WO2023204775A1

WO2023204775A1 - Herbal product for use in the prevention and/or treatment of covid-19

Info

Publication number: WO2023204775A1
Application number: PCT/TR2022/050357
Authority: WO
Inventors: Mohamed Abdualmajed Aziz AL-ZENDANI; Abdulmajid Aziz Hamoud AL-ZENDANI
Original assignee: Ipmc Group Ilac San Ve Tic Ltd Sti
Current assignee: Ipmc Group Ilac San Ve Tic Ltd Sti
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2023-10-26
Anticipated expiration: 2024-10-21

Abstract

The present invention relates to a herbal product comprising Saussurea costus root extract useful for the prevention and/or treatment of infection caused by SARS-CoV-2, namely COVID-19 disease. A pharmaceutical composition comprising the same and the method for the preparation thereof are also disclosed.

Description

HERBAL PRODUCT FOR USE IN THE PREVENTION AND/OR TREATMENT OF COVID- 19

Technical Field of the Invention

The present invention relates to a herbal product comprising Saussurea costus root extract for use in the prevention and/or treatment of infection caused by SARS-CoV-2 virus (COVID-19 disease). A pharmaceutical composition comprising the herbal product according to the invention, and preparation method thereof are also provided.

Background of the Invention

Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The first known case was identified in December 2019, and the disease has since spread worldwide, leading to an ongoing pandemic.

Most people infected with SARS-CoV-2 virus will experience mild to moderate respiratory illness and recover without requiring special treatment Older people, and those with underlying medical problems however are likely to develop serious illness.

Symptoms are variable, but often include fever, cough, headache, fatigue, breathing difficulties, and loss of smell and taste. Currently, the primary treatment of COVID-19 is symptomatic. Management of the disease includes the treatment of symptoms, supportive care, and isolation. In addition, supportive treatments, therapies, and compositions are being used and/or investigated.

WO 2021/053651 Al discloses an extract of Cocculus hirsutus, which is a tropical creeper plant, for prophylactic and/or curative treatment of an infection caused by SARS-CoV-2 virus. The document also provides a stable pharmaceutical composition comprising the said extract

KR 102240432 Bl discloses a herbal drug composition for relieving symptoms of COVID-19, wherein said drug composition comprises red pepper stalk extract as an active ingredient

Saussurea costus, also known with the names of Dolomiaea costus, Aucklandia costus, Saussurea lappa and Aucklandia lappa, is a thistle species belonging to the genus Saussurea of the Asteraceae family. The pharmaceutical name is Radix Auklandiae Lappae. Saussurea costus root comes from a perennial plant [Saussurea Lappa-Costus), that grows to about 2m in height and is native to Northern India, South West China and Pakistan. It has irregular, triangular-shaped leaves and dark blue or black florets, with brownish fruit. Saussurea costus is a medicinal plant whose extracts are known to have anti-inflammatory, hepatoprotective, anti-ulcer, anticancer, immunomodulatory and pesticidal activities. It has been traditionally used by different ethnic tribes of the Northern parts of India for treatment of various ailments like cough, cold, quartan malaria, leprosy, persistant hiccups, rheumatism, stomach ache, tooth ache, and typhoid fever (Pandey, M. M., Rastogi, S., & Rawat, A. K. S. (2007). Saussurea costus: Botanical, chemical and pharmacological review of an ayurvedic medicinal plant. Journal of Ethnopharmacology, 110(3), 379-390). It is also cited in the Chinese traditional medicine thatthis herb is often used to treat anicteric hepatitis and chronic persistent hepatitis, cholecystitis, infantile enteritis, bacillary dysentery, colic, flatulence, peptic ulcer, diarrhea, Predominant Irritable Bowel Syndrome (IBS-D), and dysmenorrhea.

The major components found in Costus roots can be listed as follows: Hepatadecatetraene, 12- methoxy-dihydrocostunolide, 22-dihydrostigmasterol, 3 -isopropropylpentanoic-acid, 3- methylbutyric-acid, 4-ethyloctanoic-acid, 7-octenoic-acid, Acetic-acid, Alkaloids, Alpha- humulene, Alpha-phellandrene, Alpha-costene, Alpha-amorphenic-acid, Alpha-amyrin-stearate, Alpha-ionone, Aplotaxene Beta-sitosterol, Beta-selinine, Beta-costene, Beta-ionone, Beta- elemene, Beta-amyrin-stearate, Betulin, Camphene, Caryophyllene, Caryophyllene-oxide, Cedrene, Cedrol, Cis-dihydroionone, Costic-acid, Costol, Constunolide Costus-acid, Costus-lactone, Dehydrocostus-lactone, Dihydrodehydrocostus-lactone, Dihydroaplotacene, Dihydrocostunolide, Dihydrocostus-lactone, EO, Friedelin, Guaia-3,9,ll-triene-12-acid, Heptanoic-acid, Hexanoic-acid, Inulin, Iszaluzanin, Kush tin, Lactones, Linalool, Leupeol, Myrcene, Naphthaline, Octanoic-acid, Oleic-acid, P-cymene, Palmitic-acid, Pentadec- 1-ene, Phellandrene, Resinoids, Saussurine, Stigmasterol, Tannin, Taraxasterol, Dihydrocostuslactone, dihydrocostunolide and alpotaxene.

WO 2011/039574 Al discloses the use of a herbal composition in the preparation of a medicament for the treatment of a subject infected with HIV, wherein the composition comprises an extract of the plant Aucklandia- (Costus Root).

There are many known therapeutic effects of Saussurea costus extracts, however its antiviral activity against infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) has yet to be brought to the literature. Furthermore, there is a continuous need to develop herbal products and pharmaceutical compositions that are effective in the prevention and/or treatment of COVID-19 disease.

In a pursuit of realizing these objectives, present invention provides a herbal product comprising Saussurea costus root extract for use in the prevention and/or treatment of infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), namely COVID-19 disease. Brief Description of the Invention

In an aspect, the present invention relates to a novel herbal product comprising Saussurea costus root extract for use in the prevention and/or treatment of COVID-19.

In an embodiment, the herbal product according to the invention is for use in a treatment comprising a dosage regimen of twice a day. Preferably, herbal product according to the invention is in unit doses of 5 ml to 10 ml.

In another aspect, the present invention provides a pharmaceutical composition comprising the herbal product according to the invention and at least one pharmaceutical excipient for use in the prevention and/or treatment of COVID-19.

Preferably, said pharmaceutical excipient is selected from the group comprising diluents, disintegrants, binders, glidants, lubricants, preservatives, coatings, plasticizers, surfactants, colorants, sweeteners, and flavors.

Preferably, said pharmaceutical composition is in a pharmaceutical dosage form selected from the group comprising solution, suspension, powder, tablet, capsule, and parenteral injection.

In a further aspect of the present invention, a method for preparing the pharmaceutical composition for use according to the present invention, comprising the steps below, is provided: treating Saussurea costus root with an aqueous medium, extracting content of the plant material, mixing the obtained plant material with at least one pharmaceutical excipient, and obtaining the pharmaceutical composition.

Preferably, the ratio of Saussurea costus root to the aqueous medium ranges from 1:20 to 1:5 (w/v). More preferably, the ratio of Saussurea costus root to the aqueous medium is 1:10 (w/v).

In an embodiment, the method for preparing the pharmaceutical composition for use according to the present invention further comprises the step of distillation of the obtained extract. Preferably, the distillation is performed at the temperature of 70°C to 90°C.

The pharmaceutical excipient according to the method of the invention is preferably selected from the group comprising diluents, disintegrants, binders, glidants, lubricants, preservatives, coatings, plasticizers, surfactants, colorants, sweeteners, and flavors. In an embodiment, the method for preparing the pharmaceutical composition for use according to the present invention further comprises the step of preparation of unit doses comprising 5 ml to 10 ml of the obtained pharmaceutical composition.

Brief Description of the Figures

Figure 1 shows the graph depicting the cytotoxic activity of Saussurea costus root extract on VERO E6 cells, as evaluated by xCELLigence RTCA MP device.

Figure 2 shows the graph depicting the antiviral activity of Saussurea costus root extract on VERO E6 cells as evaluated by xCELLigence RTCA MP device, for the dilutions of 1:64 to 1:1024.

Figure 3 shows the graph depicting the mean weight loss of two groups of K18-hACE2 transgenic mice.

Figure 4 shows the graph depicting the viral load in the lungs of K18-hACE2 transgenic mice.

Figure 5 shows the control and experimental group lung tissue samples. A) Control group lung tissue, 4x. B) Control group lung tissue, lOx. The area indicated by the arrow shows the lymphocyte infiltration areas in the interalveolar regions. C) Experimental group lung tissue, 4x. D) Experimental group lung tissue, lOx. The area indicated by the arrow shows alveoli with normal morphology.

Detailed Description of the Invention

The objective of the present invention is to provide a novel herbal product for use in the prevention and/or treatment of COVID-19 disease, i.e. SARS-CoV-2 infection. For this purpose, a herbal product comprising Saussurea costus root extract for use in the prevention and/or treatment of COVID-19 is provided.

The therapeutic uses of Saussurea costus root based on its anti-inflammatory, hepatoprotective, anti-ulcer, anticancer and immunomodulatory activities are already known in the relevant art The present inventors however, as a result of the studies and experiments they performed, have surprisingly noticed that this traditional medicinal plant root is also beneficial for the prevention and/or treatment of COVID-19, and therefore have a potential to be used as therapy or adjuvant therapy for this pandemic disease. The herbal product can also be consumed as a food supplement.

The present invention therefore presents a novel therapeutic activity of Saussurea costus root, namely anti-COVID-19 activity, that is put forth with the studies performed and explained in detail below. In this sense, a novel herbal product comprising Saussurea costus root extract for use in the prevention and/or treatment of COVID-19 disease (SARS-CoV-2 infection) is provided. The herbal product according to the invention is advantageous in that it can be easily manufactured with a simple extraction process and can be used effectively for the prevention and/or treatment of COVID-19.

The inventors have found that the dosage regimen of twice a day is suitable for an effective prevention and/or treatment of COVID-19 disease.

The dosage of the herbal product of the invention will vary depending on factors such as the severity of the condition being treated, age, physical condition, body weight, diet and the like. It is noted that the following doses would be useful depending on the weight of the patient

5 ml > <60 kg

- 7.5 ml > 60-100 kg

10 ml > >100 kg

Therefore, it is beneficial thatthe product is presented in unit doses of 5 ml to 10 ml. For example, the herbal product according to the invention can be in unit dose of 5 ml, 6 ml, 7 ml, 7.5 ml, 8 ml, 9 ml or 10 ml.

In another aspect, a pharmaceutical composition comprising the herbal product according to the invention and at least one pharmaceutical excipient for use in the prevention and/or treatment of COVID-19 is provided. The pharmaceutical composition according to the invention is advantageous in that it allows improvement of the Saussurea costus root extract with the help of the pharmaceutical excipients.

The pharmaceutical excipient in the context of the invention can be selected from the group comprising diluents, disintegrants, binders, glidants, lubricants, preservatives, coatings, plasticizers, surfactants, colorants, sweeteners, and flavors.

The examplary diluents comprise sugar compounds e.g. lactose, dextrin, glucose, sucrose and sorbitol, and inorganic compounds e.g. silicates, calcium and magnesium salts, sodium or potassium chloride.

The examplary disintegrants comprise compounds which swell or dissolve in water e.g. starch, cellulose derivatives and alginates, and crosslinked polymers e.g. crosslinked polyvinylpyrrolidone (crospovidone) and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium). The examplary binders comprise mainly natural or synthetic polymers e.g. starches, sugars, sugar alcohols and cellulose derivatives, proteins e.g. gelatin, and synthetic polymers e.g. polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG).

The examplary glidants comprise colloidal anhydrous silicon and other silica compounds e.g. silica gel, fumed silica, talc, and magnesium carbonate.

The examplary lubricants comprise minerals like talc or silica, and fats, e.g. vegetable stearin, magnesium stearate or stearic acid.

The examplary preservatives comprise antioxidants like vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium, the amino acids cysteine and methionine, citric acid and sodium citrate. Various dosage forms can be suitable for the administration of the pharmaceutical composition. Therefore, pharmaceutical composition according to the invention can be in a pharmaceutical dosage form selected from the group comprising solution, suspension, powder, tablet, capsule, and parenteral injection. Preferably, the composition is in an oral dosage form.

Saussurea costus root, just as the other conventional herbal products, is generally processed with a simple extraction procedure whereby the beneficial material of the plant is extracted through a liquid medium possibly at an elevated temperature, and this liquid medium as such or its dry material is used.

Therefore, in an aspect, present invention provides a method for preparing the pharmaceutical composition according to the invention, the method comprising the steps of: treating Saussurea costus root with an aqueous medium, extracting content of the plant material, mixing the obtained plant material with at least one pharmaceutical excipient, and obtaining the pharmaceutical composition.

Saussurea costus root plant material is preferably dried before the extraction step and is sliced into pieces for effectuating the mass transfer between the plant material and the extraction medium.

Preferably, the ratio of Saussurea costus root to the aqueous medium ranges from 1:20 to 1:5 (w/v). More preferably, the ratio of Saussurea costus root to the aqueous medium is 1:10 (w/v). The inventors, further noted that lighter fraction of the Saussurea costus root content can be lost during the extraction step at an elevated temperature, and surprisingly found that the said lighter fraction would in fact be useful for the prevention and/or treatment of COVID-19.

Therefore, in an embodiment, the method for preparing the pharmaceutical composition for use according to the present invention further comprises the step of distillation of the obtained extract, wherein the distillate is obtained in a liquid form.

The distillate obtained in the method of the present invention may be mixed with at least one pharmaceutical excipient directly as such in liquid form, or it can be dried to give a solid or semisolid preparation before mixing.

In the context of the present invention, distillation is carried out once. However, even lighter and poorer fractions can be obtained by further distillations. The distillation refers to an evaporation which can be carried out at a temperature around the reflux temperature of the extraction liqueur. The temperature can be below 100°C, more preferably below 95°C, and most preferably at a temperature ranging from 70°C to 90°C. For example, the distillation is performed at the temperature of 70°C, 75°C, 80°C, 85°C or 90°C.

The distillation is preferably performed for a duration of 1 h to 8 h, and more particularly for a duration of 1 h to 3 h.

The dosage of the pharmaceutical composition of the invention will vary depending on factors such as the severity of the condition being treated, age, physical condition, body weight, diet and the like. It is noted that the following doses would be useful depending on the weight of the patient

5 ml > <60 kg

- 7.5 ml > 60-100 kg

10 ml > >100 kg

Therefore, in an embodiment, the method for preparing the pharmaceutical composition for use according to the present invention further comprises the step of preparation of unit doses comprising 5 ml to 10 ml of the pharmaceutical composition. For example, the pharmaceutical composition according to the invention can be divided in unit doses of 5 ml, 6 ml, 7 ml, 7.5 ml, 8 ml, 9 ml, or 10 ml.

The herbal product or the pharmaceutical composition comprising the same disclosed herein can be used as a therapy, adjuvant therapy or merely as a food supplement as part of a diet programme of the patient. The detailed explanation of the studies carried out in order to produce the herbal product according to the invention and to evaluate the antiviral activity of the same are presented below.

EXAMPLES

Preparation of the Herbal Product

Roots of Saussurea costus were air dried in direct sunlight until the insides of the roots were observed black. The dried roots were then sliced. The sliced roots were then ground to a powder. The powdered plant material (1 kg) was placed in 10 L of water contained in a pot equipped with a water jacketed distillation line whereby water as a coolant is continuously circulated around the said distillation line. The extraction liqueur formed with the plant material and water is heated until 90°C with a temperature-controlled heater, and kept at this temperature for about 3 hours. The vaporized liqueur passing through the distillation line is condensed and collected in a flask in liquid form. The liquid obtained with the foregoing procedure constitutes lighter fraction of the plant material in Saussurea costus roots.

Antiviral Activity Test

It is known in the relevant art that Saussurea costus is effective in relieving symptoms of some infectious diseases. However, its protective and therapeutic effects against COVID-19 disease has yet to be discovered. The aim of this test was to investigate the antiviral effect of Saussurea costus root extract on SARS-CoV-2 virus.

Antiviral activity test was performed based on impedance, by using xCELLigence MP Real-Time Cell Analysis Device. The cell culture to be used is selected as VERO E6 (ATCC CRL-1586), since this cell line has ACE2 receptor to which SARS-CoV-2 virus binds.

As of medium, DMEM (Gibco Cat #31885-023 lot #2205969) was used. 10% Fetal bovine serum (FBS, heat inactivated, Hyclone, #SV30160.03 Lot: RE00000002) and 1% antibiotic-antimycotic solution (Sigma Cat #SLBH9584V) were added to the medium.

The sample to be tested is Saussurea costus root extract in liquid form. 1:2 dilution of the sample was mixed with 2X medium, whereas 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512 and 1:1024 dilutions were mixed with IX medium. All samples were filtered using a 0.22 pm filter.

The controls were determined as below:

Control 1: Serum containing medium

Control 2: 3.1x105 PFU/ml SARS-CoV-2 VERO E6 cells were culturedin a single-use sterile 96-well E-plate ofxCELLigence RTCAMP device as 25.000 cells would be present in each well. The device was held in the incubator throughout the experiment. Cultured cells were placed in the device and let to grow for 24 hours. The samples and 3.1xl0⁵ PFU/ml SARS-CoV-2 virus were incubated at 37°C, 5% CO2 for an hour. After the incubation, the media were removed from the 96-well cell growth plate and the incubated samples were added into the wells. The cells were then incubated at 37°C, 5% CO2 for 170 hours. The experiment was terminated at the end of this period and the obtained data was analyzed using RTCA Software Pro software.

Figure 1 shows the cytotoxic activity of Saussurea costus root extract on VERO E6 cells, as evaluated by xCELLigence RTCA MP device. The data was normalized based on the time point at which the virus was added.

The electrical conductivity is converted into unitless cell index (CI) parameter by the device at 15- minute intervals. An increase in CI indicates cell viability /health, whereas a decrease indicates cell death /unhealthiness. In the presence of virus, a decrease in CI values of VERO E6 cells is expected.

In light of this information, it can be seen in the graph of Figure 1 that out of ten dilutions of Saussurea costus root extract, 1:2 to 1:34 dilutions (including 1:34 dilution) exhibited toxicity on VERO E6 cells. The further dilutions (1:64 to 1:1024) were evaluated in terms of neutralization and/or delay of viral activity.

Figure 2 shows the antiviral activity of Saussurea costus root extract as evaluated by xCELLigence RTCA MP device for the dilutions of 1:64 to 1:1024. The data was normalized based on the time point at which the virus was added.

It can be seen in the graph of Figure 2 that, when incubated with the cells upon incubation with the virus, Saussurea costus root extract totally neutralized the viral activity of SARS-CoV-2 on VERO E6 cell line atthe dilution of 1:64. Furthermore, 1:128, 1:256, 1:512 and 1:1024 dilutions delayed the effect of virus on cells for 80.6, 23, 14 and 4.7 hours, respectively.

Challenge Test

The herbal product according to the invention, which is in a liquid form, was subjected to Challenge test The duration of the test was 14 days. The aim of this test was to investigate the protective and therapeutic effects of the herbal product against SARS-CoV-2 infection in mice.

Each experimental group consisted of 10 8-10 week old male K18-hACE2 transgenic mice. "Group 1" was designed as the experimental group. This group was subjected to infection with SARS-CoV- 2 virus and was given the herbal product of the invention. Since the inventors intended to provide the herbal product of the invention to be consumed as a food supplement, which is taken twice daily on an empty stomach before meals; to evaluate the effectiveness of this dose regimen with the Challenge test, the herbal product was started to be given to mice 2 days before the infection of mice with the SARS-CoV-2 virus. On the first 3 days of the Challenge test, SARS-CoV-2 virus was given to mice in the morning for pathogenesis, while the herbal product of the invention was given in the afternoon with an interval of 4 hours. The product was continued to be administered for 3 days after the pathogenesis period was over. Therefore, the herbal product was used in "Group 1” for 8 days in total: 2 days before the pathogenesis, 3 days during pathogenesis and 3 days upon pathogenesis.

For the pathogenesis, animals were taken to BSL-3 (BioSafety Level-3) laboratory and infected intranasally with 10⁵ TCID50 SARS-CoV-2 virus in a volume of 50 pl under anesthesia for 3 days before noon. On the same days of the experiment, 4 hours later in the afternoon, the herbal product of the invention was given by gavage at a dose of 2000mg/kgper mouse. Virus inoculation was completed on the 4th day, but the administration of the extract was continued until the 8th day.

"Group 2" on the other hand was designed as the placebo control group and was infected with the SARS-CoV-2 virus without the administration of the herbal product. The pathogenesis of the animals of "Group 2” was performed in the same way as for "Group 1”.

The animals of both groups were observed for clinical symptoms and their weights were measured daily until the end of the experiment. Starting from the 8th and 9th days of the experiment, weight loss was observed in the animals in the control group, i. e. "Group 2”. Towards the end of the experiment, death was observed in the 10th animal from the experimental group (Group 1) and the 2nd and 5th animals from the control group (Group 2). No clinical symptoms were observed in the experimental group animals throughout the experiment. Daily weight follow-ups and weight losses of the animals are given in Table 1 below. As can be seen in Table 1, there was no general weight loss in the experimental group (Group 1) animals. The only live weight loss observed in this group was 10% weight loss in the 10th animal of Group 1. For "Group 2” on the other hand, 7 to 14% live weight loss was observed in 5 animals. Table 1. Weight follow-up of the animals in Groups 1 and 2

The graph depicting the mean weight loss of two groups of K18-hACE2 transgenic mice is shown in Figure 3. GraphPad 7.0 two-way ANOVA program was used in order to plot the graph. (*) 0.0332, (**) 0.0021 and (***) 0.0002 are used as P values in the graph. The comparison of the experimental groups with the control group was found to be statistically significant

Gross Pathology During the Challenge Test, the animal numbered 10 in Group 1 and animals numbered 2 and 5 in Group 2 died on the last day of the experiment due to illness as can be seen in Table 1. Remaining mice were killed by cervical dislocation method on the 10th day of the experiment, and gross pathology was performed. All organs, particularly lungs were examined during gross pathology. Lungs, heart, spleen, liver, stomach and kidneys were divided into two and collected for histopathology and virus extraction. First half of the organs were taken into tissue cassettes to be fixed in 10% formaldehyde for 48-72 h. Virus isolation was performed on the other half of the organs and the isolates were stored at -20°C. Lungs and other organs were evaluated in terms of color, tissue integrity, appearance and size. In general, the entire lung was found to be normal in the experimental group (Group 1) animals. In the control group (Group 2) animals on the other hand, pneumonia in different sizes was observed. However, no one-to-one correlation was observed for any animal in gross pathology. Animals with lesion-free lungs despite weight loss were also encountered.

Organs that have no lesions were scored 0 (zero), and organs with lesions were scored 1-5 (one to five) depending on the presence of different lesions such as edema, hyperemia and pneumonia. Animals that died before the end of the experiment were scored 5. The Gross Pathology Scores are shown in Table 2 below.

Table 2. Gross Pathology Scores of the animals in Groups 1 and 2

qPCR Viral Load Evaluation

In this test, the therapeutic effect of neutralizing antibody was examined. Viral RNA was obtained from the collected lungs and Real Time PGR was used to determine the viral load. There is an inverse relationship between the viral load and the obtained CT value.

The amplification curves of samples with high viral load have low CT value because they cross the threshold value early. The real time PCR CT values of the samples are given in Table 3 below. NCI and NC2 regions of SARS-CoV-2 virus were examined in viral RNAs. Animals showing signs of disease gave early CT because they possessed a high amount of virus. The Real Time PCR positive control gave CT at 27 for this study. Accordingly, values up to 11-29 were considered positive for virus. Table 3. Viral load CT values in the lungs of K18-hACE2 transgenic mice in Groups 1 and 2

The CT1 value shown in Table 3 belongs to the nucleocapsid 1 region primer of the virus, and the CT2 value belongs to the nucleocapsid 2 region primer of the virus.

Late CT values between 30-40 (approximately 100-3000 viral copies, although it differs for each study) were considered insignificant due to the very small amount of virus. Since the target signal could not be obtained in the absence of viral load, the CT values of these samples were evaluated as 0 (zero).

The graph depicting the viral load in the lungs of K18-hACE2 transgenic mice is shown in Figure 4. GraphPad 7.0 two-way ANOVA program was used in order to plot the graph. (*) 0.0332, (**) 0.0021 and (***) 0.0002 are used as P values in the graph. The comparison of the experimental groups with the control group was found to be statistically significant

Histopathology Evaluation

Lung tissue samples were taken from the experimental and control groups (Group 1 and Group 2, respectively) and fixed with 10% Formaldehyde for 48-72 hours. After fixation, the samples were washed and then dehydrated. Dehydrated tissues were prepared as paraffin blocks. Tissue sections of 5 pm thickness were taken from paraffin blocks and hematoxylin-eosin staining was performed. After the tissue sections were treated with xylol for 30 minutes, rehydration (100%- 70% alcohol and 10 minutes in distilled water) was performed. The samples were kept in hematoxylin dye for 5 minutes, then washed with tap water for 10 minutes and treated with eosin for 5 minutes. Upon eosin staining, the samples were washed with tap water again and dehydration was performed by passing through the alcohol series with increasing concentrations for 5 minutes. The samples then were cleared with xylol and sealed with entellan to be examined.

The semi-quantative evaluation was used in the examination. Inflammation status in the lungs was compared with the whole lung and a value between 0-3 was assigned to the samples. These values represent the below.

0: No inflammation.

1: Morphology is normal, but mild erythrocyte and lymphocyte infiltration around bronchioles is observed.

2: Moderate erythrocyte and lymphocyte infiltration in the lung.

3: Distorted morphology and intense erythrocyte and lymphocyte infiltration.

Figure 5 shows the microscopic images of lung tissue samples of control and experimental groups. The fact that control group showed lymphocyte infiltration areas in the interalveolar regions whereas experimental group showed alveoli with normal morphology clearly indicates that the herbal product according to the invention is effective in preventing the spread of SARS-Cov-2 virus to the lungs.

The score evaluation of the histopathological studies performed for experimental and control groups of mice is given in Table 4 and Table 5 below.

Table 4. The histopathological score evaluation of lungs of mice in Group 1

Table 5. The histopathological score evaluation of lungs of mice in Group 2

According to the data presented in Table 4 for the experimental group (Group 1), mouse no 5 which was Covid positive according to the PCR test had a score of 3 in the histopathological evaluation. In addition, 3 mice (no 1, 2 and 8) had a score of 0.5, which indicates a mild lymphocyte infiltration at the edges. The reason for this mild infiltration was thought to be the sinking of the syringe needle into the lung during intracardiac blood collection. No pathological findings were observed in the lungs of the remaining mice.

On the contrary, the results for the control group as presented in Table 5 were found to involve histopathological lesions with high scores (2 and 3) for 7 out of 10 mice. No lesion was observed in one mouse, and 2 mice had lesions scored with 1. The fact that most of the control group mice showed high histopathological scores whereas experimental group involved only one mouse with a score of 3, demonstrates the preventive and therapeutic effect of the herbal product according to the invention against COVID-19 disease.

As explained in detail above, the same treatment with the herbal product according to the invention that would be applied to humans was applied to mice in scope of the studies and tests conducted. Since the product is planned to be used as a food supplement in humans, it was also used in mice in this way. While the pathogenesis started in the control group (Group 2) on the 6th day of the experiment, there was no pathogenesis observed in the experimental group (Group 1) except for 1 mouse. This means that the animals in the control group developed COVID-19 as desired. As a result, intense losses in live weight and disease symptoms were observed in animals of the control group. For the experimental group on the other hand, 90% of the animals treated with the herbal product according to the invention were fully protected from COVID-19, and only 1 mouse developed the disease. While the sick mouse showed clinical symptoms and weakened, those who did not get sick completed the experiment in a healthy way. In addition to the results of the Challenge test, histopathological studies in the scope of the present invention also proved the effectiveness of the herbal product of the invention as explained above.

In the light of the results of the antiviral activity experiments, Challenge Test and histopathological studies, it has been put forth that the herbal product according to the present invention exhibits an antiviral activity against COVID-19 disease both in vitro and in vivo. These results clearly suggest that Saussurea costus root extract can be used in the prevention and/or treatment of

COVID-19. Present inventors, taking this fact into account, present a novel herbal product comprising Saussurea costus root extract and a pharmaceutical composition comprising thereof for use in the prevention and/or treatment of COVID-19.

Claims

1. A herbal product comprising Saussurea costus root extract for use in prevention and/or treatment of COVID-19.

2. A herbal product for use according to claim 1, wherein the product is for use in treatment comprising a dosage regimen of twice a day.

3. A herbal product for use according to claim 1, wherein the product is in unit doses of 5 ml to 10 ml.

4. A pharmaceutical composition comprising the herbal product according to any of the preceding claims and at least one pharmaceutical excipient for use in the prevention and/or treatment of COVID-19.

5. A pharmaceutical composition for use according to claim 4, wherein said pharmaceutical excipient is selected from the group comprising diluents, disintegrants, binders, glidants, lubricants, preservatives, coatings, plasticizers, surfactants, colorants, sweeteners, and flavors.

6. A pharmaceutical composition for use according to claim 4, wherein the composition is in a pharmaceutical dosage form selected from the group comprising solution, suspension, powder, tablet, capsule, and parenteral injection.

7. A method for preparing the pharmaceutical composition for use according to claim 4, comprising the steps of: treating Saussurea costus root with an aqueous medium, extracting content of the plant material, mixing the obtained plant material with at least one pharmaceutical excipient, and obtaining the pharmaceutical composition.

8. A method according to claim 7, wherein the ratio of Saussurea costus root to the aqueous medium ranges from 1:20 to 1:5 (w/v).

9. A method according to claim 8, wherein the ratio of Saussurea costus root to the aqueous medium is 1:10 (w/v).

10. A method according to claim 7, wherein the method further comprises the step of distillation of the obtained extract. A method according to claim 10, wherein the distillation is performed at a temperature between 70°C to 90°C. A method according to claim 7, wherein said pharmaceutical excipient is selected from the group comprising diluents, disintegrants, binders, glidants, lubricants, preservatives, coatings, plasticizers, surfactants, colorants, sweeteners, and flavors. A method according to claim 7, wherein the method further comprises the step of preparation of unit doses comprising 5 ml to 10 ml of the obtained pharmaceutical composition.