TWI684455B - Use of composition of active ingredients in organic extract of andrographis paniculata in treating hyperlipidemia - Google Patents

Abstract

The present invention relates to a composition of active ingredients in the organic solvent extract of Andrographis paniculata stem for the use in preventing and/or treating hyperlipidemia of Diabetes patients.

Description

The active ingredient composition of andrographis paniculata organic solvent extract reduces diabetes Application of blood lipids in patients

The present invention relates to a new use of an Andrographis paniculata organic solvent extract with a hypoglycemic active ingredient composition for reducing blood lipids in diabetic patients. More particularly, the present invention relates to a composition of an active ingredient (APai) of andrographis paniculata which mainly contains andrographolide, neo-andrographolide and deoxydehydroandrographolide to reduce triacid in blood of diabetic patients Application of glycerides and/or cholesterol.

Diabetes (Diabetes mellitus, DM) is a disease that changes the way the body converts glucose (sugars) into energy, and is characterized by hyperglycemia accompanied by disturbances in fat and protein metabolism. Dyslipidemia is a common concomitant disease of diabetics, especially obese diabetics. Dyslipidemia is a risk factor for atherosclerosis and other vascular diseases. Therefore, it is not enough to control blood sugar unilaterally in diabetic patients. It is also important to lower blood lipids.

Blood lipids refer to fats in the blood, mainly including cholesterol and triglycerides. When the concentration of cholesterol or triglycerides circulating in the blood is higher than normal, it is called hyperlipidemia. When cholesterol or triglyceride value is too high, or high density lipoprotein concentration is too low, it is also called dyslipidemia. Clinically Up to 6 to 8 percent of diabetes patients will have hypertension and hyperlipidemia, and treatment is often more complicated, and diabetic patients often have obesity, so hyperlipidemia is more likely to occur. The mechanism of diabetes combined with high blood fat is complicated. The most common types include the rise of blood triglyceride, the decrease of high density lipoprotein cholesterol (HDL-C) and the increase of low density lipoprotein cholesterol (LDL-C). The pathogenic mechanism is mainly due to the decrease of lipolytic enzyme activity that breaks down fat, and the increased ability of the liver to synthesize and release triglycerides; due to the reduction of lipolytic enzyme activity, it causes the metabolism of good cholesterol (HDL-C) The speed increases, resulting in a decrease in HDL-C in the blood.

Hyperlipidemia plays a very important role in various factors related to the formation of diabetes, such as coronary arteries, cerebrovascular, and vascular sclerosis and obstruction of the lower extremities. On the one hand, hyperlipidemia is inherently prone to changes in the above blood vessels The combination of diabetes and hyperlipidemia accelerates the occurrence of such changes, so it is necessary to strictly control hyperlipidemia when actively controlling diabetes. The treatment of hyperlipidemia is most often treated with drugs, but the US Food and Drug Administration has warned that commonly used statin cholesterol-lowering drugs may increase the risk of diabetes and memory impairment. According to the analysis of the study, among the 6 statins, Lovastatin and Simvastatin, if taken for a long time, may make the person who takes it have a 5% higher risk of diabetes than those who do not take it. Therefore, it is the main objective of the present invention to find effective lipid-lowering ingredients from conventional Chinese herbal medicines.

Andrographis paniculata ( Andrographis paniculata ) is a perennial herbaceous plant in the family Cymbidium. 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. It is widely cultivated in China, India, Southeast Asia and Taiwan. The gas is light, the taste is extremely bitter and lasts. In Malaysia, Andrographis paniculata is known as "Hempedu Bumi", which means "Earth's Bile" and one of the most bitter plants in traditional medicine. In the Five Elements Doctrine of Traditional Chinese Medicine, it is believed that bitterness comes into the heart, and andrographis paniculata can be felt immediately as long as you include a small piece of its leaf, and the kind of hard-earned bitterness seems to go straight into your heart, hence the name "Andrographis paniculata".

The medicinal parts of Andrographis paniculata are dry stems and leaves. Andrographis paniculata leaves contain diterpene lactone compounds: Deoxyandrographolide (Deoxyandrographolide) 0.1% or more, Andrographolide (Andrographolide) 1.5% or more, Neoandrographolide (Neoandrographolide) 0 .2% or more, and high andrographolide (Homoandrographolide), panicolide (Panicolide). It also contains Andrographan, Andrographon, Andrographosterin, β-sitosterol-D-glucoside and so on. In addition to andrographolide, the root also contains 5-hydroxy-7,8,2",3"-tetramethoxyflavonoid (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 etc. The whole plant still contains 14-deoxy-11-oxoandrographolide (14-Deoxy-11-oxoandrographolide), 14-deoxy-11,12-didehydroandrographolide (14-Deoxy-11, 12- didehydroandrographolide). According to preliminary analysis, it also contains phenols such as sterol saponins, sugars and condensed tannins. And the healing tissues obtained from leaves, shoots, hypocotyls, roots and germs can be isolated by culture, and three sesquiterpene lactone compounds can be obtained: Paeniculides A, B and C (Pa-niculides A, B, C).

Andrographis paniculata water extract (AP _HE ) and andrographolides (Andrographolides, A) have been published with functions of lowering blood sugar, blood fat, antibacterial, anti-inflammatory and anti-cancer. Among them, andrographolide is known to have the effect of removing heat and detoxification, anti-inflammatory and analgesic, and has a special effect on bacterial and viral upper respiratory tract infections (acute and chronic tonsillitis and sore throat), bacterial dysentery and gastroenteritis (Md. Sanower Hossain et al., The Scientific World Journal 2014: 1-28, 2014). Republic of China Patent I355933 has described an andrographolide as a tumor necrosis factor (TNF-α) antagonist and its pharmaceutical composition. In addition, andrographolide has been used as an active ingredient to prepare a pharmaceutical composition for healing wounds and/or whitening skin (Republic of China Invention Patent Publication No. 201524532).

The present invention aims at the organic solvent extract with hypoglycemic activity contained in the dried stem of Andrographis paniculata, and at the same time studies its prevention and treatment of hyperlipidemia in diabetic patients. The organic extract of the dried stem of Andrographis paniculata according to the present invention has been analyzed, and the active ingredient (APai) with hypoglycemic activity contains index compounds with specific content: Andrographolides (A), Neoandrographolide (Neoandrographolide, NA) ), and deoxydehydroandrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, DDA), has a significant effect on reducing blood lipid and cholesterol concentrations in diabetic patients.

Therefore, one aspect of the present invention relates to the use of an andrographis paniculata organic solvent extract for preparing a composition for reducing blood lipids in diabetic patients, the andrographis paniculata organic solvent extract mainly contains 180-400 mg/g andrographolide (Andrographolides, A), 40-90mg/g neoandrographolide (Neoandrographolide, NA) and 16-50mg/g deoxydehydroandrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, DDA). In a preferred embodiment of the present invention, the andrographis paniculata organic solvent extract mainly includes 230-300 mg/g andrographolide, 50-70 mg/g neo-andrographolide and 20-40 mg/g deoxydehydrogen Andrographolide.

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

In some embodiments of the present invention, the active ingredient (APai) of andrographis paniculata organic solvent extract contains 230-400 mg/g andrographolide (Andrographolides, A), 50-90 mg/g neoandrographolide (Neoandrographolide, NA) and 20-50mg/g deoxydehydroandrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, DDA). In a preferred embodiment of the present invention, the active ingredient of andrographis paniculata organic solvent extract (APai) contains 230-280mg/g andrographolide, 50-70mg/g neo-andrographolide and 20-40mg/ g Deoxydehydroandrographolide.

In some embodiments of the present invention, the andrographis paniculata organic solvent extract contains 99-100% andrographis paniculata organic solvent extract active ingredient (APai), wherein the active ingredient (APai) contains 25%-40% andrographis paniculata Lactone, 5%-9% andrographolide and 2%-5% deoxydehydroandrographolide. 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% deoxydehydroandrographolide.

Another aspect of the present invention relates to a composition for reducing blood lipids in diabetic patients, comprising an andrographis paniculata organic solvent extract having pharmacological activity for reducing blood sugar, characterized in that the organic solvent extract contains 23%-40% Andrographolide, 5%-9% new andrographolide and 2%-5% deoxydehydroandrographolide.

In some embodiments of the present invention, the composition is a pharmaceutical composition. In other embodiments of the present invention, the said composition is a health food composition. In some embodiments of the present invention, the pharmaceutical composition is used to treat or prevent hyperlipidemia in diabetic patients.

Another aspect of the invention relates to a pharmaceutical composition for reducing blood lipids in diabetic patients, comprising an andrographis paniculata organic solvent extract having pharmacological activity for lowering blood sugar and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments of the present invention, the pharmaceutical composition is used to reduce blood triglyceride concentration in diabetic patients. In other specific embodiments of the present invention, the pharmaceutical composition is used to reduce the cholesterol concentration in the serum of diabetic patients.

Figure 1 shows the multiple chromatograms of mixed standard solutions measured at 210, 232 and 254 nm.

Figure 2 shows the serum of the andrographis paniculata organic solvent extract APai of the present invention, which is repeatedly orally administered at various doses (E20, E40, E80 mg/kg) 28-days, to the serum of mice induced by nicotinic amide-streptomycin type 2 diabetes The effect of changes in the content of triglyceride (Triglyceride).

Fig. 3 shows the serum of the andrographis paniculata organic solvent extract APai of the present invention, which is repeatedly orally administered at various doses (E20, E40, E80 mg/kg) 28-days, to the serum of mice induced by nicotinic amide-streptomycin type 2 diabetes The effect of changes in cholesterol levels. Each value is expressed as an average ± SEM.

"Organic andrographis paniculata organic solvent extract" in this specification refers to the crude ethanol extract of andrographis paniculata dried stem is further extracted, concentrated and dried with organic solvents such as ethanol, n-hexane, n-butanol and ethyl acetate By. Through the activity test and analysis, the andrographis paniculata organic solvent extract of the present invention contains an active ingredient (APai) with hypoglycemic activity.

Other features and advantages of the present invention will be further exemplified and described in the following embodiment examples, and the embodiment examples are only used as an auxiliary description and are not intended to limit the scope of the present invention.

Example 1: Preparation of andrographis paniculata organic solvent extract

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

Further, using a two-solvent-HPLC analysis system, the content of the main constituent substances of the active ingredient composition (APai) of the andrographis paniculata organic solvent extract of the present invention is determined. By comparing the peak spectrum and purity of the standard and sample obtained by the photodiode array detector, it is determined that the corresponding andrographolide (A), neo-andrographolide (NA) and deoxydehydrogen in APai exist. The peak of andrographolide (DDA). FIG. 1 shows the HPLC multiple chromatograms of the active ingredient with the hypoglycemic effect of the andrographis paniculata organic solvent extract of the present invention at 210, 232 and 254 nm. The figure shows the absorption peak positions of andrographolide (A), new andrographolide (NA) and deoxydehydroandrographolide (DDA). The calculation results of the contents of A, NA and DDA in APai are shown in Table 1 below. Therefore, the content of andrographolide (A) is 235.10±1.64mg/g; the content of new andrographolide (NA) is 52.31±4.62mg/g; the content of deoxydehydroandrographolide (DDA) is 22.68±1.13mg/ g. The analysis values provided in this example will vary depending on the source, origin and season of andrographis paniculata harvested.

Example 2: Evaluation of the hypolipidemic effect of the active ingredient composition of andrographis paniculata organic solvent extract on streptozotocin STZ-induced type II diabetic mice

Male ICR mice (19-21g) were purchased from Taiwan BioLASCO Co., Ltd. The mice were fed in the animal center of the Universal University of Science and Technology, the temperature was controlled at 22±2℃, the relative humidity was 55±5%, and a 12-hour light/12-hour dark cycle was applied one week before the experiment. For the induction of type II diabetic mice, nicotine amide (Sigma, Saint Louis, MO, USA) saline solution (210 mg/kg bw) was injected intraperitoneally into mice, which will be dissolved before use after 15 minutes The mice were administered with streptozotocin (STZ) (Sigma, 180 mg/kg bw, ip injection) in citrate buffer (pH 4.5). The control group received those two carriers. During the experiment, the food intake and body weight of the experimental animals were recorded once a week. The dosage of the active ingredient composition of Andrographis paniculata organic solvent extract APai of the present invention is: group E10: APai 10 mg/kg; group E20: APai 20 mg/kg; group E40: APai 40 mg/kg; group E80: APai 80 mg/kg. Wherein, the active ingredient composition of the organic solvent extract of Andrographis paniculata prepared as described in the first example above is dissolved in Tween 20 and adjusted to a 0.5% methyl cellulose (CMC) solution.

Observe the animal's physical condition at least twice a day, and the interval between each observation is not less than 6 hours to determine the animal's health or death status. Observe the clinical signs of the test animals at least once a day, and record the toxic effects exhibited by the test animals, including the start time and process of such toxic effects. After the test, the surviving mice were sacrificed; afterwards, autopsy was performed and blood samples were taken for serum biochemical analysis. All experimental rat blood samples were left at room temperature for one hour to coagulate. The serum was separated by centrifugation at 12000 rpm for 5 minutes at 4°C in a refrigerated centrifuge.

Analysis of serum triglyceride (Triglyceride)

Triglyceride (TG) is composed of fatty acids and glycerin, accounting for 70% of VLDL and 10% of LDL. The triglycerides derived from the diet enter chylomicrons from the blood and lymphatic system into fatty tissues; the liver also synthesizes triglycerides from excess sugars, proteins, and fatty acids in case of unexpected needs. TG can be used to assess the lipid metabolism status of patients, especially the high-risk groups of secondary hyperlipidemia.

Use Randox set test group analysis. Take 500μL reaction reagent (40mM/L Pipes Buffer pH 7.6, 5.5mM/L 4-chlorophenol, 17.5mM/L magnesium ion, 0.5mM/L 4-aminopyrine, 1.0mM/L ATP, 150U/ml lipid Enzyme, 0.4U/ml glycerol-kinase, 1.5U/ml glycerol-3-phosphate oxidase, 0.5U/ml peroxidase) Add 5μL of serum, mix well and react at 20~25℃ for 5 minutes, use The spectrophotometer measures the absorbance at 500 nm.

Reaction formula of biochemical value of triglycerides

As shown in the analysis results in Figure 2, ^## P<0.001 is compared with the normal animal group (Sham group); *P<0.05, **P<0.01 is compared with the STZ induction treatment group. (One-way ANOVA followed by Scheffe's multiple range test). It was shown that the amount of triglyceride in the serum of STZ-induced diabetic mice was significantly increased, and there was a significant difference when compared with the normal Sham group, indicating that STZ-induced type II diabetic mice are at risk of causing hyperlipidemia. Treatment with Apai (E10, E20, E40, E80 mg/kg) of andrographis paniculata organic solvent extract of the present invention can significantly reduce the rise of triglyceride in the serum of type II diabetes mice induced by Nicotinamide-streptozotocin. After 28 days of oral administration, the concentration of triglyceride in the serum of diabetic mice can be restored to the level of the normal animal Sham group.

Cholesterol (Chloesterol) analysis

Use Randox set test group analysis. Take 1mL reaction reagent (0.3mM/L 4-aminoantipyrine, 6mM/L Phenol, 0.5U/ml peroxidase, 0.15U/ml cholesterol esterase, 0.1U/ml cholesterol Oxidase, 80mM/L Pipes Buffer pH 6.8) Add 10μL of serum, mix well and react at 20~25℃ for 5 minutes. Use a spectrophotometer to detect the absorbance at 500nm.

Biochemical value of total cholesterol

As shown in the analysis results of Fig. 3, ^## P<0.001 is compared with the normal animal group (Sham group). * P<0.05 was compared with STZ induction treatment group. (One-way ANOVA followed by Scheffe's multiple range test). It was shown that the serum cholesterol concentration in STZ-induced diabetic mice was significantly increased, which was significantly different from that in the normal animal Sham group, indicating that STZ-induced type II diabetic mice had increased cholesterol. However, treatment with APai (E10, E20, E40, E80 mg/kg) can reduce the increase of cholesterol in the serum of type II diabetic mice induced by nicotinamide-streptozotocin, indicating the andrographis paniculata organic solvent of the present invention The active ingredient composition of the extract, APai, can effectively reduce the clinical symptoms of hyperlipidemia in diabetic mice.

Based on the above experimental results, the active ingredient composition of the organic solvent extract of Andrographis paniculata of the present invention has the effect of reducing hyperlipidemia in mice with type 2 diabetes induced by nicotinamide-streptozotocin, including reducing the serum of diabetic mice The concentration of triglyceride and cholesterol shows that the andrographis paniculata organic solvent extract of the present invention can reduce the risk of diabetic patients complicated with hyperlipidemia and cardiovascular disease. Therefore, the active ingredient composition of the andrographis paniculata organic solvent extract of the present invention can be used in research and development and manufacture, and can simultaneously have the medicine or health food for lowering blood sugar and blood fat.

Claims (6)

Use of an Andrographis paniculata organic solvent extract containing an active ingredient (APai) for the preparation of a composition for reducing blood lipids in patients with type II diabetes, wherein the Andrographis paniculata organic solvent extract is obtained by crude ethanol from dried stems of Andrographis paniculata The extract was extracted with ethanol (95%) at room temperature for 36-72 hours for a total of 5-7 times, and n-hexane (100%) was extracted at room temperature for 12-36 hours for a total of 4-7 times, n-butanol (100% ) Extracted at room temperature for 12-48 hours a total of 7-10 times, ethyl acetate (100%) at room temperature for 12-36 hours a total of 7-10 times, through the system separation method for continuous extraction and separation, and Concentrated and dried and mainly contains 180-400mg/g andrographolide (Andrographolides, A), 40-90mg/g neoandrographolide (Neoandrographolide, NA) and 16-50mg/g deoxydehydroandrographolide (14-deoxy-11, 12-dehydrogen-andrographolide, DDA). The use according to claim 1, wherein the andrographis paniculata organic solvent extract comprises 23-30% andrographolide, 5-9% neo-andrographolide and 2-6% deoxydehydroandrographolide. The use according to claim 1, wherein the andrographis paniculata organic solvent extract comprises 23%-30% andrographolide, 4%-7% neoandrographolide and 2%-5% deoxydehydroandrographolide. The use according to claim 1, wherein the andrographis paniculata organic solvent extract is used to prepare a pharmaceutical composition for reducing blood fat in diabetes. The use according to claim 1, wherein the pharmaceutical composition comprises 20-80 mg/kg of the andrographis paniculata organic solvent extract with hypoglycemic activity. The use according to claim 1, wherein the andrographis paniculata organic solvent extract is used to prepare a health food composition.

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