Composition for promoting neutrophil proliferation as well as preparation method and application thereof

Abstract

The invention discloses a composition for promoting neutrophil proliferation and a preparation method and application thereof. The composition is prepared from the following raw materials in parts by weight: 30-40 parts of gorgon fruit, 10-20 parts of fructus alpiniae oxyphyllae, 10-20 parts of ginkgo, 10-20 parts of hawthorn, 10-20 parts of Chinese yam, 10-20 parts of liquorice, 10-20 parts of poria cocos, 10-20 parts of lotus seed and 10-20 parts of astragalus membranaceus. The traditional Chinese medicine composition completely conforms to the traditional Chinese medicine mechanism and the theory of homology of medicine and food, can tonify spleen and replenish qi, reinforce kidney and arrest seminal emission and clear damp, and has good effect of promoting neutrophile granulocyte proliferation and immune protection effect.

Description

Technical Field

[0001] This application relates to the field of immune protection technology, specifically to a composition that promotes neutrophil proliferation, its preparation method, and its uses. Background Technology

[0002] Immunity is the body's own defense mechanism, its ability to recognize and eliminate any foreign invaders (viruses, bacteria, etc.), process aging, damaged, dead, and degenerated cells, and recognize and deal with mutated and virus-infected cells. Modern immunology believes that boosting immunity is the body's physiological response to recognize and eliminate "foreign" substances. The immune system performs this function, and there are various ways to enhance immunity, such as consuming beneficial foods, especially for children who need to pay close attention to strengthening their immunity.

[0003] Neutrophils, also known as polymorphonuclear leukocytes (PMNs), are the most abundant cell type in human blood. They are produced in large quantities in the bone marrow, with approximately 10¹¹ neutrophils produced per day. Neutrophils are important effector cells of the innate immune system. They enter the circulatory system, migrate to tissues, and can rapidly capture and kill invading pathogens.

[0004] With the fast pace of modern life and changes in dietary structure, children's health issues are receiving increasing attention. Children are in a critical stage of growth and development, and their immune systems are not yet fully mature, making them susceptible to external pathogens such as viruses and bacteria, leading to problems such as recurrent colds, indigestion, and allergies. Therefore, developing healthy beverages with immunomodulatory functions, effectively promoting neutrophil proliferation, and suitable for children has become an important research direction in the fields of food science and nutrition.

[0005] Traditional immune-boosting products suffer from insufficient efficacy, poor taste, and low acceptance among children. Therefore, it is necessary to develop an immune protection product that is effective, tastes better, and is more widely accepted. Summary of the Invention

[0006] This invention provides a composition for promoting neutrophil proliferation, its preparation method, and its uses. The composition for promoting neutrophil proliferation is scientifically formulated and fully conforms to the mechanisms of traditional Chinese medicine and the theory of food and medicine sharing the same origin. It can invigorate the spleen and replenish qi, strengthen the kidneys and astringe essence, and remove dampness. It has a good effect on promoting neutrophil proliferation and immune protection.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: The composition for promoting neutrophil proliferation is made from the following raw materials in parts by weight: The ingredients are: 30-40 parts Euryale ferox, 10-20 parts Alpinia oxyphylla, 10-20 parts Ginkgo biloba, 10-20 parts Crataegus pinnatifida, 10-20 parts Dioscorea opposita, 10-20 parts Glycyrrhiza uralensis, 10-20 parts Poria cocos, 10-20 parts Nelumbo nucifera, and 10-20 parts Astragalus membranaceus. Experiments have shown that the above ingredients, in their specific weight proportions, produce a synergistic effect, especially in promoting the proliferation of immune cells (such as neutrophils), where the effect is significantly better than that of single herbs or other combinations.

[0008] Preferably, the preparation method of its active ingredient includes the following steps: (1) Weigh out ginkgo, hawthorn, yam, licorice, lotus seed, and astragalus according to the weight parts. Add water equivalent to 6-10 times the total weight of the medicinal materials and decoct 1-3 times, each time for 1-3 hours. Filter and combine the filtrates to obtain the extract. Concentrate the extract to 40-60% of the total weight of the extracted medicinal materials. Cool to room temperature and add ethanol to make the alcohol content 40-80%. Let stand, filter, recover the ethanol from the supernatant, and then concentrate under reduced pressure to a relative density of 1.10-1.15 at 50℃ to obtain the extract paste. (2) After drying Poria cocos, use an ultra-micro pulverizer to break down the cell walls and pulverize it through a 250-320 mesh sieve to collect the fine powder for later use. (3) Grind Alpinia oxyphylla into coarse powder, soak it, and then extract it by steam distillation. Collect the distillate, separate the upper volatile oil, and obtain Alpinia oxyphylla volatile oil. Encapsulate the Alpinia oxyphylla volatile oil with β-cyclodextrin to obtain volatile oil inclusion complex. (4) The extract, Poria cocos powder, and volatile oil inclusion complex are mixed to obtain the final product.

[0009] In step (2), the drying temperature of Poria cocos is 40-50℃.

[0010] In step (3), when the volatile oil of Alpinia oxyphylla is encapsulated, the weight ratio of the volatile oil of Alpinia oxyphylla to β-cyclodextrin is 1:6-8.

[0011] During the encapsulation process, the temperature should be 40-50℃ and the time should be 1-2 hours.

[0012] Its ointment contains the following additives in parts by weight: 400-600 parts of fructooligosaccharides and 50-80 parts of honey.

[0013] Preferably, the preparation method of the ointment includes the following steps: The honey and fructooligosaccharides are heated and melted, then mixed with the active ingredients and boiled to obtain the final product.

[0014] The cooking temperature is 70-90℃, and the cooking time is 1-3 hours.

[0015] The present invention also discloses the use of the above-mentioned traditional Chinese medicine composition in the preparation of health products or medicines for the prevention and / or adjuvant treatment of immunodeficiency.

[0016] The formula of the present invention is composed of traditional Chinese medicines such as Euryale ferox, Ginkgo biloba, Hawthorn, Chinese yam, Licorice root, Poria cocos, Lotus seed, Alpinia oxyphylla, etc. In the formula, Euryale ferox is the monarch drug. It is of neutral nature, sweet and astringent in taste, and belongs to the spleen and kidney meridians. It benefits the kidney and consolidates essence, invigorates the spleen and stops diarrhea, and removes dampness and stops leukorrhea. Astragalus membranaceus, Chinese yam, Poria cocos, and Lotus seed are ministerial drugs. Astragalus membranaceus is known as the king of qi-tonifying herbs. It invigorates the spleen and replenishes the middle, replenishes qi and consolidates the exterior, directly infuses energy into the spleen and stomach, and enhances the overall functional power. It is the main force for "opening up the source". Chinese yam nourishes both qi and yin, invigorates the spleen and nourishes the stomach, promotes the production of body fluid and nourishes the lungs, and benefits the kidney and consolidates essence. It can not only assist Euryale ferox in astringency but also assist Astragalus membranaceus in qi-tonifying. Moreover, its medicinal property is mild and can tonify the three energizers evenly. Poria cocos invigorates the spleen and tranquillizes the mind, promotes diuresis and eliminates dampness. While tonifying, it expels the pathogenic dampness in the body, clears obstacles for the tonifying herbs, and prevents "retaining pathogens inside like closing the door". This reflects the compatibility wisdom of "purging while tonifying". Lotus seed invigorates the spleen and stops diarrhea, benefits the kidney and consolidates essence, nourishes the heart and soothes the mind. Its efficacy is similar to that of Euryale ferox, synergistically strengthening the astringency and spleen-invigorating power of the monarch drug, and can convey the tonifying effect to the heart to soothe the mind and stabilize the will. Alpinia oxyphylla, Ginkgo biloba, and Hawthorn are adjuvant drugs. Alpinia oxyphylla warms the kidney, consolidates essence, reduces urination, warms the spleen, stops diarrhea, and controls salivation. Its nature is warm, can warm and tonify the yang of the spleen and kidney, enhance the qi-transforming function, and help the spleen and stomach better transform water and grains. It has an astringent effect on enuresis and salivation caused by spleen-kidney yang deficiency. Ginkgo biloba relieves cough and asthma, astringes and stops leukorrhea, consolidates essence and reduces urination. Its main effects are on the lungs and lower jiao, assisting the monarch drug to strengthen the astringency and consolidation property of the whole formula. Hawthorn promotes digestion and removes stagnation, promotes qi movement and dispels stasis. Among many tonifying and astringent herbs, adding Hawthorn can eliminate food stagnation, promote the transformation of the middle jiao, and prevent greasiness from obstructing the stomach, making the tonifying effect not stagnant. This is an important "unblocking" role in the formula. Licorice root is the guiding drug. It invigorates the spleen and replenishes qi, clears heat and detoxifies, reduces phlegm and relieves cough, relieves spasm and stops pain, and harmonizes all the drugs. Licorice root can not only assist in invigorating the spleen and replenishing qi but also, with its sweet and neutral nature, harmonize the biases of all the drugs in the formula, enabling each drug to cooperate in combat, with the medicinal power being mild and lasting. The core idea of this formula is "invigorating the spleen and replenishing qi, consolidating the kidney and astringing essence, and concurrently removing dampness". It is a typical formula mainly for strengthening healthy qi and consolidating the root. Its focus is on enhancing the body's own physiological functions rather than directly combating exogenous pathogens. While tonifying and astringing, adjuvant drugs for promoting qi movement and eliminating stagnation are used to regulate the qi movement in the middle jiao, making the "upward, downward, outward, and inward movements" return to order, thereby ensuring the smooth generation and distribution of "qi, blood, and body fluid", achieving the purpose of non-stagnant tonification and treating both the symptoms and the root causes. In traditional Chinese medicine theory, the function of immune protection is mainly related to "healthy qi". "Huangdi Neijing" states: "When healthy qi exists inside, pathogens cannot invade." "Healthy qi" is equivalent to the body's disease resistance, self-healing ability, and stable regulatory ability. The strength of healthy qi is most closely related to the three viscera of the spleen, kidney, and lungs. The spleen is the foundation of acquired constitution and the source of qi and blood production: when the spleen is weak, qi and blood are insufficient, and healthy qi cannot be generated. The kidney is the foundation of congenital constitution and the place where essence is stored: kidney essence is the material basis of life activities, can transform into primordial qi, and is the root of healthy qi. The lung governs qi throughout the body and is externally related to the skin and hair: lung qi is in charge of the defensive function on the body surface and is the first line of defense against exogenous pathogens. This formula precisely focuses on strengthening the spleen and kidney and supplementing the lungs to achieve the purpose of enhancing healthy qi. Therefore, invigorating the spleen and replenishing qi is the most core and fundamental way to strengthen healthy qi (enhance immunity).

[0017] Basis for formulating prescriptions from the perspective of Traditional Chinese Medicine: (1) Strengthening the foundation and reducing consumption (fundamental solution): This invention uses Alpinia oxyphylla to warm and tonify the Yang of the spleen and kidneys, providing the most fundamental "energy" (vital energy) for the immune system. Through astringent ingredients such as Euryale ferox and lotus seeds, it firmly protects the body's essential substances (such as kidney essence and Qi and blood generated by the spleen), preventing their unnecessary loss. This is like a family; it not only needs to earn money (tonify Qi), but also needs to save money (strengthen the foundation) so that it has a solid foundation and the ability to withstand risks.

[0018] (2) Eliminating dampness and clearing the pathways (key step): Poria cocos promotes diuresis and eliminates dampness, which is of utmost importance. Dampness is like "silt" in the body, which can hinder the circulation of qi and blood and the absorption of medicine. Clearing dampness is equivalent to dredging the river channel, so that the yang qi and essence that are replenished can be smoothly distributed throughout the body and exert their effects. Without this step, simply tonifying and astringing may lead to "closing the door to let in the enemy" or being too rich and greasy and causing stomach upset.

[0019] (3) Strengthen the defense and enhance the synergistic effect: After solving the fundamental problem, Astragalus and Ginkgo strengthen the lung defense, Dioscorea solidifies the spleen and stomach, and Hawthorn promotes the flow of qi, blood and body fluids, making the whole immune protection system more complete and efficient.

[0020] This formula uses Euryale ferox, Astragalus membranaceus, Dioscorea opposita, Poria cocos, and Nelumbo nucifera as its core ingredients to invigorate the spleen and replenish qi, and to strengthen the kidneys and astringe essence, aiming to enhance the body's vital energy from the perspective of traditional Chinese medicine theory. It creatively introduces Alpinia oxyphylla and employs volatile oil encapsulation technology to effectively mask its unpleasant odor while introducing its warming and tonifying effects on the spleen and kidneys, thus solving the technical challenge of balancing efficacy and taste. Hawthorn promotes digestion and eliminates food stagnation, aiding digestion and preventing the cloying and greasy feeling of other tonifying herbs from hindering stomach absorption.

[0021] The pharmacodynamic experimental modeling of this invention utilizes zebrafish, which are the smallest animals currently known to possess both specific and nonspecific immunity, making them suitable for high-throughput evaluation of immunomodulatory efficacy. Intravenous injection of high doses of vinorelbine into zebrafish induces immunodeficiency. Using transgenic green fluorescent neutrophil zebrafish, a significant decrease in neutrophil fluorescence intensity was observed under a fluorescence microscope in immunodeficient zebrafish. Similarly, using transgenic green fluorescent macrophage zebrafish, a significant decrease in macrophage fluorescence intensity was observed under a fluorescence microscope in immunodeficient zebrafish. Furthermore, using transgenic red fluorescent T-cell zebrafish, a significant decrease in T-cell fluorescence intensity was observed under a fluorescence microscope in immunodeficient zebrafish.

[0022] The principle of the zebrafish immunoprotection experiment: Chloramphenicol is a broad-spectrum antibiotic. Due to its high toxicity, it can inhibit bone marrow hematopoiesis, causing allergic reactions and aplastic anemia. In zebrafish, this results in immune damage manifested by a decrease in the number of neutrophils in the tail. Therefore, the protective effect of the test substance on the immune system can be evaluated by observing the number of neutrophils in the zebrafish tail.

[0023] The advantages of this invention are as follows: (1) The traditional Chinese medicine composition of the present invention is relatively safe. The raw materials used are all traditional Chinese medicines that are both food and medicine. It is safe, has no toxic side effects, is suitable for conditioning and treating children's constitution, and has no adverse effects on children's growth and development.

[0024] (2) This invention masks unpleasant tastes by encapsulating volatile oils. Steam distillation is used to extract the volatile oil from Alpinia oxyphylla, and the extraction rate is much higher than that of traditional water extraction.

[0025] (3) Improve the traditional water extraction and alcohol precipitation process to increase the extraction rate; use cell wall breaking and pulverizing treatment to treat Poria cocos, so that its polysaccharide solubility is greatly increased from 25.3% in water extraction to 84.5%, and the technical effect is significantly improved.

[0026] (4) Compared with other immune-boosting drinks, the present invention uses zebrafish to verify efficacy, and the results are reliable, providing solid and reliable experimental data support for the product's functional claims.

[0027] (5) Compared with traditional decoctions or Chinese patent medicines, the ointment of the present invention is easier for children to take because of its better taste, which can greatly improve children's medication compliance; compared with acupoint application or pediatric massage, the ointment preparation is easier for patients to take at home, thereby reducing the number of visits and improving patients' medical compliance.

[0028] (6) Cost control: By improving the process, this invention not only increases the effective ingredients, but also makes it applicable to industrial production and saves energy costs.

[0029] (7) In terms of safety: Through the drug resistance experiment of zebrafish, it was found that the maximum drug resistance concentration is 2400 μg / mL, and the safety range is large.

[0030] This invention possesses outstanding substantive features and significant advancements. Compared to the comparative example using traditional water extraction processes, this invention, through a unique combination of processes such as "Poria cocos cell wall breaking and pulverization" and "Encapsulation of Alpinia oxyphylla volatile oil," produces unexpected technical effects: 1) the dissolution rate of the core active ingredient, Poria cocos polysaccharide, is increased from 25.3% to 84.5%; 2) the extraction rate of Alpinia oxyphylla volatile oil is increased from 1.2% to 2.8%, and its unpleasant taste problem is solved; 3) zebrafish experiments confirm that, at the same dosage, the product of this invention significantly outperforms the comparative example in promoting neutrophil proliferation. These effects are unpredictable to those skilled in the art based on existing technology. Attached Figure Description

[0031] Figure 1 The effect of different concentrations of the test sample on the survival rate of zebrafish juveniles in Experiment Example 1. Note: Compared with the control group, *p<0.05, **p<0.01, ****p<0.0001; Figure 2 The results of tail neutrophil staining in each drug administration group in Experiment Example 1; Figure 3 The number of neutrophils in each drug administration group in Experiment 1 (n=10) is shown. Note: Compared with the control group, ****P<0.001; compared with the model group, ##P<0.01 ###P<0.001. Detailed Implementation

[0032] The present invention will now be described in detail through specific embodiments. Example 1

[0033] The immune protective composition is made from the following raw materials: 30kg of Euryale ferox seeds, 10kg of Alpinia oxyphylla seeds, 10kg of Ginkgo biloba seeds, 10kg of hawthorn fruit, 10kg of Dioscorea opposita seeds, 10kg of Glycyrrhiza uralensis root, 10kg of Poria cocos, 10kg of Nelumbo nucifera seeds, and 10kg of Astragalus membranaceus.

[0034] The preparation method of its active ingredient includes the following steps: (1) Weigh out ginkgo, hawthorn, yam, licorice, lotus seed, and astragalus according to their weight. Add water equivalent to 6 times the total weight of the herbs and decoct once for 1 hour each time. Filter and combine the filtrates to obtain the extract. Concentrate the extract to 40% of the total weight of the herbs. Cool to room temperature and add ethanol to make the alcohol content reach 40%. Let stand, filter, recover the ethanol from the supernatant, and then concentrate under reduced pressure to a relative density of 1.10 at 50°C to obtain the extract paste. (2) After drying Poria cocos at 40℃, use an ultra-micro pulverizer to break the cell wall and pulverize it. Pass it through a 25-mesh sieve and collect the fine powder of Poria cocos for later use. (3) Grind Alpinia oxyphylla into coarse powder, soak it, and then extract it by steam distillation. Collect the distillate, separate the upper volatile oil, and obtain Alpinia oxyphylla volatile oil. Encapsulate the Alpinia oxyphylla volatile oil with β-cyclodextrin. The weight ratio of Alpinia oxyphylla volatile oil to β-cyclodextrin is 1:6. The encapsulation temperature is 40℃ and the time is 1 hour to obtain the volatile oil inclusion complex.

[0035] (4) The active ingredient is obtained by mixing the extract, Poria cocos powder, and volatile oil inclusion complex.

[0036] The above-mentioned active ingredients are used to prepare an ointment, with the following additives: 400 kg of fructooligosaccharides and 50 kg of honey. The preparation method of the ointment includes the following steps: The honey and fructooligosaccharides are heated and melted, then mixed with the active ingredients and boiled to obtain the final product.

[0037] The cooking temperature is 70℃ and the cooking time is 1 hour.

[0038] Experimental Example 1 demonstrates that the composition of the present invention can significantly reverse chloramphenicol-induced neutropenia in zebrafish, indicating that the composition has an immunoprotective effect that promotes neutrophil proliferation. Example 2

[0039] The immune protective composition is made from the following raw materials: 40kg of Euryale ferox seeds, 20kg of Alpinia oxyphylla seeds, 20kg of Ginkgo biloba seeds, 20kg of hawthorn fruit, 20kg of Dioscorea opposita seeds, 20kg of Glycyrrhiza uralensis root, 20kg of Poria cocos, 20kg of Nelumbo nucifera seeds, and 20kg of Astragalus membranaceus.

[0040] The preparation method of its active ingredient includes the following steps: (1) Weigh out ginkgo, hawthorn, yam, licorice, lotus seed, and astragalus according to their weight. Add water equivalent to 10 times the total weight of the herbs and decoct 3 times, each time for 3 hours. Filter and combine the filtrates to obtain the extract. Concentrate the extract to 40-60% of the total weight of the herbs. Cool to room temperature and add ethanol to make the alcohol content reach 80%. Let stand, filter, recover the ethanol from the supernatant, and then concentrate under reduced pressure to a relative density of 1.15 at 50℃ to obtain the extract paste. (2) After drying Poria cocos at 50℃, use an ultra-micro pulverizer to break the cell wall and pulverize it. Pass it through a 320-mesh sieve and collect the fine powder of Poria cocos for later use. (3) Grind Alpinia oxyphylla into coarse powder, soak it, and then extract it by steam distillation. Collect the distillate, separate the upper volatile oil, and obtain Alpinia oxyphylla volatile oil. Encapsulate the Alpinia oxyphylla volatile oil with β-cyclodextrin. The weight ratio of Alpinia oxyphylla volatile oil to β-cyclodextrin is 1:8. The encapsulation temperature is 50℃ and the time is 2 hours to obtain the volatile oil inclusion complex.

[0041] (4) The active ingredient is obtained by mixing the extract, Poria cocos powder, and volatile oil inclusion complex.

[0042] The above-mentioned active ingredients are used to prepare an ointment, with the following additives: 600 kg of fructooligosaccharides and 80 kg of honey. The preparation method of the ointment includes the following steps: The honey and fructooligosaccharides are heated and melted, then mixed with the active ingredients and boiled to obtain the final product.

[0043] The cooking temperature is 90℃ and the cooking time is 3 hours. Example 3

[0044] The immune protective composition is made from the following raw materials: 30kg of Euryale ferox seeds, 10kg of Alpinia oxyphylla seeds, 10kg of Ginkgo biloba seeds, 10kg of hawthorn fruit, 10kg of Dioscorea opposita seeds, 10kg of Glycyrrhiza uralensis root, 10kg of Poria cocos, 10kg of Nelumbo nucifera seeds, and 10kg of Astragalus membranaceus.

[0045] The preparation method of its active ingredient includes the following steps: (1) Weigh out ginkgo, hawthorn, yam, licorice, lotus seed, and astragalus according to weight. Add water and decoct twice. The first time, add water equivalent to 8 times the total weight of the herbs. The second time, add water equivalent to 6 times the total weight of the herbs. Each decoction lasts for 2 hours. Filter and combine the filtrates to obtain the extract. Concentrate the extract to 50% of the total weight of the herbs. Cool to room temperature and add ethanol to make the alcohol content reach 50%. Let stand, filter, recover the ethanol from the supernatant, and then concentrate under reduced pressure to a relative density of 1.12 at 50°C to obtain the extract paste. (2) After drying Poria cocos at 45℃, use an ultra-micro pulverizer to break down the cell walls and pulverize it through a 300-mesh sieve to collect the fine powder for later use. (3) Grind Alpinia oxyphylla into coarse powder, soak it, and then extract it by steam distillation. Collect the distillate, separate the upper volatile oil, and obtain Alpinia oxyphylla volatile oil. Encapsulate the Alpinia oxyphylla volatile oil with β-cyclodextrin. The weight ratio of Alpinia oxyphylla volatile oil to β-cyclodextrin is 1:7. The encapsulation temperature is 45℃ and the time is 1.5 hours to obtain the volatile oil inclusion complex. The inclusion results of the volatile oil from Alpinia oxyphylla are shown in Tables 1 and 2 below: Table 1 Encapsulation rate of volatile oil from Alpinia oxyphylla: serial number Inclusion sample size (g) Weight after fluid replacement (g) Absorbance (A) Average concentration of essential oil in the supernatant (mg / mL) Encapsulation rate (%) Average encapsulation yield (%) Overall score 11 0.4023 32.4239 1.06991.0692 1.36 43.40 12 0.4006 32.9212 1.22521.2243 1.56 46.76 44.05 9.89 13 0.4012 32.8339 1.17701.1729 1.50 42.00 21 0.4026 32.9794 1.03271.0330 1.31 46.93 22 0.4017 32.8773 1.01461.0162 1.29 47.89 48.24 10.58 23 0.4014 32.9192 1.09261.0927 1.39 49.88 31 0.4 32.8895 1.04971.0494 1.34 52.83 32 0.4007 33.0369 1.04971.0494 1.33 52.83 54.11 11.38 33 0.4011 32.894 1.09521.0942 1.39 59.21 41 0.4005 32.5924 1.00591.0027 1.28 34.63 42 0.4001 32.4757 0.92430.9219 1.17 38.69 41.72 9.35 43 0.4008 32.5574 1.03811.0379 1.32 44.76 51 0.4004 31.5640 1.07721.0777 1.37 54.71 52 0.4014 31.6983 1.07041.0710 1.36 55.84 55.28 11.06 53 0.4006 31.8221 0.99931.0000 1.27 51.51 61 0.4001 32.3324 1.08031.0797 1.37 32.77 62 0.4003 31.8432 1.17541.1744 1.50 43.44 42.92 9.68 63 0.4004 31.8690 1.11191.1109 1.41 42.39 71 0.4001 32.7949 1.13331.1329 1.44 49.23 72 0.4001 32.8881 1.16321.1638 1.48 50.44 49.84 9.76 73 0.4006 31.9475 1.13001.1296 1.44 47.57 81 0.4001 31.7941 1.23861.2371 1.58 36.82 82 0.4006 32.5714 1.14281.1424 1.45 34.90 35.02 7.66 83 0.4005 32.4129 1.21751.2175 1.55 33.33 91 0.4010 31.9081 1.13961.1394 1.45 45.21 92 0.4014 31.9648 1.01861.0182 1.29 39.57 44.95 9.19 93 0.4001 1.8764 1.20581.2049 1.54 44.68 .

[0046] Table 2. Yield of volatile oil from Alpinia oxyphylla encapsulation: serial number Temperature (°C) Time (h) β-Cyclodextrin mass (g) Mass (g) of water containing dissolved cyclodextrin Alpinia oxyphylla volatile oil (g) Mass of the obtained inclusion compound (g) Encapsulation yield (%) Average yield of encapsulation (%) 11 40 1 1.001 21.06 0.1900 0.9756 81.91 12 41 1 1.005 21.06 0.2060 0.9894 81.70 81.81 13 41 1 1.005 20.73 0.2164 0.9757 79.88 21 40 1.5 1.203 21 0.2095 1.2087 85.57 22 40 1.5 1.203 22.09 0.2025 1.208 85.95 85.72 23 40 1.5 1.200 22.87 0.2095 1.2071 85.64 31 40 2 1.401 22.87 0.2009 1.2674 1.3853 32 40 2 1.403 32.6 0.2015 1.3581 84.64 85.40 33 40 2 1.405 29.3 0.2027 1.3853 84.64 41 51 1 1.202 19.03 0.2035 0.8854 63.00 42 51 1 1.204 26.66 0.2035 1.0774 76.54 77.30 43 51 1 1.202 26.67 0.2015 1.0955 78.05 51 51 1.5 1.402 30.03 0.2031 1.2990 80.93 52 50 1.5 1.408 29.97 0.2007 1.3231 82.25 81.62 53 51 1.5 1.406 27.73 0.2021 1.3136 81.69 61 50 2 1.005 19.70 0.2055 0.7866 64.98 62 51 2 1.003 22.22 0.2088 0.9715 80.17 80.44 63 51 2 1.003 25.05 0.2020 0.9726 80.71 71 61 1 1.405 23.58 0.2077 1.1364 70.47 72 60 1 1.405 26.41 0.2064 1.1258 69.86 70.17 73 59 1 1.403 26.66 0.2007 1.0625 66.25 81 61 1.5 1.003 26.33 0.2012 0.7504 62.32 82 60 1.5 1.005 23.27 0.2065 0.7964 65.74 64.03 83 61 1.5 1.003 23.27 0.2020 0.6959 57.75 91 61 2 1.201 23.61 0.2022 1.0113 72.07 92 59 2 1.204 24.76 0.2066 1.0176 72.14 72.11 93 61 2 1.205 26.70 0.2017 0.9396 66.79 .

[0047] As shown in Tables 1 and 2, when the inclusion temperature is between 40-50℃ and the inclusion time is between 1-2 hours, the encapsulation efficiency and yield of the obtained volatile oil inclusion complex are significantly higher than those of the experimental group outside this range. Therefore, this parameter range is preferred in this invention.

[0048] (4) The active ingredient is obtained by mixing the extract, Poria cocos powder, and volatile oil inclusion complex.

[0049] The above-mentioned active ingredients are used to prepare an ointment, with the following additives: 500 kg of fructooligosaccharides and 70 kg of honey. The preparation method of the ointment includes the following steps: The honey and fructooligosaccharides are heated and melted, then mixed with the active ingredients and boiled to obtain the final product.

[0050] The cooking temperature is 80℃ and the cooking time is 2 hours.

[0051] In this embodiment, the volatile oil of Alpinia oxyphylla was extracted by steam distillation, with an extraction rate of 2.8%; and Poria cocos was processed by cell wall breaking and pulverization to achieve a polysaccharide dissolution rate of 84.5%.

[0052] Example 4 The immune protective composition is made from the following raw materials: 35kg of Euryale ferox seeds, 15kg of Alpinia oxyphylla seeds, 15kg of Ginkgo biloba seeds, 15kg of Crataegus pinnatifida seeds, 15kg of Dioscorea opposita seeds, 15kg of Glycyrrhiza uralensis seeds, 15kg of Poria cocos, 15kg of Nelumbo nucifera seeds, and 15kg of Astragalus membranaceus.

[0053] The preparation method of its active ingredient includes the following steps: (1) Weigh out ginkgo, hawthorn, yam, licorice, lotus seed, and astragalus according to weight, add water and decoct twice, each time for 2 hours, filter, combine the filtrates to obtain the extract, concentrate the extract to 60% of the total weight of the extracted medicinal materials, cool to room temperature, add ethanol to make the alcohol content reach 50%, let stand, filter, recover the ethanol from the supernatant, and then concentrate under reduced pressure to a relative density of 1.15 at 50℃ to obtain the extract paste; (2) After drying Poria cocos at 50℃, use an ultra-micro pulverizer to break the cell wall and pulverize it. Pass it through a 280-mesh sieve and collect the fine powder of Poria cocos for later use. (3) Grind Alpinia oxyphylla into coarse powder, soak it, and then extract it by steam distillation. Collect the distillate, separate the upper volatile oil, and obtain Alpinia oxyphylla volatile oil. Encapsulate the Alpinia oxyphylla volatile oil with β-cyclodextrin. The weight ratio of Alpinia oxyphylla volatile oil to β-cyclodextrin is 1:6. The encapsulation temperature is 50℃ and the time is 2 hours to obtain the volatile oil inclusion complex.

[0054] (4) The active ingredient is obtained by mixing the extract, Poria cocos powder, and volatile oil inclusion complex.

[0055] The above-mentioned active ingredients are used to prepare an ointment, with the following additives: 550 kg of fructooligosaccharides and 65 kg of honey. The preparation method of the ointment includes the following steps: The honey and fructooligosaccharides are heated and melted, then mixed with the active ingredients and boiled to obtain the final product.

[0056] The cooking temperature is 80℃ and the cooking time is 2 hours.

[0057] Comparative Example Weigh out 10 kg each of ginkgo, hawthorn, yam, licorice, poria, lotus seed, alpinia oxyphylla, and astragalus, and 30 kg of euryale seed. Place them in a multi-functional extraction tank, add water and decoct twice, 2 hours each time. Add 8 times the amount of water for the first decoction and 6 times the amount of water for the second. Combine the two decoctions, filter, and concentrate the filtrate to half the weight of the herbs. Let it cool to room temperature, add ethanol to make the alcohol content reach 50%, let it stand, filter, recover the ethanol from the supernatant, and then concentrate under reduced pressure to a relative density of 1.12 at 50°C. Set aside. Separately, add the prescribed amount of honey to a jacketed kettle and heat to melt. Then add the extract and fructooligosaccharides to the kettle and melt while stirring until completely melted. Add purified water to make up to the full volume. Heat the liquid to 80°C and cook for 2 hours. Discard the sugar paste liquid.

[0058] The comparative production process uses traditional water decoction extraction, resulting in a low extraction rate of functional components. The polysaccharide extraction rate of Poria cocos using water extraction is only 25.3%. The effective component extraction rate of Alpinia oxyphylla after decoction is only 1.2%. Furthermore, the volatile oil from Alpinia oxyphylla in the formula has a pungent taste and poor palatability.

[0059] Experiment Example 1: Verification of immunoprotective efficacy based on zebrafish experiments: 1. Preparation of test sample: The active ingredient prepared in Example 3 was used for experiments. Before use, it was diluted with embryonic water to prepare working solutions of 4800, 2400, 1200, 600, 300, and 150 μg / mL.

[0060] 2. Drug resistance test of the active ingredient in Example 3 Zebrafish juveniles at 3dpf were collected and placed in 24-well plates, divided into 7 groups: a blank control group (embryo water) and test sample groups (150, 300, 600, 1200, 2400, and 4800 μg / mL of the active ingredient from Example 3). Each group had 3 wells, with 10 fish per well. The fish were incubated at 28.5℃ and their survival was observed over 72 h to determine the range of drug resistance concentrations of the test sample to the juveniles.

[0061] Results analysis: like Figure 1 As shown, in Example 3, when the concentration of the active ingredient was ≤2400 μg / mL, the survival rate of zebrafish within 72 h was ≥90.0%, with no significant difference compared to the control group. When the concentration was >2400 μg / mL, the survival rate of zebrafish juveniles decreased (mortality rate >10%), showing a significant difference compared to the control group. P <0.05). Therefore, the high, medium and low concentrations of the active ingredient in Example 3 will be selected as the high, medium and low concentrations of the test sample group.

[0062] 3. Chloramphenicol (CHL)-induced immune injury model Zebrafish juveniles at 3 dpf were collected and placed in 24-well plates, divided into 6 groups: blank control group (embryo water), model control group (100 μg / mL CHL working solution), positive control group (5 μg / mL berberine hydrochloride working solution + 100 μg / mL CHL working solution), and test sample group (600, 1200, and 2400 μg / mL of the active ingredient working solution from Example 3 + 100 μg / mL CHL working solution). Each group had 3 wells, with 10 fish per well. The fish were incubated at 28.5℃ for 24 h, fixed overnight with 4% paraformaldehyde (PFA), and stained with 0.4% Sudan Black. The number of neutrophils from the vent to the caudal fin of the juvenile fish was observed under a microscope.

[0063] Step 4: Neutrophil staining After fixing with 4% PFA overnight, wash three times with phosphate-Tween buffer (PBST) for 5 min each time. Stain with 0.4% Sudan Black B solution for 1 h, wash with 70% ethanol until colorless, rehydrate with PBST, and finally store in 50% glycerol.

[0064] Results analysis: The results of neutrophil staining in zebrafish tails are shown in the figure. Figure 2 The number of neutrophils in the tail of zebrafish in each treatment group was observed. Figure 3 Compared with the blank control group, the model control group showed significant differences, indicating successful model establishment. Compared with the model control group, the positive control group showed significant differences, suggesting that berberine hydrochloride can reverse the CHL-induced immune damage model and has a protective effect on the immune system. Figure 3 As shown, the product of Example 3 of the present invention can significantly (P<0.01) increase the number of neutrophils in the tail of zebrafish at concentrations of 600, 1200 and 2400 μg / mL, and exhibits a clear dose-response relationship.

[0065] The results showed that all concentrations of the active ingredient from Example 3 had a protective effect against the 100 μg / mL CHL-induced immune injury model. The effective concentrations were 2400 μg / mL > 1200 μg / mL > 600 μg / mL, suggesting a dose-response relationship between the active ingredient from Example 3 and the immunoprotective effect in zebrafish.

[0066] Under the experimental conditions, the resistance concentration of the active ingredient in Example 3 to 3-day-farmed zebrafish juveniles after 72 hours was ≤2400 μg / mL. Compared with the blank control group, the 100 μg / mL chloramphenicol-induced immune damage model group showed a significant difference. P<0.0001), indicating successful modeling with 100 μg / mL chloramphenicol. Compared with the model control group, the active ingredient in Example 3 and the positive control group at concentrations of 600 μg / mL, 1200 μg / mL, and 2400 μg / mL all showed significant differences ( ). P <0.01). The results showed that the active ingredient in Example 3 had a protective effect against the 100 μg / mL chloramphenicol-induced immune injury model, and there was a certain dose-response relationship.

Claims

1. A composition for promoting neutrophil proliferation, characterized in that, Made from the following parts by weight of raw materials: 30-40 parts of Euryale ferox, 10-20 parts of Alpinia oxyphylla, 10-20 parts of Ginkgo biloba, 10-20 parts of Crataegus pinnatifida, 10-20 parts of Dioscorea opposita, 10-20 parts of Glycyrrhiza uralensis, 10-20 parts of Poria cocos, 10-20 parts of Nelumbo nucifera, and 10-20 parts of Astragalus membranaceus.

2. The composition for promoting neutrophil proliferation according to claim 1, characterized in that, The preparation method of its active ingredient includes the following steps: (1) Weigh out ginkgo, hawthorn, yam, licorice, lotus seed, and astragalus according to the weight parts. Add water equivalent to 6-10 times the total weight of the medicinal materials and decoct 1-3 times, each time for 1-3 hours. Filter and combine the filtrates to obtain the extract. Concentrate the extract to 40-60% of the total weight of the extracted medicinal materials. Cool to room temperature and add ethanol to make the alcohol content 40-80%. Let stand, filter, recover the ethanol from the supernatant, and then concentrate under reduced pressure to a relative density of 1.10-1.15 at 50℃ to obtain the extract paste. (2) After drying Poria cocos, use an ultra-micro pulverizer to break down the cell walls and pulverize it through a 250-320 mesh sieve to collect the fine powder for later use. (3) Grind Alpinia oxyphylla into coarse powder, soak it, and then extract it by steam distillation. Collect the distillate, separate the upper volatile oil, and obtain Alpinia oxyphylla volatile oil. Encapsulate the Alpinia oxyphylla volatile oil with β-cyclodextrin to obtain volatile oil inclusion complex. (4) Mix the extract, Poria cocos powder, and volatile oil inclusion complex to obtain the final product.

3. The composition for promoting neutrophil proliferation according to claim 2, characterized in that: In step (2), the drying temperature of Poria cocos is 40-50℃.

4. The composition for promoting neutrophil proliferation according to claim 2, characterized in that: In step (3), when the volatile oil of Alpinia oxyphylla is encapsulated, the weight ratio of the volatile oil of Alpinia oxyphylla to β-cyclodextrin is 1:6-8.

5. The composition for promoting neutrophil proliferation according to claim 4, characterized in that: During the encapsulation process, the temperature should be 40-50℃ and the time should be 1-2 hours.

6. The composition for promoting neutrophil proliferation according to any one of claims 2-5, characterized in that, Its ointment contains the following additives in parts by weight: 400-600 parts of fructooligosaccharides and 50-80 parts of honey.

7. The composition for promoting neutrophil proliferation according to claim 6, characterized in that, The preparation method of the ointment includes the following steps: The honey and fructooligosaccharides are heated and melted, then mixed with the active ingredients and boiled to obtain the final product.

8. The composition for promoting neutrophil proliferation according to claim 7, characterized in that: The cooking temperature is 70-90℃, and the cooking time is 1-3 hours.

9. Use of the traditional Chinese medicine composition according to any one of claims 1-8 in the preparation of health products or medicines for the prevention and / or adjunctive treatment of immunodeficiency.

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