CN105920074A - Maca extract as well as preparation method and application thereof - Google Patents
Maca extract as well as preparation method and application thereof Download PDFInfo
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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)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
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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
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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
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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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
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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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
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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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
Abstract
The invention discloses a maca extract as well as a preparation method and an application thereof. The maca extract mainly consists of the following materials in percentage by mass: 28-50% of total maca amide, 25-40% of aromatic glucosinolate, 4-12% of total saponins and 1-8% of adenosine, wherein the content of N-benzyl linoleamide in the total maca amide is 35-60% of the content of the total maca amide. The maca extract disclosed by the invention is high in stability, low in content of impurities and rich in various bio-active ingredients; the maca extract has a good effect on improving exercise-induced fatigue resistance and central fatigue resistance; and the maca extract is capable of significantly improving an anti-fatigue effect.
Description
Technical field
The invention belongs to biomedicine technical field, particularly to a kind of Maca extract and its preparation method and application.
Background technology
Lepidinm meyenii Walp (Lepdium meyenii Walpers; Maca) it is that Cruciferae (Brassicaceae) separate row Vegetable spp (Lepidium) is annual or 2 years raw herbaceous plant; originate in mountain area, Peru Andean; the place such as Yunnan Province of China, Tibet, Xinjiang and Sichuan for successfully completing the introducing and planting of Lepidinm meyenii Walp in succession, it is achieved that implantation in large scale.Rich in protein, unique aminoacid composition, multiple unsaturated fatty acid, mineral element and vitamin etc. are not only had due to Lepidinm meyenii Walp; and containing secondary metabolites such as aromatic series glucosinolate, macamide, Lepidinm meyenii Walp alkene, sterol, adenosine, saponin, imidazole alkaloids; there is plurality of health care functions and therapeutical effect, as strengthened energy, resisting fatigue, raising fertility, alleviating pressure, improve sexual function, improve that sleep, neuroprotective, enhancing immunity and antitumor etc. are many-sided to be acted on.
With the quickening pace of modern life, work and social life make people's health the most dog-tired, and people the most also face the biggest pressure, it is easy to produce feeling of fatigue, this state has a strong impact on the quality of life of people, and therefore people produce urgent demand by preventing fatigue with treatment.
Traditional Maca extract preparation method has water extraction, alcohol extracting method, alcohol-water extraction, decoction and alcohol sedimentation technique, ethanol extract from water precipitation, it is difficult to fully obtain Lepidinm meyenii Walp anti-fatigue active composition in view of Lepidinm meyenii Walp bioactive ingredients complexity, existing Maca extract there are the shortcomings such as effective component content is low, use titer is low, therefore a kind of Maca extract active constituent content it is badly in need of high, the extracting method that utilization rate is high.
Summary of the invention
For problem present in current Maca extract preparation process, in order to keep the biological activity needs of functional component in Maca extract, it is achieved fully extract the both effectiveness composition in Lepidinm meyenii Walp, it is an object of the invention to provide a kind of Maca extract and preparation method thereof.
Another object of the present invention is to provide the application of described Maca extract.
A kind of Maca extract, its total macamide mainly including 28% ~ 50% by mass fraction, the aromatic series glucosinolate of 25% ~ 40%, the total saponins of 4% ~ 12%, adenosine of 1% ~ 8%, wherein in total macamide, N-benzyl Asia oleamide content accounts for the 35% ~ 60% of total macamide content.
Relative to prior art, the total macamide of active component in described extract, aromatic series glucosinolate, total saponins, adenosine, particularly N-benzyl Asia oleamide significantly improves, and this extract all has good effect to raising resisting kinetic fatigue and anti-central fatigue.
The present invention also provides for the preparation method of described Maca extract, comprises the steps:
I. dry, pulverize after fresh for Lepidinm meyenii Walp is cut into slices, extract with low polar solvent under the conditions of solid-liquid ratio 1:5 ~ 20, filter to obtain extracting solution a1With filtering residue b1, extracting solution a1Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects the total macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, is dried, obtains extract A；
II. the filtering residue b being filtrated to get in step I1At 105 DEG C of enzyme denaturing 15 ~ 40 min, extract under the conditions of solid-liquid ratio 1:5 ~ 20 with ethanol water, filter to obtain extracting solution a2, extracting solution a2Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, dry, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 1:0.5 ~ 2 by weight, i.e. obtain described Maca extract.
Described preparation method makes total macamide be dissolved in low polar solvent, and glucosinolate etc. is dissolved in ethanol water, and inventor, for the different dissolution properties of extract, devises the preparation method of Maca extract targetedly.Described method is by the process respectively to extracting solution and filtering residue, N-benzyl Asia oleamide content in extract total macamide component is increased, meanwhile, by making described Maca extract have significant advantage compared to the like product of prior art the optimization of extract A, B mixed proportion.
Prepare above-mentioned according to described method
Preferably, one or more during low polar solvent described in step I is ethyl acetate, ether, hexamethylene, normal hexane or petroleum ether.
Preferably, column chromatography described in step I uses silicagel column or neutral alumina column.
Preferably, the solvent that solvent eluting described in step I uses is one or more in methanol, dehydrated alcohol, ethyl acetate or petroleum ether, eluting solvent polarity is ascending carry out isocratic, multistage is isocratic or gradient elution.
Preferably, the one during column chromatography described in step II uses D201, D301, D315, S-8, AB-8 type macroporous resin column or acidic alumina column.
Preferably, the solvent that solvent eluting described in step II uses is any one group in water-dehydrated alcohol-ammonium chloride, water-dehydrated alcohol-ammonium sulfate, water-dehydrated alcohol-sodium bicarbonate, water-dehydrated alcohol-sodium hydroxide, water-dehydrated alcohol-sodium chloride or water-dehydrated alcohol-potassium sulfate or at least two groups are isocratic, multistage is isocratic or gradient elution.Preferably, the concentration of wherein said ammonium chloride, ammonium sulfate, sodium bicarbonate, sodium hydroxide, sodium chloride or potassium sulfate is 0.01 ~ 5
Mol/L, described dehydrated alcohol is 0 ~ 80% with the ratio (V/V) of saline solution (ammonium chloride, ammonium sulfate, sodium bicarbonate, sodium hydroxide, sodium chloride or potassium sulfate).
Preferably, it is thus achieved that the drying mode of described extract A or B is vacuum drying, lyophilization or spray drying.
It is highly preferred that described preparation method comprises the steps:
I. will dry, pulverize to 10 ~ 20 mesh at latter 55 DEG C of fresh for Lepidinm meyenii Walp section, extract 2 ~ 3 times with low polar solvent under the conditions of solid-liquid ratio 1:5 ~ 20, temperature 50 C ~ 80 DEG C, each time 2 h ~ 3 h, filter to obtain extracting solution a1With filtering residue b1, extracting solution a1Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects the macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, is dried, obtains extract A；
II. the filtering residue b being filtrated to get in step I1At 105 DEG C of enzyme denaturing 15 ~ 40 min, extract 2 ~ 3 times under the conditions of solid-liquid ratio 1:5 ~ 20, temperature 50 C ~ 90 DEG C with 50% ~ 98% concentration ethanol aqueous solution, each time 1 h ~ 2 h, filter to obtain extracting solution a2, extracting solution a2Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, dry, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 1:0.5 ~ 2 by weight, i.e. obtain Maca extract of the present invention.
The present invention also provides for the application in improving resisting kinetic fatigue and anti-central fatigue of the described Maca extract.Preferably, the using dosage of described Maca extract is 1.0 g/kg.bw.d.
Showing through zoopery, Maca extract provided by the present invention can significantly improve resisting kinetic fatigue and anti-central fatigue effect.
There is advantages that (1) Maca extract of the present invention has high stability, rich in multiple bioactive ingredients, comprise fat-soluble and two effective sites of water solublity simultaneously, active constituent content is high；(2) the removable a large amount of impurity of the preparation method of Maca extract, significantly improve active constituent content, fully obtain Maca reactive composition, repeatability and good stability simultaneously, and raw material availability and active component yield are high, the equipment of production are adapted to strong, be suitable for industrialized production；(3) evaluated by resisting kinetic fatigue and anti-central fatigue pharmacological evaluation, Maca extract of the present invention all has good effect to raising resisting kinetic fatigue and anti-central fatigue, overcome and cannot reach resisting kinetic fatigue and the problem of anti-central fatigue simultaneously, significantly improve antifatigue effect.
Detailed description of the invention
In order to be better understood from technical scheme, describe, below in conjunction with embodiment, the technical scheme that the present invention provides in detail.
Embodiment
1
Maca extract described in the present embodiment is obtained by following preparation method, and its step includes:
I. weigh fresh of 12 kg Lepidinm meyenii Walp, cut into slices and be dried at latter 55 DEG C, pulverized 10 mesh, extract with petroleum ether (60～90 DEG C), solid-liquid ratio 1:10, Extracting temperature 70 DEG C, filters to obtain extracting solution, extract 2 times, time 2h, merges twice extracting solution, concentrating under reduced pressure every time, silica gel column chromatography on concentrated solution, with ethyl acetate-light petrol system gradient elution (10:90,25:75,35:65,50:50), collect the macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, vacuum drying, obtains extract A；
II. above-mentioned Lepidinm meyenii Walp extracts residue, naturally volatilize, 105 DEG C of enzyme denaturing 30 min, with 80% ethanol water extraction, solid-liquid ratio 1:10, Extracting temperature 70 DEG C, filter to obtain extracting solution, extract 2 times, time 2 h every time, merge twice extracting solution, concentrating under reduced pressure, concentrated solution carries out D201 macroporous resin column chromatography, by water-dehydrated alcohol-potassium sulfate system gradient, (1 mol/L potassium sulfate concentration is constant, dehydrated alcohol and saline solution volume ratio successively 0%, 20%, 40%, 60%, 80%), collect aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, vacuum drying, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 1:2 by weight, i.e. obtain the present embodiment Maca extract.
Described Maca extract detects through spectrophotometer method and high-speed liquid chromatography, mainly include by mass fraction 30.7% total macamide, the aromatic series glucosinolate of 38.9%, the total saponins of 9.1%, the adenosine of 5.8%, wherein in total macamide, N-benzyl Asia oleamide content accounts for the 44.3% of total macamide content.
Embodiment
2
The present embodiment Maca extract is obtained by following preparation method, and its step includes:
I. weigh fresh of 12 kg Lepidinm meyenii Walp, cut into slices and be dried at latter 55 DEG C, pulverized 10 mesh, extract with normal hexane, solid-liquid ratio 1:10, Extracting temperature 60 DEG C, filters to obtain extracting solution, extract 2 times, time 2 h, merges twice extracting solution, concentrating under reduced pressure every time, silica gel column chromatography on concentrated solution, with ethyl acetate-light petrol system gradient elution (10:90,25:75,35:65,50:50), collect the macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, vacuum drying, obtains extract A；
II. above-mentioned Lepidinm meyenii Walp extracts residue, naturally volatilize, 105 DEG C of enzyme denaturing 30 min, with 65% ethanol water extraction, solid-liquid ratio 1:10, Extracting temperature 70 DEG C, filter to obtain extracting solution, extract 2 times, time 2h every time, merge twice extracting solution, concentrating under reduced pressure, concentrated solution carries out acidic alumina column chromatography, by water-dehydrated alcohol-potassium sulfate system gradient, (0.05 mol/L potassium sulfate concentration is constant, dehydrated alcohol and saline solution volume ratio successively 0%, 20%, 40%, 60%, 80%), collect aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, vacuum drying, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 1:1 by weight, i.e. obtain the present embodiment Maca extract.
Described Maca extract detects through spectrophotometer method and high-speed liquid chromatography, mainly include by mass fraction 46.1% total macamide, the aromatic series glucosinolate of 34.6%, the total saponins of 8.4%, the adenosine of 5.0%, wherein in total macamide, N-benzyl Asia oleamide content accounts for the 45.4% of total macamide content.
Embodiment
3
Maca extract of the present invention is obtained by following preparation method, and its step includes:
I. weigh fresh of 12 kg Lepidinm meyenii Walp, cut into slices and be dried at latter 55 DEG C, pulverized 10 mesh, extract with normal hexane, solid-liquid ratio 1:5, Extracting temperature 60 DEG C, filters to obtain extracting solution, extract 2 times, time 2h, merges twice extracting solution, concentrating under reduced pressure every time, neutral alumina column chromatography on concentrated solution, with acetate-methanol system gradient elution (40:60,30:70,20:80,10:90), collect the macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, vacuum drying, obtains extract A；
II. above-mentioned Lepidinm meyenii Walp extracts residue, naturally volatilize, 105 DEG C of enzyme denaturing 30 min, with 65% ethanol water extraction, solid-liquid ratio 1:10, Extracting temperature 70 DEG C, filter to obtain extracting solution, extract 2 times, time 1h every time, merge twice extracting solution, concentrating under reduced pressure, concentrated solution carries out acidic alumina column chromatography, by water-dehydrated alcohol-potassium sulfate system gradient, (0.05 mol/L potassium sulfate concentration is constant, dehydrated alcohol and saline solution volume ratio successively 0%, 20%, 40%, 60%, 80%), collect aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, vacuum drying, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 1:2 by weight, i.e. obtain the present embodiment Maca extract.
Described Maca extract detects through spectrophotometer method and high-speed liquid chromatography, mainly include by mass fraction 29.3% total macamide, the aromatic series glucosinolate of 35.4%, the total saponins of 8.5%, the adenosine of 5.3%, wherein in total macamide, N-benzyl Asia oleamide content accounts for the 41.9% of total macamide content.
Embodiment
4
The present embodiment Maca extract is obtained by following preparation method, and its step includes:
I. weigh fresh of 12 kg Lepidinm meyenii Walp, cut into slices and be dried at latter 55 DEG C, pulverized 10 mesh, extract with normal hexane, solid-liquid ratio 1:10, Extracting temperature 60 DEG C, filters to obtain extracting solution, extract 3 times, time 3h, merges twice extracting solution, concentrating under reduced pressure every time, neutral alumina column chromatography on concentrated solution, with acetate-methanol system gradient elution (40:60,30:70,20:80,10:90), collect the macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, vacuum drying, obtains extract A；
II. above-mentioned Lepidinm meyenii Walp extracts residue, naturally volatilize, 105 DEG C of enzyme denaturing 30 min, with 65% ethanol water extraction, solid-liquid ratio 1:15, Extracting temperature 70 DEG C, filter to obtain extracting solution, extract 3 times, time 1h every time, merge twice extracting solution, concentrating under reduced pressure, concentrated solution carries out AB-8 type macroporous resin column column chromatography, by water-dehydrated alcohol-potassium sulfate system gradient, (1 mol/L potassium sulfate concentration is constant, dehydrated alcohol and saline solution volume ratio successively 0%, 20%, 40%, 60%, 80%), collect aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, vacuum drying, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 2:1 by weight, i.e. obtain the present embodiment Maca extract.
Described Maca extract detects through spectrophotometer method and high-speed liquid chromatography, mainly include by mass fraction 48.9% total macamide, the aromatic series glucosinolate of 26.2%, the total saponins of 5.1%, the adenosine of 1.3%, wherein in total macamide, N-benzyl Asia oleamide content accounts for the 57.2% of total macamide content.
Embodiment 1 ~ 4 also comprises other plant component not detected.
Application examples 1 Pharmacological action to mice resisting kinetic fatigue is tested
1.1 laboratory sample
Select the Maca extract that embodiment 2 prepares as given the test agent, (normal hexane extracts alternative Lepidinm meyenii Walp liposoluble extract, solid-liquid ratio 1:10, 60 DEG C are extracted 2 times, each 2 h, extract the extractum being concentrated to give), (dehydrated alcohol extracts Lepidinm meyenii Walp ethanol-soluble extractives, solid-liquid ratio 1:10, 70 DEG C are extracted 2 times, each 2 h, extract the extractum being concentrated to give), Lepidinm meyenii Walp water solubility extract (flooding, solid-liquid ratio 1:10, 70 DEG C are extracted 2 times, each 2 h, extract the extractum being concentrated to give), extract C (in embodiment 2, extract A and extract B are mixed to get by weight 1:3 ratio uniform), in extract D(embodiment 2, extract A and extract B are mixed to get by weight 3:1 ratio uniform).
1.2 laboratory animal
8 week old male mice in kunming, body weight 18g-22g.
1.3 experimental technique
Male Kunming strain mice is randomly divided into 9 groups, is matched group (normal saline), Lepidinm meyenii Walp liposoluble extract group (1.0 respectively
G/kg.bw.d), Lepidinm meyenii Walp ethanol-soluble extractives group (1.0 g/kg.bw.d), Lepidinm meyenii Walp water solubility extract group (1.0 g/kg.bw.d), extract C group (1.0 g/kg.bw.d), extract D group (1.0 g/kg.bw.d), low dose group (Maca extract of the present invention, 0.2
G/kg.bw.d), middle dosage group (Maca extract of the present invention, 0.5 g/kg.bw.d), high dose group (Maca extract of the present invention, 1.0 g/kg.bw.d), often group 16.Mice conventional illumination, drinking-water, 22-26 DEG C of raising, adapt to 5 days in Animal House environment before mouse test.Every day gavage 1 time, gavage amount is the 1% of Mouse Weight, after giving 30 days time continuously, carry out swimming with a load attached to the body experiment and serum urea, hepatic glycogen and blood lactase acid determination experiment respectively, with reference to " the anti-fatigue effect method of inspection " in country's " health food inspection and assessment technique specification ".
1.3.1
Swimming with a load attached to the body is tested
After last gives given the test agent 30 min, the mice of root of the tail portion load 5% body weight sheet lead is placed in swimming trunk went swimming.The depth of water is no less than 30 cm, water temperature 25 DEG C ± 1 DEG C, and mice starts to the dead time record from swimming, i.e. the mice burden swimming time, result is as shown in table 1.
The different Maca extract of table 1 exhausts the impact of time to mice burden swimming power
Note: compare with blank group,*P < 0.05,**P < 0.01；With liposoluble extract group,ZP < 0.05；With ethanol-soluble extractives group,CP < 0.05；With water solubility extract group,SP < 0.05；With extract C group,GP
< 0.05；With extract D group,DP
< 0.05
1.3.2 the impact on mice serum blood urea nitrogen, hepatic glycogen and blood lactase acid
(1) serum urea measures: last is to given the test agent 30
After min, not swimming with a load attached to the body 90 in the water that temperature is 30 DEG C
Min, after 60 min that have a rest, plucks eyeball blood sampling, after blood sample room temperature stands half an hour, with refrigerated centrifuger 2000
Rpm is centrifuged 10 min, takes serum standby, and with determination of urea nitrogen kit measurement serum urea nitrogen, result is as shown in table 2；
(2) hepatic glycogen measures: last is to after given the test agent 30
Min puts to death animal, takes liver and blots with filter paper after normal saline rinses, measures kit measurement hepatic glycogen with glycogen, and result is as shown in table 2；
(3) blood lactase acid measures: last is to after given the test agent 30
After min, the most do not bear a heavy burden at the water went swimming 10 that temperature is 30 DEG C
Stopping after min, pluck eyeball blood sampling, after blood sample room temperature stands half an hour, be centrifuged 10 min with refrigerated centrifuger 2000 rpm, take serum standby, measure blood blood lactase acid with lactic acid assay kit, result is as shown in table 2.
The different Maca extract impact on mice serum blood urea nitrogen, hepatic glycogen and blood lactase acid of table 2
Note: compare with blank group,*P < 0.05；；With liposoluble extract group,ZP < 0.05；With ethanol-soluble extractives group,CP < 0.05；With water solubility extract group,SP < 0.05；With extract C group,GP
< 0.05；With extract D group,DP
< 0.05
1.4 experiment conclusion
Maca extract of the present invention, result in swimming with a load attached to the body is tested is positive, serum urea nitrogen, hepatic glycogen, three biochemical indicator results of blood lactase acid are also the positive, and in Shi Yan, weight carrying swimming, hepatic glycogen content are significantly higher than and simply carry out extracting Lepidinm meyenii Walp liposoluble extract group, Lepidinm meyenii Walp ethanol-soluble extractives group, Lepidinm meyenii Walp water solubility extract group, extract C group and the extract D group obtained, and show that the Maca extract of the present invention has significantly more efficient resisting kinetic fatigue effect.
Application example 2 Pharmacological action to little mouse-anti central fatigue is tested
2.1 laboratory sample
Select the Maca extract that embodiment 2 prepares as given the test agent
2.2 laboratory animal
8 week old male mice in kunming, body weight 18g-22g.
2.3 experimental technique
Male Kunming strain mice is randomly divided into 4 groups, be respectively blank group, sleep deprivation cause central fatigue model (SD) group, 1. group (Maca extract of the present invention, 0.5 g/kg.bw.d) and 2. group (Maca extract of the present invention, 1.0
G/kg.bw.d), often group 16, gastric infusion 20 days, blank group and the solvent of sleep deprivation group gavage same volume.
2.3.1 set up sleep deprivation animal model
" multi-platform water environment " method using improvement is prepared sleep deprivation and is caused mice central fatigue (SD) model, sleep deprivation case a size of 45 cm × 20, cm × 30 cm, chain-wales a size of 3 cm × 5 cm.Dispose 12 chain-wales in each sleep deprivation case, put into 6 mices on wherein 6 chain-wales；Large platform a size of 15 cm × 5 cm, puts into 3 mices on 1 large platform, and mice can freely be taken the photograph water on platform and ingest.The water surface in sleep deprivation casing about 1 cm low compared with platform, water temperature controls, at 25 DEG C ± 1 DEG C, to change the water in case every day.
2.3.2 channel-type water maze training and test
Black rubber plate is used to make (70 cm × 40 cm
× 30 cm), the depth of water 22 cm, water temperature 25 DEG C.Homemade channel-type water maze is provided with cecum at I, II, III etc. 3, and destination county is provided with step place of safety, and mice can climb up platform safety district to avoid drowning after reaching home.After testing the 9th day gavage medicine, four groups of mices being carried out water maze training respectively, concrete training method is as follows: mice is put in destination county 10 s, and allowing it experience and recognizing is safe here；Then at A point, place dividing plate, start training with A point for starting point, allow mice free swimming, record the incubation period of its channel-type water maze laboratory, i.e. from putting into water to the time used by climb steps；If not finding terminal step person in 120 s, being directed at this, and to record its incubation period be 120 s, every day every, mice trained 2 times continuously.Testing the 10th, 11 days and dividing plate is placed in B point and C point respectively, mice carries out channel-type water maze training from this two place respectively.Test the 12nd day and allow mice from C point, continue to strengthen training 1 day.Carry out mice sleep after 13rd day gavage and deprive 72 h experiments, carry out the test of channel-type water maze, record mice incubation period in water maze and the total degree of entrance cecum I, II, III simultaneously every day.The data that each group mice swims out of incubation period and cecum error number in channel-type water maze laboratory are shown in Table 3 and table 4.
Mice water maze is swum out of preclinical impact by table 3 Maca extract
Note:#P < 0.05 compares with blank matched group, and * P < 0.05 causes central fatigue model group (SD group) with sleep deprivation and compares
The storage on memory of sleeping has very important impact, the sleep deprivation having studied display certain time may result in memory and cognitive decrease, cause brain tissue impairment, use channel-type water maze laboratory can test the change of mice learning and memory ability before and after sleep deprivation.From table 3 and table 4 result, the Maca extract of the present invention 2. group is compared to sleep deprivation model group, water maze is swum out of and is substantially reduced incubation period, and cecum errors number reduces, and shows that the Maca extract of the present invention can improve the decline of the mice learning and memory power ability that sleep deprivation causes.
The impact on mice cecum error number of table 4 Maca extract
Note:#P < 0.05 compares with blank matched group, and * P < 0.05 causes central fatigue model group (SD group) with sleep deprivation and compares
2.3.3 the mensuration of mice akrencephalon biochemical index
After sleep deprivation experiment terminates, putting to death mice with cervical dislocation after 20% urethane anesthetized mice, often group 10, quickly removes akrencephalon tissue in ice bath, and transfers in liquid nitrogen, be then saved in 80 DEG C of ultra cold storage freezers standby.Taking mice akrencephalon tissue to weigh, and put in small beaker, add the normal saline of 9 times of volume pre-coolings according to quality volume 1:9, quickly shred tissue and tissue fluid poured in homogenizer and carry out being fully ground homogenate, all operations is all quickly carried out on ice bath；By the tissue homogenate refrigerated centrifuger for preparing in 3000
Rpm low-temperature centrifugation 10 min, take supernatant 10% tissue homogenate, detection glutathion peroxidase (GSH-Px) vigor, superoxide dismutase (SOD) vigor, malonaldehyde (MDA) content and monoamine neurotransmitter 5-hydroxy tryptamine (5-HT), dopamine (DA) and norepinephrine (NE), method is all with reference to corresponding test kit description, and determination data is shown in Table 5 and table 6.
The impact on mice akrencephalon tissue oxidation resistance of table 5 Maca extract
Note:#P < 0.05 compares with blank matched group, and * P < 0.05 causes central fatigue model group (SD group) with sleep deprivation and compares
Sleep deprivation can cause energy expenditure to increase, and oxygen consumption increases, and produces substantial amounts of oxygen-derived free radicals, neurocyte is caused damage, such as ultra-oxygen anion free radical (O-2), hydrogen peroxide (H2O2) can be with the fatty acid on oxidative cell film, destroy cell membrane lipid structure and intracellular protein structure, and produce the lipid peroxide such as malonaldehyde (MDA), cause the generation of organism fatigue, and consumption and the generation of free radical along with oxygen, also that the transmission function that causes cranial nerve cell synapse is impaired, cause the generation of central fatigue.The organized enzyme removing free radical being widely present in body, as superoxide dismutase (SOD) mainly removes ultra-oxygen anion free radical (O-2), and glutathion peroxidase (GSH-Px) major catalytic H2O2Deng the decomposition of peroxide, play the effect of cytoprotective.As seen from the results in Table 5, supplement the mice of the Maca extract of the present invention, MDA content in cerebral tissue can be significantly reduced, and make the vitality restoration of antioxidase SOD and GSH-Px normal, show that Maca extract can alleviate the free radical and the lipid peroxide damage to cerebral tissue produced during mice sleep is deprived, recover normal antioxidase activity, reduce the generation of central fatigue.
Table 6 Maca extract is on the impact of monoamine neurotransmitter in mice akrencephalon
Note:#P < 0.05 compares with blank matched group, and * P < 0.05 causes central fatigue model group (SD group) with sleep deprivation and compares
The most mental or physical work makes brain nervous cell be overexcited, blood glucose equal energy source material consumption is too much; in order to protect the energy supply of neurocyte; cerebral cortex can produce protectiveness inhibitory action; cause the functional disorder of central nervous system; affect the reasonable release of excitatory neurotransmitter in cerebral tissue, cause the generation of central fatigue.5-HT is as important maincenter monoamines inhibitory neurotransmitter a kind of in brain, and its content increases and the irritability of neuron can be produced inhibitory action, thus reduces the motor capacity of body, causes the generation of fatigue；DA, as a kind of monoamines central excitatory neurotransmitter, can affect the secretion of some hormone of hypophysis, maintains moving equilibrium and the endurance of body, and DA content decline can cause the motor capacity of body to reduce；NE, as important monoamine neurotransmitters a kind of in brain, can make the hippocampal neuron in cerebral tissue excited by beta receptor, play the effect of its hypnotic.As seen from the results in Table 6, supplement the mice of the Maca extract of the present invention, cause central fatigue model group (SD group) with sleep deprivation and compare, the content of 5-HT can be significantly reduced, it is obviously improved the content of DA and NE, recovers normal level, the release of excitatory neurotransmitter in regulation cerebral tissue.
2.4 experiment conclusion
Maca extract of the present invention, can effectively alleviate in the reduction of the learning and memory power that sleep deprivation causes and free radical and lipid peroxide opposite end brain tissue impairment, cerebral tissue the central fatigue symptoms such as excitatory neurotransmitter release imbalance, show the Maca extract of the present invention have anti-in tired effect.
Obviously, embodiment described above is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art are obtained on the premise of not making creative work, broadly fall into the scope of protection of the invention.
The above; being only the detailed description of the invention of the present invention, but protection scope of the present invention is not limited thereto, any those skilled in the art of belonging to are in the technical scope that the invention discloses; the change that can readily occur in or replacement, all should contain within protection scope of the present invention.
Claims (10)
1. a Maca extract, it is characterized in that, its total macamide mainly including 28% ~ 50% by mass fraction, the aromatic series glucosinolate of 25% ~ 40%, the total saponins of 4% ~ 12%, adenosine of 1% ~ 8%, wherein in total macamide, N-benzyl Asia oleamide content accounts for the 35% ~ 60% of total macamide content.
2. the preparation method of Maca extract described in claim 1, it is characterised in that comprise the steps:
I. dry, pulverize after fresh for Lepidinm meyenii Walp is cut into slices, extract with low polar solvent under the conditions of solid-liquid ratio 1:5 ~ 20, filter to obtain extracting solution a1With filtering residue b1, extracting solution a1Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects the total macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, is dried, obtains extract A；
II. the filtering residue b being filtrated to get in step I1At 105 DEG C of enzyme denaturing 15 ~ 40 min, extract under the conditions of solid-liquid ratio 1:5 ~ 20 with ethanol water, filter to obtain extracting solution a2, extracting solution a2Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, dry, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 1:0.5 ~ 2 by weight, i.e. obtain described Maca extract.
Preparation method the most according to claim 2, it is characterised in that low polar solvent described in step I is one or more in ethyl acetate, ether, hexamethylene, normal hexane or petroleum ether.
Preparation method the most according to claim 2, it is characterised in that column chromatography described in step I uses silicagel column or neutral alumina column.
Preparation method the most according to claim 2, it is characterized in that, the solvent that solvent eluting described in step I uses is one or more in methanol, dehydrated alcohol, ethyl acetate or petroleum ether, eluting solvent polarity is ascending carry out isocratic, multistage is isocratic or gradient elution.
Preparation method the most according to claim 2, it is characterised in that column chromatography described in step II uses the one in D201, D301, D315, S-8, AB-8 type macroporous resin column or acidic alumina column.
Preparation method the most according to claim 2, it is characterized in that, the solvent that solvent eluting described in step II uses is any one group in water-dehydrated alcohol-ammonium chloride, water-dehydrated alcohol-ammonium sulfate, water-dehydrated alcohol-sodium bicarbonate, water-dehydrated alcohol-sodium hydroxide, water-dehydrated alcohol-sodium chloride or water-dehydrated alcohol-potassium sulfate or at least two groups are isocratic, multistage is isocratic or gradient elution.
Preparation method the most according to claim 2, it is characterised in that the drying mode obtaining described extract A or B is vacuum drying, lyophilization or spray drying.
9. according to the preparation method described in any one of claim 2 ~ 8, it is characterised in that comprise the steps:
I. will dry, pulverize to 10 ~ 20 mesh at latter 55 DEG C of fresh for Lepidinm meyenii Walp section, extract 2 ~ 3 times with low polar solvent under the conditions of solid-liquid ratio 1:5 ~ 20, temperature 50 C ~ 80 DEG C, each time 2 h ~ 3 h, filter to obtain extracting solution a1With filtering residue b1, extracting solution a1Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects the macamide component eluent based on N-benzyl Asia oleamide, eluent concentrating under reduced pressure, is dried, obtains extract A；
II. the filtering residue b being filtrated to get in step I1At 105 DEG C of enzyme denaturing 15 ~ 40 min, extract 2 ~ 3 times under the conditions of solid-liquid ratio 1:5 ~ 20, temperature 50 C ~ 90 DEG C with 50% ~ 98% concentration ethanol aqueous solution, each time 1 h ~ 2 h, filter to obtain extracting solution a2, extracting solution a2Concentrating under reduced pressure, concentrated solution loading carries out column chromatography, solvent eluting, collects aromatic series glucosinolate, total saponins, adenosine component eluent, eluent concentrating under reduced pressure, dry, pulverize, obtain extract B；
III. by extract A and the ratio uniform mixing of extract B 1:0.5 ~ 2 by weight, i.e. obtain Maca extract of the present invention.
10. the application in improving resisting kinetic fatigue and anti-central fatigue of the Maca extract described in claim 1.
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| CN106581116A (en) * | 2016-12-13 | 2017-04-26 | 中山大学 | Maca extract product preparation method and purpose thereof |
| CN106722965A (en) * | 2017-01-12 | 2017-05-31 | 浙江格蕾斯生物科技有限公司 | A kind of health caring product prescription for fatigue-relieving |
| CN106974955A (en) * | 2017-04-12 | 2017-07-25 | 云南省药物研究所 | A kind of Maca extract for improving sex dysfunction and its preparation method and application |
| CN107233490A (en) * | 2017-06-12 | 2017-10-10 | 拉萨净土睿健生物产业发展有限公司 | A kind of maca composition of supplementing qi and nourishing yin beautifying face and moistering lotion |
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| CN114671755A (en) * | 2022-04-24 | 2022-06-28 | 陕西嘉禾生物科技股份有限公司 | Preparation method of high-content macaene |
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| CN106581116A (en) * | 2016-12-13 | 2017-04-26 | 中山大学 | Maca extract product preparation method and purpose thereof |
| CN106722965A (en) * | 2017-01-12 | 2017-05-31 | 浙江格蕾斯生物科技有限公司 | A kind of health caring product prescription for fatigue-relieving |
| CN106974955A (en) * | 2017-04-12 | 2017-07-25 | 云南省药物研究所 | A kind of Maca extract for improving sex dysfunction and its preparation method and application |
| CN107233490A (en) * | 2017-06-12 | 2017-10-10 | 拉萨净土睿健生物产业发展有限公司 | A kind of maca composition of supplementing qi and nourishing yin beautifying face and moistering lotion |
| CN107233491A (en) * | 2017-06-12 | 2017-10-10 | 拉萨净土睿健生物产业发展有限公司 | It is a kind of to improve the antifatigue maca composition of energy |
| CN109453300A (en) * | 2018-12-29 | 2019-03-12 | 雨润慕德生物科技（连云港）有限公司 | A kind of extracting method and its application of macamide and glucosinolate |
| CN109453300B (en) * | 2018-12-29 | 2021-05-14 | 雨润慕德生物科技（连云港）有限公司 | Extraction method and application of macamide and glucosinolate |
| CN113637597A (en) * | 2021-09-23 | 2021-11-12 | 中肽生物科技(大连)有限公司 | Biological conversion method of maca glycoside |
| CN114671755A (en) * | 2022-04-24 | 2022-06-28 | 陕西嘉禾生物科技股份有限公司 | Preparation method of high-content macaene |
| CN114671755B (en) * | 2022-04-24 | 2023-07-28 | 陕西嘉禾生物科技股份有限公司 | Preparation method of high-content maca alkene |
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