TWI650124B - Use of composition of active ingredients in organic extract of andrographis paniculata in liver protection - Google Patents

Abstract

The invention relates to an organic solvent extract of Andrographis paniculata (AP) for protecting liver function of a diabetic patient, characterized in that the extract of Andrographis paniculata organic solvent is obtained by using ethanol as a crude extract of dried stem of Andrographis paniculata An organic solvent such as n-hexane, n-butanol or ethyl acetate is extracted, concentrated and dried, and contains an active ingredient (APai) mainly comprising 230-400 mg/g andrographolide (Andrographolides). , A), 50 - 90 mg / g neoandrographolide (NA) and 20 - 50 mg / g dehydrogenated andrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, DDA).

Description

Application of active ingredient composition of Andrographis paniculata organic solvent extract to protect liver function of diabetic patients

The present invention relates to a novel use of a hypoglycemic active ingredient composition of an extract of Andrographis paniculata organic solvent for protecting liver function. More particularly, the present invention relates to the use of an active ingredient (APai) composition of andrographolide organic solvent extract comprising mainly andrographolide, neoandrographolide and deoxyhydroandrographolide to protect liver function in diabetic patients. .

Andrographis paniculata is a perennial herb of the genus Jurassic. The stem is slender and dark green with longitudinal grooves and wing membranes at the corners. The stem has a square cross section. The leaves are lanceolate and glabrous. China, India, Southeast Asia and Taiwan are widely cultivated. It is mild, very bitter and long lasting. In Malaysia, Andrographis paniculata is called “Hempedu Bumi”, which means “earth bile” and one of the most bitter plants in traditional medicine. In the five elements of Chinese medicine, I think that bitterness comes into my heart, and if you wear a small piece of its leaves, you can feel it immediately. The kind of unforgettable bitterness seems to go straight into your heart, hence the name "through the heart."

The medicinal part of Andrographis paniculata is dry stems and leaves, and the penicillin leaves contain diterpene lactone compounds: Deoxyandrographolide 0.1% or more, Andrographolide (1.5%), Neoandrographolide (Neoandrographolide) ) 0.2% or more, and high andrographolide (Homoandrographolide), panisolide (Panicolide). It also contains Andrographan, Andrographon, Andrographosterin, β-sitosterol-D-glucoside and the like. In addition to the andrographolide, the root also contains 5-hydroxy-7,8,2",3"-tetramethoxylated flavonoids (Mono-o-methylwithtin), 5-hydroxy-7,8,2"-trimethyl Andrographin, 5,2"-dihydroxy-7,8-dimethoxyflavone (Panicolin), apigenin-7,4"-dimethyl ether (Apigenin-7,4"-dimethyl ether) , a1-sitosterol and KH ₂ PO ₄ and the like. Whole grass still contains l4-deoxy-ll-oxoandrographolide, 14-deoxy-11,12-didehydroandrographolide (14-Deoxy-ll,12- Didehydroandrographolide). According to preliminary analysis, it also contains phenolic substances such as sterol saponins, sugars and condensed tannins. The healing tissues obtained from leaves, shoots, hypocotyls, roots and germs can be isolated and cultured to obtain three sesquiterpene lactone compounds: lanolinide A, B and C (Pa-niculides A, B, C).

Andrographis water extract (AP _HE ) and andrographolide (Andrographolides, A) have been published to have functions of lowering blood sugar, lowering blood fat, antibacterial, anti-inflammatory and anti-cancer. Among them, andrographolide is known to have antipyretic, anti-inflammatory and analgesic effects, and has special effects on bacterial, viral upper respiratory tract infections (urgent, chronic tonsillitis and sore throat) and bacterial dysentery and gastroenteritis (Md. Sanower Hossain et al., The Scientific World Journal 2014: 1-28, 2014). The Republic of China Patent No. I355933 has described an andrographolide which is a tumor necrosis factor (TNF-α) antagonist and a pharmaceutical composition thereof. Further, a pharmaceutical composition for healing wounds and/or whitening skin has been prepared by using andrographolide as an active ingredient (Republic of China Patent 201524532).

Diabetes mellitus (DM) is a disease in which the body changes its metabolism of glucose (sugar) into energy, characterized by hyperglycemia accompanied by disorders of fat and protein metabolism. In addition to causing damage to the kidneys, heart, retina, nerves and other tissues and organs, diabetes can also cause various types of liver damage. Because of microvascular disease and microcirculatory disorder in diabetic patients, it can affect the ischemia and hypoxia of various organs in the body, and the liver is no exception. Ischemia can cause carbon dioxide accumulation in liver cells, acidosis, reduction of oxygen supply, increase of oxygen consumption, and liver transaminase. Increased activity, bilirubin metabolism disorder, severe cases can cause liver cell necrosis, especially when combined with diabetic ketoacidosis is more prone to liver damage.

The invention aims at the prevention and treatment of liver damage of diabetic patients by taking the organic solvent extract with hypoglycemic activity contained in the dry stem of Andrographis paniculata. The organic extract of the dried stem of Andrographis paniculata L. of the present invention is analyzed, wherein an active ingredient (APai) having a hypoglycemic pharmacological activity comprises a specific content of an indicator compound: Andrographolides (A), Neoandrographolide (NA) And deoxygenated androgenolactone (14-deoxy-11,12-dehydrogen-andrographolide, DDA).

Accordingly, one aspect of the present invention relates to the use of an extract of Andrographis paniculata organic solvent for preparing a composition for protecting liver function in a diabetic patient, wherein the extract of Andrographis paniculata organic solvent mainly comprises 180-400 mg/g andrographolide ( Andrographolides, A), 40-90 mg/g Neoandrographolide (NA) and 16-50 mg/g dehydrogenated andrographolide (DDA). In a preferred embodiment of the present invention, the extract of Andrographis paniculata organic solvent mainly comprises 230-300 mg/g andrographolide, 50-70 mg/g of new andrographolide and 20-40 mg/g. Oxygen dehydroandrographolide.

In some embodiments of the invention, the Andrographis paniculata organic solvent extract comprises 80-99% active ingredient (APai). In a specific embodiment of the present invention, the Andrographis paniculata organic solvent extract comprises 95-99% active ingredient (APai).

In some embodiments of the present invention, the andrographolide organic solvent extract active ingredient (APai) comprises 230-400 mg/g andrographolide (Andrographolides, A), 50-90 mg/g of new andrographolide ( Neoandrographolide, NA) and 20 - 50 mg/g dehydrogenated androgenolactone (14-deoxy-11,12-dehydrogen-andrographolide, DDA). In a preferred embodiment of the present invention, the andrographolide organic solvent extract active ingredient (APai) comprises 230-280 mg/g andrographolide, 50-70 mg/g of new andrographolide, and 20- 40 mg/g deoxygenated dehydroandrographolide.

In some embodiments of the present invention, the Andrographis paniculata organic solvent extract comprises 99-100% Andrographis paniculata organic solvent extract active ingredient (APai), wherein the active ingredient (APai) comprises 25%-40% Andrographis paniculata Lactone, 5%-9% new andrographolide and 2%-5% deoxygenated andrographolide. In a specific embodiment of the present invention, the Andrographis paniculata organic solvent extract comprises 25-30% andrographolide, 6-8% new andrographolide, and 3-4% deoxyhydroandrographolide.

Another aspect of the invention relates to a composition for protecting liver function of a diabetic patient, comprising an andrographis organic solvent extract having hypoglycemic pharmacological activity, characterized in that the organic solvent extract comprises 23%-40 % andrographolide, 5%-9% new andrographolide and 2%-5% deoxygenated andrographolide.

In some embodiments of the invention, the composition is a pharmaceutical composition. In other specific embodiments of the invention, the composition is a health food composition.

Another aspect of the present invention relates to a pharmaceutical composition for protecting liver function in a diabetic patient, comprising an andrographis organic solvent extract having hypoglycemic pharmacological activity and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments of the invention, the pharmaceutical composition is for reducing liver inflammatory conditions in a diabetic patient.

The "andrographis organic solvent extract" as referred to in the present specification means that the crude ethanol extract of the dried stem of Andrographis paniculata is further extracted, concentrated and dried by an organic solvent such as ethanol, n-hexane, n-butanol or ethyl acetate. By. Through the activity test and analysis, the Andrographis paniculata organic solvent extract of the present invention comprises an active ingredient (APai).

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Embodiment 1 Preparation of andrographis organic solvent extract.

The dried stem of Andrographis paniculata (4.0 kg) was chopped and extracted twice with 60 liters of ethanol under reflux. The combined extracts were concentrated under reduced pressure to give a dried crude extract (yield: 8.75%). The foregoing crude ethanol extract is sequentially subjected to the following organic solvents and extraction conditions: ethanol (95%) is extracted at room temperature for 36-72 hours for 5-7 times, and n-hexane (100%) is extracted at room temperature for 12- 4-7 times in 36 hours, n-butanol (100%) was extracted at room temperature for 12-48 hours for 7-10 times, and ethyl acetate (100%) was extracted at room temperature for 12-36 hours for a total of 7-10 Then, continuous extraction and separation are carried out by a system separation method, and then the obtained organic extract is concentrated and dried by a vacuum concentrator and an oven to obtain an extract powder, that is, the active ingredient of the organic solvent extract of the present invention (APai) .

Further, the content of the main constituent material of the active ingredient composition (APai) of the organic solvent extract of the present invention was determined by a two-solvent-HPLC analysis system. By comparing the peak spectrum and purity of the standards and samples obtained by the photodiode array detector, it is determined that the andrographolide (A), the new andrographolide (NA) and the deoxygenation dehydrogenation are corresponding to the presence of APai. The peak of andrographolide (DDA). BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows an HPLC multiple chromatogram of active compositions having hypoglycemic efficacy in an organic solvent extract of the present invention at 210, 232 and 254 nm. The calculation results of the contents of A, NA and DDA in the presence of APai are shown in Table 1 below. Therefore, the content of andrographolide (A) was 235.10±1.64 mg/g; the content of new andrographolide (NA) was 52.31±4.62 mg/g; the content of deoxyhydroandrographolide (DDA) was 22.68±1.13 mg/ g. The analytical values provided in this example will vary depending on the source, origin and season of the extract. Table 1. Contents of A, NA and DDA in APai

Embodiment 2 Andrographis organic solvent extract active ingredient composition for Streptozotocin STZ-induced type II Evaluation of liver protection efficacy in diabetic rats.

Male ICR mice (19-21 g) were purchased from BioLASCO Co., Ltd., Taiwan. Mice were fed an animal center at the University of Science and Technology of the United States, with a temperature regulation of 22 ± 2 ° C, a relative humidity of 55 ± 5% and a 12-hour light/12-hour dark cycle one week prior to the experiment. For the induction of type II diabetic rats, nicotinic acid amide (Sigma, Saint Louis, MO, USA) aqueous saline solution (210 mg/kg bw) was intraperitoneally injected into mice, and 15 minutes later, before use. The mice were administered to streptomycin (STZ) (Sigma, 180 mg/kg bw, ip injection) dissolved in citrate buffer (pH 4.5). The control group received the two carriers. The food intake and body weight of the experimental animals were recorded once a week during the experiment. The dosage of APai administered by the active ingredient of Andrographis paniculata organic solvent extract is: E10 group: APai 10 mg/kg; E20 group: APai 20 mg/kg; E40 group: APai 40 mg/kg; E80 group: APai 80 mg /kg. Among them, the active ingredient composition of Andrographis paniculata organic solvent extract prepared as in the above Example 1 was dissolved in Tween 20 and adjusted to a 0.5% methylcellulose (CMC) solution.

Observe the physical condition of the animal at least twice a day, with an interval of not less than 6 hours per observation to determine the health or death of the animal. The clinical signs of the test animals were observed at least once a day, and the toxic effects exhibited by the test animals were recorded, including the start time and course of the toxic effects. At the end of the experiment, the surviving mice were sacrificed; then autopsy was performed and blood samples were taken for serum biochemical analysis. All experimental mouse blood samples were allowed to stand at room temperature for one hour to cause coagulation. The serum was separated by centrifugation at 12,000 rpm for 5 minutes at 4 ^o C in a refrigerated centrifuge, and the liver biochemical index was measured by an automatic biochemical analyzer. The principles of AST and ALT were based on Reitman & Frankel (1957) and International Federal Clinical Chemistry (IFCC). , 1986 a, b) standard method.

AST analysis:

Aspartate Aminotransferase (AST), also known as Serum Glutamic-Oxalocetic Transaminase (GOT or SGOT), is mainly found in the heart, kidneys, liver, muscles and mind. When organ tissue is damaged, AST is released into the bloodstream, causing the AST of the blood to rise. Therefore, AST rise is an important indicator of liver damage.

As shown in the analysis of Figure 2, the serum levels of AST in STZ-induced diabetic rats were significantly increased, which was significantly different from that in the normal Sham group, indicating that STZ-induced type II diabetic rats had liver inflammation. Treatment with Apai (E10, E20, E40, E80 mg/kg) reduced the AST elevation in serum of type II diabetic mice induced by Nicotinamide-streptozotocin. After oral administration for 28 days at E80 mg/kg, diabetes The AST values in mouse serum were restored to be comparable to the normal animal Sham group.

ALT analysis:

Alanine Aminotransferase (ALT), also known as Serum Glutamic-Pyruvic Transaminase GPT or SGPT. ALT is an enzyme that is mainly found in liver cells. When the liver is damaged, ALT is released into the bloodstream, causing the blood's ALT to rise. Therefore, to test liver function, ALT is more specific and better judged than AST.

As shown in the analysis of Fig. 3, the amount of AST in the serum of STZ-induced diabetic rats was significantly increased, which was significantly different from that of the normal animal Sham group, indicating that STZ-induced type II diabetic rats had liver inflammation. Treatment with APai (E10, E20, E40, E80 mg/kg) reduced serum ALT elevation in mice with type II diabetes induced by nicotinamide-streptomycin and was dose-dependent. It is indicated that the active ingredient composition APai of the andrographis paniculata organic solvent extract of the present invention can effectively alleviate the clinical symptoms of liver inflammation in diabetic mice.

Liver antioxidant enzyme Crust Activity analysis of glutathione peroxidase (GSH Px) :

Observe the physical condition of the animal at least twice a day, with an interval of not less than 6 hours per observation to determine the health or death of the animal. The clinical signs of the test animals were observed at least once a day, and the toxic effects exhibited by the test animals were recorded, including the start time and course of the toxic effects. At the end of the experiment, the surviving mice were sacrificed; then autopsy was performed, liver samples were taken, minced and homogenized for biochemical analysis.

The GSH Px activity assay is based on hydrogen peroxide (H ₂ O ₂ ). Reduced glutathione (GSH) can reduce hydrogen peroxide via glutathione peroxidase (GSH Px) catalysis, while reduced glutathione becomes oxidized glutathione, then oxidized bran Glutathione is reduced to reduced glutathione by glutathione reductase (GSH Rd) and NADPH. Take 5 ml of liver cytoplasmic sample and 95 ml of 20 mM potassium phosphate buffer (pH 7.0), and add 0.8 ml of 100 mM potassium phosphate buffer solution (pH 7.0) to the reaction mixture (containing 1 mM EDTA, 1 mM NaN ₃ , 0.2 mM). NADPH, 1 U/ml GSH Rd and 1 mM GSH), let stand for 5 minutes at room temperature, add 0.1 ml of 2.5 mM hydrogen peroxide, and measure at 340 nm for three minutes (25 °C) with a spectrophotometer. The rate of NADPH reduction was calculated, and the activity of glutathione peroxidase was indirectly determined, and 5 ml of deionized water was used as a blank group.

The results in Figure 4 show that administration of STZ significantly reduced the GSH-Px concentration in the liver of the treated mice, which was significantly different from the normal Sham group. APai (E40, E80 mg/kg) can alleviate the decrease of GSH-Px in the liver of type II diabetic mice induced by nicotinamide-streptozotocin, even in the E80 mg/k treatment group, which can be increased to the normal Sham group. , indicating that APai can reduce liver damage caused by hyperglycemia.

Observation of liver histopathology ( Histopathological observation) :

The liver was harvested, and 1 cm square of liver tissue was cut at the same position of the largest right lobe and placed in 10% neutral fumarin for further hematoxylin-Eosin stain staining. In the comparison of histopathology, semi-quantitative methods of Shackelford et al. (2002) ( Toxicologic Pathology 30: 93-96, 2002), degree of hepatocyte inflammation, lipid degeneration, hepatocytes in chronic liver injury Semi-quantitative analysis of necrosis and bile duct hyperplasia. The degree of injury is classified into five grades: 1 = minimal (<1%); 2 = slight (1-25%); 3 = Moderate (26-50%); 4 = moderate/severe (51-75%); 5 = severe/severe (76-100%).

As a result, as shown in Fig. 5, treatment with the amarylaceous organic solvent extracts APai (E10, E20, E40, E80 mg/kg) of the present invention can improve liver tissue damage in STZ-induced diabetic mice.

Based on the above experimental results, the active ingredient composition of the andrographis paniculata organic solvent extract of the present invention has liver function damage in mice which are induced by nicotinamide-streptinomycin type II diabetes, including reduction of serum of diabetic rats. Liver function index and liver biochemical index can also prevent and repair liver cells damage, steatosis, necrosis and other tissue symptoms of diabetic rats, indicating that the organic solvent extract of Andrographis paniculata can reduce liver damage caused by hyperglycemia, especially The effect achieved at doses of 40 and 80 mg/kg was most pronounced. Therefore, the active ingredient composition of the Andrographis paniculata organic solvent extract of the present invention can be used for research and development, and can simultaneously have a blood sugar lowering and natural liver protecting effect medicine or health food.

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Figure 1 shows the multiple chromatograms of the mixed standard solution at and below. The figure shows the absorption peak of andrographolide (A), neoandrographolide (NA) and deoxyhydroandrographolide (DDA). position.

Figure 2 is a diagram showing the pericardium organic solvent extract APai of the present invention administered orally at 28-days in various doses (E20, E40, E80 mg/kg) for niacinamide-streptomycin-induced type II diabetic mice. The effect of changes in serum AST content. ^### P < 0.001 is compared with the normal animal group (Sham group); * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the STZ induction treatment group. (One-way ANOVA followed by Scheffe's multiple range test)

Figure 3 is a diagram showing the pericardium organic solvent extract APai of the present invention administered orally at 28-days in various doses (E20, E40, E80 mg/kg) for nicotine indoleamine-streptinomycin-induced type II diabetic mice. The effect of changes in serum ALT levels. Each value is expressed as mean ± SEM. ^### P < 0.001 is compared to the normal animal group (Sham group). * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the STZ induction treatment group. (One-way ANOVA followed by Scheffe's multiple range test)

Figure 4 is a diagram showing the pericardium organic solvent extract APai of the present invention administered orally at 28-days in various doses (E20, E40, E80 mg/kg) for niacinamide-streptomycin-induced type II diabetic mice. The effect of changes in liver GSH-Px concentration. Each value is expressed as mean ± SEM. ^### P < 0.001 is compared to the normal animal group (Sham group). * P < 0.05, ** P < 0.01 compared to the STZ induction treatment group. (One-way ANOVA followed by Scheffe's multiple range test)

Figure 5 shows the normal group of Sham (A), STZ-induced type II diabetic mice (B), and STZ-induced type II diabetic mice with APai-10 (E10 mg/kg) (C), with APai- 20 (E20 mg/kg) (D), APai-40 (E40 mg/kg) (E) and STZ+APai-80 (E80 mg/kg) (F) 28-day repeated oral administration of mice Hematoxylin-eosin staining results of liver tissue sections. The magnification is 400x.

Claims (6)

The use of an aqueous extract of Andrographis paniculata (APai) for preparing a pharmaceutical composition for protecting liver function of a type II diabetic patient, wherein the extract of Andrographis paniculata organic solvent is obtained by sequentially using ethanol as a crude extract of dried stem of Andrographis paniculata The alkane, n-butanol and ethyl acetate are extracted and separated, and concentrated and dried to obtain mainly 180-300 mg/g andrographolide (A), 40-70 mg/g neoandrographolide (NA) And 16-40 mg / g dehydrogenated andrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, DDA). The use of claim 1, wherein the andrographis organic solvent extract has hypoglycemic pharmacological activity. The use according to claim 1, wherein the andrographolide organic solvent extract comprises 23-30% andrographolide, 5-9% new andrographolide, and 2-6% deoxyhydroandrographolide. The use of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. The use according to claim 1, wherein the pharmaceutical composition comprises 20-80 mg/kg of the andrographis organic solvent extract. The use of claim 1, wherein the andrographis paniculata organic solvent extract is used to prepare a health food composition.