CN108210566A - A kind of limonin extract is preparing the application in treating diabetes medicament - Google Patents
A kind of limonin extract is preparing the application in treating diabetes medicament Download PDFInfo
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Abstract
The technical program discloses a kind of preparation method of limonin extract and its is preparing the application in treating diabetes medicament, what the limonin extract was prepared as follows：It using chinaberry leaf, Chinese Toon Leaves as raw material, extracts, separation, purifies the limonin extract formed to get mountain chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K, the limonin extract that the technical program provides is of great significance to preparing treatment diabetes medicament.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of limonin extract in treatment diabetes medicament is prepared
Application, be specifically related to limonin extract in a kind of chinaberry leaf Chinese Toon Leaves prepare treat in diabetes medicament should
With.
Background technology
Diabetes are a kind of metabolic diseases caused by hypoinsulinism or peripheral tissues are to insulin insensitivity
Disease characterized by lasting hyperglycemia state, and may cause various tissues, internal organs（Such as eye, kidney, heart, blood vessel, nerve）
Long-term damage, insufficiency or failure.According to pathogenesis difference, diabetes are divided into Type I diabetes, II type glycosuria
Disease, gestational diabetes mellitus and other types diabetes.
According to statistics, about 4.15 hundred million people suffer from diabetes in the people in whole world 20-79 Sui in 2015（Illness rate 8.8%）, separately
There are 3.18 hundred million people's impaired glucose tolerances outside（Early period illness rate 6.7%）.China is global the first big country of diabetic, 2015
Sufferer number is up to 1.096 hundred million people, and 1,300,000 people die of diabetes and its complication.It is predicted simultaneously according to IDF, if being not added with doing
In advance, global diabetic in 2040 will be up to 6.42 hundred million, and prediabetes crowd 4.81 hundred million, China patient populations will rise
To 1.54 hundred million.According to statistics, there is " 1/2 law " in diabetic at present, i.e., in global diabetic on average
Only 50% learns oneself illness.The epidemiological study being published according to 2013 professors Nian Ningguang on JAMA shows that China is sugared
The sick sufferer awareness of urine is only 30.1%.Meanwhile " quasi- diabetic " crowd may be up to 50.1% in Chinese adult.And
The U.S. shows that the ratio of " quasi- sugar people " is 37% in adult according to the survey data of CDC in 2014.
China's patient of diabetes awareness is only 30.1%, wherein only 25.8% patient obtains medical treatment, and is being treated
Patient in, blood glucose be well controlled only 39.7%, calculate accordingly, in diabetic, patient that blood glucose is controlled
Ratio is only 3.08%.In city and rural area, above-mentioned data are there are significant difference, and difference is also larger between different sexes, economical
The control situation of the female diabetic of under-developed area is very low.In the U.S., statistics shows that diabetes mellitus suffers from awareness and is about
75%, about 85.3% patient is treated using insulin or oral hypoglycemic agents.
Payment for medical care of the U.S. in 2015 in diabetes field is up to 320,000,000,000 dollars（Including insuring, treating, nursing expense
Deng）, global diabetes cost first is occupied, accounting is more than the whole world 10%.China occupies second, is 51,000,000,000 dollars.If we
Estimated according to two countries' diabetic's quantity, the year cost of the U.S. and the single diabetic of China be respectively 1092 with
465 dollars.
Domestic diabetic terminal market scale is 413.8 hundred million yuan within 2015.Increase by 6.8% on a year-on-year basis.Wherein：It is oral
Antidiabetic drug occupies 233.6 hundred million yuan of diabetic（Accounting 56.6%）, increased by 8.8% on a year-on-year basis.Wherein α-glucosidase inhibitors,
Sulfonylurea and biguanides difference accounting 30.1%, 24.1% and 14.8%.Insulin secretagogues class is total to accounting 29.2%.GLP-1 by
Body agonist and DPP-4 inhibitor accountings are only 1.4%.From the point of view of single variety, best-selling product front three be respectively acarbose,
Melbine and Repaglinide, the market share are respectively 27.9%, 14.8% and 13.2%.
At present there has been no the method for complete radical cure diabetes, control blood glucose is the core for the treatment of diabetes.For diabetes
For patient, the treatment of disease is the process of an integrated management, needs diet, movement, drug, blood sugar monitoring and health education
" five frame carriages " runs neck and neck.
Chinaberry leaf:For Meliaceae plant melia toosendan or the leaf of China tree.Summer, Qiu Cai, are dried.Record in《Dictionary of medicinal plant》.Pharmacology is made
With：The aqueous extract of China tree cotyledon（2~5%）The infringement to plant such as grasshopper, locust can be prevented, and it is nontoxic to people, animal, have
Effect ingredient is carotenoids hydrocarbon.Nature and flavor：《Guangdong Chinese medicine》II record " it is bitter, it trembles with fear, it is slightly poisonous." major function：Analgesic, desinsection.Control hernia
Gas, roundworm, treating swelling and pain by traumatic injury, furunculosis, skin eczema.《Detailed outline》：" hernia enters capsule pain, and beverage is decocted when facing hair.”《Guangdong Chinese medicine》Ⅱ：
" bitter fruit leaf grinds external application.Treating swelling and pain by traumatic injury is controlled, only knife wound bleeding.”
Chinese Toon Leaves：Meliaceae Chinese toon platymiscium Chinese toon Toona sinensis（A. Juss.） Roem.,[Cedrela sinensis
A. Juss.] leaf.Summer and autumn picking leaves.Record in《National Chinese herbal medicine compilation》.It is bitter in taste, puckery, temperature.Major function expelling wind and removing dampness,
Hemostasis and pain-relieving.Leaf and spray：Dysentery.Chinese toon root skin：For dysentery, enteritis, urethral infection is had blood in stool, metrorrhagia, leukorrhea, rheumatism
Pain in waist and lower extremities.Chinese toon fruit：Taste-blindness rate, chronic gastritis.
At home and abroad there is no the methods of complete radical cure diabetes, and the research of limonin constituents hypoglycemic is rarely reported.
So far, there is not yet reports such as the pertinent literature of limonin extract or patent in chinaberry leaf and Chinese Toon Leaves.
Invention content
The purpose of the present invention is to provide a kind of preparation methods of limonin extract in chinaberry leaf and Chinese Toon Leaves and new
Purposes.A kind of new application of limonin extract is to prepare treatment glycosuria in chinaberry leaf provided by the present invention and Chinese Toon Leaves
Application in medicine.
The present invention is achieved through the following technical solutions：
Chinaberry leaf used in the present invention is Meliaceae plant melia toosendan or the leaf of China tree, and Chinese Toon Leaves are Meliaceae Chinese toon platymiscium Chinese toon Toona
sinensis （A. Juss.）Roem., the leaf of [Cedrela sinensis A. Juss.].
The preparation method of limonin extract, is achieved by the steps of:
（1）3 parts by weight of chinaberry leaf, 1 parts by weight of Chinese Toon Leaves are taken, mixing crushes, and crosses 20 mesh sieve, is solvent with petroleum ether, and 50 DEG C are extracted
2 times, each extraction time is 3 hours, and each solvent dosage is 12 times of chinaberry leaf and Chinese Toon Leaves total weight, and filtration merges oil
Ether extracting solution, is recovered under reduced pressure petroleum ether, is concentrated and dried to obtain crude extract A, and the dregs of a decoction after petroleum ether extraction are spare；
（2）By step（1）The dregs of a decoction after petroleum ether extraction, with volume ratio 1:6 acetate-methanol mixed solvent extraction, 70
DEG C extraction 3 times, each extraction time is 2 hours, and 10 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage are filtered, closed
And acetate-methanol extracting solution, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, the medicine after acetate-methanol extraction
Slag is spare；
（3）By step（2）The dregs of a decoction after acetate-methanol extraction, with volume ratio 3:7 water-ethanol mixed solvent extraction, 80
DEG C extraction 2 times, each extraction time is 1.5 hours, 15 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration,
Merge water-ethanol extracting solution, solvent is recovered under reduced pressure, be concentrated and dried to obtain crude extract C, the dregs of a decoction after water-ethanol extraction are spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 90 DEG C are extracted 2 times, and each extraction time is 1 hour,
8 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration merges aqueous extract, is concentrated under reduced pressure into medicinal extract, dry
Crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 1:2 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 5:100 methanol-water elution, elution amount is 10 chromatography column volumes, then with volume ratio 20:100
Methanol-water elutes, and elution amount is 15 chromatography column volumes, and collected volume is than 20:100 methanol-water eluent merges, and recycling is molten
Agent, dry extract E；
（6）By step（3）Obtained crude extract C, by ratio of weight and number 1:1 uniformly mixed S8 macroporous absorbent resins-LSA21
Macroporous absorbent resin hybrid resin column, first with volume ratio 10:100 alcohol-water elution, elution amount are 3 resin column volumes, then
With volume ratio 40:100 alcohol-water elution, elution amount is 3 resin column volumes, and collected volume is than 40:100 alcohol-water is washed
De- liquid merges, recycling design, dry extract F；
（7）By weight 2:1 by step（5）Obtained extract E and step（6）Obtained extract F mixings are to get lemon hardship
Plain class extract.
The limonin extract of the technical program contains simultaneously：Mountain chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K, and mountain
Chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K weight ratio be 3:4:8.
The limonin extract of the technical program can be used for preparing treatment diabetes medicament, and treatment diabetes are effective.
The present invention for the first time to chinaberry leaf and Chinese Toon Leaves formula in limonin extract extraction prepare, using MCI gels-
The method that polyamide Mixed chromatogram columniation, hybrid resin column are combined is prepared simultaneously containing mountain chinaberry body C, mountain chinaberry body D, Maoma
The weight ratio of chinaberry lactone K is 3:4:8 limonin extract, treatment diabetes curative effect are notable.
Specific embodiment
Below by specific experiment example and embodiment to the preparation method of limonin extract in chinaberry leaf and Chinese Toon Leaves
It is described further, but is not limited to the present invention.
Embodiment 1：The preparation of limonin extract
（1）Take chinaberry leaf 3kg, Chinese Toon Leaves 1kg, mixing crushes, and crosses 20 mesh sieve, is solvent with petroleum ether, and 50 DEG C are extracted 2 times, every time
Extraction time is 3 hours, and each solvent dosage is 12 times of chinaberry leaf and Chinese Toon Leaves total weight, and filtration merges petroleum ether extract,
Petroleum ether is recovered under reduced pressure, is concentrated and dried to obtain crude extract A, the dregs of a decoction after petroleum ether extraction are spare；
（2）By step（1）The dregs of a decoction after petroleum ether extraction, with volume ratio 1:6 acetate-methanol mixed solvent extraction, 70
DEG C extraction 3 times, each extraction time is 2 hours, and 10 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage are filtered, closed
And acetate-methanol extracting solution, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, the medicine after acetate-methanol extraction
Slag is spare；
（3）By step（2）The dregs of a decoction after acetate-methanol extraction, with volume ratio 3:7 water-ethanol mixed solvent extraction, 80
DEG C extraction 2 times, each extraction time is 1.5 hours, 15 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration,
Merge water-ethanol extracting solution, solvent is recovered under reduced pressure, be concentrated and dried to obtain crude extract C, the dregs of a decoction after water-ethanol extraction are spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 90 DEG C are extracted 2 times, and each extraction time is 1 hour,
8 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration merges aqueous extract, is concentrated under reduced pressure into medicinal extract, dry
Crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 1:2 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 5:100 methanol-water elution, elution amount is 10 chromatography column volumes, then with volume ratio 20:100
Methanol-water elutes, and elution amount is 15 chromatography column volumes, and collected volume is than 20:100 methanol-water eluent merges, and recycling is molten
Agent, dry extract E；
（6）By step（3）Obtained crude extract C, by ratio of weight and number 1:1 uniformly mixed S8 macroporous absorbent resins-LSA21
Macroporous absorbent resin hybrid resin column, first with volume ratio 10:100 alcohol-water elution, elution amount are 3 resin column volumes, then
With volume ratio 40:100 alcohol-water elution, elution amount is 3 resin column volumes, and collected volume is than 40:100 alcohol-water is washed
De- liquid merges, recycling design, dry extract F；
（7）By weight 2:1 by step（5）Obtained extract E and step（6）Obtained extract F mixings are to get lemon hardship
Plain class extract.
Embodiment 2：The preparation of limonin extract
（1）Take chinaberry leaf 2kg, Chinese Toon Leaves 1kg, mixing crushes, and crosses 20 mesh sieve, is solvent with petroleum ether, and 50 DEG C are extracted 2 times, every time
Extraction time is 3 hours, and each solvent dosage is 12 times of chinaberry leaf and Chinese Toon Leaves total weight, and filtration merges petroleum ether extract,
Petroleum ether is recovered under reduced pressure, is concentrated and dried to obtain crude extract A, the dregs of a decoction after petroleum ether extraction are spare；
（2）By step（1）The dregs of a decoction after petroleum ether extraction, with volume ratio 1:5 acetate-methanol mixed solvent extraction, 70
DEG C extraction 3 times, each extraction time is 2 hours, and 10 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage are filtered, closed
And acetate-methanol extracting solution, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, the medicine after acetate-methanol extraction
Slag is spare；
（3）By step（2）The dregs of a decoction after acetate-methanol extraction, with volume ratio 3:5 water-ethanol mixed solvent extraction, 80
DEG C extraction 2 times, each extraction time is 1.5 hours, 15 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration,
Merge water-ethanol extracting solution, solvent is recovered under reduced pressure, be concentrated and dried to obtain crude extract C, the dregs of a decoction after water-ethanol extraction are spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 90 DEG C are extracted 2 times, and each extraction time is 1 hour,
8 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration merges aqueous extract, is concentrated under reduced pressure into medicinal extract, dry
Crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 1:1 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 5:100 methanol-water elution, elution amount is 10 chromatography column volumes, then with volume ratio 20:100
Methanol-water elutes, and elution amount is 15 chromatography column volumes, and collected volume is than 20:100 methanol-water eluent merges, and recycling is molten
Agent, dry extract E；
（6）By step（3）Obtained crude extract C, by ratio of weight and number 1:2 uniformly mixed S8 macroporous absorbent resins-LSA21
Macroporous absorbent resin hybrid resin column, first with volume ratio 10:100 alcohol-water elution, elution amount are 3 resin column volumes, then
With volume ratio 40:100 alcohol-water elution, elution amount is 3 resin column volumes, and collected volume is than 40:100 alcohol-water is washed
De- liquid merges, recycling design, dry extract F；
（7）By weight 2:2 by step（5）Obtained extract E and step（6）Obtained extract F mixings are to get lemon hardship
Plain class extract.
Embodiment 3：The preparation of limonin extract
（1）Take chinaberry leaf 1kg, Chinese Toon Leaves 3kg, mixing crushes, and crosses 20 mesh sieve, is solvent with petroleum ether, and 50 DEG C are extracted 2 times, every time
Extraction time is 3 hours, and each solvent dosage is 12 times of chinaberry leaf and Chinese Toon Leaves total weight, and filtration merges petroleum ether extract,
Petroleum ether is recovered under reduced pressure, is concentrated and dried to obtain crude extract A, the dregs of a decoction after petroleum ether extraction are spare；
（2）By step（1）The dregs of a decoction after petroleum ether extraction, with volume ratio 1:3 acetate-methanol mixed solvent extraction, 70
DEG C extraction 3 times, each extraction time is 2 hours, and 10 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage are filtered, closed
And acetate-methanol extracting solution, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, the medicine after acetate-methanol extraction
Slag is spare；
（3）By step（2）The dregs of a decoction after acetate-methanol extraction, with volume ratio 4:7 water-ethanol mixed solvent extraction, 80
DEG C extraction 2 times, each extraction time is 1.5 hours, 15 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration,
Merge water-ethanol extracting solution, solvent is recovered under reduced pressure, be concentrated and dried to obtain crude extract C, the dregs of a decoction after water-ethanol extraction are spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 90 DEG C are extracted 2 times, and each extraction time is 1 hour,
8 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration merges aqueous extract, is concentrated under reduced pressure into medicinal extract, dry
Crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 3:2 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 5:100 methanol-water elution, elution amount is 10 chromatography column volumes, then with volume ratio 20:100
Methanol-water elutes, and elution amount is 15 chromatography column volumes, and collected volume is than 20:100 methanol-water eluent merges, and recycling is molten
Agent, dry extract E；
（6）By step（3）Obtained crude extract C, by ratio of weight and number 2:1 uniformly mixed S8 macroporous absorbent resins-LSA21
Macroporous absorbent resin hybrid resin column, first with volume ratio 10:100 alcohol-water elution, elution amount are 3 resin column volumes, then
With volume ratio 40:100 alcohol-water elution, elution amount is 3 resin column volumes, and collected volume is than 40:100 alcohol-water is washed
De- liquid merges, recycling design, dry extract F；
（7）By weight 2:3 by step（5）Obtained extract E and step（6）Obtained extract F mixings are to get lemon hardship
Plain class extract.
Embodiment 4：Total citrin assay.
Using HPLC ELSD method, limonin constituents in limonin extract are analyzed
Content.Using YMC-Pack C18(150 mm × 4. 6 mm, 5 μm) chromatographic column, with mobile phase methanol-water-tetrahydrochysene furan
Mutter (50:44:6) it is eluted, flow velocity 1.0mL/min, 25 DEG C of column temperature；Using evaporative light scattering detector, drift tube temperature
92.0 DEG C, flow rate of carrier gas 2.2L/min.The result shows that limonin component content obtained by 1 extract of embodiment accounts for total extraction
The 85% of object, and the weight ratio of mountain chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K are 3:4:8.
Limonin constituents and content in 1 extract of table
Embodiment 5：Limonin constituent structure is identified
Limonin extract is prepared, then through 200-300 mesh silica gel column chromatographies by 1 method of embodiment, first with petroleum ether-acetic acid
Ethyl ester gradient elution（10:1-1:1）, then use acetate-methanol（10:0.5-2:1）Gradient elution thin layer tracing detection merges
Identical fraction stands crystallization, filters, and ethyl acetate or acetone recrystallization respectively obtain compound 1, compound 2, compound 3,
Be white amorphous powder, more than each compound chemical constitution through the wave spectrums such as mass spectrum and nuclear magnetic resonance means confirmation be：Mountain
Chinaberry body C（aphanamixoid C）, mountain chinaberry body D（aphanamixoid D）, Maoma chinaberry lactone K（chukvelutilide K）,
Purity is all higher than 98% through high performance liquid chromatography detection.
Embodiment 6：Limonin extractive HPLC-MS is analyzed
It is prepared by sample solution：1 limonin extract of embodiment, test solution is prepared with methanol, organic micro- with 0.45um
Hole filter membrane filtration, as test solution.
The preparation of standard solution：Homemade mountain chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K are taken, adds methanol appropriate, as
Standard solution.
Instrument：German Thermo Fischer Scientific high resolution liquid chromatography mass spectrometer.
Chromatographic condition：Waters Symmetry C18 chromatographic columns（5 μm, 250mm*4.6mm）, column temperature：25 DEG C, flow velocity 800
μ l/min, Detection wavelength：550nm, sampling volume：5ul.Mobile phase：Using acetonitrile as mobility A, using 0.1% ammonium hydroxide as mobility
B carries out gradient elution；
Mass Spectrometry Conditions：LTQ-Orbitrap XL type mass spectrums, ESI ion sources, sheath gas 50arb, 275 DEG C of capillary temperature assist gas
10arb, capillary voltage 25V, spray voltage 4.0kV.Positive ion mode scans, m/z scanning ranges 100 ~ 1000.
As a result：Standard items and sample level-one, two level total ion current coincide respectively, with reference to level-one, second order ms, molecular ion
Peak and fragment peak identify total limonin extract and contain mountain chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K totally 3 main lemons
Lemon hardship chlorins compound.
Experimental example 1：Therapeutic effect of the limonin extract to streptozotocin diabetes rat
Streptozotocin diabetes animal model modeling：By streptozotocin（STZ）It is dissolved in 0.1mol/L citrate buffer solutions (pH4.5)
In, prepared before use.Rat usual amounts are 70mg/kg (ip).It is once enough to give animal streptozotocin, cause β cells a large amount of
Damage, insulin synthesis and secretion are reduced, and are caused carbohydrate metabolism disturbance, are led to diabetes.STZ solution is sterile with 0.1 mol/L
Citric acid-sodium citrate buffer Fresh adjusts pH to 4.5, bacteria filter filtration sterilization into 2% solution.Rat Fast 10h
Intraperitoneal or tail vein disposably injects STZ solution by 60mg/kg weight, the mmol/L of interior random blood sugar >=16.7, stablizes for 24 hours
5d can be used as successful model.
Drug：1 limonin extract of embodiment, 2 limonin extract of embodiment, 3 limonin of embodiment
Class extract, 1 crude extract A of embodiment, 1 crude extract B of embodiment, 1 crude extract C of embodiment, 1 crude extract D of embodiment, embodiment 1
Extract E, 1 extract F of embodiment.Drug is prepared：It smashes, 40mg is respectively taken to be dissolved in 40ml distilled water, suspension is made.
Positive drug：Phenformin, pharmaceutical factory of Medicine Industry Inst., Shandong Prov. produce.Streptozotocin (STREPTOZOCIN,
STZ), specification：1g/ bottles, SIGMA companies.
Instrument：Kyoto, Japan II type GLUCOCARO blood glucose meters, full-automatic biochemical analyzer, 12 hole γ of U.S.'s production-put and exempt to survey
Determine instrument.
Reagent：Blood glucose electrode slice used in the blood glucose meter of capital of a country, blood fat reagent used in full automatic biochemical apparatus, Tianjin Depew company
(DPC) the insulin radioimmunological kit provided.
Animal：Wistar rats are purchased from Shandong Province's Experimental Animal Center.
The experiment of streptozotocin diabetes rat：Take Wistar rats 120, half male and half female, weight 180-200g.Stablize
During which feed 1w takes blood to survey fasting blood-glucose 2 times, using 3.7-5.3mmol/L as selected minimal standards.Wherein 10 be only used as it is normal right
According to spare.Remaining 110 with streptozotocin (STZ) modeling, 7-10d repetition measurements fasting blood-glucose after modeling, to be more than 14.0mmol/L
As model success rat.
Streptozotocin diabetes (STZ--DM) rat 110 is only randomly divided into modeling, 1 limonin of embodiment carries
Take object, 2 limonin extract of embodiment, 3 limonin extract of embodiment, 1 crude extract A of embodiment, embodiment 1 thick
Extract B, 1 crude extract C of embodiment, 1 crude extract D of embodiment, 1 extract E of embodiment, 1 extract F of embodiment, phenformin totally 11
Group, every group each 10, and set Normal group 10.1 limonin extract of embodiment, 2 limonin of embodiment carry
Take object, 3 limonin extract of embodiment, 1 crude extract A of embodiment, 1 crude extract B of embodiment, 1 crude extract C of embodiment, reality
1 crude extract D of example, 1 extract E of embodiment, 1 extract F of embodiment are applied, is administered by 2mg/kg, 9 mg/kg of phenformin group gives
Medicine, continuous oral dextrose tolerance test (1.5 g/kg), tail vein take blood system not survey 30min after empty stomach, oral glucose,
The blood glucose level of 60min, 90min, 120min calculate 60min blood glucose area line area after fasting blood-glucose and glucose gavage
(AUC)。
Result of the test：
Limonin extract has apparent reduction to act on the raising of STZ-DM rat fasting blood-glucoses, more has with modeling group
Pole significant difference.Table 2 the result shows that, each extract of limonin is significantly improved effect to STZ-DM rat glycometabolisms,
Effect is significantly reduced to blood glucose rise caused by glucose, hence it is evident that reduce blood glucose area line area, more have with modeling group
Pole significant difference.
Influence (mmol/L) of the 2 each extract of table to STZ-DM rat fasting blood-glucoses
Note：Compared with model group：*P<0.05, * * P<0.01.
Influence of each extract of limonin to STZ-DM rat Langerhans islet Instreptozotocin Induceds：To modeling group blood before glucose load
Clear insulin level illustrates that streptozotocin causes pancreaticβ-cell centainly to damage significantly lower than normal group；The technical program lemon
Each extract group serum insulin level of lemon bitter principle class is apparently higher than modeling group, illustrates each extract of limonin to STZ-DM
Pancreatic Islets beta-cellular damage has certain repair.Normal rat increases to 30min serum insulin secretions after glucose load,
There is insulin secretion peak in 60min, is gradually reduced thereafter；Each extract group of limonin is giving glucose load 30min serum
Insulin level increases rapidly, and is apparently higher than modeling group, has certain effect for the glycometabolism for improving STZ-DM.
Influence of each extract of limonin to normal rat fasting blood-glucose and serum insulin level and blood lipid metabolism：
Each extract of limonin acts on the fasting blood-glucose of normal rat without significantly reduction, to the Diagnostic Value of Fasting Serum of normal rat
Insulin does not influence significantly.Each extract of limonin does not have significant impact to the blood lipid metabolism of normal rat.
Claims (4)
1. a kind of limonin extract is preparing the application in treating diabetes medicament, it is characterised in that the lemon is bitter
The preparation method of plain class extract is:
（1）3 parts by weight of chinaberry leaf, 1 parts by weight of Chinese Toon Leaves are taken, mixing crushes, and crosses 20 mesh sieve, is solvent with petroleum ether, and 50 DEG C are extracted
2 times, each extraction time is 3 hours, and each solvent dosage is 12 times of chinaberry leaf and Chinese Toon Leaves total weight, and filtration merges oil
Ether extracting solution, is recovered under reduced pressure petroleum ether, is concentrated and dried to obtain crude extract A, and the dregs of a decoction after petroleum ether extraction are spare；
（2）By step（1）The dregs of a decoction after petroleum ether extraction, with volume ratio 1:6 acetate-methanol mixed solvent extraction, 70
DEG C extraction 3 times, each extraction time is 2 hours, and 10 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage are filtered, closed
And acetate-methanol extracting solution, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, the medicine after acetate-methanol extraction
Slag is spare；
（3）By step（2）The dregs of a decoction after acetate-methanol extraction, with volume ratio 3:7 water-ethanol mixed solvent extraction, 80
DEG C extraction 2 times, each extraction time is 1.5 hours, 15 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration,
Merge water-ethanol extracting solution, solvent is recovered under reduced pressure, be concentrated and dried to obtain crude extract C, the dregs of a decoction after water-ethanol extraction are spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 90 DEG C are extracted 2 times, and each extraction time is 1 hour,
8 times for chinaberry leaf and Chinese Toon Leaves total weight of each solvent dosage, filtration merges aqueous extract, is concentrated under reduced pressure into medicinal extract, dry
Crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 1:2 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 5:100 methanol-water elution, elution amount is 10 chromatography column volumes, then with volume ratio 20:100
Methanol-water elutes, and elution amount is 15 chromatography column volumes, and collected volume is than 20:100 methanol-water eluent merges, and recycling is molten
Agent, dry extract E；
（6）By step（3）Obtained crude extract C, by ratio of weight and number 1:1 uniformly mixed S8 macroporous absorbent resins-LSA21
Macroporous absorbent resin hybrid resin column, first with volume ratio 10:100 alcohol-water elution, elution amount are 3 resin column volumes, then
With volume ratio 40:100 alcohol-water elution, elution amount is 3 resin column volumes, and collected volume is than 40:100 alcohol-water is washed
De- liquid merges, recycling design, dry extract F；
（7）By weight 2:1 by step（5）Obtained extract E and step（6）Obtained extract F mixings are to get lemon hardship
Plain class extract.
2. a kind of preparation method of limonin extract according to claim 1, it is characterised in that the lemon is bitter
Plain class extract is simultaneously containing mountain chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K.
3. a kind of limonin extract, it is characterised in that containing weight ratio be 3:4:8 mountain chinaberry body C, mountain chinaberry body D, hair
Numb chinaberry lactone K.
4. one kind is 3 containing weight ratio:4:8 mountain chinaberry body C, mountain chinaberry body D, Maoma chinaberry lactone K limonin extract
Preparing the application in treating diabetes medicament.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021027581A1 (en) * | 2019-08-12 | 2021-02-18 | 浙江养生堂天然药物研究所有限公司 | Combination product comprising limonoid and dpp-4 inhibitors |
| WO2021027576A1 (en) * | 2019-08-12 | 2021-02-18 | 浙江养生堂天然药物研究所有限公司 | Combination product comprising limonoids and sglt-2 inhibitors |
| JP2022544299A (en) * | 2019-08-12 | 2022-10-17 | 浙江養生堂天然薬物研究所有限公司 | Combination product containing a limonoid compound and a thiazolidinedione compound |
| JP2022544517A (en) * | 2019-08-12 | 2022-10-19 | 浙江養生堂天然薬物研究所有限公司 | Combination product containing a limonoid compound and a sulfonylurea compound |
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| CN103585302B (en) * | 2013-10-31 | 2015-08-05 | 舒礼良 | The application of the extracts such as a kind of Song Sheng in preparation treatment diabetes medicament |
| CN104840504A (en) * | 2015-05-19 | 2015-08-19 | 泰山医学院 | Extraction and preparation method for limonin components |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103585302B (en) * | 2013-10-31 | 2015-08-05 | 舒礼良 | The application of the extracts such as a kind of Song Sheng in preparation treatment diabetes medicament |
| CN104840504A (en) * | 2015-05-19 | 2015-08-19 | 泰山医学院 | Extraction and preparation method for limonin components |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021027581A1 (en) * | 2019-08-12 | 2021-02-18 | 浙江养生堂天然药物研究所有限公司 | Combination product comprising limonoid and dpp-4 inhibitors |
| WO2021027576A1 (en) * | 2019-08-12 | 2021-02-18 | 浙江养生堂天然药物研究所有限公司 | Combination product comprising limonoids and sglt-2 inhibitors |
| EP4014979A4 (en) * | 2019-08-12 | 2022-10-12 | Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. | Combination product comprising limonoids and sglt-2 inhibitors |
| JP2022544297A (en) * | 2019-08-12 | 2022-10-17 | 浙江養生堂天然薬物研究所有限公司 | A combination product containing a limonoid compound and a DPP-4 inhibitor |
| JP2022544299A (en) * | 2019-08-12 | 2022-10-17 | 浙江養生堂天然薬物研究所有限公司 | Combination product containing a limonoid compound and a thiazolidinedione compound |
| JP2022544517A (en) * | 2019-08-12 | 2022-10-19 | 浙江養生堂天然薬物研究所有限公司 | Combination product containing a limonoid compound and a sulfonylurea compound |
| EP4014999A4 (en) * | 2019-08-12 | 2022-11-23 | Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. | Combination product containing limonin compound and sulfonylurea drug |
| EP4015000A4 (en) * | 2019-08-12 | 2022-11-23 | Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. | Combination product containing limonin compound and thiazolidinedione |
| JP7383124B2 (en) | 2019-08-12 | 2023-11-17 | 浙江養生堂天然薬物研究所有限公司 | Combination products containing limonoid compounds and DPP-4 inhibitors |
| JP7383125B2 (en) | 2019-08-12 | 2023-11-17 | 浙江養生堂天然薬物研究所有限公司 | Combination products containing limonoid compounds and thiazolidinedione compounds |
| JP7383126B2 (en) | 2019-08-12 | 2023-11-17 | 浙江養生堂天然薬物研究所有限公司 | Combination products containing limonoid compounds and sulfonylurea compounds |
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