Preparation method of traditional Chinese medicine preparation with hosta plantaginea flower polysaccharide component

Abstract

The invention relates to the technical field of traditional Chinese medicines, and discloses a preparation method of a traditional Chinese medicine preparation with a hosta plantaginea flower polysaccharide component, and the preparation method comprises the following steps: S100, preparing hosta plantaginea flower polysaccharide; s200, adding an equal amount of water into the crude polysaccharide extract, and carrying out in-vitro gastrointestinal bionic system digestion; the digestion time is 1 h, and collecting digestive juice after digestion is completed; centrifuging and collecting digestion supernate; and preparing the hosta plantaginea flower polysaccharide of the 1 KDa-2 KDa segment or the 100 KDa-120 KDa segment into the traditional Chinese medicine preparation. The hosta plantaginea flower polysaccharide buccal tablets or granules prepared by the invention have a relatively strong anti-inflammatory effect, the polysaccharide with high activity is prepared by adopting an in-vitro digestion method, the activity of the polysaccharide can be improved, and the polysaccharide of a specific section is selected, so that the acute and chronic pharyngitis can be effectively treated.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine, and in particular to a method for preparing a traditional Chinese medicine preparation containing hosta polysaccharide components. Background Art

[0002] Acute pharyngitis is an acute inflammation of the pharyngeal mucosa, submucosal tissue and lymphoid tissue. Its symptoms are dryness, burning, roughness, pain in the pharynx, and pain when swallowing. The pain in the pharynx can radiate to the ears and neck on both sides. If the inflammation involves the larynx, symptoms such as cough and hoarseness may occur. It is often part of an upper respiratory tract infection and can occur alone or secondary to acute rhinitis. The causes of acute pharyngitis include viruses (such as influenza viruses, adenoviruses, etc.), bacterial infections (such as streptococci, staphylococci, etc.), and physical and chemical factors, such as high temperature, dust, smoke, irritating gases, tobacco, alcohol, spicy foods, etc. Acute pharyngitis is a common disease. For acute pharyngitis caused by bacteria, antibiotics (such as amoxicillin) can be used to kill bacteria and indirectly relieve the inflammatory state. It is worth noting that although acute pharyngitis caused by viruses and physical and chemical factors is self-limiting, the attack period will still cause great pain to the patient. At present, there is still a lack of specific drugs to treat or relieve acute pharyngitis. More importantly, repeated episodes of acute inflammation can transform into chronic inflammation and then cause canceration of the nasopharyngeal mucosa. Therefore, it is of great value to develop drugs to treat acute pharyngitis.

[0003] Hosta flower is the flower of Hosta (H. plantaginea) of the genus Hosta of the Liliaceae family. Its Mongolian name is "Has-Hatahuer-Qiqige". It is a special and commonly used medicinal material in Mongolian medicine. "Chinese Materia Medica" (Mongolian Medicine Volume) records that it has the effects of clearing heat, detoxifying, relieving cough, and benefiting the throat. It is mainly used to treat sore throat, hoarseness, lung heat, and toxic heat. Mongolian medicines based on Hosta flower, such as Hosta Qingyan Shiwuwei Pills and Gaoletu-Baori-6, are used to treat sore throat, tonsillitis, acute and chronic pharyngitis, asthma, hoarseness and other diseases.

[0004] Existing shortcomings: The existing technology also has the extraction and preparation of hosta flower extracts, but the composition of the existing hosta flower extracts is relatively complex, containing flavonoids, phenylpropanoids, polysaccharides, tannins, alkaloids and other substances with low activity, low content of effective anti-inflammatory components, low therapeutic index (TI), and the effect on the treatment of acute and chronic pharyngitis is not very obvious. In addition, the hosta flower water extract contains a large amount of non-anti-inflammatory small molecules, which may cause adverse drug reactions. For example, alkaloids are prone to cause liver and kidney damage.

[0005] Therefore, we propose a method for preparing a traditional Chinese medicine preparation having Hosta polysaccharide (having high anti-inflammatory activity and no toxicity) as an ingredient. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a traditional Chinese medicine preparation containing hosta polysaccharide components, thereby solving the problems raised in the background technology.

[0007] The present application embodiment provides a method for preparing a traditional Chinese medicine preparation having a hosta polysaccharide component, comprising the following steps:

[0008] S100: preparation of Hosta polysaccharide;

[0009] S200: Prepare Chinese medicine preparations from the 1KDa-2KDa or 100KDa-120KDa segments of Hosta japonica polysaccharides.

[0010] Preferably, the specific steps of S100 are:

[0011] S101: crushing the dried hosta flower raw material with a crusher and sieving;

[0012] S102: adding anhydrous ethanol, stirring to extract fat-soluble pigments and some impurities, and collecting the precipitate by centrifugation;

[0013] S103: adding pure water to the precipitate, extracting in a 60°C water bath, and collecting the supernatant extract by centrifugation;

[0014] S104: Combine the two extracts, concentrate by vacuum rotary evaporation to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation;

[0015] S105: collecting the precipitated solid by centrifugation, i.e., the crude polysaccharide extract;

[0016] S106: adding an equal amount of water to the crude polysaccharide extract and subjecting it to in vitro gastrointestinal bionic system digestion; the digestion time is 1 hour, and after the digestion is completed, the digestion fluid is collected; and the digestion supernatant is collected by centrifugation;

[0017] S700: remove the digestion supernatant, use a peristaltic pump to pass the digestion supernatant through 1KDa, 2KDa, 4KDa, 100KDa, and 120KDa molecular retention plates respectively; the supernatant is divided into below 1KDa, 1KDa-2KDa, 2KDa-4KDa, 4KDa-100KDa, and 100KDa-120KDa; collect the 1KDa-2KDa segment and the 100KDa-120KDa segment solutions, concentrate, freeze-dry and weigh the mass of the segment.

[0018] Preferably, the specific steps of S100 are:

[0019] S101: crushing the dried Hosta flower raw material with a crusher and passing it through a 40-80 mesh sieve;

[0020] S102: adding anhydrous ethanol, stirring to extract fat-soluble pigments and some impurities, centrifuging at 6000 g for 10 min, and collecting the precipitate;

[0021] S103: adding pure water to the precipitate, extracting in a water bath at 60°C for 4 h, centrifuging at 6000 g for 10 min, and collecting the supernatant extract;

[0022] S104: Combine the two extracts, concentrate by vacuum rotary evaporation to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation;

[0023] S105: centrifuge at 8000 g for 10 min to collect the precipitated solid, i.e., the crude polysaccharide extract;

[0024] S106: adding 300 mL of water to 300 g of the crude polysaccharide extract for in vitro gastrointestinal bionic system digestion; digestion time 1 h, after digestion, collecting the digestion fluid; centrifuging at 3000 g for 10 min, collecting the supernatant;

[0025] S700: 300 mL of the unsalted supernatant was measured and peristaltic pumped to pass the supernatant through 1 KDa, 2 KDa, 4 KDa, 100 KDa and 120 KDa molecular retention plates respectively; the supernatant was divided into below 1 KDa, 1 KDa-2 KDa, 2 KDa-4 KDa, 4 KDa-100 KDa and 100 KDa-120 KDa; the 1 KDa-2 KDa segment and the 100 KDa-120 KDa segment solutions were collected, concentrated, freeze-dried and the mass of the segment was weighed.

[0026] Preferably, in S106, the experimental parameters of the in vitro gastrointestinal bionic system are as follows: saliva pH: 7, Ca 2+ The concentration is 1.5mM, salivary amylase is 150U / mL, the simulated saliva volume is 50mL, the simulated gastric fluid pH is 2.0, and Ca 2+ Concentration: 0.15mM, pepsin: 4000U / mL, gastric juice secretion rate: 2mL / min, HCl secretion rate: 90uL / min, gastric rolling extrusion frequency: 3 times / min, simulated intestinal fluid PH: 7, Ca 2+ The concentration is 0.6mM, pancreatic enzyme: 200U / mL, and the simulated intestinal fluid secretion rate is 3.75mL / min.

[0027] Preferably, in S200, the 1KDa-2KDa Hosta polysaccharide is made into polysaccharide lozenges by a tablet pressing machine.

[0028] Preferably, in S200, the 100KDa-120KDa range of Hosta polysaccharides are used to prepare Hosta polysaccharide granules.

[0029] Preferably, the specific steps of S200 are:

[0030] S201: preparing 100KDa-120KDa range Hosta polysaccharides and auxiliary materials, crushing the freeze-dried polysaccharides and auxiliary materials, and passing through a No. 5 sieve;

[0031] S202: using an equal amount mixing method, mixing the auxiliary material with the least amount first, and then increasing the amount of powder by an equal amount to mix thoroughly;

[0032] S203: Granulation: Add a certain amount of edible ethanol solution to the mixed powder to make the powder agglomerate and disperse when touched; pass through a 16-mesh sieve to obtain wet granules;

[0033] S204: Drying: Place the obtained wet granules in a constant temperature forced air drying oven at 60°C and dry for 0.5h, controlling the moisture content to not exceed 10%;

[0034] S205: Granulation: The dried granules are passed through a 16-mesh sieve again;

[0035] S206: tableting: uniform granules are passed through a tablet press to produce 0.6 g / tablet polysaccharide tablets;

[0036] S207: Sterilization: Put the lozenges into a transparent glass bottle, seal the cap, and sterilize at 160°C for 2 hours. After sterilization, wait for the temperature to drop below room temperature to obtain the finished product.

[0037] Preferably, the weight ratio of the 100KDa-120KDa hosta polysaccharide to the auxiliary materials is 6:4; the auxiliary materials include fillers, sweeteners, flavoring agents and lubricants; the filler is mannitol or microcrystalline cellulose, the sweetener is xylitol, the flavoring agent is citric acid, and the lubricant is magnesium stearate.

[0038] Preferably, the specific steps of S200 are: preparing granules by wet granulation process. Pass the hosta polysaccharide and auxiliary materials through an 80-mesh sieve for standby use, accurately weigh the corresponding components according to the prescription amount, add the auxiliary materials in equal amounts in increments to make them evenly mixed with the main drug, and add the wetting agent in an atomized form, and stir thoroughly until the soft material is formed; pass the soft material through a 16-mesh sieve to obtain wet granules, then transfer to an electric heating blast drying oven for drying, and pass through a 24-mesh sieve after cooling to room temperature to obtain the hosta polysaccharide granules.

[0039] Preferably, the weight ratio of the 1KDa-2KDa segment Hosta polysaccharide to the auxiliary material is 6:4; the auxiliary material includes a diluent and a wetting agent, the diluent is microcrystalline cellulose and mannitol, and the ratio of microcrystalline cellulose to mannitol is 3:1; the wetting agent is water and ethanol. .

[0040] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0041] The hosta polysaccharide lozenges or granules prepared by the invention have strong anti-inflammatory effects. The polysaccharide with high activity is prepared by an in vitro digestion method, which can improve the activity of the polysaccharide. The polysaccharide of specific segments is selected, and the TI of the 1KDa-2KDa segment is 456, and the TI of the 100KDa-120KDa segment is 521, which is much higher than the prior art and can effectively treat acute and chronic pharyngitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0043] Figure 1 The expression levels of NO, TNF-α, IL-1β, and IL-6 inflammatory factors in Example 1 of the present invention;

[0044] Figure 2 This is a hematoxylin and eosin (H&E) staining diagram in Example 1 of the present invention;

[0045] Figure 3 The expression levels of NO, TNF-α, IL-1β, and IL-6 inflammatory factors in Example 2 of the present invention;

[0046] Figure 4 This is a hematoxylin and eosin (H&E) staining image in Example 2 of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0048] Example 1: The present invention proposes a method for preparing a lozenge containing Hosta polysaccharide:

[0049] 1. Instruments and Materials:

[0050] 1.1 Materials:

[0051] Hosta flower.

[0052] 2 Methods:

[0053] 2.1 Preparation of Hosta polysaccharide:

[0054] 2.1 Preparation of Hosta polysaccharide (1KDa-2KDa segment):

[0055] (1) The dried sample / raw material is crushed with a crusher and passed through a 60-mesh sieve.

[0056] (2) Add anhydrous ethanol, stir to extract fat-soluble pigments and some impurities, centrifuge at 6000 g for 10 min, and collect the precipitate.

[0057] (3) Add pure water to the precipitate, extract in a 60°C water bath for 4 h, centrifuge at 6000 g for 10 min, and collect the supernatant. (4) Combine the two extracts, concentrate by vacuum rotary evaporation to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation.

[0058] (5) Centrifuge at 8000 g for 10 min to collect the precipitated solid, i.e., the crude polysaccharide extract.

[0059] (6) 300 g of crude polysaccharide extract was added to 300 mL of water and digested in vitro using a gastrointestinal biomimetic system. The experimental parameters were as follows: saliva pH: 7, Ca 2+ The concentration is 1.5mM, salivary amylase is 150U / mL, the simulated saliva volume is 50mL, the simulated gastric fluid pH is 2.0, and Ca 2+ Concentration: 0.15mM, pepsin: 4000U / mL, gastric juice secretion rate: 2mL / min, HCl secretion rate: 90uL / min, gastric rolling extrusion frequency: 3 times / min, simulated intestinal fluid PH: 7, Ca 2+ The concentration is 0.6mM, pancreatin: 200U / mL, and the simulated intestinal fluid secretion rate is 3.75mL / min. The digestion time is 1h. After digestion, the digestion fluid is collected; centrifugation (3000g, 10min) is performed and the supernatant is collected. Digestion by in vitro gastrointestinal bionic system can greatly increase the yield of two-stage active polysaccharides, and its yield is three times that of indigestion.

[0060] (7) 300 mL of the unsalted supernatant was measured and peristaltic pumped to pass the supernatant through the 1 KDa, 2 KDa, 4 KDa, 100 KDa, and 120 KDa molecular cutoff plates. The supernatant was divided into less than 1 KDa, 1 KDa-2 KDa, 2 KDa-4 KDa, 4 KDa-100 KDa, and 100 KDa-120 KDa. The 1 KDa-2 KDa solution was collected, concentrated, freeze-dried, and the mass of the section was weighed.

[0061] 2.2 Preparation of lozenges:

[0062] Screening of raw materials and auxiliary materials: The polysaccharides and auxiliary materials obtained by freeze-drying are crushed and passed through a No. 5 sieve to ensure the uniformity of the quality of the prepared lozenges.

[0063] Mixing: Use the equal mixing method, mix the auxiliary materials with the least amount first, and then add the powder in equal amounts to mix thoroughly. Granulation: Add a certain amount of edible ethanol solution to the mixed powder to make the powder agglomerate and disperse when touched. Pass through a 16-mesh screen to obtain wet granules.

[0064] Drying: Place the wet granules in a constant temperature forced air drying oven at 60°C and dry for 0.5 h, controlling the moisture content to not exceed 10%.

[0065] Granulation: Pass the dried granules through a 16-mesh sieve again.

[0066] Tableting: The uniform granules are passed through a tablet press to produce polysaccharide tablets of 0.6 g / tablet.

[0067] Sterilization: Put the lozenges into a transparent glass bottle, seal the cap, and sterilize at 160℃ for 2h. After sterilization, wait for the temperature to cool down to below room temperature to obtain the finished product.

[0068] 2.3 Evaluation indicators and standards for lozenge prescriptions:

[0069] 1. Single factor test:

[0070] (1) Investigation of the proportion of fillers: Based on preliminary experiments, it was determined that 60% polysaccharide was used as the main ingredient, and mannitol or microcrystalline cellulose was selected as the filler. The sensory evaluation of the lozenges, disintegration time, and friability index were investigated according to mannitol: microcrystalline cellulose = 1:2, 2:4, 1:1, 4:2, and 2:1, respectively, to determine the proportion and amount of fillers.

[0071] (2) Investigation of the proportion of xylitol dosage: The dosage of polysaccharide powder was determined to be 60% unchanged, and xylitol was added in amounts of 5%, 7%, 9%, 11%, and 13% as a sweetener. Fillers were added to make the system 100%, and the optimal xylitol dosage was determined based on the comprehensive score.

[0072] (3) Investigation of the proportion of citric acid dosage: The dosage of polysaccharide powder (60%) and xylitol (14%) was determined to remain unchanged, and citric acid was added in amounts of 2%, 3%, 4%, 5%, and 6% as a flavoring agent. Fillers were added to make the system 100%, and the optimal citric acid dosage was determined based on the comprehensive score.

[0073] (4) Investigation of the ratio of magnesium stearate as a lubricant: The dosage of polysaccharide powder (60%), xylitol (14%) and citric acid (5%) was kept unchanged, and the addition of magnesium stearate at 0.4%, 0.5%, 0.6%, 0.7% and 0.8% as a lubricant was investigated. A filler was added to make the system 100%. The optimal dosage of citric acid was determined based on the comprehensive score.

[0074] (5) Effect of different volume fractions of ethanol on granulation: The dosage ratio of polysaccharide powder 60%, xylitol 14%, citric acid 5%, and the ratio of the two fillers (mannitol: microcrystalline cellulose) = 5:3 was determined to remain unchanged. During granulation, 55%, 65%, 75%, 85%, and 95% edible ethanol were used to examine the granulation conditions.

[0075] 2. Comprehensive score of polysaccharide lozenges:

[0076] The comprehensive scoring method combining multiple indicators is used to optimize the tableting formula parameters, including the total scores of sensory score, friability, and disintegration time. The comprehensive score is the sum of the scores of each indicator. If the sensory indicator is conducive to the molding of the product, 0.1 times of its value is taken as the evaluation value; the two indicators of friability and disintegration time are not conducive to the molding of the product, and the reciprocal of the friability value and 100 times the reciprocal of the disintegration time limit are taken as the scoring values. The size of the comprehensive score directly reflects the molding quality of the tablet candy. Comprehensive score calculation = 0.1 × sensory score + 100 / disintegration time limit + 1 / friability.

[0077] (1) Sensory evaluation People who have undergone sensory training were randomly selected to score the products based on four aspects.

[0078] (2) The disintegration time is determined according to the disintegration time test method (General Chapter 0921) in the 2020 edition of the Chinese Pharmacopoeia [1].

[0079] (3) The friability is determined according to the friability test method (General Chapter 0923) in the 2020 edition of the Chinese Pharmacopoeia [1].

[0080] Through scoring calculation, the prescription of Hosta polysaccharide lozenges is 600 g (60%) of polysaccharide powder, 140 g (14%) of xylitol, 5 g (5%) of citric acid, 20.2 g (20.2%) of two fillers in total, the ratio (mannitol: microcrystalline cellulose) = 5:3, 0.8 g (0.8%) of magnesium stearate, and 95% edible ethanol for granulation.

[0081] 3. Efficacy evaluation:

[0082] 3. In vivo evaluation of polysaccharide treatment for acute pharyngitis:

[0083] 3.1 Experimental methods:

[0084] Thirty-five SD rats were divided into four groups (weight 180-220g, half male and half female), namely control group, model group, positive drug group, model group, low-dose group, and high-dose group. The model group, low-dose group, and high-dose group were sprayed with 25% ammonia water into the pharynx of the rats using a laryngeal sprayer (spread area 150mm 2 ), twice a day, once in the morning and once in the afternoon, 3 sprays each time (total volume is about 0.2ml), spray continuously for 3 days. The control group was treated with normal saline in the same way. After the model was successfully established, normal saline was given to the control group and the model group, and the low-dose and high-dose groups (lozenges) were given doses of 0.09g / kg / day and 0.27g / kg / day, respectively. After euthanasia, the throat and serum were collected.

[0085] 3.1.1 Detection of NO and TNF-α, IL-1β, IL-6 inflammatory factor expression levels:

[0086] ELISA kits (Jiangsu Jingmei Biotechnology Co., Ltd.) were used to detect the levels of TNF-α, NO, IL-1β, and IL-6 in serum according to the instructions of the kits.

[0087] The results are as follows Figure 1 As shown in the results, compared with the control group, the release level of serum NO in the model group was significantly reduced, and the release levels of TNF-α, IL-1β, and IL-6 were significantly increased. After treatment with low- and high-dose polysaccharides from Hosta, the release level of NO was significantly increased, and the levels of TNF-α, IL-1β, and IL-6 were significantly reduced, and they were dose-dependent (P<0.05). Among them, there was no significant difference in the improvement of inflammatory cytokines in the pharyngeal tissue of AP model rats between the high-dose group and the positive group of Hosta polysaccharide (P>0.05). In summary, Hosta polysaccharide lozenges have a strong anti-inflammatory effect (the effect of treating acute pharyngitis).

[0088] 3.2.2 Hematoxylin and eosin (H&E) staining:

[0089] The histopathological changes of pharyngeal tissues of rats in each group were detected by hematoxylin and eosin (H&E) staining. The residual fixative of the pharyngeal tissue was washed, dehydrated with ethanol, and the tissue was transparent and waxed. After the paraffin solidified, it was stored in a -4℃ refrigerator. The tissue wax block was sliced, and the slice thickness was 4-5μm. The slices were flattened on a slide in 45℃ water, baked, and baked at 65℃ for 1h. The baked slices were dewaxed and hydrated, stained with hematoxylin stain for 5-10min, rinsed with distilled water, differentiated with 1% hydrochloric acid aqueous solution, rinsed with distilled water, then blued with 0.2% ammonia aqueous solution, rinsed, and stained with eosin for 3-5min; rinsed with distilled water. Then dehydrated and transparent, dried, and finally sealed with neutral gum. The slices were examined under a microscope to observe the mucosal epithelial cells, submucosal glandular hyperplasia, inflammatory cell infiltration, and congestion.

[0090] like Figure 2 As shown in the figure, H&E staining showed that compared with the control group, the model group rats had obvious pathological damage in the pharyngeal tissue. The rats had desquamation of mucosal epithelial cells, heavy inflammatory cell infiltration, and pharyngeal congestion. Compared with the model group rats, these pathological changes in the pharyngeal tissue of rats were significantly improved after treatment with the positive drug group, low-dose HPFP and high-dose HPFP. Among them, the positive drug and high-dose groups improved more significantly, and the low-dose group improved less than the positive drug and high-dose groups, but the degree of damage was lower than that of the model group. In summary: Hosta polysaccharide lozenges have a strong anti-inflammatory effect (the effect of treating acute pharyngitis).

[0091] Example 2: The present invention proposes a method for preparing granules containing Hosta polysaccharide components:

[0092] 1. Instruments and Materials:

[0093] 1.1 Materials:

[0094] Hosta flower.

[0095] 2 Methods:

[0096] 2.1 Preparation of Hosta polysaccharide (100KDa-120KDa segment):

[0097] (1) The dried sample / raw material is crushed with a crusher and passed through a 60-mesh sieve.

[0098] (2) Add anhydrous ethanol, stir to extract fat-soluble pigments and some impurities, centrifuge at 6000 g for 10 min, and collect the precipitate.

[0099] (3) Add pure water to the precipitate, extract in a 60°C water bath for 4 h, centrifuge at 6000 g for 10 min, and collect the supernatant. (4) Combine the two extracts, concentrate by vacuum rotary evaporation to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation.

[0100] (5) Centrifuge at 8000 g for 10 min to collect the precipitated solid, i.e., the crude polysaccharide extract.

[0101] (6) 300 g of crude polysaccharide extract was added to 300 mL of water and digested in vitro using a gastrointestinal biomimetic system. The experimental parameters were as follows: saliva pH: 7, Ca 2+ The concentration is 1.5mM, salivary amylase is 150U / mL, the simulated saliva volume is 50mL, the simulated gastric fluid pH is 2.0, and Ca 2+ Concentration: 0.15mM, pepsin: 4000U / mL, gastric juice secretion rate: 2mL / min, HCl secretion rate: 90uL / min, gastric rolling extrusion frequency: 3 times / min, simulated intestinal fluid PH: 7, Ca 2+ The concentration was 0.6 mM, pancreatin: 200 U / mL, and the simulated intestinal fluid secretion rate was 3.75 mL / min. The digestion time was 1 hour, and after digestion, the digestion fluid was collected; centrifugation (3000 g, 10 min) was performed and the supernatant was collected.

[0102] (7) 300 mL of the unsalted supernatant was measured and peristaltic pumped to pass the supernatant through the 1KDa, 2KDa, 4KDa, 100KDa, and 120KDa molecular retention plates. The supernatant was divided into less than 1KDa, 1KDa-2KDa, 2KDa-4KDa, 4KDa-100KDa, and 100KDa-120KDa. The 100KDa-120KDa solution was collected, concentrated, freeze-dried, and the mass of the segment was weighed. (The 100KDa-120KDa segment was used to prepare granules)

[0103] 2.2 Preparation of granules:

[0104] The granules were prepared by wet granulation. The hosta polysaccharide and auxiliary materials were sieved through an 80-mesh sieve for later use. The corresponding ingredients were accurately weighed according to the prescription amount. The auxiliary materials were added in equal amounts and gradually mixed with the main drug. The wetting agent was added in an atomized form and stirred thoroughly until the soft material was formed. The soft material was sieved through a 16-mesh sieve to obtain wet granules, which were then transferred to an electric blast drying oven for drying. After cooling to room temperature, the granules were sieved through a 24-mesh sieve to obtain the hosta polysaccharide granules.

[0105] 2.3 Granule prescription evaluation indicators and standards:

[0106] 2.3.1 Forming rate: The particle forming rate is determined by double sieving method, with No. 1 sieve as the upper sieve and No. 5 sieve as the lower sieve. An appropriate amount of particles are placed in the upper sieve, covered and flapped back and forth for 3 minutes. The particles that cannot pass through No. 1 sieve and the powder that passes through No. 5 sieve are weighed and the particle forming rate is calculated. The particle forming rate score is forming rate × 100. Calculation formula: forming rate = 1-M1 / M × 100%, where M is the total mass of the particles, and M1 is the mass of the powder that does not pass through No. 1 sieve and the particles that pass through No. 5 sieve.

[0107] 2.3.2 Angle of repose: The angle of repose is tested by the limited bottom surface method. Slowly inject enough particles to fill the tray along the funnel wall above the angle of repose tester until the particles cover the tray and form a cone. The acute angle between the inclined surface of the cone and the horizontal surface at the bottom is the angle of repose α. When 0≤α≤30, 100 points are given, 30<α≤40, 80 points are given, and α>40, 60 points are given.

[0108] 2.3.3 Dissolution rate: Accurately weigh 0.5 g of dry Hosta polysaccharide in a test tube, add 5 mL of hot water, vortex for 5 min, centrifuge at 3500 r / min for 10 min, discard the supernatant, dry the residue at 60°C to a constant mass, accurately weigh the residue mass, calculate the particle dissolution rate, and the particle dissolution rate score is dissolution rate × 100. The calculation formula is: dissolution rate = (total mass of particles - mass of residue) / total mass of particles × 100%.

[0109] 2.3.4 Moisture absorption rate: Place the desiccator containing the supersaturated NaCl solution at room temperature for 48 hours to make the relative humidity 75%. Accurately weigh 0.5g of polysaccharide powder and granules and spread them in a weighing bottle that has been dried to a constant mass, with a thickness not exceeding 5mm. Dry at 60°C to a constant mass, accurately weigh, open the lid and place in a desiccator, accurately weigh at 0, 6, 12, 24, 36, 48, 60, and 72 hours, and calculate the moisture absorption rate. Calculation formula: Moisture absorption rate = (mass after moisture absorption - mass before moisture absorption) / mass before moisture absorption × 100%.

[0110] 2.3.5 Comprehensive score: The molding rate (A), angle of repose (B) and dissolution rate (C) of the granules are used as evaluation indicators for comprehensive scoring. The three have the same weight. The comprehensive score = A / 3+B / 3+C / 3.

[0111] 2.4 Single factor method to screen granule prescription process:

[0112] 2.4.1 Diluent: Select microcrystalline cellulose, mannitol and two other excipients, mix each excipient with y-Hosta scabra polysaccharide at a mass ratio of 3:1, and use the moisture absorption rate at a temperature of 25°C and a relative humidity of 75% as the evaluation index to select suitable excipients.

[0113] 2.4.2 Diluent ratio: The ratio of polysaccharide to diluent in the prescription affects the properties of the intermediate material. If the proportion of polysaccharide is too high, the viscosity of the soft material will be too high to be sieved. Conversely, it will make the soft material difficult to shape.

[0114] 2.4.3 Wetting agent concentration: Hosta polysaccharide is a water extract. Water can induce its viscosity, while alcohol inhibits its viscosity. Water and ethanol are used as wetting agents in the prescription to adjust the viscosity of the material. The effects of different volume fractions of ethanol (50%, 60%, 70%, 80%, 90%) on the soft material and particles are investigated. Granulation and scoring are performed, and the appropriate wetting agent concentration is selected according to the scoring.

[0115] 2.4.4 Wetting agent dosage: Using 75% ethanol as the wetting agent, the effects of different wetting agent dosages (7.5%, 12.5%, 17.5%, 22.5%, 27.5%) on the soft material and granules were investigated. Granulation and scoring were performed, and the appropriate wetting agent dosage was selected based on the scoring results.

[0116] 2.4.5 Drying temperature: Investigate the effect of different temperatures (60, 70, 80°C) on the drying effect of particles. Place the wet particles in a blast drying oven, set the corresponding temperature, dry for 1 hour, observe the condition of the particles, test the moisture content of the particles, and select the appropriate drying temperature according to the condition of the particles.

[0117] 2.4.6 Drying time: Take 60℃ as the drying temperature, examine the effect of different times (60, 75, 90min) on the drying effect of the particles, place the wet particles in a blast drying oven, set the corresponding drying time, observe the condition of the particles, test the moisture content of the particles, and determine the appropriate drying time based on the condition of the particles.

[0118] 2.5 Star-point design-response surface methodology to optimize prescription:

[0119] Based on the single factor experiment, the central composite design-response surface optimization formula was adopted, and the molding rate, angle of repose, and dissolution rate were used as the evaluation indicators of the granule preparation process. The ratio of Hosta polysaccharide to diluent, wetting agent concentration, and wetting agent dosage were investigated. Three batches of process validation were carried out according to the formula optimized by central composite design-response surface methodology, and the molding rate, angle of repose, and solubility were measured. The comprehensive score and average relative error were calculated to evaluate the feasibility of the model.

[0120] 2.6 Granule Quality Evaluation:

[0121] 2.6.1 Properties: Take 3 batches of granule samples and check their appearance, shape, color, odor, etc., and observe whether the granules are dry, uniform, of uniform color, and whether they are damp, softened, agglomerated, or deliquescent.

[0122] 2.6.2 Particle size: Take 2g of each of three batches of granule samples and calculate the particle forming rate. If the particle forming rate is greater than 85%, the granules are considered qualified.

[0123] 2.6.3 Moisture content: According to the requirements of "0104 Granules" in the 2020 edition of the Chinese Pharmacopoeia, the moisture content of the granules is determined by the drying method. Take 2g of each of the three batches of granules and spread them flat in a weighing bottle that has been dried to a constant mass. After accurate weighing, put them in an oven and dry them to a constant mass. Record the mass of the granules after drying. Calculate the moisture content (%) based on the reduction in moisture mass before and after drying. Granules with moisture content <8.0% are considered qualified. The calculation formula is: Moisture content = (total mass of granules - mass of granules after drying) / total mass of granules × 100%.

[0124] 2.6.4 Solubility: Take 2g of each of three batches of granules and test their solubility. If all the granules are dissolved or slightly turbid, the solubility is considered acceptable.

[0125] 2.6.5 Determination of total sugar content in granules: The phenol-sulfuric acid method was used to determine the content of Hosta polysaccharide in granules. (1) Drawing of glucose standard curve: Accurately weigh 0.020 0g of anhydrous glucose in a beaker, add ultrapure water to dissolve, and transfer to a volumetric flask to make up to 100mL, to obtain a glucose reference solution with a mass concentration of 0.2mg / mL. Accurately measure 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0mL of the reference solution in a test tube, and add ultrapure water to 2mL. Accurately pipette 1.0mL of the above solution into a test tube, add 1.0mL of 6% phenol solution, vortex mix, add 5.0mL of concentrated sulfuric acid, vortex mix, let stand at room temperature for 30min, and measure the absorbance value at a wavelength of 490nm. Draw a standard curve with the reference concentration as the horizontal axis and the absorbance value as the vertical axis. (2) Determination of polysaccharide content: Take 1.0 g of Hosta polysaccharide granules, add ultrapure water and sonicate to dissolve, filter, transfer the filtrate to a volumetric flask and make up to 100 mL, shake well to obtain the test solution, add reagents in sequence for color development, measure the absorbance at a wavelength of 490 nm, and substitute the absorbance into the standard curve to calculate the polysaccharide content.

[0126] 3.1.1 Detection of NO and TNF-α, IL-1β, IL-6 inflammatory factor expression levels:

[0127] ELISA kits (Jiangsu Jingmei Biotechnology Co., Ltd.) were used to detect the levels of TNF-α, NO, IL-1β, and IL-6 in serum according to the instructions of the kits.

[0128] The results are as follows Figure 3 As shown in the results, compared with the control group, the release level of serum NO in the model group was significantly reduced, and the release levels of TNF-α, IL-1β, and IL-6 were significantly increased. After treatment with low- and high-dose polysaccharides from Hosta, the release level of NO was significantly increased, and the levels of TNF-α, IL-1β, and IL-6 were significantly reduced, and they were dose-dependent (P<0.05). Among them, there was no significant difference in the improvement of inflammatory cytokines in the pharyngeal tissue of AP model rats between the high-dose group and the positive group of Hosta polysaccharide (P>0.05). In summary, Hosta polysaccharide lozenges have a strong anti-inflammatory effect (the effect of treating acute pharyngitis).

[0129] 3.2.2 Hematoxylin and eosin (H&E) staining

[0130] The histopathological changes of pharyngeal tissues of rats in each group were detected by hematoxylin and eosin (H&E) staining. The residual fixative of the pharyngeal tissue was washed, dehydrated with ethanol, and the tissue was transparent and waxed. After the paraffin solidified, it was stored in a -4℃ refrigerator. The tissue wax block was sliced, and the slice thickness was 4-5μm. The slices were flattened on a slide in 45℃ water, baked, and baked at 65℃ for 1h. The baked slices were dewaxed and hydrated, stained with hematoxylin stain for 5-10min, rinsed with distilled water, differentiated with 1% hydrochloric acid aqueous solution, rinsed with distilled water, then blued with 0.2% ammonia aqueous solution, rinsed, and stained with eosin for 3-5min; rinsed with distilled water. Then dehydrated and transparent, dried, and finally sealed with neutral gum. The slices were examined under a microscope to observe the mucosal epithelial cells, submucosal glandular hyperplasia, inflammatory cell infiltration, and congestion.

[0131] The results are as follows Figure 4 As shown:

[0132] H&E staining showed that compared with the control group, the model group rats had obvious pathological damage in the pharyngeal tissue. The rats had desquamation of mucosal epithelial cells, heavy inflammatory cell infiltration, and pharyngeal congestion. Compared with the model group rats, these pathological changes in the pharyngeal tissue of rats were significantly improved after treatment with the positive drug group, low-dose HPFP and high-dose HPFP. Among them, the positive drug and high-dose groups improved more significantly, and the low-dose group improved less than the positive drug and high-dose groups, but the degree of damage was lower than the model group. In summary: Hosta polysaccharide lozenges have a strong anti-inflammatory effect (the effect of treating acute pharyngitis).

[0133] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine preparation containing Hosta odorata polysaccharide, characterized in that: The following steps are involved: S100: preparation of Hosta polysaccharide; S200: Prepare Chinese medicine preparations from the 1KDa-2KDa or 100KDa-120KDa segments of Hosta japonica polysaccharides.

2. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 1, characterized in that: The specific steps of S100 are: S101: crushing the dried hosta flower raw material with a crusher and sieving; S102: adding anhydrous ethanol, stirring to extract fat-soluble pigments and some impurities, and collecting the precipitate by centrifugation; S103: adding pure water to the precipitate, extracting in a 60°C water bath, and collecting the supernatant extract by centrifugation; S104: Combine the two extracts, concentrate by vacuum rotary evaporation to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation; S105: collecting the precipitated solid by centrifugation, i.e., the crude polysaccharide extract; S106: adding an equal amount of water to the crude polysaccharide extract and subjecting it to in vitro gastrointestinal bionic system digestion; the digestion time is 1 hour, and after the digestion is completed, the digestion fluid is collected; and the digestion supernatant is collected by centrifugation; S700: remove the digestion supernatant, use a peristaltic pump to pass the digestion supernatant through 1KDa, 2KDa, 4KDa, 100KDa, and 120KDa molecular retention plates respectively; the supernatant is divided into below 1KDa, 1KDa-2KDa, 2KDa-4KDa, 4KDa-100KDa, and 100KDa-120KDa; collect the 1KDa-2KDa segment and the 100KDa-120KDa segment solutions, concentrate, freeze-dry and weigh the mass of the segment.

3. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 2, characterized in that: The specific steps of S100 are: S101: crushing the dried Hosta flower raw material with a crusher and passing it through a 40-80 mesh sieve; S102: adding anhydrous ethanol, stirring to extract fat-soluble pigments and some impurities, centrifuging at 6000 g for 10 min, and collecting the precipitate; S103: adding pure water to the precipitate, extracting in a water bath at 60°C for 4 h, centrifuging at 6000 g for 10 min, and collecting the supernatant extract; S104: Combine the two extracts, concentrate by vacuum rotary evaporation to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation; S105: centrifuge at 8000 g for 10 min to collect the precipitated solid, i.e., the crude polysaccharide extract; S106: adding 300 mL of water to 300 g of the crude polysaccharide extract for in vitro gastrointestinal bionic system digestion; digestion time 1 h, after digestion, collecting the digestion fluid; centrifuging at 3000 g for 10 min, collecting the supernatant; S700: 300 mL of the unsalted supernatant was measured and peristaltic pumped to pass the supernatant through 1 KDa, 2 KDa, 4 KDa, 100 KDa and 120 KDa molecular retention plates respectively; the supernatant was divided into below 1 KDa, 1 KDa-2 KDa, 2 KDa-4 KDa, 4 KDa-100 KDa and 100 KDa-120 KDa; the 1 KDa-2 KDa segment and the 100 KDa-120 KDa segment solutions were collected, concentrated, freeze-dried and the mass of the segment was weighed.

4. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 2 or 3, characterized in that: In S106, the experimental parameters of the in vitro gastrointestinal bionic system are as follows: saliva pH: 7, Ca 2+ The concentration is 1.5mM, salivary amylase is 150U / mL, the simulated saliva volume is 50mL, the simulated gastric fluid pH is 2.0, and Ca 2+ Concentration: 0.15mM, pepsin: 4000U / mL, gastric juice secretion rate: 2mL / min, HCl secretion rate: 90uL / min, gastric rolling extrusion frequency: 3 times / min, simulated intestinal fluid PH: 7, Ca 2+ The concentration is 0.6mM, pancreatic enzyme: 200U / mL, and the simulated intestinal fluid secretion rate is 3.75mL / min.

5. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 1, characterized in that: In the S200, the 1KDa-2KDa polysaccharide from Hosta japonica is made into polysaccharide tablets by a tablet pressing machine.

6. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 1, characterized in that: In the S200, the 100KDa-120KDa range of Hosta polysaccharides are used to prepare Hosta polysaccharide granules.

7. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 5, characterized in that: The specific steps of S200 are: S201: preparing 100KDa-120KDa range Hosta polysaccharides and auxiliary materials, crushing the freeze-dried polysaccharides and auxiliary materials, and passing through a No. 5 sieve; S202: using an equal amount mixing method, mixing the auxiliary material with the least amount first, and then increasing the amount of powder by an equal amount to mix thoroughly; S203: Granulation: Add a certain amount of edible ethanol solution to the mixed powder to make the powder agglomerate and disperse when touched; pass through a 16-mesh sieve to obtain wet granules; S204: Drying: Place the obtained wet granules in a constant temperature forced air drying oven at 60°C and dry for 0.5h, controlling the moisture content to not exceed 10%; S205: Granulation: The dried granules are passed through a 16-mesh sieve again; S206: tableting: uniform granules are passed through a tablet press to produce 0.6 g / tablet polysaccharide tablets; S207： Sterilization: Put the lozenges into a transparent glass bottle, seal the cap, and sterilize at 160℃ for 2h. After sterilization, wait for the temperature to cool down to below room temperature to obtain the finished product.

8. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 7, characterized in that: The weight ratio of the 100KDa-120KDa segment Hosta polysaccharide to the auxiliary materials is 6:4; the auxiliary materials include fillers, sweeteners, flavoring agents and lubricants; the filler is mannitol or microcrystalline cellulose, the sweetener is xylitol, the flavoring agent is citric acid, and the lubricant is magnesium stearate.

9. The method for preparing a Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 6, characterized in that: The specific steps of S200 are: preparing granules by wet granulation process. Passing the hosta polysaccharide and auxiliary materials through an 80-mesh sieve for use, accurately weighing the corresponding components according to the prescription amount, adding the auxiliary materials in equal amounts in increments to make them evenly mixed with the main drug, and adding the wetting agent in an atomized form, stirring thoroughly until the soft material is formed; passing the soft material through a 16-mesh sieve to obtain wet granules, then transferring them to an electric heating blast drying oven for drying, cooling to room temperature, and passing through a 24-mesh sieve to obtain granules, thus obtaining the hosta polysaccharide granules.

10. The method for preparing a traditional Chinese medicine preparation containing Hosta odorata polysaccharide according to claim 9, characterized in that: The weight ratio of the 1KDa-2KDa segment Hosta polysaccharide to the auxiliary materials is 6:4; the auxiliary materials include a diluent and a wetting agent, the diluent is microcrystalline cellulose and mannitol, and the ratio of microcrystalline cellulose to mannitol is 3:1; the wetting agent is water and ethanol.

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