AU2018100291A4 - Hepatoprotective composition - Google Patents

Abstract

The present invention relates to a composition for protecting liver from alcoholic liver disease, and its use as a medicament and healthy food. The composition comprises a 5 therapeutically effective amount of a whole blood collection from a mammal and a pharmaceutically or dietarily acceptable excipient. Preferably, the whole blood collection is from a porcine animal, such as domestic pig (Sus scrofa domestica). The invention is suitable for prevention or treatment of alcoholic liver disease, such as for prevention or treatment of alcoholic steatosis and alcoholic fatty liver.

Description

The present invention relates to a composition for protecting liver from alcoholic liver disease, and its use as a medicament and healthy food. The composition comprises a therapeutically effective amount of a whole blood collection from a mammal and a pharmaceutically or dietarily acceptable excipient. Preferably, the whole blood collection is from a porcine animal, such as domestic pig (Sus scrofa domestica). The invention is suitable for prevention or treatment of alcoholic liver disease, such as for prevention or treatment of alcoholic steatosis and alcoholic fatty liver.

2018100291 06 Mar 2018

HEPATOPROTECTIVE COMPOSITION

FIELD OF THE INVENTION

The present invention relates to a composition for protecting liver from alcoholic liver disease.

BACKGROUND OF THE INVENTION

Drinking alcoholic drinks is an ancient activity that constantly evolves with the civilization and culture of human beings. Moderate drinking can help to relieve emotions and have functions of promoting interpersonal communication and transferring family affection and friendship in the social process. Ethanol is the main chemical component in all kinds of alcoholic drinks. Moderate ethanol is not toxic to the human body. However, the metabolic rate of ethanol in human body is limited. Only a small part of alcohol ingested into the mouth is excreted out of body through lungs respiration or sweat gland, and is absorbed by human body mainly in the jejunal section of small intestine. About 90% of the alcohol absorbed will enter the liver for metabolism. Alcohol in the liver undergoes three fermentation systems of alcohol dehydrogenase, microsomal ethanol oxidizing system and catalase to be oxidized into acetaldehyde. The resulting acetaldehyde will be subjected to aldehyde dehydrogenase (ALDH) action to be converted into acetic acid. The acetic acid then in the form of acetyl-CoA undergoes tricarboxylic cycle to generate heat.

However, long-term or excessive alcohol intake can easily lead to alcoholic liver disease (ALD). At the outset, steatosis and alcoholic fatty liver appear, developing into alcoholic hepatitis, liver fibrosis, and even leading to cirrhosis. The main clinical symptoms of ALD include nausea, vomiting, jaundice and possibly liver enlargement and tenderness, and may be complicated by liver failure and upper gastrointestinal

2018100291 06 Mar 2018 bleeding. The pathological mechanism of alcoholic liver disease is very complicated, and generally believed that it is mainly a result of interaction of various factors such as oxidative stress, inflammatory response and nutritional imbalance induced directly or indirectly by the metabolic process of ethanol and its derivatives. First of all, acetaldehyde derived from the metabolic process of alcohol will damage the structures and functions of various organelles and enzymes in the liver cells, causing direct damage to the liver. Further, aldehydes can be combined with a number of proteins in the body to form highly immunogenic acetaldehyde adducts, which may stimulate the body's immune system to induce immunoreactive liver damage. Acetaldehyde may also impair β-oxidation of mitochondrial fatty acids, induce lipid peroxidation reaction, inhibit biosynthesis of glutathione (GSH), and diminish the antioxidant function of peroxidase. In particular, when alcohol is taken in large quantities over a short period of time, the alcohol concentration in blood is so high as to activate the metabolism of alcohol in the microsomal ethanol oxidizing system, which will produce a large amount of reactive oxygen species (ROS) during the process to attack the hepatocytes. ROS may cause liver damage through many ways, mainly by mitochondrial DNA depletion and lipid peroxidation so as to initiate hepatocyte apoptosis and necrosis and lead to depletion of reduced glutathione (GSH) to reduce the protective function of the liver against peroxidative damage. In addition, in the process of acetaldehyde metabolism into acetic acid, increased ratio of NADH/NAD+ in the hepatocytes results in the reduction of oxidative capacity of hepatocytes to fatty acids and the consequent reduction of the tricarboxylic acid cycle activity, causing the accumulation of fat in the liver and the occurrence of fatty liver, and also causing the number of mitochondria to increase to consume too much oxygen, resulting in liver cells to be prone to necrosis and fibrosis owing to hypoxia.

In China, the proportion of alcoholic liver disease patients over the overall liver disease

2018100291 06 Mar 2018 inpatients has been on the rise in recent years, from 4.2% in 1991 to 21.3% in 1996, while the proportion of alcoholic cirrhosis to total liver cirrhosis has risen from 10.8 % in 1999 to 24.0% in 2003.

Therefore, there is a strong need for compositions and medical uses that can protect the liver against alcoholic liver disease.

SUMMARY OF THE INVENTION

Now, the inventor of the present application have found that the whole blood collection from a mammal, especially the whole blood collection from a Suidae animal, such as the whole blood collection from domestic pig (Sus scrofa domestica), can improve alcoholic liver disease. To assess the effects of the whole blood collection on alcoholic liver disease, the inventor refer to the standard method proposed by Lieber et al. (de la, Μ. Η. P., Lieber, C. S., DeCarli, L. M., French, S. W., Lindros, K. 0., Jarvelainen, H.,

Bode, C., Parlesak, A., and Bode, J. C. (2001). Models of alcoholic liver disease in rodents: a critical evaluation. Alcohol Clin. Exp. Res. 25, 254S-261S) to establish a mouse model in which alcoholic liver disease is induced by long-term feeding of alcoholic diet. This application evaluated the liver condition of the animal model in terms of biochemical indices of liver function, antioxidative enzyme activity and tissue pathology, thereby confirming that said whole blood collection is effective in treating alcoholic liver disease and capable of being used as a complementary or alternative medicine for treating alcoholic liver disease.

Therefore, according to the first aspect of the invention, a hepatoprotective composition is provided, comprising a therapeutically effective amount of a whole blood collection from a mammal and a pharmaceutically or dietarily acceptable excipient.

2018100291 06 Mar 2018

In the second aspect of the invention is to provide the use of a composition in manufacturing a medicament for preventing or treating alcoholic liver disease in a subject, wherein the composition comprises a therapeutically effective amount of s whole blood collection from a mammal and a pharmaceutically or dietarily acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWING

FIG.l shows histopathological images showing the stained sections of liver tissue samples obtained from the mouse model.

DETAILED DESCRIPTION OF THE INVENTION

The term whole blood collection as used herein may refer to blood collected from an animal body and not subjected to a substantial separation process, including plasma, hemocytes with plasma as the carrier, as well as substances such as carbohydrates, proteins, minerals and hormones dissolved or suspended in the plasma. The term whole blood collection as used herein encompasses the newly collected fresh blood, the degraded products and derivatives thereof, as well as the whole blood samples that have been diluted with an appropriate buffer, and subjected to appropriate concentration to remove water and/or add additives such as anticoagulants (e.g., heparin, EDTA,

ACD-A), protease inhibitors, antibiotics and preservatives. According to the invention, under the premise of essentially reserving the main ingredients of the whole blood collection, the whole blood collection can undergo additional processing steps such as heating, filtration or chromatography to remove or deactivate the unwanted ingredients. These unwanted ingredients include, but are not limited to, toxic chemicals, allergens and pathogens. In a preferred embodiment, the collected blood is further subjected to a conventional lyophilization process such that the whole blood collection is in the form of a lyophilized powder.

2018100291 06 Mar 2018

According to the invention, the whole blood collection is obtained from a mammal, preferably from a non-human mammal, including but not limited to horses, cattle, pigs, goats, dogs, cats, mice , rats, guinea pigs, gerbils, hamsters, rabbits, chimpanzees, and rhesus monkeys. In a preferred embodiment, the whole blood collection is from pigs. The physical structures and functions of pigs in physiology, biochemistry, anatomy, metabolism, bone development, cardiovascular system, immune system and digestive system are similar to those of human and, thus, it has been regarded as the best donor for medical research and human xenotransplantation. As will be described below, the term pig as used herein is meant any animal belonging to the Suidae family, including but not limited to the wild boar (Sus scrofa), domestic pig (Sus scrofa domestica), mini swine (Sus Scrofa domestica var. mino guizhounensis Yu.), warthog (Phacochoerus africanus) and peccary (Pecari tajacu). In a more preferred embodiment, the pig is selected from an animal of genus Sus, preferably a domestic pig (Sus scrofa domestica), including but not limited to the domestic pig breeds such as Landrace, Yorkshire, Duroc Kanto, Hampshire and Berkshire, as well as the domestic pig breeds interbred from the above domestic pig, such as the LYD white pigs raised on a large scale in Taiwan area which are interbred from Landrace, Yorkshire and Duroc Kanto. The collection process of the whole blood collection includes any method that can collect the whole blood of a mammal without substantially destroying the ingredients of the whole blood collection. With domestic pig as an example, methods for collecting a small amount of blood include, but are not limited to, ear vein blood collection and precaval vein blood sampling, and a large scale whole blood collection can be made by sacrificing animals.

The term alcoholic liver disease used herein may refer to a series of liver damage conditions caused by sustained intake of alcoholic beverages or foods. Preferably, one or more of these conditions are selected from the group consisting of alcoholic steatosis,

2018100291 06 Mar 2018 alcoholic fatty liver, alcoholic steatohepatitis, hepatocellular ballooning, alcoholic hepatic fibrosis and alcoholic hepatic cirrhosis. The alcoholic liver disease can generally be diagnosed by a medical specialist, veterinarian or other clinical medical personnel. In a more preferred embodiment, the alcoholic liver disease is selected from the group consisting of alcoholic steatosis and alcoholic fatty liver.

As will be described below, the application simulated alcoholic liver disease by the conventional mouse model proposed by Lieber-DeCarli. This conventional animal model involves the long-term feeding of experimental animals with alcohol-containing fluid feed, in which 36% of the carbohydrate contents based on the total energy required by the animals are replaced with ethanol, which is used for the analog of liver damage caused by long-term intake of alcohol. Compared to the large amount of acute alcohol intake, this experimental animal model of chronic alcohol intake is considered more similar to human alcohol intake behavior.

As shown in the examples below, this animal model successfully induced alcoholic fatty liver. Fatty liver is mainly caused by the abnormal accumulation of triglyceride droplets in hepatocytes and is an early pathological symptom of alcoholic liver disease. The alcoholic fatty liver is also considered to be a result of abnormality of the lipid metabolism in the body. As shown in Example 5 below, feeding mice with fluid alcoholic feed led to a rise of liver function damage indices: serum AST and ALT. In addition, Example 7 below shows that the alcohol treatment may cause fatty infiltration and histopathological changes in the liver. Administration of the composition disclosed herein effectively improved the liver function indices and reversed liver pathological changes and hepatomegaly phenomena. These results indicate that the composition disclosed herein has a hepatoprotective activity for protecting liver from the damage caused by alcohol.

2018100291 06 Mar 2018

Long-term intake of alcohol can lead to lipid metabolism imbalance in the body, including decreased lipid oxidation and increased triglyceride (TG) synthesis, thereby resulting in increased blood lipids and fatty liver. As shown in Example 4 below, feeding mice with fluid alcoholic feed led to a significant increase in the liver TG content. Administration of the composition disclosed herein, with a low, medium or high dose, significantly reduced the liver TG content. The composition disclosed herein effectively ameliorated the liver TG accumulation, indicating that it has hepatoprotective function for protecting liver from the damage caused by alcohol.

During the metabolic process of alcohol in the liver, a large amount of free radicals are generated, resulting in an oxidative stress. The oxidative stress may interfere with and destroy the body's antioxidative defense system, leading to imbalance between free radical generation and antioxidative capability and mass production of lipid peroxides, eventually causing alcoholic liver disease. As shown in Example 6 below, feeding mice with fluid alcoholic feed led to a significant decrease in the total glutathione (GSH) content in the liver and a significant increase in catalase (CAT) activity. The significant increase in CAT activity may be due to the production of a large amount of hydrogen peroxide during the metabolism of alcohol, which is a compensatory response requiring

CAT enzyme to remove hydrogen peroxide. GSH is one of the main antioxidant substances in the cells. The total GSH decrease indicates that alcohol does cause an increase in oxidative stress in the liver cells. In general, long-term intake of alcohol caused disturbances in the antioxidative defense system of mice and led to an increase in oxidative stress, which in turn resulted in liver damage. Administration of the composition disclosed herein reduced oxidative stress, and protected liver from oxidative damage.

2018100291 06 Mar 2018

Accordingly, in one aspect of the invention is to provide a hepatoprotective composition, comprising a therapeutically effective amount of a whole blood collection from a mammal, and a pharmaceutically or dietarily acceptable excipient. In another aspect of the invention is to provide use of the aforementioned composition for manufacturing a medicament for preventing or treating alcoholic liver disease in a subject.

The term hepatoprotective as used herein means that the composition described herein is capable of maintaining the viability and vitality of liver cells, maintaining or even regaining the liver function, or alleviating or improving the liver damage caused by long-term alcohol ingestion. The term hepatoprotective encompass preventing alcoholic liver disease from occurring in a subject and/or treating alcoholic liver disease after its emergence in the subject. In this regard, the term “preventing” includes reducing the severity/intensity of, or initiation of, alcoholic liver disease. The term “treating” includes alleviation or relief of at least one clinical symptom of alcoholic liver disease after its emergence. However, the term hepatoprotective should not be interpreted as always having 100% protection against alcoholic liver disease, but rather meaning that it can substantially alleviate or relieve at least one clinical symptom of alcoholic liver disease that has occurred or may occur in a subject.

The whole blood collection may be formulated in combination with a pharmaceutically or dietarily acceptable excipient to produce a hepatoprotective composition. The term pharmaceutically or dietary acceptable excipient as used herein means an inert substance used as a carrier for the whole blood collection, which is not toxic, irritating, pyrogenic, antigenic and hemolytic to the applied subject and free from substantial pharmacological activity without reducing the beneficial effects caused by the whole blood collection. These excipients include, but are not limited to, solvents, surfactants, disintegrants, binders, diluents, lubricants, stabilizers, antioxidants, flavoring agents,

2018100291 06 Mar 2018 sweeteners, coloring agents, absorption enhancers, plasticizers, pH adjusting agents, osmotic pressure adjusting agents and thickeners. The pharmaceutically or dietarily acceptable excipients as used herein may refer to those known by the persons with ordinary skill in the art (please refer to Remington: The Science and Practice of

Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999)).

The hepatoprotective composition disclosed herein can be administrated to a subject via any suitable route to allow the composition to contact a target cell or tissue. The routes of administration include, but are not limited to, a topical, enteral or parenteral route, such as an oral, intravenous, subcutaneous, intratumoral, intramuscular, intraperitoneal, transdermal, intrathecal, or intracerebral route. Administration can be either rapid as by injection, or over a period of time as by slow infusion or administration of a slow release formulation. The term subject as used herein is intended to cover human and non-human mammals. Non-human mammals include livestock, companion animals, laboratory animals, and non-human primates. Non-human subjects include, but are not limited to, horses, cattle, pigs, goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, minks, rabbits, chimpanzees, and rhesus monkeys. In a preferred embodiment, the subject is human, especially a human patient with alcoholic liver disease or at risk of alcoholic liver disease, e.g., a patient suffering from alcoholic fatty liver.

In a preferred embodiment, the hepatoprotective composition is prepared for oral administration. The whole blood collection may be co-formulated with a suitable excipient, and pressed into tablets, pills, or encapsulated into solid capsule or soft

2018100291 06 Mar 2018 capsule. For example, the excipient may be selected from maltodextrins, starches, syrup, lactose, mannitol, sorbitol, sucrose, dextrose, gum acacia, gelatin, calcium phosphate, hydroxypropyl methylcellulose, microcrystalline cellulose, calcium sulfate dehydrate, calcium lactate trihydrate, glycine, kaolin, calcium hydroxide, talc, alginates, stearates, sodium stearyl fumarate, hydrogenated vegetable oil, higher fatty acids and alkali and alkaline earth salts thereof, glycerol, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycols, polyvinylpyrrolidone, methoxy polyethylene glycols, sodium oleate, glyceryl behenate, sodium lauryl sulfate and colloidal silica and the like.

The whole blood collection in the hepatoprotective composition is administered to a subject in a therapeutically effective amount to elicit the a beneficial biological or medicinal response that is being sought in a cell, tissue, system, animal or human by a researcher, veterinarian, medical doctor or other clinician and preferably to stabilize, ameliorate, alleviate or relieve a condition or symptom in the subject, such as improving liver function indices, reducing hepatomegaly, reducing accumulation of triglycerides in the liver, and improving liver steatosis. Although the therapeutically effective amount is generally determined by the observed effect that it has, in comparison with the effect which is observed on the similar patients who are administrated with compositions that do not include the whole blood collection disclosed herein (i.e., the control group), the actual dosage is calculated on the basis of the selected particular administration route. Further refinement of the calculations necessary to determine the appropriate dosage for administration is routinely made by those of ordinary skill in the art. When formulated into an oral dosage form administrable to a human patient, the whole blood collection is preferably administered daily, weekly or twice a week, at an amount ranging from 0.1 mg/kg body weight to 100 mg/kg body weight, preferably from 1 mg/kg to 50 mg/kg body weight, such as 10 mg/kg to 25 mg/kg body weight.

2018100291 06 Mar 2018

In one embodiment, the hepatoprotective composition may further contain other active ingredients that are beneficial to the liver so as to be formulated into healthy foods and pharmaceuticals that are suitable for individual ingestion and can ensure both safety and efficacy. In another embodiment, the hepatoprotective composition consists essentially of the whole blood collection and the acceptable excipients described above. The phrase “consist essentially of’ or “consisting essentially of”, with respect to the constitutive elements of the hepatoprotective composition defined in the claims, means that the composition contains the indicated elements and may contain additional elements only if the additional elements do not materially alter the basic and novel characteristics of the invention. In still another embodiment, the hepatoprotective composition consists of and, therefore, contains only the whole blood collection in combination with the pharmaceutically or dietarily acceptable excipient described above.

The following examples are for illustrative purposes only and do not limit the scope of the invention. The experimental results shown in each example are expressed as mean value ± standard deviation.

Preparative Example 1: Preparation of Lyophilized Powder of Whole Blood

Collection

500 ml of whole blood was taken from LDY pigs raised by Taiwan Sugar Corporation and loaded in an empty bottle containing 75 ml of anticoagulant acid citrate dextrose solution-A (ACD-A) and 3 ml gentamicin and stored at a low temperature. The pig whole blood was aliquoted into glass vials (125 ml/vial) for lyophilization and frozen at

-80°C for 24 hours, followed by lyophilization to obtain lyophilized powder of whole blood collection.

2018100291 06 Mar 2018

Example 1: Animal Model of Alcoholic Liver Disease

Six-week-old male C57BL/6J (B6) mice (BioLASCO Taiwan Co., Ltd.) were fed with a general chew diet to 10 weeks of age. Ambient temperature of the mice feeding environment was controlled at 22 ± 2°C; light and dark cycle times are 12 hours, respectively; free access to drinking water and feed, and the body weight was recorded once a week. Then, experimental model was conducted using fluid alcohol feed to induce alcoholic fatty liver damage. Prior to the experiment, the C57BL/6J (B6) mice were randomly divided into five groups. The alcohol content in the fluid alcohol feed was gradually increased one week before the experiment to adapt the experimental animals to the fluid alcohol feed. The experimental groups were as shown in Table 1 below:

Table 1. Experimental Group

Groups	ad libitum	gavage
C group (control group)	Fluid normal feed	Deionized water
TO group (alcohol-induced liver damage group)	Fluid alcohol feed	Deionized water
T2.5 group	Fluid alcohol feed	@2.5 mg/kg weight, lyophilized powder from preparative Example 1
T6.25 group	Fluid alcohol feed	@ 6.25 mg/kg weight, lyophilized powder from preparative Example 1
T12.5 group	Fluid alcohol feed	@12.5 mg/kg weight, lyophilized powder from preparative Example 1

The fluid alcohol feed was Lieber-DeCarli Regular Liquid Diet Ethanol (product

2018100291 06 Mar 2018 number 710260, containing 5.11% ethanol), purchased from Dytes Inc., Philadelphia, USA, and the normal fluid feed was Lieber-DeCarli Regular Liquid Diet Contrl (product number 710027), purchased from Dytes Inc., Philadelphia, USA, both with a caloric density of 1.0 kcal/mL. The feed supply was based on free ingestion. When experiment began, all animals were fed through oral tube with deionized water or the whole blood lyophilized powder prepared in Preparative Example 1, at 10 am in the morning in each day for 4 weeks, and fresh fluid feed was replaced at 4 pm daily and the intake amount was recorded. The body weight of the mice was monitored every two days.

After 4 weeks of feeding, the mice were weighted, and sacrificed to take blood from orbital or heart for separating serum to test for following biochemical indicators of blood lipid and liver function: triglycerides, cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Liver samples were taken by laparotomy, and weighted to calculate the liver weight/body weight ratio. A tissue block of about 1 cm³ was cut from each right liver lobe and fixed in 10% neutral formalin solution. Paraffin-embedded sections were stained with H&E for histopathological observation. The remaining liver was divided into six parts according to the anatomical locations for measuring the GSH content and catalase enzyme activity, as well as the changes in liver triglyceride and cholesterol contents, respectively.

Example 2: Body Weight Changes in Mice

The average initial body weight of ten week old mice was about 25 grams. After a week of adaptation, the weights of the respective groups of mice increased by about 1-2 grams, and the average weight was about 26-27 grams. In the experiments, because the alcohol concentration of the fluid alcohol feed was as high as 5.1%, mice began to ingest food less. Therefore, the control group is changed to adopt pair-feeding mode for

2018100291 06 Mar 2018 feeding. A week later, the body weights of the respective mouse group were reduced by about 2-3 grams with average body weight of about 23-24 grams. The body weights in the second and third weeks thereafter and final body weights maintained at the same level in all groups. There was no significant difference between the groups (p> 0.05).

5_Table 2, Changes in Body Weight

Body weight (gram)

Groups	N	Initial body weight	Zero week	1st week	2nd week	3rd week	Final body weight
C	11	24.9±1.8	27.4±2.0	23.5±2.5	24.1 ±2.1	25.6±2.1	26.5±1.6
TO	7	24.8±0.8	26.4±1.1	23.H1.2	23.H1.9	26.7±1.3	26.8±2.1
T2.5	9	25.0±0.9	26.7±1.4	24.7±1.5	25.6±1.6	26.5±1.9	26.4±2.2
T6.25	11	25.0±l.l	26.H1.1	23.8±2.3	24.3±1.4	24.7±3.1	25.8±2.5
T12.5	11	25.H1.3	27.0±1.3	24.5±1.9	24.5±1.7	25.7±1.1	25.5±1.2

Example 3: Tissue Weight of Mice

The absolute weights and relative weights of heart, spleen, lung, kidney and epididymal white adipose tissue (EWAT) were not significantly different among all groups (p>

0.05), but it was found that the absolute weight and relative weight of the liver in alcohol treatment group (TO group) were significantly greater than the control group (C group); namely, the mice of alcohol treatment group (TO group) showed hepatomegaly phenomenon (p> 0.05). Significant improvement in hepatomegaly was evident in mice group treated with different doses, especially in the mice group treated with the high dose of the whole blood lyophilized powder prepared in preparative Example 1 (T12.5 group) (p> 0.05).

2018100291 06 Mar 2018

Table 3. Absolute Weight of Tissue

Groups	N	Liver	Heart	Spleen	Lung	Kidney	EWAT
				Weight (gram)
C	11	0.98±0.08	0.12±0.01	0.06±0.01	0.14±0.01	0.29±0.03	0.58±0.15
TO	7	1.09±0.08	0.12±0.01	0.05±0.02	0.14±0.01	0.30±0.02	0.45±0.13
T2.5	9	1.00±0.15	0.1l±0.02	0.05±0.01	0.14±0.02	0.30±0.04	0.59±0.20
T6.25	11	0.97±0.10	0.1l±0.02	0.05±0.01	0.14±0.01	0.30±0.02	0.52±0.27
T12.5	11	0.94±0.11	0.11±0.01	0.05±0.01	0.13±0.01	0.30±0.03	0.49±0.16

Table 4, Relative Weight of Tissue

Groups	N	Liver	Heart	Spleen	Lung	Kidney	EWAT
			gram/100 gram body weight
C	11	3.86±0.25	0.47±0.04	0.23±0.02	0.55±0.05	1.16±0.08	2.26±0.52
TO	7	4.48±0.51	0.50±0.07	0.22±0.05	0.57±0.04	1.23±0.10	1.8H0.42
T2.5	9	4.08±0.51	0.46±0.05	0.2H0.04	0.58±0.05	1.22±0.10	2.39±0.75
T6.25	11	4.15±0.36	0.47±0.10	0.23±0.05	0.60±0.10	1.29±0.20	2.12±1.02
T12.5	11	3.97±0.40	0.48±0.05	0.19±0.07	0.56±0.05	1.26±0.13	2.06±0.65

Example 4: Triglyceride and Cholesterol Contents in Serum and Liver (1) Determination of serum triglyceride (TG) content

Commercially available detection kit was adopted (Randox Laboratories Ltd., UK, kit number TR418). Serially diluted reference solutions (12.5-200 mg/dL) and the serum samples obtained in Example 1 after appropriate dilution were added at 10 μΐ/well in a

96-well microtiter plate, and 200 μΐ of reaction reagent was added for reaction at room temperature for 10 minutes and then the absorbance at 500 nm was measured. A standard curve was plotted based on the absorbance values of the serially diluted reference solutions, and the serum TG contents were obtained by interpolating the

2018100291 06 Mar 2018 absorbance values of the serum samples onto the standard curve.

(2) Determination of serum cholesterol

Commercially available detection kit was adopted (Randox Laboratories Ltd., UK, kit number CH280). Serially diluted reference solutions (12.5-200 mg/dL) and the serum samples obtained in Example 1 after appropriate dilution were added at 10 μΐ/well in a 96-well microtiter plate, and 200 pi of reaction reagent was added for reaction at room temperature for 10 minutes and then the absorbance at 500 nm was measured. A standard curve was plotted based on the absorbance values of the serially diluted reference solutions, and the serum cholesterol contents were obtained by interpolating the absorbance values of the serum samples onto the standard curve.

(3) Preparation of liver lipid extract

The lipid extract method proposed by Folch et al. (Folch et al. J. Biol. Chem. 226:, 497,

509 (1957)) was adopted. 0.1 grams liver obtained in Example 1 was placed in a glass vial with a small amount of extraction solution (CHCUMeOH = 2:1) added to be pulverized by a homogenizer, followed by filtration with a filter paper and using volumetric flask to quantitatively extract solution to 10 ml.

(4) Determination of liver triglyceride content pi of liver extract was placed in a glass test tube and stood in a laminar-flow hood, allowing the solvent to completely evaporate. Then, a commercially available detection kit (Randox Laboratories Ltd., UK, kit number TR418) was used according to the same protocol for measuring the serum TG content.

(5) Determination of liver cholesterol content

100 μΐ of liver extract was placed in a glass test tube and stood in a laminar-flow hood

2018100291 06 Mar 2018 to allow the solvent to completely evaporate, followed by addition of 10μ1 of Triton X-100. Then, a commercially available detection kit (Randox Laboratories Ltd., UK, kit number CH280) was used according to the same protocol for measuring the serum cholesterol content.

The results are shown in Table 5 below.

Table 5. Triglyceride and Cholesterol Content in Serum and Liver

Groups	N	TG	cholesterol
mg/ liter serum	mg/ gram liver weight	mg/ total liver weight	mg/ liter serum	mg/ gram liver weight	mg/ total liver weight
C	11	50.3±15.5	6.63±2.50	6.44±2.16	98.4±20.9	2.19±0.64	2.14±0.62
TO	7	69.5±24.3	13.23±2.69	14.47±3.21	88.7±7.4	2.33±0.44	2.57±0.60
T2.5	9	68.0±32.1	9.42±1.6	9.49±2.74	91.6±7.1	2.10±0.25	2.08=1=0.31
T6.25	11	52.H17.1	11.09±3.85	10.64±3.47	82.4±23.4	2.22±0.32	2.14±0.31
T12.5	11	58.4=1=15.4	8.99±2.48	8.48±2.61	92.2±10.3	2.25±0.37	2.12±0.49

Table 5 shows that the alcohol treatment (TO group) resulted in an increase of TG level in mouse serum, whereas medium and high doses of the whole blood collection obtained in Preparative Example 1 (T6.25 and T12.5 groups) could reduce the degree of lipid increase caused by alcohol intake. In addition, the alcohol treatment (TO group) significantly resulted in accumulation of TG in mouse liver (p <0.05), indicating that the alcohol treatment caused fatty liver in mice. Administration of different doses of the whole blood collection obtained in Preparative Example 1 significantly improved the hepatic TG (p <0.05), indicating that the whole blood collection obtained in Preparative Example 1 has the potential to improve fatty liver. As for the mice serum and liver cholesterol levels in the respective groups, there was no significant difference between

2018100291 06 Mar 2018 the groups (p> 0.05).

Example 5: Serum AST and ALT Enzyme Activity

In this Example, AST and ALT enzyme activities in the serum samples obtained in

Example 1 were used as indices of liver damage to evaluate the effects of the whole blood collection obtained in Preparative Example 1 on the improvement of hepatic damage induced by alcohol in mice. Commercially available detection kits (Randox Laboratories Ltd., UK, kit numbers AL1268 and AS 1267) were used to detect biochemical indices AST (or GOT) and ALT (or GPT). The detection was based on the method proposed by Reitman and Frankel (Reitman, S, and Frankel, A., 1957, Am J Clin Pathol. 28 (1): 56-63) and the International Federation of Clinical Chemistry (IFCC). 1986. J. Clin Chem. Biochem. 24: 481-495; and Internation Federation of Clinical Chemistry (IFCC). 1986. J. Clin. Chem. Biochem. 24: 497-510). The results are shown in Table 6 below.

_Table 6. Serum AST/ALT Enzyme Activity_

Groups	Specimens number	Unit/liter (U/L)
AST	ALT
C	11	40.0±9.2	26.8±8.3
TO	7	59.9±19.2	36.8±13.2
T2.5	9	56.5±12.2	29.8±6.8
T6.25	11	49.3±15.3	27.8±8.6
T12.5	11	55.2±12.8	29.8±6.4

Table 6 shows that alcohol treatment (TO group) resulted in an increase in AST and ALT activities in mouse serum (p <0.05). After administration of the whole blood lyophilized powder obtained in Preparative Example 1, ALT enzyme activity was significantly reduced, especially in the T6.25 group. The results indicate that the whole

2018100291 06 Mar 2018 blood collection obtained in Preparative Example 1 has the potential to maintain liver function in the case of alcohol-induced chronic liver damage.

Example 6: Assessment of Liver Antioxidative Enzyme Activity

Approximately 0.1 grams fresh liver sample obtained in Example 1 was taken to add into an ice-cold homogenization buffer (0.01 M potassium phosphate buffer, pH 7.4, 1.15% KC1) and a tissue homogenizer was used to grind the liver sample into a homogenate, which was in turn filtered with gauze and adjusted to 10% (w/v) with the buffer. Centrifugal separation was done at 1000^xg at 4°C for 30 minutes to obtain a homogeneous suspension of liver tissue. After appropriate dilution, 10 μΐ of the diluted samples and serially diluted bovine serum albumin (Sigma-Aldrich, St. Louis, Missouri, USA) reference solutions were taken into a 96-well microtiter plates for enzyme-linked immunosorbent assay (ELISA). Then, 200 μΐ of Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) was added in each well for reaction for 5 minutes at room temperature, and an ELISA reader was employed to measure the absorbance at 595 nm. The protein concentrations of the respective hepatic homogenate samples were calculated based on the standard curve plotted.

Commercially available kits (Cayman Chemical, Ann Arbor, MI, kit numbers 703002 and 707002) were used to measure total glutathione (GSH) content and catalase (CAT) enzyme activity in liver. The results are shown in Table 7 and Table 8 below.

Table 7. GSH Content of Liver

Groups	Specimens		Total GSH content
	number	nmol/	μ mole/	pmole/
		mg protein	gram liver weight	total liver weight
C	11	31.2+14.7	6.14+1.77	6.11+1.99
TO	7	20.5+4.1	5.02+0.99	5.49+1.24

2018100291 06 Mar 2018

T2.5	9	20.9+3.2	5.55+1.12	5.42+0.71
T6.25	11	22.2+3.6	5.54+0.74	5.34+0.79
T12.5	11	22.3+5.3	5.87+1.24	5.48+1.34

The total GSH content shown in Table 7 included both reduced GSH and oxidized GSH. Alcohol treatment (TO group) resulted in a significant decrease in total GSH (p <0.05) in mouse liver. GSH is the main antioxidant in cells. Alcohol treatment led to a decrease 5 in the total amount of GSH, indicating that alcohol led to increased oxidative stress in liver cells. Administration of medium and high doses of the whole blood collection obtained in Preparative Example 1 resulted in an increase in total GSH. This indicates that the whole blood collection obtained in Preparative Example 1 has the potential to diminish the oxidative stress and protect the liver against oxidative damage.

_Table 8. Liver CAT Enzyme Activity_

Specimens	CAT enzyme activity
Groups	number	nmol/minutes/ mg protein	mmole/minutes/ gram liver weight	mmole/minutes/ total liver weight
C	11	429.7+164.0	87.4+26.3	85.1+22.7
TO	7	599.4+157.7	148.0+4.44	161.0+47.2
T2.5	8	601.1+227.6	155.7+53.2	159.0+65.2
T6.25	11	549.0+217.6	135.7+49.8	132.5+55.4
T12.5	11	499.2+168.8	131.2+43.0	122.9+44.1

CAT is one of the major antioxidative enzymes in cells, and its main function is to convert intracellular hydrogen peroxide into water and oxygen. Table 8 shows that alcohol treatment (TO group) led to a significant increase in CAT activity in mouse liver (p <0.05). During alcohol metabolism, a large amount of hydrogen peroxide is generated, which needs to be removed by CAT. CAT activity is the highest in

2018100291 06 Mar 2018 alcohol-treated mice, which may be a compensatory response of hepatocytes to oxidative stress to promote CAT gene expression to remove hydrogen peroxide. Administration of medium and high doses of the whole blood collection obtained in Preparative Example 1 (T6.25 and T12.5 groups) resulted in a decline in CAT activity, indicating that the whole blood collection obtained in Preparative Example 1 has the potential to inhibit the liver's capability of generating hydrogen peroxide, thereby reducing the CAT expression due to the compensatory response.

Example 7: Liver Histopathological Examination

The liver tissue obtained in Example 1 was dehydrated and cleared with various concentrations of ethanol (30, 50, 70, 95, 99.5%) and xylene. Then, xylene was replaced by hot paraffin solution, and finally the tissue was embedded with paraffin. The finished paraffin specimens were sectioned in 5 pm thickness using a microtome, and the sections were placed into clean glass slides and dried at 37°C for further pathological staining.

In order to observe the extent of hepatic cell damage, fat accumulation, necrosis and the like, H&E staining was performed on the liver tissue in order to evaluate the hepatic fat accumulation. The tissue sections were placed in xylene for 30 minutes for dewaxing, and then sequentially reconstituted in 99.5, 95, 70, 50, and 30% ethanol for 30 minutes, respectively, followed by soaking in distilled water for 10 minutes for the subsequent staining. The tissue sections were soaked in hematoxylin for 30 seconds for staining cell nuclei, and rinsed with distilled water for several minutes. Then, the tissue sections were stained by eosin for 2 to 5 minutes, and rinsed with distilled water for several minutes. The completed staining process was followed by a dehydration process by sequentially placing in 50, 70, 95 and 100% ethanol twice, each for 30 seconds, and then proceeding with hyalinization twice with xylene and sealing with sealing glue. The

2018100291 06 Mar 2018 images of the stained sections are shown in Fig.l.

Figure 1 shows that the liver tissue of the Control group was intact, in which the boundary between cells can be easily observed, and the internal structure of the cells was clean and free of impurity or vacuoles. The liver cells of the alcohol treatment (TO group) exhibited considerable pathological changes, such as ballooning degeneration and fatty droplet accumulation presenting a bright droplet-like form, and the boundary between the cells became blurring. After administration with the whole blood collection obtained in Preparative Example 1, it can be observed that the liver pathological change has significantly improved, especially the number and size of the fatty droplets significantly reduced (T2.5 group, T6.25 group and T12.5 group). The results show that the whole blood collection obtained in Preparative Example 1 has the potential to improve hepatic steatosis.

While the invention has been described with reference to the preferred embodiments above, it should be recognized that the preferred embodiments are given for the purpose of illustration only and are not intended to limit the scope of the present invention and that various modifications and changes, which will be apparent to those skilled in the relevant art, may be made without departing from the spirit and scope of the invention.

2018100291 06 Mar 2018

Claims (5)

1. We claim:

   1. A hepatoprotective composition, comprising:

   a therapeutically effective amount of a whole blood collection from a mammal and a

   5 pharmaceutically or dietarily acceptable excipient.
2. 2. The hepatoprotective composition according to claim 1, wherein the whole blood collection is from an animal of genus Sus.

   10
3. 3. The hepatoprotective composition according to anyone of the preceding claims, wherein the whole blood collection is from a domestic pig (Sus scrofa domestica).
4. 4. The hepatoprotective composition according to anyone of the preceding claims, wherein the whole blood collection is in the form of lyophilized powder.
5. 5. The hepatoprotective composition according to anyone of the preceding claims, wherein the pharmaceutically or dietarily acceptable excipient is selected from the group consisting of maltodextrins, starches, syrup, lactose, mannitol, sorbitol, sucrose, dextrose, gum acacia, gelatin, calcium phosphate, hydroxypropyl methylcellulose,

   20 microcrystalline cellulose, calcium sulfate dehydrate, calcium lactate trihydrate, glycine, kaolin, calcium hydroxide, talc, alginates, stearates, sodium stearyl fumarate, hydrogenated vegetable oil, higher fatty acids and alkali and alkaline earth salts thereof, glycerol, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycols, polyvinylpyrrolidone, methoxy polyethylene glycols, sodium

   25 oleate, glyceryl behenate, sodium lauryl sulfate and colloidal silica.

   1/1 oo o

   <N

   Mar <N o

   o oo o

   <N (A) Control (B)T0 (C) T2.5 (D) T6.25 (E) T12.5

   FIG 1