CN115282216A - Composition with potential Alzheimer disease prevention and treatment functions - Google Patents
Composition with potential Alzheimer disease prevention and treatment functions Download PDFInfo
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- CN115282216A CN115282216A CN202211122250.3A CN202211122250A CN115282216A CN 115282216 A CN115282216 A CN 115282216A CN 202211122250 A CN202211122250 A CN 202211122250A CN 115282216 A CN115282216 A CN 115282216A
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- fruit powder
- olive fruit
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Abstract
The invention provides a composition with potential Alzheimer disease prevention and treatment functions, namely the composition for preventing and treating Alzheimer disease is exerted by combined use of olive fruit powder and a medlar extract, wherein the olive fruit powder contains not less than 30% of polyphenol and not less than 5% of hydroxytyrosol; the content of flavonoids in fructus Lycii extract is not less than 15%. The composition of the olive fruit powder and the medlar extract has obvious M3 type acetylcholine receptor agonistic activity after being verified by M3 type acetylcholine receptor agonistic experiment, and shows that the composition has the function of preventing and treating Alzheimer disease and the potential function of preventing and treating Alzheimer disease.
Description
Technical Field
The invention belongs to the technical field of preparation and application of extracts of medicinal and edible substances, and particularly relates to a composition with potential Alzheimer disease prevention and treatment functions, namely the composition for preventing and treating Alzheimer disease by combined use of olive fruit powder and a wolfberry extract.
Background
Senile dementia is one of invisible killers threatening human life health after tumors and cardiovascular and cerebrovascular diseases, and mainly comprises Alzheimer disease (also called presenile dementia, AD), vascular Dementia (VD), mixed dementia and other dementias, wherein the presenile dementia is the main type and accounts for more than 60 percent of all dementias. Alzheimer's disease clinically manifests as a decline in memory due to a decline in cognitive function. With the aging of the world population, the incidence of senile dementia is increasing dramatically. Therefore, it is a common challenge for all humans to actively prevent alzheimer's disease, to find effective alzheimer's disease prevention foods, health foods and drugs, to alleviate the pain of patients, and to improve their quality of life.
The medlar is a rare traditional Chinese medicine resource used as both medicine and food in China, and the medlar is recorded in Shen nong's herbal classic to be taken for a long time and to be light, not old, cold-resistant and summer-heat-resistant. The compendium of materia medica records that the medlar is aging resistant and supplements essence; commonly known as 'charpy'. Modern research shows that: the fructus Lycii has effects of protecting nerve cells, and preventing and treating Alzheimer Disease (AD). The lycium barbarum polyphenol is one of the material bases for exerting the anti-AD effect.
The olive is a famous tree species for both woody oil and fruits in the world, contains rich and high-quality edible vegetable oil, is considered to be edible oil which is most suitable for human nutrition in the oil discovered so far, and is known as 'fragrant soft gold'. Olive polyphenols are a major active substance in olives. The research of the Australian scholars finds that the polyphenol substances in the olea europaea can inhibit the beta amyloid protein of harmful cranial nerves from accumulating in the brain, thereby preventing the Alzheimer disease.
Aiming at the great demand of preventing and treating the presenile dementia in the field of human life health, the invention combines the potential effects of the olive fruit powder and the medlar extract in preventing and treating the presenile dementia, develops a composition with the effect of preventing and treating the alzheimer disease, and aims to improve the memory of presenile dementia patients and improve the life quality of the patients.
Disclosure of Invention
The invention aims to provide a composition with potential Alzheimer disease prevention and treatment functions, namely the composition for preventing and treating Alzheimer disease is exerted by combined use of olive fruit powder and medlar extract, so that the defects of the prior art are overcome.
The olive fruit powder and wolfberry extract composition with the effect of preventing and treating the Alzheimer disease, provided by the invention, has the advantages that the content of polyphenol in the olive fruit powder is not lower than 30%, and the content of hydroxytyrosol is not lower than 5%; the content of flavonoids in the fructus Lycii extract is not less than 15%;
in the olive fruit powder and wolfberry extract composition, the weight ratio of the olive fruit powder to the wolfberry extract is (5).
In the olive fruit powder and medlar extract composition, the preparation method of the medlar extract comprises the following steps:
1) Extraction: putting the medlar powder into an extraction tank, adding 70% ethanol according to the material-liquid ratio of 1;
2) Separation: adding inorganic salt and deionized water into the extracting solution, wherein the concentration of ethanol in a final solution system is 25-35%, the concentration of the inorganic salt is 17-25%, and the system temperature is 15-35 ℃; standing to make the solution into two phases, and separating to obtain an upper phase;
the inorganic salt comprises (NH) 4 ) 2 SO 4 ，NaH 2 PO 4 ，K 2 CO 3 ，K 2 HPO 4 ，Na 2 SO 4 And K 3 PO 4 Any one or more of them, preferably K 2 CO 3 With 35% ethanol concentration, K 2 CO 3 The concentration was 21% and the temperature was 35 ℃.
3) Enrichment: evaporating the upper phase under reduced pressure, pumping the concentrated solution into a macroporous adsorption resin column, eluting with water, eluting with 70% ethanol, and collecting 70% ethanol eluate; wherein the resin filler is: d101, the diameter-height ratio of the tree column is 1:10, the sample loading flow rate is 3BV/h, and the elution flow rate is 4BV/h.
4) And (3) drying: and (3) carrying out reduced pressure rotary evaporation on the 70% ethanol eluent, recovering ethanol, pumping into a spray dryer, carrying out air inlet temperature of 125 ℃ and air outlet temperature of 95 ℃, and collecting dry powder to obtain the medlar extract. Rutin is used as a reference substance, and the content of flavone is detected to be more than or equal to 25 percent by a spectrophotometry method.
In the olive fruit powder and medlar extract composition, the preparation method of the olive fruit powder comprises the following steps:
1) Extraction: breaking cell wall of fructus Oleae Europaeae with high speed shearing machine, breaking, placing in subcritical extraction tank, adding 12 times of deionized water, adding 3% cellulase, extracting at 60 deg.C for 1 hr, extracting at 180 deg.C for 0.5 hr, filtering, and collecting extractive solution.
2) Separation: the extract passes through a tubular centrifuge at the rotating speed of 16000 r/min, and centrifugate is collected; passing the centrifugate through ultrafiltration membrane with molecular weight of 3500Da at pressure of 10-20bar and temperature of 30-40 deg.C, and collecting the filtrate.
3) Enrichment: concentrating the permeate under reduced pressure, pumping into macroporous adsorbent resin column, eluting with water, eluting with 60% ethanol, and collecting 60% ethanol eluate. The resin filler is: LSA-21, the ratio of the diameter to the height of the tree column is 1:10, the sample loading flow rate is 2BV/h, and the elution flow rate is 4BV/h.
4) And (3) drying: concentrating the 60% ethanol eluate under reduced pressure, and spray drying the concentrated solution at air inlet temperature of 135 deg.C and air outlet temperature of 95 deg.C. Collecting powder to obtain olive fruit powder. The content of polyphenol in the olive fruit powder is more than or equal to 30 percent as determined by ferrous tartrate colorimetry, and the content of hydroxytyrosol in the olive fruit powder is more than or equal to 5 percent as determined by high performance liquid chromatography.
The composition of the olive fruit powder and the medlar extract has obvious M3 type acetylcholine receptor agonistic activity through M3 type acetylcholine receptor agonistic experiment verification, and shows that the composition has the effect of preventing and treating Alzheimer disease and the potential effect of preventing and treating presenile dementia.
Drawings
FIG. 1: graph of agonist assay results for CHO-K1/M3 cell calcium flux assay, wherein OP is olive fruit powder, GP is wolfberry extract, OGP-B is a combination of olive and wolfberry extract 1. Results are expressed as mean ± SD, n =5, and represent the comparison between the administered groups, P <0.05 and P <0.01.
FIG. 2: graph of agonist assay results for CHO-K1/M3 cell calcium flux assay, wherein OGP-A is a combination of Olea Europaea and Lycium barbarum extract 5, OGP-B is a combination of Olea Europaea and Lycium barbarum extract 1, OGP-C is a combination of Olea Europaea and Lycium barbarum extract 1. Results are expressed as mean ± SD, n =5, and indicates a comparison between each dosing group and the blank group, P <0.05 and P <0.01.
Detailed Description
The medlar extract is extracted by 70 percent ethanol, so that not only can flavonoid glycoside or aglycone be extracted, but also monosaccharide, oligosaccharide and polysaccharide with low molecular weight in medlar can be extracted. When inorganic salt is added to form two phases, the ethanol phase of the upper phase is rich in flavonoid compounds, and the salt phase of the lower phase is rich in carbohydrate substances.
The olive fruit powder is extracted by subcritical water, the activity of enzyme is ensured at 60 ℃, the enzyme deactivation effect is achieved at 180 ℃, hydrogen bonds of subcritical water are opened or weakened, the polarity of water can be changed within a large range by controlling the temperature and pressure of the subcritical water, and therefore continuous extraction of polyphenol effective components with different polarities from water-soluble components to fat-soluble components in the olive oil is achieved. Improves the extraction rate and content of polyphenol in olive fruit powder.
The following detailed description of the embodiments of the present invention is provided, but the present invention is not limited to the following description:
example 1:
1) Preparation of extract of Lycium barbarum
Weighing 25kg of 20 mesh wolfberry powder, placing in a 400L extraction tank, adding 300L of 60% ethanol, starting circulating water, reflux extracting for 2h, duplex filtering to remove wolfberry residue, adding 186L and K deionized water into 260L of extractive solution 2 CO 3 Keeping at 35 deg.C and standing for 0.5h to obtain two phases, and separating to obtain upper phase. Double-effect concentrating, loading onto D101 resin column at 3BV/h, eluting with 4BV water, and eluting with 6BV 70% ethanol solution at 4BV/hEluting, collecting 70% ethanol eluate, concentrating, pump spray drying, air inlet temperature of 125 deg.C, air outlet temperature of 95 deg.C, collecting dry powder, with yield of 1.8% and flavone content of 27.3%.
2) Preparation of olive fruit powder
Breaking cell wall of 50 kg fructus Oleae Europaeae with high speed shearer, breaking, placing in subcritical extraction tank, adding 600L deionized water, adding 1.5 kg cellulase, extracting at 60 deg.C for 1h, extracting at 180 deg.C for 0.5h, filtering, passing the extractive solution through tubular centrifuge with rotation speed of 16000 rpm, collecting centrifugate, passing through ultrafiltration membrane with molecular weight of 3500Da, pressure of 13 + -2 bar, temperature of 35 deg.C, and collecting permeate. Concentrating the permeate under reduced pressure to 50L, pumping into LSA-21 resin column, eluting with 4BV water at flow rate of 2BV/h, and eluting with 60% ethanol solution at flow rate of 4BV/h. Collecting 60% ethanol eluate, concentrating under reduced pressure to recover ethanol, pumping the concentrated solution into spray dryer at flow rate of 25kg/h, air inlet temperature of 135 deg.C, and air outlet temperature of 95 deg.C. Collecting powder to obtain olive fruit powder. The yield was 8.7%. The content of polyphenol in the olive fruit powder is 35.72 percent through ferrous tartrate colorimetric method determination, and the content of hydroxytyrosol through high performance liquid chromatography determination is 7.92 percent.
According to the weight ratio, the olive fruit powder and the medlar extract are respectively as follows: 5, mixing uniformly the olive fruit powder and the medlar extract composition OGP-A, the composition OGP-B and the composition OGP-C, wherein the ratio of 1 to 1 is.
Example 2: evaluation of the activity of M3 type acetylcholine receptor agonist.
1. Materials and reagents:
sample preparation: olive fruit powder (OP), wolfberry extract (GP), olive fruit powder and wolfberry extract composition (OGP-A, OGP-B, OGP-C). CHO-K1/M3 stable cell line (M00259, genscript); calcium Kit (R8141, MD).
2. The method comprises the following steps:
1) 10000 cells/well in 384-well plates, 20 ul/well medium (F12K +10% FBS), 5% CO at 37% 2 The incubation was carried out overnight.
2) The dye working solution of Fluo-4 was prepared by mixing probenecid stock solution (500 mM) with FluAnd the uo-4 stock solution is prepared by mixing the raw materials according to the ratio of 1. The dye solution (20 ul/well) was added to the cells in 384 well plates and CO was passed through at 37 deg.C 2 Incubate for 60 minutes.
3) Sample working solutions of the respective concentrations were prepared as described above, and detection buffer (HBSS +20mM HEPES, ph = 7.4) was added.
4) The 384 well plates were removed from the incubator and equilibrated at room temperature for 10 min in the absence of light.
5) The source plate and cell plate were transferred to a FLIPR for fluorescent signal detection for 120 seconds.
6) Raw data for analysis was obtained for each well by subtracting the minimum fluorescence from the maximum fluorescence.
7) Maximum-minimum RFU values were plotted against compound concentration and dose-response curves were obtained.
As can be seen from FIG. 1, the olive fruit powder and the Chinese wolfberry extract have significant M3 receptor agonistic activity at 10mg/mL, 50mg/mL and 100mg/mL, respectively, and are in a concentration-dependent relationship. The olive fruit powder and the medlar extract have good Alzheimer disease prevention and treatment activity. The compound composition OGP-B of the olive fruit powder and the wolfberry extract 1 has similar M3 receptor agonistic activity to the single components of the olive fruit powder and the wolfberry extract at concentrations of 10mg/mL and 50mg/mL, but at a concentration of 100mg/mL, the compound composition OGP-B of the olive fruit powder and the wolfberry extract 1 has significantly higher M3 receptor agonistic activity than the single extracts of the olive fruit powder and the wolfberry extract, suggesting that the combination of the olive fruit powder and the wolfberry extract 1 has better alzheimer disease prevention and treatment activity at a high concentration.
Under the same concentration (10 mg/mL or 50 mg/mL), the M3 receptor agonistic activities of OGP-A, OGP-B and OGP-C are similar, and the results in figure 2 show that the olive and the medlar extract have good Alzheimer disease prevention and treatment activities within the mass ratio of 1.
The composition provided by the invention can be used for preparing candies or other functional foods, and the preparation method of one kind of candies comprises the following steps:
weighing 20.0kg of olive fruit powder and an extract OGP-1 of medlar, adding a proper amount of gelatin, glycerol, sorbitol, fructo-oligosaccharide and edible pigment, and preparing the gel candy by the processes of proportioning, pressing, shaping, drying, sorting, polishing, packaging and the like.
Another functional food is prepared by the following steps:
weighing 10.0kg of olive fruit powder and wolfberry extract OGP-2, 2.0kg of xylitol, 1.0kg of microcrystalline cellulose, 50g of aspartame and 800g of citric acid, fully mixing, adding 50g of talcum powder, preparing a soft material, granulating, finishing granules, tabletting, and obtaining the olive fruit powder, wolfberry extract and deer blood powder composition tabletting candy with the weight of 1.0 g/tablet.
Claims (10)
1. A composition of olive fruit powder and wolfberry extract is characterized in that the content of polyphenol in the olive fruit powder is not less than 30%, and the content of hydroxytyrosol is not less than 5%; the content of flavonoids in fructus Lycii extract is not less than 15%.
2. The composition of claim 1, wherein the weight ratio of olive fruit powder to wolfberry extract is 5.
3. The composition of claim 1 or 2, wherein the extract of lycium barbarum is prepared by:
1) Extraction: adding 70% ethanol into fructus Lycii powder, extracting, and filtering to obtain extractive solution;
2) Separation: adding inorganic salt and deionized water into the extracting solution to ensure that the concentration of ethanol in a final solution system is 25-35 percent and the concentration of the inorganic salt is 17-25 percent; standing to make the solution into two phases, and separating to obtain an upper phase;
3) Enrichment: evaporating the upper phase under reduced pressure, pumping the concentrated solution into a macroporous adsorption resin column, eluting with water, eluting with 70% ethanol, and collecting 70% ethanol eluate;
4) And (3) drying: and (3) carrying out reduced pressure rotary evaporation on the 70% ethanol eluent, recovering ethanol, pumping into a pump, and carrying out spray drying to obtain the medlar extract.
4. The composition of claim 3, wherein said inorganic salt comprises (NH) 4 ) 2 SO 4 ，NaH 2 PO 4 ，K 2 CO 3 ，K 2 HPO 4 ，Na 2 SO 4 And K 3 PO 4 Any one or more of them.
5. The composition of claim 3 wherein the inorganic salt is K 2 CO 3 Ethanol concentration 35%, K 2 CO 3 The concentration was 21% and the temperature was 35 ℃.
6. The composition according to claim 1 or 2, characterized in that said olive fruit powder is prepared by the following process:
1) Extraction: breaking cell wall of fructus Oleae Europaeae with high speed shearer, breaking, placing in subcritical extraction tank, adding deionized water and cellulase for extraction, filtering, and collecting extractive solution;
2) Separation: centrifuging the extracting solution, passing the centrifugate through an ultrafiltration membrane, and collecting the permeate;
3) Enrichment: concentrating the permeate under reduced pressure, pumping into macroporous adsorbent resin column, eluting with water, eluting with 60% ethanol, and collecting 60% ethanol eluate;
4) And (3) drying: concentrating the 60% ethanol eluate under reduced pressure, and spray drying the concentrated solution to obtain fructus Canarii albi powder.
7. The composition of claim 6, wherein said ultrafiltration membrane has a molecular weight of 3500Da.
8. Use of a composition according to claim 1 or 2 for the preparation of a preparation for the control of alzheimer's disease.
9. A functional food comprising the composition of claim 1 or 2.
10. The functional food according to claim 9, wherein the functional food is a candy.
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