Preparation method of total alkaloid from lotus plumule and medical application thereof

Technical Field

The present invention relates to method for preparing total alkaloid Plumula thereof in reducing blood lipid application of the medicament.

Background Art

In total alkaloid is Plumula liensinine, isoliensinine, methylliensinine of the alkali mixture the main component.

Plumula the nymphaeaceae plant lotus Nelumbo   nucifera   Gaertn. In mature seeds of drying young leaf and radicle. Flavor myoporum, cold, centering, the kidney, research shows that, as the main ingredient of the Plumula liensinine, isoliensinine, methylliensinine and osmanthus defattde such alkaloids, Hyperin, flavonoid yun fragrant glucoside. It has effects of lowering blood pressure, anti-arrhythmic, inhibit platelet aggregation, anti-oxidation and removal of active free radical, myocardial ischemia, laidback smooth muscle, tonified, anticancer, the protection of the brain ischemia damage, improving acute lung injury and pulmonary fibrosis, central inhibition, hypoglycemic, pharmacological action such as for treating prostate hyperplasia.

The literature reports with anti-tumor function Plumula, and the active site of the anti-tumor effect of the total alkaloid rather than total flavonoids and total polysaccharide. HepG2 on human liver cancer cells, human stomach cancer cell SGC-7901, human breast cancer cell MCF-7 and human colon cancer cells of the RKO IC₅₀ to 0.055-0.131 mg/mL. According to literature reports Neferine methylliensinine extensive chemotherapy has the role of the curative effect, and the side effect is relatively small.

Existing Plumula medical use relevant patents extract 5 a: "the Plumula Nelumbinis and its alkloids and a new use of derivative thereof" CN103070927A public number , "Plumula or its extract new use of" CN102091131A public number , "the Plumula Nelumbinis and its extract new use of" public number C N101862374A, "Lotus plumule and its usage part extract and use thereof" CN1593598A public number , "a Plumula alkaloid extract method for preparing the same and its in preparation of medicine for cerebrovascular disease application in" public number CN1670026A.

Content of the invention

The present invention provides a method for preparing total alkaloid Plumula and its medical use. The use of the traditional Chinese medicine Plumula purpose, after highly soaks mellowly raises , macroporous resin column separation method, the preparation of a has strong activity of reducing blood lipid extract Plumula.

The invention relates to a method for preparing total alkaloid Plumula, comprising the following steps:

Extracting: the coarse powder is pulverized into Plumula, using 8-12 volume times 75-85% methanol or ethanol diyestion 2-3 time, each cold soaking 48 hours, electric agitator stirring 2-3 hours, speed 400-500r/min, filtration, filtrate is combined, pressure-reducing recovery mellowly to solution specific gravity of 1.1, the heart soaks mellowly extract aqueous solution shall be Lotus;

Separation: heating plumula the heart soaks mellowly extract aqueous solution, weak polar solvent extraction for degreasing, adjusting by acid solution pH=1-2 water, standing 24h, filtering to remove the flavonoid compound or other ingredient, adjusted with an alkaline solution filtrate pH=10-11, through the macroporous adsorbent resin column adsorption, water washing in addition to heteroatoms, reuse pH=3 the 70-80% ethanol to elute, collect ethanol eluent, to neutral, decompression recovering ethanol, drying, to total alkaloid Plumula crude product;

Purification of:re-crystallization with ethanol, to total alkaloid Plumula;

Preparation by the method of total alkaloids Plumula yield > 1.0%, purity > 90%.

The invention is used for degreasing the weak polar solvent comprises a petroleum ether, ethyl ether, cyclohexane.

The acid solution of the present invention by using dilute hydrochloric acid, dilute sulfuric acid or dilute phosphoric acid solution.

The invention the alkali solution adopting NaOH, Na₂ CO₃ alkali solution or KOH.

The invention said macroporous adsorbent column models include the D101, D201, HPD100, D4020 or AB8.

The preparation method of this invention the total alkaloid Plumula the application of preparing bypolipidemic.

When the invention is used for preparing hypolipidemic, oral or gastrointestinal edministration are safe, in oral circumstances, it can be of any conventional form, such as granules, tablets, capsule, pill, syrup, oral solution, oral emulsion, : when the when the drug gastronintestinal edministration, may take any conventional form, for example, injection: such as intravenous injection, ointment, transdermal, inhalant, and the like.

The present invention is formed by preparing bypolipidemic the effective component and the solid or liquid excipient is formed with a, here the use of a solid or liquid excipient is well known in this field, for example below a few specific, powder is taken orally the powders, the excipient with lactose, starch, slurry dextrin, calcium carbonate, synthetic or natural aluminum sulfate, magnesium oxide, magnesium stearate, sodium bicarbonate, dried yeast, and the like; the excipient solution of a water, glycerin, 1, 2-propylene glycol, single syrup, ethanol, ethylene glycol, polyethylene glycol, sorbitol, etc.; the excipient can use grease with acid, hydrous lanolin, vaseline, glycerin, the bee is sacrificial , vegetable wax , liquid paraffin, resin, hydroghobing high-level sacrificial or hydrophilizing agent arranged.

The beneficial effects of this invention, total alkaloid Plumula can be used for preparing hypolipidemic, with prominent characteristic of curative effect. The dosage of the active substance according to the mode of administration, patient's age and weight and serious condition extent and other similar factors but, for oral administration quantity : 30-50mg/time, two times a day: injection 30-50mg/time, one time every day.

Mode of execution

Embodiment 1

Extracting: the coarse powder is pulverized into Plumula, with 8 times of volume 75% methanol lixiviating 2 times, each cold soaking 48 hours, electric agitator stirring 2 hours, speed 400r/min, filtration, filtrate is combined, pressure-reducing recovery mellowly to solution specific gravity of 1.1, the heart soaks mellowly extract aqueous solution shall be Lotus;

Separation: heating plumula the heart soaks mellowly extract aqueous solution, using petroleum ether extracting and degreasing, water layer with diluted hydrochloric acid solution to adjust pH=1, standing 24h, ingredients such as filtered to remove the flavonoid compound, with the filtrate solution to adjust NaOH pH=10, through the macroporous adsorbent resin column D101 adsorption, water washing in addition to heteroatoms, pH=3 for the 70% ethanol to elute, collect ethanol eluent, to neutral, decompression recovering ethanol, drying, to total alkaloid Plumula crude product;

Purification of:re-crystallization with ethanol, to total alkaloid Plumula;

Preparation by the method of total alkaloids Plumula yield > 1.0%, purity > 90%.

Embodiment 2

Extracting: the coarse powder is pulverized into Plumula, with 10 times volume 80% methanol lixiviating 3 times, each time the cold soaking 48 hours, electric agitator stirring 2 hours, speed 450r/min, filtration, filtrate is combined, pressure-reducing recovery mellowly to solution specific gravity of 1.1, the heart soaks mellowly extract aqueous solution shall be Lotus;

Separation: heating plumula the heart soaks mellowly extract aqueous solution, weak polar solvent extraction for degreasing, water regulating pH=1.5 by dilute hydrochloric acid, and 24h, ingredients such as filtered to remove the flavonoid compound, with the filtrate solution to adjust NaOH pH=10.5, through the macroporous adsorbent resin column D101 adsorption, water washing in addition to heteroatoms, pH=3 for the 75% ethanol to elute, collect ethanol eluent, to neutral, decompression recovering ethanol, drying, to total alkaloid Plumula crude product;

Purification of:re-crystallization with ethanol, to total alkaloid Plumula;

Preparation by the method of total alkaloids Plumula yield > 1.0%, purity > 90%.

Embodiment 3

Extracting: the coarse powder is pulverized into Plumula, with 12 times of volume 85% methanol lixiviating 3 times, each time the cold soaking 48 hours, electric agitator stirring 3 hours, speed 500r/min, filtration, filtrate is combined, pressure-reducing recovery mellowly to solution specific gravity of 1.1, the heart soaks mellowly extract aqueous solution shall be Lotus;

Separation: heating plumula the heart soaks mellowly extract aqueous solution, weak polar solvent extraction for degreasing, water layer with diluted hydrochloric acid solution to adjust pH=2, standing 24h, ingredients such as filtered to remove the flavonoid compound, with the filtrate solution to adjust NaOH pH=11, through the macroporous adsorbent resin column D101 adsorption, water washing in addition to heteroatoms, pH=3 for the 80% ethanol to elute, collect ethanol eluent, to neutral, decompression recovering ethanol, drying, to total alkaloid Plumula crude product;

Purification of:re-crystallization with ethanol, to total alkaloid Plumula;

Preparation by the method of total alkaloids Plumula yield > 1.0%, purity > 90%.

In the above-mentioned examples 1-3 in, also can be used for leaching of ethanol, defatted the weak polar solvent can also be used for ether, cyclohexane, acid solution with dilute sulfuric acid or dilute phosphoric acid solution, alkali solution adopting Na₂ CO₃ alkali solution or KOH, macroporous resin column models include the D201, HPD100, D4020 or AB8, the manner of replacing, one of ordinary skill in the art can be completed without creative work.

The preparation method of this invention the total alkaloid Plumula the application of preparing bypolipidemic.

By reducing fat of the lower side of the active test further shows that the effect of this invention.

The test is the mouse and RAT rolloffs drug pharmacological experiment of the relevant animal model, the decreasing blood fat from the total alkaloid Plumula a pilot study for pharmocodynamics aspects.

1, total alkaloid Plumula to the impact of the content of mouse's fat:

Materials and methods

Animals

ICR mouse, 72 only, the female half, weight 18-20g. Bought from jlu basis center of medical experiment animals, animal certificate number : SCXK-(Kyrgyz)-2003-0001.

Drugs and reagent

Total alkaloid Plumula, character: white powder, water, bitter, lot number: 20131203, jlu regeneration medical Institute of science to provide; fat khang capsule, specifications: 300 mg/granulata, lot number 20130605, Beijing University wbl biotechnology limited production; cholesterol, specifications: biochemical reagent, 25g/bottle, lot number: 20130516, tianjin kuangfu fine chemical research Institute; cholic acid sodium, specifications: biochemical reagent, lot number: 20130525, Beijing chemical reagent Company; lard, provided by the research; serum total cholesterol (TC) content determination reagent kit, lot number: 20130501, in biological science and technology Company limited north controls ;

Triglyceride content determination kit (TG), lot number: 20131831, in biological science and technology Company limited north controls ; (HDL-cho) high-density lipoprotein cholesterol content determination reagent kit, lot number: 20130171, in biological science and technology Company limited north controls ; (LDL-cho) low-density lipoprotein cholesterol content determination reagent kit, lot number: 20130171, shares in biotechnology north controls limited Company's products.

Instrument

Enzyme, type:   A-5082 PECAN, Austria   SUNRISE the Company's product.

Method

High lipid feed preparation method is as follows: reference literature^[1], in 88.7% in the feed the basis of adding 10% lard, 1% cholesterol and 0.3% cholic acid sodium. The lard govind dissolving, adding cholesterol, cholic acid sodium, is poured into the feed after mixing, mix, add right amount of warm water mix thoroughly, in the square plate, the thickness is about 1.5 cm, wall paper knife for divided into 2 cm × 3 cm-size small, is the 80 [...] constant temperature drying box for drying.

Dispensing method: the administration dose according to each group respectively, the body weight of animals, calculate the number of days of administration requirements, precision stability, to add a small amount of distilled water, grind homogeneous, add distilled water to the required concentration can be.

Purchase animals adapt to the environment after 3 days, random according to body weight is divided into: the normal control group; hyperlipidic model set; positive control drug Xuezhikang, capsules (240 mg/kg); total alkaloid Plumula high-dose group (80 mg/kg), dose group in (40 mg/kg) and a low dose group (20 mg/kg); a total of 6 groups, each group of 12 only. The male-female half.

In the course of experiment, conventional feed animal ability of normal control group, the other groups of animals are ability high lipid feed. Laboratory animal medicine each morning intragastrically to 1 time. Intragastrically volume is 20 ml/kg. Normal control group and hyperlipidic model group of animal intragastrically distilled water, are given corresponding for medicine group drug solution. Continuous administration 2 weeks, last 1 in the evening to, animal jejunitas water is 16 hours, morrows eyeball blood taking 0.5 ml, centrifugal (3000r/min) separation of serum; by enzyme colorimetric method for determining the content of the serum total cholesterol (TC), combines enzyme colorimetric determination (TG) content of serum triglycerides, phosphotungstic acid-magnesium precipitation method by determining serum high density lipoprotein cholesterol content (HDL-cho), polyethylene sulfuric acid precipitation method by determining serum low density lipoprotein cholesterol content (LDL-cho). Index data in order to average standard deviation of plus or minus Expressed, statistical processing method using interblock t-test.

Results and discussion

Experimental results indicate that, in MICE in manufacturing the high lipid feed raises continuously 2 weeks later, serum of the animal models TC, TG, a normal control group content is LDL-cho obviously rise, the normal control group content TC 3.38±0 . 51mmol/L, model group is 6.99±0 . 90mmol/L (P < 0.01); the normal control group content TG 0.71±0 . 21mmol/L, model group is 0.87±0 . 22mmol/L (P < 0.05); the normal control group content LDL-cho 0.31±0 . 29mmol/L, model group is 1.96±1 . 11mmol/L (P < 0.01); serum of the animal models HDL-cho content is obviously reduced, normal control group for 4.10±1 . 71mmol/L, model group is 2.52±0 . 83mmol/L (P < 0.05); serum of the animal models with TC (TC/HDL-cho) HDL-cho the ratio of the content of the obviously rise, the normal control group 0.95±0 . 25, the models 3.06±1 . 62 (P < 0.01); high blood fat animal model results show that has already formed.

Two weeks after the administration, the total alkaloid Plumula 80 mg/kg the drug dose group of animal serum content TC (5.35±0 . 76mmol/L) are obviously less than hyperlipidic model set (6.99±0 . 90mmol/L) (P < 0.01); the group of animal serum TG content (0.49±0 . 14mmol/L) also a hyperlipidic model set (0.87±0 . 22mmol/L) is obviously reduced (P < 0.01); the group of animal serum HDL-cho content (3.86±1 . 01mmol/L) a model set (2.52±0 . 83mmol/L) obviously rise, corresponding administration group of animal serum HDL-cho TC the ratio of the content (1.53±0 . 41) is model set (3.06±1 . 62) reduce (P < 0.05).

Total alkaloid Plumula 40 mg/kg the drug dose group of animal serum TC, TG, LDL-cho, HDL-cho content and TC/HDL-cho animal model set ratio with no significant difference compared with the; total alkaloid Plumula 20 mg/kg the drug dose group of animal serum TC, TG, LDL-cho, HDL-cho content and TC/HDL-cho ratio compared with the model group there is no significant difference of animals.

The above-mentioned test results show that, the total alkaloids Plumula can obviously reduce the content of fat model mouse serum TC, HDL-cho content of serum, the serum TC and the ratio of the content HDL-cho (TC/HDL-cho) is reduced.

Each group of animal TC, TG, LDL-cho, HDL-cho content and TC/HDL-cho ratio shown in table 1.

2, total alkaloid Plumula to the impact of the RAT blood fat content:

Materials and methods

Wistar RAT animal, male, 72 only, weight 170g-210g. Bought from jlu based medical animal center, animal certificate number : SCXK-(Kyrgyz)-2003-0001.

Drugs and reagent

Total alkaloid Plumula, character: white powder, water, bitter, lot number: 20131203, jlu regeneration medical Institute of science to provide; fat khang capsule, specifications: 300 mg/granulata, lot number 20130605, Beijing University wbl biotechnology limited production; cholesterol, specifications: biochemical reagent, 25g/bottle, lot number: 20130516, tianjin kuangfu fine chemical research Institute; cholic acid sodium, specifications: biochemical reagent, lot number: 20130525, Beijing chemical reagent Company; lard, provided by the research; serum total cholesterol (TC) content determination reagent kit, lot number: 20130501, in biological science and technology Company limited north controls ; caprylic triglyceride (TG) content determination reagent kit, lot number: 201391831, in biological science and technology Company limited north controls ; (HDL-cho) high-density lipoprotein cholesterol content determination reagent kit, lot number: 20130171, in biological science and technology Company limited north controls ; (LDL-cho) low-density lipoprotein cholesterol content determination reagent kit, lot number: 20130171, shares in biotechnology north controls limited Company's products.

Instrument:

Enzyme, type:   A-5082 PECAN, Austria   SUNRISE the Company's product.

Method:

High lipid feed preparation method is as follows: reference literature (li Yi Kui , wang Qin Mao , week Golden yellow. Pharmacological experimental methodology of traditional Chinese medicine, Shanghai: Shanghai scientific and technical Publishing house, 1991, 6:398), in 88.7% in the feed the basis of adding 10% lard, 1% cholesterol and 0.3% cholic acid sodium. The lard govind dissolving, adding cholesterol, cholic acid sodium, is poured into the feed after mixing, mix, add right amount of warm water mix thoroughly, in the square plate, the thickness is about 1.5 cm, wall paper knife for divided into 2 cm × 3 cm-size small, is the 80 [...] constant temperature drying box for drying.

Dispensing method: the administration dose according to each group respectively, the body weight of animals, calculate the number of days of administration requirements, precision stability, to add a small amount of distilled water, grind homogeneous, add distilled water to the required concentration can be.

The animals, first feeding observation, its adaptation to environment 1 week. Animal RAT cuts the tail , measured TC; TG ; and HDL-cho LDL-cho content, according to the blood lipid level random is divided into: the normal control group; hyperlipidic model set; positive control drug Xuezhikang, capsule 120 mg/kg group; is the total alkaloid Plumula 40 mg/kg, 20 mg/kg and 10 mg/kg dose group three administration. A total of 6 groups, each group of 12 only. In addition to section 1 group of animal ability of conventional feed, as the normal compared with the outer; the remaining 2-6 high-cholesterol feed both the ability, to throws the appetite : 25g/mouse/day. Each group of animals every day to ig, ig volume is 10 ml/kg; normal control and a hyperlipidic model group of animals, such as distilled water each day the capacity of the field; to consecutive 2 weeks. After the last administration, fasting (not forbidden water) 16 hours, shear RAT tail, separation of serum, measuring serum total cholesterol (TC) triglyceride (TG); high-density lipoprotein cholesterol (HDL-cho); low-density lipoprotein cholesterol content and (LDL-cho) TC/HDL-cho ratio. To continue administration to 4 weeks. For the final 1 time after administration, animals (not forbidden water) jejunitas 16 hours, shear RAT tail, separation of serum, the same measuring the above-mentioned various indicators. Add or subtract to the data included in standard long life Said; statistical processing method using interblock t-test.

Results and discussion

Before the test, the blood lipid level of each group of animals is more even, without distribution between groups (P > 0.05) differences obvious. Feed raises by Gao Zhi 2 weeks later, serum of the animal models TC, TG content obviously rise control than normal (are P < 0.01, P < 0.05), normal HDL-cho obviously reduces the content of the control group (P < 0.05), normal TC/HDL-cho the control group (P < 0.001) is significantly reduced. Note at this moment hyperlipidermia animal model has been formed. The total alkaloid Plumula the three administration in the high dose group, 40 mg/kg dose group of animal serum TC is obviously lower than the content of fat model group (P < 0.05), 20 mg/kg and 10 mg/kg dose group TC/HDL-cho a model group (P < 0.01) is significantly reduced. Continue to ability high lipid feed to 4 week, 40 mg/kg, 20 mg/kg and 10 mg/kg three dose groups of animal serum TC of high lipid content is significantly lower than model set (P < 0.001, P < 0.01 and P < 0.05), 40 mg/kg and 20 mg/kg two dose group of animal serum TG of high lipid content is significantly lower than model group (P < 0.05). 40 mg/kg dose group of animals is significantly higher than that of the serum HDL-cho content of fat model group (P < 0.05). The corresponding 40 mg/kg, 20 mg/kg and 10 mg/kg three dose groups TC/HDL-cho ratio of the serum of the animal is obviously reduced (P < 0.001, P < 0.01). The above-mentioned test results show that, the total alkaloids Plumula preventive administration 4 week, can obviously inhibit the formation of the RAT hyperlipidemia, can be reduced and TC TG content, the content HDL-cho, TC HDL-cho the ratio of the content is reduced. Detailed results are shown in table 2-table 4.