CN105920074B - Maca extract and preparation method and application thereof - Google Patents
Maca extract and 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 and a preparation method and application thereof. The extract mainly comprises 28-50% of total macamides, 25-40% of aromatic glucosinolates, 4-12% of total saponins and 1-8% of adenosine by mass, wherein the content of N-benzyl linoleamide in the total macamides accounts for 35-60% of the content of the total macamides. The maca extract disclosed by the invention is high in stability, few in impurities, rich in various bioactive components, good in effect of improving both sports fatigue resistance and central fatigue resistance, and remarkably improved in fatigue resistance.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a maca extract and a preparation method and application thereof.
Background
Maca (Lepdium meyenii Walpers, Maca) is an annual or biennial herbaceous plant of Lepidium genus (Lepidium) of Brassicaceae family (Brassicaceae), is originally produced in Peru Andes mountainous area, and successfully completes the introduction and cultivation of Maca in Yunnan, Tibet, Xinjiang, Sichuan and other places in China, thereby realizing large-scale planting. The maca not only contains rich protein, unique amino acid composition, various unsaturated fatty acids, mineral elements, vitamins and the like, but also contains secondary metabolites such as aromatic glucosinolate, macamide, macaene, sterol, adenosine, saponin, imidazole alkaloid and the like, and has various health-care functions and treatment effects, such as various effects of enhancing energy, resisting fatigue, improving fertility, relieving pressure, improving sexual function, improving sleep, protecting nerves, enhancing immunity, resisting tumors and the like.
With the acceleration of life rhythm, people are increasingly tired in labor and social life, people are also under great mental stress and easily feel tired, and the state seriously affects the life quality of people, so people have urgent need for the prevention and treatment of fatigue.
The traditional preparation method of the maca extract comprises a water extraction method, an alcohol-water extraction method, a water extraction and alcohol precipitation method and an alcohol extraction and water precipitation method, and the anti-fatigue active ingredients of maca are difficult to obtain fully, so that the existing maca extract has the defects of low content of active ingredients, low use value and the like, and therefore an extraction method with high content of the active ingredients and high utilization rate is urgently needed.
Disclosure of Invention
Aiming at the problems existing in the preparation process of the traditional maca extract, in order to keep the biological activity requirement of the functional components in the maca extract and realize the full extraction of the functional components in the maca, the invention aims to provide the maca extract and the preparation method thereof.
Another object of the present invention is to provide the use of said maca extract.
A maca extract mainly comprises, by mass, 28-50% of total maca amide, 25-40% of aromatic glucosinolate, 4-12% of total saponin and 1-8% of adenosine, wherein the content of N-benzyl linoleamide in the total maca amide accounts for 35-60% of the content of the total maca amide.
Compared with the prior art, the active ingredients of the extract, namely total macamides, aromatic glucosinolates, total saponins and adenosines, particularly N-benzyl linoleamide, are remarkably improved, and the extract has good effects of improving the exercise-induced fatigue resistance and the central fatigue resistance.
The invention also provides a preparation method of the maca extract, which comprises the following steps:
i, slicing fresh maca roots, drying, crushing, leaching with a low-polarity solvent under the condition that the material-liquid ratio is 1: 5-20, filtering to obtain an extracting solution a1 and a filter residue b1, concentrating the extracting solution a1 under reduced pressure, loading the concentrated solution into a sample, performing column chromatography, eluting with the solvent, collecting a total maca amide component eluent which takes N-benzyl linoleamide as a main body, concentrating the eluent under reduced pressure, and drying to obtain an extract A;
II, inactivating enzyme of the filter residue B1 obtained by filtering in the step I at 105 ℃ for 15-40 min, leaching with ethanol water solution under the condition that the material-liquid ratio is 1: 5-20, filtering to obtain an extracting solution a2, concentrating the extracting solution a2 under reduced pressure, loading the concentrated solution to a column for chromatography, eluting with a solvent, collecting aromatic glucosinolate, total saponin and adenosine component eluent, concentrating the eluent under reduced pressure, drying and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 1: 0.5-2 to obtain the maca extract.
The preparation method enables the total macamides to be dissolved in a low-polarity solvent, glucosinolates and the like to be dissolved in an ethanol water solution, and the inventor designs a preparation method of the maca extract with pertinence to different dissolution properties of the extract. According to the method, the extraction solution and the filter residue are respectively treated, so that the content of N-benzyl linoleamide in the total macamide component of the extract is increased, and meanwhile, the maca extract has remarkable advantages compared with similar products in the prior art by optimizing the mixing ratio of the extract A, B.
Prepared according to the method
Preferably, the low-polarity solvent in step I is one or more of ethyl acetate, diethyl ether, cyclohexane, n-hexane or petroleum ether.
Preferably, the column chromatography in the step I adopts a silica gel column or a neutral alumina column.
Preferably, the solvent used for eluting in step i is one or more of methanol, absolute ethanol, ethyl acetate or petroleum ether, and the polarity of the eluting solvent is isocratic, multistage isocratic or gradient elution from small to large.
Preferably, the column chromatography in the step II adopts one of D201, D301, D315, S-8, AB-8 type macroporous resin columns or acidic alumina columns.
Preferably, the solvent used for eluting in step ii is any one group or at least two groups of isocratic, multistage isocratic or gradient elution of water-absolute ethyl alcohol-ammonium chloride, water-absolute ethyl alcohol-ammonium sulfate, water-absolute ethyl alcohol-sodium bicarbonate, water-absolute ethyl alcohol-sodium hydroxide, water-absolute ethyl alcohol-sodium chloride or water-absolute ethyl alcohol-potassium sulfate. Preferably, the concentration of the ammonium chloride, the ammonium sulfate, the sodium bicarbonate, the sodium hydroxide, the sodium chloride or the potassium sulfate is 0.01-5 mol/L, and the ratio (V/V) of the absolute ethyl alcohol to the salt solution (the ammonium chloride, the ammonium sulfate, the sodium bicarbonate, the sodium hydroxide, the sodium chloride or the potassium sulfate) is 0-80%.
Preferably, the drying means to obtain the extract a or B is vacuum drying, freeze drying or spray drying.
More preferably, the preparation method comprises the following steps:
slicing fresh maca roots, drying at 55 ℃, crushing to 10-20 meshes, leaching for 2-3 times with a low-polarity solvent at a material-liquid ratio of 1: 5-20 and a temperature of 50-80 ℃, filtering to obtain an extracting solution a1 and a filter residue b1, carrying out reduced pressure concentration on the extracting solution a1, loading a concentrated solution to a sample, carrying out column chromatography, eluting with the solvent, collecting maca amide component eluent which takes N-benzyl linoleamide as a main body, carrying out reduced pressure concentration on the eluent, and drying to obtain an extract A;
II, inactivating enzyme of the filter residue B1 obtained by filtering in the step I at 105 ℃ for 15-40 min, leaching for 2-3 times by using 50-98% ethanol water solution at the material-liquid ratio of 1: 5-20 and the temperature of 50-90 ℃ for 1-2 h each time, filtering to obtain an extracting solution a2, concentrating the extracting solution a2 under reduced pressure, loading the concentrated solution to a column for column chromatography, eluting with a solvent, collecting aromatic glucosinolate, total saponin and adenosine component eluent, concentrating the eluent under reduced pressure, drying and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 1: 0.5-2 to obtain the maca extract.
The invention also provides application of the maca extract in improving exercise-induced fatigue resistance and central fatigue resistance. Preferably, the maca extract is used in a dose of 1.0g/kg.
Animal experiments show that the maca extract provided by the invention can obviously improve the effects of resisting sports fatigue and resisting central fatigue.
The invention has the following beneficial effects: (1) the maca extract disclosed by the invention has high stability, is rich in various bioactive components, simultaneously contains two fat-soluble effective parts and two water-soluble effective parts, and has high content of the effective components; (2) the preparation method of the maca extract can remove a large amount of impurities, remarkably improve the content of effective components, fully obtain the maca active components, has good repeatability and stability, high utilization rate of raw materials and high yield of the active components, has strong adaptability to production equipment, and is suitable for industrial production; (3) through pharmacological experimental evaluation of exercise-induced fatigue resistance and central fatigue resistance, the maca extract disclosed by the invention has good effects on improving the exercise-induced fatigue resistance and the central fatigue resistance, overcomes the problem that the exercise-induced fatigue resistance and the central fatigue resistance cannot be simultaneously achieved, and remarkably improves the fatigue resistance.
Detailed Description
In order to better understand the technical scheme of the invention, the technical scheme provided by the invention is described in detail by combining the embodiment.
Example 1
The maca extract of the embodiment is obtained by the following preparation method, and the steps comprise:
weighing 12kg of fresh maca roots, slicing, drying at 55 ℃, crushing, sieving by a 10-mesh sieve, leaching with petroleum ether (60-90 ℃), extracting at 70 ℃ according to a material-liquid ratio of 1:10, filtering to obtain an extracting solution, extracting for 2 times, wherein the time is 2 hours each time, combining the two extracting solutions, concentrating under reduced pressure, subjecting the concentrated solution to silica gel column chromatography, performing gradient elution with an ethyl acetate-petroleum ether system (10:90, 25:75, 35:65 and 50:50), collecting maca amide component eluent which takes N-benzyl linoleamide as a main body, concentrating the eluent under reduced pressure, and drying in vacuum to obtain an extract A;
naturally volatilizing the maca extraction residues, inactivating enzyme at 105 ℃ for 30min, extracting with 80% ethanol water solution at the material-liquid ratio of 1:10 at 70 ℃, filtering to obtain an extracting solution, extracting for 2 times, each time for 2h, combining the extracting solutions, concentrating under reduced pressure, performing D201 macroporous resin column chromatography on the concentrated solution, performing gradient (the concentration of 1mol/L potassium sulfate is constant, and the volume ratio of the absolute ethanol to the saline water solution is 0%, 20%, 40%, 60% and 80%) on water-absolute ethanol-potassium sulfate system, collecting aromatic glucosinolate, total saponin and adenosine component eluent, concentrating the eluent under reduced pressure, drying under vacuum, and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 1:2 to obtain the maca extract.
The maca extract is detected by a spectrophotometer method and a high-speed liquid chromatography method, and mainly comprises 30.7% of total maca amide, 38.9% of aromatic glucosinolate, 9.1% of total saponin and 5.8% of adenosine by mass, wherein the content of N-benzyl linoleamide in the total maca amide accounts for 44.3% of the content of the total maca amide.
Example 2
The maca extract of the embodiment is obtained by the following preparation method, and the steps comprise:
weighing 12kg of fresh maca roots, slicing, drying at 55 ℃, crushing, sieving by a 10-mesh sieve, leaching by using N-hexane, extracting at the material-liquid ratio of 1:10 and the extraction temperature of 60 ℃, filtering to obtain an extracting solution, extracting for 2 times, each time being 2 hours, combining the extracting solutions, concentrating under reduced pressure, subjecting the concentrated solution to silica gel column chromatography, performing gradient elution by using an ethyl acetate-petroleum ether system (10:90, 25:75, 35:65 and 50:50), collecting maca amide component eluent which takes N-benzyl linoleamide as a main body, concentrating the eluent under reduced pressure, and drying under vacuum to obtain an extract A;
naturally volatilizing the maca extraction residues, inactivating enzyme at 105 ℃ for 30min, extracting with 65% ethanol water solution, wherein the material-liquid ratio is 1:10, the extraction temperature is 70 ℃, filtering to obtain extracting solution, extracting for 2 times, each time is 2h, combining the extracting solutions, concentrating under reduced pressure, performing acidic alumina column chromatography on the concentrated solution, performing water-absolute ethyl alcohol-potassium sulfate system gradient (the concentration of 0.05mol/L potassium sulfate is unchanged, and the volume ratio of absolute ethyl alcohol to saline water solution is 0%, 20%, 40%, 60% and 80%) in sequence, collecting aromatic glucosinolate, total saponin and adenosine component eluent, concentrating the eluent under reduced pressure, drying under vacuum, and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 1:1 to obtain the maca extract.
The maca extract is detected by a spectrophotometer method and a high-speed liquid chromatography method, and mainly comprises 46.1% of total maca amide, 34.6% of aromatic glucosinolate, 8.4% of total saponin and 5.0% of adenosine by mass, wherein the content of N-benzyl linoleamide in the total maca amide accounts for 45.4% of the content of the total maca amide.
Example 3
The maca extract is obtained by the following preparation method, and the steps of the maca extract comprise:
weighing 12kg of fresh maca roots, slicing, drying at 55 ℃, crushing and sieving by a 10-mesh sieve, leaching by using N-hexane, extracting at the material-liquid ratio of 1:5 and the extraction temperature of 60 ℃, filtering to obtain an extracting solution, extracting for 2 times, extracting for 2 hours each time, combining the extracting solutions, concentrating under reduced pressure, performing neutral alumina column chromatography on the concentrated solution, performing gradient elution by using an ethyl acetate-methanol system (40: 60, 30:70, 20:80 and 10:90), collecting maca amide component eluent which takes N-benzyl linoleamide as a main body, concentrating the eluent under reduced pressure, and drying under vacuum to obtain an extract A;
naturally volatilizing the maca extraction residues, inactivating enzyme at 105 ℃ for 30min, extracting with 65% ethanol water solution, wherein the material-liquid ratio is 1:10, the extraction temperature is 70 ℃, filtering to obtain an extracting solution, extracting for 2 times, each time is 1h, combining the extracting solutions, concentrating under reduced pressure, performing acidic alumina column chromatography on the concentrated solution, performing water-absolute ethyl alcohol-potassium sulfate system gradient (the concentration of 0.05mol/L potassium sulfate is constant, and the volume ratio of absolute ethyl alcohol to saline water solution is 0%, 20%, 40%, 60% and 80% in sequence), collecting aromatic glucosinolate, total saponin and adenosine component eluent, concentrating the eluent under reduced pressure, drying under vacuum, and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 1:2 to obtain the maca extract.
The maca extract is detected by a spectrophotometer method and a high-speed liquid chromatography method, and mainly comprises 29.3% of total maca amide, 35.4% of aromatic glucosinolate, 8.5% of total saponin and 5.3% of adenosine by mass, wherein the content of N-benzyl linoleamide in the total maca amide accounts for 41.9% of the content of the total maca amide.
Example 4
The maca extract of the embodiment is obtained by the following preparation method, and the steps comprise:
weighing 12kg of fresh maca roots, slicing, drying at 55 ℃, crushing and sieving by a 10-mesh sieve, leaching by using N-hexane, extracting at the material-liquid ratio of 1:10 and the extraction temperature of 60 ℃, filtering to obtain an extracting solution, extracting for 3 times, extracting for 3 hours each time, combining the extracting solutions, concentrating under reduced pressure, performing neutral alumina column chromatography on the concentrated solution, performing gradient elution by using an ethyl acetate-methanol system (40: 60, 30:70, 20:80 and 10:90), collecting maca amide component eluent which takes N-benzyl linoleamide as a main body, concentrating the eluent under reduced pressure, and drying under vacuum to obtain an extract A;
naturally volatilizing the maca extraction residues, inactivating enzyme at 105 ℃ for 30min, extracting with 65% ethanol water solution at the material-liquid ratio of 1:15 at 70 ℃, filtering to obtain an extracting solution, extracting for 3 times, each time for 1h, combining the extracting solutions, concentrating under reduced pressure, carrying out AB-8 type macroporous resin column chromatography on the concentrated solution, carrying out water-absolute ethyl alcohol-potassium sulfate system gradient (the concentration of 1mol/L potassium sulfate is unchanged, and the volume ratio of absolute ethyl alcohol to saline water solution is 0%, 20%, 40%, 60% and 80%) in sequence, collecting aromatic glucosinolate, total saponin and adenosine component eluates, concentrating the eluates under reduced pressure, drying under vacuum, and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 2:1 to obtain the maca extract.
The maca extract is detected by a spectrophotometer method and a high-speed liquid chromatography method, and mainly comprises 48.9% of total maca amide, 26.2% of aromatic glucosinolate, 5.1% of total saponin and 1.3% of adenosine by mass, wherein the content of N-benzyl linoleamide in the total maca amide accounts for 57.2% of the content of the total maca amide.
Examples 1-4 also included other plant components that were not tested.
Application example 1 pharmacological action experiment on mice for resisting exercise-induced fatigue
1.1 Experimental samples
The maca extract prepared in example 2 is selected as a test sample, and a maca fat-soluble extract (obtained by leaching the maca with n-hexane in a material-liquid ratio of 1:10 at 60 ℃ for 2 times, and extracting and concentrating the maca extract at 2 hours each time), a maca alcohol-soluble extract (obtained by leaching the maca with absolute ethanol in a material-liquid ratio of 1:10 at 70 ℃ for 2 times, and extracting and concentrating the maca extract at 2 hours each time), a maca water-soluble extract (obtained by leaching the maca with water in a material-liquid ratio of 1:10 at 70 ℃ for 2 times, and extracting and concentrating the maca extract at 2 hours each time), an extract C (obtained by uniformly mixing the extract A and the extract B in a weight ratio of 1:3 in example 2) and an extract D (obtained by uniformly mixing the extract A and the extract B in a weight ratio of 3.
1.2 Experimental animals
The male Kunming mouse of 8 weeks old weighs 18g-22 g.
1.3 Experimental methods
Male mice of the Kunming species were randomly divided into 9 groups of control group (normal saline), maca fat-soluble extract group (1.0g/kg.bw.d), maca alcohol-soluble extract group (1.0g/kg.bw.d), maca water-soluble extract group (1.0g/kg.bw.d), extract C group (1.0g/kg.bw.d), extract D group (1.0g/kg.bw.d), low dose group (inventive maca extract, 0.2g/kg.bw.d), medium dose group (inventive maca extract, 0.5g/kg.bw.d), high dose group (inventive maca extract, 1.0g/kg.bw.d), 16 mice per group, respectively. Mice were raised under normal light and water at 22-26 ℃ and acclimatized in the animal room environment for 5 days prior to the mice's experiments. The stomach is perfused for 1 time every day, the perfused amount is 1 percent of the weight of the mouse, and after 30 days of continuous administration, the weight-bearing swimming test and the serum urea, liver glycogen and blood lactic acid measurement test are respectively carried out according to the anti-fatigue function test method in the national health food test and evaluation technical standard.
1.3.1 weight bearing swimming test
30min after the last administration of the test sample, the mice with 5% weight lead skin loaded in the tail root are placed in a swimming box for swimming. The water depth was not less than 30cm, the water temperature was 25 ℃. + -. 1 ℃, and the time from the start of swimming to death of the mice, i.e., the time for the mice to swim with a load, was recorded, and the results are shown in Table 1.
TABLE 1 Effect of different maca extracts on the exhaustion time of mice during swimming under load
Note: p compared to blank control group<0.05，**P<0.01; and a fat-soluble extract group, wherein,ZP<0.05; and an alcohol-soluble extract group, and a pharmaceutically acceptable salt thereof,CP<0.05; and a water-soluble extract group, wherein,SP<0.05; and the group C of the extract is mixed,GP<0.05; and the group D of the extract is mixed,DP<0.05
1.3.2 Effect on serum Urea Nitrogen, liver glycogen and blood lactic acid in mice
(1) And (3) serum urea determination: swimming for 90min in water at 30 ℃ after 30min of last time of sample feeding, picking up eyeballs for blood sampling after 60min of rest, standing blood samples for half an hour at room temperature, centrifuging for 10min at 2000rpm of a refrigerated centrifuge, taking serum for later use, and measuring serum urea nitrogen by using a urea nitrogen measurement kit, wherein the results are shown in Table 2;
(2) liver glycogen assay: killing the animal 30min after the last test sample, rinsing the liver with physiological saline, sucking dry with filter paper, and measuring hepatic glycogen with a glycogen measuring kit, wherein the results are shown in table 2;
(3) and (3) blood lactic acid determination: 30min after the last sample feeding, then swimming in water at 30 ℃ for 10min without load, stopping, taking the eyeball and collecting blood, standing the blood sample for half an hour at room temperature, centrifuging for 10min at 2000rpm of a refrigerated centrifuge, taking serum for later use, and measuring blood lactic acid by using a lactic acid measuring kit, wherein the results are shown in Table 2.
TABLE 2 Effect of different maca extracts on serum urea nitrogen, liver glycogen and blood lactic acid in mice
Note: p compared to blank control group<0.05; (ii) a And a fat-soluble extract group, wherein,ZP<0.05; and an alcohol-soluble extract group, and a pharmaceutically acceptable salt thereof,CP<0.05; and a water-soluble extract group, wherein,SP<0.05; and the group C of the extract is mixed,GP<0.05; and the group D of the extract is mixed,DP<0.05
1.4 conclusion of the experiment
The maca extract has positive results in a load swimming experiment, the results of three biochemical indexes of serum urea nitrogen, hepatic glycogen and blood lactic acid are also positive, and the load time and the hepatic glycogen content in the experiment are obviously higher than those of a maca fat-soluble extract group, a maca alcohol-soluble extract group, a maca water-soluble extract group, an extract C group and an extract D group which are obtained by simple extraction, so that the maca extract has a more effective exercise-induced fatigue resistance effect.
Application example 2 pharmacological action experiment on mice for resisting central fatigue
2.1 Experimental samples
The maca extract prepared in example 2 was used as a test sample
2.2 Experimental animals
The male Kunming mouse of 8 weeks old weighs 18g-22 g.
2.3 Experimental methods
Male mice of Kunming species were randomly divided into 4 groups, namely a blank control group, a sleep deprivation induced central fatigue model (SD) group, a first group (inventive maca extract, 0.5g/kg.bw.d) and a second group (inventive maca extract, 1.0g/kg.bw.d), 16 mice per group were subjected to intragastric administration for 20 days, and the blank group and the sleep deprivation group were intragastric administered with the same volume of vehicle.
2.3.1 establishing sleep deprivation animal model
An improved 'multi-platform water environment' method is adopted to prepare a mouse central fatigue (SD) model caused by sleep deprivation, the size of a sleep deprivation box is 45cm multiplied by 30cm multiplied by 20cm, and the size of a small platform is 3cm multiplied by 5 cm. 12 small platforms are arranged in each sleep deprivation box, and 6 mice are placed on 6 small platforms; the size of the large platform is 15cm multiplied by 5cm, 3 mice are placed on 1 large platform, and the mice can freely take water and feed on the platform. The water surface in the sleep deprivation box body is about 1cm lower than the platform, the water temperature is controlled to be 25 +/-1 ℃, and the water in the box is replaced every day.
2.3.2 channel Water maze training and testing
Is made of black rubber plates (70cm multiplied by 40cm multiplied by 30cm), the water depth is 22cm, and the water temperature is 25 ℃. The self-made channel type water maze is provided with 3 blind ends such as I, II, III, and the final point is provided with a step safety zone, and the mouse can climb up to the platform safety zone after reaching the final point to avoid drowning. After the gavage drug was administered on day 9 of the experiment, the water maze training was performed on four groups of mice, and the specific training method was as follows: mice were placed at endpoint for 10s, allowed to feel and know that this was safe; then, placing a partition board at the point A, starting training by taking the point A as a starting point, enabling the mouse to swim freely, and recording the latency period of the channel type water maze experiment, namely the time from putting into water to climbing up the steps; if no end step is found within 120s, it is guided to that location and its latency is recorded as 120s, with 2 consecutive sessions per mouse per day. The partition plates are respectively placed at the point B and the point C on the 10 th day and the 11 th day of the experiment, and the mice respectively start from the two points to carry out the channel type water maze training. Mice were allowed to stand from point C on day 12 of the experiment and continued for 1 day of consolidation training. And (3) carrying out a mouse sleep deprivation experiment for 72h after the gavage on day 13, simultaneously carrying out a channel type water maze test every day, and recording the incubation period of the mouse in the water maze and the total times of entering the cecum I, II and III. Data for latency and number of blind errors in the channeled water maze experiment for each group of mice are shown in tables 3 and 4.
Table 3 effect of maca extract on mouse water maze migration latency
Note:#P<0.05 comparison with blank control group<0.05 compared with the group of Central fatigue caused by sleep deprivation (SD group)
The storage of memory is influenced by sleep, researches show that memory and cognitive function decline can be caused by sleep deprivation for a certain time, brain tissue damage is caused, and changes of learning and memory abilities of mice before and after sleep deprivation can be tested by adopting a channel type water maze experiment. As can be seen from the results in tables 3 and 4, compared with the sleep deprivation model group, the maca extract of the invention has the advantages that the water maze emergence latency is remarkably reduced, and the number of blind errors is reduced, which indicates that the maca extract of the invention can improve the reduction of the learning and memory abilities of mice caused by sleep deprivation.
Table 4 effect of maca extract on mouse blind error number
Note: # P <0.05 compared with the blank control group, and # P <0.05 compared with the sleep deprivation-induced central fatigue model group (SD group)
2.3.3 measurement of Biochemical indicators of telencephalon tissue of mice
After the sleep deprivation experiment, the mice were sacrificed by cervical dislocation after anesthesia of the mice with 20% urethane, 10 mice per group, telencephalon tissues were rapidly taken out in ice bath and transferred to liquid nitrogen, and then stored in an ultra-low temperature refrigerator at-80 ℃ for later use. Weighing mouse telencephalon tissue, putting the mouse telencephalon tissue into a small beaker, adding 9 times of precooled physiological saline according to the mass volume of 1:9, quickly shearing the tissue, pouring tissue liquid into a homogenizer for fully grinding and homogenizing, and quickly carrying out all operations on ice bath; centrifuging the prepared tissue homogenate at 3000rpm for 10min by a refrigerated centrifuge, taking the supernatant to obtain 10% of tissue homogenate, and detecting the activity of glutathione peroxidase (GSH-Px), the activity of superoxide dismutase (SOD), the content of Malondialdehyde (MDA) and monoamine neurotransmitters 5-hydroxytryptamine (5-HT), Dopamine (DA) and Norepinephrine (NE), wherein the method refers to corresponding kit instructions, and the determination data are shown in tables 5 and 6.
TABLE 5 Effect of maca extract on antioxidant capacity of the telencephalon tissue of mice
Note:#P<0.05 comparison with blank control group<0.05 and central fatigue due to sleep deprivationComparison of type groups (SD group)
Sleep deprivation results in increased energy expenditure, increased oxygen consumption, and the production of large amounts of oxygen radicals, which cause damage to nerve cells, such as superoxide anion free radicals (O)-2.) Hydrogen peroxide (H)2O2) Can oxidize fatty acid on cell membrane, destroy lipid structure of cell membrane and protein structure in cell, and produce lipid peroxide such as Malondialdehyde (MDA), etc., resulting in body fatigue, and with consumption of oxygen and generation of free radicals, it will also result in impaired transmission function of brain nerve cell synapse, resulting in central fatigue. Free radical scavenging active enzymes such as superoxide dismutase (SOD) widely present in the body primarily scavenge superoxide anion free radicals (O)-2·) While glutathione peroxidase (GSH-Px) catalyzes H primarily2O2And the decomposition of peroxide plays a role in protecting cells. The results in table 5 show that the mice supplemented with the maca extract of the present invention can significantly reduce the MDA content in brain tissue and restore the activities of antioxidant enzymes SOD and GSH-Px to normal, indicating that the maca extract can alleviate the damage of free radicals and lipid peroxides generated during sleep deprivation of the mice to brain tissue, restore normal antioxidant enzyme activity, and reduce the occurrence of central fatigue.
TABLE 6 Effect of maca extract on monoamine neurotransmitters in mouse telencephalon
Note: # P <0.05 compared with the blank control group, and # P <0.05 compared with the sleep deprivation-induced central fatigue model group (SD group)
The brain nerve cells are over excited and energy substances such as blood sugar and the like are excessively consumed by long-term mental or physical work, and in order to protect the energy supply of the nerve cells, the cerebral cortex can generate protective inhibition, so that the dysfunction of the central nervous system is caused, the reasonable release of excitatory neurotransmitter in brain tissues is influenced, and the central fatigue is caused. 5-HT is an important central monoamine inhibitory neurotransmitter in brain, and the increase of the content of the 5-HT can inhibit the excitability of neurons, thereby reducing the motor capacity of the body and causing the occurrence of fatigue; DA is used as a monoamine central excitatory neurotransmitter, can influence the secretion of certain hormones in pituitary, maintains the motor balance and endurance of the organism, and the reduction of DA content can cause the reduction of the motor capacity of the organism; NE, an important central monoamine neurotransmitter in the brain, excites hippocampal neurons in brain tissue via β receptors, and exerts its sleep-promoting effect. As can be seen from the results in table 6, the mice supplemented with the maca extract of the present invention significantly decreased the 5-HT content, significantly increased the DA and NE content, restored to normal levels, and regulated the release of excitatory neurotransmitters in brain tissue, as compared to the sleep deprivation induced central fatigue model group (SD group).
2.4 conclusion of the experiment
The maca extract can effectively relieve the central fatigue symptoms such as the reduction of learning and memory caused by sleep deprivation, the damage of free radicals and lipid peroxides to brain tissues, the release imbalance of excitatory neurotransmitters in the brain tissues and the like, and shows that the maca extract has the effect of resisting the central fatigue.
It is to be understood that the above-described embodiments are only a few, and not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (8)
1. A maca extract is characterized by mainly comprising 28-50% of total maca amide, 25-40% of aromatic glucosinolate, 4-12% of total saponin and 1-8% of adenosine by mass, wherein the content of N-benzyl linoleamide in the total maca amide accounts for 35-60% of the content of the total maca amide;
the preparation method of the maca extract comprises the following steps:
i, slicing fresh maca roots, drying, crushing, leaching with a low-polarity solvent under the condition that the material-liquid ratio is 1: 5-20, filtering to obtain an extracting solution a1 and a filter residue b1, concentrating the extracting solution a1 under reduced pressure, loading the concentrated solution into a sample, performing column chromatography, eluting with the solvent, collecting a total maca amide component eluent which takes N-benzyl linoleamide as a main body, concentrating the eluent under reduced pressure, and drying to obtain an extract A; the low-polarity solvent is one or more of ethyl acetate, diethyl ether, cyclohexane, n-hexane or petroleum ether;
II, inactivating enzyme of the filter residue B1 obtained by filtering in the step I at 105 ℃ for 15-40 min, leaching with ethanol water solution under the condition that the material-liquid ratio is 1: 5-20, filtering to obtain an extracting solution a2, concentrating the extracting solution a2 under reduced pressure, loading the concentrated solution to a column for chromatography, eluting with a solvent, collecting aromatic glucosinolate, total saponin and adenosine component eluent, concentrating the eluent under reduced pressure, drying and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 1: 0.5-2 to obtain the maca extract.
2. The maca extract according to claim 1, wherein the column chromatography in step I is performed by using a silica gel column or a neutral alumina column.
3. The maca extract according to claim 1, wherein the solvent used for elution in step I is one or more of methanol, absolute ethanol, ethyl acetate or petroleum ether, and the polarity of the elution solvent is in isocratic or gradient elution from small to large.
4. The maca extract as claimed in claim 1, wherein in step ii, the column chromatography is performed by using one of a D201, D301, D315, S-8, AB-8 type macroporous resin column or an acidic alumina column.
5. The maca extract according to claim 1, wherein the solvent used for elution in step ii is any one or at least two groups of isocratic or gradient elution selected from the group consisting of water-absolute ethanol-ammonium chloride, water-absolute ethanol-ammonium sulfate, water-absolute ethanol-sodium bicarbonate, water-absolute ethanol-sodium hydroxide, water-absolute ethanol-sodium chloride and water-absolute ethanol-potassium sulfate.
6. A maca extract according to claim 1, wherein the extract A or B is obtained by drying under vacuum, freeze-drying or spray-drying.
7. A maca extract according to any one of claims 2 to 6, comprising the following steps:
slicing fresh maca roots, drying at 55 ℃, crushing to 10-20 meshes, leaching for 2-3 times with a low-polarity solvent at a material-liquid ratio of 1: 5-20 and a temperature of 50-80 ℃, filtering to obtain an extracting solution a1 and a filter residue b1, carrying out reduced pressure concentration on the extracting solution a1, loading a concentrated solution to a sample, carrying out column chromatography, eluting with the solvent, collecting maca amide component eluent which takes N-benzyl linoleamide as a main body, carrying out reduced pressure concentration on the eluent, and drying to obtain an extract A; the low-polarity solvent is one or more of ethyl acetate, diethyl ether, cyclohexane, n-hexane or petroleum ether;
II, inactivating enzyme of the filter residue B1 obtained by filtering in the step I at 105 ℃ for 15-40 min, leaching for 2-3 times by using 50-98% ethanol water solution at the material-liquid ratio of 1: 5-20 and the temperature of 50-90 ℃ for 1-2 h each time, filtering to obtain an extracting solution a2, concentrating the extracting solution a2 under reduced pressure, loading the concentrated solution to a sample for column chromatography, eluting with a solvent, collecting aromatic glucosinolate, total saponin and adenosine component eluent, concentrating the eluent under reduced pressure, drying and crushing to obtain an extract B;
and III, uniformly mixing the extract A and the extract B according to the weight ratio of 1: 0.5-2 to obtain the maca extract.
8. Use of the extract of maca according to claim 1 for enhancing resistance to exercise-induced fatigue and central fatigue.
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| 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 |
| 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 |
| 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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