Antiviral Traditional Chinese Medicine composition and preparation method and use i'hereof

Abstract

An antiviral traditional Chinese medicine composition, a preparation method therefor and a pharmaceutical application thereof. The composition uses Herba Taraxaci, Radix Stemonae, Pseudobulbus Cremastrae Seu Pleiones, Radix Puerariae, Rhizoma Atractylodis Macrocephalae, and Radix Cynanchi Atrati as main active pharmaceutical ingredients, may also optionally and additionally use Cortex Lycii Radicis and Cortex Mori as active pharmaceutical ingredients, and is prepared into a pharmaceutically acceptable dosage form according to needs. The medicine composition is effective against influenza A virus, H1N1, H7N7 and H9N2 viruses, Zika virus, dengue virus I, dengue virus II, and Chikungunya virus.

Claims

1. A method for preparing an antiviral herbal medicine composition, comprising: 1) extracting Herba Taraxaci, Radix Stemonae and Radix Cynanchi Atrati by alcohol to give an alcohol extract; 2) combining the residues obtained during the preparation of the alcohol extract of step 1) with Radix Puerariae, Rhizoma Atractylodis Macrocephalae, and Pseudobulbus Cremastrae Seu Pleiones, extracting the mixture thus obtained by water, concentrating the extract liquid, causing precipitation in the concentrated extract liquid by adding an alcohol, and collecting the supernatant as a water extract; and 3) combining the alcohol extract obtained in step 1) and the water extract obtained in step 2) to obtain the antiviral herbal medicine composition after optional post processing; wherein the amount of each of the raw material is as follows: Herba Taraxaci 30-70 parts by weight, Radix Stemonae 20-40 parts by weight, Pseudobulbus Cremastrae Seu Pleiones 20-40 parts by weight, Radix Cynanchi Atrati 20-50 parts by weight, Radix Puerariae 20-50 parts by weight, and Rhizoma Atractylodis Macrocephalae 20-60 parts by weight.

2. The method of claim 1, wherein the alcohol is 40-95% ethanol solution in water.

3. The method of claim 1, wherein the amount of Herba Taraxaci is 30-60 parts by weight, 35-55 parts by weight, or 40-50 parts by weight; the amount of Radix Stemonae is 25-35 parts by weight, or 25-30 parts by weight; the amount of Pseudobulbus Cremastrae Seu Pleiones is 25-35 parts by weight, or 25-30 parts by weight; the amount of Radix Cynanchi Atrati is 20-45 parts by weight, 20-40 parts by weight, or 25-35 parts by weight; the amount of Radix Puerariae is 20-45 parts by weight, 20-40 parts by weight, or 25-35 parts by weight; and the amount of Rhizoma Atractylodis Macrocephalae is 20-55 parts by weight, 20-50 parts by weight, or 30-40 parts by weight.

4. The method of claim 1, comprising the following steps: step 1), extracting Herba Taraxaci, Radix Stemonae, Radix Cynanchi Atrati and Cortex Mori by alcohol to give an alcohol extract, wherein the amount of Cortex Mori is 20-60 parts by weight, 20-55 parts by weight, 20-50 parts by weight, or 25-40 parts by weight; and step 2), combining the residues obtained during the preparation of the alcohol extract of step 1) with Radix Puerariae, Rhizoma Atractylodis Macrocephalae, Pseudobulbus Cremastrae Seu Pleiones and Cortex Lycii, extracting the mixture thus obtained by water, concentrating the extract liquid, causing precipitation in the concentrated extract liquid by adding an alcohol, and collecting the supernatant as a water extract, wherein the amount of Cortex Lycii is 20-60 parts by weight, 20-55 parts by weight, 20-50 parts by weight, or 30-40 parts by weight.

5. An antiviral herbal medicine composition produced by a method comprising the following steps: 1) extracting Herba Taraxaci, Radix Stemonae and Radix Cynanchi Atrati by alcohol to give an alcohol extract; 2) combining the residues obtained during the preparation of the alcohol extract of step 1) with Radix Puerariae, Rhizoma Atractylodis Macrocephalae, and Pseudobulbus Cremastrae Seu Pleiones, extracting the mixture thus obtained by water, concentrating the extract liquid, causing precipitation in the concentrated extract liquid by adding an alcohol, and collecting the supernatant as a water extract; and 3) combining the alcohol extract obtained in step 1) and the water extract obtained in step 2) to obtain the antiviral herbal medicine composition after optional post processing; wherein the amount of each of the raw material is as follows: Herba Taraxaci 30-70 parts by weight, Radix Stemonae 20-40 parts by weight, Pseudobulbus Cremastrae Seu Pleiones 20-40 parts by weight, Radix Cynanchi Atrati 20-50 parts by weight, Radix Puerariae 20-50 parts by weight, and Rhizoma Atractylodis Macrocephalae 20-60 parts by weight.

6. An oral pharmaceutical formulation comprising the herbal medicine composition according to claim 5 and a pharmaceutically acceptable carrier or adjuvant.

7. A method of treating or preventing a viral infectious disease or condition in a patient in need thereof, comprising orally administering to the patient an effective amount of the herbal medicine composition of claim 5; wherein the patient is a mammal.

8. The method of claim 7, wherein the viral infectious disease or condition is a disease or condition caused by Zika virus, dengue virus, or chikungunya virus, or a combination thereof.

9. The method of claim 7, wherein the viral infectious disease or condition is a disease or condition caused by influenza A virus.

10. The method of claim 7, wherein the mammal is a human being.

11. The antiviral herbal medicine composition of claim 5, wherein the amount of Herba Taraxaci is 30-60 parts by weight, 35-55 parts by weight, or 40-50 parts by weight; the amount of Radix Stemonae is 25-35 parts by weight, or 25-30 parts by weight; the amount of Pseudobulbus Cremastrae Seu Pleiones is 25-35 parts by weight, or 25-30 parts by weight; the amount of Radix Cynanchi Atrati is 20-45 parts by weight, 20-40 parts by weight, or 25-35 parts by weight; the amount of Radix Puerariae is 20-45 parts by weight, 20-40 parts by weight, or 25-35 parts by weight; and the amount of Rhizoma Atractylodis Macrocephalae is 20-55 parts by weight, 20-50 parts by weight, or 30-40 parts by weight.

12. The antiviral herbal medicine composition of claim 5, wherein Cortex Mori is additionally extracted in step 1), Cortex Lycii is additionally combined and extracted in step 2), and wherein the amount of Cortex Lycii is 20-60 parts by weight, 20-55 parts by weight, 20-50 parts by weight, or 30-40 parts by weight, and the amount of Cortex Mori is 20-60 parts by weight, 20-55 parts by weight, 20-50 parts by weight, or 25-40 parts by weight.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graphical representation showing the results of experiments in which the traditional Chinese medicinal composition of Example 1 prevents the replication of dengue type II virus in Vero cells. The y-axis marked as “Den 2 E1/1000 β-actin” shows the copy of the dengue type II virus in Vero cells, which is calculated as the copy number of the E1 gene in dengue type II virus per 1000 β-actin proteins; while for the x-axis, “Mock” represents virus control group (cell+culture); “0” represents virus control group (cell+virus+culture), and “Drug” represents the tested drug. “**” means p<0.01; “***” means p<0.001.

(2) FIG. 2 is a plot showing the cytotoxicity of the traditional Chinese medicinal composition of Example 1 in Vero cells tested by the LDH method. The “% Cytotoxicity” in the y-axis means the percentage of cytotoxicity, the “Lysis Buffer” in the x-axis represents cell lysate, “cells only” means that only cells are present, and “Drug” represents the drug tested.

(3) FIG. 3 is a graphical representation showing the results of experiments in which the traditional Chinese medicinal composition of Example 2 prevents the binding of Zika virus to Vero cells. The y-axis marked as “ZIKV E1/β-actin” shows the binding of Zika virus to Vero cells, which is calculated as the copy number of the E1 gene in Zika virus per a β-actin protein; while for the x-axis, “Mock” represents virus control group (cell+culture); “0” represents virus control group (cell+virus+culture), and “Drug” represents the tested drug. “*” means p<0.05.

(4) FIG. 4 is a plot showing the results of plaque forming assay testing the inhibitory effect of the traditional Chinese medicinal composition of Example 4 against chikungunya virus. The y-axis marked as “Number The plaque” shows the number of the plaque, the y-axis marked as “Plaque neutralization (%)” represents the plaque neutralization percentage, while the “Drug” in the x-axis represents the drug tested.

(5) FIG. 5 is a graphical representation showing the results of in vitro experiments in which the traditional Chinese medicinal composition of Example 5 prevents the binding of chikungunya virus to cellular receptors. The y-axis marked as “CHIKV E1/β-actin” shows the binding of chikungunya virus to Vero cells, which is calculated as the copy number of the E1 gene in chikungunya virus per a β-actin protein; while for the x-axis, “Mock” represents virus control group (cell+culture); “0” represents virus control group (cell+virus+culture), and “Drug” represents the tested drug. “*” means p<0.05.

(6) FIG. 6 is a plot showing the results of plaque forming assay testing the inhibitory effect of the traditional Chinese medicinal composition of Example 6 against Zika virus. The y-axis marked as “Plaque neutralization (%)” represents the plaque neutralization percentage, while the “Drug” in the x-axis represents the drug tested.

(7) FIG. 7 is a graphical representation showing the results of experiments in which the traditional Chinese medicinal composition of Example 6 reduces the level of Zika virus in blood. The y-axis marked as “ZIKV E1/100 β-actin” shows the copy of the Zika virus in blood of mice, which is calculated as the copy number of the E1 gene in Zika virus per 100 β-actin proteins; while for the x-axis, “Control” represents control group, “Medium” represents the drug group with medium dose, “High” represents the drug group with high dose; “d2” means day 2, and “d4” means day 4.

(8) FIG. 8 is a graphical representation showing the results of experiments in which the traditional Chinese medicinal composition of Example 6 inhibits the replication of Zika virus in spleen, brain and uterus. The y-axis marked as “ZIKV E1/100 β-actin” shows the copy of the Zika virus in blood of mice, which is calculated as the copy number of the E1 gene in Zika virus per 100 β-actin proteins; while for the x-axis, “Control” represents control group, “Medium” represents the drug group with medium dose, “High” represents the drug group with high dose; “Spleen” means spleen, “Brain” means brain, and “Uterus” means uterus.

(9) FIG. 9 is a graphical representation showing the effect of the traditional Chinese herbal composition of Example 6 on the body weight of mice. The y-axis marked by “wt in grams” shows the body weight of the mouse (in gram); while for the x-axis, “Before” means before administration, “after” means after administration; “Control” represents control group, “Medium” represents the drug group with medium dose, and “High” represents the drug group with high dose.

(10) FIG. 10 is a graph showing the schedule of an experiment illustrating the antipyretic effects of the Traditional Chinese Medicine composition of Example 6 in an animal model of fever. The “BW” means body weight and “Anal TEMP” means rectal temperature.

(11) FIG. 11 is a graph showing the schedule of an experiment testing the toxicity of the Traditional Chinese Medicine composition of Example 6 in rhesus.

(12) FIG. 12 shows the fingerprints of the Traditional Chinese Medicine composition of Examples 1-6.

EXAMPLES

(13) The embodiments of the present invention will be further described in detail below by way of exemplary Examples. It is understood that the Examples described herein are merely illustrative and are not intended to limit the scope of the claims. Any modification made within the spirit and principal of the present invention, and any improvement made or equivalents obtained according to common technical knowledge and conventional means in the art should be included in the scope of the claims.

(14) The numbers reported in the following Examples are as accurate as possible, but those skilled in the art will understand that each number should be understood as an approximate value rather than an absolutely accurate number due to unavoidable measurement errors and variation in operational parameters. For example, due to an error caused by weighing apparatus, the weight values for each materials in each composition in the Examples should be understood as having a ±2% or ±1% error.

Example 1

(15) The formulation of the Traditional Chinese Medicine composition of Example 1 was as follows:

(16) Herba Taraxaci: 470 g

(17) Radix Stemonae: 280 g

(18) Pseudobulbus Cremastrae Seu Pleiones: 280 g

(19) Radix Cynanchi Atrati: 315 g

(20) Radix Puerariae: 315 g

(21) Rhizoma Atractylodis Macrocephalae: 380 g

(22) Herba Taraxaci was collected from Hebei, China; Radix Cynanchi Atrati was collected from Henan, China, Radix Puerariae was collected from Hebei, China, Radix Stemonae was collected from Guangxi, China, Pseudobulbus Cremastrae Seu Pleiones was collected from Sichuan, China, and Rhizoma Atractylodis Macrocephalae was collected from Zhejiang, China.

(23) The process for preparing the Traditional Chinese Medicine composition of Example 1 was as follows:

(24) (1) 470 g Herba Taraxaci, 280 g Radix Stemonae and 315 g Radix Cynanchi Atrati were subjected to extraction with 8×60% ethanol for 1.5 h and the obtained mixture was filtered; and then 8×60% ethanol was added to extract for another 1.5 h, the obtained mixture was filtered; and the filtrates obtained in these two extraction processes were combined to give an alcohol extract;
(2) the residues obtained during the preparation of the alcohol extract were combined with 315 g Radix Puerariae, 280 g Pseudobulbus Cremastrae Seu Pleiones and 380 g Rhizoma Atractylodis Macrocephalae, the mixture thus obtained was subjected to extraction for 2 hours with 10× water and filtered; then the remaining mixture was again subjected to extraction for 2 hours with 10× water and filtered, the filtrates obtained in these two extraction processes were combined and concentrated to obtain a thick paste; 90% ethanol was added to the thick paste and stirred to achieve a final concentration of about 75 ethanol % and a water extract was obtained by taking the supernatant after a 24 hours settling; and
(3) the alcohol extract obtained in step 1) and the water extract obtained in step 2) were combined, concentrated and spray dried to give the Traditional Chinese Medicine composition of Example 1. The resulting composition was a brown powder, the weight of which was about 0.34 kg, and it was used directly in the following in vivo and in vitro experiments.

(25) In the following the Traditional Chinese Medicine composition of Example 1 (referred to as “tested drug” or “tested substance”) was studied in various pharmacodynamics experiments to verify its efficacy.

(26) 1. In Vitro Experiment Using the Composition of Example 1 Testing its Inhibition of Dengue Type II Virus (Dengue-2) Calculated by Copy Number

(27) Vero cells (African green monkey kidney cells, available from the American Type Culture Collection (ATCC), CCL-81) were plated in a 6-well plate at 6×10.sup.5/well and incubated under 5% CO.sub.2 at 37° C. for 24 hours and monolayer was formed. The tested substance was dissolved in PBS buffer, filter sterilized, and diluted to 1.0 mg/ml and 0.1 mg/ml. The dengue type II virus (obtained from the American Type Culture Collection ATCC, VR-1584) and 0.1 mg/ml or 1.0 mg/ml tested substance were added to Vero monolayer, and incubated at 37° C. for 24 hours. Quantitative analysis of intracellular viral replication (dengue type II E gene) was conducted by qPCR, see Paul A M, Shi Y, Acharya D, et al. Delivery of Antiviral Small Interfering RNA with Gold Nanoparticles Inhibits Dengue Virus Infection in vitro. [J]. Journal of General Virology, 2014, 95(8):1712-22 for details.

(28) The results of the PCR experiment showed that the tested substance can significantly inhibit the replication of the dengue type II virus in vitro (see FIG. 1).

(29) 2. Lactate Dehydrogenase (LDH) Assay Testing the Cytotoxicity of the Composition of Example 1 in Vero Cells

(30) The LDH Cytotoxicity Detection Kit.sup.PLUS kit (purchased from Roche Diagnostics) was used for this test. The protocol is described in the instructions in the kit. The procedure is briefly described as follows: 100 μl Vero cells (obtained from the American Type Culture Collection (ATCC), CCL-81) was plated in 96-well cell culture plate at 4×10.sup.4/well and two replicate wells were included (i.e., duplicates for each experiment). The plates were incubated overnight under 5% CO.sub.2 at 37° C. The tested substance was dissolved in PBS buffer, sterilized by filtration, and diluted to 1.0, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg/ml, respectively. 10 μL tested substances of various concentrations (2.0, 1.0, 0.25, 0.125, 0.0625, 0.03125, 0.0156 mg/ml) were added to the cell culture plate, and 10 μL PBS was used as a blank control (two duplicate wells were included). The plates were incubated for 24 hours and 48 hours under 5% CO.sub.2 at 37° C. After incubation, the LDH concentration in the medium was measured by ELx808 Ultramicroplate Reader (Bio Tek) at 450 nm, and cytotoxicity was calculated according to the instructions in the kit.

(31) The results showed that no evident cytotoxicity was observed in Vero cells after incubation for 48 hours with 2 mg/ml tested substance (see FIG. 2). These results rule out the possibility that the inhibition of dengue virus by the tested substance is due to cytotoxicity, and further confirm that the tested substance can inhibit dengue virus infection in vitro.

Example 2

(32) The formulation of the Traditional Chinese Medicine composition of Example 2 was as follows:

(33) Herba Taraxaci: 680 g

(34) Radix Stemonae: 390 g

(35) Pseudobulbus Cremastrae Seu Pleiones: 400 g

(36) Radix Cynanchi Atrati: 480 g

(37) Radix Puerariae: 440 g

(38) Rhizoma Atractylodis Macrocephalae: 540 g

(39) The origins of the above herbal raw materials are the same as the origins described in Example 1. The process for preparing the Traditional Chinese Medicine composition of Example 2 was as follows:

(40) (1) 680 g Herba Taraxaci, 390 g Radix Stemonae and 480 g Radix Cynanchi Atrati were subjected to extraction with 10×60% ethanol for 1.5 h and the obtained mixture was filtered; and then 10×60% ethanol was added to extract for another 1.5 h, the obtained mixture was filtered; and the filtrates obtained in these two extraction processes were combined to give an alcohol extract;
(2) the residues obtained during the preparation of the alcohol extract were combined with 440 g Radix Puerariae, 400 g Pseudobulbus Cremastrae Seu Pleiones and 540 g Rhizoma Atractylodis Macrocephalae, the mixture thus obtained was subjected to extraction for 2 hours with 8× water; after filtration the remaining mixture was again subjected to extraction for 2 hours with 8× water and filtered, the filtrates obtained in these two extraction processes were combined and concentrated to obtain a thick paste; 90% ethanol was added to the thick paste and stirred to achieve a final concentration of about 75 ethanol % and the water extract was obtained by taking the supernatant after a 24 hours settling; and
(3) the alcohol extract obtained in step 1) and the water extract obtained in step 2) were combined, concentrated and spray dried to give the Traditional Chinese Medicine composition of Example 2. The resulting composition was a brown powder, the weight of which was about 0.5 kg, and it was used directly in the following in vivo and in vitro experiments.

(41) Then, the Traditional Chinese Medicine composition of Example 2 (referred to as “tested drug” or “tested substance”) was studied in various pharmacodynamics experiments to verify its efficacy.

(42) 1. In Vitro Pharmacodynamics Study Testing the Inhibition of Zika Virus (ZIKV) by the Composition of Example 2

(43) The origin and the handling of the Vero cells are the same as described in Example 1. Vero cells were plated in a 6-well plate at 6×10.sup.5/well and incubated under 5% CO.sub.2 at 37° C. for 24 hours and monolayer was formed. The Zika virus (provided by CDC Arbovirus Branch) at 1 MOI and 0.1 mg/ml or 1.0 mg/ml tested substance solution in water were combined and added to the Vero monolayer, and incubated at 4° C. for 1 hour. At this temperature, the virus is able to bind to the receptors on cell surface but cannot enter into cytoplasm. After the incubation, the cells were washed with culture medium at 4° C. to remove the unbound virus. The cells were collected and total RNA was extracted using Trizol, single-strand cDNA was synthesized. Quantitative analysis of coat protein gene and house-keeping gene β-actin was conducted by real-time qPCR, see Acharya D, Bastola P, Le L, et al. An Ultrasensitive Electrogenerated Chemiluminescence-based Immunoassay for Specific Detection of Zika Virus [J]. Scientific Reports, 2016, 6:32227 for details.

(44) The results showed that the tested substance at 0.1 mg/ml or 1.0 mg/ml can inhibit the binding of about 40% or about 80% Zika virus respectively to the receptors (see FIG. 3).

Example 3

(45) The formulation of the Traditional Chinese Medicine composition of Example 3 was as follows:

(46) Herba Taraxaci: 310 g

(47) Radix Stemonae: 200 g

(48) Pseudobulbus Cremastrae Seu Pleiones: 190 g

(49) Radix Cynanchi Atrati: 210 g

(50) Radix Puerariae: 210 g

(51) Rhizoma Atractylodis Macrocephalae: 250 g

(52) The origins of the above herbal raw materials are the same as the origins described in Example 1. The process for preparing the Traditional Chinese Medicine composition of Example 3 was as follows:

(53) (1) 310 g Herba Taraxaci, 200 g Radix Stemonae and 210 g Radix Cynanchi Atrati were subjected to extraction with 10×60% ethanol for 1.5 h and the obtained mixture was filtered; and then 10×60% ethanol was added to extract for another 1.5 h, the obtained mixture was filtered; and the filtrates obtained in these two extraction processes were combined to give an alcohol extract;
(2) the residues obtained during the preparation of the alcohol extract were combined with 210 g Radix Puerariae, 190 g Pseudobulbus Cremastrae Seu Pleiones and 250 g Rhizoma Atractylodis Macrocephalae, the mixture thus obtained was subjected to extraction for 2 hours with 8× water; after filtration the remaining mixture was again subjected to extraction for 2 hours with 8× water and filtered, the filtrates obtained in these two extraction processes were combined and concentrated to obtain a thick paste; 90% ethanol was added to the thick paste and stirred to achieve a final concentration of about 80 ethanol % and the water extract was obtained by taking the supernatant after a 24 hours settling; and
(3) the alcohol extract obtained in step 1) and the water extract obtained in step 2) were combined, concentrated and spray dried to give the Traditional Chinese Medicine composition of Example 3. The resulting composition was a brown powder, the weight of which was about 0.23 kg, and it was used directly in the following in vivo and in vitro experiments.

(54) In the following the Traditional Chinese Medicine composition of Example 3 (referred to as “tested drug” or “tested substance”) was studied in various pharmacodynamics experiments to verify its efficacy.

(55) 1. In Vitro Pharmacodynamics Study Testing the Inhibition of Influenza H7N7 Virus by the Composition of Example 3

(56) 1) Determination of Toxicity Concentration 50% (TCID.sub.50) of Virus on MDCK Cells by

(57) CPE Method 100 μL MDCK cells (canine kidney epithelial cells, provided by the Office of High-tech Research on Animal Biological Formulations, Institute of Animal Husbandry and Veterinary, Beijing Academy of Agriculture and Forestry) were seeded in the wells of a 96-well plate at 2×10.sup.4 cells/well, and incubated at 37° C. for 24 h, and monolayer was formed. An avian influenza virus H7N7 strain (provided by Institute of Animal Husbandry and Veterinary, Beijing Academy of Agriculture and Forestry) was diluted to from 10.sup.−3 to 10.sup.−12 by 10× serial dilution (totally 10 concentrations) and added to the wells (8 wells for each concentration) and incubated at 37° C. The cells were monitored daily using an inverted microscope for cytopathic effect (CPE). The level of cytopathic effect (CPE) was recorded. The toxicity concentration 50% (TCID.sub.50) of virus was calculated by Reed-Muench method.

(58) After being infected with virus, the cells appeared to be swollen, netting, shrinking, and aggregated. The TCID.sub.50 of avian influenza virus H7N7 was determined to be 10.sup.−4.19/0.1 ml.

(59) 2) Determination of the Inactivation of Avian Influenza Virus H7N7 by the Tested Substance

(60) 100 μL MDCK cells were seeded in the wells of a 96-well plate at 2×10.sup.4 cells/well, and incubated under 5% CO.sub.2 at 37° C. for 24 h, and monolayer was formed. The supernatant was discarded, solutions of the tested substance at various concentrations were mixed with an equal volume of 100TCID.sub.50 influenza virus in the test tube and allowed to react for 6 hours, and then the mixture was seeded to the plate. The experiment included normal cell control group, positive control drug group (amantadine hydrochloride, available from Sigma) and virus control group. After absorption for 2 h, the supernatant was discarded, and a cell maintenance medium was added, and incubation was continued at 37° C., under 5% CO.sub.2 in an incubator. The cytopathic effect was recorded and the cell viability was determined by MTT staining. The experiment was repeated 2 times. The data were subjected to Probit regression analysis using the statistical software SPSS13.0 to calculate inhibitory concentration 50% (IC.sub.50).

(61) The results are shown in Table 3-1, wherein inhibitory concentration 50% (IC.sub.50) of the tested drug was measured to be 410 μg/mL and 447 μg/mL, demonstrating that the tested substance is capable of inactivating avian influenza virus H7N7.

(62) TABLE-US-00001 TABLE 3-1 inhibitory activity of the tested substance on H7N7 virus (x ± SD) Concentration OD value Inhibition IC50 Group (μg/ml) (A) (/%) (μg/ml) Tested substance 1250 1.24 ± 0.15 1.18 ± 0.14 88 83 410 447 625 0.85 ± 0.11 0.82 ± 0.08 59 56 312.5 0.66 ± 0.16 0.62 ± 0.06 45 42 156.25 0.57 ± 0.04 0.49 ± 0.13 38 31 78.12 0.30 ± 0.06 0.23 ± 0.01 18 12 39.06 0.24 ± 0.03 0.20 ± 0.01 13 9 Adamantanamine 312.5 1.15 ± 0.09 1.09 ± 0.10 81 76 62 66 156.25 1.01 ± 0.07 0.98 ± 0.09 71 68 78.12 0.86 ± 0.08 0.81 ± 0.04 60 55 39.06 0.55 ± 0.04 0.57 ± 0.07 36 38

Example 4

(63) The formulation of the Traditional Chinese Medicine composition of Example 4 was as follows:

(64) Herba Taraxaci: 560 g

(65) Radix Stemonae: 320 g

(66) Pseudobulbus Cremastrae Seu Pleiones: 350 g

(67) Radix Cynanchi Atrati: 360 g

(68) Radix Puerariae: 380 g

(69) Rhizoma Atractylodis Macrocephalae: 460 g

(70) The origins of the above herbal raw materials are the same as the origins described in Example 1. The process for preparing the Traditional Chinese Medicine composition of Example 4 was as follows:

(71) (1) 560 g Herba Taraxaci, 320 g Radix Stemonae and 360 g Radix Cynanchi Atrati were subjected to extraction with 10×60% ethanol for 1.5 h and the obtained mixture was filtered; and then 10×60% ethanol was added to extract for another 1.5 h, the obtained mixture was filtered; and the filtrates obtained in these two extraction processes were combined to give an alcohol extract;
(2) the residues obtained during the preparation of the alcohol extract were combined with 380 g Radix Puerariae, 350 g Pseudobulbus Cremastrae Seu Pleiones and 460 g Rhizoma Atractylodis Macrocephalae, the mixture thus obtained was subjected to extraction for 2 hours with 8× water; after filtration the remaining mixture was again subjected to extraction for 2 hours with 8× water and filtered, the filtrates obtained in these two extraction processes were combined and concentrated to obtain a thick paste; 90% ethanol was added to the thick paste and stirred to achieve a final concentration of about 80 ethanol % and the water extract was obtained by taking the supernatant after a 24 hours settling; and
(3) the alcohol extract obtained in step 1) and the water extract obtained in step 2) were combined, concentrated and spray dried to give the Traditional Chinese Medicine composition of Example 4. The resulting composition was a brown powder, the weight of which was about 0.4 kg, and it was used directly in the following in vivo and in vitro experiments.

(72) In the following the Traditional Chinese Medicine composition of Example 4 (referred to as “tested drug” or “tested substance”) was studied in various pharmacodynamics experiments to verify its efficacy.

(73) 1. In Vitro Pharmacodynamics Study Testing the Inhibition of Chikungunya Virus (CHIKV) by the Composition of Example 4

(74) The origin and the handling of the Vero cells are the same as described in Example 1. Chikungunya virus was provided by the Connecticut Agricultural Test Station. Chikungunya virus (about 150 PFU) was incubated for 1 hour at room temperature with freshly prepared aqueous solutions of tested substance of 2.0, 1.0, 0.5, 0.25, and 0.125 mg/ml, respectively, after which the solutions were added to Vero monolayer and incubated at 37° C. for 1 hour and then the formation of plaques was evaluated. For details, see Acharya D, Paul A M, Anderson J F, et al. Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity. [J]. Plos Neglected Tropical Diseases, 2015, 9(10):e0004139.

(75) The results in the plaque neutralization test showed that the tested substance can suppress Chikungunya virus in a dose-dependent manner, and the EC.sub.50 value was 282.6 μg/ml (FIG. 4).

Example 5

(76) The formulation of the Traditional Chinese Medicine composition of Example 5 was as follows:

(77) Herba Taraxaci: 370 g

(78) Radix Stemonae: 220 g

(79) Pseudobulbus Cremastrae Seu Pleiones: 230 g

(80) Radix Cynanchi Atrati: 270 g

(81) Radix Puerariae: 270 g

(82) Rhizoma Atractylodis Macrocephalae: 280 g

(83) The origins of the above herbal raw materials are the same as the origins described in Example 1. The process for preparing the Traditional Chinese Medicine composition of Example 5 was as follows:

(84) (1) 370 g Herba Taraxaci, 220 g Radix Stemonae and 270 g Radix Cynanchi Atrati were subjected to extraction with 10×60% ethanol for 1.5 h and the obtained mixture was filtered; and then 10×60% ethanol was added to extract for another 1.5 h, the obtained mixture was filtered; and the filtrates obtained in these two extraction processes were combined to give an alcohol extract;
(2) the residue obtained during the preparation of the alcohol extract were combined with 270 g Radix Puerariae, 230 g Pseudobulbus Cremastrae Seu Pleiones and 280 g Rhizoma Atractylodis Macrocephalae, the mixture thus obtained was subjected to extraction for 2 hours with 8× water; after filtration the remaining mixture was again subjected to extraction for 2 hours with 8× water and filtered, the filtrates obtained in these two extraction processes were combined and concentrated to obtain a thick paste; 90% ethanol was added to the thick paste and stirred to achieve a final concentration of about 80 ethanol % and the water extract was obtained by taking the supernatant after a 24 hours settling; and
(3) the alcohol extract obtained in step 1) and the water extract obtained in step 2) were combined, concentrated and spray dried to give the Traditional Chinese Medicine composition of Example 5. The resulting composition was a brown powder, the weight of which was about 0.28 kg, it was used directly in the following in vivo and in vitro experiments.

(85) In the following the Traditional Chinese Medicine composition of Example 5 (referred to as “tested drug” or “tested substance”) was studied in various pharmacodynamics experiments to verify its efficacy.

(86) 1. In Vitro Pharmacodynamics Study Testing the Inhibition of Chikungunya Virus (CHIKV) by the Composition of Example 5

(87) The origin and the handling of the Vero cells are the same as described in Example 1. The origin of the chikungunya virus is the same as described in Example 4. Vero cells were plated in a 6-well plate at 6×10.sup.5/well and incubated under 5% CO.sub.2 at 37° C. for 24 hours and monolayer was formed. The chikungunya virus and 0.1 mg/ml or 1.0 mg/ml tested substance solution in water were combined and added to the monolayer, and incubated at 4° C. for 1 hour. At this temperature, the virus is able to bind to the receptors on cell surface but cannot enter into cytoplasm. After the incubation, the cells were washed with culture medium at 4° C. to remove the unbound virus. The cells were collected and total RNA were extracted using Trizol, single-strand cDNA was synthesized. Quantitative analysis of coat protein gene and house-keeping gene β-actin of chikungunya virus was conducted by real-time qPCR, see Acharya D, Paul A M, Anderson J F, et al. Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity. [J]. Plos Neglected Tropical Diseases, 2015, 9(10):e0004139 for details.

(88) The results show that the tested substance at 0.1 mg/ml or 1.0 mg/ml can inhibit some of the bindings of CHIKV virus to the receptors on Vero cells (see FIG. 5).

Example 6

(89) The formulation of the Traditional Chinese Medicine composition of Example 6 was as follows:

(90) Herba Taraxaci: 480 g

(91) Radix Stemonae: 290 g

(92) Pseudobulbus Cremastrae Seu Pleiones: 290 g

(93) Radix Cynanchi Atrati: 320 g

(94) Radix Puerariae: 320 g

(95) Rhizoma Atractylodis Macrocephalae: 385 g

(96) Cortex Mori: 320 g

(97) Cortex Lycii: 385 g

(98) Herba Taraxaci was collected from Hebei, China; Radix Cynanchi Atrati was collected from Henan, China; Radix Puerariae was collected from Hebei, China; Radix Stemonae was collected from Guangxi, China; Pseudobulbus Cremastrae Seu Pleiones was collected from Sichuan, China; Rhizoma Atractylodis Macrocephalae was collected from Zhejiang, China; Cortex Mori was collected from Shandong, China; and Cortex Lycii was collected from Ningxia autonomous region, China.

(99) The process for preparing the Traditional Chinese Medicine composition of Example 6 was as follows:

(100) (1) 480 g Herba Taraxaci, 290 g Radix Stemonae, 320 g Radix Cynanchi Atrati and 320 g Cortex Mori were subjected to extraction with 8×60% ethanol for 1.5 h and the obtained mixture was filtered; and then 8×60% ethanol was added to extract for another 1.5 h, the obtained mixture was filtered; and the filtrates obtained in these two extraction processes were combined to give an alcohol extract;
(2) the residue obtained during the preparation of the alcohol extract were combined with 320 g Radix Puerariae, 290 g Pseudobulbus Cremastrae Seu Pleiones 385 g Rhizoma Atractylodis Macrocephalae and 385 g Cortex Lycii, the mixture thus obtained was subjected to extraction for 2 hours with 10× water; after filtration the remaining mixture was again subjected to extraction for 2 hours with 10× water and filtered, the filtrates obtained in these two extraction processes were combined and concentrated to obtain a thick paste; 90% ethanol was added to the thick paste and stirred to achieve a final concentration of about 70 ethanol % and the water extract was obtained by taking the supernatant after a 36 hours settling; and
(3) the alcohol extract obtained in step 1) and the water extract obtained in step 2) were combined, concentrated and spray dried to give the Traditional Chinese Medicine composition of Example 6. The resulting composition was a brown powder, the weight of which was about 0.48 kg, it was used directly in the following in vivo and in vitro experiments.

(101) In the following the Traditional Chinese Medicine composition of Example 6 (referred to as “tested drug” or “tested substance”) was studied in various pharmacodynamics experiments to verify its efficacy.

(102) 1. In Vitro Pharmacodynamics Study Testing the Inhibition of Influenza H9N2 Virus by the Composition of Example 6

(103) 1.1 Determination of Toxicity Concentration 50% (TCID.sub.50) of Virus on MDCK Cells by CPE Method

(104) 100 μL MDCK cells (canine kidney epithelial cells, provided by Institute of Animal Husbandry and Veterinary, Beijing Academy of Agriculture and Forestry) were seeded in the wells of a 96-well plate at 2×10.sup.4 cells/well, and incubated at 37° C. for 24 h, and monolayer was formed. Avian influenza virus H9N2 (provided by Beijing Academy of Agriculture and Forestry) was diluted to from 10.sup.−3 to 10.sup.−12 by 10× serial dilution (totally 10 concentrations) and added to the wells (8 wells for each concentration) and incubated at 37° C. The cells were monitored daily using an inverted microscope for cytopathic effect (CPE). The infection dose 50% (TCID.sub.50) of virus was calculated by Reed-Muench method.

(105) After infected with virus, the cells appeared to be swollen, netting, shrinking, and aggregated. The TCID.sub.50 of avian influenza virus H9N2 was determined to be 10.sup.−3.5/0.1 ml.

(106) 1.2 Determination of Inhibitory Concentration 50% (TC.sub.50) of MDCK Cells by the Tested Substance Using MTT Method

(107) 100 μL MDCK cells were seeded in the wells of a 96-well plate at 2×10.sup.4 cells/well, and incubated under 5% CO.sub.2 at 37° C. for 24 h, and monolayer was formed. The supernatant was discarded, solutions of the tested substance at 10000, 5000, 2500, 1250, 625, 312.5, 156.25, 78.12, and 39.06 μg/mL in water were added (4 wells per each concentration) and a cell control group was included (cell+culture medium). Amantadine (available from Sigma) was used as the positive control drug and its concentration was 312.5, 156.25, 78.12, and 39.06 μg/mL (4 wells per each concentration). After 3 days of observation, MTT was added to stain the cells for 4 hours, the supernatant was discarded and DMSO was added to dissolve for 0.5 hours, and OD 570 nm value was determined by Microplate Reader (see Table 6-1). The data were subjected to Probit regression analysis using the statistical software SPSS13.0 to calculate inhibitory concentration 50% (IC.sub.50) for the drugs.

(108) $Survival rate (%) = \frac{Average OD value of the test group}{Average OD value of the cell control group} \times 100 %$

(109) Upon the Probit regression analysis of the data by SPSS13.0, inhibitory concentration 50% (TC.sub.50) of the tested drug on MDCK cells was determined to be 1596 μg/mL. The maximum non-toxic concentration of the tested drug on MDCK cells was determined to be 1250 μg/mL, at which the cell morphology was the same as the control cells. In contrast, the maximum non-toxic concentration of amantadine was 312.5 μg/mL.

(110) TABLE-US-00002 TABLE 6-1 toxicity of the tested substance on MDCK cells (x ± SD) Concentration OD value Survival rate Group (μg/ml) (A) (/%) tested substance 10000 0.24 ± 0.03 20 5000 0.139 ± 0.01 12 2500 0.197 ± 0.03 17 1250 0.89 ± 0.02 75 625 0.93 ± 0.11 79 312.5 0.94 ± 0.01 80 156.25 1.11 ± 0.14 95 78.12 1.18 ± 0.11 100 39.06 1.18 ± 0.14 100 amantadine 312.5 0.97 ± 0.03 82 156.25 0.96 ± 0.05 81 78.12 1.05 ± 0.02 89 39.06 1.20 ± 0.04 100 blank 0 1.18 ± 0.16 100
1.3 Determination of the Inactivation of Avian Influenza Virus H9N2 by the Tested Substance

(111) 100 μL MDCK cells were seeded in the wells of a 96-well plate at 2×10.sup.4 cells/well, and incubated under 5% CO.sub.2 at 37° C. for 24 h, and monolayer was formed. The supernatant was discarded, solutions of the tested substance at various concentrations were mixed with an equal volume of 100TCID.sub.50 influenza virus in the test tube and allowed to react for 6 hours, and then the mixture was seeded to the plate. The experiment included a normal cell control group, positive control drug group (amantadine, available from Sigma) and virus control group. After absorption for 2 h, the supernatant was discarded, and a cell maintenance medium was added, and incubation was continued at 37° C., under 5% CO.sub.2 in an incubator. The cytopathic effect was recorded and the cell viability was determined by MTT staining. The experiment was repeated 2 times. The data were subjected to Probit regression analysis using the statistical software SPSS13.0 to calculate inhibitory concentration 50% (IC.sub.50).

(112) The results are shown in Table 6-2, wherein the inhibitory concentration 50% (IC.sub.50) of the tested drug was measured to be 380.5 μg/mL, demonstrating that the tested substance is capable of inactivating avian influenza virus H9N2.

(113) TABLE-US-00003 TABLE 6-2 inhibitory activity of the tested substance of H9N2 virus (x ± SD) Concen- tration OD value Inhibition IC50 Group (μg/ml) (A) (/%) (μg/ml) tested 1250 0.75 ± 0.03 0.76 ± 0.03 77 76 385 376 substance 625 0.66 ± 0.02 0.69 ± 0.02 66 68 312.5 0.54 ± 0.02 0.58 ± 0.02 53 55 156.25 0.43 ± 0.04 0.43 ± 0.04 41 42 78.12 0.38 ± 0.03 0.42 ± 0.06 34 38 39.06 0.29 ± 0.02 0.32 ± 0.06 24 27 amantadine 312.5 0.83 ± 0.02 0.84 ± 0.16 86 83 63 66 156.25 0.71 ± 0.07 0.68 ± 0.15 72 66 78.12 0.64 ± 0.05 0.46 ± 0.03 65 42 39.06 0.37 ± 0.02 0.32 ± 0.03 34 27
2. In Vitro Pharmacodynamics Study Testing the Inhibition of Zika Virus (ZIKV) by the Composition of Example 6

(114) Vero cells (African green monkey kidney cells, obtained from the American Type Culture Collection (ATCC), CCL-81) were plated at 6×10.sup.5/well in a 6-well plate and incubated at 5% CO.sub.2 and 37° C. for 24 hours and monolayer was formed; the tested substance was dissolved in PBS buffer and filter sterilized, and diluted to 1.0, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg/ml, respectively. Zika virus (provided by CDC Arbovirus Branch) and the tested substance diluted with DMEM cell culture medium (1% L-glutamine, 1% penicillin/streptomycin and 10% FBS) to different concentrations were mixed and incubated for 1 hour at room temperature. Then, the mixture was added to wells containing Vero monolayer cells, and incubated at 37° C. and 5% CO.sub.2 for 1 hour to allow the virus to permeate into the cells or be taken up by the cells. After the virus were removed, the cells were plated on a 1% agarose gel and incubated at 37° C. and 5% CO.sub.2 for 4 days. After staining with 0.3% neutral red, the plate was taken out and the staining medium was removed; the plate was put back into the incubator for 1-2 h, and then the uncolored plaque on red background was clearly observed on a white backboard. The average number of plaques for each concentration was counted.

(115) This In vitro experiment showed that the tested substance is able to inhibit Zika virus effectively, and the half effective dose of the tested substance to inhibit the formation of Zika virus plaque was determined to be 193.1 μg/ml (see FIG. 6).

(116) 3. In Vivo Pharmacodynamics Study Testing Zika Virus (ZIKV) Infection of Female Mice by the Composition of Example 6

(117) To study the inhibitory effect of the tested substance on Zika virus infection in mice, C57BL/6 female mice (5 weeks old, obtained from The Jackson Laboratory (Bar Harbor, Me.)) were weighed on the first day of the experiment and each mouse was intraperitoneally injected with 2 mg IFNR1 antibody (MAR1-5A3). On day 2, each mouse was infected by intraperitoneally injecting 1×10.sup.4 PFU Zika virus (provided by the CDC Arbovirus Branch) and the mice were divided into three groups (10 per group): placebo group (Control), medium dose group (Medium) and the high dose group (High). 4 hour after infection, the placebo group was administered via oral gavage 100 μl sterile water, the medium dose group was administered via oral gavage 100 μl water solution of tested substance at a dose of 0.75 g/kg, the high dose group was administered via oral gavage 100 μl water solution of tested substance at a dose of 1.50 g/kg, and the administrations were conducted once per day for 6 days.

(118) On day 2 and day 4 after Zika virus infection, orbital bloods were collected from mice for RNA extraction, cDNA synthesis and mRNA qPCR analysis of Zika virus and mouse β-actin (the method is the same as in Example 2). The qPCR results showed that the tested substance significantly inhibited the replication of Zika virus in the bloods of mice at high dose on day 2 after infection (p<0.05), and showed a tendency of inhibition on day 4 after infection (see FIG. 7).

(119) The morbidity and mortality of the mice were observed daily. All the animals appeared normal expect for one animal in the placebo group, which died between day 3 an day 4. On day 6 after Zika virus infection, all mice were weighed and sacrificed. The spleen, brain and uterus of each mouse were taken, and total RNA in spleen, brain and uterus was extracted, followed by cDNA synthesis and mRNA qPCR analysis of Zika virus and mouse β-actin (the method is the same as described in Example 2). The results showed (see FIG. 8) that tested substance at high dose (1.5 g/kg) can significantly inhibit the infection of Zika virus in the spleen; and the infection of Zika virus in brain and uterus had the tendency to be inhibited although not significant (it is believed that this is likely caused by experimental outliers).

(120) To evaluate the potential toxic effects of the tested substance on mice, the body weights before and after the experimentation were compared and the results showed that the tested substance had no significant effect on body weight, indicating that the tested substance had no side effects on mice (see FIG. 9).

(121) 4. Study on the Antipyretic Effect of the Composition of Example 6 Via Oral Administration on Rats

(122) 4.1 Test Animals

(123) SD rats (Srrague Dawley Rats) were purchased from Chengdu Dashuo Experimental Animal Co., Ltd with a license number of SCXK (Chuan) 2013-24 and a certificate number of 0018409. 15 females and 15 male rats were used in the experiment. The body weights at the time of purchase were 150-170 g, the ages were 8-9 weeks, and the body weights during the experiment were 180-200 g.

(124) Inclusion Criteria:

(125) 1) Body weight: animals weighed in the range of 0.18-0.20 kg were selected.

(126) 2) The rectal temperatures of the animals were measured once per day at the same time point for three days before the start of the experiment, and animals having rectal temperature of 37.5-38.5° C. with changes no more than 0.5° C. were selected as the rats used for the model.
4.2 Test Design and Periods

(127) The acclimation period was 3 days, and the observation period after administration was 1 day; the day of administration was indicated by D1. See FIG. 10 for detailed schedule.

(128) 4.3 Experiment

(129) In this experiment, SD rats were injected subcutaneously with 20% dry yeast suspension (5 ml.Math.kg.sup.−1) as a heat source to establish fever model for drug efficacy evaluation. 30 healthy SD rats were selected and randomly divided into 6 groups: tested substance 4 g.Math.kg.sup.−1 group, tested substance 2 g.Math.kg.sup.−1 group, tested substance 1 g.Math.kg.sup.−1 group, positive control aspirin 100 mg.Math.kg.sup.−1 group, fever model group, and blank control group (5 animals per group). Aspirin was from BAYER corp. 0.5 h and 4.5 h after the injection of pyrogen, administration was done by gavage (totally 2 times). Rectal temperatures were measured 1 h before and 1, 2, 3, 4, 5, 6, 7, 8, 9 hours after the injection of pyrogen (measurement was done by holding the animal tight while it is awake and taking rectal temperature by inserting an OMRON electronic thermometer MC-347 coated with paraffin into the rectum about 4 cm), changes of rectal temperature were recorded and animals were observed for symptoms and signs.

(130) Data processing: the rectal temperature measured 1 h before the injection of pyrogen was used as the baseline value and paired T test was used to analyze changes of the rectal temperature before and after the administration in the animals in each group, and one-way analysis of variance was used to statistically analyze each drug group and the fever model group, wherein P<0.05 indicates statistical significant difference.

(131) 4.4 Results and Analysis

(132) During the experiment period, no drug related abnormalities were observed in animals in each group in terms of feces, behavior, activity, and body color.

(133) Rectal temperatures of the rats are shown in Table 6-3 and summarized below.

(134) Blank control group: body temperature at each time point fluctuated within the normal range.

(135) Fever model group (5 rats): compared to the baseline values, the body temperatures of the rats at 1-4 hours after injection of pyrogen maintained within the normal range, the rectal temperature started to show a significant tendency of increasing 5 hours after the infection (P<0.01), and significant increase was observed at 6, 7, 8, and 9 hours (P<0.01). Therefore, changes of rectal temperature at 5, 6, 7, 8 and 9 hours after injection of the pyrogen were analyzed as the efficacy endpoint.

(136) Aspirin 100 mg.Math.kg.sup.−1 group (5 rats, equivalent to 2-4× clinical dose, which is 0.3 g-0.6 g/human/time): compared to the baseline values, the rectal temperatures of the rats at 5-8 hours (4 time points) after injection of pyrogen maintained within the normal range, body temperatures started to show a significant tendency of increasing 9 hours after the infection (P<0.05); compared to the fever model group, rectal temperatures were significantly decreased at 5, 6, 7, and 8 hours after the injection of pyrogen (P<0.01), the rectal temperatures of the rats began to increase at 9 hours. The above results indicate that aspirin 100 mg.Math.kg.sup.−1 can maintain the effect of reducing the body temperature for 4 hours starting from 4.5 h after the injection of the pyrogen.

(137) Tested substance 4 g.Math.kg.sup.−1 group (5 rats): compared to the baseline values, the rectal temperatures of the rats at 5-9 hours (5 time points) after injection of pyrogen maintained within the normal range; compared to the fever model group, rectal temperatures were significantly decreased at 5, 6, 7, 8 and 9 hours after the injection of pyrogen (P<0.01). The above results indicate that the antipyretic effect reduced the body temperature rapidly at 4.5 h after the injection of the pyrogen and the effect was maintained for 5 hours. The antipyretic effect was comparable to that of the aspirin 100 mg.Math.kg.sup.−1 and lasted longer than aspirin 100 mg.Math.kg.sup.−1 group (by 1 hour).

(138) Tested substance 2 g.Math.kg.sup.−1 group (5 rats): compared to the baseline values, the body temperatures at 5 hours after the injection of pyrogen were the same as baseline, but increased significantly or very significantly at 6, 7, 8 and 9 hours (P<0.05 or P<0.01); compared to the fever model group, the body temperature has the tendency of decreasing at 5, 6, 8 and 9 hours after the injection of pyrogen, and significantly decreased at 7 hours (P<0.01).

(139) Tested substance 1 g.Math.kg.sup.−1 group: compared to the baseline value, the body temperatures at 5, 6, 7, 8, and 9 hours after the injection of pyrogen were significantly or very significantly increased (P<0.05 or P<0.01); compared to the fever model group, rectal temperatures of the animals were comparable at 5, 6, 7, 8, and 9 hours after the injection of pyrogen.

(140) TABLE-US-00004 TABLE 6-3 Effect of the tested substance on the rectal temperature in dry yeast induced fever in rats (° C.) time Baseline- After the injection of pyrogen group value.sup.a 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 9 h Blank control group 37.6 ± 37.4 ± 37.3 ± 37.9 ± 37.6 ± 37.4 ± 37.5 ± 37.4 ± 37.4 ± 37.7 ± (n = 5) 0.3 0.3 0.4 0.4 0.3 0.2 0.3 0.3 0.3 0.2 Fever model group 37.7 ± 37.6 ± 37.4 ± 37.7 ± 37.9 ± 38.4 ± 39.1 ± 39.5 ± 39.5 ± 39.2 ± (n = 5) 0.5 0.3 0.3 0.3 0.4 0.5** 0.5*** 0.3*** 0.3*** 0.3*** Aspirin 100 mg/kg 37.8 ± 38.0 ± 37.5 ± 37.7 ± 37.8 ± 38.1 ± 38.1 ± 38.3 ± 38.5 ± 38.8 ± group 0.4 0.3 0.2 0.3 0.2 0.6 0.4.sup.# 0.5.sup.## 0.5.sup.## 0.4* (n = 5) Tested substance 37.9 ± 37.9 ± 37.5 ± 37.7 ± 37.9 ± 37.4 ± 37.3 ± 38.0 ± 38.4 ± 38.1 ± 4 g/kg group 0.5 0.6 0.4 0.4 0.3 0.5.sup.## 1.3.sup.### 1.2.sup.### 0.9.sup.### 0.9.sup.## (n = 5) Tested substance 37.8 ± 37.4 ± 37.6 ± 37.8 ± 38.0 ± 38.0 ± 38.9 ± 38.9 ± 39.2 ± 39.1 ± 2 g/kg group 0.2 0.2 0.2 0.3 0.2 0.5 0.3*** 0.3***.sup.## 0.4*** 0.4*** (n = 5) Tested substance 37.9 ± 37.7 ± 37.7 ± 38.0 ± 38.1 ± 38.7 ± 38.8 ± 39.3 ± 39.5 ± 39.0 ± 1 g/kg group 0.5 0.3 0.2 0.4 0.3 0.3* 0.2* 0.3* 0.3** 0.5** (n = 5) Notes: 1. .sup.#p < 0.05, .sup.##p < 0.01, .sup.###p < 0.001 each drug group vs. fever model group. 2. *p < 0.05, **p < 0.01, ***p < 0.001 each drug group vs. baseline value. 3. .sup.aindicates the rectal temperature of the animal measured 1 h before the injection of pyrogen.
4.5 Conclusions

(141) Aspirin 100 mg.Math.kg.sup.−1 group (equivalent to 2-4× clinical dose, which is 0.3 g-0.6 g/human/time) significantly reduced the body temperature (5-8 hours after injection of pyrogen), and the effect was maintained for 4 hours.

(142) Tested substance 4 g.Math.kg.sup.−1 significantly reduced the body temperature (5-9 hours after the injection of the pyrogen), indicating that the antipyretic effect is comparable to that seen in aspirin 100 mg.Math.kg.sup.−1 group, and the therapeutic effect is fast and maintained for 5 hours, which outlasted aspirin 100 mg.Math.kg.sup.−1 by one hour. Tested substance 2 g.Math.kg.sup.−1 significantly reduced the body temperature, and the efficacy and duration of the therapeutic effect were reduced compared to aspirin 100 mg.Math.kg.sup.−1. In conclusion, the antipyretic effect of the tested substance is clearly dose-related and is considered to be related to administration.

(143) On one hand, the antipyretic effect of the Traditional Chinese Medicine composition of the present disclosure helps to enhance the resistibility of the patient to the virus, thereby further enhancing the antiviral efficacy of the Traditional Chinese Medicine composition of the present disclosure. On the other hand, since viral infection is usually accompanied by fever, the antipyretic effect of the Traditional Chinese Medicine composition of the present disclosure shows its efficacy not only in terms of antiviral ability but also in the significant amelioration of the clinical symptoms in patients infected by the virus.

(144) 5. Toxicity Test of the Composition of Example 6 in Healthy Rhesus Monkeys by Repeated Oral Administration for 30 Days

(145) 5.1 Test Animals

(146) Variety: rhesus monkey

(147) Level: Normal level, tested to be qualified before the experimentation, including physical examination, Mycobacterium tuberculosis, parasite, Salmonella, Shigella and B virus tests.

(148) Number and Sex: 3 male, 5 female

(149) Weight: 2.2-3.5 kg

(150) Identification: stainless steel number plate on the neck ring and cage card.

(151) Supplier: Ya'an Primed Biotechnology Co., Ltd., Sichuan, China

(152) License number: SCXK (Chuan) 2014-27

(153) Certificate number: 0016929

(154) 5.2 Test Method

(155) 5.2.1 Test Design and Periods

(156) The quarantine period was 39 days, and nasal feeding was for 30 days, the day of the first administration was indicated as D0. See FIG. 11 for test design.

(157) 5.2.2 Animal Grouping and Dose Design Grouping criteria: body weight.

(158) Grouping method: stratified randomization.

(159) Group design: tested substance group and placebo group, total two groups, 3 animals in the test group and 5 animals in the placebo group.

(160) Dose of the tested substance: 15× clinical equivalent dose for monkeys, which is also 18× pharmacodynamic equivalent dose (mouse 0.75 mg/kg) for monkeys.

(161) Based on the effective dose for mice, a dose equivalent to 15× human dose was used for the toxicity test, which was 3375 mg/kg (see the table below).

(162) TABLE-US-00005 TABLE 6-4 Group design Dosage Group Group code (mg/kg) Number of the animal Tested substance group A 3375 3 Placebo group C 0 5
5.2.3 Administration
Route of administration: nasal feeding for 30 days.
Frequency of administration: once per day for 30 days.
Calculation of the dose: the dose for a 10-day period was calculated according to the body weight measured during the previous 10-day period.
Time of administration: around 08:00-09:00 AM every day, delayed for 30 min if a blood sampling was scheduled.
5.2.4 Tests
5.2.4.1 Observation of Clinical Symptoms
Frequency: once per day.
Method: observing before the cage
Objects: skin, back hair, eyes, ear, nose, mouth cavity, chest, abdomen, genitourinary organs, limbs, etc., as well as breathing, exercise, urinary, defecation and behavioral changes.
5.2.4.2 Determination of Food Intake
Period: from quarantine period to the end of the administration.
Feeding schedule: fed once at 7:45˜8:30 AM and 2:00˜3:00 PM respectively; for the first week during the drug administration period, 150 g in the morning and 100 g in the afternoon; for the second week during the drug administration period, 200 g in the morning and 150 g in the afternoon; the remaining feed was removed at 7:40˜8:00 the next morning.
Dosage: 200 g-400 g/animal/day. Reason: the average daily feed intake for rhesus monkeys is about 200 g-400 g/animal/day. The feed during this test was gradually increased: for the first week, 150 g in the morning and 100 g in the afternoon; for the second week, 200 g in the morning and 150 g in the afternoon, ad libitum.
Method of measuring food intake: record the amount of food given, the amount of discarded food and the remaining amount of the food in the food container. Food intake=food given—discarded food—remaining food in the food container. The amount percentage of the feed was determined semi-quantitatively as 0%, 25%, 50%, 75% and 100%, and the daily food intake was calculated as the amount of the daily feed multiplied by the amount percentage.
5.2.4.3 Weight Measuring
Time: before feeding.
Frequency: 2 times during quarantine period (before grouping), and once every 10 days, a total of 5 times.
Method: animals were taken out by skilled workers while they were awake, and weighed on a large animal scale (TCS-150).
5.2.4.4 Biochemical Test of Blood
Frequency: 2 times before administration period, once every 10 days during the administration period, 5 times in total.
Sampling method: the animals were fasted overnight before blood collection, and the blood collection was done at 08:00-08:30 the next day with no anesthesia. 1.0 ml blood was taken through the forearm vein; after the collection, sterile dry cotton ball was used to gently press the blood collection site. Coagulation promoting tube was used for the collection, serum was separated at 5000 rpm for 10 min at 4° C. for use in the biochemical tests. Markers tested: total cholesterol (CHO), aspartate aminotransferase (AST), alanine aminotransferase (ALT), ALT/AST, glucose (GLU), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total protein (TP), albumin (ALB), globulin (GLO), ALB/GLO, triglyceride (TG), glutamyltransferase (y-GGT), high density lipoprotein (HDL-C), and low density lipoprotein (LDL-C) (see Table 6-5 for details). Detection method: all markers were detected by Roche Cobas C501.

(163) TABLE-US-00006 TABLE 6-5 Biochemical test of blood Marker tested unit Test method aspartate aminotransferase IU/L colorimetry (AST) alanine aminotransferase (ALT) IU/L IFCC rate assay total cholesterol (CHO) mmol/L Enzyme colorimetry, 1 point endpoint test fructosamine μmol/L rate assay glucose (GLU) mmol/L hexokinase method, 2 points endpoint test total bilirubin (TBIL) μmol/L Diazotization, 2 points endpoint test direct bilirubin (DBIL) μmol/L Diazotization, 2 points endpoint test creatine phosphokinase (CK) g/L Colorimetry, rate assay urea nitrogen (BUN) mmol/L Colorimetry, rate assay creatinine(CR-S) μmol/L rate assay uric acid (URIC) μmol/L Enzyme colorimetry alkaline phosphatase (ALP) IU/L Colorimetry, rate assay total protein(TP) g/L Colorimetry, 2 points endpoint test albumin (ALB) g/L Colorimetry, 2 points endpoint test Triglyceride (TG) mmol/L Enzyme colorimetry γ-glutamyltransferase (GGT) IU/L Enzyme colorimetry high density lipoprotein (HDL) mmol/L Enzyme colorimetry low density lipoprotein (LDL) mmol/L Enzyme colorimetry
5.2.4.5 Haematological Indexes Test
Frequency: 2 times before administration period, once every 10 days during the administration period, 5 times in total.
Sampling method: the animals were fasted overnight before blood collection, and the blood collection was done at 08:00-08:30 the next day with no anesthesia. 1.0 ml blood was taken through the forearm vein; after the collection, sterile dry cotton ball was used to gently press the blood collection site. Blood samples were treated with anticoagulant EDTAK2 and used for haematological indexes test.
Detection indexes: see Table 6-6. Detection methods: the indexes were tested using ADVIA 2120i.

(164) TABLE-US-00007 TABLE 6-6 Haematological indexes test Index Unit Method Red blood cell count (RBC) 10.sup.12/L two-dimensional laser White blood cell count (WBC) 10.sup.9/L two-dimensional laser Hemoglobin (Hb) g/L cyanomethemoglobin Hematocrit (HCT) % two-dimensional laser Mean corpuscular volume (MCV) fL two-dimensional laser Mean corpuscular hemoglobin (MCH) pg two-dimensional laser Mean corpuscular hemoglobin g/L two-dimensional laser concentration (MCHC) Cell hemoglobin concentration mean g/L two-dimensional laser (CHCM) Red blood cell volume distribution width % two-dimensional laser (RDW) Mean HGB distribution width g/L two-dimensional laser Mean RBC hemoglobin concentration g/L two-dimensional laser Mean RBC hemoglobin content Pg two-dimensional laser Red blood cell CH distribution width Pg two-dimensional laser Platelet total number (PLT) 10.sup.9/L two-dimensional laser Plateletcrit (PCT) % two-dimensional laser Mean platelet volume (MPV) fL two-dimensional laser Platelet volume distribution width (PDW) fL two-dimensional laser Neutrophil count (NEUT) 10.sup.9/L peroxidase staining + two-dimensional laser Neutrophil count percentage (NEUT %) % peroxidase staining + two-dimensional laser Lymphocyte count* (LYMPH) 10.sup.9/L peroxidase staining + two-dimensional laser Lymphocyte count percentage* (LYMPH) % peroxidase staining + two-dimensional laser Number of eosinophils (EOS) 10.sup.9/L peroxidase staining + two-dimensional laser Percentage of eosinophils (EOS %) % peroxidase staining + two-dimensional laser Number of basophils (BASO) 10.sup.9/L peroxidase staining + two-dimensional laser Percentage of basophils (BASO %) % peroxidase staining + two-dimensional laser Number of monocytes (MONO) 10.sup.9/L peroxidase staining + two-dimensional laser Percentage of monocytes (MONO %) % peroxidase staining + two-dimensional laser Large unstained cell ratio % peroxidase staining + two-dimensional laser
5.2.5 Data Processing

(165) The results were presented for each animal. Measurement data such as body weight are expressed as “Mean±SD”.

(166) 5.3 Results and Analysis

(167) 5.3.1 Influence on General Activity and Food Intake

(168) Animals in the placebo group did not show abnormalities after administrations. After the tested substance was administered, no apparent abnormalities were observed in test animals. No abnormalities were observed in terms of activities and water drinking, etc.; no abnormal secretions were seen in mouth, eyes and nose; no abnormalities were found in terms of hair colour and breathing. The animals were in good general condition.

(169) 5.3.2 Influence on Weight

(170) See the below table for the influence of tested substance on the weight of rhesus monkeys. After administered for 10 days, a transient weight reduction was observed due to stress response to the nasal administration (3/3), followed by weight increase throughout the administration period. No dosing-related body weight change was observed.

(171) TABLE-US-00008 TABLE 6-7 Influence of administrating tested substance for 30 days on body weight (kg, Mean ± SD) Animal Body weight (kg) Group No. fe/m baseline.sup.a D −6 D 10 D 20 D 30 GROUP A 6890 female 3.22 3.32 3.26 3.32 3.32 tested 6642 female 2.56 2.72 2.54 2.58 2.60 substance 6500 female 2.94 3.22 3.06 3.12 3.26 (3375 mg/kg) Mean ± SD 2.91 ± 0.33 3.09 ± 0.32 2.95 ± 0.37 3.01 ± 0.38 3.06 ± 0.40 n = 3 GROUP C 6043 male 2.78 3.06 3.08 3.16 3.08 placebo 6343 male 2.22 2.62 2.64 2.78 2.72 group 6347 male 3.02 3.32 3.22 3.32 3.28 n = 5 6872 female 2.90 3.08 3.08 3.22 3.12 6880 female 2.98 2.94 3.10 3.18 3.18 Mean ± SD 2.78 ± 0.33 3.00 ± 0.26 3.02 ± 0.22** 3.13 ± 0.21** 3.08 ± 0.21 Notes: 1. .sup.a34 days before administration (D −34)
5.3.3 Influence on Biochemical Indexes

(172) The influence of the tested substance on biochemical indexes in rhesus are shown in Table 6-8 to Table 6-25. There were no obvious abnormalities in the biochemical indexes of the animals in the placebo group. There were no dosing related abnormalities in the blood biochemical indexes of the animals in the test group.

(173) TABLE-US-00009 TABLE 6-8 Influence of administrating tested substance for 30 days on ALT (Mean ± SD) ALT (IU/L) Group Animal No. fe/m baseline.sup.a D 0 D 10 D 20 D 30 GROUP A 6890 female 39.2 26.4 33.8 34.2 24.7 tested 6642 female 20.0 24.6 24.6 25.7 23.0 substance 6500 female 40.0 25.8 24.8 27.0 22.8 (3375 mg/kg) Mean ± SD 33.1 ± 11.3 25.6 ± 0.9 27.7 ± 5.3 29.0 ± 4.6 23.5 ± 1.0 n = 3 GROUP C 6043 male 41.1 23.0 22.6 22.3 24.1 placebo 6343 male 44.5 57.1 60.4 48.1 106.6 group 6347 male 24.0 10.7 10.0 10.3 14.3 n = 5 6872 female 34.5 23.5 22.3 20.9 20.7 6880 female 270.9 60.2 37.1 58.7 71.8 Mean ± SD 83.0 ± 105.3 34.9 ± 22.3 30.5 ± 19.3 32.1 ± 20.4 47.5 ± 40.2 Notes: .sup.a34 days before administration (D −34);

(174) TABLE-US-00010 TABLE 6-9 Influence of administrating tested substance for 30 days on AST (Mean ± SD) AST (IU/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 47.9 28.2 27.1 27.2 20.2 tested 6642 female 38.6 33.9 35.1 33.5 31.3 substance 6500 female 39.3 24.7 25.5 26.9 22.5 (3375 mg/kg) Mean ± SD 41.9 ± 5.2 28.9 ± 4.6 29.2 ± 5.1 29.2 ± 3.7 24.7 ± 5.9 n = 3 GROUP C 6043 male 59.0 32.4 38.4 29.7 32.9 placebo 6343 male 41.6 30.8 36.9 34.4 44.4 group 6347 male 52.2 31.3 40.1 35.1 30.8 n = 5 6872 female 43.8 30.3 30.4 25.9 24.1 6880 female 105.4 41.4 36.9 35.4 37.5 Mean ± SD 60.4 ± 26.1 33.2 ± 4.6 36.5 ± 3.7 32.1 ± 4.2 33.9 ± 7.6 Notes: .sup.a34 days before administration (D-34);

(175) TABLE-US-00011 TABLE 6-10 Influence of administrating tested substance for 30 days on ALP (Mean ± SD) ALP (IU/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 519 508 430 463 477 tested 6642 female 585 724 530 506 525 substance 6500 female 420 594 506 512 418 (3375 mg/kg) Mean ± SD 508 ± 83 609 ± 109 489 ± 52 494 ± 27 473 ± 54 n = 3 GROUP C 6043 male 383 479 445 453 445 placebo 6343 male 437 478 512 456 473 group 6347 male 317 460 426 422 378 n = 5 6872 female 532 640 625 544 487 6880 female 573 528 713 652 732 Mean ± SD 448 ± 105 517 ± 73 544 ± 122 505 ± 94 503 ± 135 Notes: .sup.a34 days before administration (D-34);

(176) TABLE-US-00012 TABLE 6-11 Influence of administrating tested substance for 30 days on GGT (Mean ± SD) GGT (IU/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 104 113 92 99 94 tested 6642 female 96 146 91 87 98 substance 6500 female 58 74 63 69 62 (3375 mg/kg) n = 3 Mean ± SD 86 ± 25 111 ± 36 82 ± 16 85 ± 15 85 ± 20 GROUP C 6043 male 56 81 74 84 86 placebo 6343 male 83 123 119 131 136 group 6347 male 56 74 66 74 72 n = 5 6872 female 107 111 113 104 111 6880 female 106 92 109 118 118 Mean ± SD 82 ± 25 96 ± 20 96 ± 24 102 ± 23 105 ± 26 Notes: .sup.a34 days before administration (D-34);

(177) TABLE-US-00013 TABLE 6-12 Influence of administrating tested substance for 30 days on TBIL (Mean ± SD) TBIL (μmol/L)) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 4.2 3.6 3.0 3.8 2.7 tested 6642 female 4.0 2.8 4.0 2.6 4.7 substance 6500 female 4.7 1.8 4.0 6.5 1.8 (3375 mg/kg) n = 3 Mean ± SD 4.3 ± 0.4 2.7 ± 0.9 3.7 ± 0.6 4.3 ± 2.0 3.1 ± 1.5 GROUP C 6043 male 3.3 2.9 2.5 2.7 2.7 placebo 6343 male 2.9 2.7 2.1 3.7 2.2 group 6347 male 2.8 4.6 5.7 5.0 4.1 n = 5 6872 female 2.4 1.5 1.7 1.3 2.7 6880 female 5.1 5.8 3.8 3.9 5.3 Mean ± SD 3.3 ± 1.1 3.5 ± 1.7 3.2 ± 1.6 3.3 ± 1.4 3.4 ± 1.3 Notes: .sup.a34 days before administration (D-34);

(178) TABLE-US-00014 TABLE 6-13 Influence of administrating tested substance for 30 days on TP (Mean ± SD) TP (g/L)) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 80.1 73.7 72.5 73.2 74.1 tested 6642 female 75.3 85.8 78.6 74.4 80.9 substance 6500 female 73.1 73.2 73.1 72.2 73.8 (3375 mg/kg) n = 3 Mean ± SD 76.2 ± 3.6 77.6 ± 7.1 74.7 ± 3.4 73.3 ± 1.1 76.3 ± 4.0 GROUP C 6043 male 70.1 67.9 69.1 70.3 70.4 placebo 6343 male 77.5 70.6 68.7 65.3 69.6 group 6347 male 77.8 78.3 76.8 76.2 78.5 n = 5 6872 female 75.3 73.1 72.4 68.2 69.1 6880 female 67.0 68.9 72.3 67.6 71.2 Mean ± SD 73.5 ± 4.8 71.8 ± 4.2 71.9 ± 3.3 69.5 ± 4.1 71.8 ± 3.9 Notes: .sup.a34 days before administration (D-34);

(179) TABLE-US-00015 TABLE 6-14 Influence of administrating tested substance for 30 days on ALB (Mean ± SD) ALB (g/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 53.3 49.6 50.2 50.6 51.8 tested 6642 female 46.4 55.5 52.5 51.4 52.6 substance 6500 female 49.9 49.1 49.6 50.4 49.8 (3375 mg/kg) Mean ± SD 49.9 ± 3.5 51.4 ± 3.6 50.8 ± 1.5 50.8 ± 0.5 51.4 ± 1.4 n = 3 GROUP C 6043 male 50.8 48.2 50.4 51.5 51.6 placebo 6343 male 48.0 44.7 43.8 44.0 46.0 group 6347 male 49.2 49.6 50.9 52.1 51.6 n = 5 6872 female 45.7 44.0 45.6 42.9 43.4 6880 female 44.3 45.0 47.6 46.2 46.1 Mean ± SD 47.6 ± 2.6 46.3 ± 2.5 47.7 ± 3.0 47.3 ± 4.2 47.7 ± 3.7 Notes: a34 days before administration (D-34);

(180) TABLE-US-00016 TABLE 6-15 Influence of administrating tested substance for 30 days on BUN (Mean ± SD) BUN (μmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 6.9 5.3 6.4 5.5 5.4 tested 6642 female 6.4 3.6 4.3 4.1 3.2 substance 6500 female 8.8 5.3 6.2 6.3 6.3 (3375 mg/kg) n = 3 Mean ± SD 7.4 ± 1.3 4.7 ± 1.0 5.6 ± 1.2 5.3 ± 1.1 5.0 ± 1.6 GROUP C 6043 male 9.2 6.1 5.9 6.1 5.5 placebo 6343 male 15.3 6.0 7.3 5.8 5.8 group 6347 male 4.4 5.6 6.8 5.6 5.3 n = 5 6872 female 4.0 5.8 4.8 6.4 5.1 6880 female 6.3 6.7 4.1 4.6 4.3 Mean ± SD 7.8 ± 4.6 6.0 ± 0.4 5.8 ± 1.3 5.7 ± 0.7 5.2 ± 0.6 Notes: .sup.a34 days before administration (D-34);

(181) TABLE-US-00017 TABLE 6-16 Influence of administrating tested substance for 30 days on CR-S (Mean ± SD) CR-S (μmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 46 49 60 57 52 tested 6642 female 48 48 61 57 51 substance 6500 female 46 46 56 63 52 (3375 mg/kg) n = 3 Mean ± SD 47 ± 1 48 ± 2 59 ± 3 59 ± 3 52 ± 1 GROUP C placebo group 6043 male 48 44 48 58 51 n = 5 6343 male 67 53 50 49 51 6347 male 40 64 65 62 58 6872 female 33 35 44 42 42 6880 female 45 45 56 47 54 Mean ± SD 47 ± 13 48 ± 11 53 ± 8 52 ± 8 51 ± 6 Notes: 1. .sup.a34 days before administration (D-34);

(182) TABLE-US-00018 TABLE 6-17 Influence of administrating tested substance for 30 days on CK (Mean ± SD) CK (μmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 118 95 86 89 76 tested 6642 female 159 215 144 166 203 substance 6500 female 129 104 82 113 79 (3375 mg/kg) Mean ± SD 135 ± 21 138 ± 67 104 ± 35 123 ± 39 119 ± 72 n = 3 GROUP C 6043 male 194 118 137 111 140 placebo 6343 male 288 142 153 182 172 group 6347 male 185 89 76 104 103 n = 5 6872 female 133 106 229 116 103 6880 female 386 138 146 160 165 Mean ± SD 237 ± 100 119 ± 22 148 ± 55 135 ± 34 137 ± 33 Notes: 1. .sup.a34 days before administration (D-34);

(183) TABLE-US-00019 TABLE 6-18 Influence of administrating tested substance for 30 days on URIC (Mean ± SD) URIC (μmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 1 2 5 2 3 tested 6642 female 6 2 5 2 2 substance 6500 female 3 2 4 4 3 (3375 mg/kg) Mean ± SD 3 ± 3 2 ± 0 5 ± 1 3 ± 1 3 ± 1 n = 3 GROUP C 6043 male 2 3 5 4 3 placebo 6343 male 5 5 4 3 2 group 6347 male 3 4 6 5 3 n = 5 6872 female 1 1 3 3 2 6880 female 3 2 4 3 3 Mean ± SD 3 ± 1 3 ± 2 4 ± 1 4 ± 1 3 ± 1 Notes: .sup.a34 days before administration (D-34);

(184) TABLE-US-00020 TABLE 6-19 Influence of administrating tested substance for 30 days on DBIL (Mean ± SD) DBIL (μmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 1.5 1.2 0.3 1.3 1.4 tested 6642 female 1.4 0.8 1.1 1.0 2.0 substance 6500 female 1.6 0.9 1.3 2.7 1.6 (3375 mg/kg) n = 3 Mean ± SD 1.5 ± 0.1 1.0 ± 0.2 0.9 ± 0.5 1.7 ± 0.9 1.7 ± 0.3 GROUP C 6043 male 1.2 0.9 0.2 0.5 1.1 placebo 6343 male 1.1 0.8 0.4 1.0 1.1 group 6347 male 1.2 1.7 0.9 0.9 1.7 n = 5 6872 female 0.8 0.7 0.8 0.5 0.9 6880 female 1.7 1.8 1.1 1.4 1.6 Mean ± SD 1.2 ± 0.3 1.2 ± 0.5 0.7 ± 0.4 0.9 ± 0.4 1.3 ± 0.3 Notes: 1. .sup.a34 days before administration (D-34);

(185) TABLE-US-00021 TABLE 6-20 Influence of administrating tested substance for 30 days on FPG (Mean ± SD) FPG (mmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 4.82 4.56 4.11 5.05 4.48 tested 6642 female 3.32 5.30 5.43 6.00 5.18 substance 6500 female 4.56 4.52 3.75 6.14 4.75 (3375 mg/kg) Mean ± SD 4.23 ± 0.80 4.79 ± 0.44 4.43 ± 0.88 5.73 ± 0.59 4.80 ± 0.35 n = 3 GROUP C 6043 male 3.81 4.06 3.50 4.38 4.32 placebo 6343 male 5.59 4.59 3.98 3.87 3.92 group 6347 male 5.09 3.85 3.73 3.86 4.28 n = 5 6872 female 4.64 4.14 4.04 4.78 4.15 6880 female 4.54 2.74 4.57 4.16 4.57 Mean ± SD 4.73 ± 0.66 3.88 ± 0.69 3.96 ± 0.40 4.21 ± 0.39 4.25 ± 0.24 Notes: .sup.a34 days before administration (D-34);

(186) TABLE-US-00022 TABLE 6-21 Influence of administrating tested substance for 30 days on FRA (Mean ± SD) FRA (μmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 218 196 178 199 190 tested 6642 female 219 236 207 219 220 substance 6500 female 207 207 188 224 196 (3375 mg/kg) n = 3 Mean ± SD 215 ± 7 213 ± 21 191 ± 15 214 ± 13 202 ± 16 GROUP C 6043 male 203 202 193 202 191 placebo 6343 male 205 204 199 194 191 group 6347 male 232 231 205 257 210 n = 5 6872 female 199 189 166 187 183 6880 female 183 191 196 182 198 Mean ± SD 204 ± 18 203 ± 17 192 ± 15 204 ± 30 195 ± 10 Notes: .sup.a34 days before administration (D-34);

(187) TABLE-US-00023 TABLE 6-22 Influence of administrating tested substance for 30 days on TC (Mean ± SD) TC (mmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 3.06 3.34 2.83 2.73 2.54 tested 6642 female 2.68 4.24 3.26 3.17 3.84 substance 6500 female 1.98 2.29 2.31 2.72 2.36 (3375 mg/kg) Mean ± SD 2.57 ± 0.55 3.29 ± 0.98 2.80 ± 0.48 2.87 ± 0.26 2.91 ± 0.81 n = 3 GROUP C 6043 male 1.57 2.24 2.24 2.46 2.52 placebo 6343 male 3.89 3.70 3.26 3.39 3.13 group 6347 male 2.65 3.25 3.40 3.39 3.37 n = 5 6872 female 2.44 2.47 2.81 2.63 2.92 6880 female 2.37 2.85 3.38 3.09 3.29 Mean ± SD 2.58 ± 0.84 2.90 ± 0.59 3.02 ± 0.50 2.99 ± 0.43 3.05 ± 0.34 Notes: .sup.a34 days before administration (D-34);

(188) TABLE-US-00024 TABLE 6-23 Influence of administrating tested substance for 30 days on LDL-c (Mean ± SD) LDL-c (mmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 1.60 1.76 1.41 1.42 1.20 tested 6642 female 1.29 1.92 1.25 1.30 1.60 substance 6500 female 1.02 0.96 0.96 1.21 0.75 (3375 mg/kg) Mean ± SD 1.30 ± 0.29 1.55 ± 0.51 1.21 ± 0.23 1.31 ± 0.11 1.18 ± 0.43 n = 3 GROUP C 6043 male 0.57 0.96 0.97 1.14 1.14 placebo 6343 male 2.10 1.78 1.60 1.73 1.27 group 6347 male 1.49 1.67 1.71 1.81 1.75 n = 5 6872 female 1.32 1.22 1.47 1.41 1.45 6880 female 1.48 1.65 1.99 1.85 1.99 Mean ± SD 1.39 ± 0.55 1.46 ± 0.35 1.55 ± 0.38 1.59 ± 0.30 1.52 ± 0.35 Notes: .sup.a34 days before administration (D-34);

(189) TABLE-US-00025 TABLE 6-24 Influence of administrating tested substance for 30 days on HDL-c (Mean ± SD) HDL-c (mmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 1.51 1.72 1.51 1.42 1.51 tested 6642 female 1.44 2.69 2.15 2.03 2.40 substance 6500 female 0.93 1.46 1.39 1.78 1.74 (3375 mg/kg) Mean ± SD 1.29 ± 0.32 1.96 ± 0.65 1.68 ± 0.41 1.74 ± 0.31 1.88 ± 0.46 n = 3 GROUP C 6043 male 1.06 1.26 1.24 1.38 1.57 placebo 6343 male 1.87 2.15 1.81 1.94 2.08 group 6347 male 1.38 1.83 1.84 1.81 1.91 n = 5 6872 female 1.23 1.37 1.46 1.46 1.65 6880 female 0.96 1.35 1.56 1.44 1.50 Mean ± SD 1.30 ± 0.36 1.59 ± 0.38 1.58 ± 0.25 1.61 ± 0.25 1.74 ± 0.24 Notes: .sup.a34 days before administration (D-34);

(190) TABLE-US-00026 TABLE 6-25 Influence of administrating tested substance for 30 days on TG (Mean ± SD) TG (mmol/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.57 0.42 0.58 0.63 0.62 tested 6642 female 0.60 0.44 0.45 0.49 0.48 substance 6500 female 0.67 0.68 0.80 0.27 0.65 (3375 mg/kg) Mean ± SD 0.61 ± 0.05 0.51 ± 0.14 0.61 ± 0.18 0.46 ± 0.18 0.58 ± 0.09 n = 3 GROUP C 6043 male 0.44 0.97 0.96 0.57 0.72 placebo 6343 male 0.56 0.50 0.52 0.39 0.36 group 6347 male 0.45 0.70 0.60 0.50 0.37 n = 5 6872 female 0.48 0.52 0.48 0.55 0.70 6880 female 0.75 0.79 0.40 0.43 0.42 Mean ± SD 0.54 ± 0.13 0.70 ± 0.20 0.59 ± 0.22 0.49 ± 0.08 0.51 ± 0.18 Notes: .sup.a34 days before administration (D-34);
5.3.4 Influence on Haematological Indexes

(191) Influence of the tested substance on haematological in rhesus are shown in Table 6-26 to Table 6-53. There were no obvious abnormalities in the haematological indexes of the animals in the placebo group and the animals in the test group.

(192) TABLE-US-00027 TABLE 6-26 Influence of administrating tested substance for 30 days on white cell (Mean ± SD) White cell WBC (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 9.49 8.85 5.47 8.17 7.60 tested 6642 female 11.80 11.97 6.64 11.24 6.90 substance 6500 female 6.58 6.06 4.85 5.06 6.20 (3375 mg/kg) Mean ± SD 9.29 ± 2.62 8.96 ± 2.96 5.65 ± 0.91 8.16 ± 3.09 6.90 ± 0.70 n = 3 GROUP C 6043 male 9.62 6.45 6.06 6.75 5.78 placebo 6343 male 13.14 11.13 13.01 10.91 9.69 group 6347 male 7.57 7.00 5.15 6.32 4.41 n = 5 6872 female 7.16 9.58 8.02 6.09 6.51 6880 female 10.55 13.50 8.38 9.79 10.29 Mean ± SD 9.61 ± 2.42 9.53 ± 2.92 8.12 ± 3.04 7.97 ± 2.22 7.34 ± 2.55 Notes: .sup.a34 days before administration (D-34);

(193) TABLE-US-00028 TABLE 6-27 Influence of administrating tested substance for 30 days on granulocytes, absolute (Mean ± SD) granulocytes, absolute GRAN (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 4.64 3.93 2.08 3.14 3.40 tested 6642 female 5.69 5.68 2.45 5.05 2.17 substance 6500 female 2.77 2.01 1.60 1.83 2.84 (3375 mg/kg) Mean ± SD 4.37 ± 1.48 3.87 ± 1.84 2.04 ± 0.43 3.34 ± 1.62 2.80 ± 0.62 n = 3 GROUP C 6043 male 5.53 3.11 2.16 2.51 2.28 placebo 6343 male 4.37 1.91 4.96 2.20 1.11 group 6347 male 2.97 1.53 1.25 1.29 0.72 n = 5 6872 female 3.78 4.55 4.79 2.43 2.62 6880 female 4.45 5.67 1.61 2.36 3.67 Mean ± SD 4.22 ± 0.94 3.35 ± 1.75 2.95 ± 1.78 2.16 ± 0.50 2.08 ± 1.19 Notes: .sup.a34 days before administration (D-34);

(194) TABLE-US-00029 TABLE 6-28 Influence of administrating tested substance for 30 days on lymphocytes, absolute (Mean ± SD) lymphocytes, absolute LYMF (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 4.43 4.36 3.17 4.56 3.78 tested 6642 female 5.27 5.69 3.92 5.73 4.36 substance 6500 female 3.48 3.75 3.01 3.02 2.96 (3375 mg/kg) Mean ± SD 4.39 ± 0.90 4.60 ± 0.99 3.37 ± 0.49 4.44 ± 1.36 3.70 ± 0.70 n = 3 GROUP C 6043 male 3.45 2.92 3.38 3.74 3.12 placebo 6343 male 7.86 8.38 7.60 7.75 8.07 group 6347 male 4.14 4.92 3.52 4.63 3.39 n = 5 6872 female 2.98 4.42 2.93 3.17 3.45 6880 female 5.43 6.73 6.02 6.62 5.77 Mean ± SD 4.77 ± 1.96 5.47 ± 2.12 4.69 ± 2.03 5.18 ± 1.94 4.76 ± 2.14 Notes: 1. .sup.a34 days before administration (D-34);

(195) TABLE-US-00030 TABLE 6-29 Influence of administrating tested substance for 30 days on number of monocytes (Mean ± SD) number of monocytes (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.20 0.28 0.06 0.22 0.17 tested 6642 female 0.33 0.30 0.09 0.22 0.16 substance 6500 female 0.12 0.09 0.04 0.05 0.08 (3375 mg/kg) Mean ± SD 0.22 ± 0.11 0.22 ± 0.12 0.06 ± 0.03 0.16 ± 0.10 0.14 ± 0.05 n = 3 GROUP C 6043 male 0.29 0.12 0.10 0.14 0.11 placebo 6343 male 0.53 0.44 0.22 0.43 0.19 group 6347 male 0.25 0.33 0.19 0.24 0.13 n = 5 6872 female 0.21 0.17 0.13 0.21 0.19 6880 female 0.24 0.38 0.18 0.29 0.22 Mean ± SD 0.30 ± 0.13 0.29 ± 0.14 0.16 ± 0.05* 0.26 ± 0.11 0.17 ± 0.05 Notes: 1. .sup.a34 days before administration (D-34);

(196) TABLE-US-00031 TABLE 6-30 Influence of administrating tested substance for 30 days on number of eosinophils (Mean ± SD) number of eosinophils (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.12 0.21 0.10 0.18 0.19 tested 6642 female 0.30 0.16 0.11 0.11 0.11 substance 6500 female 0.14 0.16 0.17 0.12 0.28 (3375 mg/kg) Mean ± SD 0.19 ± 0.10 0.18 ± 0.03 0.13 ± 0.04 0.14 ± 0.04 0.19 ± 0.09 n = 3 GROUP C 6043 male 0.29 0.26 0.36 0.30 0.23 placebo 6343 male 0.08 0.26 0.16 0.41 0.21 group 6347 male 0.08 0.08 0.04 0.05 0.09 n = 5 6872 female 0.15 0.38 0.12 0.22 0.22 6880 female 0.35 0.59 0.46 0.37 0.50 Mean ± SD 0.19 ± 0.12 0.31 ± 0.19 0.23 ± 0.18 0.27 ± 0.14 0.25 ± 0.15 Notes: 1. .sup.a34 days before administration (D-34);

(197) TABLE-US-00032 TABLE 6-31 Influence of administrating tested substance for 30 days on number of basophils (Mean ± SD) number of basophils (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.02 0.01 0.00 0.01 0.01 tested 6642 female 0.03 0.02 0.00 0.01 0.01 substance 6500 female 0.01 0.01 0.00 0.00 0.00 (3375 mg/kg) Mean ± SD 0.02 ± 0.01 0.01 ± 0.01 0.00 ± 0.00 0.01 ± 0.01 0.01 ± 0.01 n = 3 GROUP C 6043 male 0.01 0.01 0.00 0.01 0.00 placebo 6343 male 0.03 0.03 0.01 0.01 0.02 group 6347 male 0.01 0.02 0.01 0.01 0.00 n = 5 6872 female 0.00 0.01 0.01 0.00 0.00 6880 female 0.00 0.01 0.01 0.02 0.02 Mean ± SD 0.01 ± 0.01 0.02 ± 0.01 0.01 ± 0.00 0.01 ± 0.01 0.01 ± 0.01 Notes: 1. .sup.a34 days before administration (D-34);

(198) TABLE-US-00033 TABLE 6-32 Influence of administrating tested substance for 30 days on number of large unstained cells (Mean ± SD) number of large unstained cells LUC (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.08 0.07 0.05 0.06 0.06 tested 6642 female 0.17 0.12 0.07 0.13 0.09 substance 6500 female 0.07 0.04 0.03 0.03 0.03 (3375 mg/kg) Mean ± SD 0.11 ± 0.06 0.08 ± 0.04 0.05 ± 0.02 0.07 ± 0.05 0.06 ± 0.03 n = 3 GROUP C 6043 male 0.05 0.03 0.05 0.05 0.04 placebo 6343 male 0.27 0.10 0.06 0.12 0.09 group 6347 male 0.12 0.11 0.14 0.09 0.07 n = 5 6872 female 0.03 0.04 0.03 0.04 0.03 6880 female 0.07 0.11 0.09 0.13 0.12 Mean ± SD 0.11 ± 0.10 0.08 ± 0.04 0.07 ± 0.04 0.09 ± 0.04 0.07 ± 0.04 Notes: 1. .sup.a34 days before administration (D-34);

(199) TABLE-US-00034 TABLE 6-33 Influence of administrating tested substance for 30 days on neutrophil percentage (Mean ± SD) neutrophil percentage NEUT (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 48.9 44.4 38.0 38.5 44.7 tested 6642 female 48.2 47.4 37.0 44.9 31.4 substance 6500 female 42.1 33.2 33.0 36.2 45.9 (3375 mg/kg) Mean ± SD 46.4 ± 3.7 41.7 ± 7.5 36.0 ± 2.6 39.9 ± 4.5 40.7 ± 8.0 n = 3 GROUP C 6043 male 57.5 48.3 35.7 37.2 39.5 placebo 6343 male 33.3 17.2 38.1 20.1 11.4 group 6347 male 39.3 21.9 24.3 20.4 16.4 n = 5 6872 female 52.8 47.5 59.7 39.9 40.2 6880 female 42.2 42.0 19.2 24.1 35.7 Mean ± SD 45.0 ± 9.9 35.4 ± 14.7 35.4 ± 15.7 28.3 ± 9.5 28.6 ± 13.7 Notes: 1. .sup.a34 days before administration (D-34);

(200) TABLE-US-00035 TABLE 6-34 Influence of administrating tested substance for 30 days on lymphocyte percentage (Mean ± SD) lymphocyte percentage LYMPH (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 46.7 49.2 58.1 55.8 49.7 tested 6642 female 44.7 47.6 59.0 51.0 63.1 substance 6500 female 52.8 61.9 62.1 59.7 47.8 (3375 mg/kg) Mean ± SD 48.1 ± 4.2 52.9 ± 7.8 59.7 ± 2.1 55.5 ± 4.4 53.5 ± 8.3 n = 3 GROUP C 6043 male 35.8 45.3 55.8 55.3 54.0 placebo 6343 male 59.8 75.3 58.4 71.0 83.2 group 6347 male 54.8 70.4 68.4 73.2 76.8 n = 5 6872 female 41.6 46.2 36.6 52.2 53.0 6880 female 51.5 49.9 71.9 67.6 56.1 Mean ± SD 48.7 ± 9.8 57.4 ± 14.3 58.2 ± 13.8 63.9 ± 9.5 64.6 ± 14.3 Notes: 1. .sup.a34 days before administration (D-34);

(201) TABLE-US-00036 TABLE 6-35 Influence of administrating tested substance for 30 days on monocyte percentage (Mean ± SD) monocyte percentage MONO (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 2.1 3.1 1.2 2.6 2.2 tested 6642 female 2.8 2.5 1.3 1.9 2.3 substance 6500 female 1.8 1.5 0.8 1.1 1.3 (3375 mg/kg) Mean ± SD 2.2 ± 0.5 2.4 ± 0.8 1.1 ± 0.3 1.9 ± 0.8 1.9 ± 0.6 n = 3 GROUP C 6043 male 3.0 1.9 1.7 2.1 1.8 placebo 6343 male 4.0 4.0 1.7 3.9 2.0 group 6347 male 3.3 4.8 3.7 3.8 3.0 n = 5 6872 female 2.9 1.7 1.7 3.5 2.9 6880 female 2.2 2.8 2.2 3.0 2.1 Mean ± SD 3.1 ± 0.7 3.0 ± 1.3 2.2 ± 0.9 3.3 ± 0.7 2.4 ± 0.6 Notes: 1. .sup.a34 days before administration (D-34);

(202) TABLE-US-00037 TABLE 6-36 Influence of administrating tested substance for 30 days on eosinophils percentage (Mean ± SD) eosinophils percentage EOS (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 1.3 2.4 1.9 2.2 2.4 tested 6642 female 2.5 1.3 1.6 1.0 1.6 substance 6500 female 2.1 2.6 3.5 2.3 4.5 (3375 mg/kg) Mean ± SD 2.0 ± 0.6 2.1 ± 0.7 2.3 ± 1.0 1.8 ± 0.7 2.8 ± 1.5 n = 3 GROUP C 6043 male 3.0 4.0 5.9 4.4 4.0 placebo 6343 male 0.6 2.3 1.2 3.8 2.2 group 6347 male 1.0 1.1 0.8 0.9 2.0 n = 5 6872 female 2.1 4.0 1.5 3.7 3.4 6880 female 3.3 4.4 5.4 3.8 4.9 Mean ± SD 2.0 ± 1.2 3.2 ± 1.4 3.0 ± 2.5 3.3 ± 1.4 3.3 ± 1.2 Notes: 1. .sup.a34 days before administration (D-34);

(203) TABLE-US-00038 TABLE 6-37 Influence of administrating tested substance for 30 days on basophils percentage (Mean ± SD) basophils percentage BASO (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.2 0.1 0.0 0.1 0.1 tested 6642 female 0.3 0.2 0.1 0.1 0.2 substance 6500 female 0.1 0.1 0.0 0.1 0.0 (3375 mg/kg) Mean ± SD 0.2 ± 0.1 0.1 ± 0.1 0.0 ± 0.1 0.1 ± 0.0 0.1 ± 0.1 n = 3 GROUP C 6043 male 0.1 0.1 0.0 0.2 0.1 placebo 6343 male 0.2 0.3 0.1 0.1 0.2 group 6347 male 0.1 0.3 0.3 0.2 0.1 n = 5 6872 female 0.0 0.1 0.1 0.1 0.0 6880 female 0.0 0.1 0.1 0.2 0.2 Mean ± SD 0.1 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 Notes: 1. .sup.a34 days before administration (D-34);

(204) TABLE-US-00039 TABLE 6-38 Influence of administrating tested substance for 30 days on large unstained cell percentage (Mean ± SD) large unstained cell percentage LUC (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.9 0.8 0.8 0.8 0.8 tested 6642 female 1.5 1.0 1.0 1.2 1.3 substance 6500 female 1.1 0.7 0.7 0.6 0.4 (3375 mg/kg) Mean ± SD 1.2 ± 0.3 0.8 ± 0.2 0.8 ± 0.2 0.9 ± 0.3 0.8 ± 0.5 n = 3 GROUP C 6043 male 0.6 0.4 0.9 0.7 0.7 placebo 6343 male 2.0 0.9 0.5 1.1 0.9 group 6347 male 1.6 1.6 2.7 1.4 1.7 n = 5 6872 female 0.5 0.5 0.4 0.7 0.5 6880 female 0.7 0.8 1.1 1.3 1.2 Mean ± SD 1.1 ± 0.7 0.8 ± 0.5 1.1 ± 0.9 1.0 ± 0.3 1.0 ± 0.5 Notes: 1. .sup.a34 days before administration (D-34);

(205) TABLE-US-00040 TABLE 6-39 Influence of administrating tested substance for 30 days on red cells (Mean ± SD) Red cells RBC (10.sup.12/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 5.54 5.42 5.67 5.31 5.25 tested 6642 female 5.12 5.92 5.01 5.00 4.64 substance 6500 female 5.49 5.33 4.98 5.21 4.98 (3375 mg/kg) Mean ± SD 5.38 ± 0.23 5.56 ± 0.32 5.22 ± 0.39 5.17 ± 0.16 4.96 ± 0.31 n = 3 GROUP C 6043 male 4.50 5.40 5.43 5.62 5.40 placebo 6343 male 5.16 4.84 5.06 4.93 5.18 group 6347 male 5.43 6.06 5.62 5.62 5.61 n = 5 6872 female 5.16 5.41 5.35 5.16 5.18 6880 female 5.67 5.58 5.75 5.81 5.93 Mean ± SD 5.18 ± 0.44 5.46 ± 0.44 5.44 ± 0.27 5.43 ± 0.37 5.46 ± 0.32 Notes: 1. .sup.a34 days before administration (D-34);

(206) TABLE-US-00041 TABLE 6-40 Influence of administrating tested substance for 30 days on hemoglobin concentration (Mean ± SD) Hemoglobin concentration HGB (g/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 138 134 143 134 131 tested 6642 female 137 159 138 137 128 substance 6500 female 129 126 121 128 124 (3375 mg/kg) Mean ± SD 135 ± 5 140 ± 17 134 ± 12 133 ± 5 128 ± 4 n = 3 GROUP C 6043 male 122 126 128 132 127 placebo 6343 male 130 123 130 124 130 group 6347 male 138 150 145 143 142 n = 5 6872 female 125 131 130 126 127 6880 female 140 142 146 146 149 Mean ± SD 131 ± 8 134 ± 11 136 ± 9 134 ± 10 135 ± 10 Notes: 1. .sup.a34 days before administration (D-34);

(207) TABLE-US-00042 TABLE 6-41 Influence of administrating tested substance for 30 days on hematocrit (Mean ± SD) Hematocrit HCT (fL) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 41.5 40.0 41.3 38.8 39.1 tested 6642 female 43.3 48.6 40.0 39.3 38.4 substance 6500 female 38.4 37.7 35.1 37.2 36.1 (3375 mg/kg) Mean ± SD 41.1 ± 2.5 42.1 ± 5.7 38.8 ± 3.3 38.4 ± 1.1 37.9 ± 1.6 n = 3 GROUP C 6043 male 33.7 39.4 39.3 40.6 39.9 placebo 6343 male 42.3 40.3 40.3 38.6 41.5 group 6347 male 42.9 47.9 42.8 43.0 44.1 n = 5 6872 female 37.8 39.4 38.0 36.7 37.5 6880 female 43.4 43.4 43.6 42.2 44.3 Mean ± SD 40.0 ± 4.2 42.1 ± 3.6 40.8 ± 2.4 40.2 ± 2.6 41.5 ± 2.9 Notes: 1. .sup.a34 days before administration (D-34);

(208) TABLE-US-00043 TABLE 6-42 Influence of administrating tested substance for 30 days on mean corpuscular volume (Mean ± SD) Mean corpuscular volume MCV (fL) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 74.9 73.8 72.9 73.1 74.5 tested 6642 female 84.5 82.0 79.9 78.6 82.8 substance 6500 female 70.1 70.8 70.4 71.4 72.5 (3375 mg/kg) Mean ± SD 76.5 ± 7.3 75.5 ± 5.8 74.4 ± 4.9 74.4 ± 3.8 76.6 ± 5.5 n = 3 GROUP C 6043 male 74.9 72.8 72.4 72.3 74.0 placebo 6343 male 82.0 83.4 79.7 78.2 80.1 group 6347 male 79.0 79.0 76.2 76.6 78.6 n = 5 6872 female 73.4 72.8 71.1 71.1 72.3 6880 female 76.5 77.8 75.9 72.6 74.8 Mean ± SD 77.2 ± 3.4 77.2 ± 4.5 75.1 ± 3.4 74.2 ± 3.1 76.0 ± 3.3 Notes: 1. .sup.a34 days before administration (D-34);

(209) TABLE-US-00044 TABLE 6-43 Influence of administrating tested substance for 30 days on mean corpuscular hemoglobin (Mean ± SD) Mean corpuscular hemoglobin MCH (pg) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 24.9 24.8 25.2 25.2 25.0 tested 6642 female 26.7 26.9 27.5 27.4 27.6 substance 6500 female 23.5 23.6 24.2 24.5 24.8 (3375 mg/kg) Mean ± SD 25.0 ± 1.6 25.1 ± 1.7 25.6 ± 1.7 25.7 ± 1.5 25.8 ± 1.6 n = 3 GROUP C 6043 male 27.1 23.3 23.6 23.5 23.5 placebo 6343 male 25.1 25.4 25.6 25.2 25.1 group 6347 male 25.3 24.8 25.7 25.4 25.2 n = 5 6872 female 24.2 24.2 24.3 24.4 24.5 6880 female 24.6 25.4 25.4 25.2 25.1 Mean ± SD 25.3 ± 1.1 24.6 ± 0.9 24.9 ± 0.9 24.7 ± 0.8 24.7 ± 0.7 Notes: 1. .sup.a34 days before administration (D-34);

(210) TABLE-US-00045 TABLE 6-44 Influence of administrating tested substance for 30 days on mean corpuscular hemoglobin concentration (Mean ± SD) Mean corpuscular hemoglobin concentration MCHC (g/l) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 332 336 346 344 336 tested 6642 female 316 328 345 349 334 substance 6500 female 335 334 344 343 342 (3375 mg/kg) Mean ± SD 328 ± 10 333 ± 4 345 ± 1 345 ± 3 337 ± 4 n = 3 GROUP C 6043 male 362 320 326 324 317 placebo 6343 male 306 305 322 323 313 group 6347 male 321 314 338 332 321 n = 5 6872 female 330 332 341 343 338 6880 female 322 326 335 347 335 Mean ± SD 328 ± 21 319 ± 10 332 ± 8 334 ± 11 325 ± 11 Notes: 1. .sup.a34 days before administration (D-34);

(211) TABLE-US-00046 TABLE 6-45 Influence of administrating tested substance for 30 days on red blood cell distribution width (Mean ± SD) Red blood cell distribution width RDW (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 12.6 12.4 12.4 12.8 12.9 tested 6642 female 12.5 12.0 12.2 12.6 13.6 substance 6500 female 12.2 12.4 13.0 13.4 13.1 (3375 mg/kg) Mean ± SD 12.4 ± 0.2 12.3 ± 0.2 12.5 ± 0.4 12.9 ± 0.4 13.2 ± 0.4 n = 3 GROUP C 6043 male 12.4 11.6 11.8 11.5 11.6 placebo 6343 male 14.0 12.0 11.9 12.1 12.1 group 6347 male 13.8 12.1 11.9 11.8 12.1 n = 5 6872 female 12.0 11.5 11.9 11.4 12.1 6880 female 12.5 11.8 12.0 11.8 11.7 Mean ± SD 12.9 ± 0.9 11.8 ± 0.3 11.9 ± 0.1 11.7 ± 0.3 11.9 ± 0.2 Notes: 1. .sup.a34 days before administration (D-34);

(212) TABLE-US-00047 TABLE 6-46 Influence of administrating tested substance for 30 days on mean HGB distribution width (Mean ± SD) Mean HGB distribution width HDW (g/l) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 20.8 21.9 21.0 22.0 22.5 tested 6642 female 21.0 22.0 22.6 23.9 23.2 substance 6500 female 22.6 23.0 22.7 22.9 24.3 (3375 mg/kg) Mean ± SD 21.5 ± 1.0 22.3 ± 0.6 22.1 ± 1.0 22.9 ± 1.0 23.3 ± 0.9 n = 3 GROUP C 6043 male 19.4 20.3 20.1 19.7 20.5 placebo 6343 male 18.4 18.6 18.4 18.5 18.7 group 6347 male 20.6 20.5 21.1 21.3 21.6 n = 5 6872 female 22.3 23.0 23.2 23.9 23.9 6880 female 20.2 21.4 20.9 22.0 21.5 Mean ± SD 20.2 ± 1.5 20.8 ± 1.6 20.7 ± 1.7 21.1 ± 2.1 21.2 ± 1.9 Notes: 1. .sup.a34 days before administration (D-34);

(213) TABLE-US-00048 TABLE 6-47 Influence of administrating tested substance for 30 days on mean RBC hemoglobin concentration (Mean ± SD) Mean RBC hemoglobin concentration CHCM (g/l) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 316 328 333 332 329 tested 6642 female 298 320 331 339 320 substance 6500 female 322 334 336 337 333 (3375 mg/kg) Mean ± SD 312 ± 12 327 ± 7 333 ± 3 336 ± 4 327 ± 7 n = 3 GROUP C 6043 male 295 310 314 312 302 placebo 6343 male 288 287 304 307 298 group 6347 male 305 307 320 314 306 n = 5 6872 female 316 327 335 331 325 6880 female 306 311 319 337 320 Mean ± SD 302 ± 11 308 ± 14 318 ± 11 320 ± 13 310 ± 12 Notes: 1. .sup.a34 days before administration (D-34);

(214) TABLE-US-00049 TABLE 6-48 Influence of administrating tested substance for 30 days on mean RBC hemoglobin content (Mean ± SD) Mean RBC hemoglobin content CH (Pg) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 23.6 24.2 24.2 24.2 24.5 tested 6642 female 25.1 26.2 26.4 26.5 26.3 substance 6500 female 22.5 23.6 23.6 24.0 24.1 (3375 mg/kg) Mean ± SD 23.7 ± 1.3 24.7 ± 1.4 24.7 ± 1.5 24.9 ± 1.4 25.0 ± 1.2 n = 3 GROUP C 6043 male 22.1 22.5 22.7 22.5 22.3 placebo 6343 male 23.5 23.9 24.2 24.0 23.9 group 6347 male 24.0 24.2 24.3 24.0 24.0 n = 5 6872 female 23.2 23.7 23.8 23.5 23.4 6880 female 23.4 24.1 24.2 24.4 23.9 Mean ± SD 23.2 ± 0.7 23.7 ± 0.7 23.8 ± 0.7 23.7 ± 0.7 23.5 ± 0.7 Notes: 1. .sup.a34 days before administration (D-34);

(215) TABLE-US-00050 TABLE 6-49 Influence of administrating tested substance for 30 days on red blood cell CH distribution width (Mean ± SD) Red blood cell CH distribution width CHDW (Pg) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 3.07 3.14 3.15 3.20 3.27 tested 6642 female 3.26 3.22 3.32 3.37 3.38 substance 6500 female 2.98 3.23 3.24 3.38 3.36 (3375 mg/kg) Mean ± SD 3.10 ± 0.14 3.20 ± 0.05 3.24 ± 0.09 3.32 ± 0.10 3.34 ± 0.06 n = 3 GROUP C 6043 male 2.70 2.67 2.72 2.64 2.65 placebo 6343 male 3.26 3.05 3.01 3.01 2.97 group 6347 male 3.31 3.03 2.98 2.95 2.94 n = 5 6872 female 3.03 3.05 3.11 2.99 3.03 6880 female 3.21 3.15 3.21 3.24 3.10 Mean ± SD 3.10 ± 0.25 2.99 ± 0.18 3.01 ± 0.18 2.97 ± 0.21 2.94 ± 0.17 Notes: 1. .sup.a34 days before administration (D-34);

(216) TABLE-US-00051 TABLE 6-50 Influence of administrating tested substance for 30 days on platelet (Mean ± SD) Platelet PLT (10.sup.9/L) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 405 463 554 469 459 tested 6642 female 478 501 493 364 422 substance 6500 female 356 394 389 393 431 (3375 mg/kg) Mean ± SD 413 ± 61 453 ± 54 479 ± 83 409 ± 54 437 ± 19 n = 3 GROUP C 6043 male 252 351 284 314 339 placebo 6343 male 312 322 358 278 406 group 6347 male 285 344 322 315 348 n = 5 6872 female 344 379 322 283 243 6880 female 377 304 317 281 310 Mean ± SD 314 ± 49 340 ± 29 321 ± 26 294 ± 19 329 ± 59 Notes: 1. .sup.a34 days before administration (D-34);

(217) TABLE-US-00052 TABLE 6-51 Influence of administrating tested substance for 30 days on plateletcrit (Mean ± SD) Plateletcrit PCT (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 0.35 0.39 0.44 0.37 0.39 tested 6642 female 0.44 0.44 0.40 0.32 0.35 substance 6500 female 0.28 0.33 0.32 0.30 0.34 (3375 mg/kg) Mean ± SD 0.36 ± 0.08 0.39 ± 0.06 0.39 ± 0.06 0.33 ± 0.04 0.36 ± 0.03 n = 3 GROUP C 6043 male 0.19 0.25 0.21 0.22 0.25 placebo 6343 male 0.29 0.31 0.30 0.28 0.37 group 6347 male 0.28 0.37 0.32 0.33 0.34 n = 5 6872 female 0.29 0.31 0.30 0.28 0.23 6880 female 0.30 0.26 0.27 0.22 0.26 Mean ± SD 0.27 ± 0.05 0.30 ± 0.05 0.28 ± 0.04 0.27 ± 0.05 0.29 ± 0.06 Notes: 1. .sup.a34 days before administration (D-34);

(218) TABLE-US-00053 TABLE 6-52 Influence of administrating tested substance for 30 days on platelet distribution width (Mean ± SD) Platelet distribution width PDW (%) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 57.9 55.1 49.0 48.4 49.5 tested 6642 female 55.6 55.4 46.0 51.5 45.5 substance 6500 female 49.4 49.4 51.1 49.7 48.1 (3375 mg/kg) Mean ± SD 54.3 ± 4.4 53.3 ± 3.4 48.7 ± 2.6 49.9 ± 1.6 47.7 ± 2.0 n = 3 GROUP C 6043 male 55.5 50.3 48.4 58.3 52.5 placebo 6343 male 52.4 53.5 57.1 52.2 51.2 group 6347 male 68.8 77.6 68.1 76.8 67.3 n = 5 6872 female 54.9 56.5 67.7 68.3 63.6 6880 female 55.2 65.8 62.5 54.8 60.4 Mean ± SD 57.4 ± 6.5 60.7 ± 11.1 60.8 ± 8.2 62.1 ± 10.3 59.0 ± 7.0 Notes: 1. .sup.a34 days before administration (D-34);

(219) TABLE-US-00054 TABLE 6-53 Influence of administrating tested substance for 30 days on mean platelet volume (Mean ± SD) Mean platelet volume MPV (fL) Group Animal No. fe/m baseline.sup.a D0 D10 D20 D30 GROUP A 6890 female 8.5 8.5 7.9 7.9 8.4 tested 6642 female 9.1 8.7 8.0 8.9 8.3 substance 6500 female 8.0 8.4 8.3 7.6 7.9 (3375 mg/kg) Mean ± SD 8.5 ± 0.6 8.5 ± 0.2 8.1 ± 0.2 8.1 ± 0.7 8.2 ± 0.3 n = 3 GROUP C 6043 male 7.7 7.1 7.5 7.1 7.3 placebo 6343 male 9.1 9.7 8.5 9.9 9.2 group 6347 male 9.7 10.9 10.0 10.4 9.7 n = 5 6872 female 8.5 8.2 9.5 9.8 9.5 6880 female 7.9 8.6 8.7 8.0 8.5 Mean ± SD 8.6 ± 0.8 8.9 ± 1.5 8.8 ± 1.0 9.0 ± 1.4 8.8 ± 1.0 Notes: 1. .sup.a34 days before administration (D-34);
5.4 Conclusions

(220) Under the conditions of this experiment, the dose of the tested substance was 15 times of the clinical equivalent dose for monkey, and 18 times of the pharmacodynamical (0.75 mg/kg mouse) equivalent dose for monkey (3375 mg/kg). After 30 days, no toxicity was observed in rhesus monkeys, indicating that the safety of the tested substance.

Example 7

(221) In order to perform characterization and the quality control for the Traditional Chinese Medicine composition of the present disclosure, the inventor has established the fingerprint of the Traditional Chinese Medicine composition of the present disclosure by ultra-high performance liquid chromatography (UHPLC) after a lot of trials. The analysis conditions were as follows:

(222) Instruments and reagents: Ultimate3000 liquid chromatograph system (Thermo) and Chromeleon 7.2 chromatography workstation, ultrapure water machine (molar 1810D cell type), ultrasound system (Jiemen JP-100ST).

(223) Acetonitrile and methanol are chromatographically pure (Fisher) and formic acid is chromatographically pure (Fisher).

(224) Preparation of test solution: the Traditional Chinese Medicine composition to be tested was weighed accurately, dissolved in 80° C. hot water, and subjected to ultrasonic extraction for 60 min; the supernatant was taken after centrifugation and filtered through 0.22 μm microporous membrane, and was formulated into 5 mg/mL solution.

(225) Determination of fingerprint: 1 μL test solution was pipetted accurately into the ultra-high performance liquid chromatograph, and the chromatograms were recorded.

(226) The operating conditions for the chromatography were as follows:

(227) Column: Waters ACQUITY UPLC BEH C18 2.1×150 mm 1.7 μm column;

(228) Mobile phase: mobile phase A: 0.05% formic acid in water, mobile phase B: acetonitrile;

(229) Gradient elution:

(230) 0-18 min, 3% B-18% B; 18-23 min, 18% B-40% B; 23-28 min, 40% B-100% B; 28-30 min, 100% B-100% B; 30-32 min, 100% B-3% B;

(231) Temperature: 25° C.;

(232) Flow rate: 0.3 mL/min;

(233) Detection wavelength: 254 nm;

(234) Injection volume: 1 uL.

(235) The above chromatographic conditions were used for the UHPLC fingerprint analysis of the traditional Chinese medicinal compositions of Example 1 to Example 6. The spectra are shown in FIG. 12. The results show that the UHPLC spectra of the Traditional Chinese Medicine compositions of the present disclosure having the raw materials with amounts defined in the present disclosure have very good consistency, with five obvious characteristic peaks as shown in the Table below:

(236) TABLE-US-00055 TABLE 7-1 UHPLC fingerprint data of the traditional Chinese medicinal compositions of Example 1 to Example 6 retention retention retention retention retention time, no. 1 time, no.2 time, no. 3 time, no.4 time, no. 5 peak (min) peak (min) peak (min) peak (min) peak (min) Example 1 14.540 15.297 15.530 17.177 18.230 Example 2 14.537 15.293 15.527 17.173 18.230 Example 3 14.510 15.263 15.493 17.140 18.200 Example 4 14.530 15.290 15.523 17.17 18.223 Example 5 14.523 15.280 15.513 17.157 18.200 Example 6 14.530 15.290 15.523 17.163 18.213

(237) That is, the retention times of the five characteristic peaks (no. 1, 2, 3, 4, and 5) are about 14.5±0.1, 15.3±0.1, 15.5±0.1, 17.2±0.1, and 18.2±0.1 (min), respectively. Based on these characteristic peaks, the Traditional Chinese Medicine composition according to the present disclosure can be identified easily and they can also be used for quality control.

(238) While the Examples have been described in detail hereinabove, it is understood that modifications and combinations may be made in the form and details without departing from the spirit of the present invention. It will be appreciated that, without departing from the disclosure and the claims as interpreted in broad sense, various changes may be made to the Examples described herein.

Antiviral Traditional Chinese Medicine composition and preparation method and use i'hereof

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