Preparation method of Chinese olive polyphenol and application of Chinese olive polyphenol

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and discloses a preparation method of Chinese olive polyphenol and application of the Chinese olive polyphenol.The Chinese olive polyphenol is prepared by the steps that Chinese olives are washed, denucleated and then sequentially subjected to vacuum low-temperature drying, smashing and screening treatment, and Chinese olive powder is obtained; then extracting the Chinese olive components in an intermittent ultrasonic manner by taking ethanol as a solvent, centrifuging the extracted substances, performing reduced-pressure suction filtration, and freeze-drying the filtrate to obtain Chinese olive extract powder, namely Chinese olive polyphenol; according to the Chinese olive polyphenol effervescent tablet and the preparation method thereof, Chinese olive polyphenol is applied to development and research of drugs for resisting the chronic obstructive pulmonary disease, and a new idea of Chinese olive polyphenol in research and development of new drugs, food and health care products for resisting the chronic obstructive pulmonary disease is developed. Meanwhile, the Chinese olive polyphenol is prepared into the Chinese olive polyphenol effervescent tablet, the sweet aftertaste of Chinese olives is reserved while the active effect is reserved, the taste is good, and the development and utilization rate of Chinese olive resources is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and relates to a preparation method of Chinese olive polyphenols and the application of Chinese olive polyphenols. Background Art

[0002] After tobacco is burned, harmful substances in cigarette smoke will enter the human body through the respiratory system and cause a series of oxidative stress, inflammatory responses and immune changes, etc., resulting in damage to the respiratory system, digestive system, circulatory system and reproductive system, etc. Among them, chronic obstructive pulmonary disease (COPD), as a common chronic respiratory disease, is closely related to cigarette smoke exposure. COPD is characterized by a variety of clinical manifestations, mainly manifested in emphysema and chronic bronchitis, and has become a heavy medical burden worldwide. According to the statistics of the WHO in 2019, among all causes of death, the death caused by COPD ranks the 3rd, and the situation is very serious. At present, the drugs commonly used for the treatment of COPD include glucocorticoids, bronchodilators, theophylline and phosphodiesterase inhibitors, etc. These drugs often have relatively high side effects when used for a long time. Therefore, developing natural drugs with low toxicity, few side effects and high efficiency to prevent or treat COPD is the focus of current research.

[0003] Chinese olive, also known as olive (Chinese olive), white olive, is the mature and dried fruit of the olive plant Canarium album (Lour.) Raeusch of the Burseraceae family native to China. It is distributed in many provinces (autonomous regions) such as Fujian, Sichuan, Yunnan and Guangdong in China, and is one of the first batch of foods homologous between medicine and food announced in China. Chinese olive contains rich active substances such as vitamins, terpenoids and polyphenols. It is recorded in books such as Compendium of Materia Medica and Chinese Pharmacopoeia that it has the effects of clearing the throat and relieving sore throat, and dispelling alcohol and detoxifying. At present, domestic and foreign scholars' research on Chinese olive focuses on its antiviral activity and auxiliary hypoglycemic activity, etc., while the relevant research on the influence of Chinese olive on COPD is relatively lacking. In addition, there are few types of related products of Chinese olive on the market, mainly including tablets, granules and pills. These medicaments only retain the medicinal value of Chinese olive, but do not well take into account the edible value of Chinese olive as a food homologous between medicine and food. Therefore, further development of Chinese olive products also needs to conduct more in-depth research. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies in the prior art, and provide an application of Chinese olive polyphenols with strong antioxidant properties, which can inhibit smoke-induced oxidative stress and inflammation of lung epithelial cells, and a Chinese olive polyphenol effervescent tablet and its preparation method.

[0005] In order to achieve the above purpose, the present invention is realized through the following technical solutions: An application of Chinese olive polyphenols, wherein the Chinese olive polyphenols are used for preparing a drug or an effervescent tablet for treating COPD.

[0006] Further, the Chinese olive polyphenol is prepared by the following method:

[0007] Step (1): Wash the Chinese olives, remove the pits, and then successively perform vacuum low-temperature drying, pulverization, and screening to obtain Chinese olive powder.

[0008] Step (2): According to the material-liquid ratio of Chinese olive powder to ethanol solvent of 1:10 - 25 (g / ml), add the Chinese olive powder to the ethanol solvent, and extract the Chinese olive powder in the way of ultrasonic 1 s - intermittent 1 s. The ultrasonic power is 300 W - 500 W, the temperature is 30 - 50 °C, and the extraction time is 10 - 60 min. Centrifuge the extracted material and perform vacuum filtration under reduced pressure. The filtrate is freeze-dried to obtain Chinese olive extract powder, that is, Chinese olive polyphenol.

[0009] Further, in step (1), the temperature of the vacuum low-temperature drying is 40 °C.

[0010] Further, in step (1), the screening is through an 80-mesh sieve.

[0011] Further, in step (2), the ethanol has a volume concentration of 70% - 95%.

[0012] Further, the polyphenol content of the Chinese olive extract powder in step (2) is 110.51 mg / g.

[0013] Further, the Chinese olive polyphenol is used to prepare an effervescent tablet for treating chronic obstructive pulmonary disease. Among them, the effervescent tablet includes raw materials with the following weight ratios: 5 - 7.7 parts of Chinese olive polyphenol, 40 - 52 parts of disintegrant, 1 - 3 parts of sweetener, 10 - 40 parts of filler, and 1 - 3 parts of lubricant.

[0014] Further, the disintegrant is citric acid and sodium bicarbonate, and the mass ratio of citric acid to sodium bicarbonate is 1:1.4 - 1.5.

[0015] Further, the preparation method of the effervescent tablet is as follows: Weigh the raw materials according to the ratio, mix the Chinese olive polyphenol, disintegrant, sweetener, and filler evenly, add anhydrous ethanol to make soft material, sieve, granulate, dry, size, and then mix evenly with the lubricant, and press into tablets to obtain.

[0016] The present invention has the following beneficial effects: The present invention adopts the intermittent ultrasonic cell disruption-assisted extraction technology. The extraction method of "ultrasonic - intermittent - ultrasonic" is more conducive to the dissolution of polyphenol active ingredients. The obtained Chinese olive polyphenol has a good scavenging effect on free radicals such as ABTS+ and DPPH, and has a certain effect on Fe 3+It has excellent reduction effects, showing strong antioxidant activity. In addition, olive polyphenols significantly reduce the contents of MDA and NO in smoke-induced lung epithelial Beas-2B cells, weaken the oxidative stress and inflammatory responses of lung epithelial cells, indicating that olive polyphenols can be used as potential drugs for preventing or treating chronic obstructive pulmonary disease (COPD).

[0017] The present invention applies olive polyphenols to the development research of drugs for anti-COPD, opening up new ideas for the research and development of new drugs, foods and health products of olive polyphenols in the field of anti-COPD. At the same time, the present invention prepares the olive polyphenols into effervescent tablets of olive polyphenols, which are convenient to carry and can be instantaneously dissolved and drunk while retaining the active efficacy, reduce the astringency of olives while retaining the aftertaste of olives and have a good taste, improve the development and utilization rate of olive resources, and further enhance the medicinal value and edible value of olives as foods homologous to medicine and food.

[0018] In the preparation of the extracted effervescent tablets of olive polyphenols of the present invention, on the basis of the intermittent ultrasonic method, the content of olive polyphenols and the disintegrant are further optimized to obtain an appropriate ratio to obtain effervescent tablets of olive polyphenols with a higher sensory score, which not only ensures the activity of the effervescent tablets but also has a better taste; in addition, the present invention provides a preparation method of effervescent tablets of olive polyphenols, and this preparation method further ensures the taste. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the content diagram of the olive polyphenols extracted in Example 1 and Comparative Examples 1-3;

[0020] Figure 2 is the ABTS+ free radical scavenging ability diagram of the olive polyphenols extracted in Example 1 and Comparative Examples 1-3;

[0021] Figure 3 is the DPPH free radical scavenging ability diagram of the olive polyphenols extracted in Example 1 and Comparative Examples 1-3;

[0022] Figure 4 is the Fe 3+ reduction ability diagram of the olive polyphenols extracted in Example 1 and Comparative Examples 1-3;

[0023] Figure 5 is the total antioxidant diagram of the olive polyphenols extracted in Example 1 and Comparative Examples 1-3;

[0024] Figure 6 is the diagram of the influence of the olive polyphenols extracted in Example 1 and Comparative Examples 1-3 on the survival rate of Beas-2B cells;

[0025] Figure 7 is the diagram of the NO release of Beas-2B cells induced by cigarette smoke extract (CSE) by the olive polyphenols extracted in Example 1 and Comparative Examples 1-3;

[0026] Figure 8 It is a graph showing the release of MDA in Beas-2B cells induced by cigarette smoke extract (CSE) by the green olive polyphenols extracted from Example 1 and Comparative Examples 1-3;

[0027] Figure 9 It is a single-factor test graph of each factor in the preparation process of the green olive polyphenol effervescent tablets. Specific Embodiments

[0028] The present invention will be further described below in conjunction with embodiments, but it shall not be used as a basis for limiting the present invention.

[0029] I. Sources of Materials and Reagents

[0030] Gallic acid, ABTS, DPPH standards: Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity: 99%;

[0031] Folin-Ciocalteu reagent: Purchased from Shanghai Yuanye Bio-Technology Co., Ltd., AR grade;

[0032] Sodium nitrite, potassium ferrocyanide, trichloroacetic acid, potassium persulfate: Purchased from Shanghai Macklin Biochemical Co., Ltd., AR grade;

[0033] Green olive raw materials, purchased from the olive orchard in Minqing County, Fuzhou City, Fujian Province. The specific variety is Sandalwood olive, produced in Fuzhou, Fujian;

[0034] Ethanol: Purchased from Shanghai Titan Technology Co., Ltd., AR grade;

[0035] 1640 culture medium, PBS buffer: Purchased from Nanjing KeyGen Biotech Co., Ltd.;

[0036] Griess reagent, CKK8 kit, MDA detection kit: Purchased from Shanghai Beyotime Biotechnology Co., Ltd.;

[0037] Citric acid, sodium bicarbonate, stevioside, mannitol, PEG6000: Purchased from Tianjin Yongli Chemical Co., Ltd., food grade.

[0038] II. Instruments

[0039] Swing-type high-speed crusher model DFY-1000C, manufacturer Wenling Lind Machinery Co., Ltd.;

[0040] Ultrasonic cell crusher model JY92-II DN, manufacturer Ningbo Xinzhi Biotechnology Co., Ltd.;

[0041] Multifunctional microplate reader model Multiskan FC, manufacturer Thermo Fisher Scientific;

[0042] The powder tablet press model is NL-15, and the manufacturer is Tianjin Nuolei Xinda Technology Co., Ltd.

[0043] Example 1

[0044] This example provides a method for preparing olive polyphenols:

[0045] (1) Wash the olives, remove the pits, and then carry out vacuum drying at 40 °C, pulverization, and screening treatment in sequence to obtain olive powder.

[0046] (2) Using 20 ml of 70% ethanol as the solvent, perform intermittent ultrasonic cell disruption-assisted extraction on 1 g of olive powder ("ultrasonic for 1 s - intermittent for 1 s" cycle). The temperature of ultrasonic extraction is 45 °C, the ultrasonic time is 30 min, and the ultrasonic power is 400 W. Centrifuge the material after ultrasonic-assisted extraction and perform vacuum filtration under reduced pressure. The filtrate is freeze-dried to obtain olive extract powder (the polyphenol content is determined to be 110.51 mg / g by the Folin-Ciocalteu method), that is, olive polyphenols.

[0047] This example also provides an olive polyphenol effervescent tablet, which is prepared from the following raw materials in the following weight ratios: 7.7 parts of olive polyphenols, 2.7 parts of stevioside, 35.6 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:1.4.

[0048] This example also provides a method for preparing the olive polyphenol effervescent tablet, and its steps include: weighing the raw materials according to the ratio, taking the raw materials other than the lubricant, mixing them evenly, adding anhydrous ethanol to make soft materials, sieving, granulating, drying, sizing, and then mixing with the lubricant evenly. The amount of tablets pressed in each group is 20 tablets, and the weight of each tablet is 1 g. Press the tablets to obtain.

[0049] Example 2

[0050] The difference from Example 1 is: an olive polyphenol effervescent tablet, which is prepared from the following raw materials in the following weight ratios: 5 parts of olive polyphenols, 2.7 parts of stevioside, 38.3 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:1.4.

[0051] Example 3

[0052] The difference from Example 1 is: an olive polyphenol effervescent tablet, which is prepared from the following raw materials in the following weight ratios: 7.5 parts of olive polyphenols, 2.7 parts of stevioside, 35.8 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:1.4.

[0053] Comparative Example 1

[0054] The operation steps of Comparative Example 1 are different from those of Example 1 only in that: the extraction method of the olive polyphenols is direct ultrasonic extraction, and the remaining parameters and operations are exactly the same. The detected polyphenol content and the evaluation of the anti-chronic obstructive pulmonary disease activity are as Figures 1-8 shown.

[0055] Comparative Example 2

[0056] The operation steps of Comparative Example 2 are different from those of Example 1 only in that: the extraction method of the olive polyphenols is intermittent ultrasonic cell disruption-assisted extraction ("ultrasonic for 0.5 s - intermittent for 0.5 s" cycle), and the remaining parameters and operations are exactly the same. The detected polyphenol content and the evaluation of the anti-chronic obstructive pulmonary disease activity are as Figures 1-8 shown.

[0057] Comparative Example 3

[0058] The operation steps of Comparative Example 3 are different from those of Example 1 only in that: the extraction method of the olive polyphenols is intermittent ultrasonic cell disruption-assisted extraction ("ultrasonic for 1.5 s - intermittent for 1.5 s" cycle), and the remaining parameters and operations are exactly the same. The detected polyphenol content and the evaluation of the anti-chronic obstructive pulmonary disease activity are as Figures 1-8 shown.

[0059] Comparative Example 4

[0060] The difference from Example 1 is that: direct powder compression: the olive polyphenols, citric acid, sodium bicarbonate, stevioside, mannitol, and PEG6000 powders are mixed evenly in a certain proportion and directly compressed into tablets, and the remaining parameters and operations are exactly the same.

[0061] Comparative Example 5

[0062] The difference from Example 1 is that: granulation and compression of acid and base separately: citric acid, olive polyphenols, mannitol, and stevioside are mixed evenly, and anhydrous ethanol is added to make a soft material. The soft material should be such that it can be kneaded into a ball and dispersed when pinched. The soft material is sieved through a 20-mesh sieve to granulate, dried, and then sized; separately, sodium bicarbonate is mixed evenly with olive polyphenols, mannitol, and stevioside, and the above operations are repeated. A certain proportion of acid granules and base granules are mixed evenly with PEG6000 powder and compressed into tablets, and the remaining parameters and operations are exactly the same.

[0063] Comparative Example 6

[0064] The operation steps of Comparative Example 6 are different from those of Example 1 only in that: the effervescent tablets of olive polyphenols are prepared from the following raw materials in the following weight ratios: 10 parts of olive polyphenols, 2.7 parts of stevioside, 33.3 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:1.4. The remaining parameters and operations are exactly the same.

[0065] Comparative Example 7

[0066] The difference in the operation steps between Comparative Example 7 and Example 1 is only that: the olive polyphenol effervescent tablets are prepared from the following raw materials in the following weight ratios: 12.5 parts of olive polyphenols, 2.7 parts of stevioside, 30.8 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:1.4. The remaining parameters and operations are exactly the same.

[0067] Comparative Example 8

[0068] The difference in the operation steps between Comparative Example 8 and Example 1 is only that: the olive polyphenol effervescent tablets are prepared from the following raw materials in the following weight ratios: 15 parts of olive polyphenols, 2.7 parts of stevioside, 28.3 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:1.4. The remaining parameters and operations are exactly the same.

[0069] Comparative Example 9

[0070] The difference in the operation steps between Comparative Example 9 and Example 1 is only that: the olive polyphenol effervescent tablets are prepared from the following raw materials in the following weight ratios: 7.7 parts of olive polyphenols, 2.7 parts of stevioside, 35.6 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 2:1. The remaining parameters and operations are exactly the same.

[0071] Comparative Example 10

[0072] The difference in the operation steps between Comparative Example 10 and Example 1 is only that: the olive polyphenol effervescent tablets are prepared from the following raw materials in the following weight ratios: 7.7 parts of olive polyphenols, 2.7 parts of stevioside, 35.6 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1.5:1. The remaining parameters and operations are exactly the same.

[0073] Comparative Example 11

[0074] The difference in the operation steps between Comparative Example 11 and Example 1 is only that: the olive polyphenol effervescent tablets are prepared from the following raw materials in the following weight ratios: 7.7 parts of olive polyphenols, 2.7 parts of stevioside, 35.6 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:1. The remaining parameters and operations are exactly the same.

[0075] Comparative Example 12

[0076] The operating steps of Comparative Example 12 are different from those of Example 1 only in that: the olive polyphenol effervescent tablets are prepared from the following raw materials in the following weight ratios: 7.7 parts of olive polyphenol, 2.7 parts of stevioside, 35.6 parts of mannitol, 2 parts of PEG6000, and 52 parts of citric acid and sodium bicarbonate with a mass ratio of 1:2. The remaining parameters and operations are exactly the same.

[0077] 1. Determination of olive polyphenol content and verification of biological activity test

[0078] The olive polyphenols prepared in Example 1 and Comparative Examples 1 to 3 were respectively subjected to the following tests to prove the biological activity of the olive polyphenols provided by the present invention.

[0079] 1.1 Determination of olive polyphenol content

[0080] Accurately weigh the gallic acid standard product, and use anhydrous ethanol to prepare gallic acid standard solutions with different concentrations. Take 0.2 mL of the gallic acid standard solution, add 1 mL of distilled water and 0.2 mL of Folin-Ciocalteu reagent in sequence, mix well and let stand at room temperature for 3 min, then add 0.6 mL of 7.5% Na2CO3 solution, mix well and let stand for 40 min, and measure its absorbance at a wavelength of 765 nm. Use the concentration of the gallic acid standard solution as the abscissa and the absorbance value as the ordinate to establish a gallic acid standard curve.

[0081] The sample to be tested is prepared into a test sample solution of 1 mg / mL with anhydrous ethanol, and is measured according to the above method. The total phenol concentration in the sample solution is obtained by substituting the absorbance into the standard curve, and the total polyphenol content of the sample is calculated according to the following formula: P = C × V × N / M

[0082] In the formula, P—the total polyphenol content of the sample to be tested (calculated as gallic acid equivalent), mg / g; C—the total polyphenol concentration of the extract sample solution obtained by substituting into the standard curve, mg / ml; V—the volume of the extract sample solution, ml; N—dilution factor; M—the mass of the sample to be tested, g.

[0083] The results are as Figure 1As shown in the figure. The total phenol content of Example 1 was the highest, reaching 110.51 mg / g, while that of Comparative Example 1 was the lowest, indicating that the ultrasonic method had a significant impact on the extraction rate of olive polyphenols. Among them, the total phenol content obtained by intermittent ultrasonic cell disruption-assisted extraction (Example 1, Comparative Example 2, and Comparative Example 3) was higher than that obtained by direct ultrasonic extraction (p < 0.05). Compared with Comparative Example 2 ("ultrasonic 0.5 s - intermittent 0.5 s" cycle), the extraction rate of olive polyphenols in Example 1 ("ultrasonic 1 s - intermittent 1 s" cycle) was significantly improved (p < 0.05), indicating that appropriately extending the ultrasonic and intermittent time helped to fully exert the cavitation effect of ultrasonic waves. However, when the ultrasonic and intermittent time was further extended (Comparative Example 3 - "ultrasonic 1.5 s - intermittent 1.5 s" cycle), the extraction rate decreased again. Therefore, the intermittent ultrasonic cell disruption-assisted extraction method with a "ultrasonic 1 s - intermittent 1 s" cycle was finally used to extract olive polyphenols. At this time, the content of olive polyphenols obtained by extraction was the highest. This extraction method could not only alleviate the overheating of the instrument caused by continuous ultrasound and prevent the appearance of a hollow phenomenon in the solution, but also, the appropriate ultrasonic working time could fully exert the cavitation effect of ultrasonic waves and significantly improve the extraction rate of olive polyphenols.

[0084] 1.2 Determination of the scavenging ability of olive polyphenols against ABTS+ free radicals

[0085] After mixing 2.45 mmol / L potassium persulfate and 7.0 mmol / L ABTS solution in a volume ratio of 1:1 evenly, it was placed in the dark at room temperature overnight for 12 - 16 h. Before use, it was diluted with absolute ethanol to an absorbance value of 0.7 ± 0.02 at a wavelength of 734 nm, which was the ABTS working solution. The samples to be tested were dissolved and diluted into different concentrations of test solutions (1000, 500, 200, 100, 50, 25, 12.5, 6.25 μg / mL). 50 μL of the test solution was taken and mixed evenly with 150 μL of the ABTS working solution, reacted for 30 min under dark conditions, and its absorbance value was measured at a wavelength of 734 nm. Methanol was used instead of the sample solution as the blank, and VC was used instead of the sample solution as the positive control. After parallel determination three times for each concentration, the average value was taken, and the scavenging rate of the sample against ABTS+ free radicals was calculated according to the following formula:

[0086] Scavenging rate of ABTS+ free radicals (%) = (A0 - A1) / A0 × 100%, where A0 is the absorbance of the blank; A1 is the absorbance after the reaction of the sample (or VC) with the ABTS working solution.

[0087] The results are as Figure 2 shown, IC 50The lower the value, the stronger the free radical scavenging ability. Example 1 has the strongest ABTS+ free radical scavenging ability, while Comparative Example 1 has the weakest free radical scavenging ability, which also corresponds to the polyphenol content it contains. The higher content of olive polyphenols in Example 1 shows stronger ABTS+ free radical scavenging activity.

[0088] 1.3 Determination of the scavenging ability of olive polyphenols against DPPH free radicals

[0089] Dissolve the sample to be tested and dilute it into test solutions with different concentrations (1000, 500, 200, 100, 50, 25, 12.5, 6.25 μg / mL). Pipette 100 μL of the test solution and add it to 100 μL of 0.2 mmol / L DPPH solution, then mix well. Let it stand in the dark at room temperature for 30 min, and measure the absorbance at a wavelength of 517 nm. Use methanol instead of the sample solution as the blank, and use VC instead of the sample solution as the positive control. After parallel determination three times for each concentration, take the average value. Calculate the scavenging rate of the sample against DPPH free radicals according to the following formula:

[0090] Scavenging rate of DPPH free radicals (%) = (A0 - A1) / A0 × 100%, where A0 is the absorbance of the blank; A1 is the absorbance after the reaction of the sample (or VC) with the DPPH solution.

[0091] The results are as Figure 3 shown. Similar to the ABTS+ free radical scavenging activity, Example 1 has the strongest DPPH free radical scavenging ability, while Comparative Example 1 has the weakest DPPH free radical scavenging ability. The higher content of olive polyphenols in Example 1 also shows stronger DPPH free radical scavenging activity.

[0092] 1.4 Determination of the reducing ability of olive polyphenols against Fe 3+

[0093] Take 200 μL of test solutions with different concentrations (500, 200, 100, 50, 25, 12.5, 6.25 μg / mL), and successively add 500 μL of PBS buffer solution (0.2 M, pH 6.6), 500 μL of 1% potassium ferricyanide solution. Mix well and place it in a water bath at 50 °C for 30 min. After taking it out, quickly cool it. Then add 500 μL of 10% trichloroacetic acid, let it stand for 10 min, take 500 μL of the supernatant, add 500 μL of distilled water and 100 μL of 0.1% ferric chloride solution, mix evenly and let it stand for 10 min, and measure the absorbance at a wavelength of 700 nm. Parallel determination is carried out three times for each concentration.

[0094] The results are as Figure 4 ​As shown. As the concentration increases, the absorbance of each sample also increases. According to the slope of the straight line, the reducing ability of the sample can be seen. The slope of Example 1 is significantly higher than that of Comparative Examples 1-3, showing the strongest Fe 3+ reducing ability. Fe 3+ The order of the strength of the Fe reducing ability is: Example 1 > Comparative Example 3 > Comparative Example 1 > Comparative Example 2.

[0095] 1.5 Determination of the total antioxidant capacity of green olive polyphenols

[0096] Take 200 μL of test sample solutions with different concentrations (500, 200, 100, 50, 25, 12.5, 6.25 μg / mL), and then add 200 μL of 0.6 mol / L concentrated sulfuric acid, 28 mmol / L ammonium molybdate, and 4 mmol / L sodium phosphate respectively. After mixing, place it in a water bath at 95 °C for 90 min. After cooling, measure the absorbance at 695 nm, and measure each concentration in parallel three times.

[0097] The results are as Figure 5 shown. As the concentration increases, the absorbance of each sample also increases. The absorbance of Example 1 at each concentration is significantly higher than that of Comparative Examples 1-3. The order of the strength of the total antioxidant capacity is: Example 1 > Comparative Example 2 > Comparative Example 3 > Comparative Example 1.

[0098] 1.6 Determination of the effect of green olive polyphenols on the release of NO from lung epithelial cells Beas-2B induced by cigarette smoke extract

[0099] Draw a NO standard curve using NaNO2 as the standard product. Inoculate Beas-2B cells in the logarithmic growth phase into a 96-well plate (1×104 cells / well). After culturing for 24 h, discard the culture medium. Respectively set up a blank control group, a cigarette smoke extract CSE model group (treated with 4% CSE), and a sample treatment group (4% CSE + 100 μg / mL sample). Continue to culture for 24 h. Take 100 μL of the supernatant into a 96-well plate, mix it with 100 μL of the prepared Griess reagent (Griess A reagent: Griess B reagent = 1:1), react for 10 min, measure the absorbance at 540 nm, and substitute it into the standard curve to calculate the NO concentration. Among them, the final concentration of the added green olive polyphenols is 100 μg / mL. CCK8 is used to measure the cell survival rate: Treat Beas-2B cells with different samples (final concentration 100 μg / mL). After incubating for 24 h, measure and calculate the survival rate according to the operation instructions of the CCK-8 kit.

[0100] The smoke generated by cigarette combustion contains various harmful components such as nicotine, coal tar, and benzo[a]pyrene, which can stimulate Beas-2B human lung epithelial cells to produce inflammation. After being stimulated by the smoke, the cells will produce oxidative stress and secrete a large amount of inflammatory mediators and cytokines, including NO, prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α), etc. The results of the effects of Example 1 and Comparative Examples 1-3 on the survival rate of Beas-2B cells and the release amount of NO induced by cigarette smoke extract (CSE) are as Figures 6-7 shown. Compared with the control group, the content of NO in the CSE model group was significantly increased (p < 0.05), indicating that the model was successfully constructed. After sample treatment, Example 1 and Comparative Examples 1-3 both significantly reduced the content of NO (p < 0.05). Among them, the release amount of NO in Example 1 was the lowest and the effect was the most significant. The CCK8 results showed that at the experimental concentration (100 μg / mL), Example 1 and Comparative Examples 1-3 had no obvious cytotoxicity (p > 0.05), that is, olive polyphenols could significantly inhibit the release of NO without affecting the survival rate of Beas-2B cells, which indicated that olive polyphenols had strong anti-inflammatory activity and could relieve the inflammatory reaction of chronic obstructive pulmonary disease caused by smoke.

[0101] 1.7 Determination of the effect of olive polyphenols on the release of MDA in Beas-2B cells of lung epithelial cells induced by cigarette smoke extract

[0102] A standard curve was drawn with MDA as the standard product. Beas-2B cells in the logarithmic growth phase were seeded in 96-well plates (1×104 cells / well). After culturing for 24 h, the culture medium was discarded. The blank control group, the cigarette smoke extract CSE model group (treated with 4% CSE), and the sample treatment group (4% CSE + 100 μg / mL sample) were set up respectively, and then cultured for another 24 h. The supernatant was discarded and the protein was extracted. The content of MDA was determined according to the operation instructions of the MDA detection kit of Beyotime.

[0103] Beas-2B cells will produce oxidative stress after being stimulated by cigarette smoke. Among them, MDA is an aldehyde lipid peroxidation product produced by cells under external stimulation and is an important marker of cell oxidative stress injury. The results of the effects of Example 1 and Comparative Examples 1-3 on the MDA content of Beas-2B cells induced by cigarette smoke extract (CSE) are as Figure 8 shown. Example 1 significantly reduced the MDA content in Beas-2B cells stimulated by CSE (p < 0.05), while there was no significant difference in the MDA content between Comparative Examples 1-3 and the model group (p > 0.05), indicating that the olive polyphenols in Example 1 had a certain alleviating and protective effect on the oxidative stress injury of lung epithelial cells caused by cigarette smoke.

[0104] 2. Effervescent tablets of olive polyphenols and its preparation method

[0105] 2.1 Investigation of tableting process

[0106] The green fruit polyphenol effervescent tablets of comparative example 4, which were directly compressed by powder, were easy to stick to the pulp and had serious moisture absorption. Although the disintegration time was relatively short, the comprehensive sensory score was low; the effervescent tablets of comparative example 5, which were granulated and compressed by acid and alkali separately, had a complete surface, but large spots and different colors and gloss; the effervescent tablets of embodiment 1, which were granulated and compressed by acid and alkali mixing, had a complete and smooth surface, were not sticky, had small spots, and had a short disintegration time, and had the highest comprehensive sensory score.

[0107] 2.2 Verification of the activity of green fruit polyphenol effervescent tablets

[0108] The green fruit polyphenol effervescent tablets prepared in Example 1 and Comparative Examples 1 to 12 were subjected to ABTS+ free radical and DPPH free radical scavenging ability determination, Fe3+ reducing ability and total antioxidant capacity determination tests to prove the biological activity of the green fruit polyphenol effervescent tablets prepared by the present invention. The test methods are the same as the methods described in 1.1, 1.2, 1.3 and 1.4 above. In addition, the green fruit polyphenol effervescent tablets prepared in Example 1 and Comparative Examples 1 to 12 were subjected to comprehensive sensory evaluation, and the sensory scoring standards were the same as Tables 1 and 2. The results are shown in Table 3. The different ultrasonic methods in Comparative Examples 1-3 affected the extraction rate and content of green fruit polyphenols, and also affected the activity of the effervescent tablets. In comparison, Example 1 has the strongest antioxidant capacity, and the ultrasonic method of "ultrasound 1s-intermittent 1s" cycle is more sufficient for the extraction of green fruit polyphenols, and also retains the special flavor of green fruit to a large extent, and its comprehensive sensory score is also the highest. In Comparative Examples 6-8, as the main bioactive ingredient in the effervescent tablet, the amount of green fruit polyphenol added is increased successively, and its antioxidant activity is also increased accordingly. However, the excessive amount of green fruit polyphenol added makes the sourness and astringency of green fruit polyphenol gradually prominent, and the soup color after the effervescent tablet disintegrates is gradually turbid, and the comprehensive sensory score shows a downward trend, and the taste is not good. Comparative Example 9 increases the proportion of citric acid in the acid-base ratio. Compared with Example 1, although citric acid can synergistically contribute to a certain antioxidant effect, it significantly increases the sourness of the effervescent tablet, because there are a large amount of phenolic acid in green fruit polyphenol, increasing the content of citric acid will cause the effervescent tablet to have a heavier overall sour taste and uncomfortable taste. In Comparative Example 10, the proportion of citric acid in the acid-base ratio is reduced, and the antioxidant activity is improved to a certain extent, and the sour taste is improved, but the overall taste is still not good. Comparative Examples 11-12 further reduced the proportion of citric acid in the acid-base ratio, and the sour taste was significantly improved, but the citric acid ratio was low and the acid-base ratio was not coordinated, resulting in a prolonged disintegration time of the effervescent tablet, a small amount of foaming and insufficient disintegration, and a relatively low comprehensive sensory score. In summary, based on the comprehensive biological activity and sensory scores, the effervescent tablet of Example 1 not only showed strong antioxidant activity, but also had a better taste, while retaining the active efficacy, it also reduced the sour and astringent feeling of the green fruit, and retained the sweetness of the green fruit.

[0109] Table 1 Statistical Table of Weights of Sensory Evaluation Factors

[0110]

[0111] Table 2 Comprehensive Sensory Evaluation Criteria for Olive Fruit Polyphenol Effervescent Tablets

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[0113] Table 3 Results of Activity and Comprehensive Sensory Scores of Olive Fruit Polyphenol Effervescent Tablets

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[0115]

[0116] The above has shown and described the basic principles, main features and advantages of the present invention. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. An application of green fruit polyphenols, characterized in that: The green fruit polyphenols are used for preparing medicines or effervescent tablets for treating chronic obstructive pulmonary disease.

2. The use of green fruit polyphenols according to claim 1, characterized in that: The green fruit polyphenols are prepared by the following method: Step (1), washing the green fruit, removing the core, and then vacuum low-temperature drying, crushing, and screening to obtain green fruit powder; Step (2), according to the solid-liquid ratio of green fruit powder to ethanol solvent of 1:10-25, the green fruit powder is added to the ethanol solvent, and the green fruit powder is extracted by ultrasonic 1s-intermittent 1s, the ultrasonic power is 300W-500W, the temperature is 30-50°C, the extraction time is 10-60min, the extracted material is centrifuged and filtered under reduced pressure, and the filtrate is freeze-dried to obtain green fruit extract powder, i.e., green fruit polyphenols.

3. The use of green fruit polyphenols according to claim 1, characterized in that: The temperature of vacuum low-temperature drying in step (1) is 40°C.

4. The use of green fruit polyphenols according to claim 1, characterized in that: The sieving in step (1) is through an 80-mesh sieve.

5. The use of green fruit polyphenols according to claim 1, characterized in that: In step (2), the volume concentration of ethanol is 70%-95%.

6. The use of green fruit polyphenols according to claim 1, characterized in that: The polyphenol content of the green fruit extract powder in step (2) is 110.51 mg / g.

7. The use of green fruit polyphenols according to claim 1, characterized in that: The green fruit polyphenols are used to prepare effervescent tablets for treating chronic obstructive pulmonary disease, wherein the effervescent tablets include the following raw materials in weight ratio: 5 to 7.7 parts of green fruit polyphenols, 40 to 52 parts of disintegrants, 1 to 3 parts of sweeteners, 10 to 40 parts of fillers, and 1 to 3 parts of lubricants.

8. The use of green fruit polyphenols according to claim 8, characterized in that: The disintegrants are citric acid and sodium bicarbonate, and the mass ratio of citric acid to sodium bicarbonate is 1:1.4-1.

5.

9. The use of green fruit polyphenols according to claim 7, characterized in that: The preparation method of the effervescent tablet is as follows: weigh the raw materials according to the ratio, mix the green fruit polyphenols, disintegrant, sweetener and filler, add anhydrous ethanol to make a soft material, sieve, granulate, dry, granulate, mix with lubricant, and press into tablets.

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