CN109985045A - Betulinic acid prepares the purposes for the treatment of leptin resistance related drugs as leptin sensitizer - Google Patents
Betulinic acid prepares the purposes for the treatment of leptin resistance related drugs as leptin sensitizer Download PDFInfo
- Publication number
- CN109985045A CN109985045A CN201910389301.0A CN201910389301A CN109985045A CN 109985045 A CN109985045 A CN 109985045A CN 201910389301 A CN201910389301 A CN 201910389301A CN 109985045 A CN109985045 A CN 109985045A
- Authority
- CN
- China
- Prior art keywords
- leptin
- betulinic acid
- mouse
- fat
- treatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
Landscapes
- Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Diabetes (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Medicines Containing Plant Substances (AREA)
- Child & Adolescent Psychology (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Epidemiology (AREA)
Abstract
The purposes for treating leptin resistance related drugs is prepared as leptin sensitizer the invention discloses a kind of betulinic acid.The present invention gives betulinic acid treatment, detection weight, Serum leptin levels, liver tg, insulin tolerance and carbohydrate tolerance test and blood lipids index using high in fat, ob/ob and db/db obesity mice as research object.The results show that betulinic acid can significantly mitigate high-fat adiposity mouse weight, improve leptin resistance, reduce liver tg, improve insulin resistance, blood lipid is reduced；Ob/ob and db/db mouse gives betulinic acid treatment without loss of weight and improves glycolipid metabolism effect, and cellular level confirms that betulinic acid combines the expression that can enhance p-STAT3 with leptin.The present invention treats obesity, nonalcoholic fatty liver, diabetes B and hyperlipidemia as leptin sensitizer for betulinic acid and provides foundation, meanwhile, new orientation treatment is provided to develop more treatments and the biological active constituents from natural medicines of leptin resistance related disease.
Description
Technical field
The invention belongs to technical field of medicine preparation, in particular to a kind of betulinic acid is thin as the preparation treatment of leptin sensitizer
Element resists the purposes of related drugs.
Background technique
Leptin (leptin) is the protein product of Expression of Obese Gene, mainly plays and makees and in conjunction with leptin receptor
With.Leptin receptor is widely present in the histoorgans cell surface such as hypothalamus, liver, kidney, heart and lung, and leptin mainly passes through
In conjunction with hypothalamic leptin receptor play its central nervous system effect: lose weight, reduce appetite, reduce Energy intaking and
Increase energy consumption；The peripheral action of leptin includes adjusting glycometabolism balance, inhibit Fatty synthesis and promoting lipolysis, is participated in
Hematopoiesis and the adjusting of immune function promote growth etc..In conjunction with leptin receptor extracellular domain part, activation downstream tyrosine swashs leptin
Enzyme JAK2 makes JAK2 phosphorylation, and the JAK2 of phosphorylation can make intracellular section of protein phosphorylation of leptin receptor, and then activate downstream transcription
Factor S TAT3 and STAT5 plays physiological function in turn.There is hyperleptinaemia more in overweight people, generally existing leptin resistance, simply
Say to be exactly that effect to leptin is insensitive or reactionless.Therefore, leptin resistance, enhancing leptin sensibility are corrected for obesity
Treatment and improvement Abnormality of Glycolipid Metabolism have very important significance.
With the continuous improvement of people's living standards, fat disease incidence is continuously increased, obesity is considered as 21 century stream
Row disease.The World Health Organization points out that the death of at least 2,800,000 people can every year in the global problem of obesity report of publication in 2014
It is attributed to overweight or fat.Obesity is not only a kind of independent disease, and leads to diabetes B, blood fat disorder, non-alcoholic
Fatty liver, a series of important risk factor of diseases related generations of metabolism such as cardiovascular disease.Obesity has become the current mankind
One of public health challenge of the most serious faced.According to heating power principle of dynamics, treating obesity will be from reduction Energy intaking
And (or) increases energetic supersession and set about.The method for theoretically treating obesity is very simple, but actually many method treatment obesities
Validity is very limited.Clinically the fat method of received treatment has much at present, including movement, diet and reduction energy
Amount intake or the drug therapy absorbed etc. would generally take operative treatment for extremely fat patient.Regrettably, only very
Few some people can be by adhering to moving and (or) dieting maintaining normal type, though operation weight loss effect is good, operation is held
The raw incidence of the risk and complicated by postoperative of load is all very big.Currently, a kind of small side of and risk prominent still without weight loss effect
Method is applied to clinical Bariatric.
Non-alcoholic fatty liver disease (non-alcoholic fatty liver disease, NAFLD) is a kind of no excess
History of drinking history, with liver cell diffusivity steatosis and fat store up based on clinical pathology syndrome.NAFLD is a kind of and metabolism
Closely related disease, the illness rate of western countries NAFLD have been raised to 20-30%, and the disease incidence of obese people NAFLD is even
More than 50%.In recent years, with the continuous improvement of people's living standards, the illness rate of China some areas adult NAFLD also
More than 15%.NAFLD is a kind of most common chronic liver disease, with obesity, hyperinsulinemia, diabetes B and cardiovascular and cerebrovascular
Event is closely related, and quite a few patient can progress to fatal End-stage liver disease, it has also become global important public to defend
One of raw problem.NFALD not only seriously affects personal health status, but also also brings along to social public medical system huge
Burden.The most basic performance of NAFLD is exactly the excessive accumulation of triglycerides in liver cell, the triglycerides source in liver cell
In the intake and the synthesis of itself of liver cell.On the other hand, the decrease of liver fat acid beta-oxidation and the decrease of VLDL secretion
It can promote the deposition of liver lipids.Problem, which occurs, for any of the above-described link all can cause triglycerides in the over-deposit of liver.Needle
The treatment of NAFLD patient is had been a hot spot of research.Current Primary Care includes correcting bad life style, control drink
Food, movement, weight-reducing etc.；Class of medications includes using slimming drugs, insulin sensitizer, fat regulation medicine, liver protection anti-inflammatory drug etc.,
But still there is not the traditional Chinese and western medicine that NAFLD conventional therapy is formally directly used in by examination & approval without the specific medicament of evidence-based medicine EBM verifying yet
Drug.Influence and NAFLD in view of metabolic syndrome correlative factor to NAFLD patient's long term prognosis inherently belong to constantly
The process disease of deterioration, it would therefore be highly desirable to develop the effective traditional Chinese and western medicine drug for being applicable in NAFLD treatment applied to clinic.
Diabetes B is a kind of chronic metabolic disease, mostly occurs in 40 years old or more the middle-aged and the old, present 2 type glycosuria
The age of onset of disease is intended to rejuvenation.With the continuous improvement of people's living standards, change and the society of living habits
The disease incidence of the raising of aging degree, diabetes B is continuously increased, it has also become after cardiovascular disease and malignant tumour
Another seriously endangers the important Chronic Non-Communicable Diseases of human health.Cut-off was to 2011, global about 3.66 hundred million people
With diabetes, it is contemplated that the year two thousand thirty, global diabetes total number of persons is up to 5.22 hundred million people.Diabetes and its various acute and chronics are simultaneously
Crippling and lethal caused by hair disease has become the big disease in third place in the world of current threat human health, seriously affects glycosuria
The quality of life of patient.Meanwhile bring social pressures and financial burden also increasingly aggravate.Have become that the whole world is important public to be defended
One of raw problem.Therefore, finding effective treatment method becomes social urgent problem to be solved.
Blood lipid is neutral fat (triacylglycerol and cholesterol) and lipoid (phosphatide, glycolipid, sterol, steroids) in blood plasma
General name, be widely present in human body, be the necessary substance of life cells basic metabolism.Main component in blood lipid is that trigalloyl is sweet
Oil and cholesterol, wherein triacylglycerol participant energy i (in vivo) is metabolized, and the main synthetic cell serous coat of cholesterol, steroid hormone
And bile acid.Hyperlipidemia is that one or more of lipid structures are unbalance in the blood plasma due to caused by body fat metabolic disorder
Disease.Its diagnostic criteria be blood total cholesterol (TC) > 5.72mmol/L and/or low density lipoprotein cholesterol (LDL-C) >
3.12mmol/L and (or) Triglycerides in Serum (TG) > 1.70mmol/L.The rapid development of China's economy and people's life water
It is flat to improve so that cerebrovascular disease incidence of disease is continuously increased, and hyperlipidemia be induce the significant risk of cardiovascular and cerebrovascular disease because
Element, blood lipid one of the Critical policies up to standard for being considered as preventing and treating cardiovascular and cerebrovascular disease.Improve dietary structure and drug therapy is blood lipid
Main path up to standard, however, the blood lipid compliance rate in China is not high, therefore, control blood lipid is that a society faces very
Severe problem.
Betulinic acid (Betulinic Acid, BA, also known as betulinic acid) is a kind of natural pentacyclic triterpenoid, is deposited
It is in many plants, such as the bark of silver birch, the root of compositae plant Aplotaxis auriculata (Salgssurea lappa Clarke),
The pharmacotoxicological effect of betulinic acid includes antitumor, antiviral, anti-inflammatory, anti parasitic etc..Up to the present, betulinic acid can be used as
The drug that leptin sensitizer treats obesity, nonalcoholic fatty liver, diabetes B and hyperlipidemia is not yet reported.At this
In invention, the obesity mice that inventor uses high fat diet to induce as disease animal model, model by high fat diet induction from
And make mouse that obese state, nonalcoholic fatty liver be presented, and impaired glucose tolerance and hyperlipidemia occur, while having used thin
The ob/ob obesity mice of plain gene defect and the db/db mouse of leptin receptor gene defect are as disease animal model, comparison three
The difference of the fat mouse models phenotype of kind.This three kinds fat mouse models are widely used in the research of metabolic disease.
Betulinic acid in treatment tumour, the effect in terms of antiviral, anti-infective and anti parasitic by called optical imaging, for
Metabolic disease therapeutic effect is still unknown.The present invention is directed to the Mice model of obesity and tool of creation are fed by high lipid food
There is ob/ob the and db/db Mice model of obesity of gene defect, comprehensive careful weight for observing disease mice and in glycolipid metabolism
The phenotype of aspect, specifically having inquired into betulinic acid improves the effect of related disease as leptin sensitizer, clinically to support with leptin
The treatment of the disease of decorrelation provides certain evidence, has important scientific value and medical significance, while can also be out
The active ingredient of Chinese herbs for sending out leptin sensitizer more provides valuable experience.
Summary of the invention
The present invention is intended to provide betulinic acid prepares the purposes for the treatment of leptin resistance related drugs as leptin sensitizer.Birch
Acid may be used as correlation and the leptins such as preparation treatment obesity, nonalcoholic fatty liver (NAFLD), diabetes B and hyperlipidemia
Resist the drug of related disease.By the obese model mouse of application betulinic acid processing, there is significant loss of weight and improve liver
Dirty glycolipid metabolism effect.
The purpose of the present invention is achieved through the following technical solutions:
The present invention relates to a kind of betulinic acids to treat disease related to leptin resistance as the preparation of leptin (leptin) sensitizer
The purposes of the drug of disease.
The betulinic acid is by being used to prepare treatment antiobesity agents, the non-alcohol for the treatment of as leptin (leptin) sensitizer
Property fatty liver (NAFLD) drug, treatment diabetes B drug and treatment one or more of hyperlipidemia.
The invention further relates to a kind of betulinic acids in the drug that preparation treats using leptin (leptin) sensitizer as target spot
Purposes.
In one embodiment of the invention, using the betulinic acid as active constituent, in addition pharmaceutically acceptable auxiliary material
Or complementary ingredient is prepared into pharmaceutical preparation.
In the pharmaceutical preparation, the content of betulinic acid is >=98% (being measured with HPLC method or other methods), applies agent
Amount is 20-5000mg/kg.
The pharmaceutical preparation is selected from one of tablet, pulvis, granule, capsule, oral solution, sustained release agent.
The invention further relates to a kind of treatments using leptin sensitizer as the pharmaceutical preparation of the disease of target spot, and the pharmaceutical preparation is
Using betulinic acid as active constituent, also contain pharmaceutically acceptable auxiliary material or complementary ingredient.
Inventive principle of the invention is: betulinic acid (Betulinic Acid, BA) is a kind of natural pentacyclic triterpene
Compound is present in the root including compositae plant Aplotaxis auriculata and the medium many plants of Japanese birch bark, has very strong pharmacology living
Property and wide spectrum range, and it is highly-safe.But betulinic acid treats disease related to leptin resistance as leptin (leptin) sensitizer
Disease is not yet reported.Betulinic acid (with HPLC method measured) of the present invention with content >=98%, application dose 150mg/kg,
It is added into high lipid food and mouse is fed.It is intended to create disease mice model by High-fat diet, is birch
Acid treatment related disease provides certain evidence, has important scientific value and medical significance.Meanwhile betulinic acid being added general
Logical forage feed ob/ob (leptin shortage) and two kinds of Mice model of obesity of db/db (leptin receptor defect) compare betulinic acid to three
The difference of kind Mice model of obesity effect phenotype, illustrates the pharmacotoxicological effect target spot of betulinic acid, to develop more leptins
(leptin) active ingredient of Chinese herbs of sensitizer provides valuable experience, is the application discovery with huge clinical landscapes.
Compared with prior art, the invention has the following beneficial effects:
It has been reported that leptin (leptin) sensitizer it is relatively fewer.Leptin receptor itself without tyrosine kinase activity, but
It can be coupled with JAK tyrosine kinase.PTP1B can be such that leptin receptor associated kinase JAK2 dephosphorylation inactivates, and lead to leptin receptor
Response cannot be generated to leptin, so as to cause leptin resistance.PTP1B specific inhibitor is studied with the effect of enhanced sensitivity leptin, at
The hot spot in field is developed for slimming drugs in recent years.Although there is many natural PTP1B inhibitor all to show with clinical application valence
Value prospect, but there is presently no the PTP1B inhibitor of clinical use ratified by FDA, reason may be due to inhibitor
Activity it is low or selectivity it is bad.Firstly, natural PTP1B catalyst structure domain has very high charge, in addition, PTP1B with
The certain similitude of the active structure and binding site of other PTPs, this causes its inhibitor low to the activity of PTP1B and selection
Property it is bad, therefore, at present design PTP1B inhibitor oral drugs become one very have challenge task.
Metreleptin is the agonist of leptin receptor, and acquisition FDA approval listing is for congenital or acquired within 2014
The treatment of body fat dysbolism；Then obtain within 2017 the treatment that EMA approval is used for familial local fat dysbolism.
Metreleptin once entered the development project as universal antiadipositas drug object, but since patient's body generates antibody evidence
Occur and it is halted.In recent years, finding a kind of novel leptin (leptin) sensitizer becomes the hot spot of slimming medicine research field,
Celastrol and withaferin A is the novel leptin sensitizer of two classes, facilitates the tolerance for overcoming leptin.Leptin
(leptin) sensitizer can be used as the new therapeutic agent of the metabolic diseases such as treatment obesity, therefore develop the leptin of high-efficiency low-toxicity
(leptin) leptin sensitizer has major clinical significance.
The present invention provides betulinic acid by pressing down as leptin (leptin) sensitizer by the research to betulinic acid
The fat process of high fat diet inducing mouse processed reduces Fatty synthesis, improves liver lipids deposition, corrects insulin resistance and height
Pionemia, the treatment for obesity, nonalcoholic fatty liver, diabetes B, hyperlipidemia and leptin resistance related disease provide
New direction.
Detailed description of the invention
Fig. 1 is the molecular structural formula of betulinic acid；
Fig. 2 is to give control high lipid food and high lipid food feeding 2 months containing betulinic acid under the same conditions
The mouse weight (a) of C57BL/6J；Control normal diet is given under the same terms and the normal diet containing betulinic acid feeds 1
The ob/ob mouse weight (b) of the moon；Control normal diet is given under the same terms and the normal diet containing betulinic acid feeds 1
The db/db mouse weight (c) of the moon；
Fig. 3 is to compare mouse group high in fat and+BA mouse group serum leptin level high in fat；
Fig. 4 is to compare mouse group high in fat and+BA mouse group liver tg high in fat level (a)；Ob/ob control mice group
It is horizontal (b) with BA group liver tg；Db/db control mice group and BA group liver tg are horizontal (c)；
Fig. 5 is to compare mouse group high in fat and+BA mouse group insulin tolerance test (a) high in fat and carbohydrate tolerance test (b) knot
Fruit；Ob/ob control mice group and BA group insulin tolerance test (c) and carbohydrate tolerance test (d) result；Db/db control mice group
With BA group insulin tolerance test (e) and carbohydrate tolerance test (f) result；
Fig. 6 is to compare mouse group high in fat and+BA mouse group serum total cholesterol content detection (a) high in fat；Ob/ob mouse pair
According to group and BA group serum total cholesterol content detection (b)；Db/db control mice group and BA group serum total cholesterol content detection
(c)；
Fig. 7 is to compare mouse group high in fat and+BA mouse group serum LDL cholesterol content detection (a) high in fat；
Ob/ob control mice group and BA group serum LDL cholesterol content detection (b)；Db/db control mice group and BA group
Serum LDL cholesterol content detection (c)；
Fig. 8 is to compare mouse group high in fat and+BA mouse group serum triglyceride content detection high in fat；Ob/ob control mice
Group and BA group serum triglyceride content detection (b)；Db/db control mice group and BA group serum triglyceride content detection (c)；
Fig. 9 is Primary adipocyte control group, BA processing group, leptin processing group and BA+leptin processing group phosphorylation
STAT3 expression.
Specific embodiment
Below with reference to embodiment, the invention will be further described:
Experimental animal: C57BL/6J mouse, ob/ob mouse and db/db mouse, 6~8 week old, healthy SPF grades of male, purchase
In Beijing Vital River Experimental Animals Technology Co., Ltd.；Environment temperature is 22 ± 0.5 DEG C, 12 hours/12 hours light and shade alternatings.
Feed is provided by Shanghai Fan Bo Bioisystech Co., Ltd, and wherein high lipid food heat group becomes 60% fat, 20% carbon water
Compound, 20% protein；High in fat plus betulinic acid feed fills betulinic acid according to the relationship of trial test mouse weight and food-intake
Divide and mix into above-mentioned high lipid food, it is ensured that the betulinic acid of every 1kg mouse weight feed about 150mg.
Experimental data indicates that experimental group and control group are compared with mean ± standard error, * P < 0.05, * * p < 0.01, * * * p <
0.001, n=15.
Instrument: blood glucose meter and test paper are purchased from Omron automation (China) Co., Ltd.
Drug and reagent: betulinic acid (it is 98% that HPLC method, which measures content) is purchased from Nanjing Guang Run company (Fig. 1)；Serum Leptin Levels
It detects ELISA kit and is purchased from R&D company；Liver and serum triglyceride detection kit are purchased from Sigma company；The total gallbladder of serum
Sterol detection kit is purchased from Sigma company；Serum LDL cholesterol detection kit is purchased from Sigma company；Phosphorus
It is acidified STAT3 antibody and is purchased from CST company.
Specifically, the present invention uses 6~8 week old, healthy SPF grades of male, the mouse model of C57BL/6J, by experiment mice
It is randomly divided into control group high in fat and+BA intervention group high in fat, gives control high lipid food and the high lipid food containing betulinic acid respectively
It feeds 8 weeks, periodic monitoring weight during this period.Wherein the content of betulinic acid is >=98% (being measured with HPLC method), application dose
For 150mg/kg.After modeling success, at high fat diet the 9th week, insulin tolerance test (ITT) is implemented to two groups of mouse, it is high in fat
It feeds the 10th week, dextrose tolerance test (GTT) is implemented to two groups of mouse.Before putting to death mouse, with the mode for plucking eyeball and taking blood
Two groups of Mouse whole bloods are taken, serum is left and taken after whole blood centrifugation, detects serum indices；Mouse liver is left and taken, liver glycerol is detected
Three ester contents.As a result, it has been found that the obesity mice group weight of betulinic acid processing is significantly lower than high in fat group, leptin resistance, liver are improved
Dirty tissue lipid metabolism is obviously improved, while correcting for insulin resistance；Serum total cholesterol, low density lipoprotein cholesterol,
And triglycerides makes moderate progress in betulinic acid processing group.Ob/ob mouse and db/db mouse after giving betulinic acid treatment not
There is above-mentioned phenotype.Mouse primary fat cell is extracted, is found after being handled respectively with BA, leptin and BA+leptin, BA+
Leptin processing group expression of STAT 3 phosphorylation amount highest.Therefore, betulinic acid can be used as leptin (leptin) sensitizer, be used as
The drug of preparation treatment and leptin resistance related disease.
Embodiment 1
The influence of obesity mice, ob/ob mouse and db/db mouse weight that betulinic acid induces high fat diet:
After a week through normal diet adaptable fed, mouse is randomly divided into two groups, every group 15, guarantees that two groups of mouse are flat
Equal original body mass is identical.High lipid food and high in fat plus betulinic acid forage feed two months is given respectively, weighs mouse every three days
Weight.Two groups of mouse original body mass indifferences start mouse high in fat on the 7th day in administration and high in fat plus betulinic acid mouse weight are opened
There is difference in beginning, and high in fat plus betulinic acid group weight is lower than high in fat group (P < 0.05), and changes over time difference and be gradually increased,
After administration 20 days, high in fat plus betulinic acid group weight is significantly lower than high in fat group (P < 0.01), after administration one month, two groups of Mice Bodies
Method of double differences value more increases (P < 0.001) (Fig. 2 a).
After a week through normal diet adaptable fed, ob/ob mouse is randomly divided into two groups, every group 15, guarantees two groups
The mouse original body mass that is averaged is identical.Normal diet and general food plus betulinic acid forage feed two months are given respectively, are weighed every three days
The weight of mouse.Two groups of mouse weights indifference (Fig. 2 b) always.
After a week through normal diet adaptable fed, db/db mouse is randomly divided into two groups, every group 15, guarantees two groups
The mouse original body mass that is averaged is identical.Normal diet and general food plus betulinic acid forage feed two months are given respectively, are weighed every three days
The weight of mouse.Two groups of mouse weights indifference (Fig. 2 c) always.
Experimental result shows, betulinic acid can significantly inhibit the body weight increase of high fat diet mouse, and to ob/ob mouse and
Db/db mouse weight illustrates that betulinic acid by acting on leptin and (or) leptin receptor, increases leptin sensibility and plays without influence
Weight loss effect.
Embodiment 2
The influence for the obesity mice Serum leptin levels that betulinic acid induces high fat diet:
Serum leptin levels reduction is the one of the important signs that leptin resistance is eased.Therefore, it takes respectively high in fat and high
Rouge dosing group mice serum detects Serum leptin levels with ELISA method.
The specific detection method is as follows: preparing mice serum leptin standard items first, in accordance with doubling dilution method, (kit mentions
For)；Prepare 1 × HRP Wash Buffer 500ml, is used for board-washing；Plate is taken, 300 μ 1 × HRP of l Wash are added in every hole
Buffer is cleaned three times, spare；It is separately added into 30 μ l Assay Buffer into background hole, gauge orifice, adds into sample well
Enter 40 μ l Assay Buffer, then is separately added into 30 μ l Matrix Solution into background hole, gauge orifice；Next, back
10 μ l Assay Buffer are added in Jing Kongzhong, and 10 μ l mouse Leptin standard items of gradient dilution are added in gauge orifice, and sample well is added
10 μ l test serums；50 μ l Antiserum Solution, sealing plate are added in every hole, and room temperature shakes (400-500rpm) 2 hours；
Liquid in hole is removed, 1 × HRP Wash Buffer, 300 μ l is cleaned three times；100 μ l Detection are added in every hole
Antibody, sealing plate, room temperature shake (400-500rpm) 1 hour；Remove liquid in hole, 1 × HRP Wash Buffer300 μ l
Cleaning three times；100 μ l Enzyme Solution, sealing plate are added in every hole, and room temperature shakes (400-500rpm) 30 minutes；Remove hole
Interior liquid, 1 × HRP Wash Buffer, 300 μ l are cleaned six times；100 μ l Substracte Solution, envelope is added in every hole
Plate is protected from light room temperature concussion (400-500rpm) 5-20 minutes；Finally, 100 μ l Stop Solution, upper machine testing is added in every hole
(dominant wavelength: 450nm, commplementary wave length: 590nm).
Compared with common mouse high in fat, betulinic acid group mice serum leptin high in fat is substantially reduced, and difference has statistics
Meaning (P < 0.001) (Fig. 3).
Experimental result shows that high fat diet mice serum leptin is decreased obviously after betulinic acid processing, side light birch
Wood acid can improve the leptin resistance of high-fat adiposity mouse.
Embodiment 3
Influence of the betulinic acid to mouse liver tissue high in fat:
Caused by nonalcoholic fatty liver (NAFLD) is typically due to liver lactone deposition, and triglycerides is wherein most
Important index.Therefore, mouse liver tissue is taken to carry out liver TG measurement at the end of experiment.
Specific assay method is as follows: carrying out tissue homogenate first, the 5% of 500ul is added in the EP pipe of 1.5ml
NP40 takes out about 30mg liver specimens and is placed on the inside (recording specific weight).Ice bath is on beveller with 45s*60HZ grinding two
It is secondary that homogenate is made.Sample supernatants are fully transferred to 1.5mL centrifuge tube afterwards.Then by the water-bath 5 minutes of 95 degree of the EP pipe of 1.5ml
(having white precipitate to be formed) is placed at room temperature for 10 minutes；95 degree water-bath 5 minutes again, then it is placed at room temperature for 10 minutes.Maximum (top) speed
13300g is centrifuged 2 minutes, room temperature.In the EP pipe for shifting supernatant about 100ul to new 0.6ml after centrifugation.With phosphoglycerol
Enzymatic measurement surveys TG.It is arranged blank tube three, wherein 2 μ L distilled waters are added, setting calibration pipe is single, wherein middle be added 2 schools μ L
Quasi- product are added 2 μ L serum samples in sample tube, 125 μ L R1,37 DEG C of water-bath 5min after mixing are added in each enzyme mark hole；Then
62.5 μ L R2 are added in each hole, mix, 37 DEG C of water-bath 5min.Wherein TG ratio at dominant wavelength 550nm, commplementary wave length 660nm
Color, TC colorimetric at dominant wavelength 546nm, commplementary wave length 660nm.With distilled water school zero when calculating, each pipe absorbance is read.It calculates public
Formula: TG (mmol/L)=(sample tube absorbance-blank tube absorbance)/(calibration pipe absorbance-blank tube absorbance) x2.26
(calibration object concentration), TC (mmol/L)=(sample tube absorbance-blank tube absorbance)/(calibration pipe absorbance-blank tube extinction
Degree) x5.17 (calibration object concentration).
Compared with common mouse high in fat, betulinic acid group mouse liver triglycerides (TG) level high in fat is remarkably decreased, difference
With statistical significance (P < 0.01) (Fig. 4 a), ob/ob mouse (Fig. 4 b) and db/db mouse (Fig. 4 c) liver tg without
Notable difference.
Experimental result shows that betulinic acid can reduce liver esters deposition by increasing leptin sensibility, treats non-alcohol
Property fatty liver (NAFLD).
Embodiment 4
Influence of the betulinic acid to three kinds of fat mouse islets element tolerances and glucose tolerance:
After a week through normal diet adaptable fed, mouse is randomly divided into two groups, every group 15, guarantees that two groups of mouse are flat
Equal original body mass is identical.It feeds 8 weeks, after modeling success, at high fat diet the 9th week, insulin tolerance examination is implemented to two groups of mouse
It tests (ITT), feeds the 10th week, dextrose tolerance test (GTT) is implemented to two groups of mouse.
ITT test: the same day it is early 8 when change padding and hungry mouse, by insulin according to the dosage of 1U/kg mouse when afternoon 13
It prepares spare, while mouse is weighed and being numbered, get syringe, blood glucose meter and test paper ready.Tail, which is cut, when afternoon 14 takes blood,
Detect fasting blood-glucose；It is injected intraperitoneally according to 10ml/kg insulin dose, measures blood glucose after 15,30,60,90,120min
And it records；Return cage to food after the completion of test.
GTT test: changing padding and hungry mouse when proxima luce (prox. luc) 18, by glucose according to the dosage of 1g/kg mouse when next day 8
It prepares spare, while mouse is weighed and being numbered, get syringe, blood glucose meter and test paper ready.Start to cut tail when 9 and take blood,
Detect fasting blood-glucose；It is injected intraperitoneally according to 10ml/kg glucose dose, measures blood glucose after 15,30,60,90,120min
And it records；Return cage to food after the completion of test.
Compared with common mouse high in fat, betulinic acid group mouse islets element tolerance high in fat and glucose tolerance are superior to control height
Rouge group mouse (Fig. 5 a, b), difference have statistical significance (p < 0.001 * P < 0.05, * * p < 0.01, * * *)；Ob/ob mouse (figure
5c, d) and db/db mouse (Fig. 5 e, f) insulin tolerance and glucose tolerance no significant difference.
Experimental result shows that betulinic acid can significantly improve insulin resistance by increasing leptin sensibility, reduces blood glucose.
Embodiment 5
Influence of the betulinic acid to three kinds of fat mice serum total cholesterols, low density lipoprotein cholesterol and triglycerides:
Serum total cholesterol (TC) measuring method is as follows: mainly being detected using ultraviolet spectrophotometry.To blank tube
4 μ l distilled waters of middle addition, 4 μ l standard items are added into standard pipe, and 4 μ l mice serums are added in sample cell, and reagent R1 is added
Next reagent R2 125 μ l, 37 DEG C of water-bath 5min, dominant wavelength 550nm, commplementary wave length is added in 250 μ l, 37 DEG C of water-bath 5min
The upper machine testing of 660nm.Calculation formula is total cholesterol (mmol/L)=(sample tube absorbance-blank tube absorbance)/(standard pipe
Absorbance-blank tube absorbance) * calibration object concentration.
Serum LDL cholesterol (LDL) measuring method is as follows: mainly being examined using ultraviolet spectrophotometry
It surveys.3 μ l distilled waters are added into blank tube, 3 μ l standard items are added into standard pipe, 3 μ l mice serums are added in sample cell,
Be added reagent R1 150 μ l, 37 DEG C of water-bath 5min, next be added reagent R2 50 μ l, 37 DEG C of water-bath 5min, dominant wavelength 600nm,
Machine testing on commplementary wave length 700nm.Calculation formula is low density lipoprotein cholesterol (mmol/L)=(sample tube absorbance-blank
Pipe absorbance)/(standard pipe absorbance-blank tube absorbance) * calibration object concentration.
Serum triglyceride (TG) measuring method is as follows: mainly being detected using ultraviolet spectrophotometry.To blank tube
3 μ l distilled waters of middle addition, 3 μ l standard items are added into standard pipe, and 3 μ l mice serums are added in sample cell, and reagent R1 is added
Next reagent R2 125 μ l, 37 DEG C of water-bath 5min, dominant wavelength 550nm, commplementary wave length is added in 250 μ l, 37 DEG C of water-bath 5min
The upper machine testing of 660nm.Calculation formula is triglycerides (mmol/L)=(sample tube absorbance-blank tube absorbance)/(standard pipe
Absorbance-blank tube absorbance) * calibration object concentration.
High fat diet mice serum total cholesterol (Fig. 6 a), the low density lipoprotein cholesterol (figure handled by betulinic acid
7a) and triglycerides (Fig. 8 a) concentration is declined (p < 0.001)；Ob/ob mouse (Fig. 6 b, 7b, 8b) and db/db mouse (figure
6c, 7c, 8c) control group and medication group no significant difference.
The experimental results showed that high in fat plus betulinic acid group mice serum total cholesterol, low density lipoprotein cholesterol and glycerol
Three esters relatively compare high in fat group of mouse and are reduced, and betulinic acid, without influence, illustrates betulinic acid to ob/ob mouse and db/db mouse
The blood lipid of high fat diet obesity mice can be improved by increasing leptin sensibility, treat hyperlipidemia.
Embodiment 6
The influence that betulinic acid expresses leptin receptor downstream signaling molecule:
Specific operation process is as follows: mouse subcutaneous fat precursor extracted, six orifice plates are spread, inducer is added, it will be subcutaneous
Preadipocyte induction is mature fat cell, and cell is divided into four groups, respectively control group (Con), BA group (BA),
Leptin group (Lep10) and BA+leptin group (B+L10), BA dosage are 10 μM, and the dosage of leptin is 10nM, dosing object thorn
Cell is received after swashing 30 minutes, extracts total protein of cell, is tested with Western blot to detect pSTAT3 (p-
STAT3) expression (Fig. 9).
Experimental result shows that BA+leptin group can obviously be enhanced compared with leptin group with the expression of pSTAT3, and BA can
To promote leptin receptor downstream signaling molecule STAT3 phosphorylation, illustrates that BA increases leptin sensibility, be leptin sensitizer.
In conclusion the high-fat adiposity mouse group of betulinic acid processing can lose weight, Serum Leptin Levels content, liver are reduced
Triglycerides is reduced, and improves insulin resistance；Serum total cholesterol, low density lipoprotein cholesterol and triglycerides are in birch
Sour processing group makes moderate progress, the enhancing of leptin sensibility, and betulinic acid is unobvious to ob/ob mouse and the effect of db/db mouse.It says
Bright betulinic acid can be used as leptin (leptin) sensitizer really to be played and loses weight, and is reduced Serum leptin levels, is corrected non-wine
Essence fatty liver improves insulin resistance, treats hyperlipidemia, which is the application hair with huge clinical landscapes
It is existing.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (6)
1. a kind of betulinic acid is in the purposes as leptin sensitizer preparation treatment and the drug of leptin resistance related disease.
2. purposes according to claim 1, which is characterized in that the betulinic acid is by being used to make as leptin sensitizer
In standby treatment antiobesity agents, treatment nonalcoholic fatty liver drug, treatment diabetes B drug and treatment hyperlipidemia
It is one or more of.
3. a kind of purposes of betulinic acid in the drug that preparation treats using leptin sensitizer as target spot.
4. purposes described in any one of claim 1 to 3, which is characterized in that using the betulinic acid as active constituent, add
Upper pharmaceutically acceptable auxiliary material or complementary ingredient are prepared into pharmaceutical preparation.
5. purposes according to claim 4, which is characterized in that in the pharmaceutical preparation, the content of betulinic acid is >=98%,
Its application dose is 20-5000mg/kg.
6. purposes according to claim 4, which is characterized in that the pharmaceutical preparation is selected from tablet, pulvis, granule, glue
One of capsule, oral solution, sustained release agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910389301.0A CN109985045A (en) | 2019-05-10 | 2019-05-10 | Betulinic acid prepares the purposes for the treatment of leptin resistance related drugs as leptin sensitizer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910389301.0A CN109985045A (en) | 2019-05-10 | 2019-05-10 | Betulinic acid prepares the purposes for the treatment of leptin resistance related drugs as leptin sensitizer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109985045A true CN109985045A (en) | 2019-07-09 |
Family
ID=67136489
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910389301.0A Pending CN109985045A (en) | 2019-05-10 | 2019-05-10 | Betulinic acid prepares the purposes for the treatment of leptin resistance related drugs as leptin sensitizer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109985045A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113599502A (en) * | 2021-07-22 | 2021-11-05 | 廖儒佳 | Composition of withaferin A and leptin and application of composition in preparation of medicine for preventing and/or treating hemorrhagic stroke |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018017940A1 (en) * | 2016-07-21 | 2018-01-25 | Baylor College Of Medicine | Use of betulinic acid for the treatment or prevention of nonalcoholic steatohepatitis and nonalcoholic fatty liver disease |
| CN108478582A (en) * | 2018-05-30 | 2018-09-04 | 上海市内分泌代谢病研究所 | Application of the betulinic acid as aliphatic acid synthase inhibitor |
| CN108771682A (en) * | 2018-05-30 | 2018-11-09 | 上海市内分泌代谢病研究所 | Betulinic acid promotes the application of fatty acid oxidation |
-
2019
- 2019-05-10 CN CN201910389301.0A patent/CN109985045A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018017940A1 (en) * | 2016-07-21 | 2018-01-25 | Baylor College Of Medicine | Use of betulinic acid for the treatment or prevention of nonalcoholic steatohepatitis and nonalcoholic fatty liver disease |
| CN108478582A (en) * | 2018-05-30 | 2018-09-04 | 上海市内分泌代谢病研究所 | Application of the betulinic acid as aliphatic acid synthase inhibitor |
| CN108771682A (en) * | 2018-05-30 | 2018-11-09 | 上海市内分泌代谢病研究所 | Betulinic acid promotes the application of fatty acid oxidation |
Non-Patent Citations (3)
| Title |
|---|
| AKRAM AHANGARPOUR 等: "The effect of betulinic acid on leptin, adiponectin, hepatic enzyme levels and lipid profiles in streptozotocin-nicotinamide-induced diabetic mice", 《RESEARCH IN PHARMACEUTICAL SCIENCES》 * |
| CELIO L.DE MELO 等: "Betulinic Acid, a Natural Pentacyclic Triterpenoid, Prevents Abdominal Fat Accumulation in Mice Fed a High-Fat Diet", 《J. AGRIC. FOOD CHEM.》 * |
| 陈珠 等: "肾茶功效相关药理作用研究进展", 《药物资讯》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113599502A (en) * | 2021-07-22 | 2021-11-05 | 廖儒佳 | Composition of withaferin A and leptin and application of composition in preparation of medicine for preventing and/or treating hemorrhagic stroke |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cheraghpour et al. | Hesperidin improves hepatic steatosis, hepatic enzymes, and metabolic and inflammatory parameters in patients with nonalcoholic fatty liver disease: A randomized, placebo‐controlled, double‐blind clinical trial | |
| Zhu et al. | A newly identified polysaccharide from Ganoderma atrum attenuates hyperglycemia and hyperlipidemia | |
| Xiong et al. | Antiobesity and antihyperglycemic effects of ginsenoside Rb1 in rats | |
| Ibrahim et al. | Butanol fraction of Parkia biglobosa (Jacq.) G. Don leaves enhance pancreatic β-cell functions, stimulates insulin secretion and ameliorates other type 2 diabetes-associated complications in rats | |
| Wan et al. | Panax notoginseng reduces atherosclerotic lesions in ApoE-deficient mice and inhibits TNF-α-induced endothelial adhesion molecule expression and monocyte adhesion | |
| Najmi et al. | Effect of Nigella sativa oil on various clinical and biochemical parameters of metabolic syndrome | |
| Guo et al. | Guava leaf extracts promote glucose metabolism in SHRSP. Z-Leprfa/Izm rats by improving insulin resistance in skeletal muscle | |
| Balakrishnan et al. | Moringa concanensis Nimmo ameliorates hyperglycemia in 3T3-L1 adipocytes by upregulating PPAR-γ, C/EBP-α via Akt signaling pathway and STZ-induced diabetic rats | |
| Bhavsar et al. | Effect of saponins from Helicteres isora on lipid and glucose metabolism regulating genes expression | |
| Xu et al. | Scutellarin protects against diabetic cardiomyopathy via inhibiting oxidative stress and inflammatory response in mice | |
| CN109364269A (en) | A kind of composition, evaluation method and its preparation predicted and treat diabetes B | |
| Kamani et al. | Efficacy of Emilia coccinea aqueous extract on inhibition of α-amylase enzyme activity and insulin resistance in dexamethasone treated-rats | |
| CN108014118B (en) | A kind of purposes of notoginsenoside Ft1 | |
| Lin et al. | Aqueous extract of Solanum nigrum attenuates Angiotensin-II induced cardiac hypertrophy and improves cardiac function by repressing protein kinase C-ζ to restore HSF2 deSUMOlyation and Mel-18-IGF-IIR signaling suppression | |
| CN105012329B (en) | It is a kind of for treating the drug of type-II diabetes | |
| JPS58172317A (en) | 26-hydroxycholesterol medicine for unstriated muscle cell proliferation inhibition and cerebrotendinous xanthomatosis detection | |
| CN109985045A (en) | Betulinic acid prepares the purposes for the treatment of leptin resistance related drugs as leptin sensitizer | |
| CA3022247C (en) | Composition for treating diabetic disease | |
| CN108771682A (en) | Betulinic acid promotes the application of fatty acid oxidation | |
| CN101879151A (en) | Application of emodin in preparing medicaments for treating P2X3 mediated neuropathic pains/nerve system diseases | |
| Yu et al. | Atractylodin alleviates cancer anorexia-cachexia syndrome by regulating NPY through hypothalamic Sirt1/AMPK axis-induced autophagy | |
| Alshehri | Antidiabetic activity of cissus rotundifolia plant growing in Saudi Arabia | |
| Pan et al. | Association of gliclazide and left ventricular mass in type 2 diabetic patients | |
| US20080026084A1 (en) | Method and composition for management of weight and blood sugar | |
| CN106727599A (en) | A kind of blocking agent of the protein activation of lymphocyte antigen 96 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190709 |
|
| RJ01 | Rejection of invention patent application after publication |