CN115844962A

CN115844962A - Application of Rosa roxburghii extract in preparation of preparation for preventing or treating novel coronavirus infection

Info

Publication number: CN115844962A
Application number: CN202211204325.2A
Authority: CN
Inventors: 高秀丽; 唐丽; 张硕; 张敏; 王鹏娇; 孙小东
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2023-03-28

Abstract

The invention discloses application of roxburgh rose and roxburgh rose extract in preparation of a preparation for resisting novel coronavirus infection, and the roxburgh rose extract can play a role in preventing and treating the excessive activation of pulmonary complement and blood coagulation cascade. The fructus Rosae Normalis extract is prepared by mixing fructus Rosae Normalis 10g with 80% ethanol 100ml, ultrasonic extracting at 50 deg.C and 400W power for 2 times, recovering ethanol from the filtrate, extracting with ethyl acetate, and recovering ethyl acetate.

Description

Application of Rosa roxburghii extract in preparation of preparation for preventing or treating novel coronavirus infection

The invention belongs to the field of the following:

the invention relates to a new application of roxburgh rose, in particular to an application of roxburgh rose extract, roxburgh rose juice and roxburgh rose fruit in preparing a preparation for preventing or treating novel coronavirus infection.

Background

COVID-19 is a rapidly developing health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to primary lung injury, patients with COVID19 often develop a pro-coagulant state caused by viral-induced endothelial dysfunction, cytokine storm, and excessive activation of the complement cascade. The innate immune response is the first line defense mechanism against pathogen invasion. In early infections, pathogens contribute to local complement activation and inflammatory responses. Depending on the strength of the initial injury and the time of exposure, excessive activation or dysregulation of the complement cascade may occur, which may cause collateral damage to cells and tissues, further leading to multiple organ dysfunction. They are also a necessary condition for triggering specific adaptive immune responses. The complement pathway is the basis of innate host immunity and has a key role in defending against pathogens. Products of the complement pathway are present and activate the entire alveolar-capillary membrane. The alveolar epithelium serves as the first barrier to inhaled pathogens, while the vascular endothelium amplifies the response. The complement system remains mildly active under normal equilibrium conditions, but is strengthened by endothelial stress caused by viral infections, bleeding and trauma. Covi-19 associated coagulopathy is a complex pathological process caused by thromboinflammation, endothelial injury, and excessive activation of the complement and coagulation cascade. Thus, activation of the complement cascade is one of the earliest immune responses to infection. Studies have also shown that excessive complement activation contributes to dysregulation of the host immune response, and also leads to serious consequences of infection. Therefore, the inhibition of the excessive activation of pulmonary complement and blood coagulation cascade of a new coronary patient can obviously reduce the serious morbidity of the new coronary.

Rosa roxburghii Trtt English name: roxburgh) is the fruit of Rosa roxburghii which is a deciduous shrub plant of Rosaceae. The fertilizer is widely distributed in warm zones and subtropical zones, mainly distributed in provinces such as Guizhou, yunnan, hunan and the like in China, wherein Guizhou resources are most abundant. The roxburgh rose has high nutritive value and medicinal value, the content of vitamin C in the pulp of the roxburgh rose is higher than that of the fruits, the content of vitamin C in every 100g of the pulp is 2054-2725 mg, and the content of vitamin C is 500 times higher than that of apples and pears, 100 times higher than that of oranges and 100 times higher than that of kiwi fruits, and 9 times higher than that of kiwi fruits; the content of vitamin P is extremely high, and every 100g of pulp contains 5980-12895 mg of vitamin P, which is 120 times higher than that of oranges and 150 times higher than that of vegetables. The fruit king is called "Wei C Huang". It is also rich in vitamin B1, B2, E, K1 and other 16 kinds of trace elements, and has 46 times higher function than wild jujube and 2.4 times higher total flavone content than ginkgo leaf. Rosa roxburghii is also known as a green rare fruit with long life and cancer prevention, contains anti-cancer substances and SOD anti-aging substances, and also has the effects of strengthening spleen, promoting digestion, removing food stagnation, invigorating qi, and strengthening kidney. The fermentation of rosa roxburghii tratt is the hot spot of the current research direction. The active ingredients of the fermented product are fully reserved and can be reserved for a long time at normal temperature.

At present, no research shows that the roxburgh rose has a prevention and treatment effect on the novel coronavirus infection, the invention researches the possible prevention and treatment effect of the roxburgh rose and the roxburgh rose extract on the novel coronavirus infection, and provides a new research direction for exploring the potential mechanism of the roxburgh rose and the roxburgh rose extract.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of roxburgh rose and roxburgh rose extract in preparing a preparation for preventing or treating novel coronavirus infection.

The invention is realized by adopting the following technical scheme:

use of Rosa roxburghii Tratt extract in preparing preparation for preventing or treating novel coronavirus infection is provided.

The preparation method of the roxburgh rose extract comprises the following steps:

(1) The preparation method of the roxburgh rose extract comprises the following steps: cleaning fructus Rosae Normalis, crushing, adding 60-90% ethanol, mixing at a ratio of 100g, 600mL-1000mL, and ultrasonically extracting at 40-70 deg.C for 0.5-4 hr for 2-4 times at 200-500W to obtain product A;

(2) Filtering product A, collecting filtrate, recovering ethanol from the filtrate under reduced pressure by using a rotary evaporator, extracting for 2-5 times with ethyl acetate with volume ratio of 1.5-2, and recovering ethyl acetate extraction part to obtain fructus Rosae Normalis extract.

In the step (1): cleaning a proper amount of fructus Rosae Normalis, crushing, adding 80% ethanol, mixing, wherein the ratio of fructus Rosae Normalis to 80% ethanol is 100g, 800mL, and ultrasonically extracting at 50 deg.C under 400W power for 2 times, each time for 1 hr, to obtain product A.

In the step (2): filtering product A, collecting filtrate, recovering ethanol from the filtrate under reduced pressure by using a rotary evaporator, extracting for 3 times by using ethyl acetate with a volume ratio of 1.

Application of fructus Rosae Normalis natural juice in preparing preparation for preventing or treating novel coronavirus infection is provided.

The preparation method of the fructus Rosae Normalis natural juice comprises cleaning fructus Rosae Normalis, squeezing, and filtering to remove fructus Rosae Normalis residue to obtain fructus Rosae Normalis natural juice.

A preparation for preventing or treating infection of new coronavirus comprises fructus Rosae Normalis, fructus Rosae Normalis extract or fructus Rosae Normalis juice.

A preparation for preventing or treating novel coronavirus infection is prepared from fructus Rosae Normalis, fructus Rosae Normalis extract or fructus Rosae Normalis juice.

The preparation is oral preparation or capsule.

The invention has the beneficial effects that:

1. the Rosa roxburghii Tratt extract (RRTP) is a main active ingredient in an underutilized functional medicinal and edible homologous plant, and researches show that the RRTP can obviously improve pathological injury of lung tissues and permeability of pulmonary capillaries, inhibit inflammatory reaction of the lung tissues, acute-phase protein and S-100 calcium binding protein, inhibit over-activation of complement and blood coagulation cascade, possibly have a synergistic prevention effect on novel coronavirus infection, and regulate disordered lipid metabolism and amino acid metabolism.

2. The research shows that RRTP has obvious advantages in the aspects of supplement and adjuvant therapy of novel coronavirus infection ALI, and discloses a complex improvement mechanism of RRTP to ALI for the first time, thereby providing a breakthrough for development and demonstration of RRTP adjuvant therapy ALI and novel coronavirus infection.

Drawings

FIG. 1: lung tissue HE staining of each group of mice;

FIG. 2: protein concentration of BALF in each group of mice (. P <0.05,. P <0.01,. P < 0.001) compared to model group;

FIG. 3: inflammatory factor detection assay of lung tissue in each group of mice (, compared to model group, P <0.05, P <0.01, P < 0.001);

FIG. 4: relative content heatmap of 83 different proteins;

FIG. 5: GO enrichment analysis of differential proteins;

FIG. 6: domain enrichment analysis of differential proteins figure 4D:

FIG. 7: KEGG pathway enrichment analysis of differential proteins;

FIG. 8: relative content heatmap of differential metabolites;

FIG. 9: KEGG pathway enrichment analysis of differential metabolites;

FIG. 10: typical total ion flow pattern for RRTP.

In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.

Detailed Description

Example 1:

the preparation method of the roxburgh rose extract comprises the following steps: taking 100g of roxburgh rose fruit, cleaning, crushing, adding 1000mL of 80% ethanol, mixing, carrying out ultrasonic extraction for 2 times at 50 ℃ with 400W power, each time for 1 hour, filtering, collecting filtrate, recovering ethanol from the filtrate under reduced pressure by using a rotary evaporator, extracting for 3 times by using ethyl acetate with the volume ratio of 1.

The usage and dosage are as follows: the Rosa roxburghii Tratt extract is 3-4 g/day.

The efficacy is as follows: preventing or treating novel coronavirus infections.

Example 2:

the preparation method of the roxburgh rose extract oral liquid comprises the following steps: taking 20g of sucrose, dissolving in 100ml of distilled water, taking 2g of the roxburgh rose extract obtained in the embodiment 1, uniformly mixing, sterilizing and filling to obtain the roxburgh rose extract oral liquid.

The usage and dosage are as follows: the Rosa roxburghii extract oral liquid is 100 ml/day.

Example 3:

the preparation method of the roxburgh rose extract comprises the following steps: taking 100g of roxburgh rose fruit, cleaning, crushing, adding 800mL of 70% ethanol, mixing, carrying out ultrasonic extraction for 3 times at the temperature of 60 ℃ and under the power of 200W for 1 hour each time, filtering, collecting filtrate, recovering ethanol from the filtrate under reduced pressure by using a rotary evaporator, extracting for 2 times by using ethyl acetate with the volume ratio of 1.

The preparation method of the roxburgh rose extract capsule comprises the following steps: pulverizing sucrose 20g, sieving with 100 mesh sieve, mixing with fructus Rosae Normalis extract 20g, vacuum drying at low temperature for sterilization, pulverizing, sieving with 100 mesh sieve, filling, and packaging to obtain fructus Rosae Normalis extract capsule.

The usage and dosage are as follows: fructus Rosae Normalis extract capsule 4 granules/time, 3 times/day.

Example 4:

the preparation method of the roxburgh rose extract comprises the following steps: taking 100g of roxburgh rose fruit, cleaning, crushing, adding 900mL of 90% ethanol, mixing, carrying out ultrasonic extraction for 4 times at 40 ℃ and with the power of 500W, each time for 0.5 hour, filtering, collecting filtrate, recovering ethanol from the filtrate under reduced pressure by using a rotary evaporator, extracting for 5 times by using ethyl acetate with the volume ratio of 1.

The preparation method of the roxburgh rose juice comprises the following steps: taking 100g of roxburgh rose fruit, cleaning, squeezing juice, filtering to remove roxburgh rose dregs to obtain 200ml of roxburgh rose raw juice.

The usage and dosage are as follows: 150ml-200 ml/day of Rosa roxburghii juice.

The efficacy is as follows: preventing or treating new coronavirus infection.

Example 5:

the preparation method of the superfine powder and the whole powder of fresh and dry roxburgh rose fruit comprises the following steps: cleaning fresh or dried fructus Rosae Normalis, vacuum drying, pulverizing, and sieving with 5000-10000 mesh sieve to obtain superfine powder of fresh or dried fructus Rosae Normalis; cleaning fresh or dried fructus Rosae Normalis, vacuum drying, pulverizing, and sieving with 100-200 mesh sieve to obtain fine powder of fresh or dried fructus Rosae Normalis.

The usage and dosage are as follows: the superfine powder of fresh or dried fructus Rosae Normalis is 100-300 g/day. The fine powder of fresh or dried fructus Rosae Normalis is 100-300 g/day.

The efficacy is as follows: preventing or treating new coronavirus infection.

A large number of analysis and verification experiments are carried out, and the following results are the experimental research results of the invention:

1. mechanism experiment

1.1 Experimental materials

1.1.1 Experimental animals

SPF grade (no specific pathogen) BALB/c male mice 40, 4-6 weeks old, weight 20-25g, purchased from Changsha, and certification numbers: SCXK (Xiang) 2019-0013. The mice are raised in SPF-level animal rooms of the centers of microbiological and biochemical and pharmaceutical engineering of Guizhou medical university, the raising environment is natural light circulation illumination with light and shade alternately for 12 hours respectively, the indoor environment temperature is 24 +/-2 ℃, the relative humidity is 40-60%, drinking water and cages are sterilized, and feeds and padding are sterilized by irradiation. The acclimation period was 7 days, and the animals had free access to drinking water. The experimental protocol was approved by the animal ethics committee of the university of medical, guizhou.

1.1.2 Experimental reagents

Rosa roxburghii extract (the Rosa roxburghii extract of example 1); dexamethasone sodium phosphate injection (sui patent pharmaceuticals gmbh); lipopolysaccharide LPS (Escherichia coli055: B5, sigma Co., USA); BCA protein quantification kit (solibao biotechnology limited); ELISA kits for TNF-alpha, IL-6, IL-8, IL-10, IL-1 beta and the like (Xinbo Sheng Biotech Co.).

1.1.3 Experimental instruments

UHPLC-HESI-Q-Exactive Plus Orbitrap-MS (Saimei fly)

1.2 Experimental methods

1.2.1 Acute Lung Injury (ALI) modeling

30min after the last administration, mice were injected intraperitoneally with 10% chloral hydrate, anesthetized on a 45 degree plate with the back facing. Irradiating the laryngeal mouth part of the mouse in vitro by using a white lamp, opening the oral cavity of the mouse, slightly pulling the tongue out of the oral cavity by using tweezers, fixing, observing the opening and closing of the glottis of the mouse under the irradiation of light, pushing an LPS solution (2 mg/kg) aiming at the opening and closing part of the glottis, and completely covering the nostrils at two sides of the mouse to block the upper respiratory tract of the mouse breathing through the nostrils, so that the ventilation can only pass through the oral cavity, the throat to the trachea and the left and right main bronchus.

1.2.2 Experimental groups and dosing

40 mice were randomly divided into 4 groups (n = 10): normal saline control group (Normal group), ALI Model group (Model group), positive dexamethasone group (DEX group), and Rosa roxburghii extract administration group (RRTP group).

(1) Normal group: the stomach is irrigated for 1 time every day with the same volume of normal saline (10 mL/kg/d) for 7 consecutive days, and 50ul of normal saline with the same volume is dripped into the air passage after the last 30 min.

(2) Model group: the gavage is carried out for 1 time every day, the normal saline (10 mL/kg/d) with the same volume is gavaged, the normal saline with the same volume of 50ul is dripped into the air passage after the last 30min for 7 consecutive days.

(3) DEX group: the same volume of normal saline (10 mL/kg/d) is perfused into the stomach for the first 4 days, dexamethasone (2 mg/kg) is injected into the abdominal cavity for the last three days, and LPS is dripped into the airway after the last 30 min.

(4) RRTP group: the preparation is administered by intragastric administration 1 time per day, 10mL/kg/d fructus Rosae Normalis extract preparation is administered for 7 days, and LPS is dripped into airway after the last 30 min.

1.2.3 specimen Collection

After mice are instilled into LPS air passages and stimulated for 24 hours, the eyeballs of all groups of mice are taken for blood and killed, whole blood is put into a heparinized blood collection tube and centrifuged at 4000r/min at 4 ℃ for 10min, and supernatant fluid, namely blood plasma, is collected and stored at-80 ℃. Fixing a mouse in a supine position on an operation board, longitudinally cutting the skin of the neck, carrying out blunt dissection on subcutaneous tissues, exposing the trachea and the lung, separating the trachea, cutting a small opening on the trachea, taking out the complete lung, slowly injecting 1mL of precooled sterile normal saline into the lung for lavage, gradually enlarging the lung of the mouse, slowly recovering bronchoalveolar lavage fluid (BALF), slowly injecting the obtained fluid into the lung, staying for 1min, slowly pumping back, repeating the lavage for 3 times to ensure that the recovery rate is more than 90 percent, finally recovering BALF after 1 injection, and placing the BALF in a 2mL centrifuge tube (placed in an ice bath).

1.2.4 pathological evaluation of Lung tissues-HE staining

Washing lung tissues of a mouse by using cold PBS, then placing the mouse lung tissues in 10% neutral formalin for fixation for 24 hours, then sequentially placing the mouse lung tissues in 50% alcohol for 12 hours, 70% alcohol for 4 hours, 80% alcohol for 30min, 90% alcohol for 30min, 95% alcohol for 45min, absolute alcohol for 45min and absolute alcohol for 45min, then adding xylene for transparence, wax dipping and embedding to prepare paraffin specimens, and slicing (5 mu m) and then carrying out HE staining, wherein the method comprises the following specific steps:

(1) The sections were stained with hematoxylin for 5min and rinsed with running water for 1min.

(2) Treating with 1% hydrochloric acid alcohol for 20s, and washing with running water for 1min.

(3) The blue promoting solution is used for treating for 30s and the washing is carried out for 1min by running water.

(4) Treating with 1% eosin solution for 3min, and washing with running water for 1min.

(5) Dehydrating with 85% ethanol for 20s; dehydrating with 90% ethanol for 30s; dehydrating with 95% ethanol for 1min for 2 times.

(6) Xylene for 2 minutes.

(7) And (5) sealing by using neutral gum.

(8) The pathological changes of the lung tissue were observed under an optical microscope and the photograph was taken for retention.

1.2.5BCA method for determining protein concentration in BALF

Unfreezing the BALF supernatant, determining the protein concentration in BALF by using a BCA method, operating according to the kit instruction, and calculating the protein concentration of the sample to be detected according to the protein standard and a standard curve drawn by the OD value.

1.2.6 measurement of inflammatory cytokines in Lung tissue of mice

The concentrations of TNF-alpha, IL-1 beta, IL-6 and IL-10 in the lung tissues of each group of mice were determined separately and exactly according to the ELISA kit instructions.

1.2.7 Lung tissue Metabolic analysis

Extracting endogenous metabolites of lung tissue with an organic reagent. Briefly, 100mg lung tissue samples were extracted with 0.5mL of pre-cooled aqueous methanol acetonitrile (2, 2.

<xnotran> UHPLC-HESI-Q-Exactive Plus Orbitrap-MS ZORBAX Eclipse Plus C18 (2.1*100mm,1.8 μm; (Agilent Technologies, CA, USA) . (0.1% , A) (0.1% , B), 0.3mL/min. :0-2.5min,2-2%B;2.5-5min,2-40%B;5-12min,40-100%B;12-16.1min,100-100%B;16-16.1min,100-2%B;16.1-19min,2-2%B. . 3.5/2.8kV (+/-), 100-1500m/z. 320 ℃, 350 ℃. 3s. Compound Discoverer3.2 , , . m/z, SIMCA-P14.1 (Umetrics, AB, umea, ) , (PCA) (OPLS-DA). HMDB (http:// www.hmdb.ca /) KEGG (http:// www.kegg.ca /) , MS2 . , MetaboAnalyst5.0 RRTP ALI , </xnotran> Pathways where the impact value was >0.1 were considered significant.

1.2.8 Lung tissue proteomics analysis

Lung tissue was ground into cell powder with liquid nitrogen, mixed rapidly with four volumes of lysis buffer (1% protease inhibitor cocktail, 8M urea) and shaken well. Sonicate three times on ice using a high intensity sonicator, then centrifuge at 12000rpm for 10 minutes at 4 ℃. Finally, the supernatant was obtained and the protein concentration was determined according to the instructions of the BCA kit. For digestion, 5mM dithiothreitol was added to the solution, followed by incubation at 56 ℃ for 30 minutes, followed by alkylation with 11mM iodoacetamide in the absence of light for 15 minutes. The samples were diluted with 100mm teab to a urea concentration of less than 2M and finally subjected to a second digestion with trypsin/protein mass ratios of 1. After trypsinization, desalting with Strata X C18 SPE (Phenomenex), vacuum drying, 0.5MTEAB reconstitution, and detection based on TMT kit. The polypeptides in lung samples were separated by high pH reverse phase High Performance Liquid Chromatography (HPLC) using an Agilent 300extended C18 column (4.6X 250mm,5 μm). Briefly, the polypeptide was separated into 60 fractions within 1h using 8% to 32% acetonitrile (pH 9.0) as a gradient, and 6 fractions were synthesized and dried by vacuum centrifugation.

The polypeptides were dissolved in solvent A and separated using the EASY-nLC1000UPLC system (Thermo Fisher Scientific). Gradient elution conditions for solvent A (0.1% formic acid, 2% acetonitrile) and solvent B (0.1% formic acid, 90% acetonitrile) were 0-4min,7-11% B;4-53min,11-32% by weight B;53-57min,32-80% by weight B;57-60min,80% B. The flow rate was maintained at 500nL/min. The isolated polypeptides were detected on Orbitrap Exploris TM480 (Thermo Fisher Scientific) with a nano electrospray ion source. The polypeptide was dissolved in solvent A and directly loaded onto a home-made reverse phase analytical column (length 25cm, diameter 100 μm). The polypeptides were separated on an EASY-nLC1000UPLC system (Thermo Fisher Scientific) at a constant flow rate of 450nL/min with a 7% -25% gradient of solvent B, increasing to 80% in 24 min, 8min, 25% -35%, 4min, and finally 4 min. MS data is acquired by adopting a DDA mode. The electrospray voltage was 2.3kV. When the scanning range is 400-1200 m/z, the resolution of the primary mass spectrum is 60000; at a scan start of 110m/z, the secondary mass spectral resolution was set to 15000 and TurboTMT was set to Off. To improve the efficiency of mass spectrometry, the Automatic Gain Control (AGC) is set to 100% and the intensity threshold is 5E4ions/s. MS/MS data were retrieved using a Proteome scanner (v.2.4.1.15) with an ion mass error of 10ppm and a fragment ion mass error of 0.02Da.

1.2.9 data processing and statistical analysis

Data were statistically analyzed using GraphPad prism9.0 software. Statistical comparisons between groups were performed using one-way ANOVA. P <0.05 had statistical differences.

1.3 Experimental results and analysis

1.3.1 Rosa Roxburghii extract significantly reduced LPS-induced pathological damage to lung tissue in ALI mice

Morphological changes and histological changes of lung tissue 24h after LPS injection were evaluated using HE assay. As shown in fig. 1, there was no apparent morphological damage to the lungs of normal mice. The results show that oral pharyngeal inhalation of saline does not induce additional inflammatory responses. The ALI model mouse induced by LPS has obvious inflammatory cell infiltration, pulmonary congestion and alveolar wall thickening, and the pathological lung of the ALI model mouse is suggested to be serious. Notably, the use of RRTP and DEX interventions can significantly reduce the severity of histopathological examination results.

1.3.2 Rosa Roxburghii extract significantly reduced LPS-induced BALF protein concentration in ALI mice

ALI increases the permeability of capillaries in the lung, causing massive extravasation of proteins and other substances, so that the concentration of total protein in BALF is an important index for reflecting the permeability of capillaries in lung injury. The effect of RRTP on LPS-induced ALI mouse lung capillary permeability was observed by BCA assay of protein concentration in BALF. The results show that RRTP can obviously improve the capillary permeability.

1.3.3 Rosa Roxburghii extract significantly reduces the concentration of inflammatory factors in lung tissue of ALI mice induced by LPS

Proinflammatory cytokines are important components of the onset of ALI and play an important role in the onset of ALI. Therefore, we tested inflammatory cytokines in lung tissue to investigate the protective effect of RRTP on LPS-treated ALI mice. As can be seen from FIG. 3, the levels of cytokines in lung tissues of rats in the model group were significantly higher than those in the normal group (P < 0.05) except for IL-10. Notably, RRTP and DEX pretreatment inhibited the elevation of TNF-alpha, IL-1 beta and IL-6 and the decline of IL-10 in ALI mice compared to the model group, suggesting that RRTP protected the lung inflammation in ALI mice by inhibiting the release of proinflammatory factors.

1.3.4 Rosa Roxburghii extract intervenes in post-proteomic changes in ALI mice

To fully elucidate the proteomic regulation effect of RRTP on ALI mice, we analyzed lung tissue samples from the model and RRTP groups using a TMT-marker based quantitative proteomic technique. A total of 5730 proteins were identified, of which 5518 were quantified. Proteins with fold change values >1.2 or <1/1.2 were considered significant (P < 0.05). The model group differentially expressed 83 proteins from the RRTP group, 27 of which were up-regulated and 56 of which were down-regulated (fig. 4).

Furthermore, we performed GO enrichment analysis on 83 differentially expressed proteins (fig. 5). In terms of molecular function, serine-type endopeptidase inhibitor activity, chemotactic agent activity, receptor ligand activity, receptor modulator activity and toll-like receptor 4 binding are the most enriched 10 terms, while the most enriched cellular component terms refer to the extracellular space, extracellular region, cytoplasmic vesicle lumen and platelet alpha granule. It is clear that most of the biological process items are associated with inflammatory responses, such as bacterial responses, acute phase responses, regulation of cytokine production, acute inflammatory responses, and negative regulation of hydrolase activity. Furthermore, to further clarify the structure and function of the proteins, we performed protein domain enrichment analysis, which showed that the differential proteins were mainly associated with serpin (serine protease inhibitor), lipocalin/cytosolic fatty acid binding protein family and S-100/ICaBP type calcium binding domain (fig. 6), indicating that these types of proteins play an important role in the efficacy of RRTP in the prevention and treatment of ALI. KEGG pathway analysis showed that complement and coagulation cascades are the most enriched pathways, followed by antigen processing presentation and cholesterol metabolism (figure 7). The complement system is a non-specific defense mechanism of the body against pathogens and is an intermediary and important component of innate immunity. Complement interacts with the blood coagulation system and participates in pathological processes such as inflammation and thrombosis. Thus, proteomic functional enrichment analysis indicates that protein regulation of ALI mice by RRTP is primarily associated with the inflammatory process and complement and coagulation cascades of the immune system.

1.3.5 Regulation of metabolism of ALI mice by Roxburgh Rose extract

UHPLC-HESI-Q-ExactivePlusOrbitrap-MS metabolic spectrum analysis data is analyzed by PCA and does not contain grouping information so as to estimate the overall distribution of the lung tissue sample and the reliability of the model. In the absence of molecular selection, the lung tissue metabolome data of the RRTP group was well isolated from the model group mice, and the other groups were isolated to some extent. Furthermore, in combination with the above results, RRTP plays an important role in the intervention and regulation of abnormalities in lung metabolism in ALI mice. And establishing pairwise comparison by adopting a supervision mode OPLS-DA, and observing the difference of metabolic spectra between the model group and the RRTP group. The reliability and the degree of fitting of the OPLS-DA model were examined by 200 permutations. The R2 and Q2 values are both smaller than the original values, and the values of the Q2 regression line and the intersection point of the longitudinal axis are both smaller than zero. The result shows that the OPLS-DA model is stable and reliable and has no overfitting. In summary, the model is applicable to metabolite prediction. In the OPLS-DA model, variables with high contribution to the grouping (VIP > 1) and P <0.05 were selected as potential biomarkers.

From the secondary mass spectral information, RRTP reversed the changes of 24 endogenous metabolites, 5 of which were down-regulated and 19 of which were up-regulated (fig. 8). Of the 24 potential biomarkers, more than 50% were lipids and nearly 30% were amino acids, and these endogenous metabolites were significantly enriched in 5 metabolic pathways of phenylalanine, tyrosine and tryptophan biosynthesis, taurine and hypotaurine metabolism, arachidonic acid metabolism, tyrosine metabolism, and retinol metabolism (fig. 9). Early intervention of RRTP was suggested to regulate the disordered amino acid and lipid metabolism in ALI mice.

1.4 prevention and treatment effects of Rosa roxburghii Tratt extract on ALI mice and mechanism summary thereof

1.4.1 inhibition of pulmonary inflammatory response

An imbalance of proinflammatory and anti-inflammatory responses is an important cause of ALI, and regulation of the body's uncontrolled inflammatory response is one of the key mechanisms for preventing and treating ALI. After RRTP early intervention, inflammatory factors TNF-alpha, IL-1 beta and IL-6 in lung tissues of ALI mice are obviously inhibited, and anti-inflammatory factors IL-10 are obviously increased, which suggests that RRTP protects ALI by relieving inflammatory reaction of lung tissues. In addition, the total protein concentration in BALF is an important index of capillary permeability after lung injury, and our results show that RRTP can obviously reduce the protein concentration in lung tissues of ALI mice, can relieve the capillary permeability of the lung, and simultaneously RRTP also obviously reduces the severity of pathological change of the lung tissues. Taken together, these results suggest that RRTP-mediated lung protection may be involved in the maintenance of lung morphology and inhibition of inflammatory responses.

1.4.2 inhibition of acute phase proteins and S-100 calcium binding proteins

We detected 6 active acute phase proteins and 2S 100 calcium binding proteins in the rest of the DEPs in RRTP and model groups, which are involved in the early immune response to LPS infection. After RRTP early intervention, the expression of acute phase protein and S100 calcium binding protein in lung tissue of ALI mice is most obviously reduced, namely alpha-1-acid glycoprotein 1 (ORM 1), alpha-1-acid glycoprotein 2 (ORM 2), serum amyloid A-1 (SAA 1), serum amyloid A-2 (SAA 2), serum amyloid A-3 (SAA 3), serum Amyloid P Component (APCS), S100A9 and S100A4. In particular, ORM2, as an acute phase reactant, is a sensitive index reflecting the degree of inflammation and the degree of tissue damage, and is also an index for evaluating the therapeutic effect. Novel coronavirus infection is an unprecedented global threat and can cause lung injury such as cytokine storm, blood coagulation disorder, immune dysfunction, lung epithelial cell apoptosis and the like. In part similar to LPS induced pathological changes in ALI. In fact, it was found that SAA might be a valuable indicator of disease severity in patients with COVID-19; decreased levels of SAA levels give a better prognosis compared to patients with an increasing trend. In addition, a proinflammatory warning protein S100A9 associated with various inflammation-related diseases is highly expressed in fatal COVID-19 patients, and inhibition of S100A9 can reduce LPS-induced lung injury. Interestingly, in this study, we found that, as previously described, both acute phase proteins and S100 calcium binding proteins were elevated in lung tissues of ALI mice, while their levels decreased after RRTP-dried. The above results indicate that RRTP can alleviate ALI by inhibiting the acute phase protein and S100 calcium binding protein.

1.4.3 inhibition of complement and coagulation cascade activation

In our results, SERPINE1, SERPINA1A, serpinaf 2, SERPINA1D, and SERPINA11B, and other serine protease inhibitors, were involved in the coagulation cascade, and the expression of the above proteins was significantly reduced in the RRTP group as compared with the model group. The coagulation system plays a crucial role in the immune response against infection, preventing tissue damage and promoting repair of damaged sites. However, in the immune response against infection, excessive activation of the coagulation cascade often exacerbates the production of proinflammatory cytokines, and coagulation-induced thrombin is also activated, further exacerbating the inflammatory response. Thus, our results indicate that ALI-related coagulation and the accompanying immune/inflammatory processes are strongly induced in ALI mice, and that over-activation of complement and coagulation cascades is effectively inhibited following early RRTP intervention. In particular, SERPINE1, a major inhibitor of plasminogen activation, has been shown to be involved in the pathological process of inflammatory lung diseases and higher levels have been detected in many lung diseases, e.g., in lung tissue and BALF samples from patients with ALI/Acute Respiratory Distress Syndrome (ARDS), asthma, idiopathic Pulmonary Arterial Hypertension (PAH), chronic obstructive pulmonary disease, and cove-19, it is not surprising that early RRTP intervention-induced SERPINE1 deficiency would provide protection against excessive fibrin accumulation and lipopolysaccharide-induced lung injury, given that an increase in SERPINE1 levels is associated with many lung diseases.

1.4.4 modulation of macrophage and lipid metabolism

Compared with the model group, 24 metabolites in the lung tissue of mice in the RRTP group are obviously changed. Metabolic changes help regulate macrophage activation and gain function appropriate to tissue damage or specific circumstances. Our metabolomic data show that various lipids such as lipid B4, alpha-tocotrienol, palmitoleic acid, eicosapentaenoic acid, docosahexaenoic acid, beta-pinene and arachidonic acid are deregulated in LPS-induced ALI mice. Most of them are associated with macrophage function and expression is down-regulated. In particular, lipoprotein B4 is an important lipid component of neuroprotective signals that can locally inhibit inflammation and exert neuroprotective effects in acute injury, and inhibition of this key metabolite exacerbates injury-induced injury. Eicosapentaenoic acid and docosahexaenoic acid are omega-3 (n-3) polyunsaturated fatty acids with immunomodulatory and anti-inflammatory effects that modulate systemic inflammatory responses in ALI patients, relieve oxygenation and prognosis. Compared with the model group, the RRTP group has higher lipoxin B4, eicosapentaenoic acid and docosahexaenoic acid, and the RRTP can relieve and improve ALI mouse lipid disorder. It is worth noting that arachidonic acid metabolism is one of the important pathways for RRTP to regulate ALI mouse metabolism change, and is also one of the main lipid metabolism pathways for generating inflammation mediators and inducing inflammation. It has been demonstrated that arachidonic acid levels are elevated and arachidonic acid metabolism is disturbed in LPS-induced ALI mice, consistent with our findings. Interestingly, arachidonic acid levels decreased and arachidonic acid metabolism was inhibited after administration, suggesting that RRTP can indirectly regulate macrophage function by improving abnormal lipid metabolism in ALI mice, and promote remission of lung tissue damage.

1.4.5 Regulation of amino acid metabolism

In addition to lipid metabolites, amino acids are also one of the major metabolites of ALI mouse RRTP that alter lung disease, including lysine, lysergine, tyrosine, desleucine, and S- (PGA 1) -glutathione. Numerous studies have established the unique cytoprotective properties of dietary ergothioneine, which has been shown to reduce inflammation, prevent ALI, prevent endothelial dysfunction and hinder pulmonary fibrosis. In addition, cell immune ergothioneine and tyrosine of which tyrosine and derivatives can influence airway inflammation are active endogenous amino acids in ALI mice, participate in various physiological processes of immunization, inflammation and the like of the ALI mice, and RRTP can reverse the amino acids to be close to a normal level, which shows that RRTP can effectively intervene in amino acid metabolic disorder of the ALI mice.

1.5 summary

In conclusion, RRTP is a main active component in an underutilized functional medicine-food homologous plant, can obviously improve the lung tissue pathological injury and the lung capillary permeability of an ALI mouse, inhibits the lung tissue inflammatory reaction, acute-phase protein and S-100 calcium binding protein, inhibits the over-activation of complement and coagulation cascade, and has a synergistic prevention effect on ALI. And regulating disturbed lipid metabolism and amino acid metabolism. The research shows that RRTP has obvious advantages in the aspect of ALI supplementation and adjuvant therapy, and the RRTP discloses a complex improvement mechanism of the ALI for the first time, thereby providing a breakthrough for the development and demonstration of RRTP adjuvant therapy of the ALI and COVID-19.

2. Identification of Rosa roxburghii extract components

2.1UHPLC-ESI-Q-Exactive Plus orbital trap mass spectrometry

The potential active ingredients of the RRTP extract were detected by using Thermo Vanqish Horizon UHPLC system (Sammerfo) and using Hypersil Gold C18 chromatography column (2.1 x 100mm,1.9 μm). The most preferred mobile phase is H2O (0.1% formic acid, A) and acetonitrile (0.1% formic acid, B), the mobile phase is 0-3min,2-2% B;3-23min,2-98% by weight B;23-26min,98-98% B;26-26.1min,98-2% by weight B;26.1-28min,2-2%, the flow rate is 0.3mL/min, the column temperature is 40 ℃, and the sample size is 2 μ L. MS collection was performed using the ESI-Q-active Plus Orbitrap System (Sammer fly) and samples were analyzed in both positive and negative modes. Mass spectrum parameters were 3.5/2.5kV (+/-), spray voltage; 350 ℃, evaporator temperature; 320 ℃, capillary temperature; fullms-ddms2 general methods; 100-1500m/z, scanning range; in 70000, resolution (MS 1); in 17500, resolution MS/MS; 20,40,60, step Normalized Collision Energy (NCE). Raw data were imported into Compound discover 3.2 software, setting the relative molecular mass deviation to less than 5ppm. The results were analyzed based on the exact relative molecular mass of the Xcalibur software, primary and secondary mass spectral fragmentation information, in combination with the standard spectral and fragmentation information from databases such as m/zcoud, m/z Vault, masslist, moNA, chemispider, etc. and literature. And improving the confidence coefficient of the result by adopting an advanced recognition algorithm mzLogic, and performing fragment-assisted inference and verification by using Mass frontier7.0 software to recognize the effective components of the RRTP extract.

2 preliminary results of identification

55 potential active ingredients (shown in table 1) are preliminarily identified by mass spectrometry, wherein 22 flavonoids, 6 amino acids, 6 phenolic acids, 9 triterpenes, 3 phenylpropanoids, 7 organic acids, 1 tannin and 1 coumarin are contained in the potential active ingredients. These abundant phenolic compounds may play an essential role in the prevention and treatment of ALI.

Preliminary identification of the active components of RRTP suggests that these compounds with different activities determine the anti-ALI effect of RRTP. Quinic acid and its derivatives, catechin and its derivatives, procyanidins, quercetin, chlorogenic acid, luteolin, taxifolin, vitamin C, and other components with higher content have been shown to alleviate ALI symptoms by inhibiting inflammation or modulating immune system. Of these, catechol, one of the most abundant components in RRTP, has been shown to significantly inhibit coronavirus replication and enhance adaptive immunity, and has great potential in treating COVID-19, suggesting that RRTP may have potential for preventing inflammation and immune disorders associated with COVID-19 in addition to preventing and treating ALI. We have previously found ellagic acid in RRTP and have been shown to significantly reduce inflammation and lung tissue damage in ALI mice. These abundant active ingredients with anti-inflammatory and immunomodulatory properties provide a material composition basis for RRTP to play a role in the prevention and treatment of ALI. (see FIG. 6)

TABLE 1 initial identification of potential effective components in RRTP extracts by UHPLC-ESI-Q-active + Orbitrap-MS method

2.3 content of 7 indices in Rosa roxburghii extract

The results of the content of 7 indices in the rosa roxburghii tratt extract can be seen in table 2:

TABLE 2 determination of the content of active principles in RRTP extracts

Claims

1. Use of Rosa roxburghii Tratt extract in preparing preparation for preventing or treating novel coronavirus infection is provided.

2. A method of preparing a rosa roxburghii tratt extract according to claim 1, wherein: the preparation method of the roxburgh rose extract comprises the following steps:

(2) Filtering product A, collecting filtrate, recovering ethanol from the filtrate under reduced pressure by using a rotary evaporator, extracting for 2-5 times by using ethyl acetate with a volume ratio of 1.

3. A method of preparing a rosa roxburghii tratt extract according to claim 2, wherein: in the step (1): cleaning a proper amount of fructus Rosae Normalis, crushing, adding 80% ethanol, mixing, wherein the ratio of fructus Rosae Normalis to 80% ethanol is 100g, 800mL, and ultrasonically extracting at 50 deg.C under 400W power for 2 times, each time for 1 hr, to obtain product A.

4. A method of preparing a rosa roxburghii tratt extract according to claim 2, wherein: in the step (2): filtering product A, collecting filtrate, recovering ethanol from the filtrate under reduced pressure by using a rotary evaporator, extracting for 3 times by using ethyl acetate with a volume ratio of 1.

5. Application of fructus Rosae Normalis natural juice in preparing preparation for preventing or treating novel coronavirus infection is provided.

6. The method for preparing raw juice of Rosa roxburghii Tratt as claimed in claim 5, wherein: the preparation method of the fructus Rosae Normalis natural juice comprises cleaning fructus Rosae Normalis, squeezing, and filtering to remove fructus Rosae Normalis residue to obtain fructus Rosae Normalis natural juice.

7. Application of fructus Rosae Normalis natural juice in preparing preparation for preventing or treating novel coronavirus infection is provided.

8. A formulation for the prevention or treatment of infection against a novel coronavirus, characterized by: the preparation comprises fructus Rosae Normalis, fructus Rosae Normalis extract or fructus Rosae Normalis juice.

9. A formulation for the prevention or treatment of infection against a novel coronavirus, characterized by: the preparation is mainly prepared from roxburgh rose, roxburgh rose extract or roxburgh rose juice.

10. The agent for the prevention or treatment of infection by a novel coronavirus according to claim 8 or 9, wherein: the preparation is an oral preparation or a capsule.