Guishaodihuang pill and preparation method thereof

Abstract

The present invention relates to the technical field of Guishao Dihuang Pill preparation, and discloses a Guishao Dihuang Pill and a preparation method thereof. The Guishao Dihuang Pill comprises the following components in parts by weight: 35-45 parts of Chinese Angelica sinensis; 35-45 parts of white peony root wine; 150-170 parts of Rehmannia root prepared; 75-85 parts of cornus fruit wine; 55-65 parts of peony bark; 75-85 parts of Chinese yam; 55-65 parts of Poria cocos; and 55-65 parts of Alisma orientalis. Each pill contains 5-20 mg of vitamin C and 2-10 mg of vitamin E, with the addition ratio of vitamin C to vitamin E being 3:1. By adding vitamin C and vitamin E to the Guishao Dihuang Pill, an innovative formula is formed that takes traditional Chinese medicine as the main body and combines new excipients. The synergistic antioxidant effect of vitamins C and E is utilized to effectively protect the active ingredients in the pill from oxidation or degradation, thereby enhancing the immune regulation function.

Description

Technical Field

[0001] The invention relates to the technical field of Guishao Dihuang pill preparation, in particular to a Guishao Dihuang pill and a preparation method thereof. Background Art

[0002] Guishao Dihuang Wan (Gui Shao Di Huang Wan) is a classic Chinese medicine formula, known for its yin-tonifying and kidney-invigorating properties, nourishing blood and regulating menstruation. It is widely used to treat immunosuppressive disorders and improve sub-health conditions. However, existing preparation methods primarily rely on traditional water extraction and alcohol precipitation processes, resulting in low extraction efficiency for the active ingredients in the medicinal material (such as catalpol, paeoniflorin, and polysaccharides). In particular, some heat-sensitive ingredients are susceptible to decomposition or degradation at high temperatures, resulting in loss of the active ingredients. Furthermore, due to the wide variation in polarity among the active ingredients, traditional processes hinder the targeted extraction of multiple components, resulting in significant deficiencies in the product's efficacy. Optimizing extraction techniques to improve the extraction and retention of active ingredients has become an urgent challenge.

[0003] In terms of formulation stability, the existing Guishao Dihuang Pills production process is relatively extensive, and the active ingredients in the product are easily oxidized or degraded during storage, resulting in a decrease in efficacy. Although traditional excipients such as refined honey can delay the degradation of ingredients to a certain extent, they have limited effects in protecting easily oxidized ingredients and improving the stability of pills. At the same time, the lack of precise parameter control of the extraction, concentration and molding links during the preparation process can easily lead to inconsistent quality between batches. Especially in the purification of the extract, conventional concentration and coarse filtration methods in the existing technology are difficult to effectively remove impurities and enrich key ingredients, further limiting the efficacy and market competitiveness of the preparation.

[0004] Furthermore, while the traditional formulation of Guishao Dihuang Wan can exert certain immunomodulatory effects, its antioxidant capacity and potential for enhancing efficacy are insufficient. Studies have shown that antioxidant capacity is crucial for enhancing immune function, while the antioxidant capacity of traditional Chinese medicine itself is relatively limited. In modern Chinese medicine formulations, the introduction of functional excipients is an effective way to enhance efficacy and product competitiveness, but Guishao Dihuang Wan currently lacks the synergistic benefits of functional ingredients such as vitamins. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention provides a Guishao Dihuang Pill and a preparation method thereof, which solves the problem that the existing Guishao Dihuang Pill has a relatively extensive process and the active ingredients in the product are easily oxidized or degraded during storage, resulting in a decrease in efficacy.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A Guishao Dihuang Pill, comprising the following components in parts by weight:

[0007] Angelica: 35-45 parts;

[0008] White peony root with wine: 35-45 parts;

[0009] Rehmannia root: 150-170 parts;

[0010] Wine-boiled cornus fruit: 75-85 parts;

[0011] Paeonia suffruticosa root bark: 55-65 parts;

[0012] Chinese yam: 75-85 servings;

[0013] Poria: 55-65 parts;

[0014] Alisma: 55-65 parts;

[0015] Each pill contains 5 to 20 mg of vitamin C and 2 to 10 mg of vitamin E, and the pills are prepared by the following process:

[0016] The process includes the following steps: pretreatment of medicinal materials: cleaning, slicing, and drying the medicinal materials; multi-stage temperature-controlled extraction of active ingredients; concentration and purification of the extract; addition and mixing of auxiliary materials; and preparation molding.

[0017] Furthermore, the synergistic effects of Chinese herbal medicine components:

[0018] The Rehmannia root, white peony root and angelica in the formula are all classic medicinal materials for nourishing yin and blood, and tonifying the kidneys. They have the functions of enhancing immunity and regulating endocrine; Paeonia suffruticosa and Cornus officinalis further balance the overall effect by clearing heat, nourishing yin, and consolidating the body; Chinese yam, Poria cocos and Alisma orientalis play the role of strengthening the spleen and replenishing qi, promoting absorption, and improving the overall utilization rate of the medicine.

[0019] Each component achieves the comprehensive effect of enhancing immune function through multi-target and multi-pathway pharmacological actions.

[0020] Functions of new materials:

[0021] Vitamin C: It has significant antioxidant function, can eliminate free radicals in the body, protect the stability of active ingredients of traditional Chinese medicine in the body, promote the synthesis of collagen, and enhance the body's immune response.

[0022] Vitamin E: It is a fat-soluble antioxidant that can protect the cell membrane structure, enhance the body's antioxidant capacity, regulate cellular immune function, and work synergistically with vitamin C to enhance the overall efficacy.

[0023] The addition of vitamins C and E not only enhances the immunomodulatory effect of the preparation, but also protects the stability of the active ingredients of traditional Chinese medicine during the preparation process and extends the shelf life of the product.

[0024] Preferably, the addition ratio of vitamin C and vitamin E is 3:1.

[0025] Further experiments have shown that a 3:1 ratio of vitamin C to vitamin E (e.g., 15mg of vitamin C and 5mg of vitamin E per pill) achieves optimal synergistic antioxidant and immunomodulatory effects. Vitamin C rapidly scavenges free radicals in body fluids, while vitamin E, in its lipid-soluble environment, protects cell membranes. This ratio not only maximizes the antioxidant effects of both, but also reduces gastrointestinal discomfort potentially associated with vitamin C overdose, optimizing dosage safety.

[0026] Preferably, the pills are water-honeyed pills, which are prepared by adding 35 to 50 g of refined honey to every 100 g of medicinal material powder.

[0027] Preferably, the pills are small honey pills, which are prepared by adding 80 to 110 g of refined honey to every 100 g of medicinal material powder.

[0028] Furthermore, the choice of refined honey: refined honey is a traditional Chinese medicine pill-making auxiliary material, which plays the role of bonding and delaying the release of drugs. It also has certain nourishing effects and can complement the main medicinal effects of Guishao Dihuang Pills.

[0029] Water-honeyed pills are suitable for patients who need quick results. Honeyed pills have a better taste and are convenient to take. Small honeyed pills are more suitable for long-term use due to their higher honey content and are conducive to the preservation of the medicine.

[0030] The following steps are involved:

[0031] Wash the herbs and slice them into slices. The thickness of slices of Radix Rehmanniae is 0.5-2.0 mm, and the thickness of slices of other herbs is 1.0-2.0 mm.

[0032] The multi-stage temperature-controlled extraction was carried out under the following conditions: the first stage temperature was 50-55°C, the extraction time was 1.5-2 hours, and the pH was adjusted to 4.0-4.5; the second stage temperature was 65-75°C, the extraction time was 2-3 hours, and the pH was adjusted to 5.5-6.5; the third stage temperature was 85-95°C, and the extraction time was 1-1.5 hours;

[0033] The extracts were combined and concentrated to a solid content of 60-80 mg / mL, and purified by ultrafiltration and nanofiltration membranes;

[0034] Add vitamin C and vitamin E, spray-dry, prepare powder, and mix with refined honey to make pills;

[0035] Preparation molding: The mixed liquid is spray-dried to form powder, mixed with refined honey to form pills, and dried to obtain the finished product.

[0036] Furthermore, low-temperature drying can avoid the loss of heat-sensitive components, such as catalpol and paeoniflorin in Rehmannia glutinosa. Slicing can increase the surface area of ​​the medicinal material and improve extraction efficiency. Multi-stage temperature-controlled extraction can maximize the retention of multiple components by separating the release of active ingredients at different temperatures:

[0037] In the first stage, heat-sensitive components (such as paeoniflorin and ferulic acid) are extracted and the pH is adjusted to acidic to reduce component degradation;

[0038] The second stage releases polar components (such as paeonol and loganin);

[0039] The third stage involves high-temperature extraction of heat-resistant macromolecules (such as Rehmannia glutinosa polysaccharides), using high temperature to promote their dissolution. Vacuum decompression and concentration can reduce the damage to the ingredients caused by high temperature. Ultrafiltration and nanofiltration can efficiently remove impurities while retaining the active ingredients to ensure product purity and efficacy. Vitamins C and E are evenly dispersed in a solution state to avoid fluctuations in efficacy caused by uneven addition. Spray drying can quickly convert the concentrate into a uniform powder to avoid degradation of active ingredients caused by long-term high-temperature treatment.

[0040] Preferably, the thickness of the medicinal material slices is 0.5 to 1.0 mm, the drying temperature is 40 to 50° C., and the drying time is 8 to 12 hours.

[0041] Preferably, during the first stage of extraction, the weight-to-volume ratio of the medicinal material to water is 1:10 to 1:12.

[0042] Preferably, the concentration step is carried out at a reduced pressure of 0.08 to 0.1 MPa and a concentration temperature of 45 to 55°C.

[0043] Preferably, the spray drying temperature range is 70-90° C., and the spray pressure is 0.2-0.5 MPa.

[0044] Preferably, the ratio of adding the refined honey during the pill making process is 35 to 110 g per 100 g of medicinal material powder.

[0045] Furthermore, the thickness of the medicinal material and the drying conditions directly affect the extraction efficiency. Slicing increases the surface area, while low-temperature drying avoids the loss of heat-sensitive components.

[0046] Controlling the ratio of medicinal materials to water can improve the solvent's extraction capacity and ensure uniform concentration of the extract;

[0047] The reduced pressure concentration conditions effectively reduce the risk of thermal degradation of ingredients while concentrating the active ingredients;

[0048] Optimization of drying parameters ensures uniform powder particles and avoids high temperature damage;

[0049] The ratio of refined honey affects the uniformity and formability of pills, providing a guarantee for the stability of the preparation.

[0050] The present invention provides a Guishao Dihuang Pill and a preparation method thereof, which has the following beneficial effects:

[0051] 1. The present invention adds vitamin C and vitamin E to Guishao Dihuang Pills, creating an innovative formula based on traditional Chinese medicine combined with novel excipients. This innovative formula utilizes the synergistic antioxidant effects of vitamins C and E to effectively protect the active ingredients in the pills from oxidation or degradation, thereby enhancing immune regulation. Compared to existing traditional approaches that rely solely on the compatibility of traditional Chinese medicines, this invention overcomes the bottleneck of insufficient antioxidant capacity of traditional Chinese medicine ingredients and significantly improves the overall efficacy of the drug.

[0052] 2. The present invention adopts a multi-stage temperature-controlled extraction process, optimizes the extraction efficiency of active ingredients according to different temperature conditions, and extracts in low, medium and high temperature stages, accurately retaining the key components of different medicinal materials such as catalpol, paeoniflorin and polysaccharides. In traditional preparation methods, the problem of loss of heat-sensitive components due to single high-temperature extraction is relatively common. The improvement of the present invention avoids these problems, significantly improves the extraction rate of effective ingredients, and the technical solution has higher controllability.

[0053] 3. The present invention uses a combination of ultrafiltration membrane and nanofiltration membrane technology to effectively remove impurities and large molecular weight ineffective components, while enriching active ingredients with moderate molecular weight. The problems of low purity of conventional extracts after concentration and poor drug stability in the prior art are solved. The purification technology solution of the present invention not only improves the purity of the preparation, but also extends the shelf life of the product, providing a feasible path for large-scale production.

[0054] 4. The present invention uses a combination of ultrafiltration membrane and nanofiltration membrane technology to effectively remove impurities and large molecular weight ineffective components, while enriching active ingredients with moderate molecular weight. The problems of low purity of conventional extracts after concentration and poor drug stability in the prior art are solved. The purification technology solution of the present invention not only improves the purity of the preparation, but also extends the shelf life of the product, providing a feasible path for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Flow chart of the method of the present invention. DETAILED DESCRIPTION

[0056] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0057] Please see the attached Figure 1 : Example 1

[0058] Preparation of Guishao Dihuang Pills and its basic process parameters

[0059] Pretreatment of medicinal materials

[0060] Weigh 40g of Chinese Angelica, 40g of white peony root with wine, 160g of Rehmannia root, 80g of cornus officinalis with wine, 60g of peony bark, 80g of yam, 60g of Poria cocos, and 60g of Alisma orientalis.

[0061] The medicinal materials were rinsed with deionized water (temperature 40°C) to remove surface impurities.

[0062] Slice Rehmannia root to a thickness of 0.5 mm, and slice the other herbs to a thickness of 1 mm. Place the sliced ​​herbs in a drying oven at 45°C for 10 hours to ensure the moisture content is below 10%.

[0063] Multi-stage temperature controlled extraction

[0064] Phase I: All medicinal materials were mixed in proportion, 10 times the amount of deionized water was added, the extraction temperature was set to 55°C, the pH was adjusted to 4.2 (0.3% citric acid was added), the stirring speed was controlled at 120 rpm, the extraction time was 1.8 hours, and the extract was filtered.

[0065] The second stage: add 8 times the amount of 70% ethanol solution to the medicinal residue, raise the temperature to 70℃, extract for 2.5 hours, adjust the pH to 5.8 (add 0.2% sodium citrate buffer), stir at 150 rpm, and filter the extract II.

[0066] The third stage: add 6 times the amount of deionized water to the residue, raise the temperature to 90°C, extract for 1.2 hours, maintain the stirring speed at 180 rpm, and filter the extract III.

[0067] Concentration and purification

[0068] The extracts I, II, and III were combined, placed in a vacuum concentrator, and concentrated at 45°C and 0.1 MPa to a total solid content of 65 mg / mL.

[0069] Use ultrafiltration membrane (molecular weight cutoff 1-5kDa) to remove impurities, and then use nanofiltration membrane (molecular weight cutoff 200-500Da) to enrich the effective ingredients to obtain a clarified concentrate.

[0070] Auxiliary material addition and molding

[0071] Add 10 mg / portion of vitamin C pills and 3 mg / portion of vitamin E pills to the concentrate, stir thoroughly, and use a spray dryer to prepare a powder (temperature 80°C, spray pressure 0.3 MPa).

[0072] The dried medicinal powder and refined honey are mixed in a ratio of 1:0.4 to make water-honey pills. After the pills are formed, they are dried at 50°C until the moisture content is less than 8% to obtain the final product.

[0073] Example 2

[0074] Guishao Dihuang Pills with improved ratio of vitamin C and vitamin E

[0075] Processing of medicinal materials

[0076] Weigh 38g of Chinese angelica, 42g of white peony root with wine, 165g of Rehmannia root, 78g of cornus officinalis with wine, 60g of peony bark, 78g of yam, 62g of Poria cocos, and 60g of Alisma orientalis.

[0077] After cleaning the medicinal materials, the slices are all 1mm thick and dried at a low temperature of 40°C for 9 hours to ensure that the moisture content of the medicinal materials is less than 8%.

[0078] Extraction process

[0079] Stage 1: Add 12 times the amount of deionized water, set the extraction temperature to 50°C, the extraction time to 1.5 hours, adjust the pH to 4.0 (add 0.2% citric acid solution), stir at 100 rpm, and filter the extract I.

[0080] The second stage: add 9 times the amount of 70% ethanol solution to the medicinal residue, adjust the temperature to 68°C, and keep it for 3 hours. Adjust the pH to 6.2 (add 0.3% sodium citrate solution), and filter the extract II.

[0081] The third stage: add 7 times the amount of pure water to the medicinal residue, set the temperature to 92°C, extract for 1 hour, stir at 200 rpm, and filter the extract III.

[0082] Concentration, purification and excipient addition

[0083] The three-stage extracts were combined and concentrated at 50°C and 0.08 MPa under reduced pressure to a total solid content of 70 mg / mL.

[0084] The ultrafiltration and nanofiltration operations were the same as in Example 1. 15 mg / portion of vitamin C pills and 5 mg / portion of vitamin E pills (ratio 3:1) were added to the concentrate and stirred thoroughly.

[0085] Preparation molding

[0086] The powder is spray-dried at 85°C and 0.4 MPa. The powder is then mixed with refined honey in a ratio of 1:0.9 to form small honey pills. After forming, the pellets are dried to a moisture content of 6%.

[0087] Example 3

[0088] Guishao Dihuang Wan improves the extraction process accuracy

[0089] Medicinal material pretreatment

[0090] Weigh 40g of Chinese Angelica, 40g of white peony root with wine, 160g of Rehmannia root, 80g of cornus officinalis with wine, 60g of peony bark, 80g of yam, 60g of Poria cocos, and 60g of Alisma orientalis.

[0091] The medicinal materials were washed and sliced ​​into slices with a thickness of 0.8 mm, and dried at 45°C for 10 hours. The moisture content of the medicinal materials was less than 10%.

[0092] Multi-stage temperature controlled extraction

[0093] Stage 1: Place the medicinal material in an extraction tank and add 11 times the volume of pure water. Set the extraction temperature to 52°C, adjust the pH to 4.5, extract for 1.7 hours, and filter the extract.

[0094] The second stage: add 8 times the amount of 75% ethanol solution to the medicinal residue, extract at 70℃, extract for 2.2 hours, and filter the extract II.

[0095] The third stage: add 6.5 times the amount of deionized water to the medicinal residue, set the temperature to 89°C, extract for 1.3 hours, and filter the extract III.

[0096] Concentration and purification

[0097] The extracts I, II and III were combined and concentrated at 48°C and 0.09 MPa, until the solid content reached 80 mg / mL.

[0098] Impurities are removed by ultrafiltration membrane, and then key components are enriched by nanofiltration membrane. The total active ingredient extraction rate of the concentrate is increased by 30% compared with traditional methods.

[0099] Molding process

[0100] Add 20 mg / portion of vitamin C pills and 6 mg / portion of vitamin E pills (3.3:1 ratio) to the concentrate and stir evenly. Control the temperature of the spray drying equipment at 75°C and the spray pressure at 0.35 MPa. After preparing the powder, mix it with refined honey in a ratio of 1:0.6 to make water-honey pills, and dry them to a moisture content of 7%.

[0101] Example 4

[0102] Guishao Dihuang Wan with improved stability and extended shelf life

[0103] Medicinal material processing

[0104] The weighing ratio of the medicinal materials is the same as that in Example 1, the slice thickness is 1 mm, the drying temperature is 40° C., and the drying time is 10 hours.

[0105] Multi-segment extraction

[0106] The first stage extraction temperature was 55°C, the time was 2 hours, and the pH was adjusted to 4.3.

[0107] The second stage temperature was 70°C, the duration was 2.8 hours, and the pH was 6.0.

[0108] The temperature of the third stage was 91° C. and the time was 1.2 hours. The extracts from the three stages were combined and concentrated to a solid content of 65 mg / mL.

[0109] Adding auxiliary materials

[0110] Add 12mg / portion of vitamin C pills and 4mg / portion of vitamin E pills to the concentrate. The synergistic effect of the two can delay the oxidation of the active ingredients. The spray drying temperature is 80℃ to obtain the powder, which is mixed with refined honey in a ratio of 1:0.5 to make honey pills. The moisture content of the product is controlled at 6%, and the shelf life is extended to 24 months after sealed packaging.

[0111] Comparative Example 1 (corresponding to Example 1, without using multi-stage temperature control extraction technology)

[0112] Medicinal material pretreatment

[0113] The types and proportions of medicinal materials were the same as those in Example 1. After washing and slicing (all with a thickness of 1 mm), they were dried at 45° C. for 10 hours.

[0114] Extraction process

[0115] The medicinal materials were added with 12 times the amount of pure water at one time, the extraction temperature was 90°C, the time was 3 hours, no segmented temperature control operation was performed, the extract was filtered to obtain a crude extract.

[0116] Concentration and purification

[0117] The extract was directly concentrated (temperature 55°C, reduced pressure 0.1 MPa) to a total solid content of 65 mg / mL without ultrafiltration and nanofiltration separation steps, and the extract was directly used to prepare pills.

[0118] Auxiliary material addition and molding

[0119] Add 10 mg / portion of vitamin C pills and 3 mg / portion of vitamin E pills to the concentrate, mix well and then spray-dry (temperature 85°C). Mix the obtained powder with refined honey in a ratio of 1:0.4 to make water-honey pills, and dry to obtain the finished product.

[0120] Comparative Example 2 (corresponding to Example 2, without the introduction of vitamin C and vitamin E)

[0121] Medicinal material pretreatment

[0122] The types, proportions, cleaning and slice thickness of the medicinal materials were consistent with those in Example 2, and the drying conditions were the same.

[0123] Extraction process

[0124] The multi-stage temperature-controlled extraction process is consistent with Example 2, including three stages of temperature-controlled extraction:

[0125] Stage 1: 50°C, 1.5 hours, pH adjusted to 4.0;

[0126] Stage 2: 70°C, 3 hours, pH adjusted to 6.2;

[0127] Stage 3: 92°C, 1 hour.

[0128] Concentration and purification

[0129] The extracts were combined and concentrated in vacuo to a total solid content of 70 mg / mL. The ultrafiltration and nanofiltration separation processes were the same as in Example 2.

[0130] Molding process

[0131] The spray-dried medicinal powder is directly mixed with refined honey in a ratio of 1:0.9 to prepare small honey pills without the addition of excipients such as vitamin C and vitamin E.

[0132] Comparative Example 3 (corresponding to Example 3, adjusting the purification process parameters)

[0133] Medicinal material pretreatment

[0134] The types, proportions and slice thicknesses of the medicinal materials were consistent with those in Example 3, and the drying conditions were the same.

[0135] Extraction process

[0136] The multi-stage temperature-controlled extraction process is consistent with Example 3, including three stages of extraction:

[0137] Stage 1: 52°C, 1.7 hours, pH adjusted to 4.5;

[0138] Stage 2: 70°C, 2.2 hours;

[0139] Stage 3: 89°C, 1.3 hours.

[0140] Concentration and purification

[0141] The extracts were combined and vacuum concentrated to a solid content of 80 mg / mL. During the purification process, only traditional coarse filtration was used, without ultrafiltration or nanofiltration. Impurities in the extracts were not further removed and the product was directly transferred to the next molding process.

[0142] Auxiliary material addition and molding

[0143] Add 20mg / portion of vitamin C pills and 6mg / portion of vitamin E pills to the concentrate, mix well and spray dry. Mix the powder with refined honey in a ratio of 1:0.6 to make water-honey pills.

[0144] Comparative Example 4 (corresponding to Example 4, adjusting the refining ratio and molding parameters)

[0145] Medicinal material pretreatment

[0146] The types, proportions and slice thicknesses of the medicinal materials were consistent with those in Example 4, and the drying conditions were the same.

[0147] Extraction process

[0148] The multi-stage temperature-controlled extraction process is consistent with that in Example 4:

[0149] Stage 1: 55°C, 2 hours;

[0150] Stage 2: 70°C, 2.8 hours;

[0151] Stage 3: 91°C, 1.2 hours.

[0152] Concentration and purification

[0153] The extraction liquid combination, vacuum concentration, ultrafiltration and nanofiltration operations were consistent with Example 4. The total solid content was 65 mg / mL.

[0154] Auxiliary material addition and molding

[0155] Vitamin C (12mg / pill) and Vitamin E (4mg / pill) were added to the concentrate. The spray-dried powder was mixed with refined honey in a ratio of 1:0.2. The pills were not fully formed during the pill-making process, resulting in some pills with a rough surface and uneven moisture content after drying.

[0156] Comparative Example 5 (corresponding to Example 1, changing the spray drying process parameters)

[0157] Medicinal material pretreatment

[0158] The types, proportions and slice thicknesses of the medicinal materials were consistent with those in Example 1, and the drying conditions were the same.

[0159] Extraction process

[0160] The multi-stage temperature-controlled extraction process was consistent with that in Example 1, including three-stage extraction temperature and time settings.

[0161] Concentration and purification

[0162] The extracts were combined and concentrated in vacuo to a total solid content of 65 mg / mL. The ultrafiltration and nanofiltration separation steps were the same as in Example 1.

[0163] Auxiliary material addition and molding

[0164] Vitamin C 10mg / portion pills and vitamin E 3mg / portion pills were added to the concentrate, and the spray drying process parameters were adjusted to: drying temperature 95℃, spray pressure 0.5MPa. After drying, the powder particles were too fine and some powders agglomerated, affecting the molding. The powder and refined honey were mixed in a ratio of 1:0.4, the surface viscosity of the water-honeyed pills increased, and the drying was incomplete.

[0165] Experiment 1: Comparison of active ingredient extraction rates

[0166] Objective: To verify whether the multi-stage temperature-controlled extraction process of the present invention can improve the extraction rate of active ingredients (such as catalpol, paeoniflorin, and polysaccharides).

[0167] Experimental methods:

[0168] Sample preparation

[0169] The extracts were prepared using Example 1, Example 3, Comparative Example 1, and Comparative Example 3, and the three-stage extraction (or corresponding one-time extraction) was completed using the respective extraction processes.

[0170] The extracts were combined and concentrated to a total solid content of 60 mg / mL.

[0171] Test indicators

[0172] Catalpol content determination: High performance liquid chromatography (HPLC) was used with a C18 column, a mobile phase of methanol and water (50:50, v / v), and a detection wavelength of 280 nm.

[0173] Determination of paeoniflorin content: HPLC was used with a C18 column, acetonitrile-water (30:70, v / v) as the mobile phase, and the detection wavelength was 230 nm.

[0174] Determination of polysaccharide content: The phenol-sulfuric acid method was used with a detection wavelength of 490 nm, and the content was calculated using a glucose standard curve.

[0175] Experimental setup

[0176] Experimental groups: Example 1 and Example 3.

[0177] Control group: Comparative Example 1 and Comparative Example 3.

[0178] Each group was tested 3 times and the average value was taken.

[0179] Experiment 2: Comparison of formulation stability

[0180] Objective: To evaluate the protective effect of vitamin C and vitamin E on the active ingredients in the preparation and to verify the stability of the product during storage.

[0181] Experimental methods:

[0182] Sample preparation

[0183] Small honey pills were prepared using Example 2 (containing vitamins C and E) and Comparative Example 2 (excluding vitamins C and E), respectively, and stored after being formed according to their respective processes.

[0184] Experimental setup

[0185] Under accelerated aging conditions of 40°C and 75% relative humidity, the active ingredient content and oxidative degradation of the samples were tested at 0 days, 7 days, 14 days, 30 days, and 60 days.

[0186] Test indicators

[0187] Degradation rates of catalpol and paeoniflorin: HPLC was used to determine the content of active ingredients at each storage time point, and the degradation rates were calculated by comparing with the initial values.

[0188] Antioxidant capacity determination: The antioxidant properties of the product were evaluated by DPPH free radical scavenging ability test, with the detection wavelength of 517nm.

[0189] Moisture content: Karl Fischer titration was used to detect the changes in moisture content in the pills.

[0190] Experiment 3: Comparison of Extract Purity

[0191] Objective: To verify whether the ultrafiltration and nanofiltration technologies in the present invention can significantly improve the purity of the extract and the enrichment efficiency of key active ingredients.

[0192] Experimental methods:

[0193] Sample preparation

[0194] Example 1 and Comparative Example 1 were used for comparison, and the extract was purified by using an ultrafiltration-nanofiltration separation process and a traditional coarse filtration method, respectively.

[0195] The two groups of extracts were concentrated to 60 mg / mL and then analyzed.

[0196] Experimental setup

[0197] The comparative experiment was set up into two groups: the extract of Example 1 (ultrafiltration to nanofiltration separation) and the extract of Comparative Example 1 (conventional coarse filtration).

[0198] Test indicators

[0199] Total impurity content: The ratio of macromolecules and impurities in the residue (1 kDa fraction) was determined gravimetrically.

[0200] Catalpol and paeoniflorin content: HPLC was used to determine the enrichment of the two components in the extract.

[0201] Color and clarity: The clarity of the extracts was compared by optical density measurement (wavelength 450 nm).

[0202] Experiment 4: Comparison of immune regulation ability

[0203] Objective: To verify whether the preparation of the present invention has a better regulatory effect on the immune function of mice than the control preparation.

[0204] Experimental methods:

[0205] Animal experiment design

[0206] 48 mice were randomly divided into the following groups, with 8 mice in each group:

[0207] Example 1 group (multi-stage temperature control + vitamins C and E);

[0208] Example 2 group (multi-stage temperature control + vitamins C and E);

[0209] Comparative Example 1 (single high-temperature extraction, without vitamins C and E);

[0210] Comparative Example 2 (multi-stage temperature-controlled extraction, without vitamins C and E);

[0211] Blank control group (no treatment).

[0212] Experimental setup

[0213] The mice were gavaged with the corresponding preparations of the above groups every day (dose of 0.2 g / kg body weight) for 14 consecutive days.

[0214] Immune indexes were tested on the 15th day.

[0215] Test indicators

[0216] Thymus and spleen index: The thymus and spleen of mice were collected and their weight / body weight (mg / g) was calculated.

[0217] Serum IL-2 and IFN-γ levels: Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of immune-related factors in serum.

[0218] T lymphocyte proliferation rate: The MTT method was used to detect the proliferation ability of mouse spleen cells under ConA stimulation.

[0219] Experiment 1: Comparative experiment on active ingredient extraction rate

[0220] Purpose of the experiment

[0221] The effect of multi-stage temperature-controlled extraction technology on improving the extraction efficiency of active ingredients such as catalpol, paeoniflorin, and polysaccharides was verified, and the necessity and scientificity of extraction process optimization were demonstrated by comparing the examples with the comparative examples.

[0222] Sample preparation

[0223] Example 1 and Example 3: According to the multi-stage temperature-controlled extraction process, three stages of extraction were completed: the first stage was low-temperature extraction (50-55°C), the second stage was medium-temperature extraction (65-75°C), and the third stage was high-temperature extraction (85-95°C). The extracts were combined and concentrated to a solid content of 60 mg / mL.

[0224] Comparative Example 1: A single high-temperature extraction (90°C) was used for 3 hours without staged extraction. The extract was concentrated to 60 mg / mL.

[0225] Comparative Example 3: A three-stage temperature-controlled extraction was performed without ultrafiltration and nanofiltration. The extract was directly coarsely filtered and then concentrated to 60 mg / mL.

[0226] Sample testing

[0227] Catalpol content was determined by HPLC using a C18 column with a mobile phase of methanol and water (50:50, v / v) and a detection wavelength of 280 nm.

[0228] Determination of paeoniflorin content: HPLC method was used with a C18 column, the mobile phase was acetonitrile-water (30:70, v / v), and the detection wavelength was 230 nm.

[0229] Determination of polysaccharide content: Phenol-sulfuric acid method was used. Phenol solution and sulfuric acid were added to develop color, and the absorbance was measured at a wavelength of 490 nm.

[0230] Each sample was measured three times in parallel and the average value was taken.

[0231] Experimental data

[0232] Table 1 Comparison of active ingredient extraction rates between examples and comparative examples Experimental Summary

[0233] This experiment revealed the significant advantages of multi-stage temperature-controlled extraction technology in extracting active ingredients. The data showed that the extraction efficiencies of catalpol and paeoniflorin in the examples were generally high, while in Comparative Example 1, due to the use of a single high-temperature extraction process, catalpol decomposition and paeoniflorin loss were particularly significant. This also shows that heat-sensitive components require precise temperature control, and a single extraction technology is difficult to balance the stability and release of multiple components. This result proves the scientific nature of the multi-stage temperature-controlled extraction scheme, which retains more heat-sensitive components through temperature control and pH adjustment.

[0234] Judging from the polysaccharide extraction results, the Example also has significant advantages over Comparative Example 3. Although Comparative Example 3 showed slight improvements in the extraction of certain components, the lack of a purification step resulted in a high level of impurities, ultimately affecting the concentration and quality of the polysaccharides. In contrast, the use of ultrafiltration and nanofiltration in the Example effectively reduced the interference of large molecular impurities and improved the enrichment of polysaccharides. This combined process further improved the purity of the extract through separation and enrichment, laying the foundation for the drug's efficacy.

[0235] This is not just a comparison of numbers, but a logical verification. High-temperature extraction is simple, but the damage to the medicinal ingredients is also obvious. The multi-stage extraction technology adopted in the embodiment not only avoids the degradation of heat-sensitive components, but also provides more suitable extraction conditions for large molecular components. This design concept not only solves the shortcomings of traditional processes, but also provides a modern and scientific solution for the efficient utilization of active ingredients.

[0236] Experiment 2: Comparative experiment on formulation stability

[0237] Purpose of the experiment

[0238] By conducting accelerated aging tests on the pills prepared in Example 2 and Comparative Example 2, the protective effects of vitamins C and E on the active ingredients in the preparations were evaluated, and the stability improvement effect of the preparations of the present invention was verified.

[0239] Sample preparation

[0240] Example 2: Guishao Dihuang Pills containing vitamin C (15 mg / pill) and vitamin E (5 mg / pill) were prepared and made into small honey pills according to the process, with the moisture content controlled at 6%.

[0241] Comparative Example 2: Guishao Dihuang Wan was prepared according to the comparative example process, without adding vitamin C and E, and the remaining steps were the same.

[0242] Aging test

[0243] The samples were stored in an accelerated aging chamber at 40°C and 75% relative humidity, and the sampling time was 0 day, 7 days, 14 days, 30 days, and 60 days.

[0244] The contents of catalpol and paeoniflorin were detected at each time point, and the antioxidant capacity (DPPH free radical scavenging rate) was measured. Each group of samples was measured in parallel three times.

[0245] Test Method

[0246] Determination of catalpol and paeoniflorin content: The same as Experiment 1, determined by HPLC.

[0247] DPPH free radical scavenging rate: After adding 0.1 mM DPPH solution, the absorbance was measured at a wavelength of 517 nm and the scavenging rate was calculated.

[0248] Determination of moisture content: Karl Fischer titration method was used, 2 g of sample was taken each time for determination, and the average value was calculated.

[0249] Experimental data

[0250] Time (days) Sample number Catalpol content (mg / mL) Paeoniflorin content (mg / mL) DPPH clearance rate (%) Moisture content (%) 0 Example 2 2.36 1.92 86.5 6.1 Comparative Example 2 2.31 1.88 85.8 6.0 7 Example 2 2.30 1.87 83.4 6.3 Comparative Example 2 2.19 1.76 78.2 6.2 14 Example 2 2.22 1.78 81.2 6.4 Comparative Example 2 2.06 1.62 72.9 6.5 30 Example 2 2.10 1.62 76.5 6.5 Comparative Example 2 1.82 1.34 65.1 6.7 60 Example 2 1.95 1.48 71.8 6.7 Comparative Example 2 1.60 1.10 56.2 6.9 Experimental Summary

[0251] The protective effects of vitamins C and E in the preparation are very intuitive. Under accelerated aging conditions, the decrease in the content of catalpol and paeoniflorin in the examples is much lower than that in the comparative examples. In particular, after storage for 30 days, the examples still retain a relatively high content, while the comparative examples have begun to decline significantly. The key here is that vitamin C provides a rapid antioxidant barrier for the active ingredients in the aqueous phase, while vitamin E stabilizes the overall oxidative environment inside the pills through its fat-soluble properties. The combination of these two makes the entire system perform better under oxidative stress.

[0252] This is further supported by the DPPH free radical scavenging rate. In the Examples, the rate of decline in antioxidant capacity slowed significantly, while the clearance rate of the Comparative Example had already dropped significantly after 14 days. For traditional Chinese medicine pills, antioxidant performance is directly related to maintaining efficacy. The Comparative Example gradually lost its effectiveness during storage, clearly unable to meet the modern consumer demand for efficient and stable pharmaceuticals. The Examples address this core issue by introducing functional excipients.

[0253] The changes in moisture content are also very convincing. The moisture control in the examples is more stable, while the comparative examples, due to the lack of sufficient protective excipients, experience slight fluctuations in moisture content in the later stages, which may further accelerate the degradation of the ingredients. It is precisely these detailed optimizations that demonstrate the stability advantage of the present invention. The experimental results not only demonstrate the synergistic effect of vitamins C and E, but also clarify the scientific nature and reliability of the formulation design of the present invention.

[0254] Experiment 3: Comparative experiment on immune regulation ability

[0255] Purpose of the experiment

[0256] Through animal experiments, the effects of Guishao Dihuang Pills prepared in the embodiment and the comparative example in enhancing immune function were compared, focusing on evaluating the synergistic effect of vitamins C and E and the effect of multi-stage temperature-controlled extraction technology on immune regulation, and verifying the contribution of the present invention to improving immunity.

[0257] Experimental procedures

[0258] Animal grouping and treatment

[0259] Forty-eight healthy Kunming mice (♂) aged 8 weeks, weighing approximately 20 ± 2 g, were randomly divided into the following six groups, with 8 mice in each group:

[0260] Blank control group: The mice were given an equal amount of normal saline, 0.2 mL per day by gavage.

[0261] Group 1 of Example 1 was gavaged with the Guishao Dihuang Wan solution (0.2 g / kg) prepared in Example 1 every day.

[0262] Group 2 of Example 2 was gavaged daily with the Guishao Dihuang Wan solution (0.2 g / kg, containing vitamins C and E) prepared in Example 2.

[0263] Comparative Example 1 group: The Guishao Dihuang Wan solution (0.2 g / kg, single high-temperature extraction) prepared in Comparative Example 1 was orally administered every day.

[0264] Comparative Example 2 group: The Guishao Dihuang Wan solution (0.2 g / kg, without vitamin C and E) prepared in Comparative Example 2 was orally administered every day.

[0265] Comparative Example 3 group: The Guishao Dihuang Wan solution (0.2 g / kg, without purification step) prepared in Comparative Example 3 was orally administered every day.

[0266] Experimental cycle

[0267] After 14 days of continuous gavage, the mice were fasted for 12 hours and samples were collected to detect immune-related indicators.

[0268] Experimental testing

[0269] Spleen index: The spleen of the mouse was removed, weighed, and spleen weight / body weight (mg / g) was calculated.

[0270] Thymus index: remove the thymus, weigh it, and calculate the thymus weight / body weight (mg / g).

[0271] Serum IL-2 and IFN-γ levels: Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of IL-2 and IFN-γ in serum (pg / mL).

[0272] T lymphocyte proliferation rate: Mouse spleen cells were obtained and stimulated with ConA (final concentration 5 μg / mL). The proliferation capacity was determined by MTT assay, and the OD value reflected the proliferation rate.

[0273] Experimental data

[0274] Table 3. Comparison of immunomodulatory ability between examples and comparative examples Experimental Summary

[0275] This experiment further verified the immunomodulatory advantages of the present invention through an animal model. The spleen index and thymus index of mice added with vitamins C and E in Example 2 were significantly higher than those of the control group and the blank control group, indicating that the excipients have a positive effect on the development and functional enhancement of immune organs, especially in the improvement of the spleen index. The performance of the embodiment is particularly outstanding. This also confirms that the combination of multi-stage temperature-controlled extraction technology and excipient design can maximize the retention and utilization of the active ingredients of traditional Chinese medicine, while the control group with single high-temperature extraction cannot achieve such an effect.

[0276] Judging from the detection data of IL-2 and IFN-γ, the serum cytokine level of Example 2 is significantly better than that of Example 1, and further higher than all the comparative examples. This shows that the synergistic effect of vitamins C and E is not only antioxidant, but also can enhance the overall effect by regulating the immune pathway. The high level of IFN-γ also suggests that this improved pill has a strong stimulating effect on cellular immunity, which is consistent with the proliferation results of T cells during antigen activation. Among the comparative examples, especially Comparative Example 2 where no functional excipients were introduced, the immune indicators are significantly insufficient compared with the examples, indicating that the existing technology cannot solve the loss of active ingredients during extraction and stability.

[0277] Finally, the results for T cell proliferation rate are also very interesting. Example 2 achieves the best stimulation effect, reflecting the potential promotion of immune cell activity by vitamin excipients. However, in Comparative Example 3, due to the high amount of impurities in the extract, the cell culture environment is limited, and the proliferation rate is much lower than that of the examples. This comparison highlights the impact of the extraction and purification process on the actual effect of the product. The present invention, through reasonable process optimization and excipient design, solves the shortcomings of traditional Guishao Dihuang Wan in enhancing immune function from a mechanistic perspective. This is not only a technical improvement, but also a complete application innovation.

[0278] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Guishao Dihuang Wan, characterized in that: Pretreatment of medicinal materials: weigh 40g of Chinese Angelica, 40g of white peony root, 160g of Rehmannia root, 80g of cornus fruit, 60g of peony bark, 80g of Chinese yam, 60g of Poria, and 60g of Alisma. The medicinal materials were rinsed with deionized water at 40°C to remove surface impurities; sliced ​​Rehmannia root with a thickness of 0.5 mm, and the remaining medicinal materials were sliced ​​with a thickness of 1 mm; after slicing, the slices were placed in a drying oven at 45°C for 10 hours to ensure that the moisture content of the medicinal materials was less than 10%; Multi-stage temperature controlled extraction: Stage 1: All medicinal materials were mixed in proportion, 10 times the amount of deionized water was added, the extraction temperature was set to 55°C, the pH was adjusted to 4.2, the stirring speed was controlled at 120 rpm, the extraction time was 1.8 hours, and the extract was filtered. The second stage: 8 times the amount of 70% ethanol solution was added to the medicinal residue, the temperature was raised to 70 ° C, the extraction time was 2.5 hours, the pH was adjusted to 5.8, the stirring speed was 150 rpm, and the extract was filtered II; The third stage: add 6 times the amount of deionized water to the residue, raise the temperature to 90℃, extract for 1.2 hours, maintain the stirring speed at 180 rpm, and filter the extract III; Concentration and purification: Combine extracts I, II, and III, place in a vacuum concentrator, and concentrate at 45°C and 0.1 MPa to a total solid content of 65 mg / mL. Use an ultrafiltration membrane with a molecular weight cutoff of 1 to 5 kDa to remove impurities, and then use a nanofiltration membrane with a molecular weight cutoff of 200 to 500 Da to enrich the active ingredients to obtain a clarified concentrate. Addition of excipients and molding: Add 10 mg / portion of vitamin C pills and 3 mg / portion of vitamin E pills to the concentrate; after stirring evenly, prepare the powder through spray drying equipment, the spray drying temperature is 80°C, and the spray pressure is 0.3 MPa; the dry powder is mixed with refined honey in a ratio of 1:0.4 to make water-honeyed pills. After the pills are formed, they are dried at 50°C until the moisture content is less than 8% to obtain the final product.

2. A method for preparing Guishao Dihuang Wan, characterized in that: For the treatment of medicinal materials, weigh 38g of Chinese Angelica, 42g of white peony root with wine, 165g of Rehmannia root, 78g of cornus fruit with wine, 60g of peony bark, 78g of Chinese yam, 62g of Poria, and 60g of Alisma. After cleaning the herbs, the slices are all 1mm thick and dried at 40°C for 9 hours to ensure that the moisture content of the herbs is less than 8%. Extraction process: Stage 1: Add 12 times the amount of deionized water, set the extraction temperature to 50°C, the extraction time to 1.5 hours, adjust the pH to 4.0, stir at 100 rpm, and filter the extract I; Stage II: Add 9 times the amount of 70% ethanol solution to the medicinal residue, adjust the temperature to 68°C, and wait for 3 hours until the pH is adjusted to 6.

2. Filter the extract II. The third stage: add 7 times the amount of pure water to the medicinal residue, set the temperature to 92℃, extract for 1 hour, stir at 200rpm, and filter the extract III; Concentration, purification and excipient addition: Combine the three-stage extracts I, II, and III and concentrate them at 50°C and 0.08 MPa to a total solid content of 70 mg / mL; An ultrafiltration membrane with a molecular weight cutoff of 1 to 5 kDa is used to remove impurities, and then a nanofiltration membrane with a molecular weight cutoff of 200 to 500 Da is used to enrich the active ingredients to obtain a clarified concentrate; Add 15mg / portion of vitamin C pills and 5mg / portion of vitamin E pills to the concentrate and stir thoroughly; Formulation: The drug powder is obtained by spray drying equipment at a drying temperature of 85°C and a spray pressure of 0.4 MPa. The powder is mixed with refined honey in a ratio of 1:0.9 to form small honey pills. After forming, the product is dried and the moisture content is controlled at 6%.

3. A preparation method of Guishao Dihuang Wan, characterized in that: Pre-process the medicinal materials: weigh 40g of Chinese Angelica, 40g of white peony root, 160g of Rehmannia root, 80g of cornus fruit, 60g of peony bark, 80g of Chinese yam, 60g of Poria, and 60g of Alisma. The herbs were washed and sliced ​​to a thickness of 0.8 mm and dried at 45°C for 10 hours until the moisture content was less than 10%. Multi-stage temperature controlled extraction: Stage 1: Place the medicinal material in an extraction tank, add 11 times the amount of pure water, set the extraction temperature to 52°C, adjust the pH to 4.5, extract for 1.7 hours, and filter the extract I; The second stage: add 8 times the amount of 75% ethanol solution to the medicinal residue, extract at 70℃ for 2.2 hours, and filter the extract II; The third stage: add 6.5 times the amount of deionized water to the medicinal residue, set the temperature to 89 ° C, extract for 1.3 hours, and filter the extract III; Concentration and purification: Combine extracts I, II, and III, concentrate at 48°C and reduce the pressure to 0.09 MPa until the solid content reaches 80 mg / mL. Use ultrafiltration to remove impurities, and then use nanofiltration to concentrate the key components. The total active ingredient extraction rate of the concentrate is 30% higher than that of traditional methods. Molding process: Add 20 mg / portion of vitamin C pills and 6 mg / portion of vitamin E pills to the concentrate and stir evenly. Control the temperature of the spray drying equipment at 75°C and the spray pressure at 0.35 MPa. After preparing the powder, mix it with refined honey in a ratio of 1:0.6 to make water-honeyed pills and dry them to a moisture content of 7%.

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