CN111544487B - Application of litchi rind extract mainly containing polymer polyphenol in preparation of sucrase inhibitor - Google Patents
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Abstract
The invention discloses an application of litchi rind extract mainly containing polymer polyphenol in preparation of a sucrase inhibitor, and belongs to the technical field of biology. The litchi rind extract mainly containing polymer polyphenol disclosed by the invention has a good inhibition effect on sucrase, and the sucrase is applied to weight reduction, blood sugar reduction and dental caries prevention.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of litchi rind extract mainly containing polymer polyphenol in preparation of a sucrase inhibitor.
Background
The sucrase inhibitor can be used as medicine, health product or food for preventing and treating obesity and diabetes: the use of sucrose has been well established and people, especially young children, have been hard to shake their particular taste dependence on the sweetness of sucrose. The sucrase inhibitor can inhibit activity of sucrase in gastrointestinal tract, and slow digestion and absorption of sucrose in food, thereby inhibiting increase of blood glucose concentration, and can be effectively used in combination with diet therapy of diabetes patients. In obese patients, it can reduce the conversion of sugar to fat, delay the emptying of the intestinal tract, and increase the consumption of fat to reduce body weight. The sucrase inhibitor plays the effects of reducing weight and blood sugar by inhibiting sucrase, and is discharged from the body through gastrointestinal tract, so that the sucrase inhibitor does not need to enter a blood circulation system and act on the brain center, does not inhibit appetite while reducing weight and blood sugar, has no side effect when the dosage is very high, and accords with the weight-reducing principle of the world health organization. The sucrase inhibitor can be used for preventing dental caries: the sucrase in saliva is not derived from normal oral tissue of human body, but is secreted by acid-producing bacteria in oral cavity, such as Streptococcus mutans. The sucrase can hydrolyze sucrose into glucose and fructose, and provides a substrate for sugar catabolism in dental plaque, so as to facilitate smooth sugar metabolism. Sucrose has been shown to be an important cariogenic carbohydrate, in plaque, sucrase activity represents the rate of sucrose utilization by bacteria in plaque, and the ability of plaque to synthesize sucrase reflects the amount of plaque cariogenic potential. Therefore, the inhibition of the activity of the invertase of the streptococcus mutans, the reduction of the nutrient supply and the sugar metabolism of the bacteria, and the reduction of the cariogenic capacity of the cariogenic bacteria are of great significance.
The litchi is a fruit of a normally green plant of the soapberry family, is mainly produced in places such as Guangdong, guangxi and Fujian provinces in China, and currently, the annual output of the litchi in China is about 160 million tons, which accounts for more than 80% of the total world output of the litchi. The deep processing of litchi produces peel waste of about 20% of fresh fruit quality, and litchi peel as a large amount of agricultural product waste is not effectively developed and utilized so far.
Therefore, the application of litchi rind extract mainly containing polymer polyphenol in the preparation of sucrase inhibitor is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the invention provides an application of litchi rind extract mainly containing polymer polyphenol in preparing sucrase inhibitor.
In order to achieve the purpose, the invention adopts the following technical scheme:
an application of litchi rind extract mainly containing polymer polyphenol in preparing sucrase inhibitor is provided.
Further, the preparation method of the litchi rind extract mainly containing polymer polyphenol comprises the following steps:
(1) Extracting fresh litchi pericarp and 8 times volume concentration 80% ethanol water solution at room temperature for 2 times, soaking for 7d each time, filtering, and mixing filtrates;
(2) Centrifuging the filtrate at 3500rpm for 10min, and collecting supernatant to obtain medicinal liquid;
(3) Purifying the medicinal liquid by column chromatography with XDA-7 macroporous resin, and eluting with 60% ethanol water solution to obtain extract eluate; the conditions of column chromatography purification are as follows: the mass concentration of the sample loading liquid is 3.5mg/mL, the pH value of the liquid medicine is 4.5, and the sample loading speed is 4BV/h; the flow rate of the eluent is 6BV/h;
(4) And (3) concentrating the extract eluent under the conditions of vacuum degree of 95kPa, heating bath temperature of 40 ℃ and cooling water temperature of 15 ℃ under reduced pressure, and performing spray drying on the obtained reflux liquid at a feeding rate of 51mL/min, an air inlet temperature of 160 ℃, an air exhaust temperature of 60 ℃, a material liquid concentration of 22% and an atomizer rotation speed of 21000r/min to obtain the litchi rind extract.
Furthermore, the litchi rind extract mainly containing polymer polyphenol is applied to preparing the medicines for losing weight, reducing blood sugar and preventing decayed teeth.
The specific application method of the litchi rind extract mainly comprising polymer polyphenol comprises the following steps: taking litchi rind extract mainly containing polymer polyphenol, adding or not adding adjuvants, and making into various preparations by conventional method; can be made into capsule, tablet, granule or water solution.
According to the technical scheme, compared with the prior art, the invention discloses the application of the litchi rind extract mainly containing polymer polyphenol in preparing the sucrase inhibitor, wherein the litchi rind contains a large amount of polyphenol compounds, and the polyphenol compounds have various biological activities of resisting oxidation, resisting tumors, preventing cardiovascular diseases, delaying senescence and the like. The research of the invention proves that the litchi rind extract mainly containing polymer polyphenol has good inhibition effect on sucrase, and the sucrase has application in weight reduction, blood sugar reduction and dental caries prevention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the content ratio of polar parts of litchi rind extract of the present invention;
FIG. 2 is a graph showing the inhibition curve of litchi rind extract with polymer polyphenol as main component on sucrase of small intestine of pig in different concentrations;
FIG. 3 is the inhibiting curve of litchi rind extract with polymer polyphenol as main component in different concentrations on saccharose of streptococcus mutans.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The preparation method of the litchi rind extract comprises the following specific steps:
extracting fresh litchi pericarp and 8 times volume concentration 80% ethanol water solution at room temperature for 2 times, soaking for 7d each time, filtering, and mixing filtrates; centrifuging the filtrate at 3500rpm for 10min, and collecting supernatant to obtain medicinal liquid; purifying the medicinal liquid by column chromatography with XDA-7 macroporous resin, and eluting with 60% ethanol water solution to obtain extract eluate; the conditions of column chromatography purification are as follows: the mass concentration of the sample loading liquid is 3.5mg/mL, the pH value of the liquid medicine is 4.5, and the sample loading speed is 4BV/h; the flow rate of the eluent is 6BV/h; and (3) carrying out reduced pressure concentration on the extract eluent under the conditions of vacuum degree of 95kPa, heating bath temperature of 40 ℃ and cooling water temperature of 15 ℃, carrying out spray drying on the obtained reflux liquid, wherein the feeding rate is 51mL/min, the air inlet temperature is 160 ℃, the air exhaust temperature is 60 ℃, the material liquid concentration is 22%, and the rotation speed of an atomizer is 21000r/min, so as to obtain the litchi rind extract.
The Folin-phenol colorimetric method is adopted for determination, and the polyphenol content is 77.8%.
Example 2
The separation of components of the litchi rind extract obtained in example 1 was studied, and the specific procedure was as follows:
the litchi rind extract obtained in example 1 is extracted by n-hexane and ethyl acetate/anhydrous ether (1:1) step by step to obtain an n-hexane phase, an ethyl acetate/anhydrous ether phase and a water phase of the litchi rind extract leaching liquor, and the total phenol content in the three phases is respectively measured, so that the result shows that the total phenol content of the water phase is the highest, the total phenol content of the ethyl acetate/anhydrous ether phase is the second and the n-hexane phase is the smallest (figure 1). The water phase contains phenolic substances which are polymers of polyphenol, and the normal hexane phase and the ethyl acetate/anhydrous ether phase are oligomers and monomers of polyphenol. Therefore, the litchi rind extract is mainly composed of polymers of polyphenol, and is secondarily composed of oligomers and monomers, wherein the polymer polyphenol accounts for about 79%.
Example 3 inhibitory Effect of litchi rind extract based on Polymer Polyphenol on sucrase in porcine small intestine (one) test reagents were prepared:
(1) preparation of 0.01mol/L PBS solution at pH = 7.0:
by adding 61.0ml of 0.2mol/L disodium hydrogenphosphate, 39.0ml of 0.2mol/L sodium dihydrogenphosphate and then 0.8% sodium chloride solution to 1L, 0.01mol/L PBS buffer having pH =7.0 was obtained.
0.2mol/L disodium hydrogen phosphate: taking Na 2 HPO 4 ·12H 2 Adding 500ml of distilled water into 35.81g of O, fully dissolving and storing;
0.2mol/L sodium dihydrogenphosphate: taking NaH 2 PO 4 ·2H 2 Adding 500ml of distilled water into 15.6g of O, fully dissolving and storing;
preparation of 0.8% sodium chloride: 4g of NaCl is taken and 500ml of distilled water is added, and the NaCl is fully dissolved and then is stored.
(2) Preparation of sucrase for pig small intestine: the pig small intestine was thoroughly washed with pre-cooled 0.01mol/L PBS buffer pH =7.0, the contents were stored in a centrifuge tube, the pig small intestine was then broken open, the inner surface of the small intestine was scraped with a clean glass slide, the scrapings were mixed with the above contents, centrifuged at 4 ℃ at 4000r/min for 30min, and the supernatant was stored as an enzyme stock at-20 ℃ for future use.
(3) Preparing 0.1mol/L sucrose solution: 34.2 g of sucrose was dissolved in 100ml of 0.01mol/L PBS buffer solution with pH =7.0 to prepare a 0.1mol/L sucrose solution, and the solution was stored after being sufficiently dissolved.
(4) And (3) measuring the enzyme activity of the pig small intestine sucrase: respectively measuring 0.2mL of zymogen solution diluted by 10 times, 0.2mL of 0.1mol/L sucrose solution and 0.4mL of 0.01mol/L PBS buffer solution with the pH =7.0, respectively keeping the temperature at 37 ℃ for 10min, then uniformly mixing, adding into the same test tube, reacting at the constant temperature for 30min, then detecting the glucose concentration by using a glucose kit produced by Nanjing construction, and calculating the activity of the porcine small intestine sucrase according to the method of the kit.
(5) Preparing litchi rind extract solution mainly containing polymer polyphenol: dissolving litchi rind extract mainly containing polymer polyphenol in 0.01mol/L PBS buffer solution with pH =7.0 to obtain litchi rind extract solutions mainly containing polymer polyphenol with various concentrations.
(II) determination of inhibitory activity of litchi rind extract mainly containing polymer polyphenol on sucrase in small intestine of pig
Blank group: 0.2mL of a 10-fold diluted porcine small intestine sucrase stock solution, 0.2mL of a 0.1mol/L sucrose solution and 0.6mL of a 0.01mol/L PBS buffer solution with the pH =7.0 are respectively measured and are respectively kept at the constant temperature for 10min at 37 ℃, then are uniformly mixed and added into the same test tube, react for 30min at the constant temperature, and then the glucose concentration is detected by using a glucose kit produced by Nanjing.
Measurement group: respectively measuring 0.2mL of 10-time diluted stock solution of the porcine small intestine sucrase, 0.1mol/L of 0.2mL of sucrose solution, 0.2mL of litchi peel extract solution with different concentrations and mainly comprising polymer polyphenol and 0.01mol/L of PBS buffer solution with pH =7.0, respectively keeping the temperature at 37 ℃ for 10min, then uniformly mixing, adding into the same test tube, reacting at the constant temperature for 30min, and detecting the glucose concentration by using a glucose kit produced by Nanjing.
Control group: respectively measuring 0.1mol/L sucrose solution 0.2mL,0.2mL litchi rind extract solution with different concentrations and mainly comprising polymer polyphenol and 0.01mol/L PBS buffer solution with pH =7.0 0 0.6mL, respectively keeping the temperature at 37 ℃ for 10min, then uniformly mixing, adding into the same test tube, reacting for 30min under the constant temperature condition, and then detecting the glucose concentration by using a glucose kit produced by Nanjing construction.
The inhibition rate of the litchi rind extract mainly containing polymer polyphenol on the porcine small intestine sucrase is calculated at different concentrations, and the result is shown in figure 2. The results in FIG. 2 show that the inhibitory effect on sucrase in pig small intestine is continuously improved when the concentration of the extract is continuously increased, and the inhibitory rate of litchi rind extract mainly containing polymer polyphenol on sucrase in pig small intestine is 75.1% when the concentration is 1000ng/ml, so that litchi mainly containing polymer polyphenol can be seenThe branch bark extract has good inhibition effect on sucrase of small intestine of pig. The half Inhibitory Concentration (IC) of litchi peel extract mainly containing polymer polyphenol on sucrase in small intestine of pig is calculated by SPSS17.0 calculation software 50 ) 421.6ng/ml.
Example 4 inhibitory Effect of litchi rind extract based on Polymer Polyphenol on Streptococcus mutans sucrase
Preparing a test reagent:
(1) Preparing bacterial liquid: inoculating the recovered Streptococcus mutans strain in TSB liquid culture medium, culturing at 37 deg.C for 24 hr, centrifuging at 3000r/min for 15min, collecting bacteria, washing with sterile saline for 2 times, and adjusting to appropriate concentration with sterile saline for use.
(2) Preparing litchi rind extract solution mainly containing polymer polyphenol: dissolving litchi rind extract mainly containing polymer polyphenol in TSB liquid culture medium containing 2% sucrose to obtain litchi rind extract solution mainly containing polymer polyphenol with various concentrations.
(II) determination of inhibition activity of litchi rind extract on streptococcus mutans sucrase by using polymer polyphenol as main component
Blank group: adding 1ml of streptococcus mutans bacterial liquid into 10ml of TSB liquid culture medium containing 2% sucrose, culturing at 37 ℃ for 24h, centrifuging at 3000r/min for 15min, taking supernatant, and detecting the glucose concentration by using a glucose kit produced by Nanjing.
Measurement group: adding 1ml of Streptococcus mutans bacterial liquid into 10ml of litchi rind extract solution with various concentrations and mainly polymer polyphenol, culturing at 37 deg.C for 24h, centrifuging at 3000r/min for 15min, collecting supernatant, and detecting glucose concentration with glucose kit produced by Nanjing.
The inhibition rate of different concentrations of litchi rind extract mainly containing polymer polyphenol on streptococcus mutans sucrase is calculated, and the result is shown in figure 3. The results in FIG. 3 show that the inhibitory effect on sucrase of Streptococcus mutans is continuously improved when the concentration of the extract is continuously increased, and the inhibition rate of litchi rind extract mainly containing polymer polyphenol on sucrase of small intestine of pig is 72.2% when the concentration is 120 mug/ml, so that the sucrase of small intestine of pig can be inhibitedSo that the litchi rind extract mainly containing polymer polyphenol has good inhibition effect on streptococcus mutans sucrase. The half Inhibition Concentration (IC) of litchi rind extract mainly containing polymer polyphenol on the sucrase of streptococcus mutans is calculated by SPSS17.0 calculation software 50 ) 52.1. Mu.g/ml.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (1)
1. The application of the litchi rind extract mainly containing polymer polyphenol in preparing the sucrase inhibitor is characterized in that the preparation method of the litchi rind extract mainly containing polymer polyphenol comprises the following steps:
(1) Extracting fresh litchi pericarp and 8 times volume concentration 80% ethanol water solution at room temperature for 2 times, soaking for 7d each time, filtering, and mixing filtrates;
(2) Centrifuging the filtrate at 3500rpm for 10min, and collecting supernatant to obtain medicinal liquid;
(3) Purifying the medicinal liquid by column chromatography with XDA-7 macroporous resin, and eluting with 60% ethanol water solution to obtain extract eluate; the conditions of column chromatography purification are as follows: the mass concentration of the sample loading liquid is 3.5mg/mL, the pH value of the liquid medicine is 4.5, and the sample loading speed is 4BV/h; the flow rate of the eluent is 6BV/h;
(4) Concentrating the extract eluent under reduced pressure under the conditions of vacuum degree of 95kPa, heating bath temperature of 40 ℃ and cooling water temperature of 15 ℃, and performing spray drying on the obtained reflux liquid at a feeding rate of 51mL/min, an air inlet temperature of 160 ℃, an air exhaust temperature of 60 ℃, a feed liquid concentration of 22% and an atomizer rotation speed of 21000r/min to obtain the litchi rind extract;
the sucrase inhibitor is applied to the preparation of the medicament for preventing the decayed teeth.
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