TWI902267B - Use of sting-free propolis alcohol extract for preventing kidney and liver damage and inhibiting kidney inflammation, and pharmaceutical composition thereof - Google Patents

Abstract

This invention relates to the use of a sting-free propolis alcohol extract for the prevention of kidney and liver damage and the inhibition of kidney inflammation, and its pharmaceutical composition. Animal experiments were conducted to test the efficacy of the propolis alcohol extract to demonstrate that the sting-free propolis alcohol extract can effectively increase the body's antioxidant enzymes and inhibit the expression of type II cyclooxygenase (COX-2) caused by inflammation, thereby achieving the purpose of inhibiting inflammation. The pharmaceutical composition contains an effective amount of active ingredient and a pharmaceutically acceptable carrier. Appropriate use of this drug can help slow down or prevent tissue fibrosis.

Description

Uses of sting-free propolis alcohol extract for the prevention of kidney and liver damage and the inhibition of kidney inflammation, and its pharmaceutical compositions.

This invention relates to the use of a non-sting propolis alcohol extract for the prevention of kidney and liver damage and the inhibition of kidney inflammation, and to a pharmaceutical composition of the non-sting propolis alcohol extract.

Early-stage prostate cancer relies on androgens for survival and reproduction. Therefore, eliminating androgen secretion or reducing androgen receptor (AR) expression in patients has become a primary approach to hormone therapy for prostate cancer. Many studies have indicated that propolis alcohol extracts and their purified forms can effectively inhibit the growth of various human cancer cells, but no studies have been found on sting-free propolis.

Free radicals are unstable atoms or groups with unpaired electrons, formed by metabolism within the body or by external factors such as radiation, ultraviolet rays, and smoking. The generation of free radicals can lead to cellular oxidation and increase the accumulation of lipid peroxides, damaging cell structure and causing disease. If the body's antioxidant defense system can be enhanced to decompose reactive oxygen species (ROS), cellular damage can be effectively reduced, delaying aging and death. The antioxidant defense system in organisms can be divided into primary and secondary defense systems. Primary defense systems, such as vitamin C, vitamin E, and antioxidant enzymes, can directly eliminate or inhibit reactive oxygen species (ROS). Secondary defense systems include lipases, proteases, and DNA repair enzymes, which are responsible for eliminating the oxidative products of reactive oxygen species, repairing damaged cells, and replenishing free radical derivatives that cannot be eliminated by the primary defense system. Coenzyme Q10 (CoQ10) is a fat-soluble antioxidant that can regenerate vitamin E within lipoproteins, promote cellular respiration, and play a crucial role in scavenging free radicals. It is currently used for cardiovascular diseases and neurodegenerative diseases. Alpha-lipoic acid, an antioxidant, can directly scavenge free radicals (ROS) and is used to treat neuropathy, cataracts, and diabetes. Given the high stress and irregular eating and sleeping habits of modern life, and considering the importance of health, functional foods that combat aging and fatigue have become a trend. Recent medical research has shown that aging and fatigue are closely related to free radicals and antioxidants. If the body's antioxidant system cannot eliminate excessive free radicals, the body accumulates excessive oxidative damage, causing an imbalance in antioxidant mechanisms. This ultimately affects physiological functions, leading to aging, gene mutations, disease, and even death. Traditional antioxidant drugs often have numerous side effects, such as nausea, vomiting, gastrointestinal pain, and skin rashes. Drug interactions may occur, and differences in bioavailability can lead to high drug burdens.

During exercise, the organism will cause muscle soreness, stiffness, weakness and other uncomfortable symptoms due to nutrient consumption and accumulation of lactic acid. Bioflavonoids are known to strengthen capillaries and are therefore useful in treating blood stasis, varicose veins, and hemorrhoids. Flavonoids have anti-inflammatory, antibacterial, anti-cancer and other effects, and can slow down the release of histamine from mast cells. Since histamine is a key chemical that causes allergic reactions, flavonoids can have anti-allergic effects by reducing histamine. In patients with liver and kidney disease, liver/kidney fibrosis is a common feature of most patients with chronic liver/kidney disease, regardless of the initial cause of liver/kidney injury. Taking the liver as an example, the activation of hepatic stellate cells (HSCs) is the main cause of liver fibrosis. As the degree of liver damage increases, the expression of α-smooth muscle actin (α-SMA) also increases, leading to excessive deposition of extracellular matrix (ECM) and the phenomenon of fibrosis. Currently, drugs and methods used to inhibit liver fibrosis include anti-fibrotic drugs, antiviral drugs, and lifestyle modifications, such as acetylcysteine anti-fibrotic drugs and antiviral drugs for treating hepatitis B, which improve patients' diet and lifestyle. However, the disadvantages are that drug treatment may have side effects and adverse reactions, such as hepatotoxicity, and lifestyle modifications require long-term adherence by patients, requiring greater self-discipline and willpower.

The search for natural treatments that are highly adaptable to the human body, and the subsequent improvement and enhancement of treatment efficiency, are of great importance to those engaged in this field, and require redesign and research for effective application.

The main purpose of this invention is to provide a sting-free propolis alcohol extract for the prevention of kidney and liver damage and the inhibition of kidney inflammation. Animal experiments are used to test the efficacy of the propolis alcohol extract to demonstrate that the sting-free propolis alcohol extract can effectively increase the body's antioxidant enzymes and inhibit inflammation.

To achieve the above objectives, the sting-free propolis was provided by Sanchi Ecological Bee Farm (Taoyuan, Taiwan). Its alcohol extract was obtained by extracting with 100 ml of ethanol for 2 hours, and the ethanol extract layer was then evaporated under vacuum to obtain a dark brown paste-like substance used in this invention. The sting-free propolis extract contains at least one type of flavonoid compound, and this flavonoid compound contains at least one propolinin (Propolin G), with protocatechuic acid (PCA) as its main component.

The purpose of this invention is to provide a pharmaceutical composition comprising an effective amount of an active ingredient and a pharmaceutically acceptable carrier, wherein the dosage of the pharmaceutical composition, based on the active ingredient compound, is approximately 1.2 to 12 mg/kg body weight per day.

Another objective of this invention is that the main active ingredient of sting-free propolis, flavonoids, has good antioxidant and anti-inflammatory effects. It mainly reduces the accumulation of extracellular matrix (ECM) and decreases the expression of Smad2/3 proteins by inhibiting the activity of α-smooth muscle actin (α-SMA) and collagen Iα1 (Col Iα1), thereby inducing cell apoptosis.

Another objective of this invention is to increase the activity of antioxidant enzymes (SOD) and glutathione peroxidase (GPx), reduce the levels of creatinine (CRTN) and blood urea nitrogen (BUN), and decrease the expression of type II epoxidase COX-2 in renal tissue.

Another objective of this invention is to provide various delivery forms, using any suitable method to formulate the composition into different forms of administration such as oral administration and subcutaneous injection.

To achieve the above-mentioned objectives and effects, the technical means and methods adopted in this invention are described in detail below with reference to the preferred embodiments of this invention, so as to facilitate a complete understanding.

Alcohol extraction of propolis is a known technique, but most of the propolis in related patents comes from Brazil, mainland China, and Malaysia, and most of them only conduct antibacterial or cellular experiments. The propolis in this invention comes from a sting-free propolis species endemic to Taiwan, and the active ingredients obtained by alcohol extraction are then tested on mice.

The sting-free propolis used in this invention was provided by Sanchi Ecological Bee Farm (Taoyuan, Taiwan). 100 grams of sting-free propolis was extracted with 100 ml of ethanol for 2 hours (refluxing three times). The ethanol extract was evaporated under vacuum to obtain a dark brown paste-like substance. This sting-free propolis extract contains at least one type of flavonoid compound, and this flavonoid compound contains at least one propolinin (Propolin G), with protocatechuic acid (PCA) as its main component.

Flavonoids in sting-free propolis were analyzed using reversed-phase high-performance liquid chromatography (HPLC). The HPLC system consisted of a Shimadzu LC-20AT solvent delivery system and an SPD-M20A photodiode array detector at a wavelength of 280 nm. The HPLC system used a SIL-20A autosampler for sample injection. Separation was performed on a TSK-GEL ODS-100S column (maintained at 25°C) at a flow rate of 1.0 mL/min using 50% ethyl acetate (EA) as the mobile phase, followed by a 10-minute eluent flush to ensure column equilibration and return to normal baseline and starting conditions.

The animal experiments of this invention used the aristolochic acid nephropathy (AAN) test to determine whether the propolis alcohol extract was effective in protecting against acute kidney and liver damage. This experiment used C57BL/6J mice. Before administering propolis, the initial body weight of each group of mice was measured, and the study groups were divided into: 1. Normal control group, administered only with ethanol solvent (5% EtOH); 2. Aristolochic acid administered intraperitoneally (i.p.) at 2.5 mg/kg body weight; 3. Aristolochic acid (AA) plus propolis at 20 and 200 mg/kg body weight (experimental groups); 4. Administered only propolis; After being fed once daily for 28 consecutive days, the liver antioxidant enzymes of each group of mice were measured. The results are shown in Figure 1.

Figure 1(A) and Figure 1(B) show the performance of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the plasma of the above-mentioned animals. The plasma samples show that long-term administration of AA does indeed reduce the activity of SOD and GPx, while pre-administration of propolis (20 mg/kg) via syringe can increase the activity of SOD and GPx in the plasma of mice with kidney injury.

Refer to Figure 2, which shows the effect of propolis (20 mg/kg) on AAN nephritis mice and the observation of type II cyclooxygenase (COX-2) expression using the Western ink dot method. Since current research shows that NF-κB and AP-1, two transcription factors, can regulate the expression of COX-2, the effects of NF-κB and AP-1 on propolis's effect on AAN nephritis mice can be indirectly understood by observing the level of COX-2 protein expression in the kidneys. The experimental results show that pre-administration of propolis (20 mg/kg) can reduce COX-2 protein expression (β-actin as the control group).

The animal urine test of this invention measures the levels of creatinine (CRTN) and blood urea nitrogen (BUN). Before conducting the above-mentioned animal experiments on mice, the initial levels of creatinine and BUN in their urine are recorded. Immediately after the administration of propolis, the final levels of creatinine and BUN in the mouse urine are recorded. The creatinine (CRTN) determination experiment utilizes the principle of colorimetric determination using a creatinine detection kit (QuantiChrom™ Creatinine Assay Kit (DICT-500)). After a quantitative amount of urine is added to the creatinine detection kit, the picrates and creatinine react to produce a red compound. The absorbance is measured at a wavelength of 510 nanometers, and then the creatinine concentration is calculated. The blood urea nitrogen (BUN) determination experiment utilizes the urea test kit (QuantiChrom™ Urea Assay Kit (DIUR-500)) and colorimetric determination principle. The urea test kit is based on the modified Jung method. After a quantitative amount of urine is added to the urea test kit, the colorimetric reagent reacts with urea to produce a colored complex. The absorbance is measured at a wavelength of 520 nanometers, and then converted to obtain the urea nitrogen concentration. The average values of the experimental results for creatinine and urea nitrogen are shown in Figures 3(A) to 3(D). The creatinine concentrations in Figures 3(A) and 3(B) are calculated using the following formula: The urea nitrogen concentrations in Figures 3(C) and 3(D) are calculated using the following formula: .

The results in Figures 3(A) to 3(D) show that when propolis was administered at a dose of 20 mg/kg to kidney-damaged mice, it effectively reduced the levels of creatinine (CRTN) and blood urea nitrogen (BUN), thereby achieving a kidney-protective effect.

The liver protein analysis of this invention utilizes Western ink dot analysis to detect the protein expression levels of α-smooth muscle actin (α-SMA) and collagen Iα1 (Col Iα1) in mouse liver. First, before the experiment, mice were divided into three groups and administered propolis at 0 mg/kg body weight via syringe: 1. Normal control group: administered only solvent (5% EtOH); 2. Negative control group: administered 2.5 mg/kg body weight of aristolochic acid via intraperitoneal (i.p.); 3. Experimental group: administered 20 and 200 mg/kg body weight of aristolochic acid (AA) plus propolis. After feeding once daily for 28 consecutive days, creatinine (CRTN) and blood urea nitrogen (BUN) levels were tested. Mice were then sacrificed, and their livers were surgically removed. Total protein was extracted, and 50 mg of the obtained protein was analyzed by electrophoresis at 120 volts using a 1.5 mm thick discontinuous acrylamide gel, with a 5% acrylamide gel on top and a 12% acrylamide gel on the bottom. Next, the colloid was transferred onto a PVDF membrane. After shaking and washing three times for 10 minutes each time with TBST buffer (24.22 g Tris, 87.75 g NaCl, 10 mL Tween 20, diluted with water to 1 liter), primary antibody was added and the membrane was incubated at 4°C for one day. The next day, the membrane was shaken and washed three times for 10 minutes each time with TBST buffer. Secondary antibody was then added, and the membrane was incubated at room temperature for one hour. Finally, the membrane was shaken and washed three times for 10 minutes each time with TBST buffer. The PVDF membrane was then coated with enhanced chemiluminescence (ECL). After the reaction, the intensity of the cold light was detected by a fully automated cold light analyzer. The results are shown in Figure 2, with β-actin as the internal control group.

The results in Figure 4 show that, compared with the control group mice that were not given propolis, the propolis-treated mice showed significantly reduced protein expression of α-smooth muscle actin (α-SMA) and collagen Iα1 (Col Iα1), indicating that propolis can prevent kidney damage by controlling the protein expression of α-smooth muscle actin (α-SMA) and collagen Iα1 (Col Iα1).

This invention discloses a sting-free propolis pharmaceutical composition for preventing kidney and liver damage and inhibiting kidney inflammation. The composition comprises an effective amount of an active ingredient and a pharmaceutically acceptable carrier. The active ingredient is selected from a compound of formula (I) or its pharmaceutically acceptable salts and esters, while the carrier is a diluent or excipient. This pharmaceutical composition can enhance the protein expression of antioxidant enzymes SOD and glutathione peroxidase GPx and inhibit the protein expression of type II epoxidase COX-2, as well as reduce urinary creatinine (CRTN) and blood urea nitrogen (BUN) levels, and inhibit α-smooth muscle actin (α-SMA), collagen Iα1 (Col Iα1), or Smad2/3. The protein's performance helps to slow down or prevent the process of tissue fibrosis, and pathological section results show that it does not cause renal toxicity in low doses (20 mg/kg) of non-stinged propolis alcohol extract.

The pharmaceutical composition of this invention can be administered at different frequencies, such as once a day, multiple times a day, or once every few days, depending on the needs of the target. For example, when used to suppress exercise fatigue, the dosage of the drug, calculated based on the compound of formula (I), is approximately 10 mg/kg body weight to approximately 80 mg/kg body weight per day, preferably approximately 15 mg/kg body weight to approximately 70 mg/kg body weight per day, where the unit "mg/kg body weight" refers to the amount of drug required per kilogram of body weight.

The pharmaceutical composition of this invention can be used in veterinary and human medicine, and can be administered in any form and by any suitable manner, such as oral, subcutaneous, intravenous, or intra-articular injection, but is not limited thereto. For example, the delivery form is selected from the following group: intravenous injection, nasal drops, inhaled medication, sublingual tablets, sitz baths, local injections, topical skin preparations, oral administration, eye drops, topical ointments, intramuscular injections, subcutaneous injections, intradermal injections, sustained-release formulations, and controlled-release formulations. Taking the preparation of a drug formulation suitable for oral administration as an example, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier that will not adversely affect the activity of rutin, such as: solvents, oily solvents, thinners, stabilizers, absorption delayers, disintegrants, emulsifiers, antioxidants, binders, lubricants, humectants, etc. The composition can be formulated into a form suitable for oral administration using any suitable method, such as: tablets, capsules, granules, powders, fluid extracts, solutions, syrups, suspensions, emulsions, and tinctures, etc. As for the dosage form suitable for subcutaneous, intravenous, or intra-articular injection, the pharmaceutical composition of the present invention may contain one or more components such as isotropic solutions, salt buffers (such as phosphate buffers or citrate buffers), solubilizers, emulsifiers, and other carriers to prepare intravenous infusions, emulsion intravenous infusions, dry powder injections, suspension injections, or dry powder suspension injections.

The pharmaceutical composition of this invention may, as needed, contain flavoring agents, coloring agents, and other additives to improve the taste and visual appeal of the resulting drug. Appropriate amounts of preservatives, antiseptics, antibacterial agents, and antifungal agents may also be added to improve the shelf life of the resulting drug. Furthermore, as needed, the pharmaceutical composition of this invention may contain one or more other active ingredients to further enhance its efficacy or increase the flexibility and compatibility of the formulation. For example, the pharmaceutical composition of this invention may contain one or more of the following active ingredients: vitamins, whey protein, muscle relaxants, nonsteroidal anti-inflammatory drugs, and other active ingredients, provided that these other active ingredients do not adversely affect the efficacy of rutin.

The above detailed description is only for the purpose of describing the preferred feasible embodiments of the present invention. However, these embodiments are not intended to limit the scope of the patent application of the present invention. All other equivalent variations and modifications made without departing from the technical spirit disclosed in the present invention should be included in the scope of the patent application of the present invention.

In conclusion, the above-mentioned non-stinging propolis alcohol extract and its pharmaceutical composition for preventing kidney and liver damage and inhibiting kidney inflammation are indeed effective and purposeful. Therefore, this invention is a highly practical invention. To meet the requirements for an invention patent application, this application is hereby filed in accordance with the law. We hope that the patent committee will grant this application as soon as possible to protect the inventor's hard work. If the patent committee has any questions, please do not hesitate to write to us for guidance. The inventor will do his best to cooperate. We appreciate your assistance.

Graph 1 (A) shows the percentage of superoxide dismutase (SOD) activity relative to phosphate-buffered saline (PBS). Graph 1 (B) shows the percentage of glutathione peroxidase (GPx) activity relative to phosphate-buffered saline (PBS). Graph 2 shows the protein expression of COX-2 relative to the β-actin control group. Graph 3 (A) shows the plasma creatinine (CRTN) concentration. Graph 3 (B) shows the urine creatinine (CRTN) concentration. Graph 3 (C) shows the plasma urea nitrogen (BUN) concentration. Figure 3 (D) shows the concentration of blood urea nitrogen (BUN) in urine. Figure 4 shows the expression of α-smooth muscle actin (α-SMA) and collagen Iα1 (Col Iα1) in the aristolochic acid nephropathy (AN) experiment using sting-free propolis extract.

Claims (2)

A sting-free propolis alcohol extract for the prevention of kidney and liver damage and the inhibition of kidney inflammation, wherein the sting-free propolis alcohol extract is extracted with an ethanol solvent, and wherein the sting-free propolis extract contains at least one class of flavonoid compounds, including at least one propolisin (Propolin G). As in claim 1, the main component of this type of flavonoid compound is protocatechuic acid (PCA).