.5, and can be applied to medicines.

Drawings

FIG. 1 is a standard graph of an embodiment of the present invention;

FIG. 2 is a bar chart of the primary screen concentration in an embodiment of the invention;

FIG. 3 is a toxicity test chart of 11# bergenia in an embodiment of the present invention;

FIG. 4 is a toxicity test chart of the 43# kola in the example of the present invention;

FIG. 5 is a graph of inhibition of 11# bergenia in an embodiment of the present invention;

FIG. 6 is a graph showing the inhibition rate of the 43# kola in the example of the present invention;

FIG. 7 is an image of cells in an embodiment of the invention;

FIG. 8 is a scatter plot of the activity of bergenia 11 and bergenia 43 in the examples of the present invention.

Detailed Description

Example 1

The invention provides a water leaching active matter for inhibiting the infection of a variant strain of COVID-19XBB 1.5 and application thereof, wherein the water leaching active matter for inhibiting the infection of the variant strain of COVID-19XBB 1.5 is a kola seed water leaching active matter;

finding out effective active substances capable of interfering ACE2-RBD protein interaction (PPI) and inhibiting SARS-CoV-2 virus infection, screening 64 common Chinese herbal medicines by using a COVID-19 serum neutralizing antibody fluorescence immunochromatography kit, and performing preliminary activity evaluation; subsequently, an in vitro infection model was established using the covd-19 xbb1.5 variant and Vero E6 cells; the anti-infective ability of the active water extract was then verified using this model;

materials and experimental methods

Experimental materials

The herbs used are obtained from the local market of traditional Chinese medicines and special precautions are taken to ensure their cleanliness; thoroughly cleaning the plant surface, and removing any dirt or soil residues by using purified water; subsequently evaporating the moisture from the plant surface under controlled room temperature conditions; subsequently performing an identification process by a plant taxonomy expert; any unidentifiable herbs were excluded and finally 64 herbs were selected for the subsequent study stage;

SARS-CoV-2 S1 protein (40591-V08H), human ACE2 protein (10108-H08H), COVID-19 neutralizing antibody (40591-MM 43), COVID-19 nucleoprotein antibody (40143-R004) are all purchased from Beijing Fangda Biotechnology Co., ltd. (Beijing, china);

XBB1.5 variant and Vero-E6 cells were supplied by the Guangzhou customs technical center (Guangzhou, china);

immunochromatographic test papers are manufactured and provided by Qinghai altemide biotechnology Co., ltd (Qinghai Xining China);

the dry fluorescence immunoassay instrument is provided by Qinghai altemiet biotechnology Co., ltd (Qinghai Xining China);

method for preparing herbal extract

The herbal medicine is air-dried in a drying room at room temperature for five days, then, the herbal medicine is sheared into sections with the length of 0.5 cm by using scissors and placed in a nylon net bag, after weighing the herbal medicine, purified water which is 30 times of the weight of the herbal medicine is added into an extractor, and once the water is boiled, the herbal medicine is extracted for 2 hours; subsequently, drying the water extract at 60 ℃ with an electric heating drying oven to obtain dried water extract powder; then, redissolving the dried powder in purified water, filtering to remove insoluble substances, and drying again by using a drying oven at 60 ℃; after complete drying, we stored the powders in sealed glass bottles and marked with label paper, finally we stored them in a dry environment at room temperature;

preliminary screening of herbal water extracts

We evaluated the effectiveness of different herbal extracts in inhibiting ACE2 and S1 protein interactions using immunochromatography; the colloidal gold labeled antibody kit for semi-quantitatively detecting the neutralizing antibody of serum COVID-19 developed by Qinghai Aike river research biotechnology Co., ltd has obtained the CE certification of the medical instrument in the Netherlands; based on the related technology, the Qinghai Aike river research biotechnology limited company provides a detection kit of the COVID-19 serum neutralizing antibodies marked with fluorescent microspheres for us, and aims to preliminarily quantify the screening results of us;

we designed a T-line that exploits the interaction between ACE2 protein and S1 protein; the step involves labeling Eu fluorescent microbeads on S1 protein, then uniformly distributing on pretreated glass fiber membrane and thoroughly drying; subsequently, ACE2 protein is immobilized at a designated T-line position on nitrocellulose membrane; furthermore, we created a C-line by exploiting the interaction between natural chicken IgY antibodies and goat anti-chicken IgY antibodies; the chicken IgY antibody is marked with Eu fluorescent microbeads and uniformly dispersed on the pretreated glass fiber membrane; finally, goat anti-chicken IgY antibody is firmly immobilized at the C-line position on the nitrocellulose membrane; individual test strips are carefully assembled into separate plastic housings; the Qinghai Audi Biotechnology Co., ltd. Carries out careful quality inspection of the production batch, and all sample specimens meet the strict company standard;

as shown in fig. 1, to analyze the quantitative performance of the reagent paper tape and establish a standard graph, we performed the following steps; the purchased S1 neutralizing antibodies were used as a reference, diluted in the sample treatment solution of the reagent paper tape to obtain various concentrations approaching the recommended IC50 values in the antibody specification; after uniform mixing, antibodies with different concentrations are applied to sample holes of the reagent paper tape; then, the test strip was placed horizontally without moving for 15 minutes; measuring fluorescence values corresponding to the T line and the C line, respectively, using a fluorescence immunoassay analyzer of qinghai audi me biotechnology limited company; regression analysis was performed using log2 (T-line fluorescence value/C-line fluorescence value) as a dependent variable, log2 (neutralizing antibody concentration) as an independent variable, to obtain a standard curve; this standard curve enables us to determine the concentration of S1 neutralizing antibodies equivalent to the activity of the unknown sample; we refer to this activity assessment measure as equivalent antibody concentration; from the standard graph, we can continue to measure equivalent antibody concentrations for different herbal extracts; we accurately weighed the water extracts of all herbs and dissolved them in the sample treatment solution attached to the reagent paper tape at a concentration of 3mg/mL; then, the prepared sample is applied to a sample hole of the immunochromatographic test paper, and the test paper tape is placed on a horizontal platform and kept stand for 15 minutes; finally, we used a fluorescence immunoassay to test and record the resulting equivalent antibody concentrations, as shown in table 1, allowing semi-quantitative assessment of all test sample activities;

watch 1Screening the Biological Activity of 64 Traditional Tibetan Medicines

Wherein the concentration of 11# bergenia and 43# kom is obviously higher than that of other herbal medicines;

high performance liquid chromatography detection of 11# bergenia and 43# kov

HPLC analysis is carried out by adopting a Hanbang high performance liquid chromatography system with a dual-wavelength ultraviolet detector; the chromatographic separation was carried out using a Kromasil CN column (5 μm, 10.0X1250 mm); the mobile phase consists of water-formic acid (A; 100:0.1, volume ratio) and acetonitrile-formic acid (B; 100:0.1, volume ratio) and is separated from 0% to 100% B solution in 0 to 40 minutes using a gradient elution procedure; the flow rate was set at 4.0mL/min; the compound was detected at 210nm wavelength by binding to 254nm wavelength, and the column temperature was maintained at 55 ℃;

cell culture

The method of culturing Vero-E6 cells involves several steps; first, a complete medium such as Dulbecco's Modified Eagleketone Medium (DMEM) (Gibco, new York, U.S.) is prepared, and 10% Fetal Bovine Serum (FBS) (Gibco, new York, U.S.) is added; next, the frozen Vero-E6 cells were thawed in a 37℃water bath until only one small ice crystal remained; transferring the cells into a sterile round bottom tube, gently adding pre-warmed complete medium; centrifuging the round bottom tube at a lower speed, precipitating the cells, and carefully removing the supernatant; suspending the cell pellet in complete medium and transferring to T25 or T75 cell flasks (BKMAN, long sand, china); the flask was placed in a 37 ℃ and 5% co2 incubator; periodically feeding the cells with fresh whole medium and monitoring their growth and fullness; when the cells reached a degree of confluence of 80-90%, washing with PBS and digestion with pancreatin-EDTA solution; fresh complete medium is added to stop pancreatin activity and the cell suspension is transferred to a new bottle or petri dish;

cytotoxicity test of 11# bergenia and 43# kola

As shown in fig. 3-4, the experimental setup included three groups: sample treatment group, cell control group (normal cells), blank control group (no cells); samples were serially diluted three-fold starting at 1000 μg/mL for a total of 10 different concentrations; each concentration was tested in three duplicate wells; vero-E6 cells were cultured in 96-well plates and the supernatant removed; then, 100. Mu.L of the corresponding sample dilution was added to each well and incubated at 37℃and 5% CO2 for 24 hours; after the incubation is finished, detecting by using a CCK-8 reagent; the supernatant of the sample dilution was discarded, and 100. Mu.L of DMEM medium without fetal bovine serum was added to each well, and Kong Chuwai of the blank group; in addition, 10. Mu.L of CCK-8 solution was added to each well and the plates were incubated at 37℃and 5% CO2 for 2 hours; finally, absorbance at 450nm was measured using a spectrophotometer; generating a cell viability curve to determine a maximum safe concentration of the sample;

antiviral Activity Using live Virus test samples

The synbiotic source and quantification of the novel coronavirus XBB1.5 variant, the complete genome sequence of which is predetermined by Guangzhou customs technical center; the complete genomic sequence has been uploaded to the Nextclone database (https:// clones. Next strain. Org /) for comparison to determine the classification of the target virus; the virus used in this study was indeed the covd-19 xbb1.5.7 variant;

vero E6 cells were cultured in 96-well bottom flat bottom cell culture plates at a density of 2 x 104 cells per well; after 12 hours, when the cells reached 90% density, the supernatant was discarded; then, a 96-well round bottom plate was prepared, and 180. Mu.L of serum-free DMEM was added to each well; in row a, 20 μl of virus stock solution was added to each well, followed by 10-fold dilutions of 8 wells each; then, the diluted virus solution was added to wells from which the cell supernatant had been discarded, and 100. Mu.L of each well was added; plates were incubated at 37℃and 5% CO2 for 72 hours; observing and recording cytopathic effects (CPE), in particular the number of wells displaying CPE; subsequently, TCID50 is calculated using the Reed-Muench method;

activity verification experiment

As shown in fig. 7, to establish the drug incubation medium, 11# bergenia and 43# kola liq water extract actives were carefully diluted to achieve concentrations of 0.33, 0.11, 0.037, 0.012 and 0.0041mg/ml using D2 medium (dmem+2% fbs); the experiments included three different groups: a control group of cells only, a virus-infected group, and a drug-virus interacting group; in each group, three replicates of each drug dilution were used; before Vero E6 cells are infected with virus, the cell culture supernatant is carefully replaced with the appropriate drug incubation medium, and 100 μl is added per well; subsequently, the cells were incubated in an environment of 37 ℃ with 5% co2 for 2 hours;

after pretreatment of the cell culture plates in the BSL-3 laboratory, the supernatant was discarded; omicron xbb.1.5 virus was diluted in D2 medium (dmem+2% fbs) at a fold ratio of infection degree of 0.2; drug treatment media were prepared by diluting the kola-seed water extract actives to concentrations of 0.66, 0.22, 0.073, 0.024 and 0.0081mg/ml in D2 medium (dmem+2% fbs); these five drug concentrations were used as treatment sites; then, mixing the diluted virus with the medicine with corresponding concentration in an equal volume; the mixture was added to the cell culture plate in a volume of 50 μl per well; cells were incubated at 37℃and 5% CO2 concentration for 1 hour; after incubating the virus-containing medium for 1 hour, the supernatant was discarded; subsequently, 100 μl of D2 medium (dmem+2% fbs) with the appropriate drug concentration was added to each well; next, the plates were incubated at 37℃and 5% CO2 concentration for 24 hours;

200. Mu.L of 4% PFA fixative was added per well in BSL-3 laboratory to inactivate viruses and fix cells; after fixing at room temperature for more than 1 hour, the fixing solution was sucked off, and 200. Mu.L of 4% PFA fixing solution was added; sterilizing the exterior surface of the cell culture plate in preparation for subsequent analysis; the fixed cells in the wells were thoroughly washed with PBS buffer and then treated with Triton-X100; next, the cells were incubated with a rabbit monoclonal antibody directed against SARS-CoV-2 core protein for 1 hour at room temperature; after a thorough washing step to remove any excess primary antibody, the cells were incubated with Alexa Fluor 488-labeled anti-rabbit IgG for an additional 1 hour; finally, DAPI was used to stain nuclei as a control for cell counts; quantitatively evaluating the fluorescence intensity in the cell culture plates using a Celigo system and capturing cell images, as described in fig. 7, to determine the level of viral infection;

experimental results

Establishment of screening method

Selecting Eu fluorescent microspheres to respectively mark the S1 protein and the chicken IgY antibody as shown in figure 1, and performing a chromatographic experiment; eu fluorescent microspheres labeled with S protein form bright fluorescent lines at the T-line position when interacting with ACE2 coated T-line; similarly, eu fluorescent microspheres labeled with chicken IgY produce striking fluorescence when interacting with the C-line coated with sheep anti-chicken IgY antibodies; in the presence of inhibitors, binding between S protein and ACE2 is blocked, resulting in a decrease in fluorescence of the T line; however, the fluorescence on line C remains unchanged and unaffected, regardless of the sample liquid;

for semi-quantitative analysis, it is essential to establish a standard curve for the fluorescent test paper; for this purpose, purified recombinant novel coronavirus S1 protein neutralizing antibodies are used; the antibody is diluted with a sample solution of a reagent strip to obtain six different concentrations ranging from 1 to 60 μg/mL; then applying each diluted antibody solution to a sample well of a reagent strip; after 15 minutes incubation, fluorescence values were recorded; the collected data are linear as shown in fig. 1; the standard curve equation shows that the correlation coefficient is very high, and the R2 value is expressed as 0.97, so that the correlation is good; this standard curve equation can be used for subsequent semi-quantitative analysis;

screening of active aqueous extracts

In the preliminary screening experiments, all water leached active samples were used at the same concentration of 3mg/mL, each sample repeated twice; table 1 and fig. 2 show that the 11# bergenia and 43# kola water extract actives exhibited abnormal activity superior to 1000 μg/mL neutralizing antibodies; subsequently, as shown in fig. 8, the inhibitory activities of 11# bergenia and 43# kochia were repeatedly tested, starting from an initial concentration of 3mg/mL, to a final concentration of 0.25, 0.13, 0.063, 0.031, 0.016, 0.0078 and 0.0039mg/mL, to verify the preliminary screening results; as shown in fig. 5-6, the antibody concentration of the kola-fruit water extraction active was greater than 1000 μg/mL for a concentration of 3mg/mL; with decreasing concentration, the inhibitory activity decreases; the kola-seed water extract active substance shows concentration-activity effect in terms of its inhibition characteristics, and its inhibition effect has good reproducibility.

Claims

1. A water leaching active for inhibiting infection by a covd-19 xbb1.5 variant, characterized by: the water extract active for inhibiting the infection of the variant strain of the COVID-19XBB 1.5 is the water extract active of the kola seed.

2. A water leaching active for inhibiting infection by a covd-19 xbb1.5 variant according to claim 1, wherein: the water leaching method of the kola vine fruit water leaching active matter comprises the following steps:

step three: weighing, adding purified water 30 times the weight of the herbal medicine into an extractor for extraction, and extracting the herbal medicine for 2 hours once the water is boiled;

3. Use of a water leaching active for inhibiting infection of a covd-19 xbb1.5 variant according to claim 1, characterized in that: the kombucha water extract active substance is used as an inhibitor of the infection of a variant strain of COVID-19XBB 1.5 and is applied to medicines.

4. Use of a water leaching active for inhibiting infection of a covd-19 xbb1.5 variant according to claim 1, characterized in that: the application of the kola seed water extraction active substance in preventing and treating the infection of a variant strain of COVID-19XBB 1.5.