CN108210600A - A kind of preparation method and applications of limonin extract - Google Patents
A kind of preparation method and applications of limonin extract Download PDFInfo
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- CN108210600A CN108210600A CN201711438784.6A CN201711438784A CN108210600A CN 108210600 A CN108210600 A CN 108210600A CN 201711438784 A CN201711438784 A CN 201711438784A CN 108210600 A CN108210600 A CN 108210600A
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- extract
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- leaf
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- chinaberry
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- 239000000284 extract Substances 0.000 title claims abstract description 80
- VHLJDTBGULNCGF-UHFFFAOYSA-N Limonin Natural products CC1(C)OC2CC(=O)OCC23C4CCC5(C)C(CC(=O)C6OC56C4(C)C(=O)CC13)c7cocc7 VHLJDTBGULNCGF-UHFFFAOYSA-N 0.000 title claims abstract description 49
- KBDSLGBFQAGHBE-MSGMIQHVSA-N limonin Chemical compound C=1([C@H]2[C@]3(C)CC[C@H]4[C@@]([C@@]53O[C@@H]5C(=O)O2)(C)C(=O)C[C@@H]2[C@]34COC(=O)C[C@@H]3OC2(C)C)C=COC=1 KBDSLGBFQAGHBE-MSGMIQHVSA-N 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- 239000003814 drug Substances 0.000 claims abstract description 16
- 241001156382 Lansium Species 0.000 claims abstract description 15
- 235000006662 Lansium Nutrition 0.000 claims abstract description 15
- 238000010828 elution Methods 0.000 claims description 43
- 239000000287 crude extract Substances 0.000 claims description 39
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- 244000089795 Clausena lansium Species 0.000 claims description 31
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- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 25
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/58—Meliaceae (Chinaberry or Mahogany family), e.g. Azadirachta (neem)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/75—Rutaceae (Rue family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The technical program discloses a kind of preparation method and applications of limonin extract, what the limonin extract was prepared as follows：Using Maoma chinaberry leaf, small Chinese wampee leaf, Chinese Toon Leaves as raw material, extraction, separation, purifying, up to the limonin extract that small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F are formed, 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 preparation method and applications of limonin extract, tool
Body is to be related to the preparation method of limonin extract in a kind of Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves and its controlled in preparation
Treat the application in diabetes medicament.
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.
Maoma chinaberry leaf：To be Meliaceae melia plant Maoma chinaberry (Chukrasia tabularis var. velutina
(Wall) King) leaf.Maoma chinaberry, deciduous tree, tree crown umbrella shape, tree performance are carried out.Even number pinnate compound leaf, 10-18 pieces of leaflet；It is small
Leaf alternate, papery, ovate-elliptic, base portion is anisopleual, and two sides is by pubescence.Panicle basidixed, flower yellow band purple, virtue
It is fragrant.Capsule is subsphaeroidal or oval, rough surface.The end of spring and the beginning of summer at florescence, fruiting period autumn.
Small Chinese wampee leaf：For the small Calusena lansium of Rutaceae（Clausena emarginata Huang）Leaf.Nature and flavor：It is bitter, pungent, it is micro-
Temperature.It is toxic.Major function ventilating the lung and relieving cough, promoting qi circulation and relieving pain, clearing and activating the channels and collaterals.Cold headache, cough due to wind-cold evil are cured mainly, migraine is had a stomachache,
Neuralgia, toothache, rheumathritis, traumatic injury.
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, lumbago, skelalgia of rheumatism.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 the pertinent literature of limonin extract or patent etc. in Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves
Report.
Invention content
The purpose of the present invention is to provide one kind of limonin extract in Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves
Preparation method and new application.Limonin extract in Maoma chinaberry leaf provided by the present invention, small Chinese wampee leaf and Chinese Toon Leaves
A kind of new application is to prepare the application in treating diabetes medicament.
The present invention is achieved through the following technical solutions：
Maoma chinaberry leaf used in the present invention is Meliaceae melia plant Maoma chinaberry (Chukrasia tabularis var. velutina
(Wall) King) leaf；Small Chinese wampee leaf is the small Calusena lansium of Rutaceae（Clausena emarginata Huang）Leaf；Chinese Toon Leaves
For Meliaceae Chinese toon platymiscium Chinese toon Toona sinensis（A. Juss.）Roem.,[Cedrela sinensis A. Juss.]
Leaf.
The preparation method of limonin extract, is achieved by the steps of:
（1）2 parts by weight of Maoma chinaberry leaf, 3 parts by weight of small Chinese wampee leaf, 4 parts by weight of Chinese Toon Leaves are taken, mixing crushes, and crosses 20 mesh sieve, uses
Hexamethylene is solvent, and 60 DEG C are extracted 3 times, and each extraction time is 2.5 hours, and each solvent dosage is Maoma chinaberry leaf, small Calusena lansium
12 times of leaf and Chinese Toon Leaves total weight, filtration merge hexamethylene extracting solution, hexamethylene are recovered under reduced pressure, is concentrated and dried to obtain crude extract
A, the dregs of a decoction after hexamethylene extraction are spare；
（2）By step（1）The dregs of a decoction after hexamethylene extraction, with volume ratio 1:5 Ethyl formate-methanol mixed solvent extraction, 75
DEG C extraction 4 times, each extraction time be 3.5 hours, each solvent dosage be Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges Ethyl formate-methanol extract liquid, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, Ethyl formate-
The dregs of a decoction after methanol extraction are spare；
（3）By step（2）The dregs of a decoction after Ethyl formate-methanol extraction, with volume ratio 4:6 water-ethanol mixed solvent extraction, 50
DEG C extraction 7 times, each extraction time is 3 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges water-ethanol extracting solution, solvent is recovered under reduced pressure, and is concentrated and dried to obtain crude extract C, the medicine after water-ethanol extraction
Slag is spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 80 DEG C are extracted 2 times, and each extraction time is 1 hour,
Each solvent dosage is 8 times of Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight, and filtration merges aqueous extract, is concentrated under reduced pressure
To medicinal extract, dry crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 2:1 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 3:97 methanol-water elution, elution amount is 5 chromatography column volumes, then with volume ratio 20:80 first
Alcohol-water elution, elution amount are 12 chromatography column volumes, and collected volume is than 20:80 methanol-water eluent merges, recycling design,
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-NKA9
Macroporous absorbent resin hybrid resin column, first with volume ratio 9:91 alcohol-water elution, elution amount is 4 resin column volumes, then is used
Volume ratio 50:50 alcohol-water elution, elution amount is 2 resin column volumes, and collected volume is than 50:50 alcohol-water eluent
Merge, 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：Small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element
B, mountain chinaberry body F, and the weight ratio of small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F are 2:2:3:4.
The limonin extract of the technical program can be used for preparing treatment diabetes medicament, and treatment diabetes are effective.
During the present invention is for the first time formulated Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves prepared by the extraction of limonin extract,
The method being combined using MCI gels-polyamide Mixed chromatogram columniation, hybrid resin column is prepared simultaneously containing small Calusena lansium element
B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F weight ratio be 2:2:3:4 limonin extract, treatment sugar
Urine disease is significant in efficacy.
Specific embodiment
Limonin in Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves is extracted below by specific experiment example and embodiment
The preparation method of object is described further, but is not limited to the present invention.
Embodiment 1：The preparation of limonin extract
（1）Maoma chinaberry leaf 2kg, small Chinese wampee leaf 3kg, Chinese Toon Leaves 4kg are taken, mixing crushes, and crosses 20 mesh sieve, is solvent with hexamethylene,
60 DEG C are extracted 3 times, and each extraction time is 2.5 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves gross weight
12 times of amount, filtration merge hexamethylene extracting solution, hexamethylene are recovered under reduced pressure, and crude extract A are concentrated and dried to obtain, after hexamethylene extraction
The dregs of a decoction it is spare；
（2）By step（1）The dregs of a decoction after hexamethylene extraction, with volume ratio 1:5 Ethyl formate-methanol mixed solvent extraction, 75
DEG C extraction 4 times, each extraction time be 3.5 hours, each solvent dosage be Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges Ethyl formate-methanol extract liquid, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, Ethyl formate-
The dregs of a decoction after methanol extraction are spare；
（3）By step（2）The dregs of a decoction after Ethyl formate-methanol extraction, with volume ratio 4:6 water-ethanol mixed solvent extraction, 50
DEG C extraction 7 times, each extraction time is 3 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges water-ethanol extracting solution, solvent is recovered under reduced pressure, and is concentrated and dried to obtain crude extract C, the medicine after water-ethanol extraction
Slag is spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 80 DEG C are extracted 2 times, and each extraction time is 1 hour,
Each solvent dosage is 8 times of Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight, and filtration merges aqueous extract, is concentrated under reduced pressure
To medicinal extract, dry crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 2:1 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 3:97 methanol-water elution, elution amount is 5 chromatography column volumes, then with volume ratio 20:80 first
Alcohol-water elution, elution amount are 12 chromatography column volumes, and collected volume is than 20:80 methanol-water eluent merges, recycling design,
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-NKA9
Macroporous absorbent resin hybrid resin column, first with volume ratio 9:91 alcohol-water elution, elution amount is 4 resin column volumes, then is used
Volume ratio 50:50 alcohol-water elution, elution amount is 2 resin column volumes, and collected volume is than 50:50 alcohol-water eluent
Merge, 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）Maoma chinaberry leaf 3kg, small Chinese wampee leaf 2kg, Chinese Toon Leaves 1kg are taken, mixing crushes, and crosses 20 mesh sieve, is solvent with hexamethylene,
60 DEG C are extracted 3 times, and each extraction time is 2.5 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves gross weight
12 times of amount, filtration merge hexamethylene extracting solution, hexamethylene are recovered under reduced pressure, and crude extract A are concentrated and dried to obtain, after hexamethylene extraction
The dregs of a decoction it is spare；
（2）By step（1）The dregs of a decoction after hexamethylene extraction, with volume ratio 1:5 Ethyl formate-methanol mixed solvent extraction, 75
DEG C extraction 4 times, each extraction time be 3.5 hours, each solvent dosage be Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges Ethyl formate-methanol extract liquid, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, Ethyl formate-
The dregs of a decoction after methanol extraction are spare；
（3）By step（2）The dregs of a decoction after Ethyl formate-methanol extraction, with volume ratio 4:6 water-ethanol mixed solvent extraction, 50
DEG C extraction 7 times, each extraction time is 3 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges water-ethanol extracting solution, solvent is recovered under reduced pressure, and is concentrated and dried to obtain crude extract C, the medicine after water-ethanol extraction
Slag is spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 80 DEG C are extracted 2 times, and each extraction time is 1 hour,
Each solvent dosage is 8 times of Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight, and filtration merges aqueous extract, is concentrated under reduced pressure
To medicinal extract, dry crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 2:5 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 3:97 methanol-water elution, elution amount is 5 chromatography column volumes, then with volume ratio 20:80 first
Alcohol-water elution, elution amount are 12 chromatography column volumes, and collected volume is than 20:80 methanol-water eluent merges, recycling design,
Dry extract E；
（6）By step（3）Obtained crude extract C, by ratio of weight and number 1:3 uniformly mixed S8 macroporous absorbent resins-NKA9
Macroporous absorbent resin hybrid resin column, first with volume ratio 9:91 alcohol-water elution, elution amount is 4 resin column volumes, then is used
Volume ratio 50:50 alcohol-water elution, elution amount is 2 resin column volumes, and collected volume is than 50:50 alcohol-water eluent
Merge, 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 3：The preparation of limonin extract
（1）Maoma chinaberry leaf 2kg, small Chinese wampee leaf 2kg, Chinese Toon Leaves 5kg are taken, mixing crushes, and crosses 20 mesh sieve, is solvent with hexamethylene,
60 DEG C are extracted 3 times, and each extraction time is 2.5 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves gross weight
12 times of amount, filtration merge hexamethylene extracting solution, hexamethylene are recovered under reduced pressure, and crude extract A are concentrated and dried to obtain, after hexamethylene extraction
The dregs of a decoction it is spare；
（2）By step（1）The dregs of a decoction after hexamethylene extraction, with volume ratio 1:5 Ethyl formate-methanol mixed solvent extraction, 75
DEG C extraction 4 times, each extraction time be 3.5 hours, each solvent dosage be Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges Ethyl formate-methanol extract liquid, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, Ethyl formate-
The dregs of a decoction after methanol extraction are spare；
（3）By step（2）The dregs of a decoction after Ethyl formate-methanol extraction, with volume ratio 4:6 water-ethanol mixed solvent extraction, 50
DEG C extraction 7 times, each extraction time is 3 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges water-ethanol extracting solution, solvent is recovered under reduced pressure, and is concentrated and dried to obtain crude extract C, the medicine after water-ethanol extraction
Slag is spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 80 DEG C are extracted 2 times, and each extraction time is 1 hour,
Each solvent dosage is 8 times of Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight, and filtration merges aqueous extract, is concentrated under reduced pressure
To medicinal extract, dry crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 2:4 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 3:97 methanol-water elution, elution amount is 5 chromatography column volumes, then with volume ratio 20:80 first
Alcohol-water elution, elution amount are 12 chromatography column volumes, and collected volume is than 20:80 methanol-water eluent merges, recycling design,
Dry extract E；
（6）By step（3）Obtained crude extract C, by ratio of weight and number 1:4 uniformly mixed S8 macroporous absorbent resins-NKA9
Macroporous absorbent resin hybrid resin column, first with volume ratio 9:91 alcohol-water elution, elution amount is 4 resin column volumes, then is used
Volume ratio 50:50 alcohol-water elution, elution amount is 2 resin column volumes, and collected volume is than 50:50 alcohol-water eluent
Merge, 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 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 (48:47:5) it is eluted, flow velocity 1.0mL/min, 25 DEG C of column temperature；Using evaporative light scattering detector, drift tube temperature
91.0 DEG C, flow rate of carrier gas 2.1L/min.The result shows that limonin component content obtained by 1 extract of embodiment accounts for total extraction
The 78% of object, and the weight ratio of small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F are 2:2:3:4.
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（10:0.5-1:7）, then with Ethyl formate-methanol（10:0.3-2:1）Gradient elution thin layer tracing detection closes
And identical fraction, crystallization is stood, is filtered, ethyl acetate or acetone recrystallization respectively obtain compound 1, compound 2, compound
3rd, compound 4 are white amorphous powder, more than each compound chemical constitution through the wave spectrums means such as mass spectrum and nuclear magnetic resonance
It confirms and is：Small Calusena lansium element B（clauemargine B）, new shellfish erythrophloeum ferdii element A（libiguin A）, new shellfish erythrophloeum ferdii element B（libiguin
B）, mountain chinaberry body F（aphanamixoid F）, 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 small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F are taken, is added
Appropriate methanol, 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 small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry
Body F totally 4 main limonoids.
Experimental example 1：Hypoglycemic effect experimental study
1 material and instrument
1.1 SPF grades of animal Kunming mouses, half male and half female, 18~22 g.
1.2 drugs and reagent
Drug：1 limonin extract of embodiment, 2 limonin extract of embodiment, 3 limonin of embodiment carry
Object, 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 is taken to extract
Object E, 1 extract F of embodiment；Metformin hydrochloride tablet, Zhonghui Pharmaceutical Co., Ltd., Beijing；Alloxan is public for U.S. sigma
Take charge of product, import packing；Sodium chloride injection, medicine company Group Co., Ltd of Nanning Baihui；The detection examination of blood glucose GOD-PAP methods
Agent box, Maike Tech Co., Ltd., Sichuan Prov..Glucose, Chengdu Ke Long chemical reagents factory.
1.3 instrument
UV-1901 dual-beams ultraviolet-uisible spectrophotometer (Beijing Pu Xi all purpose instruments Co., Ltd)；ST16R types are desk-top cold
Freeze centrifuge (Thermo Scien-tific companies of the U.S.)；HH-8 digital displays thermostat water bath (Changzhou Guohua Electric Appliance Co., Ltd.)
。
2 methods
The foundation and administration of 2.1 Experimental Diabetes of Mice models
Normal Kunming mouse is taken, after being deprived of food but not water 12h, is injected intravenously the alloxan physiological saline that 65mg/kg is newly prepared
Solution, after 72h, fasting blood sugar is prohibited in detection, and blood sugar concentration ＞ 12mmol/L then think modeling success, qualified mouse is taken to divide at random
It is 11 groups, every group 10, i.e. model group (give and wait capacity pure water)；Melbine group (0.6g/kg)；Medicine group (embodiment 1
Limonin extract, 2 limonin extract of embodiment, 3 limonin extract of embodiment, embodiment 1 slightly carry
Object A, 1 crude extract B of embodiment, 1 crude extract C of embodiment, 1 crude extract D of embodiment, 1 extract E of embodiment, 1 extract of embodiment
F) (5mg/kg), totally 11 groups.Another take is only used as blank control group (give and wait capacity pure water) with batch normal mouse 10.Each group
Animal gastric infusion once a day, continuous 28d.The 1h after administration administration in the 7th, 14,21 and 28 day, retroorbital venous clump
Blood is taken, centrifuges serum, blood glucose is detected according to kit method.
Influence of the 2.2 limonin extracts to diabetic mice sugar tolerance
Animal is administered and grouping is the same as " 2.1 " item.After last time takes blood, immediately to each group animal gavage glucose solution 6g/kg,
30,60,120min after gavage, retroorbital venous clump takes blood, centrifuges serum, the behaviour of kit (GOD-PAP methods)
Make step and measure blood sugar concentration with method.
2.4 statistical analysis technique experimental results are represented with x ± s.Data analysis is carried out using SPSS13.0, is examined with t
Compare two group differences, P ＜ 0.05 think significant difference.
3 results
Influence of the 3.1 limonin extracts to diabetic mice fasting blood-glucose
Influence experimental result of the limonin extract to blood glucose in diabetic mice is shown in Table 2.It is injected intravenously after alloxan respectively
The fasting blood glucose level of group animal is significantly higher than blank group.After treatment 1-4 weeks, 1 limonin extract group animal of embodiment
Blood glucose level is substantially less than model group.
Influence (x ± s) of the 2 limonin extract of table to blood glucose in diabetic mice
Note：Compared with same time blank group,##P ＜ 0.01；Compared with same time model group,*P ＜ 0.05,**P ＜ 0.01,
Influence of the 3.2 limonin extracts to diabetic mice sugar tolerance
30min after the glucose of gavage 6g/kg, the level of postprandial blood sugar of each group mouse significantly increase.The blood glucose of naive animals
Level is significantly replied in postprandial 60min, close to normal level during 120min；But model group animal blood glucose is postprandial 120
Min is still maintained at higher level, and Glucose Tolerance is abnormal；1 limonin extract of embodiment, 2 limonin of embodiment
Class extract, 3 limonin extract of embodiment, 1 crude extract B of embodiment, 1 crude extract C of embodiment, 1 extract of embodiment
E, 1 extract F of embodiment can significantly inhibit the raising of postprandial blood sugar, and make blood glucose during postprandial 120min close to before the meal
Level can effectively reply Glucose Tolerance.
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）2 parts by weight of Maoma chinaberry leaf, 3 parts by weight of small Chinese wampee leaf, 4 parts by weight of Chinese Toon Leaves are taken, mixing crushes, and crosses 20 mesh sieve, uses
Hexamethylene is solvent, and 60 DEG C are extracted 3 times, and each extraction time is 2.5 hours, and each solvent dosage is Maoma chinaberry leaf, small Calusena lansium
12 times of leaf and Chinese Toon Leaves total weight, filtration merge hexamethylene extracting solution, hexamethylene are recovered under reduced pressure, is concentrated and dried to obtain crude extract
A, the dregs of a decoction after hexamethylene extraction are spare；
（2）By step（1）The dregs of a decoction after hexamethylene extraction, with volume ratio 1:5 Ethyl formate-methanol mixed solvent extraction, 75
DEG C extraction 4 times, each extraction time be 3.5 hours, each solvent dosage be Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges Ethyl formate-methanol extract liquid, solvent is recovered under reduced pressure, is concentrated and dried to obtain crude extract B, Ethyl formate-
The dregs of a decoction after methanol extraction are spare；
（3）By step（2）The dregs of a decoction after Ethyl formate-methanol extraction, with volume ratio 4:6 water-ethanol mixed solvent extraction, 50
DEG C extraction 7 times, each extraction time is 3 hours, and each solvent dosage is Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight
15 times, filtration merges water-ethanol extracting solution, solvent is recovered under reduced pressure, and is concentrated and dried to obtain crude extract C, the medicine after water-ethanol extraction
Slag is spare；
（4）By step（3）The dregs of a decoction after water-ethanol extraction, are extracted with water, and 80 DEG C are extracted 2 times, and each extraction time is 1 hour,
Each solvent dosage is 8 times of Maoma chinaberry leaf, small Chinese wampee leaf and Chinese Toon Leaves total weight, and filtration merges aqueous extract, is concentrated under reduced pressure
To medicinal extract, dry crude extract D；
（5）By step（2）Obtained crude extract B, by ratio of weight and number 2:1 uniformly mixed MCI gels-polyamide mixing
Chromatographic column, first with volume ratio 3:97 methanol-water elution, elution amount is 5 chromatography column volumes, then with volume ratio 20:80 first
Alcohol-water elution, elution amount are 12 chromatography column volumes, and collected volume is than 20:80 methanol-water eluent merges, recycling design,
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-NKA9
Macroporous absorbent resin hybrid resin column, first with volume ratio 9:91 alcohol-water elution, elution amount is 4 resin column volumes, then is used
Volume ratio 50:50 alcohol-water elution, elution amount is 2 resin column volumes, and collected volume is than 50:50 alcohol-water eluent
Merge, 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 small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F.
3. a kind of limonin extract, it is characterised in that containing weight ratio be 2:2:3:4 small Calusena lansium element B, Xin Beige
Lignin A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F.
4. one kind is 2 containing weight ratio:2:3:4 small Calusena lansium element B, new shellfish erythrophloeum ferdii element A, new shellfish erythrophloeum ferdii element B, mountain chinaberry body F
Limonin extract is preparing the application in treating diabetes medicament.
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| CN201711438784.6A CN108210600A (en) | 2017-12-27 | 2017-12-27 | A kind of preparation method and applications of limonin extract |
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Cited By (3)
| 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 |
| EP4015000A4 (en) * | 2019-08-12 | 2022-11-23 | Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. | Combination product containing limonin compound and thiazolidinedione |
| EP4014999A4 (en) * | 2019-08-12 | 2022-11-23 | Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. | Combination product containing limonin compound and sulfonylurea drug |
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| CN105012452A (en) * | 2014-04-30 | 2015-11-04 | 周亚伟 | New application of clausena lansium leaves |
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| CN105012452A (en) * | 2014-04-30 | 2015-11-04 | 周亚伟 | New application of clausena lansium leaves |
Non-Patent Citations (2)
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| 侯文彬；等: "2014年新天然活性化合物简介", 《中草药》 * |
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Cited By (3)
| 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 |
| EP4015000A4 (en) * | 2019-08-12 | 2022-11-23 | Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. | Combination product containing limonin compound and thiazolidinedione |
| EP4014999A4 (en) * | 2019-08-12 | 2022-11-23 | Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. | Combination product containing limonin compound and sulfonylurea drug |
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Application publication date: 20180629 |