CN113813204B - New use of hawk tea in anti-saccharification and anti-saccharification product and preparation method thereof - Google Patents

New use of hawk tea in anti-saccharification and anti-saccharification product and preparation method thereof Download PDF

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CN113813204B
CN113813204B CN202111041955.8A CN202111041955A CN113813204B CN 113813204 B CN113813204 B CN 113813204B CN 202111041955 A CN202111041955 A CN 202111041955A CN 113813204 B CN113813204 B CN 113813204B
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hawk tea
glycation
hawk
tea
saccharification
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CN113813204A (en
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易帆
李丽
董银卯
何一凡
陈春宇
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Beijing Technology and Business University
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Abstract

The invention relates to new anti-saccharification application of hawk tea and an anti-saccharification product and a preparation method thereof, belonging to the technical field of plant extraction. The invention provides a new anti-saccharification application of hawk tea. In another aspect, the invention provides an anti-glycation article characterized by comprising an anti-glycation active ingredient; the action target point of the anti-saccharification active ingredient is selected from the group consisting of fructosamine, protein carbonylation reaction paths, AGEs, and reaction paths for crosslinking non-enzymatic glycosylation reaction intermediates and proteins; the anti-glycation active component comprises glede tea extract. The third aspect of the invention provides a preparation method of an anti-glycation product, which is characterized in that hawk tea extract is used as an anti-glycation active ingredient of the anti-glycation product. Experiments prove that the hawk tea extract has better effects than positive control aminoguanidine in inhibiting fructosamine and protein carbonylation and protein crosslinking.

Description

New use of hawk tea in anti-saccharification and anti-saccharification product and preparation method thereof
Technical Field
The invention belongs to the technical field of plant extraction, and particularly relates to new anti-saccharification application of hawk tea, an anti-saccharification product thereof and a preparation method thereof.
Background
Hawk tea LitseacoreanaLevlvar lanuginose (Migo) Yang et P.H. Huang is the tea obtained by sun drying tender leaves of the twig of Mao Baopi camphor, is a special product of asbestos county in Sichuan province, and is a geographic mark product of China. Locally, it is called hawk tea, also called old tea. The leaf of the hawk tea is elliptical, green in face and white in back, so the hawk tea is also called white tea, contains various components such as amino acid, mineral elements, vitamin C, vitamin B1, vitamin B2, flavonoids and polyphenols, and has the characteristics of high iron, high selenium, high zinc and high chromium, thereby being beneficial to improving the functions of a hematopoietic system and an endocrine system; other microelements such as rubidium, phosphorus, zinc, etc. have health promoting effects on cranial nerve and cardiovascular and cerebrovascular diseases.
Non-enzymatic glycosylation (nonenzymatic glycosylation, NEG) is defined as a complex series of non-enzymatic reactions occurring between glucose and proteins, the end products of which are advanced glycosylation end products AGEs. The non-enzymatic glycosylation reaction of proteins proceeds similarly to free radical chain reactions, roughly comprising three stages, early, medium and final, with fructosamine, active carbonyl compounds and advanced glycosylation end products AGEs, respectively.
Advanced glycation end products AGEs generated by the non-enzymatic glycosylation of proteins mainly affect human bodies, and the formation and aggregation of the AGEs in the bodies can cause occurrence of a plurality of diseases, are closely related to pathogenesis of senile diseases such as diabetes, nephropathy, alzheimer disease and aging, and can cause pathological changes of the bodies in a mode of directly affecting cell and tissue functions and combining with specific receptors to change the protein and cell functions. In addition, AGEs can cause skin problems such as wrinkles, pigmentation, and aging by affecting the amount of melanin produced by the skin, the function of collagen and elastin, the adhesion of skin cells, the process of cell growth, and the like.
With increasing importance of skin aging problems, research into anti-glycation is also increasing. Many chemical components and plant components with anti-glycation effect have been found, wherein the chemical components such as aminoguanidine and analogues thereof, melatonin, thiol compounds and the like, and the plant components such as radix puerariae extract, ginseng extract, quercetin, curcumin and the like can inhibit non-enzymatic glycosylation, so that AGEs are reduced, and the effect of delaying aging is achieved.
The reported effects of the hawk tea relate to blood sugar reduction, antioxidant activity, blood fat reduction, thirst quenching, summer heat relieving, digestion promoting, distention relieving, detoxification, detumescence, refreshing, intelligence improving, eyesight improving, stomach strengthening, blood stasis dissipating, pain relieving, diarrhea stopping, hiccups stopping and the like, however, the application of the hawk tea in the aspect of anti-saccharification (fructosamine inhibition, carbonylation, AGEs, protein crosslinking) has not been reported in the field.
Disclosure of Invention
Based on the above-mentioned blank of the prior art in the field, the present invention provides the use of hawk tea in inhibiting fructosamine, or in inhibiting carbonylation, or in inhibiting AGEs.
The technical scheme of the invention is as follows:
new use of hawk tea in anti-glycation is provided.
The anti-glycation is selected from the group consisting of fructosamine inhibition, carbonylation inhibition, AGEs inhibition, protein cross-linking inhibition;
the hawk tea is preferably an hawk tea extract;
preferably, the hawk tea extract is preferably an aqueous extract of hawk tea.
The water extract of the hawk tea refers to a product obtained by extracting the hawk tea in water at a high temperature;
preferably, the high temperature extraction is performed at an extraction temperature of 50-100deg.C, preferably 80deg.C;
the extraction time is 1h-24h, preferably 2h;
preferably, the hawk tea is crushed and sieved before high-temperature extraction;
preferably, the ratio of eagle tea to water used in the high temperature extraction is 1-7 g:1-500 ml, preferably 1 g:50 ml.
Preferably, the product obtained by high-temperature extraction is filtered, concentrated and freeze-dried to obtain the hawk tea extract.
The anti-glycation effect concentration of Litsea coreana extract is 10-5000 μg/ml, preferably 10 μg/ml,50 μg/ml,500 μg/ml, 5000 μg/ml.
An anti-glycation article comprising an anti-glycation active ingredient; the action target point of the anti-saccharification active ingredient is selected from the group consisting of fructosamine, protein carbonylation reaction paths, AGEs, and reaction paths for crosslinking non-enzymatic glycosylation reaction intermediates and proteins; the anti-glycation active component comprises glede tea extract.
The anti-glycation effective concentration of the hawk tea extract is 10-5000 mug/ml, preferably 10 mug/ml, 50 mug/ml, 500 mug/ml, 5000 mug/ml;
the hawk tea extract refers to a product obtained by extracting hawk tea in water at high temperature;
preferably, the high temperature extraction is performed at an extraction temperature of 50-100deg.C, preferably 80deg.C;
the extraction time is 1h-24h, preferably 2h;
preferably, the hawk tea is crushed and sieved before high-temperature extraction;
preferably, the ratio of the eagle tea to the water in the high-temperature extraction is 1-7g to 1-500ml, preferably 1g to 50ml;
preferably, the product obtained by high-temperature extraction is filtered, concentrated and freeze-dried to obtain the hawk tea extract.
The anti-saccharification product is as follows: skin care products, cosmetics, medicines, or health care products;
the dosage forms of the anti-saccharification product are powder, liquid, capsule, tablet, granule, injection, pill and atomization agent;
preferably, the anti-glycation article further comprises an adjuvant; the auxiliary materials are preferably selected from the following components: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders.
A preparation method of an anti-saccharification product is characterized in that hawk tea extract is used as an anti-saccharification active ingredient of the anti-saccharification product.
The hawk tea extract refers to a product obtained by extracting hawk tea in water at high temperature;
preferably, the high temperature extraction is performed at an extraction temperature of 50-100deg.C, preferably 80deg.C;
the extraction time is 1h-24h, preferably 2h;
preferably, the hawk tea is crushed and sieved before high-temperature extraction;
preferably, the ratio of eagle tea to water used in the high temperature extraction is 1-7 g:1-500 ml, preferably 1 g:50 ml.
The invention provides a new application of hawk tea in the anti-saccharification field for the first time. The invention takes hawk tea as a research object, adopts a tea drinking mode (namely water extraction) to extract the active ingredients of the hawk tea, detects the total flavonoid content in the active ingredients, researches the in-vitro anti-saccharification activity of the aqueous extract of the hawk tea, and detects the inhibition rate of fructosamine, medium-term protein carbonylation and protein crosslinking of the early products of non-enzymatic glycosylation reaction at three concentrations of 5000 mug/mL, 500 mug/mL and 50 mug/mL of the aqueous extract of the hawk tea, and fully proves the anti-saccharification activity of the hawk tea through a large number of experiments.
The invention is proved by full experiments: the inhibition rate of the in-vitro non-enzymatic glycosylation of the hawk tea extract is in positive phase correlation with the concentration of the extract, and the higher the concentration of the hawk tea extract is, the higher the inhibition rate of the in-vitro non-enzymatic glycosylation is. FIG. 7 shows that the extract of Litsea coreana shows an inhibition of greater than 90% at a maximum concentration of 5000 μg/mL; under the conditions that the mass concentration is 500 mug/mL and 50 mug/mL, the inhibition rate of the hawk tea extract is far higher than that of aminoguanidine at the same concentration.
The whole anti-glycation effect of the hawk tea can be primarily judged by an in-vitro inhibition non-enzymatic glycosylation experiment, but the non-enzymatic glycosylation reaction is approximately divided into three stages, the anti-glycation inhibition stage of the hawk tea cannot be determined, in addition, the crosslinking reaction can occur in the non-enzymatic glycosylation reaction process mainly by intermediate products (active carbonyl compounds) and proteins, mainly collagen and other long-life proteins, so that the tissue mechanical properties of extracellular proteins are influenced, the physiological functions of intracellular proteins can be changed, and the formation of polymerized or crosslinked lipofuscin-like pigment or fluorescent substances is induced, so that the gradual accumulation of lipofuscin in the body is caused. The meaning of "protein crosslinking" in the present invention is: the intermediate (reactive carbonyl compound) during the non-enzymatic glycosylation reaction reacts with the cross-linking of the protein. Inhibiting protein cross-linking may also promote anti-glycation effects to some extent. Therefore, the invention further detects the influence of hawk tea extracts with different concentrations (5000 mug/mL, 500 mug/mL and 50 mug/mL) on the content of fructosamine, intermediate protein carbonylation and protein crosslinking which are early products of the non-enzymatic glycosylation reaction, and further proves the anti-saccharification effect of the hawk tea.
In terms of fructosamine inhibition, as shown in fig. 2, the inhibition of the hawk tea extract on fructosamine showed concentration dependence, and the inhibition rate of the hawk tea extract on fructosamine was significantly higher than that of the positive control aminoguanidine of the same concentration at each concentration of 5000 μg/mL,500 μg/mL and 50 μg/mL (fig. 2).
In the protein carbonylation test experiment, as shown in fig. 3, hawk tea has higher inhibition effect on protein carbonylation than the positive control aminoguanidine at the same concentration, and has higher inhibition effect at the concentration of 5000 mug/mL. The hawk tea extract has a protein carbonylation inhibition of more than 100% in 5000 mug/mL and 500 mug/mL, and presumably, the solution prepared by the hawk tea extract is too dark at the two concentrations, so that the hawk tea extract at the concentration is not suitable for detection, and the hawk tea extract has a certain inhibition effect on protein carbonylation at 50 mug/mL.
In inhibiting protein cross-linking, as shown in FIG. 4, negative inhibition was exhibited at a concentration of 5000 μg/mL, probably because the hawk tea extract was affected too deeply by color, resulting in inaccurate data, indicating that the hawk tea extract at this concentration was not optimal in inhibiting protein cross-linking; the inhibition rate of the hawk tea extract on protein crosslinking is obviously higher than that of positive control aminoguanidine at the same concentration in 500 mug/mL and 50 mug/mL.
Drawings
FIG. 1 is a rutin standard curve in Experimental example 1 of the present invention.
FIG. 2 is a bar graph showing the inhibition of fructosamine by Litsea coreana extract in experimental example 3 of the invention.
FIG. 3 is a bar graph showing the inhibition of protein carbonylation by hawk tea in experimental example 3 of the invention.
FIG. 4 is a bar graph showing the inhibition of protein cross-linking by hawk tea in experimental example 3 of the present invention.
Detailed Description
The following describes the present invention in further detail with reference to specific examples, but is not intended to limit the scope of the present invention.
Sources of biological materials
The hawk tea used in the experimental example of the present invention is commercially available.
Example 1 New use of Litsea coreana tea of the invention for anti-glycation
The embodiment of the group provides new anti-saccharification application of hawk tea.
The anti-saccharification application of the hawk tea is provided for the first time. Those skilled in the art can use hawk tea in any anti-glycation related product, method based on the description and teachings of the present invention. Any of the activities of using the hawk tea in anti-glycation related products (e.g., raw materials or ingredients of anti-glycation products relate to hawk tea), preparing (e.g., hawk tea is involved in preparing anti-glycation products), producing (use or addition of hawk tea in the production of anti-glycation products), using (use of hawk tea for anti-glycation), serving (use of hawk tea related products or ingredients in the relevant anti-glycation services to customers in medical/cosmetic/health/wellness institutions), selling (selling anti-glycation products containing components of hawk tea) falls within the scope of the present invention.
In specific embodiments, the anti-glycation is selected from the group consisting of fructosamine inhibition, carbonylation inhibition, AGEs inhibition, protein cross-linking inhibition;
the hawk tea is preferably an hawk tea extract;
preferably, the hawk tea extract is preferably an aqueous extract of hawk tea.
In some embodiments, the aqueous extract of hawk tea refers to the product obtained by high temperature extraction of hawk tea in water;
preferably, the high temperature extraction has an extraction temperature of 50-100 ℃, preferably 80 ℃, and the advantage of using this extraction temperature is: on the premise of not damaging the effective substances in the tea, the effective components in the tea are extracted as much as possible, and the extracted effective components are more similar to the effective components which can be obtained by daily tea brewing.
The extraction time is 1h-24h, preferably 2h; the benefit of using this extraction time is: on the premise of ensuring the efficiency, the effective components in the tea are extracted as much as possible, and the pollution of the tea possibly caused by long-time extraction is avoided.
Preferably, the hawk tea is crushed and sieved before high-temperature extraction;
preferably, the ratio of the eagle tea to the water in the high-temperature extraction is 1-7g to 1-500ml, preferably 1g to 50ml; the advantages of using this dosage ratio are: under the condition of being closer to actual tea making, the effective substances in the tea are fully extracted, and the difficulty brought to the subsequent concentration due to excessive use of water is avoided.
Preferably, the product obtained by high-temperature extraction is filtered, concentrated and freeze-dried to obtain the hawk tea extract.
The detection proves that the main component of the water extract of the hawk tea is flavonoid substances.
In specific embodiments, the anti-glycation onset concentration of the hawk tea extract is 10-5000 μg/ml, preferably 10 μg/ml,50 μg/ml,500 μg/ml, 5000 μg/ml.
Group 2 example, anti-saccharification articles of the present invention
The present set of embodiments provides an anti-glycation article. In all embodiments of the present group, the anti-glycation articles share the following common features: the anti-glycation article comprises an anti-glycation active ingredient; the action target point of the anti-saccharification active ingredient is selected from the group consisting of fructosamine, protein carbonylation reaction paths, AGEs, and reaction paths for crosslinking non-enzymatic glycosylation reaction intermediates and proteins; the anti-glycation active component comprises glede tea extract.
In specific embodiments, the anti-glycation onset concentration of the hawk tea extract is 10-5000 μg/ml, preferably 10 μg/ml,50 μg/ml,500 μg/ml, 5000 μg/ml;
in a more specific embodiment, the hawk tea extract refers to the product obtained by extracting hawk tea in water at high temperature;
in a further embodiment, the high temperature extraction has an extraction temperature of 50-100 ℃, preferably 80 ℃, the benefits of using this extraction temperature are: on the premise of not damaging the effective substances in the tea, the effective components in the tea are extracted as much as possible, and the extracted effective components are more similar to the effective components which can be obtained by daily tea brewing.
The extraction time is 1h-24h, preferably 2h; the benefit of using this extraction time is: on the premise of ensuring the efficiency, the effective components in the tea are extracted as much as possible, and the pollution of the tea possibly caused by long-time extraction is avoided.
In other embodiments, the hawk tea is crushed and sieved prior to high temperature extraction;
preferably, the ratio of the eagle tea to the water in the high-temperature extraction is 1-7g to 1-500ml, preferably 1g to 50ml; the advantages of using this dosage ratio are: under the condition of being closer to actual tea making, the effective substances in the tea are fully extracted, and the difficulty brought to the subsequent concentration due to excessive use of water is avoided.
In some embodiments, preferably, the product obtained by high temperature extraction is filtered, concentrated, and lyophilized to obtain the hawk tea extract.
In embodiments that are adapted for actual production, the anti-glycation articles may be: skin care products, cosmetics, medicines, or health care products; the person skilled in the art can further make the anti-glycation products according to the invention into new products on the market, for example solid beverages,
Further, the dosage form of the anti-saccharification product is powder, liquid, capsule, tablet, granule, injection, pill and atomization agent;
preferably, the anti-glycation article further comprises an adjuvant; the auxiliary materials are preferably selected from the following components: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders.
Group 3 example, method of preparing anti-saccharification articles of the present invention
The present set of embodiments provides a method of making an anti-glycation article. All embodiments of this group share the following common features: the hawk tea extract is used as an anti-saccharification active ingredient of the anti-saccharification product.
In a specific embodiment, the hawk tea extract refers to a product obtained by extracting hawk tea in water at high temperature;
in some embodiments, preferably, the high temperature extraction has an extraction temperature of 50-100 ℃, preferably 80 ℃, the benefits of using this extraction temperature being: on the premise of not damaging the effective substances in the tea, the effective components in the tea are extracted as much as possible, and the extracted effective components are more similar to the effective components which can be obtained by daily tea brewing.
The extraction time is 1h-24h, preferably 2h; the advantage of adopting this extraction time is that under the prerequisite of guaranteeing efficiency, extract the active ingredient in the tealeaves as far as possible, and avoid extracting the pollution that probably arouses tealeaves for a long time.
In other embodiments, preferably, the hawk tea is crushed and sieved prior to high temperature extraction;
in a specific embodiment, preferably, the ratio of eagle tea to water in the high temperature extraction is 1-7 g:1-500 ml, preferably 1 g:50 ml; the advantages of using this dosage ratio are: under the condition of being closer to actual tea making, the effective substances in the tea are fully extracted, and the difficulty brought to the subsequent concentration due to excessive use of water is avoided.
The new application and the anti-saccharification effect of the hawk tea are further described in detail by experimental examples:
experimental example 1 extraction of Litsea coreana active substance
1.1 extraction of Litsea coreana active substances
Because the hawk tea is often used as tea drink, the experiment is mainly carried out by improving the tea drink mode, and extracting the effective substances of the hawk tea.
1.1.1 reagents and instruments
Electronic balance (TB-2002, beijing Sidoris instruments systems Co., ltd.); a pulverizer (JP-400B-8); ultra-low temperature preservation boxes (DW-86L 388A, sea group); freeze dryer (VIRTIS); a rotary steaming instrument; vacuum suction filter
Reagent: deionized water
1.1.2 Experimental methods
Crushing the tea leaves by a crusher, and sieving the crushed tea leaves by a 24-mesh sieve for standby. According to the feed liquid ratio of 1:50 (g/ml) ratio, 4.0g of the raw material and 200ml of the solvent (water) were weighed, extracted with distilled water (80 ℃ C., 2 h), filtered, the filtrate was taken, and the filtrate was concentrated in vacuo (60-70 ℃ C.) using a rotary evaporator. And finally, pouring the concentrated extract into a culture dish, putting into a low-temperature refrigerator (-80 ℃) to be frozen for 24 hours, taking out, drying the active substances of the hawk tea by using a freeze dryer, and weighing for later use.
1.1.3 results
The yield of the extract obtained by the hawk tea according to the water extraction mode is shown in table 1.
TABLE 1 extraction yield of Litsea coreana
1.2 detection of Total Flavonoids content in Litsea coreana tea active substance
1.2.1 major instruments and reagents
Multifunctional microplate reader (M200 PRO, TECAN); electronic balance (TB-2002, beijing Sidolis instrument systems Co., ltd.)
Rutin control (98%); ethanol (volume fraction 75%)
1.5% sodium nitrite: 10g of sodium nitrite (NaNO) is taken 2 ) Adding deionized water to 200g, and uniformly stirring;
2.10% aluminum nitrate: aluminum nitrate (Al (NO) 3 ) 3 )·9H 2 Adding deionized water to 200g of 35.27g of O, uniformly stirring, and standing;
3.1M/L sodium hydroxide: taking 4.0g of sodium hydroxide, adding 100mL of deionized water, and uniformly stirring;
1.2.2 drawing of rutin Standard Curve
Through experiments by adopting a sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method, rutin reference substance is dissolved by ethanol (volume fraction is 75 percent) to prepare rutin solution with the mass concentration of 1.0 g/L; diluting with ethanol to obtain mass concentrations of 0.0 respectively; 0.2;0.4; 0.6;0.8;1.0mg/mL of control solution; then respectively taking 1mL of rutin reference substance solutions with different concentrations in a volumetric flask (10 mL), sequentially adding deionized water to fill up to a scale mark (2.4 mL) according to the following table from left to right, standing for 10min, measuring the light absorption value (wavelength is set to 510 nm), and taking 75% ethanol as a blank control.
TABLE 2 order of reagent addition
1.2.3 detection of total flavonoids content of Litsea coreana extract
Taking 1.0mg of freeze-dried powder, dissolving in an extraction solvent to prepare a solution with the mass concentration of 1mg/mL, taking out 1mL of the solution in a 10mL volumetric flask, sequentially adding all reagents according to a table 4 for reaction, adding deionized water to fill up to a scale mark (2.4 mL), standing for 10min, and measuring the light absorption value (the wavelength is set to 510 nm) by taking the diluted solvent as a blank control. And calculating the total flavone extraction rate.
Total flavone extraction rate:
wherein C is the concentration of rutin in 1mL of freeze-dried powder, g/mL;
m-mass of lyophilized powder, g
W-the mass of tea powder weighed, g
1000- -concentration unit conversion, g- -mg
1.2.4 results and discussion
The absorbance value is taken as an ordinate, the concentration of the rutin reference substance is taken as an abscissa, and a rutin flavone content standard curve is drawn as shown in figure 1.
The total flavone content of the hawk tea extract was measured according to the method described above, and the results are shown in Table 3, in which the flavone extraction rate of the hawk tea extract is relatively low. Because of the complex ingredients in the hawk tea, the method adopted in the experiment may be interfered by other phenolic acid substances in the hawk tea, and the measured result may be higher.
TABLE 3 total flavone content and total flavone extraction yield in Litsea coreana extracts
Experimental example 2 Litsea coreana in vitro non-enzymatic glycosylation inhibition experiment
2.1 major instrumentation and reagents
Multifunctional microplate reader (M200 PRO, TECAN); an analytical balance; pipette (200. Mu.L, 1mL, 5 mL); a constant temperature box; vortex machine (QL 861, sea door, its Mobel instruments manufacturing company)
Bovine Serum Albumin (BSA); aminoguanidine sulfate hydrate (a 0309, tokyo chemical industry co.); sodium azide; fructose; na (Na) 2 HPO 4 ·12H 2 O (AR, national pharmaceutical group chemical company limited); naH (NaH) 2 PO 4 ·2H 2 O (AR, national pharmaceutical group chemical company limited);
PBS was prepared at ph=7.3-7.4, and 0.02% sodium azide was added to the PBS solution
20mg/mL bovine serum albumin solution (BSA solution): 0.5g BSA was weighed, PBS was added to volume to 25mL, and 0.45 μm aqueous filter was used for filtration:
0.5mol/L fructose solution: 2.25g of fructose is weighed, and PBS is used for constant volume to 25mL;
BSA-fructose reaction solution: 20mL of BSA solution (20 mg/mL) was mixed with 20mL of fructose solution (0.5 mol/L).
2.2 in vitro non-enzymatic glycosylation inhibition assay methods
2.2.1 Experimental procedure
The aqueous solution of hawk tea of 10mg/mL is diluted with PBS, and the solution is diluted in a gradient manner to four concentrations of 5000 mug/mL, 500 mug/mL, 50 mug/mL and 10 mug/mL. PBS was used instead of the test substance and mixed with the reaction solution to serve as a negative control group. The negative control group reflects the fact that there is no inhibition of the non-enzymatic glycosylation reaction in the system. Positive control selection is well known for aminoguanidine. Since the test substance and the positive control can emit certain fluorescence, a blank control group, i.e., the test substance (or the positive control) is mixed with PBS without adding a reaction solution, is provided.
Different test groups were prepared as incubation systems according to the ratios given in Table 4 below and incubated in a 37℃incubator protected from light.
TABLE 4 reaction System establishment
Each test group was incubated in a thermostated incubator protected from light, the fluorescence intensity of each test group was measured on day 5, excitation wavelength 370nm, emission wavelength 440nm, gain 87 (note that this value required appropriate manual adjustment according to the actual test, which value was usually adjusted so that the fluorescence reading of the final negative group was optimal at the 5-digit number), and the inhibition rate was calculated.
2.2.2 data processing
Each sample was set in 3 replicates, the detection result was an arithmetic mean of 3 determinations, and RSD (relative standard deviation) was calculated. The fluorescence values RSD of the 3 parallel measurement should be less than or equal to 3 percent, and sometimes one numerical deviation is large, so that RSD is more than 3 percent, and the arithmetic average value of the other two parallel measurement should be properly selected as a detection result. The inhibition rate was then calculated.
Calculation formula of Inhibition Ratio (IR):
the final index is expressed as mean ± standard deviation.
2.3 results and discussion
TABLE 5 Litsea coreana in vitro non-enzymatic glycosylation inhibition results (inhibition units:%)
The symbol "-" indicates no inhibition effect
The results are shown in Table 5 above, where the eagle tea extract was positively correlated over the concentration range measured, the higher the eagle tea extract concentration, the higher the in vitro non-enzymatic glycosylation inhibition rate.
The extract of hawk tea shows an inhibition rate higher than 90% under the action of 5000 mug/mL of the highest concentration; under the conditions that the mass concentration is 500 mug/mL and 50 mug/mL, the inhibition rate of the hawk tea extract is far higher than that of aminoguanidine at the same concentration. At the lowest concentration of 10 mug/mL, neither the hawk tea nor the positive control aminoguanidine significantly inhibited non-enzymatic glycosylation.
Experimental example 3 further verifying the anti-glycation Activity of Litsea coreana extract of the present invention
3.1 major instrumentation and reagents
Multifunctional microplate reader (M200 PRO, TECAN); a constant temperature box; a water bath kettle;
fructosamine detection kit and protein carbonylation detection kit are all purchased from Nanjing's institute of biological engineering (Nanjing, china)
3.2 Experimental methods
3.2.1 fructosamine content detection
The experiment aims to measure the level of early fructosamine products in the saccharification reaction process by adopting an ultraviolet spectrophotometry, and examine the inhibition capability of a sample on the early stages of non-enzymatic glycosylation reaction. Fructosamine is an early product of non-enzymatic glycosylation reaction, and has a ketoamine structure, and the ketoamine structure (fructosamine) can be subjected to reduction reaction with nitrotetrazolium blue NBT in an alkaline environment to generate formazan, and can be subjected to colorimetric reaction by taking fructosamine DMF as a standard reference substance for quantitative detection.
The experiments were performed according to the experimental method of the kit, and the establishment of the non-enzymatic glycosylation incubation system was performed with reference to table 6 in 5.2.1, and the incubation time was adjusted to 3 days.
The inhibition of fructosamine by hawk tea extract is shown in figure 2.
3.2.2 protein carbonylation assay
The experiment aims to measure the carbonylation level of protein in the middle stage of the saccharification reaction process by adopting an ultraviolet spectrophotometry, and examine the inhibition capability of a sample to the middle stage of the non-enzymatic glycosylation reaction. The carbonyl can react with 2, 4-dinitrophenylhydrazine to generate a reddish brown hydrazone substance, and the absorbance value with a characteristic absorption peak at 370nm can be measured by colorimetric determination, so that the carbonyl content can be calculated.
Test methods reference kit, establishment of non-enzymatic glycosylation incubation system reference table 6 in 5.2.1, and incubation time was adjusted to 4 days.
The inhibition of protein carbonylation by hawk tea extract is shown in figure 3.
3.2.3ThT analysis of protein Cross-Linked inhibition Rate
ThT is benzimidazole salt capable of emitting fluorescence when combined with molecules rich in beta-sheet structures, the fluorescence intensity emitted by the ThT is positively correlated with the content of the beta-sheet structures of the detected molecules, and the emission spectrum is red-shifted, so that the ThT fluorescent probe can be used for detecting whether amyloid aggregates are misfolded or not. The content of amyloid β -crosslinked structure in glycosylated BSA was determined using ThT fluorochrome method.
According to the method described in the research on the inhibition effect and mechanism of catechin on glycosidase and protein non-enzymatic glycosylation, the experiment is carried out, the THT working solution is prepared, and the solution is stored at 4 ℃ in a dark place for standby. 10. Mu.L of the solution to be measured (incubation system in 5.2.1) was mixed with 200. Mu.L of LThT working solution (diluted 10 times), and the mixture was subjected to shaking with a microplate reader, and then the fluorescence value was measured at an excitation wavelength of 440nm and an emission wavelength of 482 nm.
The inhibition of protein cross-linking by the hawk tea extract is shown in figure 4.

Claims (10)

1. Use of water extract of hawk tea in preparing anti-saccharification product; the anti-glycation is selected from the group consisting of fructosamine inhibition, carbonylation inhibition, AGEs inhibition, and protein cross-linking inhibition.
2. The use of the aqueous extract of hawk tea according to claim 1 for preparing an anti-glycation product, characterized in that the aqueous extract of hawk tea refers to the product obtained by extracting hawk tea in water at high temperature.
3. Use of the aqueous extract of hawk tea according to claim 2, characterized in that the extraction temperature of the high temperature extraction is 50-100 ℃;
the extraction time is 1h-24h.
4. Use of the aqueous extract of hawk tea according to claim 3, characterized in that the extraction temperature of the high temperature extraction is 80 ℃; the extraction time was 2h.
5. Use of an aqueous extract of hawk tea according to any one of claims 1-3 for the preparation of an anti-glycation product, characterized in that the hawk tea is crushed and sieved before high temperature extraction.
6. The use of the aqueous extract of hawk tea according to claim 5 for the preparation of an anti-glycation product, characterized in that the ratio of the hawk tea to the water used in the high temperature extraction is 1-7 g/1-500 ml.
7. The use of the aqueous extract of hawk tea according to claim 6 for the preparation of an anti-glycation product, characterized in that the ratio of the hawk tea to the water used in the high temperature extraction is 1 g/50 ml.
8. Use of the aqueous extract of hawk tea according to any one of claims 2-4, 6, 7 for the preparation of an anti-glycation product, characterized in that the hawk tea extract is obtained by filtering, concentrating and freeze-drying the product obtained by high temperature extraction.
9. Use of an aqueous extract of hawk tea according to claim 1 for the preparation of an anti-glycation product, characterized in that the anti-glycation onset concentration of the aqueous extract of hawk tea is 10-5000 μg/ml.
10. Use of an aqueous extract of hawk tea according to claim 9 for the preparation of an anti-glycation product, characterized in that the anti-glycation onset concentration of the hawk tea extract is 10 μg/ml,50 μg/ml,500 μg/ml, 5000 μg/ml.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN104920771A (en) * 2015-05-25 2015-09-23 安徽科技学院 Hawk-tea chewing gum with health-care efficacy and production method thereof
CN107737159A (en) * 2017-11-23 2018-02-27 华益药业科技(安徽)有限公司 A kind of preparation method with hypoglycemic activity Laoyin tea leaf flavone extract
CN108558837A (en) * 2018-03-12 2018-09-21 安徽农业大学 Flavanols alkaloid and its preparation method and application

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Publication number Priority date Publication date Assignee Title
CN104920771A (en) * 2015-05-25 2015-09-23 安徽科技学院 Hawk-tea chewing gum with health-care efficacy and production method thereof
CN107737159A (en) * 2017-11-23 2018-02-27 华益药业科技(安徽)有限公司 A kind of preparation method with hypoglycemic activity Laoyin tea leaf flavone extract
CN108558837A (en) * 2018-03-12 2018-09-21 安徽农业大学 Flavanols alkaloid and its preparation method and application

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