A deer foetus composition and capsules thereof

Abstract

The application discloses a kind of deer fetus compositions and its capsule, the composition includes deer fetus freeze-dried powder and fig extract, and the weight ratio of both is 1-2:1-2.The application is compatible with deer fetus and fig, deer fetus adopts vacuum freeze-drying process, fig adopts alcohol extraction process, and specific excipient is selected to prepare deer fetus capsule preparation, and the process is stable and feasible, and the prepared deer fetus capsule improves the sensory quality of product, and the retention of heat-sensitive active ingredients of deer fetus and the effective enrichment of fig active ingredients are considered, with significant effect of helping to enhance immunity, which provides a new technical approach for the depth development of deer fetus resources.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a deer placenta composition and its capsules. Background Technology

[0002] The fetus is from a female sika deer ( Cervus nippon Temminck) or red deer ( Cervus elaphus Deer placenta is a traditional Chinese medicine made from the processed and dried embryo and placenta of deer at a specific developmental stage. It is rich in nutrients and bioactive components. Modern chemical research shows that it contains 18 common amino acids, of which 8 are essential amino acids, accounting for 39.67% to 41.26% of the total content, forming the basis of protein nutrition. In terms of trace elements, deer placenta is rich in essential trace elements such as iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn), as well as basic nutrients such as nucleic acids, polypeptides, minerals, fatty acids, carbohydrates, inorganic salts, lipids, and vitamins.

[0003] Deer placenta possesses immunomodulatory, antioxidant, anti-aging, endocrine-regulating, anti-inflammatory, and analgesic effects. However, raw deer placenta has a distinct fishy and muttony odor, which can easily cause unpleasant taste when consumed directly, reducing consumer compliance. Traditional deer placenta processing often involves baking and drying combined with alcohol aging to mask the odor and facilitate storage. However, this method involves high operating temperatures, which can easily lead to the denaturation and inactivation of heat-sensitive active ingredients such as proteins and polypeptides in the deer placenta. The immunomodulatory active ingredients in figs are mainly flavonoids and phenolic compounds, which have low polarity.

[0004] Therefore, this invention improves the sensory quality of the product while preserving the heat-sensitive active ingredients of deer placenta and effectively enriching the effective ingredients of fig, thus preparing a deer placenta composition and capsules with significant immune-enhancing effects. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a deer placenta composition and its capsules. The deer placenta composition is composed of two medicinal materials: deer placenta and fig. It follows the principle of "using both warming and clearing, and combining tonification and unblocking" to achieve a synergistic effect of tonifying deficiency without promoting heat and supporting the body without causing stagnation. While improving the sensory quality of the product, it also takes into account the retention of the heat-sensitive active ingredients of deer placenta and the effective enrichment of the active ingredients of fig.

[0006] The objective of this invention is achieved through the following means: A deer placenta composition comprising freeze-dried deer placenta powder and fig extract in a weight ratio of 1-2:1-2. Preferably, the weight ratio is 1:1.

[0007] The method for preparing the above-mentioned deer placenta composition includes the following steps: (1) Vacuum freeze-drying of deer placenta: Take whole fresh deer placenta, divide it evenly along the coronal plane, cut it into pieces, spread it flat in the freeze-drying tray, place it in the cold trap of the freeze dryer, and freeze-drying process parameters are: pre-freezing temperature -55°C~-45°C, first sublimation shelf temperature -10°C~0°C, desorption drying temperature 20°C~30°C, maintain for 20 h~30 h, vacuum degree maintained at 150 μbar~250 μbar. After freeze-drying, pulverize the freeze-dried deer placenta, sieve it, and obtain freeze-dried deer placenta powder. (2) Fig extract: Take fig powder, add 10 to 20 times the weight of fig powder with 40% to 50% ethanol and reflux for extraction. Extract for 1 to 3 hours, extract 2 to 4 times, concentrate and dry to obtain fig extract. (3) Mix deer placenta freeze-dried powder and fig extract according to the ratio.

[0008] Preferably, the freeze-drying process parameters in step (1) are: pre-freezing temperature -50°C, first sublimation shelf temperature 0°C, desorption drying temperature 25°C, maintained for 30 h, and vacuum degree maintained at 200 μbar.

[0009] Preferably, in step (2), the fig powder is added and refluxed with 50% ethanol at a mass concentration of 150 times its weight for 1 hour, and the extraction is performed 3 times.

[0010] The above-mentioned deer placenta composition is formulated into a preparation with pharmaceutically acceptable excipients, preferably a capsule.

[0011] The above-mentioned deer placenta capsules are made from the following components in parts by weight: 100-200 parts of freeze-dried deer placenta powder, 100-200 parts of fig extract, 5-15 parts of croscarmellose sodium, and 5-15 parts of magnesium stearate.

[0012] The above-mentioned deer placenta capsules are prepared by weighing deer placenta freeze-dried powder, fig extract, croscarmellose sodium cellulose and magnesium stearate according to the prescription amount, mixing them evenly, and filling them into hard capsules.

[0013] The deer placenta composition described in this invention can be used in the preparation of drugs that enhance immunity.

[0014] Based on the characteristics of the active ingredients of the two types of raw materials, this invention selects specific targeted processing techniques to finally complete the compounding and encapsulation of the composition.

[0015] The main active ingredients in deer placenta are proteins, polypeptides, amino acids and nucleosides, which are highly heat-sensitive. This invention uses a vacuum freeze-drying process to directly sublimate water under low temperature and vacuum conditions, thus preserving its biological activity to the greatest extent. The resulting freeze-dried product has a loose and porous structure and good rehydration properties, which is particularly beneficial for the dissolution and absorption of subsequent formulations.

[0016] The immunomodulatory active ingredients in figs are mainly flavonoids and phenolic compounds, which are relatively low in polarity. This invention uses reflux extraction with 40%–50% low-concentration ethanol. This concentration range effectively extracts moderately polar components such as flavonoids and saponins, while also dissolving polysaccharides, thus achieving the enrichment of various active ingredients.

[0017] This formula addresses the "fundamental deficiency" issues such as kidney yang depletion and insufficient essence and blood caused by work pressure and irregular lifestyles in modern people. Deer placenta is the principal ingredient, greatly replenishing deficiencies, strengthening vital energy, and enhancing bodily functions from the root. Fig is the secondary ingredient; it is the fruit of the fig tree (Ficus carica), a plant belonging to the genus Ficus of the Moraceae family. Ficus carica The fruit of the fig tree (Limonium spp.) is sweet and neutral in nature, and enters the lung, spleen, and large intestine meridians. In this formula, it plays three key roles: adjuvant, beneficial, and synergistic. Firstly, as an adjuvant: the cool and moistening nature of fig effectively counteracts the warming and drying properties of deer placenta, preventing symptoms of "heatiness" such as dry mouth and constipation that may occur with prolonged use or in individuals with a constitution prone to heat, ensuring the entire formula is warm without being drying. Secondly, as a beneficial agent: entering the lung meridian clears heat and moistens the lungs, helping the deer placenta to replenish its vital energy and improving shortness of breath caused by deficiency; entering the large intestine meridian strengthens the spleen and clears the intestines, ensuring smooth bowel movements and better absorption of the tonifying ingredients, achieving nourishment without stagnation. Thirdly, as a synergistic agent: fig strengthens the spleen and generates fluids, promoting the production of qi and blood, complementing the deer placenta's replenishment of innate essence and blood. When combined with deer placenta, the deer placenta warms and nourishes the essence and blood of the lower burner (kidneys and liver), while the fig clears and moistens the dryness and heat of the upper burner (lungs) and middle burner (stomach and intestines). The two ingredients form a unique pattern of treating both the upper and lower body, regulating qi and blood, and simultaneously supporting the body's resistance and moisturizing dryness, achieving an overall effect of being warm without being drying, nourishing without being greasy, and clearing without being cold. This combination not only follows the theory of traditional Chinese medicine, but also differs from the all-encompassing formulation of pharmaceutical products, and from the simple accumulation of health ingredients in the formulations of commercially available health foods. Attached Figure Description

[0018] Figure 1 Weight monitoring results of a chemotherapy mouse model Figure 2 Spleen mass index results in a chemotherapy mouse model Figure 3 Platelet count results in a chemotherapy mouse model Figure 4 White blood cell count results in a chemotherapy mouse model Figure 5 Leukocyte classification results in a chemotherapy mouse model Figure 6 Eutectic point temperature measurement curve Figure 7 Eutectic point temperature measurement curve Figure 8 DSC curve Figure 9 freeze-drying curve Figure 10 Vacuum freeze-dried deer placenta finished product Detailed Implementation

[0019] The present invention will be further explained and illustrated below through specific embodiments: Example 1

[0020] (1) Vacuum freeze-drying of deer placenta: Take whole fresh deer placenta, evenly divide and cut it into pieces along the coronal plane, spread it flat in a freeze-drying tray, and place it in the cold trap of a freeze dryer. Set the pre-freezing temperature to -50°C, lower it to the target temperature within 30 min and maintain it for 2 h to ensure that the sample is completely frozen. After pre-freezing, enter the sublimation drying stage. The system controls the material temperature to slowly rise from -50°C to 0°C and maintain it for 30 h, and the vacuum degree is maintained at 200 μbar, so that the frozen ice crystals directly sublimate into water vapor under low temperature vacuum conditions. After sublimation drying, enter the desorption drying stage, the material temperature is gradually raised to 25°C and maintained for 30 h, and the vacuum degree is maintained at 200 μbar to remove residual bound water. The drying endpoint is monitored online by pressure rise test. After freeze-drying, pulverize the freeze-dried deer placenta, sieve it, and obtain deer placenta freeze-dried powder.

[0021] (2) Fig alcohol extraction: 15 times the amount of 50% ethanol was used to extract the fig powder by reflux for 1 hour and 3 times. The extract was then concentrated and dried to obtain the fig extract.

[0022] (3) Mix deer placenta freeze-dried powder and fig extract in a mass ratio of 1:1.

[0023] The above-mentioned method for preparing deer placenta capsules includes the following steps: Weigh out the freeze-dried deer placenta powder, fig extract, croscarmellose sodium cellulose and magnesium stearate according to the prescription, mix them evenly, and fill them into No. 0 capsules.

[0024] Example 2

[0025] (1) Vacuum freeze-drying of deer placenta: Take whole fresh deer placenta, evenly divide and cut it into pieces along the coronal plane, spread it flat in a freeze-drying tray, and place it in the cold trap of a freeze dryer. Set the pre-freezing temperature to -55°C, lower it to the target temperature within 30 min and maintain it for 2 h to ensure that the sample is completely frozen. After pre-freezing, enter the sublimation drying stage. The system controls the material temperature to slowly rise from -50°C to 0°C and maintain it for 30 h, and the vacuum degree is maintained at 200 μbar, so that the frozen ice crystals directly sublimate into water vapor under low temperature vacuum conditions. After sublimation drying, enter the desorption drying stage, the material temperature is gradually raised to 30°C and maintained for 25 h, and the vacuum degree is maintained at 200 μbar to remove residual bound water. The drying endpoint is monitored online by pressure rise test. After freeze-drying, pulverize the freeze-dried deer placenta, sieve it, and obtain deer placenta freeze-dried powder.

[0026] (2) Fig alcohol extraction: Take 20 times the amount of 45% ethanol of fig powder and reflux for extraction. The extraction time is 2 hours and the extraction is repeated twice. The mixture is then concentrated and dried to obtain fig extract.

[0027] (3) Mix deer placenta freeze-dried powder and fig extract in a mass ratio of 1:1.

[0028] The above-mentioned method for preparing deer placenta capsules includes the following steps: Weigh out the freeze-dried deer placenta powder, fig extract, croscarmellose sodium cellulose and magnesium stearate according to the prescription, mix them evenly, and fill them into No. 0 capsules.

[0029]

[0030] Experimental Example 1: Protective effect of deer placenta capsules on cyclophosphamide-induced immunosuppressed mice 1. Experimental Objective The protective effect of deer placenta capsules against chemotherapy-induced immunosuppression was evaluated using a cyclophosphamide (CTX)-induced immunosuppressed mouse model, and the effect was compared with that of deer placenta lyophilized powder alone.

[0031] 2. Test Sample Freeze-dried deer placenta powder: prepared according to the method in Example 1.

[0032] The contents of the deer placenta capsules were mixed evenly according to the prescription in Example 1 (deer placenta freeze-dried powder: fig extract = 1:1).

[0033] 3. Laboratory animals Fifty female Balb / c mice, aged 6-8 weeks and weighing 18-22 g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd., with production license No.: SCXK (Shanghai) 2017-0005. The mice were housed in a SPF barrier environment with free access to food and water. The license No. for the use of experimental animals is SYXK (Shanghai) 2020-0009.

[0034] 4 Main Reagents and Instruments for Experiment Cyclophosphamide (CTX): Baxter Oncology Gmbh, Lot No. 1L513A.

[0035] Recombinant human thrombopoietin injection (Tebo): Shenyang Sansheng Pharmaceutical Co., Ltd., Lot No. 202204029.

[0036] RPMI-1640 medium and fetal bovine serum (FBS): Gibco.

[0037] Concanavalin A (ConA) and lipopolysaccharide (LPS): Sigma.

[0038] CCK-8 kit: Meilun Biotechnology.

[0039] Sodium carboxymethyl cellulose (CMC-Na): prepared into 0.5% solution as suspending agent.

[0040] Red blood cell lysate (self-prepared): 8.291 g of NH4CL, 1.0012 g of KHCO3, 0.02923 g of EDTA, add distilled water to 1 L, adjust pH to 7.6.

[0041] Phosphate buffered saline (PBS): 8.0 g of NaCl, 1.16 g of Na2HPO4, 0.2 g of KH2PO4, 0.2 g of KCl, add distilled water to 1 L, pH 7.3.

[0042] Anti-mouse CD3-BV421, CD4-PerCP-Cy5.5, and CD8-PE fluorescent antibodies: BD.

[0043] Blood cell analyzer, flow cytometer (BD FACSCanto II), microplate reader (Thermo), and CO2 incubator.

[0044] 5 Experimental Grouping and Model Establishment Fifty mice were randomly divided into 5 groups with 10 mice in each group:

[0045] Modeling method: Except for the normal control group, the other groups were intraperitoneally injected with cyclophosphamide 75 mg / kg (prepared with physiological saline to a concentration of 7.5 mg / mL, injected at a rate of 0.1 mL / 10g body weight) on days 1, 4, and 7 of the experiment. The normal control group was intraperitoneally injected with an equal volume of physiological saline at the same time points.

[0046] 6. Sample preparation and administration Dosage conversion basis: Assuming an adult takes 4 g of deer placenta lyophilized powder orally per day, and a mouse weight of 20 g, the equivalent dose ratio based on body surface area conversion between humans and mice is 0.0026. The calculated dose per 10 g body weight for mice by gavage is: 4 × 0.0026 ÷ 20 × 10 = 0.0052 g / 10g·day (i.e., 0.52 g / kg·day).

[0047] Deer placenta freeze-dried powder suspension: Weigh the deer placenta freeze-dried powder, grind and dilute it with 0.5% sodium carboxymethyl cellulose solution to form a uniform suspension, and prepare a concentration of 0.052 g / mL (0.1 mL per 10 g body weight by gavage).

[0048] Deer placenta capsule contents suspension: Weigh the contents of the deer placenta capsules (containing deer placenta freeze-dried powder and fig extract in a 1:1 ratio), grind and dilute with 0.5% sodium carboxymethyl cellulose solution to form a uniform suspension. Calculated based on 0.052 g / mL of deer placenta freeze-dried powder, the concentration of the capsule contents is 0.104 g / mL (0.1 mL per 10 g body weight administered by gavage, containing 0.0052 g of deer placenta freeze-dried powder and 0.0052 g of fig extract).

[0049] Terbinafine injection: For adults, the daily subcutaneous injection dose is 18,000 units. Using a conversion factor of 0.0026, the dose for mice per 10 g body weight is 18,000 × 0.0026 ÷ 20 × 10 = 23.4 U / 10g·day (i.e., 2340 U / kg·day). Dilute the stock solution (15,000 U / mL) with physiological saline to 234 U / mL, and administer 0.1 mL subcutaneously per 10 g body weight.

[0050] Blank solvent control: The normal control group and the model control group were administered an equal volume (0.1 mL / 10 g) of 0.5% sodium carboxymethyl cellulose solution by gavage daily.

[0051] Dosage regimen: Starting from day 1 of the experiment, the deer placenta lyophilized powder group and the deer placenta capsule group received the corresponding suspension (0.1 mL / 10g body weight) via gastric gavage daily. The positive control group received a subcutaneous injection of terbinafine (0.1 mL / 10g body weight). The normal control and model control groups received an equal volume of 0.5% sodium carboxymethyl cellulose solution via gavage. The intervention was continued for 10 consecutive days. A No. 20 gavage needle was used for gavage, and the procedure was performed gently to avoid injury.

[0052] 7. Detection Indicators and Experimental Methods 7.1 Weight monitoring Starting from the first day of the experiment, the weight of each mouse was measured at a fixed time every morning, and the average weight and trend of each group were recorded and calculated.

[0053] 7.2 Blood cell differential analysis At the experimental endpoint (day 10), two hours after drug administration, blood was collected by enucleation and placed in sodium citrate anticoagulant tubes. After thorough mixing, the white blood cell (WBC), red blood cell (RBC), platelet (PLT) counts and hemoglobin (HGB) levels in whole blood were measured using a hematology analyzer.

[0054] 7.3 Spleen Mass Index After blood collection, mice were euthanized by cervical dislocation, and the spleen was aseptically removed. The spleen was rinsed with pre-cooled PBS to remove surface blood, blotted dry with filter paper, and weighed. The spleen index was calculated as follows: Spleen index (mg / g) = Spleen weight (mg) / Mouse body weight (g).

[0055] 7.4 Detection of Splenic Lymphocyte Proliferative Capacity Spleen cell suspension preparation: Spleens from each group of mice were collected and placed in a petri dish containing 5 mL of RPMI-1640 basal culture medium. The cells were gently ground using a syringe needle and filtered through a 200-mesh nylon mesh. The filtrate was collected in a 15 mL centrifuge tube and centrifuged at 1200 rpm for 5 minutes at 4°C. The supernatant was discarded, and the precipitate was mixed with approximately 0.6 mL of erythrocyte lysis buffer per spleen. The mixture was gently shaken and allowed to stand for 1 minute. 10 mL of PBS was added for equilibration, and the mixture was centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded, and the precipitate was washed once with PBS. Finally, the cells were resuspended in RPMI-1640 complete culture medium containing 10% FBS.

[0056] Cell counting and adjustment: Mix 20 μL of cell suspension with an equal volume of 0.4% trypan blue, and count the number of viable cells using a hemocytometer. Adjust the cell concentration to 4 × 10⁻⁶ cells / mL. 6 per mL.

[0057] Lymphocyte proliferation stimulation: Add 100 μL of cell suspension (4 × 10⁴ cells / well) to each well of a 96-well cell culture plate. 5 (1 cell / well), then add 100 μL of mitogen: ConA (final concentration 5 μg / mL) or LPS (final concentration 10 μg / mL). Set up three replicates and a blank control well without mitogen (add 100 μL of complete culture medium). Incubate the culture plate at 37°C in a 5% CO2 incubator for 48 hours.

[0058] CCK-8 assay: 6 hours before the end of culture, add 10 μL of CCK-8 solution to each well, gently shake to mix, and continue culture for another 6 hours. After culture, measure the absorbance (OD value) at 450 nm using a microplate reader. Lymphocyte proliferation rate (%) = (OD value of experimental wells - OD value of blank wells) / (OD value of normal control wells corresponding to the original stimulus - OD value of blank wells) × 100%.

[0059] 7.5 Detection of peripheral blood T lymphocyte subset proportions Peripheral blood mononuclear cell (PBMC) isolation: Take 200 μL of anticoagulated whole blood and dilute it with an equal volume of PBS. Take a 15 mL centrifuge tube, add 3 mL of Ficoll separation buffer, and slowly add the diluted blood sample to the supernatant. Centrifuge at 2000 rpm and 18°C ​​for 20 minutes, and carefully aspirate the white membrane layer into a new centrifuge tube. Add 5 mL of PBS to wash, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and repeat the washing once. Resuspend the cells in 100 μL of PBS.

[0060] Fluorescent labeling: Add anti-mouse CD3-BV421 (1 μL), CD4-PerCP-Cy5.5 (1 μL), and CD8-PE (1 μL) fluorescent antibodies respectively, mix gently, and incubate at room temperature in the dark for 20 minutes. After incubation, wash with 2 mL PBS, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and repeat once. Finally, resuspend the cells in 300 μL PBS.

[0061] Flow cytometry detection: Using a flow cytometer, first gating lymphocyte populations, then detecting CD3. + CD4 + The ratio of helper T cells (Th) to CD3⁺CD8⁺ cytotoxic T cells (CTL).

[0062] 8. Data Statistics Quantitative data are expressed as mean ± standard deviation. The results were presented in the table. One-way ANOVA was used for comparisons among multiple groups, with each group compared to the model control group. The significance level was set at P < 0.05. Statistical analysis and graphing were performed using GraphPad Prism 7 software.

[0063] 9. Experimental Results 9.1 Weight Changes There was no significant difference in average body weight among the groups on day 1 of the experiment. The model control group showed a significant decrease in body weight after CTX injection (days 1, 4, and 7), and remained lower than the normal control group on day 10 (P<0.001). The weight loss in the deer placenta lyophilized powder group and the deer placenta capsule group was slightly less than that in the model group, but there was no statistically significant difference (P>0.05), suggesting that the samples had no significant effect on improving body weight. Experimental results are shown below. Figure 1 .

[0064] 9.2 Spleen Mass Index Experimental results show (see) Figure 2 Table 2): The spleen mass index of mice in the model control group showed a decreasing trend compared with that of the normal control group. Terbinafine could alleviate the decrease in spleen mass to a certain extent, while both deer placenta freeze-dried powder and deer placenta capsules could significantly increase the spleen mass index, and both showed statistically significant differences compared with the model group. Among them, the effect of deer placenta capsules was more obvious.

[0065]

[0066] Compared with the model control group, P<0.01, *P<0.001.

[0067] 9.3 Proliferative capacity of spleen lymphocytes The experimental results are shown in Table 3. The lymphocyte proliferation capacity of the model group was significantly reduced (P<0.001 vs. normal group). The deer placenta capsule group showed a significant increase in lymphocyte proliferation rate under ConA and LPS induction (P<0.01), while there was no significant difference in the deer placenta freeze-dried powder group, indicating that the combination with fig enhanced the activation and proliferation capacity of lymphocytes.

[0068]

[0069] Compared with the model control group, **P<0.01.

[0070] 9.4 Platelet count The experimental results are shown in Figure 3 Table 4 shows that platelet count decreased significantly after CTX modeling; the positive control drug terbinafine significantly increased platelet count (P<0.001); although deer placenta lyophilized powder and deer placenta capsules showed an increasing trend, there was no statistical difference compared with the model group.

[0071]

[0072] Compared with the model control group, ***P<0.001.

[0073] 9.5 Red blood cells and hemoglobin The experimental results are shown in Table 5. The red blood cell count and hemoglobin level in the model group were significantly lower than those in the normal group (P<0.001). There were no significant differences between the drug administration groups and the model group, indicating that the test samples had no significant effect on red blood cells and hemoglobin.

[0074]

[0075] 9.6 White blood cell count and differential The main components of white blood cells include neutrophils (NEUT), lymphocytes (LYMPH), monocytes / macrophages (MONO), eosinophils (EOS), and basophils (BASO). Their cellular functions are primarily related to immune defense, such as inflammation prevention, immune responses, and allergic reactions. Chemotherapy can also lead to a decrease in white blood cell production, as shown in experimental results (see...). Figure 4 Table 6: The total white blood cell count in the model group was significantly lower than that in the normal group (P<0.001). The total white blood cell count in each treatment group showed a slight upward trend, but there was no statistically significant difference compared with the model group.

[0076]

[0077] Further white blood cell differential results showed (see) Figure 5 (Table 7) The proportion of lymphocytes in the model group decreased and the proportion of neutrophils increased compared with the normal group; the proportion of lymphocytes in the deer placenta freeze-dried powder group and the deer placenta capsule group showed an upward trend.

[0078]

[0079] 9.7 T lymphocyte subset ratio The experimental results are shown in Table 8. The proportions of Th and CTL in the model group were significantly lower than those in the normal group (P<0.001). The proportions in the deer placenta lyophilized powder group showed an increasing trend but no statistical difference. The deer placenta capsule group significantly increased the proportion of Th (P<0.01), and the proportion of CTL also showed an increasing trend, suggesting that the deer placenta capsule can effectively reverse the CTX-induced imbalance of T cell subsets, and the effect is better than that of deer placenta lyophilized powder alone.

[0080]

[0081] Compared with the model control group, *P<0.05, **P<0.01.

[0082] 10. Experimental Conclusions In a cyclophosphamide-induced immunosuppressed mouse model, deer placenta capsules significantly increased spleen mass index, enhanced ConA and LPS-induced splenic lymphocyte proliferation, and upregulated peripheral blood helper T cells (CD3+). + CD4 + The above indicators are all superior to those of the same dose of freeze-dried deer placenta powder alone, indicating that the combination of deer placenta and fig has a synergistic effect in enhancing immunity. Therefore, the deer placenta capsules prepared by this invention have significant health benefits in enhancing immunity.

[0083] Experimental Example 2: Determination of the freeze-drying process for deer placenta This invention uses the resistance method to determine the eutectic point and eutectic point of deer placenta, and the differential scanning calorimetry (DSC) method to determine its glass transition temperature. Based on the above parameters, the pre-freezing temperature, the first sublimation shelf temperature, and the desorption drying temperature are set. On this basis, freeze-drying experiments are carried out, and the rationality and feasibility of the set process parameters are evaluated with the appearance and active ingredient retention rate of the freeze-dried product as indicators.

[0084] 1. Determination of eutectic point, eutectic melting point, and glass transition temperature The resistance measurement curve is shown below. Figure 6 , Figure 7 DSC curves are shown below. Figure 8 .

[0085] Depend on Figure 6 It can be seen that during the cooling process, when the temperature drops to around -40°C, the resistance value begins to rise slowly; as the temperature decreases further, the resistance value shows a significant jump, indicating that the liquid phase in the sample has completely solidified. Figure 7 It can be seen that during the heating process, the resistance value drops sharply near -40°C, corresponding to the melting initiation temperature of the eutectic mixture. Based on the measured values ​​using the resistance method, the eutectic point of the deer placenta is determined to be -44.1°C, and the eutectic point is -42.4°C. Figure 8 The DSC curve shows that a glass transition plateau appears around 66.73°C during the heating stage, thus determining the glass transition temperature of the freeze-dried deer placenta to be 66.73°C.

[0086] 2. Setting and Verification of Freeze-drying Process Parameters Based on the above parameter measurement results and the heat and mass transfer principle of freeze-drying, the freeze-drying process parameters are set as follows: pre-freezing temperature -50°C, which is below the eutectic point and can ensure that the material is completely frozen; primary sublimation shelf temperature 0°C, at which temperature the temperature of the frozen layer inside the material is always far below the eutectic point -42.4°C, and only surface heating is used to promote the sublimation of ice, thereby improving drying efficiency while ensuring product structural stability; analytical drying temperature 25°C, which is below the glass transition temperature and can ensure the full removal of bound water in the amorphous region, thereby improving product storage stability.

[0087] The rationality and safety of this parameter setting can be verified from three dimensions: process principle, freeze-drying curve, and finished product quality. From the perspective of process principle, in the first sublimation stage of freeze-drying, the shelf acts as a heat source, and heat is conducted through the material layer to the ice-air interface. The ice sublimation process continuously absorbs heat, ensuring that the temperature of the frozen layer inside the material is always far below the set temperature of the shelf; only the surface material temperature is close to the shelf temperature. Therefore, setting the shelf temperature to 0°C can shorten the drying cycle while ensuring that the temperature of the frozen layer inside the material remains below the eutectic point of -42.4°C and without the risk of melting. From the freeze-drying history curve (… Figure 9As can be seen, the product temperature remained below 0°C throughout the entire sublimation stage, and was well below the eutectic point throughout, with no sudden temperature rise or overheating. Simultaneously, the vacuum level in the drying oven remained stable at a low vacuum, and the condenser temperature remained below -60°C, indicating that the ice sublimation process was consistently stable, the frozen layer did not melt, and there was no risk of product collapse. In terms of finished product quality, the samples were in good condition after freeze-drying. Figure 10 The material exhibited a complete structure, without collapse, bubbling, or melting marks, displaying a uniform, loose, porous structure, further validating the rationality and safety of the 0°C single sublimation temperature. The vacuum freeze-drying process was determined as follows: Whole fresh deer placenta was uniformly divided and cut into pieces along the coronal plane, spread evenly in a freeze-drying tray, and placed in the cold trap of a freeze dryer. The pre-freezing temperature was set at -50°C, and the temperature was lowered to the target temperature within 30 minutes and maintained for 2 hours to ensure complete freezing of the sample. After pre-freezing, the sublimation drying stage began. The system controlled the material temperature to slowly rise from -50°C to 0°C and maintain it for 30 hours, while the vacuum degree was maintained at 200 μbar, allowing the frozen ice crystals to directly sublimate into water vapor under low-temperature vacuum conditions. After sublimation drying, the desorption drying stage began, with the material temperature gradually increased to 25°C and maintained for 30 hours, while the vacuum degree was maintained at 200 μbar to remove residual bound water. The drying endpoint was monitored online using a pressure rise test. After freeze-drying, the freeze-dried deer placenta was pulverized, sieved, and the resulting freeze-dried deer placenta powder was sealed for later use.

[0088] Experimental Example 3: Determination of Fig Alcohol Extraction Process Using commercially available dried figs (crushed through a 50-mesh sieve) as raw material, the effects of different extraction solvents and extraction methods on the extraction efficiency of total flavonoids were compared in a preliminary experiment. Fig powder was treated with water and 50% ethanol as solvents, respectively, using reflux extraction and ultrasonic extraction. After appropriate treatment, the absorbance of the extract was measured at 359 nm with rutin as a reference standard.

[0089] The results showed that the reflux extraction method using 50% ethanol as the solvent produced the highest absorbance value, and therefore it was determined to be the basic extraction process for fig powder. See Table 9. Further optimization of the extraction parameters will be achieved through single-factor and orthogonal experiments.

[0090]

[0091] Based on the single-factor experiment, 10 g of fig powder was taken and... L 9 (3) 4 An orthogonal experimental design was used to systematically investigate the effects of ethanol concentration, solvent ratio, extraction time, and extraction times on the extraction efficiency. The extract yield, total polysaccharide content, and total flavonoid content were used as evaluation indicators to optimize and screen the best extraction process parameters. The factor level design is shown in Table 10, and the orthogonal experimental arrangement and results analysis are shown in Table 11.

[0092]

[0093]

[0094]

[0095] Using the comprehensive score as the indicator, the range analysis results showed D>A>B>C, meaning the order of influencing factors was: extraction times > ethanol concentration > solvent volume ratio > extraction time. The optimal combination of each factor and level was A3B2C1D3, i.e., ethanol concentration of 50%, ethanol volume ratio of 15 times, extraction time of 1 hour, and extraction times of 3 times. Further analysis of variance was conducted using option C as the error term.

[0096] The analysis of variance showed that none of the factors were statistically significant. The determined extraction process was as follows: reflux extraction with 15 times the volume of 50% ethanol for 1 hour, repeated 3 times.

[0097] The method for determining the total polysaccharide content in fig alcohol extract was as follows: the determination was performed in accordance with the General Chapter 0401 (ultraviolet-visible spectrophotometry) of Part IV of the 2025 edition of the Chinese Pharmacopoeia.

[0098] The method for determining the total flavonoid content in fig alcohol extract is as follows: refer to the second method (aluminum nitrate-sodium nitrite method) in the "Technical Guidelines for Testing and Evaluation of Physicochemical and Hygienic Indicators of Health Food (2020 Edition)".

[0099] 40 g of fig powder was weighed and extracted three times with 15 times the volume of 50% ethanol solution, each time for 1 hour. Three batches of verification experiments were conducted. The results showed that the verification experiment results were in good agreement with the orthogonal experiment results. Therefore, the fig ethanol extraction process was determined to be reflux extraction with 15 times the volume of 50% ethanol, extraction time of 1 hour, and extraction three times. See Table 13.

[0100]

[0101] Experimental Example 4: Screening of Capsule Formulation Composition The disintegration time test method of the General Chapter (0921) of Part IV of the 2020 edition of the Chinese Pharmacopoeia and the determination of the angle of repose of surfactant powders and particles (GB / T 11986-1989) were investigated respectively.

[0102] 1. Screening of disintegrant dosage Croscarmellose sodium was selected as the disintegrant. Results showed that the addition of croscarmellose sodium significantly shortened the disintegration time; when the dosage was increased to 3%, the disintegration time was significantly improved. Therefore, the dosage of croscarmellose sodium was determined to be 3%. (See Table 14.)

[0103]

[0104] 2. Screening of Lubricant Dosage Magnesium stearate, a commonly used lubricant, was selected. Results showed that adding magnesium stearate powder improved flowability, but the angle of repose showed no significant improvement at dosages of 1.0% and 1.5%. Considering the hydrophobic nature of magnesium stearate, the dosage was determined to be 1.0%. (See Table 15.)

[0105]

[0106] 3. Determining the Capsule Model To determine the appropriate capsule size, the theoretically required minimum volume needs to be calculated based on the tap density of the contents of the deer placenta capsule. Referring to the tap density determination method in General Chapter (0993) of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the results are shown in Table 16.

[0107] The average tap density of the mixture powder prepared by the method in Example 1 for the three batches was 0.41 g / mL, the target fill weight of each capsule was 0.30 g, and the theoretical minimum required volume was 0.73 mL. Comparing the volumes of the various capsule types, capsule No. 0 met the fill weight requirement with an appropriate margin; although capsule No. 00 had a larger volume, its larger size might cause difficulty in swallowing. Therefore, capsule No. 0 was selected.

[0108]

[0109] 4. Prescription

[0110] Deer placenta lyophilized powder and fig extract were prepared according to the method in Example 1. The deer placenta lyophilized powder, fig extract, croscarmellose sodium, and magnesium stearate were weighed according to the prescribed amounts, mixed evenly, and filled into No. 0 capsules. Three batch validation tests were conducted. The results showed that the quality was uniform across batches, with good reproducibility, and the established formulation process was stable and feasible. See Table 17.

[0111]

Claims

1. A deer placenta composition, characterized in that... The composition comprises freeze-dried deer placenta powder and fig extract in a weight ratio of 1-2:1-2.

2. The deer placenta composition according to claim 1, characterized in that... The freeze-dried deer placenta powder and fig extract are in a weight ratio of 1:

1.

3. A method for preparing the deer placenta composition according to claim 1, characterized in that... The method includes the following steps: (1) Vacuum freeze-drying of deer placenta: Take whole fresh deer placenta, divide it evenly along the coronal plane, cut it into pieces, spread it flat in the freeze-drying tray, place it in the cold trap of the freeze dryer, and freeze-drying process parameters are: pre-freezing temperature -55°C~-45°C, first sublimation shelf temperature -10°C~0°C, desorption drying temperature 20°C~30°C, maintain for 20 h~30 h, vacuum degree maintained at 150 μbar~250 μbar. After freeze-drying, pulverize the freeze-dried deer placenta, sieve it, and obtain freeze-dried deer placenta powder. (2) Fig extract: Take fig powder, add 10 to 20 times the weight of fig powder with 40% to 50% ethanol and reflux for extraction. Extract for 1 to 3 hours, extract 2 to 4 times, concentrate and dry to obtain fig extract. (3) Mix deer placenta freeze-dried powder and fig extract according to the ratio.

4. The method for preparing the deer placenta composition according to claim 3, characterized in that... The freeze-drying process parameters in step (1) are: pre-freezing temperature -50°C, first sublimation shelf temperature 0°C, desorption drying temperature 25°C, maintained for 30 h, and vacuum degree maintained at 200 μbar.

5. The method for preparing the deer placenta composition according to claim 3, characterized in that... In step (2), the fig powder is added at 150 times its weight of 50% ethanol and refluxed for 1 hour, and the extraction is repeated 3 times.

6. The deer placenta composition according to claim 1, characterized in that... The composition is formulated with pharmaceutically acceptable excipients.

7. The deer placenta composition according to claim 6, characterized in that... The preparation is a capsule.

8. A deer placenta capsule according to claim 7, characterized in that... It is made by mixing 100-200 parts of freeze-dried deer placenta powder, 100-200 parts of fig extract, 5-15 parts of croscarmellose sodium and 5-15 parts of magnesium stearate.

9. A method for preparing the deer placenta capsule according to claim 8, characterized in that... The method involves weighing out the freeze-dried deer placenta powder, fig extract, croscarmellose sodium cellulose, and magnesium stearate according to the prescription, mixing them evenly, and filling them into hard capsules.

10. The use of the deer placenta composition according to claim 1 in the preparation of a medicament for enhancing immunity.

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