CN106822599B - Dendrobium loddigesii Rolfe extract and anti-diabetic application thereof - Google Patents
Dendrobium loddigesii Rolfe extract and anti-diabetic application thereof Download PDFInfo
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- 241000229499 Dendrobium loddigesii Species 0.000 title claims abstract description 25
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- 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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8984—Dendrobium
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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- 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
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Abstract
The invention relates to a dendrobium candidum extract and an anti-diabetic application thereof. The Dendrobium loddigesii Rolfe extract is obtained by cold soaking the stem of Dendrobium loddigesii Rolfe in acetone water solution, and separating and purifying the extract with resin. Experiments prove that the dendrobium candidum extract has obvious hypoglycemic activity and can be used for treating diabetes and protecting health. The invention also provides an anti-diabetic pharmaceutical composition and a health food containing the dendrobium candidum extract.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a dendrobium candidum extract and application thereof in anti-diabetic medicines and foods.
Background
The traditional Chinese medicine for treating diabetes has a long history of thousands of years in China and has the advantage of lower toxicity compared with western medicines. Dendrobe is clinically used for treating symptoms such as fever body fluid impairment, dry mouth, polydipsia, stomach yin deficiency, poor appetite, retching, persistent asthenic fever after disease, yin deficiency, fire excess, bone steaming, overstrain, dim and unclear eyes, flaccidity of bones and muscles and the like. The dendrobium and a plurality of traditional Chinese medicines are reported to be decocted together, and the dendrobium decoction has exact curative effects on reducing blood sugar and blood fat of patients with type II diabetes and relieving insulin resistance. Dendrobium loddigesii also called Dendrobium nobile, Dendrobium candidum, Dendrobium loddigesii Rolfe, and Dendrobium loddigesii Rolfe, which are mainly distributed in Guizhou, Guangxi, Yunnan and Guangdong provinces, and collected by the Chinese pharmacopoeia since 2000, and are representative of Dendrobium nobile materials widely used in clinical practice. The prior literature discloses that certain monomers extracted from dendrobium candidum have certain alpha-glucosidase inhibitory activity, but the monomers are not the main component of the dendrobium candidum, the availability is unstable, and the in vivo anti-diabetic effect cannot be proved.
Disclosure of Invention
The invention aims to provide a dendrobium candidum extract, and an extraction method of the extract comprises the following steps: (a) cold soaking pulverized herba Dendrobii in 50-80 v/v% acetone water solution for 1-5 days, performing ultrasonic assistance for 1-2 times per day, each time for 30-40 min, and repeatedly extracting for 3-4 times; (b) concentrating the acetone-water extract obtained in the step (a), adsorbing the concentrated acetone-water extract by using reverse phase silica gel, macroporous adsorption resin or MCI resin, eluting by using water, 70%, 80%, 90% and 100% methanol or ethanol in sequence, wherein the using amount of each gradient solvent is 2-4 column volumes, and collecting 70% -90% methanol or ethanol elution parts to obtain the dendrobium candidum extract.
In another aspect of the present invention, an anti-diabetic pharmaceutical composition is provided, which comprises a therapeutically effective amount of the dendrobium candidum extract of the present invention and a pharmaceutically acceptable carrier.
The invention further provides an anti-diabetes health food, which comprises effective amount of the dendrobium candidum extract and food additives.
The invention also provides a method for preparing the dendrobium candidum extract, which comprises the steps of (a) soaking the crushed dendrobium candidum in 50-80% acetone aqueous solution for 1-5 days under the assistance of ultrasonic for 1-2 times every day, and repeatedly extracting for 3-4 times every 30-40 minutes; (b) concentrating the acetone-water extract obtained in the step (a), adsorbing the concentrated acetone-water extract by using reverse phase silica gel, macroporous adsorption resin or MCI resin, sequentially eluting by using 70%, 80%, 90% and 100% methanol or ethanol, wherein the using amount of each gradient solvent is 2-4 column volumes, and collecting 70% -90% methanol elution parts to obtain the dendrobium candidum extract.
In another aspect, the invention provides an application of the dendrobium candidum extract in preparation of a medicament or health food for treating diabetes.
The pharmaceutical composition or health food of the present invention can be prepared into appropriate dosage forms, including but not limited to solid preparations or liquid preparations such as tablets, capsules, oral liquids, soft capsules, granules, etc. The pharmaceutical composition of the present invention is used in the form of a dose per unit body weight. The dosage can be changed according to the age, weight, type and severity of the disease to be treated, and the like of the patient for one or more times.
In some embodiments, the amount of the acetone-water solution added in step (a) of the dendrobium candidum extract is 10-15 times (by mass) of the mass of the dendrobium candidum. In some embodiments, the dendrobium candidum extract of the invention is filtered after the extraction in step (a), the filtrate is taken, the acetone-water solution is recovered, and the extract is obtained by concentration.
In some embodiments, the dendrobium candidum extract of the invention is adsorbed by reverse phase silica gel in the step (b), and then eluted by water, 70%, 80%, 90% and 100% methanol sequentially. The reverse phase silica gel is a silica gel obtained by chemically reacting a normal phase silica gel (ordinary silica gel, functional group is OH) with OH substituted by OR (R is a hydrocarbon chain OR carbon chain, CH), such as C18 reverse phase silica gel, and is available from fuji, japan.
In some embodiments, the dendrobium loddigesii extract of the present invention is adsorbed by macroporous adsorbent resin in step (b), and then sequentially eluted by water, 70%, 80%, 90% and 100% ethanol. The macroporous adsorption resin is a macromolecular adsorption resin which does not contain exchange groups and has a macroporous structure, has a good macroporous network structure and a larger specific surface area, can selectively adsorb organic matters in an aqueous solution through physics, and can be selected from D101, D201, D301, HPD100, HPD400, HPD500, HPD600, AB-8 and NKA-9.
In some embodiments, the dendrobium candidum extract of the invention is adsorbed by MCI resin in the step (b), and then is sequentially eluted by water, 70%, 80%, 90% and 100% methanol. The MCI resin is selected from CHP20/P20, CHP20/P30, CHP20/P50, CHP20/P70, CHP20/P120, CHP50/P30, CHP50/P20, CHP50/P30, CSP50/P10, CHP07/P120, CMG20/P10, CMG20/P30 and CMG 20/P150. Preferably, the MCI resin is selected from CHP20/P20, CHP20/P30, CHP20/P50, CHP20/P70, CHP20/P120, CHP50/P30, CHP50/P20, CHP50/P30, CSP50/P10 and CHP 07/P120. More preferably, the MCI resin is one of CHP20/P20, CHP20/P30, CHP20/P50, CHP20/P70 and CHP 20/P120. The MCI resin is available from, for example, mitsubishi chemical corporation (japan, tokyo).
In some embodiments, the dendrobium candidum of the invention is dendrobium candidum (dendrobium candidum) of the family OrchidaceaeDendrobium loddigesii Rolfe) The stems of (1) are distributed in Guangdong, Guangxi, Yunnan, Guizhou and Hainan, preferably produced in YunnanDendrobium loddigesii A Rolfe plant.
The dendrobium candidum extract can be independently and directly applied, can be mutually combined for application, and can also be combined with other medicines including plant extracts for use in a compound mode. Animal pharmacodynamic experiments prove that the dendrobium candidum extract has definite hypoglycemic drug effect, obviously promotes insulin secretion and relieves insulin resistance.
Detailed Description
The invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention, but rather are intended to be covered by the claims.
Example 1-preparation of the extract of Dendrobium loddigesii Rolfe of the invention
Taking dried Dendrobium Loddigesii Rolfe (C. Merr.), (Dendrobium loddigesii Rolfe) Pulverizing, weighing 1000g, placing in a container, and adding VAcetone (II)：VWater (W)= 8: 1, soaking the mixture in the acetone-water solution for 3 times, soaking for 5 days for the first time, soaking for 1 day for the second time and the third time respectively, extracting by ultrasonic assistance during the soaking period, adopting 10 times of the acetone-water solution each time, filtering, taking filtrate, recovering acetone, and concentrating under reduced pressure to obtain 55g of dry extract. Dissolving the dendrobium candidum dry extract by using a proper amount of methanol, loading a sample on a C-18 reverse phase silica gel column with the volume of 10 cm' -40 cm (i.d.), then sequentially eluting by using water, 70%, 80%, 90% and 100% of methanol, wherein the gradient dosage is 5 column volumes, collecting 70% and 80% of methanol eluent, and performing reduced pressure spin drying to obtain 18 g of the dried dendrobium candidum extract.
Example 2 preparation of the extract of Dendrobium loddigesii Rolfe of the present invention
The preparation of the dendrobium candidum dry extract is the same as that in example 1. Dissolving the dendrobium candidum dry extract by using a proper amount of ethanol, loading a sample to a D101 macroporous adsorption resin adsorption column with the column volume of 10 cm' 100cm (i.d.), then sequentially eluting by using water, 70%, 80%, 90% and 100% of ethanol, wherein the using amount of each gradient solvent is 3 column volumes, and collecting 70% -90% of ethanol elution parts to obtain the dendrobium candidum extract.
Example 3 preparation of the extract of Dendrobium loddigesii Rolfe of the present invention
The preparation of the dendrobium candidum dry extract is the same as that in example 1. Dissolving the dendrobium candidum dry extract by using a proper amount of methanol, loading a sample to an MCI GEL resin adsorption column with the column volume of 10 cm' 80cm (i.d.), then sequentially eluting by using water, 70%, 80%, 90% and 100% of methanol, wherein the using amount of each gradient solvent is 3 column volumes, and collecting 70% -90% of methanol elution parts to obtain the dendrobium candidum extract.
EXAMPLE 4 preparation of pharmaceutical composition (tablet)
Taking the dendrobium candidum extract prepared by the method of example 1, 60mg, 130mg of lactose, 55mg of starch and 5mg of magnesium stearate, mixing the dendrobium candidum extract, the lactose and the starch, wetting with water, sieving and drying the wetted mixture, sieving again, adding the magnesium stearate, and tabletting the mixture, wherein each tablet is 250mg in weight and the content of the dendrobium candidum extract is 60 mg.
EXAMPLE 5 preparation of health food (Capsule)
60mg of the dendrobium candidum extract prepared by the method in the embodiment 2, 137mg of lactose and 3mg of magnesium stearate are taken. Mixing the Dendrobium loddigesii Rolfe extract with adjuvants, sieving, mixing, and encapsulating the obtained mixture into hard gelatin capsules with each capsule weight of 200mg and content of Dendrobium loddigesii Rolfe extract of 60 mg.
Experimental example 1 anti-diabetic Effect of Dendrobium loddigesii Rolfe extract
The experimental principle is as follows: the dbdb mouse is a typical animal model of type 2 diabetes mellitus due to a single gene mutation (BKS. Cg-Dock7m +/+ Leprdb/Nju), and is derived from a mutation in the Janus protein tyrosine kinase (JAK) gene site of the number 4 autosomal leptin receptor (LEPR). The dbdb mice are characterized by spontaneous type II diabetes mellitus, which is obese, hyperglycemic, and highly resistant to insulin, and in the C57BLKS background, many features can be observed, such as uncontrolled elevation of blood glucose, loss of insulin-producing beta cells, uncontrolled blood glucose levels of exogenous insulin, and elevated gluconeogenic enzyme activity. Peripheral neuropathy and cardiomyopathy can be observed in the C57BLKS-Leprdb homozygote, the healing speed is reduced, the metabolism is increased and the like, and the method is a common animal model for researching the medicament and the mechanism of the type II diabetes.
The experimental method comprises the following steps: db/db mice, 5 weeks old, after being acclimatized for one week, were randomly divided into 3 groups: model control group, Dendrobium loddigesii Rolfe extract group (prepared in examples 1-3) (100m g/kg), metformin group (130 mg/kg), 8 mice per group, and C57 mice were used as normal control group. The administration is carried out by intragastric administration every day for 9: 00-10: 00 am, the administration is carried out 7 days per week for 7 weeks, and the weight and the blood sugar value of the mouse are detected 1 time every 7 days. Mice had free access to food and water during the experiment.
Sugar tolerance test method: at 6 weeks of administration, the glucose solution (2 g/kg) was gavaged 12 hours after fasting in each group of mice, and the blood glucose levels of the mice were tested at 0.5, 1, 1.5, and 2 hours after sugar administration. The blood sugar test method comprises the following steps: test paper method is adopted. The type of the glucometer is as follows: ACCU-CHEK Performa. The type of the blood glucose test paper is as follows: ACCU-CHEK Performa.
The blood sugar reducing test results of the dendrobium candidum extract are shown in table 1, and the test results are expressed as the average value of blood sugar values of mice detected every week, wherein the administration group is a dendrobium candidum extract group, the positive medicine group is a metformin group, and the model group and the normal control group are used as the medicine group. The experimental results of the Dendrobium loddigesii Rolfe extract on the glucose tolerance of dbdb mice are shown in table 2, and the experimental results are expressed as the average value of blood glucose values of the mice in 12 hours in fasting state and the average values of blood glucose values of the mice in 0.5, 1, 1.5 and 2 hours after the glucose solution is orally taken (2 g/kg), wherein the drug administration group is a Dendrobium loddigesii Rolfe extract group, the positive drug group is a metformin group, and the model group and a normal control group are adopted.
From the experimental results in tables 1 and 2, the dendrobium candidum extract has better in-vivo blood sugar reducing effect, and the in-vivo blood sugar reducing effect is close to that of the clinical blood sugar reducing agent metformin.
TABLE 1-Dendrobium loddigesii extract dbdb mice in vivo hypoglycemic experiment
TABLE 2 influence of Dendrobium Loddigesii extract on sugar tolerance of dbdb micea
aEach oneThe oral glucose dose of experimental mice was 2 g/kg.
Claims (7)
1. The extraction method of the dendrobium candidum extract comprises the following steps:
(a) cold soaking pulverized herba Dendrobii in 50-80 v/v% acetone water solution for 1-5 days, performing ultrasonic assistance for 1-2 times per day, each time for 30-40 min, and repeatedly extracting for 3-4 times;
(b) concentrating the acetone-water extract obtained in the step (a), adsorbing the concentrated acetone-water extract by using C18 reverse phase silica gel, sequentially eluting by using water, 70%, 80%, 90%, 100% methanol or ethanol, wherein the using amount of each gradient solvent is 2-4 column volumes, and collecting 70% -90% methanol or ethanol elution parts to obtain the dendrobium candidum extract.
2. The Dendrobium loddigesii Rolfe extract of claim 1, wherein the amount of acetone aqueous solution added in step (a) is 10-15 times of the mass of Dendrobium loddigesii Rolfe.
3. The dendrobium candidum extract as claimed in claim 1, wherein the C18 reversed phase silica gel is used for adsorption in step (b), and then water, 70%, 80%, 90% and 100% methanol are used for elution sequentially.
4. An anti-diabetic pharmaceutical composition comprising a therapeutically effective amount of the extract of dendrobium candidum of any one of claims 1-3 and a pharmaceutically acceptable carrier.
5. A health food for assisting in lowering blood sugar, comprising an effective amount of the Dendrobium loddigesii Rolfe extract of any one of claims 1-3 and a food additive.
6. A method for preparing a Dendrobium loddigesii Rolfe extract, comprising the following steps:
(a) cold soaking pulverized herba Dendrobii in 50-80 v/v% acetone water solution for 1-5 days, and performing ultrasonic assistance for 1-2 times per day for 30-40 min each time; repeatedly extracting for 3-4 times;
(b) concentrating the acetone-water extract obtained in the step (a), adsorbing the concentrated acetone-water extract by using C18 reverse phase silica gel, sequentially eluting by using water, 70%, 80%, 90% and 100% methanol, wherein the using amount of each gradient solvent is 2-4 column volumes, and collecting 70% -90% methanol elution parts to obtain the dendrobium candidum extract.
7. The use of the Dendrobium loddigesii Rolfe extract according to any one of claims 1-3 in the preparation of a medicament for treating diabetes or a health food for assisting in lowering blood sugar.
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