Preparation technology of spiny jujube seed preparation, application thereof and immune activator

Abstract

The present invention provides a preparation process of a Chinese jujube seed preparation, its application and an immune activator, and belongs to the field of biomedical technology. The preparation process provided by the present invention comprises the following steps: S1, the clean Chinese jujube seed is steamed and dried, and then taken out after vacuum drying to obtain dried Chinese jujube seed; S2, the dried Chinese jujube seed is extracted twice by water decoction, and the filtrate is filtered to obtain the filtrate; S3, the filtrate is concentrated, centrifuged, and the Chinese jujube seed preparation is obtained after sterilization. The vacuum drying comprises: drying at 45°C for 2h in the first stage, drying at 50°C for 1h in the second stage, drying at 55°C for 1h in the third stage, and drying at 60°C for 1h in the fourth stage. The Chinese jujube seed preparation prepared by the preparation process provided by the present invention can further activate macrophages and enhance the immune regulation ability. The present invention can provide an experimental basis for improving the quality of Chinese jujube seed medicinal materials, provide a scientific basis for the development and utilization of Chinese jujube seed medicinal materials resources, and has important significance for the wider clinical application prospects of Chinese jujube seed.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a preparation process of a spiny jujube seed preparation and an application thereof and an immune activator. Background Art

[0002] Ziziphus jujuba seed is a traditional Chinese medicine, which is recorded in Shennong's Herbal Classic and in the medical book Treatise on Febrile and Miscellaneous Diseases. It is the dried mature seed of Ziziphus jujuba, a plant of the Rhamnaceae family, and is a dual-use medicine and food. The main production areas are in Hebei, Henan, Shaanxi, Inner Mongolia and other places. The appearance is purple-red or purple-brown, and the shape is oblate or oblate, with a smooth and shiny surface. It is mainly used to treat symptoms such as palpitations, dreaminess, physical weakness and sweating, insomnia, and sweating and body fluid. It is the first choice of tranquilizer in Chinese clinical practice. Ziziphus jujuba seed contains saponins, triterpenes, flavonoids, alkaloids, fatty acids and polysaccharides. Ziziphus jujuba seed has multiple biological activities and has a certain effect on immune activation, but the ability of Ziziphus jujuba seed-related preparations under the existing preparation process to regulate the immune system is limited.

[0003] Drying is the most common and critical processing link in the production process of Chinese herbal medicines. The drying method and drying process parameters have a very important influence on the quality of Chinese herbal medicines. Different drying methods have a significant impact on the production efficiency and active ingredient content of medicinal materials. Drying is an important part of the processing of Chinese jujube seeds and has an important impact on the quality of Chinese jujube seeds. At present, traditional drying methods such as sun drying and shade drying are often used in the processing of Chinese jujube seeds. However, methods such as hot air drying, vacuum freeze drying, and vacuum drying have shown certain advantages in drying efficiency, time consumption, and ensuring the quality of dried samples. In addition, vacuum drying can isolate the dried sample from oxygen to a certain extent, usually has a higher drying rate, and can obtain dried samples with better quality. Using vacuum drying of Chinese medicine can greatly improve the quality of Chinese medicine and the content and efficiency of active ingredients. In the vacuum drying state, moisture can evaporate at low temperature, which can avoid the denaturation of active ingredients extracted by high temperature, effectively evaporate the moisture in the medicinal materials, not only retain the active ingredients, but also reduce the corruption of medicinal materials and the growth of microorganisms, and improve the shelf life. Vacuum drying can not only improve the drying efficiency, but also prevent the active ingredients of medicinal materials from being destroyed. Experimental studies have shown that using vacuum drying technology to dry jujube seeds can retain more amino acids, fatty acids and carbohydrates.

[0004] Therefore, based on the traditional preparation process, the present invention uses vacuum drying technology to optimize it, and explores the effects of different preparation processes of Chinese jujube seeds on the immune mechanism of macrophages, providing a scientific basis for the development and utilization of Chinese jujube seeds medicinal resources, and at the same time providing clues for finding immunomodulatory drugs, which is of great significance to the broader clinical application prospects of Chinese jujube seeds. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a preparation process of a Chinese jujube seed preparation, its application and an immune activator. The preparation process provided by the present invention comprises the following steps: S1, the clean Chinese jujube seed is steamed and dried, and then taken out after vacuum drying to obtain dried Chinese jujube seed; S2, the dried Chinese jujube seed is extracted twice by water decoction, and the filtrate is filtered to obtain the filtrate; S3, the filtrate is concentrated, centrifuged, and the Chinese jujube seed preparation is obtained after sterilization. The vacuum drying includes: drying at 45°C for 2h in the first stage, drying at 50°C for 1h in the second stage, drying at 55°C for 1h in the third stage, and drying at 60°C for 1h in the fourth stage. Compared with the traditional Chinese jujube seed preparation process, the Chinese jujube seed preparation prepared by the preparation process provided by the present invention can further activate macrophages and enhance the immune regulation ability. The present invention can provide an experimental basis for improving the quality of Chinese jujube seed medicinal materials, provide a scientific basis for the development and utilization of Chinese jujube seed medicinal materials resources, and has important significance for the wider clinical application prospects of Chinese jujube seed.

[0006] the term:

[0007] The temperature of the "high heat" described in the present invention is: 100°C / 500W (heater power).

[0008] The temperature of the "slow fire" described in the present invention is: 100°C / 250W (heater power).

[0009] The temperature of "warm fire" described in the present invention is: 40-50℃.

[0010] The technical solution of the present invention includes:

[0011] In one aspect, the present invention provides a preparation process of a spiny jujube seed preparation, the preparation process comprising the following steps:

[0012] S1. Steam and dry the purified Chinese jujube seeds, vacuum dry them, and then take them out to obtain dried Chinese jujube seeds;

[0013] S2, extracting dried Chinese date seeds twice by decoction, and filtering to obtain a filtrate;

[0014] S3. The filtrate is concentrated, centrifuged, and sterilized to obtain the spiny jujube seed preparation.

[0015] The vacuum drying in step S1 includes: drying at 45° C. for 2 h in the first stage, drying at 50° C. for 1 h in the second stage, drying at 55° C. for 1 h in the third stage, and drying at 60° C. for 1 h in the fourth stage.

[0016] Specifically, the steaming in step S1 includes steaming at 37-60° C. for 30 min.

[0017] Preferably, the steaming in step S1 includes: steaming at 37°C for 30 min; or steaming at 37°C for 10 min in the first stage, steaming at 45°C for 10 min in the second stage, and steaming at 50°C for 10 min in the third stage; or steaming at 50°C for 10 min in the first stage, steaming at 55°C for 10 min in the second stage, and steaming at 60°C for 10 min in the third stage.

[0018] Further preferably, the steaming in step S1 comprises: a first stage of steaming at 37° C. for 10 min, a second stage of steaming at 45° C. for 10 min, and a third stage of steaming at 50° C. for 10 min.

[0019] Specifically, the vacuum degree of the vacuum drying in step S1 is -0.2 to -0.05 MPa.

[0020] Preferably, the vacuum degree of the vacuum drying in step S1 is -0.1 MPa.

[0021] Specifically, the decoction extraction twice described in step S2 comprises:

[0022] (1) Soak dried Chinese jujube seeds in water, decoct, collect and filter the liquid to obtain filtrate 1;

[0023] (2) Add water to the residue and boil it twice, collect the liquid and filter it to obtain filtrate 2;

[0024] (3) Combine filtrate 1 and filtrate 2 to obtain filtrate.

[0025] More specifically, the weight ratio of the dried spiny jujube kernels to water in step S2 (1) is 1:10-15.

[0026] Preferably, the weight ratio of the dried spiny jujube kernels to water in step S2 (1) is 1:10.

[0027] More specifically, the soaking time in step S2 (1) is 1-3 hours.

[0028] Preferably, the soaking time in step S2 (1) is 1 hour.

[0029] Preferably, the decocting in step S2 (1) comprises: boiling over high heat and then continuing to simmer over low heat for 30 minutes.

[0030] Preferably, the filtration in step S2 (1) is performed by filtering while hot using three layers of filter cloth.

[0031] More specifically, the weight ratio of the medicinal residue to water in step S2 (2) is 1:6-10.

[0032] Preferably, the weight ratio of the medicinal residue to water in step S2 (2) is 1:8.

[0033] Preferably, the decocting in step S2 (2) comprises: boiling over high heat and then continuing to simmer over low heat for 30 minutes.

[0034] Preferably, the filtration in step S2 (2) is performed by filtering while hot using three layers of filter cloth.

[0035] Specifically, the concentration described in step S3 is to concentrate the filtrate to a concentration of 0.5-2 g / mL in terms of crude drug amount.

[0036] Preferably, the concentration in step S3 is to concentrate the filtrate to a crude drug concentration of 1 g / mL.

[0037] Specifically, the concentration described in step S3 is concentrated by warm fire.

[0038] Specifically, the centrifugation in step S3 is performed at 12000 r / min for 10 min.

[0039] On the other hand, the present invention provides a spinach seed preparation prepared by the above preparation process.

[0040] In another aspect, the present invention provides the use of the above-mentioned Semen Ziziphi Spinosae preparation in the preparation of anti-tumor drugs.

[0041] Specifically, the tumor includes, but is not limited to, a digestive system tumor, a respiratory system tumor, a reproductive system tumor, a musculoskeletal system tumor, a nervous system tumor, an endocrine system tumor, a circulatory system tumor, a urinary system tumor or a reproductive system tumor.

[0042] Specifically, the Chinese date seed preparation achieves anti-tumor efficacy by promoting the polarization of macrophages to the M1 type, releasing pro-inflammatory factors, activating macrophages and enhancing immune regulation ability.

[0043] Preferably, the pro-inflammatory factors include COX-2, iNOS, TNF-α and / or IL-1β.

[0044] In another aspect, the present invention provides an immunostimulator, comprising the above-mentioned spinach seed preparation.

[0045] The technical effects achieved by the present invention are:

[0046] Compared with the traditional process, the Chinese date seed preparation prepared by the preparation process provided by the present invention can further promote the polarization of macrophages to M1 type, release pro-inflammatory factors COX-2, iNOS, TNF-α, IL-1β, activate macrophages and enhance immune regulation ability, thereby achieving anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1is the mRNA level of iNOS gene.

[0048] Figure 2 is the mRNA level of TNF-α gene.

[0049] Figure 3 is the mRNA level of COX-2 gene.

[0050] Figure 4 is the mRNA level of IL-1β gene. DETAILED DESCRIPTION

[0051] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0052] Example 1 New preparation process of spiny jujube seeds

[0053] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming. Steam them at 37°C, 45°C and 50°C for 10 minutes each. After steaming, dry them naturally at room temperature for 1 to 3 hours. After cooling, take them out and spread the Chinese jujube kernels flat on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows, with a total of four stages. The first stage is dried at 45°C for 2 hours, the second stage is dried at 50°C for 1 hour, the third stage is dried at 55°C for 1 hour, and the fourth stage is dried at 60°C for 1 hour. After completing the above drying continuously, take them out.

[0054] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0055] Example 2 New preparation process of spiny jujube seeds

[0056] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming at 37°C for 30 minutes. After steaming, dry them naturally at room temperature for 1 to 3 hours. After cooling, take them out and spread the Chinese jujube kernels flat on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows, with a total of four stages. The first stage is drying at 45°C for 2 hours, the second stage is drying at 50°C for 1 hour, the third stage is drying at 55°C for 1 hour, and the fourth stage is drying at 60°C for 1 hour. After completing the above drying continuously, take them out.

[0057] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0058] Example 3 New preparation process of spiny jujube seeds

[0059] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming. Steam them at 50℃, 55℃ and 60℃ for 10 minutes each. After steaming, dry them naturally at room temperature for 1h to 3h. After cooling, take them out and spread the Chinese jujube kernels on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows, with a total of four stages. The first stage is dried at 45℃ for 2h, the second stage is dried at 50℃ for 1h, the third stage is dried at 55℃ for 1h, and the fourth stage is dried at 60℃ for 1h. After completing the above drying continuously, take them out.

[0060] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0061] Comparative Example 1 Traditional preparation process of spiny jujube seeds

[0062] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0063] Comparative Example 2

[0064] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming. Steam them at 37°C, 45°C and 50°C for 10 minutes each. After steaming, dry them naturally at room temperature for 1 to 3 hours. After cooling, take them out and spread the Chinese jujube kernels on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows, with a total of four stages. The first stage is dried at 45°C for 1 hour, the second stage is dried at 50°C for 0.5 hour, the third stage is dried at 55°C for 0.5 hour, and the fourth stage is dried at 60°C for 0.5 hour. After completing the above drying continuously, take them out.

[0065] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0066] Comparative Example 3

[0067] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming. Steam them at 37°C, 45°C and 50°C for 10 minutes each. After steaming, dry them naturally at room temperature for 1 to 3 hours. After cooling, take them out and spread the Chinese jujube kernels flat on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows: dry continuously at 45°C for 5 hours. Take them out after completing the above drying.

[0068] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0069] Comparative Example 4

[0070] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming. Steam them at 37°C, 45°C and 50°C for 10 minutes each. After steaming, dry them naturally at room temperature for 1 to 3 hours. After cooling, take them out and spread the Chinese jujube kernels on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows, with a total of four stages. The first stage is dried at 45°C for 2 hours, the second stage is dried at 50°C for 1 hour, the third stage is dried at 55°C for 1 hour, and the sixth stage is dried at 60°C for 1 hour. After completing the above drying continuously, take them out.

[0071] The test solution was prepared by traditional decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil over high heat and continue to boil for 30 minutes over low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Concentrate it to 1g / mL (raw drug amount) over low heat. Centrifuge at 12000r / min for 10 minutes, sterilize it in aliquots and store it in a -80℃ refrigerator for later use.

[0072] Comparative Example 5 Preparation process of spiny jujube seeds

[0073] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming. Steam them at 37°C, 45°C and 50°C for 10 minutes each. After steaming, dry them naturally at room temperature for 1 to 3 hours. After cooling, take them out and spread the Chinese jujube kernels on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows, with a total of four stages. The first stage is dried at 40°C for 2 hours, the second stage is dried at 50°C for 1 hour, the third stage is dried at 60°C for 1 hour, and the sixth stage is dried at 70°C for 1 hour. After completing the above drying continuously, take them out.

[0074] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0075] Comparative Example 6 Preparation process of spiny jujube seeds

[0076] Select fresh, plump and moderately sized clean Chinese jujube kernels and put them into a water bath for steaming. Steam them at 37°C, 45°C and 50°C for 10 minutes each. After steaming, dry them naturally at room temperature for 1 to 3 hours. After cooling, take them out and spread the Chinese jujube kernels on a tray. Place the Chinese jujube kernels in a vacuum drying oven and evacuate them to keep the vapor pressure at -0.1MPa. The vacuum drying method adopts a gradient temperature change method. The drying time-temperature program is set as follows, with a total of four stages. The first stage is drying at 45°C for 2.5 hours, the second stage is drying at 50°C for 1.5 hours, the third stage is drying at 55°C for 1.5 hours, and the fourth stage is drying at 60°C for 1.5 hours. After completing the above drying continuously, take them out.

[0077] The test solution was prepared by traditional water decoction method. Weigh an appropriate amount of Ziziphus jujuba seeds and add 10 times the amount of water to soak for 1 hour. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Add 8 times the amount of water to the residue for a second decoction. Boil with high heat and continue to boil for 30 minutes with low heat. Pour out the solution and filter it with three layers of filter cloth while hot. Combine the two filtrates and concentrate to 1g / mL (raw drug amount) with low heat. Centrifuge at 12000r / min for 10 minutes, sterilize in aliquots and store in a -80℃ refrigerator for later use.

[0078] Experimental Example 1 RT-qPCR detection of inflammatory factor expression in macrophages

[0079] RAW264.7 cells in logarithmic growth phase and in good growth condition were cultured at 1×10 5 / mL and inoculated in a 24-well culture plate, 500μL per well, placed in a cell culture incubator at 37°C and 5% CO2, after 24 hours, discard the cell supernatant, and add 500μL of the prepared experimental group solution to each well. Set up the following groups: control group (DMEM complete medium), Example 1 group to Example 3 group (respectively added with 1mg / ml Ziziphus jujuba seeds prepared in Examples 1-3), Comparative Example 1 group to Comparative Example 6 group (respectively added with 1mg / ml Ziziphus jujuba seeds prepared in Comparative Examples 1-6), each group is set with 3 parallel duplicate wells, put the 24-well plate in a CO2 incubator for 24 hours, take it out of the incubator, and extract RNA, wherein the primers are COX-2, TNF-α, iNOS, IL-6, IL-1β and β-Actin, respectively.

[0080] 1. Extraction and concentration determination of total cell RNA

[0081] (1) RAW264.7 cells in the logarithmic phase were collected and prepared into a single-cell suspension. Then, 1×10 5 The cells were seeded in a 24-well culture plate, 500 μL of DMEM complete medium was added, and cultured in a constant temperature incubator (37°C, 5% CO2) for 24 h.

[0082] (2) The original culture medium was discarded, and 500 μL of the solution was added to each well of the control group (DMEM complete medium), Example 1 to Example 3 (1 mg / ml of Ziziphus jujuba seed prepared in Examples 1-3 were added respectively), and Comparative Example 1 to Comparative Example 6 (1 mg / ml of Ziziphus jujuba seed prepared in Comparative Examples 1-6 were added respectively). Three parallel wells were set up for each group, and the cells were incubated in a constant temperature incubator for 12 h.

[0083] (3) Discard the original culture medium and wash twice with pre-cooled PBS solution (operate on ice). Then, add 0.5 mL Trizol solution to each well, shake well, and let stand for 10 minutes.

[0084] (4) Transfer the cell lysate to a 1.5 mL centrifuge tube and place at room temperature for 5 min. Add 0.1 mL of chloroform to the centrifuge tube, cover tightly, shake vigorously for about 15 seconds, and incubate at room temperature for 5 min.

[0085] (5) After incubation, centrifuge (4°C, 12,000 rpm, 15 min). The mixed liquid will be separated into three layers: the lower layer is the red phenol-chloroform phase containing DNA and protein, the middle is the colorless mixed phase, and the upper layer is the colorless aqueous phase containing RNA. The volume of the aqueous phase is about 0.5 mL. Pipette about 0.2 mL of the upper aqueous phase into another 1.5 mL centrifuge tube.

[0086] (6) Add 0.5 mL of isopropanol to the centrifuge tube, invert to mix evenly, and incubate at room temperature for 10 min.

[0087] (7) After incubation, centrifuge (4°C, 12,000 rpm, 15 min). White gelatinous precipitate can be observed on the bottom and side walls of the tube.

[0088] (8) Carefully discard the supernatant, add 0.5 mL of 75% ethanol (prepared with DEPC water), mix well to suspend the precipitate, centrifuge (4°C, 8000 rpm, 5 min), discard the 75% ethanol, repeat twice, and naturally dry for 10-20 min to evaporate the residual ethanol.

[0089] (9) Dissolve the precipitate in 20 μL DEPC water and measure the OD260 / OD280 ratio. An OD260 / OD280 ratio between 1.8 and 2.0 indicates that the RNA purity meets the requirements.

[0090] 2. RNA reverse transcription to cDNA

[0091] According to the SPARKscript II RT Plus Kit, reverse transcription and cDNA synthesis were performed in a 20 μL reaction system. The specific steps are as follows:

[0092] (1) According to the RNA concentration to be detected, prepare the reaction system on ice as follows:

[0093] Table 1 Reverse transcription mixed reaction system

[0094]

[0095] Mix the prepared system, centrifuge it and place it in a PCR instrument, and set the reaction program: 42℃, 5 min; 4℃, ∞.

[0096] (2) After the reaction is complete, place on ice. Continue to add the ingredients shown in Table 2 directly to the same tube:

[0097] Table 2 Reverse transcription mixed reaction system

[0098]

[0099] Mix well and centrifuge, set the reaction program in the PCR instrument: 50℃, 20min; 85℃, 5min; 4℃, ∞; finally, store the obtained cDNA at -20℃ for later use.

[0100] 3. RT-PCR amplification

[0101] The present invention uses β-Actin as an internal reference, searches for the corresponding gene sequence on the NCBI website, uses PrimerPremier 5 software to design reasonable upstream and downstream primers, and submits them to Shenzhen Sangon Biotechnology Co., Ltd. for synthesis. The primer powder is diluted to 100 μM with sterile water.

[0102] According to the instructions of the TB Green® Premix™ II kit, take appropriate amounts of TB Green Premix Ex TaqII, Forward Primer, Reverse Primer, cDNA, ROX and DEPC-treated water to form a 20 μL PCR reaction system, mix gently, and perform PCR amplification, and record the CT value of the experiment. The RT-PCR primer sequences and reaction conditions are shown in Table 3.

[0103] Table 3 RT-PCR primer sequences and reaction conditions

[0104]

[0105] Note: In the table, "F" represents forward primer and "R" represents reverse primer.

[0106] The results of real-time fluorescence quantitative PCR showed that: Figure 1-Figure 4 As shown, compared with the blank control group, after stimulation with the Chinese jujube seeds prepared by the traditional process of comparative example 1, the RNA expression level of each gene in RAW264.7 cells increased slightly; after stimulation with the Chinese jujube seeds obtained by the preparation process of comparative examples 2-6, the RNA expression level of each gene in RAW264.7 cells also increased compared with comparative example 1, but the increase was not as obvious as in Examples 1-3. After the Chinese jujube seeds treated with the new process of Examples 1-3 acted on RAW264.7 cells, the mRNA expression of iNOS, TNF-α, COX-2, and IL-1β genes increased to varying degrees. The reason may be that after drying, the Chinese jujube seeds affect the physicochemical properties of traditional Chinese medicine by changing the temperature and moisture content of traditional Chinese medicine. This study can provide a scientific reference for establishing a reasonable Chinese jujube seed processing technology and provide an experimental basis for improving the quality of Chinese jujube seed medicinal materials.

[0107] The results showed that sour jujube seeds can promote the RNA expression of COX-2, iNOS, TNF-α, and IL-1β genes in RAW264.7 cells. It can be known that after treating RAW264.7 cells with sour jujube seeds, macrophages are promoted to polarize to M1 type. M1 macrophages release high levels of cytokines, such as TNF-α, IL-6, and IL-1β. They can engulf and destroy microorganisms and remove tumor cells. They have strong anti-infection, anti-tumor, and apoptotic cell and necrotic tissue removal capabilities. They can mediate Th1 immune response to kill pathogens and protect the body through antigen presentation. Polarized macrophages play a key role in the development of inflammatory response or disease and the degree of repair of tissues and organs, and have important clinical application value.

[0108] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A preparation process of a spiny jujube seed preparation, characterized in that: The preparation process comprises the following steps: S1. Steam and dry the purified Chinese jujube seeds, vacuum dry them, and then take them out to obtain dried Chinese jujube seeds; S2, extracting dried Chinese date seeds twice by decoction, and filtering to obtain a filtrate; S3, the filtrate is concentrated, centrifuged, and sterilized to obtain the spiny jujube seed preparation; The vacuum drying in step S1 includes: drying at 45°C for 2h in the first stage, drying at 50°C for 1h in the second stage, drying at 55°C for 1h in the third stage, and drying at 60°C for 1h in the fourth stage; The steaming in step S1 includes steaming at 37-60° C. for 30 min.

2. The preparation process according to claim 1, characterized in that: The steaming in step S1 includes: steaming at 37°C for 30 minutes; or steaming at 37°C for 10 minutes in the first stage, steaming at 45°C for 10 minutes in the second stage, and steaming at 50°C for 10 minutes in the third stage; or steaming at 50°C for 10 minutes in the first stage, steaming at 55°C for 10 minutes in the second stage, and steaming at 60°C for 10 minutes in the third stage.

3. The preparation process according to claim 1, characterized in that: The vacuum degree of the vacuum drying described in step S1 is -0.2 ~ -0.05MPa.

4. The preparation process according to claim 1, characterized in that: The decoction extraction twice described in step S2 comprises: (1) Soak dried Chinese jujube seeds in water, decoct, collect and filter the liquid to obtain filtrate 1; (2) Add water to the residue and boil it, collect the liquid and filter it to obtain filtrate 2; (3) Combine filtrate 1 and filtrate 2 to obtain filtrate.

5. The preparation process according to claim 1, characterized in that: The concentration described in step S3 is: concentrating the filtrate to a concentration of 0.5-2 g / mL in terms of crude drug amount.

6. The spiny jujube seed preparation obtained by the preparation process according to any one of claims 1 to 5.

7. An immune activator, characterized in that The immune activator comprises the spinosa jujube seed preparation according to claim 6.

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