CN114594177A - Method for screening anti-aging active ligand - Google Patents
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- CN114594177A CN114594177A CN202210085368.7A CN202210085368A CN114594177A CN 114594177 A CN114594177 A CN 114594177A CN 202210085368 A CN202210085368 A CN 202210085368A CN 114594177 A CN114594177 A CN 114594177A
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Abstract
The embodiment of the invention discloses a method for screening anti-aging active ligands, which comprises the steps of carrying out affinity adsorption on a moringa oleifera leaf extract and multi-target enzyme, and then carrying out high-speed centrifugation to remove unadsorbed components; then adding a solvent for desorption, carrying out chromatography-mass spectrometer analysis on the desorbed solution to obtain a corresponding chromatographic peak, and calculating the area of the chromatographic peak to obtain the anti-aging active ligand in the moringa leaves; the invention applies the ultrafiltration technology to the screening of the anti-aging active ligand of the moringa leaves, and screens out potential components with anti-aging activity from the moringa leaves by combining the high performance liquid chromatography and mass spectrum combined technology; the method has the advantages of high analysis speed, strong specificity and low sample consumption, and is beneficial to quickly screening out potential active ingredients from complex natural products.
Description
Technical Field
The invention relates to the technical field of natural medicine analysis and extraction and skin care products, in particular to a method for screening anti-aging active ligands.
Background
Moringa oleifera is a perennial deciduous tree of the genus Moringa of the family Moringaceae, native to India, and now widely planted in tropical and subtropical regions of Asia, Africa and Central America, and has received extensive attention from researchers due to its rich pharmacological activity and very high nutritional value.
In the related technology, the moringa oleifera is shown to have obvious anti-aging effect, and the screening method of the anti-aging enzyme activity ligand adopts a spectrum method. However, the inventor finds that factors such as background interference exist in the spectrum method, false negative or false positive phenomena are easy to generate, and no related technology is available for identifying and screening the anti-aging active ingredients in the moringa oleifera.
Disclosure of Invention
In view of the above, the embodiment of the invention provides a rapid and high-sensitivity method for identifying and screening anti-aging active ligands in moringa oleifera.
The embodiment of the invention provides a method for screening anti-aging active ligands, which comprises the steps of carrying out affinity adsorption on a moringa oleifera leaf extract and multi-target enzyme, and then carrying out high-speed centrifugation to remove unadsorbed components; and then adding a solvent for desorption, carrying out chromatography-mass spectrometer analysis on the desorbed solution to obtain a corresponding chromatographic peak, calculating the area of the chromatographic peak to obtain an enrichment rate, and obtaining the anti-aging active ligand according to the enrichment rate.
Further, the method for screening anti-aging active ligand comprises the following steps: (1) preparing an enzymatic reaction buffer solution;
(2) preparing a sample to be detected; weighing a proper amount of moringa oleifera leaf extract, and fully dissolving the moringa oleifera leaf extract by using an enzymatic reaction buffer solution to obtain a sample solution to be detected;
(3) The sample solution to be detected is reacted with the target enzyme in a binding way; respectively sucking a proper amount of sample solution to be detected, elastase, collagenase and hyaluronidase into a centrifuge tube, uniformly mixing, and incubating at a constant temperature; after the incubation is finished, transferring the mixed solution into an ultrafiltration centrifugal tube for high-speed centrifugation; adding an enzymatic reaction buffer solution for elution; adding a proper amount of organic solvent into the retention ligand in the ultrafiltration tube, eluting the components combined with the target enzyme, and collecting the eluent to obtain an experimental group solution; taking the inactivated target enzyme as a control group, and processing by the same method to obtain a control group solution;
(4) performing instrumental analysis on the experimental group solution and the control group solution; drying a proper amount of the sample solution to be detected, the experimental group solution and the control group solution, adding an organic solvent for dissolving, and performing chromatography-mass spectrometry to obtain chromatographic peaks of the components;
(5) calculating the enrichment rate to obtain an anti-aging active ligand of the moringa leaves; and (4) calculating the enrichment rate according to the chromatographic peak to obtain the anti-aging active ligand of the moringa leaves.
Preferably, in the step (1), the enzymatic reaction buffer is 0.1mol/L phosphate buffer or 0.05mol/L tris buffer.
Preferably, proper amounts of tris (hydroxymethyl) aminomethane, calcium chloride and sodium chloride are weighed, then proper amounts of 12mol/L hydrochloric acid are added, and a pH value is adjusted to 7.8 by using a pH meter to obtain 0.05mol/L tris (hydroxymethyl) aminomethane solution; an appropriate amount of sodium dihydrogen phosphate is taken, then an appropriate amount of 0.1mol/L sodium hydroxide solution is added, and the pH value is adjusted to 5.35 by using a pH meter to obtain 0.1mol/L sodium dihydrogen phosphate solution.
Preferably, in the step (2), a proper amount of moringa oleifera leaf extract is taken and fully dissolved by using an enzymatic reaction buffer solution to obtain a moringa oleifera leaf sample solution to be tested with the concentration of 8.0 mg/mL.
Preferably, in the step (3), 100 μ L of the sample solution to be tested, 20 μ L of 10U elastase, 40 μ L of 2U collagenase, and 40 μ L of 60U hyaluronidase are respectively taken and mixed in a 0.2mL centrifuge tube, incubated at a constant temperature of 37 ℃ for 40min, after the incubation is finished, the mixed solution is transferred to an ultrafiltration centrifuge tube with a cut-off molecular weight of 10KD, centrifuged at a revolution of 10000rpm/min for 10 min, then 200 μ L of the enzymatic buffer solution is added to wash off components which are not combined with the three target enzymes, centrifuged again at 10000rpm/min for 10 min, the filtrate is discarded, and the above step is repeated for 2 times; adding 200 mu L of organic solvent into the ultrafiltration tube, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 10000rpm/min, eluting components combined with the target enzyme, repeating the steps for 2 times, collecting and combining filtrates to obtain an experimental group solution; the control group is prepared by heating inactivated elastase, collagenase and hyaluronidase in boiling water, and performing the same steps as the experimental group to obtain the control solution.
Preferably, in the step (5), the enrichment ratio between the potential bioactive ligand and the three target enzymes in the moringa leaves is evaluated by the Binding Degree (BD); the calculation formula is as follows:
BD(％)＝(Aa-Ab)/Aa×100％
Wherein BD is the binding degree of the multi-target enzyme and the active ligand screened from the moringa leaves, AaAnd AbRespectively represents the peak area of each chromatographic peak of the moringa leaf extract treated by active enzyme and inactive enzyme.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the method for screening the anti-aging active ligand comprises the steps of carrying out affinity adsorption on a moringa oleifera leaf extract and a target enzyme, and then carrying out high-speed centrifugation to remove unadsorbed components; then adding a solvent for desorption, carrying out chromatography-mass spectrometer analysis on the desorbed solution to obtain a corresponding chromatographic peak, calculating the area of the chromatographic peak to obtain an enrichment rate, and obtaining an anti-aging active ligand according to the enrichment rate; the method provided by the embodiment of the invention is rapid and efficient, has high sensitivity without being interfered by other matrixes in the sample, is small in sample dosage, and can be used for screening and identifying the anti-aging active ligand in the moringa leaves for the first time; and provides a technical research screening method for screening anti-aging active ligands from other natural products; the anti-aging active ligand identified by the method provided by the embodiment of the invention is a plant extract, is natural, safe and non-irritant, and can be applied to the technical field of cosmetic skin care; the anti-aging active ingredient in the moringa leaves screened by the method for the first time can be applied to the technical fields of cosmetics and skin care, and also can be applied to the technical fields of health care products, medicines and the like, so that the resource utilization rate of the moringa leaves is further improved, the industrial production is facilitated, and the high-value utilization of the moringa leaves is realized.
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FIG. 1 is a schematic flow chart of a method for screening anti-aging active ligands according to an embodiment of the present invention;
FIG. 2 is an ultrafiltration screening chromatogram of Moringa oleifera leaf extract and elastase in an embodiment of the present invention;
FIG. 3 is an ultrafiltration screening chromatogram of Moringa oleifera leaf extract and collagenase according to an embodiment of the present invention;
FIG. 4 is an ultrafiltration screening chromatogram of Moringa oleifera leaf extract and hyaluronidase in an embodiment of the present invention;
FIG. 5 is an extraction chromatogram of multiple reaction monitoring anion patterns of peaks 6, 8 of a liquid chromatogram of an embodiment of the invention;
FIG. 6 is a second order mass spectrum of liquid chromatography peak 6 in an example of the present invention;
FIG. 7 is a second order mass spectrum of liquid chromatography peak 8 in an example of the present invention;
wherein: in FIG. 2, the detection wavelength is 280nm, line a represents the HPLC chromatogram of the Moringa oleifera leaf extract, and lines b and c represent the liquid chromatogram of the Moringa oleifera leaf extract after the action of elastase and inactivated elastase, respectively; in FIG. 3, the detection wavelength is 280nm, line d represents the HPLC chromatogram of the Moringa oleifera leaf extract, and lines e and f represent the liquid chromatogram of the Moringa oleifera leaf extract after it has been reacted with collagenase and inactivated collagenase, respectively; in FIG. 4, the detection wavelength is 280nm, the line g represents the HPLC chromatogram of the Moringa oleifera leaf extract, and the lines h and i represent the liquid chromatogram of the Moringa oleifera leaf extract after the action of hyaluronidase and inactivated hyaluronidase, respectively; in fig. 5, the extraction chromatogram of the multi-reaction monitoring negative ion mode of the potential anti-aging active ligand 3-caffeoylquinic acid corresponding to the liquid chromatogram peak 6 uses parent ion 352.98 as the ion for extraction, and the signal intensity is 6.19 × 105; the ion used for extraction in the extraction chromatogram of the multi-reaction monitoring negative ion mode of the potential anti-aging active ligand 4-caffeoylquinic acid corresponding to the liquid chromatogram peak 8 is parent ion 353.01, and the signal intensity is 2.11 multiplied by 105.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings.
Skin aging is one of the visible precursors of human aging, mainly due to oxidative stress and enhanced activation of proteolytic enzymes (such as elastase, collagenase and hyaluronidase) which belong to the group of Matrix Metalloproteinases (MMPs). These proteolytic enzymes hydrolyze proteins that are important in maintaining skin activity, e.g., hyaluronic acid performs a variety of functions in the skin, including retaining moisture and promoting the mechanical elasticity and flexibility of the skin. In addition, elastin plays an important role in maintaining skin elasticity, and the degradation of elastin is the major cause of skin relaxation and fine wrinkles resulting from skin aging. Meanwhile, collagen widely present in the extracellular matrix plays a key role in maintaining the flexibility, strength and elasticity of the skin. However, hyaluronidase, elastase, and collagenase can degrade hyaluronic acid, elastin, and collagen, respectively. Therefore, active ligands for hyaluronidase, elastase and collagenase are potential anti-aging active ingredients because they have anti-aging and anti-wrinkle activity on the skin.
The embodiment of the invention provides a method for screening anti-aging active ligands, which comprises the steps of carrying out affinity adsorption on a moringa oleifera leaf extract and a target enzyme, and then carrying out high-speed centrifugation to remove unadsorbed components; then adding a solvent for desorption, carrying out chromatography-mass spectrometry instrument analysis on the desorbed solution, collecting corresponding chromatographic peaks, calculating the enrichment ratio through the chromatographic peak area, and obtaining the anti-aging active ligand according to the enrichment ratio.
Referring to fig. 1, the method for screening anti-aging active ligand comprises the following steps: (1) preparing an enzymatic reaction buffer solution;
specifically, a proper amount of tris (hydroxymethyl) aminomethane, calcium chloride and sodium chloride are weighed, then a proper amount of 12mol/L hydrochloric acid is added, and a pH value is adjusted to 7.8 by using a pH meter to prepare a 0.05mol/L tris (hydroxymethyl) aminomethane solution; taking a proper amount of sodium dihydrogen phosphate, then adding a proper amount of 0.1mol/L sodium hydroxide solution, and adjusting the pH value to 5.35 by using a pH meter to prepare 0.1mol/L sodium dihydrogen phosphate solution;
(2) preparing a sample solution to be detected; weighing a proper amount of moringa oleifera leaf extract, and fully dissolving the moringa oleifera leaf extract by using an enzymatic reaction buffer solution to obtain a sample solution to be detected; the concentration of the sample solution to be detected is 8.0 mg/mL;
(3) performing affinity reaction on the sample solution to be detected and the target enzyme; respectively sucking a proper amount of sample solution to be detected, elastase, collagenase and hyaluronidase into a centrifuge tube, uniformly mixing, and incubating at a constant temperature; after the incubation is finished, transferring the mixed solution into an ultrafiltration centrifugal tube for high-speed centrifugation; adding an enzymatic reaction buffer solution for elution; adding a proper amount of organic solvent into the retention ligand in the ultrafiltration tube, eluting the components combined with the target enzyme, and collecting the eluent to obtain an experimental group solution; taking the inactivated target enzyme as a control group, and processing by the same method to obtain a control group solution;
Specifically, 100 mul of sample solution to be tested, 20 mul of 10U elastase, 40 mul of 2U collagenase and 40 mul of 60U hyaluronidase are respectively taken and evenly mixed in a 0.2mL centrifuge tube, incubation is carried out for 40min at constant temperature of 37 ℃, after the incubation is finished, the mixed solution is transferred to an ultrafiltration centrifuge tube with the molecular weight cut-off of 10KD, centrifugation is carried out for 10 min at the revolution of 10000rpm/min, then 200 mul of the enzymatic buffer solution is added to wash off components which are not combined with the three target enzymes, centrifugation is carried out for 10 min again at 10000rpm/min, filtrate is discarded, and the step is repeated for 2 times; adding 200 mu L of organic solvent into the ultrafiltration tube, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 10000rpm/min, eluting components combined with the target enzyme, repeating the steps for 2 times, and collecting and combining filtrate to obtain an experimental group solution; heating inactivated elastase, collagenase and hyaluronidase in boiling water for the control group, and performing the same steps as the experimental group to obtain a control group solution;
(4) analyzing and collecting chromatographic peak information by an instrument for the experimental group solution and the control group solution; drying a proper amount of the sample solution to be detected, the experimental group solution and the control group solution, adding an organic solvent for dissolving, and performing chromatography-mass spectrometry to obtain chromatographic peaks of the components;
Specifically, the sample solution to be tested, the experimental group solution and the control group solution are dried by a nitrogen blowing instrument and then dissolved by adding 50 mu L of pure methanol or pure acetonitrile for high performance liquid chromatography-mass spectrometry analysis;
wherein the high performance liquid chromatograph has the type of Agilent 1220Infinity II, Agilent 1260Infinity II or Agilent1290Infinity II; the chromatographic column is Waters Symmetry RP-C18 (specification of 250mm multiplied by 4.6mm, 5 μm), Agilent HC-C18 (specification of 250mm multiplied by 4.6mm, 5 μm), Athena C18-WP (specification of 150 multiplied by 4.6mm, 5 μm) or Hypersil BDS C18 (specification of 150mm multiplied by 4.6mm, 3 μm); the mobile phase is A-0.1% formic acid-water and B-acetonitrile; gradient elution procedure: 8-30% of B in 0-30 min; 30-40 minutes, 30-95% B; the percentages indicated are volume percentages; the detection wavelength is 254nm, 280nm or 360 nm; the flow rate is 0.6mL/min, 0.8mL/min or 1 mL/min; the sample amount is 5 or 10 mu L; the column temperature is 25 ℃ or 30 ℃;
the high performance liquid chromatography-mass spectrometry instrument is Thermo Fisher Scientific (Access Max) or Agilent1290 definition II 6530C; the ion source is in an electrospray ionization (ESI) positive ion mode or a negative ion mode; the spraying voltage is 3000V; the mass scanning range is 100-1000 m/z or 150-1500 m/z; the secondary spectrum scanning mode is data dependent scanning; the vaporization temperature is 250 ℃, 300 ℃ or 350 ℃; the capillary temperature is 250 ℃, 300 ℃ or 350 ℃; sheath gas pressure was 40 psi; the auxiliary air pressure was 10 psi;
(5) Calculating the enrichment rate to obtain an anti-aging active ligand of moringa leaves; and calculating the enrichment rate according to the chromatographic peak to obtain the anti-aging active ligand of the moringa leaves.
Evaluating the enrichment rate of potential bioactive ligands and the three target enzymes in the moringa leaves by using the Binding Degree (BD); the calculation formula is as follows:
BD(％)＝(Aa-Ab)/Aa×100％
wherein BD is the binding degree of the multi-target enzyme and the active ligand screened from the moringa leaves, AaAnd AbRespectively represents the peak area of each chromatographic peak of the moringa leaf extract treated by active enzyme and inactive enzyme.
Referring to the attached figures 2-7, the moringa oleifera leaf extract has 10, 8 and 14 potential active ingredients which respectively show potential affinity activities to different degrees on elastase, collagenase and hyaluronidase; for elastase, peak 8 shows a large BD value of 62.34%, followed by 59.49% of peak 10, 59.13% of peak 5, 58.91% of peak 9, 58.00% of peak 4, 36.76% of peak 2, and the like; for collagenase, peak 8 has the greatest BD value of 49.15%, followed by 37.18% of peak 2, 26.91% of peak 10, 25.83% of peak 6, and so on; for hyaluronidase, peak 6 has the highest BD value of 36.75%, followed by 27.11% peak 15, 21.87% peak 7, 16.63% peak 17, 15.03% peak 18, and so on; the BD values of each peak were significantly different, and the peak area of the active ligand after interaction with the corresponding target enzyme was larger than the peak area of the inactive ligand, indicating that there was a competitive interaction between these active ligands and the corresponding target enzyme. Wherein peak 8 has a higher affinity for both elastase and collagenase, and peak 6 has a higher affinity for hyaluronidase; peak 6 (3-caffeoylquinic acid) and Peak 8 (4-caffeoylquinic acid) are therefore potential anti-aging active ligands in Moringa oleifera leaf extract.
In the embodiment of the invention, the multi-target enzyme and the moringa oleifera leaf extract sample are incubated in a solution state, so that the natural conformation under natural conditions is simulated really, and the screened active ligand has high specificity; meanwhile, multi-target enzyme is adopted for incubation, so that the screening efficiency is further improved; the method provided by the embodiment of the invention can evaluate the strength of the combination of the multi-target enzyme and the active ligand by utilizing the combination degree, so as to determine the potential active ligand with strong combination, and provide a screening basis for subsequent pharmacological and pharmacodynamic verification; the anti-aging active ligand screened from the moringa leaves by the method provided by the embodiment of the invention has important guiding significance for the research and development of the moringa in the cosmetic skin care field in the later period.
The anti-aging active ligands 3-caffeoylquinic acid and 4-caffeoylquinic acid extracted from the moringa leaves in the embodiment of the invention can also be applied to preparing chlorogenic acid compounds, the prepared chlorogenic acid is used for preparing medicines, health products or foods for reducing uric acid, and the medicines, health products or foods are dosage forms given through gastrointestinal tracts, such as oral administration.
The anti-aging active ligands 3-caffeoylquinic acid and 4-caffeoylquinic acid extracted from the moringa leaves in the embodiment of the invention can also be applied to preparing medicines for treating and preventing metabolic syndrome, diabetes and complications thereof, cardiovascular diseases, senile dementia and hyperlipidemia.
The moringa leaf active ligands 3-caffeoylquinic acid and 4-caffeoylquinic acid screened by the method disclosed by the embodiment of the invention are not only applied to the fields of anti-aging cosmetics and skin care products, but also can be widely applied to the aspects of other medicines, health care products and the like, so that the resource utilization rate of the moringa leaves is improved, and the high-value utilization of the moringa leaves is realized.
In this document, the terms front, back, upper and lower are used to define the components in the drawings and the positions of the components relative to each other, and are used for clarity and convenience of the technical solution. It is to be understood that the use of the directional terms should not be taken to limit the scope of the claims.
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A method for screening anti-aging active ligands is characterized in that: carrying out affinity adsorption on the moringa oleifera leaf extract and a target enzyme, and then centrifuging at a high speed to remove unadsorbed components; and then adding a solvent for desorption, carrying out chromatography-mass spectrometer analysis on the desorbed solution, collecting corresponding chromatographic peaks, and calculating the area of the chromatographic peaks to obtain the anti-aging active ligand in the moringa leaves.
2. The method of claim 1, wherein the ligand has anti-aging activity, and the method comprises the following steps: the anti-aging active ligand in the moringa leaves is 3-caffeoylquinic acid and 4-caffeoylquinic acid.
3. The method of claim 1, wherein the ligand has anti-aging activity, and the method comprises the following steps: the method comprises the following steps: (1) preparing an enzymatic reaction buffer solution;
(2) preparing a sample to be detected; weighing a proper amount of moringa oleifera leaf extract, and fully dissolving the moringa oleifera leaf extract by using an enzymatic reaction buffer solution to obtain a sample solution to be detected;
(3) the sample solution to be tested is combined with the target enzyme for reaction; respectively sucking a proper amount of sample solution to be detected, elastase, collagenase and hyaluronidase into a centrifuge tube, uniformly mixing, and incubating at a constant temperature; after the incubation is finished, transferring the mixed solution into an ultrafiltration centrifugal tube for high-speed centrifugation; adding an enzymatic reaction buffer solution for elution; adding a proper amount of organic solvent into the retention ligand in the ultrafiltration tube, eluting the components combined with the target enzyme, and collecting the eluent to obtain an experimental group solution; taking the inactivated target enzyme as a control group, and processing by the same method to obtain a control group solution;
(4) analyzing and collecting chromatographic peak information by an instrument for the experimental group solution and the control group solution; drying a proper amount of the sample solution to be detected, the experimental group solution and the control group solution, adding an organic solvent for dissolving, and performing chromatography-mass spectrometry to obtain chromatographic peaks of the components;
(5) Calculating the enrichment rate to obtain an anti-aging active ligand of the moringa leaves; and (4) calculating the enrichment rate according to the chromatographic peak to obtain the anti-aging active ligand of the moringa leaves.
4. The method of claim 3, wherein the ligand has anti-aging activity, and the ligand is selected from the group consisting of: in the step (1), the enzymatic reaction buffer solution is 0.1mol/L phosphate buffer solution or 0.05mol/L tris buffer solution.
5. The method of claim 3, wherein the ligand has anti-aging activity, and the ligand is selected from the group consisting of: in the step (2), a proper amount of moringa oleifera leaf extract is taken and fully dissolved by using an enzymatic reaction buffer solution to obtain a moringa oleifera leaf sample solution to be detected with the concentration of 8.0 mg/mL.
6. The method of claim 3, wherein the ligand has anti-aging activity, and the ligand is selected from the group consisting of: in the step (3), 100 microliter of sample solution to be detected, 20 microliter of 10U elastase, 40 microliter of 2U collagenase and 40 microliter of 60U hyaluronidase are respectively taken and uniformly mixed in a 0.2mL centrifuge tube, incubation is carried out for 40min at constant temperature of 37 ℃, after the incubation is finished, the mixed solution is transferred to an ultrafiltration centrifuge tube with the molecular weight cutoff of 10KD, centrifugation is carried out for 10 min at the revolution of 10000rpm/min, then 200 microliter of the enzymatic buffer solution is added to wash out components which are not combined with the three target enzymes, centrifugation is carried out for 10 min again at 10000rpm/min, filtrate is discarded, and the step is repeated for 2 times; adding 200 mu L of organic solvent into the ultrafiltration tube, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 10000rpm/min, eluting components combined with the target enzyme, repeating the steps for 2 times, collecting and combining filtrates to obtain an experimental group solution; the control group is prepared by heating inactivated elastase, collagenase and hyaluronidase in boiling water, and the rest steps are the same as those of the experimental group to obtain the control group solution.
7. The method for screening ligands with anti-aging activity according to claim 2, wherein: the anti-aging active ligands 3-caffeoylquinic acid and 4-caffeoylquinic acid in the moringa leaves can be applied to preparation of chlorogenic acid compounds.
8. The method for screening ligands with anti-aging activity according to claim 2, wherein: the 3-caffeoylquinic acid and 4-caffeoylquinic acid in the moringa leaves are used as drug ligands for treating and preventing metabolic syndrome, diabetes and complications thereof, cardiovascular diseases, senile dementia and hyperlipidemia.
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