Bitterness-removed extract of kuding tea with anti-fatigue efficacy, preparation method and fingerprint detection method thereof

Abstract

The application discloses a kind of anti-fatigue efficacy of kuding tea bitter extract and preparation method and fingerprint detection method, belong to extract preparation and detection technical field;The extract is kuding tea after enzymolysis, water extraction, concentration, drying, and using macroporous resin column purification enrichment is prepared;When using macroporous resin column purification enrichment, first flush with pure water, then elute with 30-80% ethanol solution by volume fraction, and collect alcohol eluate.The application carries out macroporous resin purification treatment to kuding tea extract, retains its effective component to the greatest extent while reducing bitterness, can effectively improve energy and resist fatigue;Through fingerprint, the quality of product is controlled, it is favorable to the stability of product improvement, and then it is favorable to the further popularization of product.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of extract preparation and detection, and particularly relates to a bitter taste removed Ilex kudingcha extract, a preparation method and a fingerprint spectrum detection method. BACKGROUND

[0002] Kuding tea is the dry leaf of evergreen tree Ilex latifolia Thunb. of Ilex of Aquifoliaceae, and has the effects of clearing heat, detoxifying and generating fluid.

[0003] Kuding tea contains more than 200 components such as kuding saponins, amino acids, vitamin C, polyphenols, flavonoids and caffeine, wherein phenolic acid, triterpenoid saponin and flavonoids are considered as the medicinal active components in kuding tea, and chlorogenic acid is the main source of bitterness in kuding tea.

[0004] However, even though it contains more active components beneficial to health, the oral acceptance is extremely low due to the strong bitterness, so that the popularization and application are limited. At present, various beverages in the form of compound on the market are all aimed at improving the taste by masking the bitterness, and the bitterness of kuding tea has not been fundamentally solved. Therefore, how to improve the bitterness of kuding tea and retain its active components is an important problem to solve the popularization difficulty. SUMMARY

[0005] Therefore, the application discloses a bitter taste removed Ilex kudingcha extract, a preparation method and a fingerprint spectrum detection method. The kuding tea extract is prepared by combining enzymolysis and water extraction, which is beneficial to the dissolution of effective components. The kuding tea extract is purified by a macroporous resin column, so that the bitterness is reduced and the effective components are retained to the greatest extent, and the effects of enhancing energy and resisting fatigue are enhanced. The quality control of the product by the fingerprint spectrum is beneficial to the improvement of product stability, and further beneficial to the further popularization of the product.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme:

[0007] The bitter taste removed Ilex kudingcha extract with the effect of resisting fatigue is prepared by the following steps: the kuding tea is subjected to enzymolysis, water extraction, concentration, drying, and then purified and enriched by a macroporous resin column, and dried to obtain the product. The macroporous resin is a cross-linked polystyrene adsorption resin. When the purification and enrichment by the macroporous resin column are performed, the column is first washed with pure water, and then eluted with an ethanol solution with a volume fraction of 30-80%, and the alcohol eluate is collected.

[0008] The preparation method of the bitter taste removed Ilex kudingcha extract with the effect of resisting fatigue comprises the following steps:

[0009] (1) The kuding tea is crushed, and then subjected to enzymolysis with water and enzyme preparation;

[0010] (2) After enzymolysis, water is added for water extraction, Kuding tea is added for water extraction, the combined extract is filtered, concentrated, and dried to obtain an extract powder;

[0011] (3) The extract powder is configured into a solution after water is added, and then purified and enriched using a macroporous resin column:

[0012] After loading, the water is first washed away, and then a 30-80% ethanol solution is used for elution, and the alcohol eluate is collected;

[0013] The macroporous resin is a cross-linked polystyrene adsorption resin;

[0014] (4) The alcohol eluate is dried to obtain a Kuding tea bitterness-removed extract.

[0015] Preferably, in step (1),

[0016] The mass ratio of Kuding tea to water is 1:2-1:3;

[0017] The enzyme preparation is a composite enzyme preparation prepared by compounding cellulase, pectinase, and plant hydrolysis composite enzyme at a mass ratio of 2:1:1;

[0018] The enzymolysis conditions are that the enzyme preparation addition amount is 0.2%-0.3% of the dry sample amount of Kuding tea, the enzymolysis time is 40-60 min, the temperature is 45-50°C, and the pH value is 4.5-5.5.

[0019] Preferably, in step (2),

[0020] The mass ratio of Kuding tea to water is 1:6-1:12;

[0021] The extraction is performed 1-3 times, and each time is 1-3 hours;

[0022] The filtration is 80-100 mesh filtration;

[0023] The concentrated extract obtained by concentration has a specific gravity of 1.01-1.08.

[0024] Further preferably, in step (2), the mass ratio of Kuding tea to water is 1:10, the extraction is performed 2 times, each time is 2 hours, the filtration is 60 mesh filtration, and the concentrated extract obtained by concentration has a specific gravity of 1.04-1.05.

[0025] Preferably, in step (3),

[0026] The loading concentration is 3-10 mg / mL, and the loading volume is 1-3 column volumes;

[0027] The pure water washing volume is 3-5 column volumes;

[0028] The ethanol solution elution volume is 2-4 column volumes;

[0029] The loading, pure water washing and ethanol solution elution flow rate is 1.5 times the column volume / h.

[0030] Further preferably, in step (3), the loading concentration is 3-6 mg / mL, the loading volume is 1 times the column volume; the pure water washing volume is 3 times the column volume; the ethanol solution elution volume is 3 times the column volume.

[0031] Further preferably, in step (3), the ethanol solution volume fraction is 40-80%.

[0032] Preferably, in step (3),

[0033] The ratio of column height to diameter of the macroporous resin column is 1:6-1:8; the cross-linked polystyrene adsorption resin is AB-8.

[0034] In step (1) or (4), the drying is spray drying or reduced pressure drying.

[0035] The spray drying parameters are: inlet air temperature 120℃, outlet air temperature 90℃, centrifugal frequency 300Hz, feed pump 17rpm, air induction frequency 50Hz.

[0036] A fingerprint detection method of the bitterness-removed extract of Kuding tea as described above, which adopts high performance liquid chromatography to construct the fingerprint, and the chromatographic conditions are:

[0037] The chromatographic column is an octadecylsilane bonded silica gel chromatographic column.

[0038] The column temperature is 25-35℃.

[0039] The flow rate is 0.9-1.1 mL / min.

[0040] The detection wavelength is 317-337 nm.

[0041] The mobile phase A is acetonitrile, the mobile phase B is 0.4% phosphoric acid water, and the gradient elution program is:

[0042] Time / min Mobile phase A / % Mobile phase B / % 0.00～15.00 13→13 87→87 15.00～50.10 13→46 87→54 50.10～60.10 46→95 54→5 60.10～70.10 95→95 5→5 70.10～80.10 13→13 87→87 .

[0043] Preferably, the chromatographic column is C18, the specification is: 4.6x250mm, 5μm; the column temperature is 35℃; the flow rate is 1.0 mL / min; and the detection wavelength is 327 nm.

[0044] Preferably, the test sample preparation method is:

[0045] The Kuding tea extract is configured into a solution using a 50% methanol aqueous solution, sealed and ultrasonicated for 30-60 min, shaken well, filtered to obtain a test sample solution, which is used for high performance liquid chromatography detection.

[0046] Preferably, the preparation method of 0.1 mg / mL control is as follows:

[0047] Precisely take 10 mg of chlorogenic acid control, dissolve in 100 mL of 50% methanol solution, filter, shake well, and obtain.

[0048] The above detection method is applied to control the quality of products containing the bitter taste removing extract of Kuding tea.

[0049] A preparation for improving energy and resisting fatigue, comprising the bitter taste removing extract of Kuding tea as described above and other acceptable adjuvants or aids.

[0050] Further, the dosage form of the preparation includes powder, granules, tablets, capsules, soft capsules, soft candies, oral liquids, and other acceptable any dosage form.

[0051] Compared with the prior art, the bitter taste removing extract of Kuding tea and the preparation method thereof are disclosed, the preparation method is suitable for industrialization, the prepared Kuding tea extract has good water solubility (1 g of the extract can be dissolved in 1-10 ml of water), small bitterness, good taste, and good anti-fatigue activity, and has wide application and promotion prospects. And the detection can be performed through the fingerprint detection method, which is beneficial to control the product quality. BRIEF DESCRIPTION OF DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0053] Figure 1 The fingerprint spectrum of the Kuding tea extract A prepared in Example 1 (flow phase elution gradient 1 condition) is shown;

[0054] Figure 2 The fingerprint spectrum of the chlorogenic acid standard is shown (flow phase elution gradient 1 condition);

[0055] Figure 3 The fingerprint spectrum of the Kuding tea extract A prepared in Example 1 (flow phase elution gradient 2 condition) is shown;

[0056] Figure 4 The fingerprint spectrum of the Kuding tea extract A prepared in Example 1 (flow phase elution gradient 3 condition) is shown;

[0057] Figure 5 The fingerprint spectrum of the AB-8-① number Kuding tea extract prepared in Example 1 is shown;

[0058] Figure 6 AB-8-② Kuding tea extract fingerprint prepared in Example 1 is shown;

[0059] Figure 7 AB-8-③ Kuding tea extract fingerprint prepared in Example 1 is shown;

[0060] Figure 8 D101-① Kuding tea extract fingerprint prepared in Comparative Example 1 is shown;

[0061] Figure 9 D101-② Kuding tea extract fingerprint prepared in Comparative Example 1 is shown;

[0062] Figure 10 D101-③ Kuding tea extract fingerprint prepared in Comparative Example 1 is shown;

[0063] Figure 11 The chlorogenic acid standard curve is shown. DETAILED DESCRIPTION

[0064] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0065] Herein, the term "embodiment" is used as "exemplary" to describe any embodiment, which is not necessarily superior or better than other embodiments. In the performance index test of the embodiments of the present application, unless otherwise specified, the conventional test method in the art is adopted. It should be understood that the terms described in the present application are only used to describe the specific embodiments, and are not used to limit the disclosure of the present application.

[0066] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as generally understood by those of ordinary skill in the art to which the present application belongs; as the test methods and technical means not specially noted in the present application refer to the experimental methods and technical means generally used by those of ordinary skill in the art.

[0067] Example 1

[0068] Sample 1 preparation: Kuding tea extract

[0069] Take 100g of Kuding tea (dried leaves), crush and pass through an 80-mesh sieve, add 3 times the amount of water, set the enzymatic hydrolysis temperature to 50°C, adjust the pH of the material to 5.0, add 0.2% of the composite enzyme preparation based on the amount of Kuding tea, and hydrolyze for 40 minutes. After the enzymatic hydrolysis is complete, add 10 times the amount of water for the first water extraction, and then add 8 times the amount of water for the second extraction (both extractions are carried out under normal pressure with heating and reflux extraction, with a heating temperature of 100°C and a condensation reflux temperature of 4°C), and each extraction is carried out for 2 hours. Combine the extraction liquids, filter through a 100-mesh sieve, concentrate to a specific gravity of 1.04-1.05, and perform centrifugal spray drying with the following parameters: inlet air temperature 120°C, outlet air temperature 90°C, centrifugal frequency 300Hz, feed pump 17rpm, and air induction frequency 50Hz. Obtain Kuding tea extract A, about 10g, with a yield of about 10.55%.

[0070] Take 0.84g of Kuding tea extract A and dilute with water to a 3mg / mL solution. Select activated AB-8 macroporous resin (soak in ethanol for 24 hours), take a column with a diameter of 3.8cm, fill according to the ratio of diameter to height of 1:6, with a filling height of 22.8cm and a filling volume of 258cm 3 , and rinse with pure water until there is no alcohol taste.

[0071] Load the concentration at 3mg / mL and the loading volume at 1 times the column volume of 258mL (the loading and subsequent pure water washing, ethanol solution elution flow rate are all 1.5 times the column volume / h, i.e. 2 drops / s). First, wash with pure water for 3 times the column volume, and after loading, elute with pure water to obtain the original solution of 258mL (discarded); elute with water for 3 times the column volume to obtain the water eluate AB-8-① eluate; then elute with 40% ethanol solution with a volume fraction of 2 times the column volume to obtain the 40% alcohol eluate AB-8-② eluate; and then elute with 80% ethanol solution with a volume fraction of 2 times the column volume to obtain the 80% alcohol eluate AB-8-③ eluate. Evaporate AB-8-② eluate and AB-8-③ eluate to obtain AB-8-② Kuding tea extract 0.17g and AB-8-③ Kuding tea extract 0.14g.

[0072] Example 2

[0073] Sample 2 preparation: Kuding tea extract preparation

[0074] Take 1 kg of Kuding tea (dried leaves), crush and pass through an 80-mesh sieve, add 3 times the amount of water, set the enzymatic hydrolysis temperature to 50°C, adjust the pH of the material to 5.0, add 0.2% of the dry sample of Kuding tea of a composite enzyme preparation, and hydrolyze for 40 minutes. After the enzymatic hydrolysis is complete, add water to 10 times the amount of water for the first water extraction, and then add 8 times the amount of water for the second extraction (both extractions are performed by adding 10 times and 8 times the amount of water, respectively, for two extractions (normal pressure heating reflux extraction, heating temperature 100°C, condensation reflux temperature 4°C), 2 hours each time. Combine the extraction liquid, filter through a 100-mesh sieve, concentrate to a specific gravity of 1.04-1.05, and perform centrifugal spray drying with the following parameters: inlet air temperature 120°C, outlet air temperature 90°C, centrifugal frequency 300 Hz, feed pump 17 rpm, and air induction frequency 50 Hz. Kuding tea extract B is obtained with a yield of about 10.00%.

[0075] Take 28.26 g of Kuding tea extract B and dilute with water to make a 6 mg / mL solution. Select activated AB-8 macroporous resin (soaked in ethanol for 24 hours), take a column with a diameter of 10 cm, fill it according to the ratio of diameter to height of 1:6, with a filling height of 60 cm and a filling volume of about 4700 cm 3 . Wash with pure water until there is no alcohol taste. The loading concentration is 6 mg / mL, and the loading volume is 1 column volume of 4700 mL (the flow rate of loading and subsequent pure water washing and ethanol solution elution is 1.5 column volumes / h, i.e. 2 drops / s). First, wash with pure water for 3 column volumes, and elute with pure water to obtain 4700 mL of the original solution (discarded) and 14000 mL of the water wash (discarded). Replace the volume fraction of 40% ethanol solution for elution, and elute with 2 column volumes of 40% ethanol solution to obtain 40% alcohol eluate. Combine the 40% alcohol eluate, concentrate, and dry under reduced pressure, crush and pass through a 60-mesh sieve to obtain AB-8-④ Kuding tea extract about 6 g with a yield of about 21.23%.

[0076] To further demonstrate the beneficial effects of the present application and better understand the present application, the following comparative examples and experiments further illustrate the product quality and performance of the Kuding tea extract disclosed by the present application, but should not be understood as limiting the present application. The product properties obtained by other determination experiments made by those skilled in the art according to the above application content and the applications made according to the above properties are also considered to fall within the protection scope of the present application.

[0077] Comparative Example 1

[0078] Use D101 macroporous resin instead of AB-8 macroporous resin, and the rest is the same as Example 1.

[0079] Water eluent 3 times column volume 774 mL, get water eluent D101-① eluent; replace the volume fraction 40% ethanol solution for elution, elution volume is 2 times column volume, get 40% alcohol eluent D101-② eluent; then replace the volume fraction 80% ethanol solution for elution, elution volume is 2 times column volume, get 80% alcohol eluent D101-③ eluent.

[0080] D101-② eluent, D101-③ eluent evaporated to dryness, get D101-② Kuding tea extract 0.20 g, D101-③ Kuding tea extract 0.18 g.

[0081] Experiment 1 fingerprint

[0082] 1、Instrument and equipment

[0083] High performance liquid chromatograph: Shimadzu LC-20A;

[0084] Electronic balance: one hundred thousandth analytical balance (Mettler Toledo MS105DU).

[0085] 2、Reagents and materials

[0086] Methanol (Fisher chromatographic pure), acetonitrile (Fisher chromatographic pure), water (Watsons distilled water); microporous filter membrane (BOJINnylon 0.22 μm), syringe (Jiangxi Qingshantang Medical Instrument 1 mL).

[0087] 3、Chromatographic conditions

[0088] Shimadzu InertSustain AQ-C18 (4.6 x 250 mm, 5 μm), with octadecylsilane bonded silica as filler; with acetonitrile as mobile phase A, with 0.4% phosphoric acid aqueous solution as mobile phase B, respectively according to table 1-3 for gradient elution; detection wavelength is 327 nm.

[0089] Table 1 mobile phase elution gradient 1

[0090] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00～15.00 13→13 87→87 15.00～50.10 13→46 87→54 50.10～60.10 46→95 54→5 60.10～70.10 95→95 5→5 70.10～80.10 13→13 87→87

[0091] Table 2 mobile phase elution gradient 2

[0092] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00～50.00 5→95 95→5 50.00～60.00 95→95 5→5 60.00～60.10 95→5 5→95 60.10～70.10 5→5 95→95

[0093] Table 3 mobile phase elution gradient 3

[0094] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00～40.00 5→20 95→80 40.00～50.00 20→30 80→70 50.00～60.00 30→70 70→30 60.00～65.00 70→95 30→5 65.00～70.00 95→95 5→5 70.00～71.00 95→5 5→95 71.00～81.00 5→5 95→95

[0095] 4、Sample preparation

[0096] Precisely take 0.1 g of the Kuding tea extract A prepared in Example 1 in a 100 mL conical flask, add 25 mL of 50% methanol aqueous solution, ultrasonic at 35℃ for 60 min, cool to room temperature, make up the weight loss, filter, shake well, and obtain.

[0097] Precisely take 10 mg of chlorogenic acid reference substance, dissolve in 100 mL of 50% methanol aqueous solution, filter, shake well, and obtain.

[0098] 5. Sample determination

[0099] Precisely take 10 μL of the sample solution, inject into the liquid chromatograph, and determine, and obtain.

[0100] The obtained fingerprint spectrum is shown in Figure 1-4 The peaks in the spectrum are well separated under the condition of mobile phase elution gradient 1, while the peaks in the spectrum are difficult to separate under the conditions of mobile phase elution gradients 2 and 3.

[0101] Experiment 2: Fingerprint spectrum comparison

[0102] The instrument, reagent materials, and chromatographic conditions (mobile phase elution gradient 1) are the same as in Experiment 1.

[0103] Sample preparation:

[0104] Prepare AB-8-① eluent, AB-8-② eluent, and AB-8-③ eluent according to Example 1, and prepare D101-① eluent, D101-② eluent, and D101-③ eluent according to Comparative Example 1, and after concentration, use 50% methanol solution to make up to 20 mL, cool to room temperature, shake well, filter, and obtain.

[0105] Precisely take 10 μL of the sample solution, inject into the liquid chromatograph, and determine, and the results are shown in Figure 5-10 The content of chlorogenic acid is significantly increased after elution with 40% ethanol solution using AB-8, and each marker peak is significantly different from the Kuding tea extract A prepared in Example 1, indicating that the use of macroporous resin AB-8 eluted with 40% ethanol solution has a significant enrichment effect on chlorogenic acid in Kuding tea. The use of D101 resin column eluted with 40% ethanol solution and 80% ethanol solution has little effect on the Kuding tea extract.

[0106] Experiment 3: Determination of chlorogenic acid content

[0107] 1. Instruments and equipment

[0108] High performance liquid chromatograph: Agilent Technologies 1260 Infinity II;

[0109] Electronic balance: 0.1 mg analytical balance (Mettler Toledo MS 105DU).

[0110] 2. Reagents and materials

[0111] Methanol (Fisher chromatographically pure), phosphoric acid (analytically pure), water (distilled water); chlorogenic acid (Shanghai Yuan Ye Biological 5 mg); microporous filter membrane (BOJIN nylon 0.22 μm), syringe (Jiangxi Qingshantang Medical Instrument 1 mL).

[0112] 3. Chromatographic conditions

[0113] Shimadzu Shim-pack VP-ODS-C18 (4.6 x 250 mm, 5 μm) with octadecylsilane-bonded silica gel as the filler; acetonitrile-0.4% phosphoric acid solution (13:87) as the mobile phase; detection wavelength 327 nm. The theoretical plate number should not be less than 2000 calculated by the chlorogenic acid peak.

[0114] 4. Preparation of reference solution

[0115] Take the chlorogenic acid reference, accurately weigh, and place it in a brown volumetric flask. Add 50% methanol aqueous solution to prepare a solution, and it is obtained.

[0116] 5. Preparation of test solution

[0117] The Kuding tea extract A prepared in Example 1, AB-8-② Kuding tea extract, AB-8-③ Kuding tea extract, and D101-② Kuding tea extract and D101-③ Kuding tea extract prepared in Comparative Example 1 were used as samples.

[0118] Accurately weigh 0.1 g of each sample in a 25 mL conical flask, accurately add 25 mL of 50% methanol aqueous solution, weigh, and after ultrasonic treatment at 35°C for 60 min, take it out and cool to room temperature. Make up the weight loss with 50% methanol aqueous solution, shake well, filter with a 0.22 μm filter membrane, and it is obtained.

[0119] 6. Determination

[0120] Accurately pipette 10 μL of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph.

[0121] The standard curve y = 3E+07x-96757, r = 0.9999 drawn according to the reference was used. 2 Figure 11 The chlorogenic acid reference 5 mg was diluted to 10 mL with 50% methanol aqueous solution, and a 6-point standard curve was prepared by the double dilution method. The chlorogenic acid concentration was calculated by the standard curve and the test results of the test sample, and the chlorogenic acid content of each sample was calculated, and the results are shown in Table 4. ​

[0122] Table 4: Chlorogenic acid content of each sample

[0123]

[0124] From the data in Table 4 above, it can be seen that chlorogenic acid has a wide range of biological activities, such as free radical scavenging and central nervous system stimulation. As an effective component of Kuding tea extract and a marker component for quality control, after enzymatic hydrolysis and water extraction, the content of chlorogenic acid in Kuding tea extract A is 4.98%, after purification by AB-8 macroporous resin column eluted with 40% ethanol, the content of chlorogenic acid in AB-8-② Kuding tea extract is 16.68%, which is increased by 335%, while the content of chlorogenic acid in AB-8-③ Kuding tea extract purified by further elution with 80% ethanol is only 0.30%, which is very small and can be ignored. It is proved that most of the chlorogenic acid in Kuding tea extract A is purified after purification by AB-8 macroporous resin column eluted with 40% ethanol. The contents of chlorogenic acid in D101-② Kuding tea extract and D101-③ Kuding tea extract purified by D101 macroporous resin are very small, indicating that this resin column is not suitable for the purification and enrichment of effective components in Kuding tea extract A.

[0125] Experiment 4: Pharmacodynamic experiment

[0126] 1. Instruments and consumables: swimming tank (Shanghai Xinsoft Information Technology Co., Ltd.), sodium lime (Shanghai Wu Si Chemical Reagent Co., Ltd., batch number: 20200713), white vaseline (Shandong Lierkang Medical Technology Co., Ltd., batch number: 200701), electronic balance (Germany Sartorius Group), mouse gavage device (Jinan Yiyansheng Technology Development Co., Ltd.).

[0127] 2. Test samples:

[0128] Kuding tea extract A prepared in Example 1 and AB-8-④ Kuding tea extract prepared in Example 2.

[0129] Positive drug: Monster Magic Claw Black Classic Concentrate, concentrated ratio is 330 mL concentrated to 180 mL.

[0130] 3. Animals: SPF male KM mice, body weight 20±2g, 80. Provided by Beijing Vantoll Life Science and Technology Co., Ltd., license number: SCXK(Jing)20210006.

[0131] 4. Rearing conditions, grouping and drug administration:

[0132] Mice were housed in groups of 5 per cage with free access to food and water in a room with controlled temperature (21 ± 2 °C), humidity (50 ± 10%), and a 12-h light / dark cycle (lights on at 20:00, off at 8:00). All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

[0133] Mice were acclimated for one week after arrival before entering the experiment. Each group of 20 mice was divided into 3 groups according to the random block design method, as shown in Table 5.

[0134] Table 5 Experimental grouping

[0135] Group Mouse administration dose g / kg Normal control group / Positive control group 30 mL / kg Kuding tea extract A group 0.0835 AB-8-④ Kuding tea extract group 0.0835

[0136] The A group of Ilicium fortunei extract and the AB-8-④ number Ilicium fortunei extract group were both administered 0.0835 g / kg, dissolved in purified water; the normal control group was administered the same volume of normal saline by gavage. The mice were administered by gavage at a fixed time in the morning (9:00) every day, and the gavage volume was 0.1 mL·10 g -1 , and the administration lasted for 28 days.

[0137] 5. Weight-loaded swimming experiment

[0138] During the administration period, the mice were subjected to weightless swimming adaptability training once every 3 days for 5 min each time. Ten mice from each group were randomly selected, and 1 h after gavage on the 28th day, the mice were tied to the tail root with a lead wire at 7% of their body weight to cause the mice to be in a weight-loaded state, and immediately placed in a 50 cm x 40 cm x 40 cm water tank, with the water depth controlled not less than 30 cm and the water temperature controlled at 25 ± 1 °C. The time from the start of swimming to the time when the mouse could not float out of the water 10 s after sinking was recorded as the exhaustive swimming time of the mouse. The first sinking time and the exhaustive time of the mouse were recorded.

[0139] The experimental data are represented, and statistical analysis was performed using GraphPad Prism 8.02 software (GraphPad Software, Inc., San Diego, California, USA). Before testing the data parameters of all groups, normality test (Kolmogorov-Smirnov test) and variance homogeneity test (Levene test) were performed. All test parameters were compared between groups using unpaired T test (single-tailed) for pairwise comparison, and the test level was set at p < 0.05.

[0140] The results are shown in Table 6. In the weight-loaded swimming experiment of mice, compared with the normal control group, Ilicium fortunei extract A can significantly prolong the first sinking time of mice in weight-loaded swimming, and Ilicium fortunei extract A and AB-8-④ number Ilicium fortunei extract can significantly increase the exhaustive swimming time of mice in weight-loaded swimming.

[0141] Table 6 Mouse weight-loaded swimming experiment result record table

[0142] Group Administration (g / kg) Number First sinking time (s) Exhaustion swimming time (s) Normal control group / 10 29.73±8.380 71.32±11.08 Positive control group 30 mL / kg 10 53.00±8.155* 147.4±24.16** Kuding tea extract A group 0.0835 10 41.28±6.639 123.6±20.92* AB-8-④ Kuding tea extract group 0.0835 10 55.52±9.119* 168.9±50.51*

[0143] (*p < 0.05 compared with the normal control group, **p < 0.01 compared with the normal control group)

[0144] 6. Hypoxia tolerance experiment under normal pressure

[0145] After resting for 1 day after the last weight-loaded swimming experiment, 1 h after gavage administration on the 29th day, 10 mice in each group were placed in a 250 mL ground-mouth bottle containing 5 g of sodium lime, one mouse per bottle, and the bottle opening was sealed with a pre-coated vaseline plug to prevent gas leakage. The time to respiratory arrest was immediately recorded as the indicator of mouse death due to hypoxia. The hypoxia tolerance time was calculated according to the following standard: T = (T1-T0) / (V0-W0 / 0.94) x 100 (in the formula, T1 is the mouse death time, T0 is the time when the sealing starts, V0 is the effective bottle volume, W0 is the mouse weight, and 0.94 is the density of the mouse determined by the drainage method).

[0146] The experimental data are expressed as GraphPad Prism 8.02 software (GraphPad Software, Inc., San Diego, California, USA) was used for statistical analysis. Before testing the data parameters of all groups, normality test (Kolmogorov-Smirnov test) and variance homogeneity test (Levene test) were performed. All test parameters were compared between groups using unpaired T test (single-tailed) for pairwise comparison, and the test level was set at p < 0.05.

[0147] The results are shown in Table 7. Compared with the normal control group, both Kuding tea extract A and AB-8-④ Kuding tea extract can significantly prolong the hypoxia tolerance time of mice.

[0148] Table 7 Mouse hypoxia tolerance experiment result record table under normal pressure

[0149] Group Administration (g / kg) Number Hypoxia tolerance time (s) Normal control group / 10 1026±45.38 Positive control group 30 mL / kg 10 1183±57.21* Kuding tea extract A group 0.0835 10 1185±35.12** AB-8-④ Kuding tea extract group 0.0835 10 1270±58.72**

[0150] (*p < 0.05 compared with the normal control group, **p < 0.01 compared with the normal control group)

[0151] 7. Blood sample detection experiment

[0152] 7.1 Blood lactic acid and lactate dehydrogenase content determination and tissue sampling (remaining mice)

[0153] During the administration period, non-weighted swimming adaptation training was performed once every 3 days for 5 minutes. The remaining 10 mice in each group were subjected to swimming in a 50 cm x 40 cm x 40 cm water tank after 1 hour of intragastric administration on day 28, with the water depth controlled to be not less than 30 cm and the water temperature being 25 ± 1℃. After 1 hour of weighted swimming (the accurate iron wire weight ratio determined in the pre-experiment was capable of making the mouse weighted swimming slightly more than 1 hour), the mouse was taken out, and after 30 minutes, the mouse was anesthetized with isoflurane. After the eyelid reflex of the animal was detected to enter a deep anesthetic state, all animals were subjected to abdominal aortic blood sampling. The animal blood was placed at room temperature for 30 minutes, and then centrifuged at 3500 rpm for 15 minutes to obtain the supernatant, which was stored in a -80℃ refrigerator for examination.

[0154] 7.2 Muscle lactate, muscle ATP, muscle glycogen, liver glycogen, SOD and MDA content detection

[0155] The ELISA kit was used to detect the muscle lactate, muscle glycogen, liver glycogen content, SOD and MDA of the mice, and the AMP / ATP ratio was calculated. The frozen mouse quadriceps muscle tissue and liver were taken out, thawed (-20℃, 4℃), washed with pre-cooled PBS (0.01M, pH = 7.4) to remove residual blood, and cut into small pieces. The cut tissue was placed in a sterile steel bead with a corresponding volume of PBS (1:9 weight / volume ratio, 1g of tissue sample corresponding to 9mL of PBS, and records were kept), homogenized with a tissue homogenizer, frozen and thawed twice (-20℃, 4℃), and then centrifuged (13000 rpm / 10 min) to obtain the supernatant, which was detected according to the method in the relevant kit instruction manual. Within 5 minutes after the reaction was terminated, the concentration values of each standard in the test kit were input, and the optical density values (OD) of each group were measured in turn at the specified wavelength with an enzyme-labeled instrument. After obtaining the standard curve, the sample OD value was substituted into the obtained regression equation to obtain the concentration of each sample. If the sample was diluted, the final sample concentration needed to be multiplied by the dilution factor.

[0156] 7.3 Statistical method

[0157] The experimental data are represented by GraphPad Prism 8.02 software (GraphPad Software, Inc., San Diego, California, USA) was used for statistical analysis. Before testing the data parameters of all groups, normality test (Kolmogorov-Smirnov test) and variance homogeneity test (Levene test) were performed. All test parameters were compared between groups in pairs using unpaired T test (single-tailed), and the test level was set to p<0.05.

[0158] 7.4 Blood lactate and lactate dehydrogenase content

[0159] Table 8 Mouse blood lactic acid, lactic acid dehydrogenase experimental results record table

[0160]

[0161] * Compared with the normal control group p <0.05, ** Compared with the normal control group p <0.01

[0162] Table 9 Mouse blood lactic acid, lactic acid dehydrogenase experimental results record table

[0163]

[0164] # Compared with the positive control group p <0.05

[0165] Table 10 Mouse blood lactic acid, lactic acid dehydrogenase experimental results record table

[0166]

[0167] $ Compared with the A group of Ilicium fortunei water extract p <0.05

[0168] Compared with the normal control group, the positive drug (Monster Claw Black Classic) group, the A group of Ilicium fortunei extract and the AB-8-④ number Ilicium fortunei extract group can significantly reduce the blood lactic acid content of mice; the positive drug (Monster Claw Black Classic) group and the A group of Ilicium fortunei extract can significantly increase the blood lactic acid dehydrogenase content of mice. Compared with the positive control group, the AB-8-④ number Ilicium fortunei extract group more significantly reduces the blood lactic acid content of mice. There is a significant difference between the A group of Ilicium fortunei extract and the AB-8-④ number Ilicium fortunei extract group.

[0169] 7.5 Muscle lactic acid, muscle ATP, muscle glycogen, liver glycogen, superoxide dismutase (SOD) and malondialdehyde (MDA) content

[0170] Table 11 Mouse muscle lactic acid, muscle ATP, muscle glycogen, liver glycogen, SOD and MDA experimental results record table

[0171]

[0172] * Compared with the normal control group p <0.05, ** Compared with the normal control group p <0.01, *** Compared with the normal control group p <0.001 Table 12 Mouse muscle lactic acid, muscle ATP, muscle glycogen, liver glycogen, SOD and MDA experimental results record table

[0173]

[0174] # p<0.05 compared with the positive control group, ## p<0.01 compared with the positive control group, ### p<0.001 compared with the positive control group

[0175] Table 13 Mouse muscle lactic acid, muscle ATP, muscle glycogen, liver glycogen, SOD and MDA experimental result record table

[0176]

[0177] $ p<0.05 compared with the Kuding tea water extract A group, $$ p<0.01 compared with the Kuding tea water extract A group,

[0178] Compared with the normal control group, the positive drug (Monster Claw Black Classic) group, the Kuding tea extract A group and the AB-8-④ Kuding tea extract group can significantly reduce the muscle lactic acid content of the mice; the positive drug (Monster Claw Black Classic) group, the Kuding tea extract A group and the AB-8-④ Kuding tea extract group can significantly increase the muscle ATP content of the mice; the Kuding tea extract A group and the AB-8-④ Kuding tea extract group can significantly increase the muscle glycogen content of the mice; the positive drug (Monster Claw Black Classic) group and the AB-8-④ Kuding tea extract group can significantly increase the liver glycogen content of the mice; the positive drug (Monster Claw Black Classic) group and the Kuding tea extract A group can significantly increase the superoxide dismutase content of the mice; the Kuding tea extract A group can significantly reduce the malondialdehyde content of the mice.

[0179] Compared with the positive control group, the Kuding tea extract A group can more significantly increase the muscle glycogen content of the mice, and the AB-8-④ Kuding tea extract group can more significantly reduce the muscle SOD content of the mice.

[0180] In terms of muscle lactic acid content and SOD, there is a significant difference between the AB-8-④ Kuding tea extract group and the Kuding tea extract A group; in terms of malondialdehyde, there is a significant difference between the AB-8-④ Kuding tea extract group and the Kuding tea extract A group.

[0181] From the above pharmacodynamic experimental results, it can be seen that the Kuding tea subjected to enzymatic water extraction process (Kuding tea extract A group in Example 1) and the Kuding tea subjected to enzymatic water extraction followed by macroporous resin purification process (AB-8-④ Kuding tea extract in Example 2) both have the effect of prolonging the exhaustive swimming time of the mice under load compared with the normal control group, and the effect of the Kuding tea subjected to enzymatic water extraction followed by macroporous resin purification process is more significant, which can also prolong the first sinking time of the mice under load.

[0182] In the biochemical index detection, the extract of Kuding tea by enzymatic hydrolysis and water extraction and the extract of Kuding tea by enzymatic hydrolysis, water extraction and macroporous resin purification both have the effect of reducing blood lactic acid. The extract of Kuding tea by enzymatic hydrolysis, water extraction and macroporous resin purification significantly reduces the blood lactic acid content of mice, and there is a significant difference between the normal control group, the positive control group, the extract of Kuding tea by enzymatic hydrolysis and water extraction. It is suggested that the extract of Kuding tea by enzymatic hydrolysis, water extraction and macroporous resin purification has a significant effect on improving lactic acid accumulation in the body. The extract of Kuding tea by enzymatic hydrolysis, water extraction and macroporous resin purification significantly reduces the muscle lactic acid content, significantly increases the muscle ATP content, muscle glycogen content and liver glycogen content of mice, and has a significant difference in muscle lactic acid content, SOD and malondialdehyde compared with the extract of Kuding tea by enzymatic hydrolysis and water extraction. It is suggested that the extract of Kuding tea by enzymatic hydrolysis, water extraction and macroporous resin purification has a significant effect on improving muscle energy metabolism.

[0183] Through comprehensive analysis, it can be seen that the extract of Kuding tea by enzymatic hydrolysis, water extraction and macroporous resin purification has a good anti-fatigue effect, and the effect is more significant than the extract without macroporous resin purification process.

[0184] Experiment 5: Tasting bitter taste of the product

[0185] 1. Materials: disposable paper cups, marker pens, Kuding tea extract A prepared in Example 1, AB-8-④ Kuding tea extract.

[0186] 2. Experimental personnel: 12 company personnel (Qingdao Chenlan Biotechnology Co., Ltd.)

[0187] 3. Experimental steps:

[0188] Take 4g of Kuding tea extract A prepared in Example 1 and AB-8-④ Kuding tea extract and dissolve them in 2 liters of pure water. After completely dissolving, mix them evenly. Pour them into paper cups, 24 each. Mark the bottom of the paper cups with a pencil (the tasting personnel cannot see it), record cup A for Kuding tea extract A and record cup B for AB-8-④ Kuding tea extract. Randomly label the cups 1-48 on the side, place cup A in tray numbered 1 and 3, and place cup B in tray numbered 2 and 4.

[0189] 12 tasting personnel randomly take paper cups from trays 1, 2, 3 and 4, taste them and record the results. The scoring standard is:

[0190] 0 points - not bitter;

[0191] 1 point - a little bitter;

[0192] 2 points - relatively bitter;

[0193] 3 points - very bitter.

[0194] The experimental results are shown in Tables 14 and 15.

[0195] Table 14 Bitterness score record

[0196]

[0197]

[0198] Table 15 Score statistics results

[0199]

[0200]

[0201] The results show that the taste of the Ilex kudingcha extract purified by the macroporous resin is obviously improved, the bitterness is removed well, and the taste is between a little bitter to relatively bitter. The taste of the Ilex kudingcha extract without purification by the macroporous resin is relatively bitter, and the palatability is poor.

[0202] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to the above-described embodiments will be apparent to those of ordinary skill in the art in view of the foregoing description, and the general principles described herein can be implemented in additional embodiments without departing from the spirit or scope of the application. Accordingly, the application 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. A bitterless extract of Kuding tea with anti-fatigue efficacy, characterized in that, The preparation method of the bitterless extract comprises the following steps: (1) After the Kuding tea is crushed, water and an enzyme preparation are used for enzymolysis; (2) After the enzymolysis, water is added for water extraction, and the extract is obtained by filtering, concentrating and drying the combined extract; (3) The extract is dissolved in water and then purified and enriched by a macroporous resin column: After loading, the macroporous resin column is first washed with pure water, and then eluted with an ethanol solution with a volume fraction of 40%, and the alcohol eluate is collected; The macroporous resin is AB-8; (4) The bitterless extract of Kuding tea is obtained by drying the alcohol eluate; The enzyme preparation is a composite enzyme preparation prepared by compounding cellulase, pectinase and plant hydrolysis composite enzyme at a mass ratio of 2:1:1; The enzymolysis conditions are that the enzyme preparation is added in an amount of 0.2%-0.3% of the dry sample of Kuding tea, the enzymolysis time is 40-60 min, the temperature is 45-50°C, and the pH value is 4.5-5.

5.

2. The extract of unprocessed Kuding tea according to claim 1, wherein the extract has an anti-fatigue effect. In step (1), The mass ratio of Kuding tea to water is 1:2-1:

3.

3. The extract of unprocessed Kuding tea according to claim 1, wherein the extract has an anti-fatigue effect. In step (2), The mass ratio of Kuding tea to water is 1:6-1:12; The extraction time is 1-3 hours; The filtration is 80-100 mesh filtration; The concentrated extract obtained by concentration has a specific gravity of 1.01-1.

08.

4. The non-bitter extract of leaves of Kuding tea according to claim 1, wherein, In step (3), The loading concentration is 3-10 mg / mL, and the loading volume is 1-3 column volumes; The pure water washing volume is 3-5 column volumes; The ethanol solution elution volume is 2-4 column volumes; The flow rate of loading, pure water washing and ethanol solution elution is 1.5 column volumes / h; The ratio of column height to diameter of the macroporous resin column is 1:6-1:

8.

5. The bitterless extract of Kuding tea with anti-fatigue efficacy according to claim 1, characterized in that, In step (4), The drying is spray drying or reduced-pressure drying; The spray drying parameters are: inlet air temperature 120°C, outlet air temperature 90°C, centrifugal frequency 300 Hz, feed pump 17 rpm, and air induction frequency 50 Hz.

6. A formulation for improving energy and anti-fatigue, characterized in that, The preparation comprises the bitterless extract of Kuding tea according to claim 1 and other acceptable excipients; and the dosage forms of the preparation include powder, granules, tablets, capsules, soft candies, oral solutions and other acceptable dosage forms.

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