TWI754260B - Use of ovatodiolide against sars-cov-2 - Google Patents

Abstract

This subject invention is directed to a compound of formula I (Ovatodiolide) which is safe and effective to use in a pharmaceutical composition for inhibition of SARS-CoV-2. The pharmaceutical composition comprising a safe and effective amount of a compound of formula I (Ovatodiolide) or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier, which has great potential to treat SARS-CoV-2 induced COVID-19 based on a safe and effective amount of a compound of formula I (Ovatodiolide).

Description

The use of ichthyolide against novel coronavirus

The focus of the present invention is the use of Ovatodiolide (Ova) against novel coronavirus; however, in the process of research and development, the preparation, identification and analysis of the natural product Ovatodiolide, basic toxicological tests, and Ova Molecular docking simulation of the action mechanism of lactones against the new coronavirus, biochemical experiment confirmation of the anti-new coronavirus of fenugreek lactones, etc., etc., were discussed in sequence and confirmed that fenugreek lactones can be produced in an appropriate amount, safe and safe, with anti-new coronavirus The mechanism of action and actual efficacy of the virus; ichthyolide is indeed a potential natural product to inhibit the infection of the new coronavirus (SARS-CoV-2), and can be developed as a treatment for severe special infectious pneumonia (COVID-19). drug.

At present, because the world has not yet developed effective preventive vaccines or effective therapeutic drugs for the new coronavirus; the new coronavirus has spread on all continents, leading to a global pandemic of severe special infectious pneumonia. Many countries and multinational pharmaceutical companies are actively carrying out Research and develop effective vaccines or drugs.

Anisomeles indica O.Kuntze is a commonly used herbal medicine in Taiwan, also known as guest wipe grass (Guangdong Jiaoling, Meixian), golden sword grass, Ben Huxiang and so on. The Ministry of Health and Welfare of Taiwan has listed the Echinophytum in the list of raw materials that can be used in food, and the whole plant is edible. It is an annual or perennial herb of the Labiatae family. It is mainly distributed in southwestern China, India, the Philippines, Java, Indonesia, and Sumatra. It can be found in the plains and low-altitude mountainous areas of Taiwan. There are also sporadic medicinal cultivation in Hualien Yuli, Taiwan. For general folk medicinal purposes, it is collected in summer and autumn, pulling up the whole grass or cutting the ground parts, washing it, and using it fresh or sun-dried. The whole plant has antipyretic, expelling wind, dehumidification, stomachic, detoxifying, analgesic and antibacterial effects. Folk is commonly used to treat colds and fever, abdominal pain and vomiting, cholera, stomach pain, gastroenteritis, neurodermatitis, rheumatism, bone pain, muscle and bone pain, eczema, swollen poison, sores, stool poisoning, and poisonous snake bites.

The research and development team has long been engaged in the breeding of Ichthyopsis (GenBank: GU726292), and has continued to conduct a series of researches on the whole plant extract of Ichthyopsis grown on farms, especially focusing on the preparation of pure crystalline substance of Iridium lactone (Figure 2). Specific implementation of extraction, separation and purification, analysis and identification, as well as anti-inflammatory, anti-virus, anti-Helicobacter pylori, anti-cancer, anti-cancer stem cells and other pharmacological studies. In recent years, our team has completed the test experiment of the drug "Tamiflu" (Roche: Tamiflu) in the treatment of type A and type B influenza with fenugreek, and found that fenugreek extract and fenugreek inhibit influenza virus. good effect. It was recently learned at the end of 2019 that AIDS drugs have shown positive reactions to the treatment of patients with new coronavirus infection; according to the literature, it was reported that fenugreek lactone can inhibit HIV ( Fitoterapia , 2000, 71(5): 574-576 .) . In addition, existing studies have also found that ichthyolide can inhibit the gastritis caused by Helicobacter pylori in the stomach wall, and can also inhibit the inflammatory response mediated by NF-B and STAT3. Therefore, ichthyolide may also alleviate the novel coronavirus-induced gastritis. symptoms of pneumonia.

The present invention is based on the discovery of ichthyolide or a pharmaceutically acceptable salt thereof, which can be used to inhibit novel coronavirus infection, and even to treat or prevent severe special infectious pneumonia. Specifically, the present invention provides a pharmaceutical composition for inhibiting novel coronavirus infection, and even treating or preventing pneumonia, comprising a safe and effective amount of fenugreek or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable vector.

The ichthyolide of the present invention has a chemical structural formula I, and the chemical structural formula I is shown in FIG. 1 .

The present invention provides a pharmaceutical composition mainly used for inhibiting novel coronavirus infection by using ichthyolide as an active ingredient. The molecular docking results of the simulated iodine lactone to the receptor-binding domain (RBD) of the spike glycoprotein on the surface of the novel coronavirus showed that the ovale lactone (Ova) was bound to several hydrophobic amino acids (L455) of the RBD. , F456, Y489, F490) formed a hydrophobic pocket and formed hydrogen bonds with Y489 and Q493 (Figure 3). The binding site is located at the interface between the new coronavirus spike glycoprotein RBD and the human cell membrane receptor angiotensin-converting enzyme 2 (ACE2), and it is predicted that Ovalide (Ova) can block the Or interfere with the direct binding of the receptor binding domain (RBD) of the viral spike glycoprotein to the receptor (ACE2). The binding of the novel coronavirus surface spike glycoprotein to the human cell membrane receptor angiotensin-converting enzyme-2 (ACE2) is a key step in mediating virus invasion into the host. Blocking or interfering with the binding of the virus to the receptor is a potential prevention and treatment Strategy.

At the same time, it was learned that the host endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L play a key role in the fusion process of the new coronavirus. The results of simulated molecular docking also showed that Ovalide (Ova) may also bind to the catalytic pockets of endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L. Ophiolactone (Ova) binds to the hydrophobic S2 site of Cathepsin B composed of Y75, P76, A173, A200, and E245 through a hydrophobic alicyclic ring, and forms a covalent complex with the catalytic cysteine C29 through an exocyclic olefin Thereby inhibiting the activity of Cathepsin B (Fig. 4A). On the other hand, iodine lactone (Ova) binds the hydrophobic S2 site of Cathepsin L composed of L69, M70, Y72, A135, and M161 with a hydrophobic alicyclic ring, and forms an exocyclic olefin with catalytic cysteine C25. The covalent complex thus inhibited the activity of Cathepsin L (Fig. 4B). endosomal cysteine proteolytic enzyme Cathepsin B and Cathepsin L play a key role in the fusion process of 2019-nCoV. Ophiolactone (Ova) potentially blocks the fusion process of 2019-nCoV.

This study specifically implements the novel coronavirus pseudovirus inhibitory activity detection system developed by the laboratory of Professor Zhang Linqi, director of the AIDS Comprehensive Research Center of Tsinghua University, Beijing, to specifically evaluate whether ichthyolide blocks the process of novel coronavirus infection in host cells. The experimental results show that ichthyolide has a specific molecular mechanism of action that is different from that of chloroquine or Remdesivir in inhibiting the new coronavirus. inhibitory effect (Figure 5).

Ophiolactones can possess one or more opposite palm centers and thus have various stereoisomeric forms. The ichthyolide mentioned in the present invention includes all such isomers; in addition, it also includes the derivative compounds containing the main structure of ichthyolide, and the molecular mechanism of action of these derivative compounds for inhibiting the novel coronavirus is related to The invention disclosed in the present invention combines the molecular docking mechanism of the novel coronavirus surface spike glycoprotein receptor binding region, or the molecular docking mechanism of the host endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L, which have similar effects. Ophiolactone has the effect of selectively inhibiting novel coronavirus infection; due to its extremely small molecular weight, a lower dose of Ophiolactone or a pharmaceutically acceptable salt thereof can be used, together with a pharmaceutically acceptable carrier, i.e. The desired therapeutic effect can be obtained. The present invention is a pharmaceutical composition for inhibiting novel coronavirus infection, and even for treating or preventing severe special infectious pneumonia (COVID-19). Pharmaceutically acceptable carrier for the inhibition of novel coronavirus, or administration to patients with severe specific infectious pneumonia (COVID-19) symptoms to cure, restore, alleviate, alleviate, alter, treat, ameliorate, ameliorate or affect disease , symptoms of a disease, or a constitution predisposing to a disease. As used herein, "an effective amount" refers to an effective amount of ichthyolide or a pharmaceutically acceptable amount thereof. Salts are acceptable, in amounts that have inhibitory or therapeutic effects. The effective amount varies depending on the route of administration, excipient usage, and co-usage with other active agents.

"Severe special infectious pneumonia (COVID-19)" here means deadly pneumonia caused by the new coronavirus (SARS-CoV-2) invading the human body. The new coronavirus uses the spike glycoprotein on the surface of the coronavirus to recognize the angiotensin-converting enzyme-2 (ACE2) on the surface of the cell, and then infect the normal cells of the human body. A possible mechanism is that when the virus invades the body, the immune cells in the body act violently, triggering an immune storm in the body, releasing a large number of free radicals (such as free peroxidation) to denature proteins, DNA damage, and excessive production of cytokines, resulting in a large number of cells. Necrosis, severe and fatal pneumonia develops in the lungs. Ophiolactone can effectively inhibit new coronavirus infection, thereby preventing or treating severe special infectious pneumonia (COVID-19).

Phyllostachys edulis is prepared by extracting the whole grass, aerial parts, or leaves of Phyllostachys edulis with organic solvent, and separating and purifying by silica gel column; or by chemical synthesis method. For example: "Panthera japonica extract" refers to the fenugreek extract extracted from a suitable growth level. In order to obtain the Phyllostachys edulis extract, extraction techniques well known in the art can be used. For example, the dried and ground sagebrush can be suspended in a solvent or a mixture of two or more solvents for a sufficient period of time. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, acetone, ethers (eg, diethyl ether) and ethyl acetate and hexane ( hexane). Then the solid residue is removed (eg, by filtration) to obtain the eucalyptus extract solution, which can be purified by alumina, silica, silica gel column to prepare eucalyptus lactone.

In the treatment method of the present invention, ichthyolide or a pharmaceutically acceptable salt thereof may be administered simultaneously or separately, orally, parenterally, via inhalation spray or By means of an implanted reservoir. As used herein, "non-oral" refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial , intrasternal (intrasternal) subarachnoid (intrathecal), disease site (intraleaional) and intracranial (intracranial) injection and perfusion techniques. The ichthyolide and/or its pharmaceutically acceptable salts used in the present invention can be formed into a suitable pharmaceutical form together with at least one solid, liquid or semi-liquid excipient or adjuvant. Forms include, but are not limited to, lozenges, capsules, emulsions, aqueous suspensions, dispersions and solutions. Carriers commonly used for lozenges include lactose and cornstarch. A lubricating agent, such as magnesium stearate, is also typically added to the lozenge. Diluents for capsule form include lactose and dried cornstarch. When an aqueous suspension or emulsion is used for oral administration, the active ingredient may be suspended or dissolved in an oily phase combined with an emulsifying or suspending agent. If desired, certain sweetening, flavoring and coloring agents may be added. The iodine lactones or pharmaceutically acceptable salts thereof used in the present invention can also be formulated into sterile injectable compositions (eg, aqueous or oily suspensions), eg, using suitable dispersion using techniques known in the art Or wetting agents (eg Tween 80) and suspending agents. Sterile injectable formulations can also incorporate sterile injectable solutions or suspensions in non-toxic non-oral diluents or solvents such as 1,3-butanediol. Vehicles and solvents that can be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are often employed as a solvent or suspending medium (eg, synthetic mono- or di-glycerides). Fatty acids such as oleic acid and its glyceride derivatives are also useful in the preparation of injectables, which are natural pharmaceutically acceptable oils such as olive oil, castor oil, especially in their polyoxyethylated base (polyoxyethylated) variant. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. The ichthyolide or its pharmaceutically acceptable salts used in the present invention can also be formulated into an inhalation composition according to techniques well known in this technical field. For example, salt solutions can be prepared using benzyl alcohol or other suitable preservatives, adsorption promoters to enhance bioavailability, fluorocarbons or other auxiliaries well known in the art Dissolving or dispersing agent to formulate. A carrier used in a pharmaceutical composition must be "acceptable", compatible with the active ingredient of the formulation (and preferably capable of stabilizing the active ingredient) and not injurious to the patient. For example, co-solvents such as cyclodextrins, which form specific more soluble complexes with the active compound(s) of the extract, serve as pharmacological adjuvants for the delivery of the active ingredient. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose and sodium lauryl sulfate.

In addition, since antiviral agents are prone to side effects when administered to patients in high doses. It is based on the results of the series of toxicological experiments of fenugreek lactone disclosed in the present invention, including the single-dose oral acute toxicity test for rats, the 28-day feeding toxicity test for rats, the Ames test for Salmonella reverse mutation, and the in vitro mammalian test. Chromosomal abnormality analysis test of cell line, micronucleus test of peripheral blood of mice, etc., all show that ichthyolide has no genotoxicity, and provides a safe oral dose range. The pharmaceutical composition of the present invention contains a safe and effective dose of ichthyolide for inhibiting novel coronavirus infection, wherein the safe and effective dose is an average adult (60 kg body weight): within 480 mg orally per day, and continuously taken within 28 days. It is better for an average adult (60 kg body weight): 20 mg to 40 mg orally every day, and it is appropriate to take it continuously for 7 to 14 days. The specific dose administered to an individual patient depends on all possible factors, such as the activity of the specific compound used, age, weight, General health status, gender, eating status, time and route of administration, excretion rate, combination of medicinal substances, and severity of the disease to be treated, etc.

The present invention also provides the use of a composition for preparing a medicament for inhibiting coronavirus, wherein the composition comprises Ovatodiolide or a structural isomer of Ovatodiolide.

Figure 1. Structural formula of Ovatodiolide.

Fig. 2. X-ray ORTEP diagram of fenugreek lactone crystallization.

Figure 3. The complex structure of the novel coronavirus spike glycoprotein receptor binding region RBD and the human cell membrane receptor ACE2, and the docking structure of Ovalide (Ova) and RBD. Ovalide (Ova) is bound to RBD , interfere with its binding to ACE2. Labels in the figure: ACE2: Angiotensin-converting enzyme 2; nCov S-RBD: Novel coronavirus spike glycoprotein receptor binding region; Ovatodiolide (Ova): Ophiolactone.

Figure 4. The docking structure of Ophiolactone (Ova) with cysteine proteolytic enzymes Cathepsin B (A) and Cathepsin L (B), in which Ova lactone (Ova) is an exocyclic olefin with Cathepsin, respectively The catalytic cysteine C29 of B, and the catalytic cysteine C25 of Cathepsin L form a covalent bond. Labels in the figure: Ovatodiolide (Ova): iodine lactone; Cathepsin B: cysteine proteolytic enzyme B; Cathepsin L: cysteine proteolytic enzyme L.

Figure 5. The activity curve of ichthyolide in inhibiting novel coronavirus infection, IC ₅₀ =3.73μM.

In order to make the above and other objects, features, and advantages of the present invention more apparent and comprehensible, the following specific descriptions are given. The preferred embodiment is described in detail as follows:

Embodiment 1. the preparation and analysis of fenugreek lactone: get fenugreek (annual, Taiwan Hualien Yuli harvested in autumn) roughly dry leaf part (800g), put into oven (40 ℃) and carry out drying (24 hours), Can get dried echidna leaves (500g). Put the dried leaves (500g) into a 20 liter bucket (PE material), and add 10 liters of 95% alcohol to ensure that all the leaves are soaked in the solvent, then seal the bucket and store it in a cool place for 7 days. After 7 days, the 95% alcohol layer and the leaves were separated by filtration and concentrated by a rotary evaporative concentrator to obtain a yellow-green extract (10 g). Purification by alumina column chromatography (neutral alumina: 300 g). The yellow-green extract (10 g) was dissolved with 10 mL of acetone and added to the packed alumina column. The solvent extraction ratio was Hexane: Ethyl acetate/100%: 0%, and the polarity was gradually increased to Hexane: Ethyl acetate/70%: 30%. Through the screening and comparison of thin-layer chromatography (TLC) test paper, ichthyolide can flow out of the column in the third section. After the volatile substances were drained by a rotary evaporation concentrator, 15 mL of acetone was added to redissolve, and the crystal growth method of solvent diffusion was used to obtain 1.56 g of transparent echeveria lactone crystals after seven days, and the yield was about 0.3%. Identification by X-ray, NMR, IR, Mass, HPLC and other instruments. The purified crystals were identified as ichthyolide. Through drying, extraction, purification and identification, we have successfully isolated ovatodiolide from Ophiopogon japonicus, and its yield to leaves is about 3000ppm, but leaves only account for 7% of the whole plant % or less, and it is not easy to collect. Analysis and identification of iodine lactone: X-ray Crystals of ovatodiolide are Orthorhombic, space group P2 ₁ 2 ₁ 2 ₁ , with a=10.7714(3), b=12.8674(3), c=13.0829(3), V= 1813.29(8)Å ³ ,D(calculated)=1.203Mg/m ³ ,Z=4,Formula weight=328.39,Goodness-of-fit on F ² =1.056,R indices(all data): R1=0.0347,wR2 =0.0942. ORTEP diagram is depicted as Figure 2(Figure 2). ¹ HNMR (400MHz, CDCl ₃ ): 1.59(s, 3H), 1.62(m, 1H), 1.64(m, 1H), 1.72(s, 3H), 2.04 (m,1H), 2.12(m,1H), 2.19(m,1H), 2.26(dd,1H), 2.39(m,1H), 2.45(m,1H), 2.52(m,1H), 2.80( m, 1H), 2.86(dd, 1H), 4.81(m, 1H), 4.85(bd, 1H), 5.08(m, 1H), 5.12(bd, 1H), 5.57(bs, 1H), 6.12(bs , 1H), 6.98 (bs, 1H). ¹³ CNMR (125 MHz, CDCl ₃ ): 15.1, 19.3, 23.7, 24.9, 33.3, 36.3, 40.3, 42.7, 77.9, 78.8, 122.9, 125.0, 129.1, 131.2, 134.3, 134.5, 139.6, 147.4, 170.4, 173.9. FTIR (KBr pellet): 3100, 2900, 1740, 1650, 1430, 1395, 1320, 1200, 1110, 1045, 1080, 980, 960, 930, 910, 880, 860, 820, 750, 625 cm ¹ and HRMS(ESI) m/z calcd _. for C ₂₀ H ₂₄ O ) 328.1675, found 328.1672.

Example 2. Single-dose oral acute toxicity test of fenugreek to rats: This example is to test the safety test of single-dose oral acute toxicity of fenugreek to rats, and provides a reference for food safety assessment. The test is based on the Taiwan Ministry of Health and Welfare Health Food Safety Assessment-Single-dose Oral Acute Toxicity Test, USEPA (Health Effects Test Guidelines, OPPTS 870.1100, Acute oral toxicity, US EPA 712-C-98-190.In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA712-C-00-366) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No. 420: Acute Oral Toxicity-Fixed Dose Procedure, No. 423: Acute Oral Toxicity-Acute Toxic Class Method, No. 425: Acute Oral Toxicity-Up and Down Method) and other test specifications for single-dose oral acute toxicity test. In this experiment, a single-dose oral acute toxicity test of ichthyolide to rats (Sprague-Dawley, SD strain) was carried out. Ophiolactone is yellowish crystalline, and the test purity is 99.95%. During the test, it is prepared with 10% DMSO to make a solution with a concentration of 0.1g/mL. The feeding volume of each rat is 10mL/kg body weight, according to the body weight on the day. The rats were orally fed, and the final dose was 1 g/kg-body weight in total, and the rats were continuously observed for 14 days after administration. The results showed that after oral administration of ichthyolide to rats, all rats had no symptoms of poisoning or died. In terms of weekly body weight change (g), the weekly body weight and body weight gain of male and female mice in the treatment group were not significantly different from those in the control group. After the test, the blood values of male and female mice in the treatment group changed, including: total white blood cells (WBC count), total red blood cells (RBC count), hematocrit ratio (Hct), mean corpuscular volume (MCV), mean hemoglobin ( MCH), mean hemoglobin concentration (MCHC), total platelet (platelet) and leukocyte classification were not significantly abnormal. Serum liver and kidney enzyme values of male and female mice in the treatment group, including: aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN) and creatinine (creatinine) No effect. There was no significant difference between the treatment group and the control group in terms of absolute body weight (g) and organ weight percentage in adrenal gland, brain, heart, kidney, liver, spleen, thymus, testis or ovary. After examination of internal organs, the adrenal gland, brain, heart, kidney, liver, spleen, thymus, testis or ovary and other important organs in the treatment group had no macroscopic lesions. The results of histopathological examination showed that there were no histopathological changes related to the test substance in the important organs of the ichthyolide-treated group. The comprehensive test results show that the acute toxicity test of a single dose of ichthyolide 1g/kg-body weight (about 50mg/kg-body weight in terms of human applicable dose) did not cause acute poisoning or death to rats, and None of the important organs in the body caused the pathological changes related to the toxic reaction of the tissues and organs.

Embodiment 3. 28-day feeding toxicity test of fenugreek lactone to rats: this embodiment is to test the repeated dose oral toxicity safety test of fenugreek lactone to rats, so as to establish material safety data sheet (material safety data) sheet), providing a reference for the clinical safety assessment of repeated use in humans. The test is based on the 28-day feeding toxicity test (1999) and the non-clinical drug test safety regulations (2014) of the Taiwan Ministry of Health and Welfare, and complies with the US Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, OPPTS 870.1100, Repeated Dose 28 -Day Oral Toxicity Study in Rodents.In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA712-C- 00-366) and Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No.407 : Repeated Dose 28-day Oral Toxicity Study in Rodents) and other test specifications. This experiment investigates whether ichthyolide may cause potential side effects and toxicity to human body for clinical safety assessment, and conducts clinical side effect observation test of 28-day repeated dose oral toxicity of ichthyolide in rats. The ichthyolide is a yellowish crystal, and the test purity is regarded as 99.95%. The sample is prepared with 5% DMSO during the test. Rats (Sprague-Dawley, SD strain) were divided into control group (5% DMSO), low-dose group (10 mg/kg-body weight), middle-dose group (25 mg/kg body weight) and high-dose group (50 mg/kg) -body weight) and other 4 groups, 20 rats in each group, half male and half male, each rat was fed with a volume of 10 mL/kg-body weight, and the rats were orally fed according to their body weight on that day for 28 consecutive days. The test results showed that after oral administration of ichthyolide to rats for 28 days, all the rats did not suffer from poisoning symptoms or death due to the test substance. After the test, the body weight changes, feed consumption, urine, blood, serum enzyme values and organ weights of male and female mice in each treatment group were compared with those of the control group, although there were slight individual differences. There is an increase or decrease, but it is still within the normal range, or there is no dose-response correlation between groups, and it has no clinicopathological significance and has nothing to do with the test substance. There were no obvious pathological changes in the whole body organs of the rats in each group. The results of histopathological examination showed that the high-dose group did not cause any pathological changes related to organ toxicity to various organs in the body. Based on the above inspection results, ichthyolide was administered orally in the low-dose group (10mg/kg-body weight), the middle-dose group (25mg/kg-body weight) and the high-dose group (50mg/kg-body weight) respectively. After 28 days of continuous feeding to rats, it did not cause toxic reactions to various organs of male and female rats. The "No observed adverse effect level" (NOAEL) of the 28-day feeding toxicity test on rats was 50mg/kg- body weight, the equivalent human dose is about 8mg/kg-body weight.

Example 4. Ames test of typhimurium to Salmonella reverse mutation: This example is to test the Ames test of ichthyolide to reverse mutation of Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 strains, so as to Create a product safety data sheet (Material Safety Data Sheet) to provide reference for use safety assessment. The test is based on the genotoxicity study (1999) of the "Health Food Safety Assessment Method" of the Ministry of Health and Welfare of Taiwan, and the US Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, Bacterial Reverse Mutation Test, US EPA 712-C-98- 247.In: OPPTS Harmonized Test Guidelines, Series 870.5100, 1998) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No. 471: Bacterial Reverse Mutation Test, 2002) and other test specifications Mutability test. In this experiment, the Ames test of ichthyolide to reverse mutation of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 was carried out. In the test, the 1.25, 2.5 and 5 mg/plate concentrations of ichthyolide were used together with the strain for 18-20 hours, and the bacterial toxicity test was carried out. The results showed that ichthyolide below 5mg/plate had no significant toxicity to TA102 strain, but it was toxic to TA98, TA100, TA1535 and TA1537 strains; so 0.63, 1.25 and 2.5mg/plate of ichthyolide were added. Equal concentration and TA98, TA100, TA1535 and TA1537 strains were co-acted for 18 hours, and the bacterial toxicity test was carried out. The results showed that ichthyolide below 2.5mg/plate had no significant toxicity to TA98, TA100, TA1535 and TA1537 strains. The highest concentration of ichthyolide that has no significant toxicity to TA102 strain was serially diluted down by 2 times, and 5 concentrations of 0.31, 0.63, 1.25, 2.5 and 5 mg/plate were selected as the formal test of Ames; while TA98, TA100, TA1535 and The highest concentration of TA1537 strain with no significant toxicity was serially diluted down by 2 times, and 5 concentrations of 0.16, 0.31, 0.63, 1.25 and 2.5 mg/plate were selected as the formal test of Ames, and the direct and rat liver were carried out respectively. Activated enzyme extract (S9) was mixed and acted on mutant strains of Salmonella to simulate the gene variability of the metabolites of ichthyolide metabolized by liver enzymes (S9) in animals to each strain, and co-cultured for 48 hours The bacterial count was then counted. The results showed that the number of reverse mutant bacteria on bacteria was not more than 2 times greater than the number of reverse mutant bacteria in the negative control group, whether directly or after the action of S9. Based on the above results, ichthyolide has no mutagenicity in the Ames test of the reverse mutation of Salmonella, and the result of the bacterial gene mutation test is negative (non genetic mutation in Ames test).

Embodiment 5. Chromosomal abnormality analysis of in vitro mammalian cell line by ichthyolide: This example is to test ichthyolide for the analysis of chromosomal abnormality of in vitro mammalian cell line, thereby establishing product safety data (Material Safety Data Sheet), providing Use reference for security assessments. The test is based on the in vitro mammalian chromosome aberration analysis test of Taiwan Ministry of Health and Welfare Health Food, and complies with the US Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, OPPTS 870.1100, In vitro mammalian chromosome aberration test, US EPA 712-C-98-190 .In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA712-C-00-366, 1998) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No. 473: In vitro mammalian chromosome aberration test 1997) and other test specifications for genotoxicity tests. Chromosomal aberration test with mammalian cell in culture was performed in this experiment. Ophiolactone is a yellowish crystal, and it is prepared with Dimethyl sulfoxide (DMSO) in the test. The cytotoxicity test is divided into two parts, one part uses CHO-K1 cells to directly interact with lepidolide, the other part uses rat liver activating enzyme extract (S9) to simulate human metabolism, CHO-K1 cells and rat liver activating enzyme The extract (S9) and the ichthyolide are mixed and acted upon. In the test without rat liver activating enzyme extract (-S9), 5 test doses of 12.5, 15, 17.5, 20 and 25 μM were used for 24-hour cytotoxicity test. -K1 cell survival rate is about 65.6%. In addition, in the test containing rat liver activating enzyme extract (+S9), the cytotoxicity test was carried out with 5 test doses of 60, 70, 75, 80 and 90 μM for 24 hours. The survival rate of K1 cells was about 60.3%, which showed that the iodine lactone was cytotoxic to CHO-K1 cells, and this concentration was selected as the highest dose in the formal test. For the cell chromosome variation test, take samples of ichthyolide at 12.5, 15, and 17.5 μM (-S9), prepare the sample solution in each cell culture dish, and co-culture for 24 hours; After 3 hours, the number and structure of chromosomes in cells were observed after 24 hours. The results showed that under the test conditions of the S9 mixed liquid metabolic activation system, ichthyolide was 12.5, 15 and 17.5 μM (-S9) and 55, 65 and 75 μM (+S9), and other three dose groups. The frequency of chromosomal abnormalities in CHO-K1 cells did not increase significantly compared with the negative control group, and there was no significant change in the location of chromosomal aberrations in the cells. Based on the above results, it is shown that no matter whether it contains S9 mixture or not, fenugreek lactone has no mutagenic effect on the chromosome of mammalian cell line CHO-K1 in vitro.

Example 6. Micronucleus test of fenugreek lactone on peripheral blood of mice: This example is a genotoxic test for testing fenugreek lactone, so as to establish a product safety data (Material Safety Data Sheet) and provide a method for safety assessment of use. refer to. The test is based on the health food safety assessment of the Taiwan Ministry of Health and Welfare - the genotoxicity test of the mouse peripheral blood micronucleus test, and in line with the US Environmental Protection Agency (USEPA) (Mammalian Erythrocyte Micronucleus Test, In: OPPTS Harmonized Test Guidelines, Series 870.5395, EPA 712- C-98-226) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals. Section 4: Health Effects. No. 474: Mammalian Erythrocyte Micronucleus Test, 1997) and other test specifications for genotoxicity tests. In this experiment, the micronucleus test of ichthyolide on the peripheral blood of mice (ICR strain) was carried out. This test mainly tests the ratio of ichthyolide to the occurrence of micronuclei in the peripheral blood of rodents (in vivo), so as to evaluate the degree of damage caused by gene mutations that directly or indirectly induce erythrocyte chromosomes or mitosis. The test took ICR mice as the test object, and the test was divided into negative control group, positive control group (Cyclophosphamide, 60mg/kg bw ip), low dose of cyclophosphamide (0.25g/kg bw), medium dose (0.5g/kg bw) kg bw) and high-dose (1g/kg bw), 5 groups, 5 mice (male) in each group, and a single feeding of ichthyolide by gastric tube, 48 hours and 72 hours after administration of the test substance , to evaluate the incidence (‰) of reticulocytes and reticulocytes in the peripheral blood of mice. The results showed that 48 hours and 72 hours after the administration of ichthyolide, there were no toxic symptoms and body weight differences among the treatment groups. The reticulocytes in the peripheral blood of mice were stained with 0.1% Acridine orange stain, which turned orange-red under a fluorescence microscope, and yellow-green fluorescent micronuclei of about 1/20-1/5 the size of erythrocytes were seen in the reticulocytes. There was no significant difference in the number of reticulocytes and the number of micronuclei in the reticulocytes between the treatment groups at 48 hours and 72 hours, compared with the negative control group. Compared with the negative control group, the number of reticulocytes in the positive control group decreased significantly (p<0.05), and the number of micronuclei in the reticulocytes also increased significantly (p<0.05). Based on the above results, there was no significant difference in the number of reticulocytes and micronuclei in the peripheral blood of mice in each dose group of ichthyolide compared with the negative control group, and the test results were negative. Therefore, ichthyolide does not have the toxic effect of chromosomal gene mutation on peripheral erythrocytes of mice.

Example 7. Molecular docking simulation study on the binding region of the novel coronavirus surface spike glycoprotein receptor binding region of the spirochete lactone: the novel coronavirus surface spike glycoprotein and the human cell membrane receptor angiotensin-converting enzyme 2 (angiotensin-converting enzyme Enzyme 2, ACE2) binding is a key step in mediating virus invasion into the host, and blocking or interfering with the binding of virus to receptors is a potential preventive and therapeutic strategy. This example is based on molecular docking to evaluate whether Ovalide (Ova) binds to the novel coronavirus surface spike glycoprotein and blocks or interferes with its binding to the receptor molecule angiotensin-converting enzyme 2 (ACE2), to clarify Antiviral mechanism of ichthyolide. The specific implementation method is as follows: take the crystal structure (PDB code: 6M0J) of the receptor-binding domain (RBD) of the spike glycoprotein on the surface of the new coronavirus as the molecular docking receptor, and use the MOE software to add hydrogen atoms to the RBD structure. Perform energy optimization. The structure of the ligand ichthyolide (Ova) was also constructed by MOE software, and the energy optimization was performed using the standard MMFF94 molecular force field and an energy gradient of 0.0001 kcal/mol as the convergence criteria. The molecular docking module based on MOE performs molecular docking, and the energy-optimized docking structure is further optimized for energy and docking mode analysis. Molecular docking results showed that Ovalide (Ova) bound to the hydrophobic pocket formed by several hydrophobic amino acids (L455, F456, Y489, F490) of RBD, and formed hydrogen bonds with Y489 and Q493 (Figure 3). The binding site is located at the interface between the new coronavirus spike glycoprotein RBD and the receptor ACE2, and it is predicted that Ophiolactone (Ova) can block or interfere with the direct binding of the virus spike glycoprotein RBD to the receptor ACE2.

Example 8. Molecular docking simulation study on the binding of lepidolide to host endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L: fusion of host endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L to coronavirus Process plays a key role. This example is to evaluate whether ichthyolide binds and inhibits Cathepsin B and Cathepsin L based on molecular docking, so as to clarify the antiviral mechanism of ichthyolide. The specific implementation method is as follows: take the crystal structures of human endosomal cysteine proteolytic enzymes Cathepsin B and cathepsin L (PDB code: 3AI8 & 2XU1) as molecular docking receptors, use MOE software to add hydrogen atoms to the RBD structure and carry out Energy optimization. The structure of the ligand ichthyolide (Ova) was also constructed by MOE software, and the energy optimization was performed using the standard MMFF94 molecular force field and an energy gradient of 0.0001 kcal/mol as the convergence criteria. The molecular docking module based on MOE performs molecular docking, and the energy-optimized docking structure is further optimized for energy and docking mode analysis. Molecular docking results also showed that Ovalide (Ova) may also bind to the catalytic pockets of endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L. Ophiolactone (Ova) binds to the hydrophobic S2 site of Cathepsin B composed of Y75, P76, A173, A200, and E245 through a hydrophobic alicyclic ring, and forms a covalent complex with the catalytic cysteine C29 through an exocyclic olefin Thereby inhibiting the activity of Cathepsin B (Fig. 4A). On the other hand, iodine lactone (Ova) binds to the hydrophobic S2 site of Cathepsin L composed of L69, M70, Y72, A135, and M161 with a hydrophobic alicyclic ring, and forms the catalytic cysteine C25 through an exocyclic olefin. The covalent complex thus inhibited the activity of Cathepsin L (Fig. 4B). Since the endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L play a key role in the fusion process of coronaviruses, Ovalide (Ova) potentially blocks the invasion and fusion process of 2019-nCoV.

Example 9. Study on the activity of fenugreek lactone in inhibiting novel coronavirus infection: the relationship between fenugreek lactone described in Example 7 and the receptor-binding domain (RBD) of the novel coronavirus surface spike glycoprotein The results of the molecular docking simulation study, and the results of the molecular docking simulation study between the fenugreek lactone described in Example 8 and the endosomal cysteine proteolytic enzymes Cathepsin B and Cathepsin L, etc., it is predicted that fenugreek lactone can inhibit the novel coronavirus Virus infection process. This example is based on the novel coronavirus pseudovirus inhibitory activity detection system developed by the laboratory of Professor Zhang Linqi, director of the AIDS Comprehensive Research Center of Tsinghua University, Beijing, to evaluate whether ichthyolide blocks the process of novel coronavirus infection of host cells. The specific implementation method is carried out according to the two steps of new coronavirus pseudovirus construction and new coronavirus infection inhibition detection: Step 1. Utilize membrane glycoprotein deletion (Env-defective) and HIV-1 virus genome plasmid pNL4-3R expressing luciferin protein 293T cells were co-transfected with -E-luciferase, and pcDNA3.1/SARS-CoV-2 expressing the full-length surface spike glycoprotein of the novel coronavirus, and cultured in DMEM medium containing 10% fetal bovine serum for 60 hours. The culture supernatant was taken to obtain the virus solution of the novel coronavirus pseudovirus (referred to as SARS-CoV-2 virus solution). Step 2. Take a 96-well cell culture plate, and add 100 microliters of diluent diluent and 50 microliters of SARS-CoV-2 virus solution to each well (the virus concentration in 50 microliters of SARS-CoV-2 virus solution is 1×10 ⁴ TCID50/mL), so that the concentration of the anthoenolide solution in the mixed system is the corresponding dilution concentration, and incubate at 37° C. for 1 hour. An equal volume of 10% fetal bovine serum in DMEM medium was used to replace the diluent of the iodine lactone solution as a virus control. An equal volume of DMEM medium containing 10% fetal bovine serum was used to replace the SARS-CoV-2 virus fluid as a cell control. Take the cell culture plate and inoculate 100 microliters of Huh7 cell suspension in each well (the solvent used to prepare the cell suspension is DMEM medium containing 10% fetal bovine serum, and the concentration of Huh7 cells in the cell suspension is 2×10 ^{5 )} cells/mL) and incubated at 37°C for 64 hours. Aspirate and discard the supernatant, add 150 microliters of lysis solution (Microglass Biotechnology, product number T003, according to the instructions) to each well, and incubate at 37°C for 5 minutes. The cell culture plate was taken and the luciferase activity was detected. Multiple replicate wells were set up for each treatment. Inhibitory activity (%)=[1-(fluorescence intensity of test group-fluorescence intensity of cell control)/(fluorescence intensity of virus control-fluorescence intensity of cell control)]×100%. Prism 5 software was used to calculate the ichthyolide concentration at which the inhibitory activity was 50%, that is, the _IC50 value of ichthyolide (Fig. 5). The results of the study show that ichthyolide has a specific molecular mechanism of action that is different from that of chloroquine or Remdesivir in inhibiting the new coronavirus, and significantly exhibits the inhibitory effect on the new coronavirus infection at the micromolar level.

Claims (8)

Use of a composition for preparing a medicine for inhibiting novel coronavirus (SARS-CoV-2), wherein the composition comprises a safe and effective amount of Ovatodiolide, or a safe and effective amount of Ovatodiolide Structural isomers of lactones, wherein the ichthyolide has a chemical structural formula I:

The use as claimed in claim 1, wherein the composition further comprises a pharmaceutically acceptable salt or carrier of the ichthyolide or the structural isomer of the ichthyolide. The use according to claim 1, wherein the composition can be used to prevent or treat diseases caused by novel coronavirus (SARS-CoV-2). The use according to claim 1, wherein the composition can be used for the prevention or treatment of severe special infectious pneumonia (COVID-19). The use as claimed in claim 1, wherein the eucalyptus lactone is a natural compound prepared by extracting eucalyptus with an organic solvent and separating and purifying by a chromatographic column, or preparing the same fish needle by chemical synthesis Synthetic compounds in the configuration of natural herbalactones. The use according to claim 1, wherein the safe and effective amount is 60 kg of body weight Generally, adults take less than 480 mg per day, and continue to take it for less than 28 days. The use according to claim 1, wherein the safe and effective amount is 20 mg to 40 mg orally per day for an average adult with a body weight of 60 kg, and the dose is continued for 7 to 14 days. Use of a composition for preparing a medicine for inhibiting coronavirus, wherein the composition comprises Ovatodiolide, or a structural isomer of Ovatodiolide, wherein the Ovatodiolide has A chemical structure of formula I:

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