Traditional Chinese medicine compound preparation for treating aplastic anemia

Abstract

The invention provides a traditional Chinese medicine composition for treating aplastic anemia. The traditional Chinese medicine composition is prepared from monarch drugs including astragalus membranaceus, prepared rehmannia roots and hairyvein agrimony, ministerial drugs including lychee seeds, rhizoma bletillae and sliced cornu cervi, adjuvant drugs including peach kernels, carthamus tinctorius, lumbricus, rhizoma wenyujin concisum and rabdosia amethystoides, conductant drugs including liquorice and adjuvant drugs including cassia twigs. According to the traditional Chinese medicine composition, the compatibility thought of taking the bulbophyllum and the lychee seeds as the outline is created for the first time, the bletilla striata is combined with the lychee seeds for promoting the circulation of qi, and the antler slices are used for replenishing essence and benefiting marrow, so that a characteristic scheme of regulating qi and blood and dredging and resuscitation is formed. The composition can regulate the core pathogenesis of qi and blood deficiency, marrow sea insufficiency and blood stasis of aplastic anemia at multiple targets, effectively improves the bone marrow hematopoiesis function, solves the problem that the existing therapy is difficult to consider deficiency and excess inclusion, and provides a brand-new and efficient traditional Chinese medicine treatment choice for aplastic anemia.

Description

Technical Field

[0001] This invention relates to a traditional Chinese medicine compound preparation for treating aplastic anemia, belonging to the field of traditional Chinese medicine composition technology. Background Technology

[0002] Aplastic anemia (AA), an acquired bone marrow failure disease, is considered a key research focus in both hematology and traditional Chinese medicine. Since the mid-20th century, Western medicine treatment strategies have primarily focused on immunosuppressive therapy (such as anti-thymocyte globulin ATG and cyclosporine CsA) and hematopoietic stem cell transplantation (HSCT). Although these methods have achieved some efficacy in some patients, their application is limited by donor availability, economic costs, and the risk of serious complications. In recent years, with the development of molecularly targeted drugs, TPO receptor agonists and androgen drugs have also been gradually applied clinically, but their effects on improving the complete remission rate (CR) are limited, and they also have significant adverse reactions such as hepatotoxicity, androgenization, and metabolic disorders. Therefore, while existing Western medicine treatments have value in delaying disease progression, the overall efficacy is not ideal, and the relapse rate and progression risk remain high. In the AA population, some patients do not meet the diagnostic criteria for severe aplastic anemia (SAA) and are classified as non-severe aplastic anemia (NSAA). Although these patients have a milder decrease in blood cell counts, they still face the risk of disease progression and transformation, making clinical intervention essential. NSAA has a high incidence in Asia and constitutes a significant portion of the overall AA patient population. Current treatment for NSAA primarily relies on cyclosporine (CsA) monotherapy or individualized combination therapy; however, long-term complete remission rates remain low, necessitating new treatment approaches.

[0003] Against this backdrop, traditional Chinese medicine (TCM) treatment has gradually gained attention. Since the 1980s, scholars both domestically and internationally have begun exploring the adjunctive effects of qi-tonifying, blood-nourishing, kidney-tonifying, and spleen-strengthening formulas on aplastic anemia. Clinical practice and animal experiments have shown that TCM can exert its effects through multiple pathways, including: improving the bone marrow microenvironment, promoting hematopoietic stem cell proliferation and differentiation, regulating immune function, and anti-inflammatory and antioxidant responses. Previous studies have reported that TCM can steadily improve peripheral blood counts, alleviate anemia and bleeding symptoms, and improve patients' physical condition and quality of life. Overall, TCM clinical treatment of aplastic anemia still focuses on "treating from the perspective of the kidneys," but due to a lack of large-sample clinical and experimental data, the relevant efficacy evidence is still insufficient. Some patients may experience improvement in the early stages of drug efficacy, but as the treatment course lengthens, the efficacy reaches a plateau, making further breakthroughs difficult.

[0004] Chinese invention patent CN 1062144C discloses a traditional Chinese medicine preparation for nourishing bone marrow and promoting blood production. The optimal formulation is as follows: It is composed of 15g of *Codonopsis pilosula* (or 30g of *Codonopsis pilosula*), 50g of *Astragalus membranaceus*, 15g of stir-fried *Atractylodes macrocephala*, 20g of *Cornus officinalis*, 20g of tortoise shell glue (or 30g of tortoise shell), 20g of turtle shell, 30g of *Ligustrum lucidum*, 30g of *Rehmannia glutinosa*, 15g of *Morinda officinalis*, 6g of cinnamon, 18g of *Epimedium brevicornu*, 20g of deer antler glue (or 5g of deer antler), 20g of *Angelica sinensis*, 20g of donkey-hide gelatin, 30g of *Spatholobus suberectus*, 30g of *Agrimonia pilosa*, 5g of *Panax notoginseng*, and 4g of indigo naturalis. Based on modern pharmacological analysis, this formula promotes bone marrow hematopoiesis, increases blood cells and hemoglobin, promotes DNA synthesis, and regulates humoral and cellular immunity.

[0005] However, the efficacy of this blood-nourishing traditional Chinese medicine preparation for AA is not significant enough to fully meet the clinical needs of AA patients for a highly effective, safe, and long-term feasible treatment plan. Therefore, developing an innovative plan with high clinical operability and scalability has become an urgent breakthrough in this field. Summary of the Invention

[0006] The present invention aims to solve the above-mentioned problems, thereby providing a traditional Chinese medicine compound preparation for the treatment of aplastic anemia.

[0007] The technical solution of the present invention to solve the above problems is as follows:

[0008] A traditional Chinese medicine compound preparation for treating aplastic anemia, comprising the following traditional Chinese medicine components:

[0009] The principal herb is selected from at least one of Astragalus membranaceus, Rehmannia glutinosa, and Agrimonia pilosa.

[0010] The medicinal ingredients are selected from at least one of the following: litchi seed, Bletilla striata, and deer antler slices.

[0011] The adjuvant ingredients are selected from at least one of the following: peach kernel, safflower, earthworm, turmeric, and fragrant tea vegetable.

[0012] Used medicinal ingredient: Selected from licorice;

[0013] The adjuvant herb is selected from cinnamon twig.

[0014] As a preferred embodiment of the above technical solution, the mass fractions of each component are as follows: principal drug, 80-100 parts; assistant drug, 52-62 parts; adjuvant drug, 50-65 parts; guiding drug, 2-5 parts; adjuvant and guiding drug, 6-12 parts.

[0015] As a preferred embodiment of the above technical solution, the principal ingredient is a combination of Astragalus membranaceus, Rehmannia glutinosa, and Agrimonia pilosa; the assistant ingredient is a combination of Litchi chinensis seed, Bletilla striata, and deer antler slices; and the adjuvant ingredient is a combination of Prunus persica kernel, Carthamus tinctorius, Pheretima aspergillum, Curcuma longa slices, and Citrus aurantium.

[0016] As a preferred embodiment of the above technical solution, the mass fractions of each component are as follows:

[0017] Chief herbs: Astragalus membranaceus 27-33 parts, Rehmannia glutinosa 27-33 parts, Agrimonia pilosa 27-33 parts;

[0018] The following ingredients were used: 38-42 parts of lychee seed, 2-4 parts of Bletilla striata, and 13-17 parts of deer antler slices;

[0019] Medicinal ingredients: 8-10 parts peach kernel, 5-7 parts safflower, 11-13 parts earthworm, 8-10 parts turmeric, 18-22 parts fragrant tea leaves;

[0020] Medicinal herb: 2-4 parts licorice root;

[0021] Adjuvant herb: 7-11 parts of cinnamon twig.

[0022] As a preferred embodiment of the above technical solution, the mass fractions of the components are as follows:

[0023] Astragalus membranaceus 30 parts, Rehmannia glutinosa 30 parts, Agrimonia pilosa 30 parts, Litchi chinensis seed 40 parts, Bletilla striata 3 parts, Cervi cornu slices 15 parts, Prunus persica kernel 9 parts, Carthamus tinctorius 6 parts, Pheretima aspergillum 12 parts, Curcuma longa slices 9 parts, Rhizoma Citri Reticulatae 20 parts, Cinnamomum cassia twig 9 parts, Glycyrrhiza uralensis 3 parts.

[0024] As a preferred embodiment of the above technical solution, the mass fractions of the components are as follows:

[0025] Astragalus membranaceus 30g, Rehmannia glutinosa 30g, Agrimonia pilosa 30g, Litchi chinensis seed 40g, Bletilla striata 3g, Cervi cornu slices 15g, Prunus persica kernel 9g, Carthamus tinctorius 6g, Pheretima aspergillum 12g, Curcuma longa slices 9g, Rhizoma Citri Reticulatae 20g, Cinnamomum cassia twig 9g, Glycyrrhiza uralensis 3g.

[0026] In the above-mentioned technical solution of the present invention, the prescription is based on the comprehensive treatment method of "tonifying qi and nourishing blood, promoting blood circulation and unblocking collaterals, warming yang and assisting transportation, stopping bleeding and astringing yin", and provides all-round conditioning for the pathogenesis of aplastic anemia such as qi and blood deficiency, insufficient marrow and blood stasis.

[0027] Its name ("Jili Shengxue Formula") itself embodies the core diagnostic and treatment principles governing the entire formula, with "Jili" as the guiding principle. "Ji" refers to Bletilla striata in the formula, signifying its ability to "astringe and stop bleeding, preserve and generate blood," specifically controlling bleeding and stabilizing blood counts. This is crucial for managing NSAA patients prone to bleeding and blood not returning to its proper channels, and it can also repair damaged microvessels. "Li" refers to lychee seeds in the formula, signifying their ability to "dredge and unblock, promote qi and blood circulation," aiming to improve qi stagnation and blood stasis, as well as bone marrow obstruction. It is the key driving force for the overall qi and blood circulation in the formula. The two, one astringent and the other dispersing, one active and the other passive, combine strength and gentleness, addressing both the root cause and the symptoms, together forming the unique combination of "harmonizing qi and blood, and balancing astringency and dispersion."

[0028] Guided by this core idea, the entire formula adheres to the principle of "combining movement and stillness" and employs rigorous combinations of principal, assistant, adjuvant, and guide herbs to construct a treatment system that combines attack and tonification for synergistic effects.

[0029] The principal herbs—Astragalus membranaceus, Rehmannia glutinosa, and Agrimonia pilosa—work together to replenish Qi and blood, strengthen the body's resistance, and provide the driving force for blood production. Astragalus membranaceus, a superior Qi-tonifying herb, invigorates Qi, raises Yang, and strengthens the body's defenses against toxins, enhancing immunity, activating bone marrow hematopoietic stem cells, and increasing white blood cell and red blood cell levels. Rehmannia glutinosa nourishes Yin and blood, replenishes essence and marrow, and is a key herb for blood deficiency, promoting the production of red blood cells and hemoglobin. Agrimonia pilosa benefits Qi and blood, astringes and stops bleeding, and also has anti-inflammatory and antioxidant effects, supporting the stability of the hematopoietic microenvironment. These three herbs work synergistically to construct a treatment framework primarily focused on tonification, providing the driving force for blood production and laying a solid foundation for the treatment of NSAIDs.

[0030] The assistant herbs—lychee seed, Bletilla striata, and deer antler slices—work together to regulate Qi and blood circulation, protect blood, and promote tissue regeneration, assisting the principal herb in strengthening hematopoietic function from different dimensions. Lychee seed, with its bitter and warm properties, regulates Qi, soothes the liver, and disperses stagnation, aiding spleen function and promoting Qi and blood circulation; it is a key herb for the overall Qi and blood circulation in the formula. Bletilla striata, with its astringent and consolidating properties, effectively stops bleeding and is crucial for managing NSAA patients prone to bleeding and blood not returning to its proper channels. It also repairs damaged microvessels, playing a key role in protecting and consolidating blood. Deer antler slices warm and tonify kidney Yang, replenish essence and marrow, making it a suitable remedy for those with aplastic anemia suffering from "empty marrow sea," compensating for kidney deficiency and lack of essence and blood. These three herbs work synergistically, assisting the principal herb in strengthening hematopoietic function through three key aspects: regulating Qi, stopping bleeding and consolidating blood, and replenishing essence and marrow, providing strong support for the principal herb.

[0031] The adjuvant herbs—peach kernel, safflower, earthworm, turmeric, cinnamon twig (also used as an adjuvant), and fragrant tea herb—are used together to invigorate blood circulation, remove blood stasis, warm the meridians, and unblock the collaterals. Among them, peach kernel and safflower are a traditional "blood-invigorating and blood-stasis-removing" pair, helping to move stagnant blood and preventing stagnation caused by excessive tonification; earthworm unblocks the collaterals, invigorates blood circulation, has anti-inflammatory and anticoagulant properties, and helps improve bone marrow microcirculation; turmeric invigorates blood circulation, promotes qi flow, and relieves pain, addressing "blood stasis and qi obstruction"; cinnamon twig warms and unblocks the meridians, harmonizes the body's defenses, assists yang qi transformation, and promotes blood circulation; fragrant tea herb has multiple effects such as clearing heat and detoxifying, reducing swelling and dissipating nodules, and regulating immunity. This group of herbs targets NSAA accompanied by blood stasis and poor circulation, aiming to break through the pathological state of "deficiency with excess," enhance the ability to flow smoothly, and ensure that tonification does not retain pathogenic factors.

[0032] Guiding herbs: Licorice root and cinnamon twig (also serving as adjuvant herbs) work together to harmonize the effects of the other herbs. Licorice root, with its sweet, mild, and gentle properties, can coordinate the overall effects of the formula and alleviate potential toxic side effects. Cinnamon twig, in this context, further demonstrates its guiding role by "directing the herbs to their respective meridians and assisting yang and harmonizing the middle jiao," promoting the smooth flow and proper functioning of qi and blood. The combination of these two guiding herbs ensures that the formula is mild and not overly drying, synergistic and harmonious, and enhances the overall effectiveness of the treatment.

[0033] From a Western medical perspective, Astragalus membranaceus, Rehmannia glutinosa, and deer antler slices stimulate the proliferation and differentiation of bone marrow hematopoietic stem cells, increasing the production of red blood cells and platelets; Amaranthus praecox and Agrimonia pilosa regulate the proportion of T cell subsets and downregulate the levels of IFN-γ and TNF-α; Prunus persica, Carthamus tinctorius, Pheretima aspergillum, and Curcuma longa promote blood circulation and improve the bone marrow microenvironment; Bletilla striata astringes yin and stops bleeding, while Litchi chinensis seed soothes the liver and regulates qi, addressing both the symptoms and the root cause.

[0034] Another object of the present invention is to provide a method of using the above-mentioned prescription.

[0035] The technical solution is as follows:

[0036] The above-mentioned traditional Chinese medicine compound preparation is used as follows: Mix the raw materials according to the formula, add 3 to 5 times the weight of water, soak for 1 hour, boil for 30 minutes, and keep the decoction; add an equal amount of water to the dregs, continue to boil for 15 minutes, remove the dregs, combine the decoctions, and concentrate under reduced pressure to form an extract; it can also be further prepared into tablets, granules, capsules or oral liquids.

[0037] Another object of the present invention is to provide a method for preparing the above-mentioned prescription.

[0038] The technical solution is as follows:

[0039] The preparation method of the above-mentioned traditional Chinese medicine compound preparation is as follows: the medicinal materials listed in the prescription, except for deer antler slices, are extracted by decoction at least once, and the filtrates are combined and concentrated; in the post-processing step, deer antler slice solution is added, stirred evenly, and then filtered and shaped to obtain the drug preparation.

[0040] As a preferred embodiment of the above technical solution, the dosage form of the pharmaceutical preparation is an oral liquid, granules, or capsules.

[0041] In summary, the present invention has the following beneficial effects:

[0042] Preliminary laboratory and clinical observations have shown that the compound formula of this invention exhibits the following advantages:

[0043] 1. Significant hematological efficacy: After 6 months of treatment, all three blood cell counts (Hb, WBC, PLT) were significantly improved compared to baseline (p<0.001), with a total effective rate of 73.9%, of which the combined cure and significant improvement rate exceeded 40%;

[0044] 2. Strong Mechanism Support: UPLC-QE-MS analysis detected 1,177 chemical components, of which 52 were blood-entering components (such as amygdalin, luteolin, and vitexin), confirming that they have multi-target effects such as anti-inflammatory, anti-apoptotic, and hematopoietic promotion.

[0045] 3. Basic research results: Preliminary in vitro and in vivo experiments show that the Jili Shengxue formula has significant therapeutic effects on aplastic anemia and leukopenia. It restores hematopoietic function and increases white blood cell count by increasing bone marrow hematopoietic stem cell and white blood cell production in mice with drug-induced bone marrow failure and promoting the migration of neutrophils from the marginal pool to the circulating pool.

[0046] 4. Good safety profile: When used in combination with immunosuppressants, this compound did not increase liver and kidney toxicity and was well tolerated;

[0047] In summary, the "Jili Shengxue Formula" proposed in this invention can significantly improve the blood count level of patients with aplastic anemia and reduce the risk of relapse through multi-level regulation of "tonifying qi and nourishing blood, promoting blood circulation and unblocking collaterals, warming yang and assisting transportation, and stopping bleeding and astringing yin". It has high safety and good clinical application value and promotion prospects. Attached Figure Description

[0048] Figure 1 It is a line graph showing the changes in PLT, HGB, and WBC during follow-up.

[0049] Figure 2 The mean ± standard deviation of PLT, HGB, and WBC at 6 months in each treatment group;

[0050] Figure 3 It is a box plot showing the baseline and 6-month improvement of PLT, HGB, and WBC.

[0051] Figure 4 These are the base peak chromatogram and total ion current chromatogram of the sample in positive ion mode (POS) full scan mode;

[0052] Figure 5 These are the base peak chromatogram and total ion current chromatogram of the sample in positive ion mode (NEG) full scan mode;

[0053] Figure 6 These are identification diagrams for 52 compounds;

[0054] Figure 7 It is an identification table of 52 compounds;

[0055] Figure 8 This is a bar chart showing the peripheral blood leukocyte (WBC) levels of mice in different groups of bone marrow failure models;

[0056] Figure 9 This is a bar chart showing the levels of peripheral blood routine indicators (WBC, HGB, PLT) in different groups of aplastic anemia model mice;

[0057] Figure 10 Representative flow cytometry plots and quantitative analysis of hematopoietic stem cells from different groups of bone marrow failure models;

[0058] Figure 11 This is a flow cytometry plot of CD11b-positive cells induced to differentiate into neutrophils in HL-60 cells from different groups of drug-containing serum;

[0059] Figure 12 CD62L on peripheral blood neutrophils in a bone marrow failure model + Flow plot;

[0060] Figure 13This is a flow cytometry plot of representative hematopoietic stem cells in the bone marrow of aplastic anemia model mice and its quantitative analysis. Detailed Implementation

[0061] The present invention will be further explained and described below with reference to the accompanying drawings.

[0062] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims, will be protected by patent law.

[0063] Example 1: Traditional Chinese Medicine Preparation

[0064] The herbal composition is approximately 270g, and the formula is as follows (parts by weight):

[0065] Astragalus membranaceus 30 parts, Rehmannia glutinosa 30 parts, Agrimonia pilosa 30 parts, Litchi chinensis seed 40 parts, Bletilla striata 3 parts, Cervi cornu slices 15 parts, Prunus persica kernel 9 parts, Carthamus tinctorius 6 parts, Pheretima aspergillum 12 parts, Curcuma longa slices 9 parts, Rhizoma Citri Reticulatae 20 parts, Cinnamomum cassia twig 9 parts, Glycyrrhiza uralensis 3 parts.

[0066] Method of using the traditional Chinese medicine composition: Mix all raw materials according to the formula, add 3-5 times the weight of water, soak for 1 hour, decoct for 30 minutes, and retain the decoction. Add an equal amount of water to the dregs, continue to decoct for 15 minutes, remove the dregs, combine the decoctions, and concentrate under reduced pressure to obtain 160g of traditional Chinese medicine preparation.

[0067] Example 2: Study on the therapeutic effects and mechanisms of traditional Chinese medicine preparations on non-severe aplastic anemia patients.

[0068] I. Cases and Methods

[0069] 1. Inclusion criteria

[0070] 1) The patient was diagnosed with NSAA by routine blood tests, reticulocyte count and bone marrow morphology examination. The peripheral blood three lineages were reduced but did not meet the SAA / VSAA criteria.

[0071] 2) The patient received routine Western medicine treatment, mainly "Jili Shengxue Fang" combined with low-dose senna, and underwent regular follow-up and evaluation. The patient's medical records are complete.

[0072] 2. Exclusion Criteria

[0073] 1) Congenital or hereditary AA (such as Fanconi anemia, telomere disease, etc.) or clear evidence suggesting hereditary bone marrow failure syndrome;

[0074] 2) Comorbid myelodysplastic syndrome (MDS), clinical dominant stage of PNH, leukemia, and other hematological diseases;

[0075] 3) Individuals who have previously received hematopoietic stem cell transplantation (HSCT);

[0076] 4) Comorbid severe infection, active tuberculosis, or other severe systemic diseases;

[0077] 5) Those with incomplete medical records.

[0078] 3. Source of cases

[0079] Patients who received this combined treatment at the Department of Hematology, Zhejiang Provincial Hospital of Traditional Chinese Medicine from September 2024 to September 2025 were included, and complete cases meeting the diagnostic and inclusion / exclusion criteria were included.

[0080] Research Content

[0081] 1) Study endpoints: including the degree of improvement in blood counts (white blood cells, platelets, hemoglobin) at 1, 3, and 6 months, and assessment of the efficacy and safety of the three blood cell lines;

[0082] 2) Information Collection: The study will collect baseline information from patients, including demographic characteristics, disease course, past medication history, and comorbidities. Simultaneously, it will compile hematological data (peripheral blood complete blood count, reticulocyte count), bone marrow examination, liver and kidney function indicators, and combine this with follow-up data to obtain efficacy and safety evaluations.

[0083] 3) Efficacy and safety assessment: The assessment will be based on established standards, combined with a comprehensive analysis of blood count improvement and transfusion dependence outcomes. Specifically, efficacy assessment criteria will be developed with reference to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines (Trial Implementation)".

[0084] Cure: Clinical symptoms such as anemia and bleeding have basically disappeared; male patients have an Hb level ≥ 120 g / L and female patients have an Hb level ≥ 100 g / L; WBC count ≥ 4 × 10⁻⁶. 9 / L (or ANC ≥1.5×10) 9 / L), PLT count ≥ 80×10 9 / L.

[0085] Significant effect: Clinical symptoms such as anemia and bleeding are significantly relieved; male patients have an Hb level ≥120 g / L and female patients have an Hb level ≥100 g / L; WBC count ≥ 3.5×10⁻⁶. 9 / L (or ANC ≥1.5×10) 9 ( / L), PLT count increased compared to before treatment.

[0086] Effective: Clinical symptoms such as anemia and bleeding are alleviated; Hb levels increase by more than 30 g / L compared to before treatment; or PLT count increases by 20 × 10⁻⁶ compared to before treatment. 9 / L (If the patient previously relied on platelet transfusions, maintaining platelet transfusion-free status for ≥8 weeks can also be considered as meeting this criterion).

[0087] Ineffective: Clinical symptoms such as anemia and bleeding, as well as routine blood indicators, did not improve or even worsened.

[0088] Overall effective rate (%) = [(number of cured cases + number of cases with significant effect + number of effective cases) / total number of cases] × 100%.

[0089] Statistical methods: Blood counts (Hb, WBC, PLT) were collected from patients at 0, 1, 3, and 6 months of follow-up. Data were then processed and analyzed. Quantitative data were expressed as mean ± standard deviation or median (interquartiles), and categorical data were expressed as number of cases and percentages. Repeated measures ANOVA or nonparametric tests were used to assess trends in blood counts over time. Paired tests were used to compare baseline and 6-month data. Appropriate correction methods were employed for multiple comparisons. All analyses were considered statistically significant with p < 0.05. Results are presented visually in tabular and graphical formats.

[0090] II. Results

[0091] 1. Basic Case Information

[0092] During the study period, 54 non-severe aplastic anemia patients who met the case inclusion criteria were admitted, including 29 males and 25 females, with a median age of 47 (range 6-87).

[0093] Therapeutic effect analysis

[0094] 2.1 Changes in blood count

[0095] Compared with baseline, the patients' Hb, WBC, and PLT levels showed a gradual upward trend at 1, 3, and 6 months of treatment (Table 1), suggesting that "Jili Shengxue Fang" combined with conventional treatment can improve hematological parameters within a certain period of time. Paired t-test results (Table 2) showed that the levels of all three blood cell lines were significantly improved at 6 months compared with baseline (p<0.001), indicating that the treatment was statistically significant in improving anemia and thrombocytopenia.

[0096] Figure 1 The data shows the trends of PLT, HGB, and WBC levels during the 0, 1, 3, and 6-month follow-up periods. All three blood cell counts showed a gradual upward trend, with more significant improvements at 3 and 6 months, suggesting that combined therapy is more effective in the medium to long-term follow-up phases.

[0097] Figure 2 The mean ± standard deviation of the three blood cell counts at 6 months is shown for different treatment groups. 46 patients were followed up for 6 months and included in the 6-month efficacy evaluation (n=46); the remaining 8 patients were not included in the 6-month efficacy statistics because the follow-up period was less than 6 months.

[0098] Figure 3 Box plots showing baseline and 6-month blood count improvement visually illustrate the magnitude and dispersion of improvement in each treatment group. In the cured group, all three blood cell lines recovered to near or above the lower limit of normal; the markedly effective group showed significant improvement; the effective group showed limited improvement; and the ineffective group showed no significant improvement.

[0099] Table 1 shows the comparison of the differences in the three peripheral blood cell lines (Δ = follow-up − baseline) before and after treatment.

[0100] Table 1

[0101] Table 2 shows the results of paired t-tests of baseline and 6-month blood count indicators.

[0102] Table 2

[0103] t-value p-value PLT -4.03 ＜0.01 HGB -6.01 ＜0.01 WBC -5.88 ＜0.01

[0104] Table 3 shows the improvement of the three blood cell lines at 1, 3, and 6 months after treatment.

[0105] Table 3

[0106] 2.2 Efficacy Assessment

[0107] In June, the overall effective rate was 73.9%, with 15.2% cured, 26.1% showing significant improvement, and 32.6% showing improvement. Overall, the patients' anemia and bleeding symptoms were alleviated, and some patients met the clinical cure criteria. The majority of patients benefited, with the combined proportion of cured and significantly improved cases exceeding 40%, consistent with the trend of improved blood counts.

[0108] Table 4 shows the distribution of therapeutic effects and the overall effective rate (n, %) at 6 months.

[0109] therapeutic effect cure Effective efficient invalid total Number of examples 7 12 15 12 46 percentage 15.2% 26.1% 32.6% 26.1% 100%

[0110] 3. Safety assessment

[0111] No significant liver or kidney function damage or serious adverse reactions were observed during treatment. A few patients experienced mild gastrointestinal discomfort, which was relieved after symptomatic treatment and did not lead to discontinuation of the medication. Overall, this compound preparation is considered to have good safety and tolerability.

[0112] Table 5 shows the toxic and adverse reactions of androgens and immunosuppressants.

[0113] Table 5

[0114] Example 3: Analysis of potential active ingredients in traditional Chinese medicine preparations

[0115] I. Experimental Materials and Methods

[0116] 1. Experimental Materials

[0117] 1.1 Experimental Animals and Rearing Environment

[0118] Six-week-old male SD rats weighing 0.2 - 0.3 Kg were reared in the barrier environment of the Animal Experiment Research Center of Zhejiang Chinese Medical University [SYXK (Zhe) 2008 - 0116]; fed with ordinary feed and allowed free access to food and water. Ambient temperature: 20 - 22 °C, relative humidity: 40% - 60%.

[0119] 1.2 Drugs

[0120] Traditional Chinese medicine prescription: the traditional Chinese medicine preparation prepared in Example 2.

[0121] 1.3 Main Reagents

[0122] Methanol: Thermo Fisher Scientific (product number A452 - 4, fisher); Acetonitrile: Thermo Fisher Scientific (product number A998 - 4, fisher); Formic acid: Thermo Fisher Scientific (product number A117 - 50, fisher).

[0123] 1.4 Main Instruments

[0124] Ultrasonic cleaner (F - 060SD): Shenzhen Fuyang Technology Group Co., Ltd.; Vortex oscillator (TYXH - I): Shanghai Hanno Instruments Co., Ltd.; Desktop high-speed refrigerated centrifuge (TGL - 16MS): Shanghai LUXIANGYI Centrifuge Instruments Co., Ltd.; High-performance liquid chromatograph (ACQUITY UPLC I-Class): Chromatographic column (ACQUITY UPLC HSS T3 (100 mm × 2.1mm, 1.8 um)); PDA detector (ACQUity UPLC); High-resolution liquid chromatography-mass spectrometry (Thermo-Orbitrap-QEHF).<00​​​​​​​Take the following herbs for blood-nourishing formula (Astragalus membranaceus 30g, Rehmannia glutinosa 30g, Agrimonia pilosa 30g, Litchi chinensis seed 40g, Bletilla striata 3g, Cervi cornu slices 15g, Prunus persica kernel 9g, Carthamus tinctorius 6g, Pheretima aspergillum 12g, Curcuma longa slices 9g, Cinnamomum cassia twig 9g, Citrus aurantium 20g, Glycyrrhiza uralensis 3g), place them in a ceramic decoction pot, add 10 times the amount of water and soak for 30 minutes. After boiling, simmer for 15 minutes and filter out the extract. Add an appropriate amount of purified water, boil, and simmer for 10 minutes. Filter out the extract again, combine the two decoctions, and concentrate the drug concentration to 2.11g / mL. Dilute the test solution to 20g, centrifuge at 12,000g for 10 minutes at 4℃, and use the supernatant for analysis.

[0128] 2.2 Preparation of drug-containing rat serum

[0129] To prepare drug-concentrated serum, rats were administered 2 mL of the drug twice daily by gavage for 7 consecutive days. Control mice were given the same volume of physiological saline to prepare control serum. Two hours after each administration, blood was collected from the heart, allowed to stand at room temperature for 2 hours, and then centrifuged at 3000 r / min and 4℃ for 10 min to collect the serum. The serum was inactivated at 56℃ for 30 min and then stored at -80℃.

[0130] 2.3 LC-MS Analysis

[0131] 2.3.1 Sample pretreatment:

[0132] Serum Samples: Take 150 μL of serum thawed on ice and add 600 μL of ice-cold methanol-acetonitrile (2:1, v / v) containing mixed internal standards (4 μg / mL) for protein precipitation. Vortex for 1 min, sonicate in an ice-water bath for 10 min, and incubate at -40℃ for 30 min. Centrifuge at 12000 rpm for 10 min at 4℃. Take 500 μL of the supernatant and dry it under nitrogen. Redissolve the residue in 200 μL of water-methanol-acetonitrile (1:2:1, v / v / v), vortex for 1 min, sonicate for 3 min, and incubate at -40℃ overnight for further lipid removal. Centrifuge again at 12000 rpm for 10 min at 4℃, and transfer 120 μL of the supernatant to a vial with a leg-lined insert for LC-MS / MS analysis.

[0133] Traditional Chinese medicine samples: Take 100 μL of homogeneous sample and add 900 μL of aqueous solution containing a mixed internal standard (4 μg / mL). Vortex for 1 min, then sonicate in an ice-water bath for 60 min. Centrifuge at 12000 rpm for 10 min at 4℃. Dilute the supernatant 2-fold with water (containing the same concentration of mixed internal standard). Transfer 200 μL of the diluted solution to a vial with an inner liner for LC-MS / MS analysis.

[0134] 2.3.2 Liquid Chromatography-Mass Spectrometry Conditions:

[0135] Analysis was performed on a combined platform equipped with an ACQUITY UPLC I-Class HF ultra-high performance liquid chromatography system and a QE high-resolution mass spectrometer.

[0136] Chromatographic conditions: An ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) was used, with a column temperature of 45℃. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile. The flow rate was 0.35 mL / min. The injection volume was 5 μL. The PDA detector scan range was 210–400 nm.

[0137] Mass spectrometry conditions: Ion source: HESI; Sample mass spectrometry signal acquisition was performed using positive and negative ion scanning modes; Data acquisition mode: DDA; Scan mode: Full MS / dd-MS2 (TOP 8); Mass spectrometry conditions: Ion source: HESI; Sample mass spectrometry signal acquisition was performed using positive and negative ion scanning modes; Data acquisition mode: DDA; Scan mode: Full MS / dd-MS2 (TOP 8).

[0138] II. Results

[0139] 2.1 Screening of chemical components

[0140] The sample was analyzed using the above analytical conditions in both positive and negative ion modes to obtain the base peak chromatogram (BPC) and total ion chromatogram (TIC) for both modes. (See attached image.) Figure 4 and Figure 5 .

[0141] 2.2 Chemical composition identification

[0142] A total of 1,177 compounds were identified in the decoction of the drug, including 199 terpenoids, 192 flavonoids, 145 phenylpropanoids, 107 sugars and glycosides, 87 amino acids and polypeptides, 60 organic heterocyclic compounds, 51 phenols, 42 fatty acyl compounds, 37 lipids and lipid-like molecules, 19 steroids, and 238 other compounds.

[0143] A total of 52 compounds in the drug's bloodstream components were identified (see...) Figure 6 and Figure 7, among which the ones with relatively high contents are amygdalin, butoxysuccinic acid, prunasin, gastrodin, L-epiafzelechin, L-canavanine, mannoheptulose, cyclopterin, 3,3-dimethylglutarate, luteolin-7-glucuronide, and vitexin.

[0144] Example 4 Study on the Therapeutic Effect and Mechanism of Traditional Chinese Medicine Preparation on Aplastic Anemia

[0145] I. Experimental Materials and Methods

[0146] 1. Experimental Materials

[0147] 1.1 Experimental Animals and Rearing Environment

[0148] C57 mice aged 4 - 8 weeks, male, weighing 20 - 30 g, were reared in the barrier environment of the Animal Experimental Research Center of Zhejiang Chinese Medical University [SYXK(Zhe) 2008 - 0116]; fed with ordinary feed and allowed free access to food and water. Environmental temperature: 20 - 22 °C, relative humidity: 40% - 60%.

[0149] 1.2 Drugs

[0150] Traditional Chinese medicine prescription: The traditional Chinese medicine preparation prepared in Example 2.

[0151] 2 Model Establishment:

[0152] 2.1 Establishment of Bone Marrow Failure Model

[0153] C57BL / 6 mice were intraperitoneally injected with 80 mg / kg / d cyclophosphamide (CTX) for 3 days to induce leukopenia. After the model was established, the administration group was treated with drugs for 12 days.

[0154] Grouping: Blank group (ctrl), model group (mod), low-dose Jilishangshengxue Formula (JLSXF-L, 0.729 g / mL), high-dose Jilishangshengxue Formula (JLSXF-H, 1.459 g / mL), with 6 mice in each group, and each mouse was administered 0.3 mL per day.

[0155] 2.2 Establishment of Aplastic Anemia Animal Model

[0156] C57BL / 6 mice were first intraperitoneally injected with 200 mg / kg / week dextran iron (for 10 weeks). Then, all animals were subjected to whole-body irradiation ( 60 Co 6.0 Gy, 1 Gy / min). Within 4 h, 0.2 ml of thymocyte suspension (5 × 10 6 cells / ml) from DBA / 2 mice was infused via the tail vein. After the model was established, the administration group was treated with drugs for 12 - 14 days.

[0157] The study included: a blank control group (ctrl), a model group (mod), a low-dose Jili Shengxue Formula (JLSXF-L, 0.932 g / mL), and a high-dose Jili Shengxue Formula (JLSXF-H, 1.864 g / mL). Each group consisted of 6 animals, and each animal was given 0.3 mL of the formula daily.

[0158] 3. Detection Methods

[0159] 3.1 Hematological parameters detection

[0160] Whole blood was collected from the orbital sinus of mice and analyzed using a fully automated modular animal blood analyzer for a five-part differential blood test.

[0161] 3.2 Detection of peripheral blood neutrophil adhesion markers

[0162] Whole blood was collected from the orbital sinus of mice using heparinized capillaries and EDTA-containing collection tubes. After erythrocyte lysis, cells were centrifuged at 300×g for 10 minutes, and the pellet was resuspended in 500 μL of phosphate-buffered saline. After washing and centrifuging, flow cytometry antibodies CD11b, Ly-6G, and CD62L were added, and the cells were incubated at 4 °C in the dark for 30 minutes. After washing and centrifugation, the cells were resuspended in 200 μL of phosphate-buffered saline containing 0.5% BSA. Neutrophils were circled by flow cytometry, and changes in their adhesion parameters were observed.

[0163] 3.3 Hematopoietic stem cell testing

[0164] To obtain mouse bone marrow cells, a single-cell suspension was filtered through a 70 μm filter membrane. An appropriate amount of erythrocyte lysis buffer was added to treat the BMCs. The treated BMCs were counted, and the same number of cells were used for subsequent experiments. The expression of Lin, sca-1, ckit, CD150, and CD48 (MFI and percentage of positive cells) was analyzed by flow cytometry.

[0165] 4. Cell Experiments

[0166] 4.1 Selection and culture of cell lines

[0167] The human cell line HL-60 was selected as the research cell line. HL-60 cells were cultured in IMDM medium containing 1% penicillin / penicillin antibiotics (P / S) and 20% fetal bovine serum (FBS). Cells were sporulated at a rate of 1 × 10⁶ cells / year. 5 / mL were seeded in IMDM medium containing 1% P / S and 20% FBS. The culture medium was changed every 48-72 h, and cells in the logarithmic growth phase were collected for subsequent studies.

[0168] 4.2 Serum containing the herbal formula "Jili Shengxue Fang" induces HL-60 cell differentiation

[0169] Myeloid differentiation was assessed by detecting the expression of the cell surface marker CD11b using flow cytometry. HL-60 cells were treated with serum containing 2.5%, 5%, 7.5%, and 15% of the traditional Chinese medicine formula *Jili Shengxue Fang* for 5 days. After treatment, cells were collected and washed twice with ice-cold phosphate-buffered saline (PBS). Human anti-CD11b-APC antibody was added, and the cells were incubated at 4°C in the dark for 30 min. The samples were then analyzed using flow cytometry.

[0170] II. Results

[0171] 2.1 Hematological parameters

[0172] 2.1.1 Hematological parameters of the bone marrow failure model

[0173] The white blood cell count in the model group was consistently lower than that in the control group. Compared to the control group (ctrl), the WBC count in the model group (mod) was reduced by nearly 50%. At a dose of 0.8745 g / kg / day (body weight), equivalent to a human clinical dose, such as... Figure 8 As shown, the Jili Shengxue formula showed a significant increase in WBC (lymphocytes and neutrophils) in the model mice.

[0174] 2.1.2 Hematological parameters of aplastic anemia animal models

[0175] Hematological tests were performed on AA mouse models 12 days after drug administration to assess hematopoietic recovery. On day 12, compared to the normal group, all model groups showed significantly reduced levels of white blood cells (WBC), hemoglobin (HGB), and platelets (PLT). Notably, as... Figure 9 As shown, both low and high doses of the Jili Shengxue formula resulted in an increase in WBC count, with the low dose significantly affecting WBC levels; the low dose of the Jili Shengxue formula significantly increased HGB and PLT levels.

[0176] 2.2 Effects of Jili Shengxue Formula on Bone Marrow Hematopoietic Stem Cells in a Bone Marrow Failure Model

[0177] like Figure 10 As shown, after administration of the Jili Shengxue formula, the BMCs of the model mice were significantly restored. Further flow cytometry analysis revealed a significant reduction in HSPCs in the bone marrow of the model mice. This indicates that CTX can significantly reduce HSPCs in the bone marrow, thereby inhibiting hematopoietic function and affecting peripheral blood leukocytes. Notably, the Jili Shengxue formula primarily increased the number of HPCs and HSCs. The results indicate that CTX can damage HSPCs in the bone marrow, inhibiting their further proliferation and differentiation. The Jili Shengxue formula can significantly restore the number of HSPCs in the bone marrow, thereby reconstructing bone marrow hematopoiesis and enhancing the body's immune system.

[0178] 2.3 In vitro induction of myeloid cell differentiation using serum containing the herbal formula of *Jili Shengxue*.

[0179] CD11b is a marker expressed by neutrophils, monocytes, and natural killer (NK) cells, used to assess myeloid cell differentiation. Figure 11 As shown, on day 5 of treatment with serum containing the herbal formula *Jili Shengxue Fang*, flow cytometry analysis revealed a significant increase in the proportion of CD11b-positive cells, supporting the induction of neutrophil differentiation in HL-60 cells.

[0180] 2.4 Effect of Jili Shengxue Formula on CD62L Expression on Peripheral Blood Neutrophils in a Bone Marrow Failure Model

[0181] The migration of neutrophils from the circulating pool to the marginal pool is a process of activation, marked by the shedding of CD62L and the upregulation of CD11b. Figure 12 As shown, the decreased expression of the adhesion molecule CD62L leads to the mobilization of neutrophils into the blood, thereby increasing the number of neutrophils in the circulating pool.

[0182] 2.5 Effects of Jili Shengxue Formula on Bone Marrow Hematopoietic Stem Cells in Aplastic Anemia Animal Models

[0183] like Figure 13 As shown, flow cytometry analysis revealed a significant decrease in HSPCs in the bone marrow of aplastic anemia model mice. Different doses of the Jili Shengxue formula increased the number of LSKs and MMPs to varying degrees. Preliminary findings suggest that low-dose Jili Shengxue formula (0.932 g / mL) has a significant therapeutic effect on aplastic anemia model mice, improving HSPCs in the bone marrow damaged by aplastic anemia and thus promoting bone marrow hematopoiesis.

Claims

1. A traditional Chinese medicine compound preparation for treating aplastic anemia, characterized in that... Includes the following Chinese herbal ingredients: The principal herb is selected from at least one of Astragalus membranaceus, Rehmannia glutinosa, and Agrimonia pilosa. The medicinal ingredients are selected from at least one of the following: litchi seed, Bletilla striata, and deer antler slices. The adjuvant ingredients are selected from at least one of the following: peach kernel, safflower, earthworm, turmeric, and fragrant tea vegetable. Used medicinal ingredient: Selected from licorice; The adjuvant herb is selected from cinnamon twig.

2. A traditional Chinese medicine compound preparation for treating aplastic anemia according to claim 1, characterized in that, The mass fractions of each component are as follows: principal drug, 80-100 parts; assistant drug, 52-62 parts; adjuvant drug, 50-65 parts; guiding drug, 2-5 parts; adjuvant and guiding drug, 6-12 parts.

3. A traditional Chinese medicine compound preparation for treating aplastic anemia according to claim 1 or 2, characterized in that: The principal ingredient is a combination of Astragalus membranaceus, Rehmannia glutinosa, and Agrimonia pilosa; the assistant ingredient is a combination of Litchi chinensis seed, Bletilla striata, and deer antler slices; and the adjuvant ingredient is a combination of Prunus persica kernel, Carthamus tinctorius, Pheretima aspergillum, Curcuma longa slices, and Citrus aurantium.

4. A traditional Chinese medicine compound preparation for treating aplastic anemia according to claim 3, characterized in that, The mass fractions of each component are as follows: Chief herbs: Astragalus membranaceus 27-33 parts, Rehmannia glutinosa 27-33 parts, Agrimonia pilosa 27-33 parts; The following ingredients were used: 38-42 parts of lychee seed, 2-4 parts of Bletilla striata, and 13-17 parts of deer antler slices; Medicinal ingredients: 8-10 parts peach kernel, 5-7 parts safflower, 11-13 parts earthworm, 8-10 parts turmeric, 18-22 parts fragrant tea leaves; Medicinal herb: 2-4 parts licorice root; Adjuvant herb: 7-11 parts of cinnamon twig.

5. A traditional Chinese medicine compound preparation for treating aplastic anemia according to claim 3, characterized in that, The mass fractions of each component are as follows: Astragalus membranaceus 30 parts, Rehmannia glutinosa 30 parts, Agrimonia pilosa 30 parts, Litchi chinensis seed 40 parts, Bletilla striata 3 parts, Cervi cornu slices 15 parts, Prunus persica kernel 9 parts, Carthamus tinctorius 6 parts, Pheretima aspergillum 12 parts, Curcuma longa slices 9 parts, Rhizoma Citri Reticulatae 20 parts, Cinnamomum cassia twig 9 parts, Glycyrrhiza uralensis 3 parts.

6. A traditional Chinese medicine compound preparation for treating aplastic anemia according to claim 3, characterized in that, The mass fractions of each component are as follows: Astragalus membranaceus 30g, Rehmannia glutinosa 30g, Agrimonia pilosa 30g, Litchi chinensis seed 40g, Bletilla striata 3g, Cervi cornu slices 15g, Prunus persica kernel 9g, Carthamus tinctorius 6g, Pheretima aspergillum 12g, Curcuma longa slices 9g, Rhizoma Citri Reticulatae 20g, Cinnamomum cassia twig 9g, Glycyrrhiza uralensis 3g.

7. The method for preparing the traditional Chinese medicine compound preparation according to any one of claims 1 to 6, characterized in that: The medicinal materials listed in the prescription, excluding deer antler slices, are extracted by decoction at least once, and the filtrates are combined and concentrated. In the post-processing step, deer antler slice solution is added, stirred evenly, and then filtered and shaped to obtain the pharmaceutical preparation.

8. The method for preparing the traditional Chinese medicine compound preparation according to claim 7, characterized in that: The dosage form of the pharmaceutical preparation is oral liquid, granules, or capsules.

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