Application of a kind of liao's wind pill in preparation of glioma drugs

Abstract

The application discloses the application of Huaxingdan in the preparation of a glioma drug in the technical field of biology and medicine, detects the influence of Huaxingdan on the proliferation activity of glioma cells U251, U87 and A172 by using an MTT method in vitro, finds that Huaxingdan has a significant inhibitory effect on the proliferation of glioma cells, and is time and concentration dependent. The influence of Huaxingdan on the apoptosis of glioma cells U87 is detected by using a flow cytometry, and it is found that Huaxingdan can induce the apoptosis of U87 cells, and is concentration dependent. The animal experiment shows that Huaxingdan can effectively inhibit the growth of glioma cells in vivo, and the HE staining shows that Huaxingdan has no toxicity in vivo, and can be used as a drug for preventing and treating glioma.

Description

Technical Field

[0001] This invention relates to the fields of biology and medicine, specifically to the application of Liao's Huafeng Dan in the preparation of drugs for glioma. Background Technology

[0002] Liao's Wind-Dispelling Pills, manufactured by Guizhou Wansheng Pharmaceutical Co., Ltd., are currently used for: calming wind and relieving spasms, clearing phlegm and opening the orifices. They are indicated for wind-phlegm obstruction, stroke hemiplegia, epilepsy, facial nerve palsy, and facial paralysis. The main ingredients include over 20 traditional Chinese medicinal herbs such as Aconitum carmichaelii, Arisaema heterophyllum, Pinellia ternata, Aconitum carmichaelii, Perilla frutescens, Bombyx mori, Scorpion, Atractylodes lancea, Gastrodia elata, Schizonepeta tenuifolia, and Cinnabar. The pills are vermilion in color, with a brownish-yellow cross-section; they have a strong aroma and a pungent taste.

[0003] Gliomas are the most common primary malignant tumors of the central nervous system, characterized by a high recurrence rate. The lack of effective treatment strategies leads to high mortality and short survival. Typically, only 5.5% of patients survive for 5 years after diagnosis, with a median overall survival of approximately 14.5–16.6 months. Despite significant advances in surgery, radiotherapy, and chemotherapy, the prognosis for gliomas remains quite poor. Temozolomide is the first-line chemotherapy drug for gliomas, but its efficacy is often limited by drug resistance. Summary of the Invention

[0004] The present invention aims to provide the application of Liao's Huafeng Dan in the preparation of glioma drugs, in order to address the difficulty in treating glioma and to find new drugs with significant therapeutic effects on glioma.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the application of Liao's Huafengdan in the preparation of glioma drugs.

[0006] The formula of Liao's Wind-Dispelling Pill includes: 170-190g of motherwort, 155-165g of perilla leaf, 70-75g of silkworm, 32-40g of scorpion, 34-42g of processed arisaema, 33-43g of atractylodes, 22-31g of realgar, 68-76g of borax, 8-14g of croton oil, 1-4g of artificial musk, 32-40g of borneol, 70-75g of gastrodia, 15-20g of schizonepeta, 2-5g of sandalwood, and 21-27g of cinnabar.

[0007] The herbal formula includes: 120-130g of ox bile, 25-29g of Aconitum carmichaelii, 26-30g of Pinellia ternata, 27-30g of Arisaema heterophyllum, 26-30g of Aconitum carmichaelii, 12-16g of Curcuma longa, and 0.10-0.17g of Massa fermentata.

[0008] Furthermore, Liao's Huafeng Dan can be directly prepared into a formulation for glioma treatment, wherein the formulation can be any one of the pharmaceutically permissible tablets, injections, capsules, oral liquids, or drop pills.

[0009] This invention investigated the effect of Huafengdan on the proliferation of glioma cells U251, U87, and A172 in vitro using the MTT assay. The results showed that Huafengdan significantly inhibited glioma cell proliferation in a time- and concentration-dependent manner. Flow cytometry was used to examine the effect of Huafengdan on apoptosis of U87 glioma cells, revealing that Huafengdan induced apoptosis in U87 cells in a concentration-dependent manner. Animal experiments demonstrated that Huafengdan effectively inhibited the growth of glioma cells in vivo, and HE staining confirmed its lack of toxicity in vivo, suggesting its potential as a drug for the prevention and treatment of gliomas.

[0010] The beneficial effects of this invention are as follows: Compared with the existing uses of Huafengdan, the inventors discovered that Huafengdan has a good in vitro and in vivo inhibitory effect on gliomas, thus discovering a new use for this drug. Furthermore, Huafengdan is a marketed drug that can be directly applied to humans without further clinical safety assessments, showing great application potential and rapidly increasing the market share of this product. Attached Figure Description

[0011] Figure 1 The results show the inhibitory effects of Liao's Huafengdan on the growth of glioma cells U251, U87, and A172.

[0012] Figure 2 It is a flow cytometry detection of apoptosis in U87 glioma cells;

[0013] Figure 3 This is a test of the inhibitory effect of Liao's Huafeng Pill on the growth of glioma in vivo;

[0014] Figure 4 This is an in vivo toxicity test of Liao's Huafeng Dan. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0016] Example 1

[0017] In vitro activity assay of Liao's Huafengdan in inhibiting glioma cells:

[0018] 1. Experimental materials

[0019] ① Preparation of sample solutions: Grind Huafengdan pills into powder, dissolve in DMSO, and sonicate and filter to prepare a stock solution with a concentration of 100 mg / mL. Prepare sample solutions of different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL).

[0020] ② Preparation of 5% MTT solution: Weigh 0.5g of MTT powder, add 100ml of physiological saline, dissolve at 60℃, and store at 4℃.

[0021] ③ Cell lines: U251, U87, and A172 cells were provided by the Guizhou Provincial Natural Products Research Center Laboratory and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2.

[0022] ④ DMEM medium (Hycone); fetal bovine serum (FBS) (Zhejiang Tianhang Biotechnology); tetramethyl azolidinyl ether salt (MTT) (Solarbio); dimethyl sulfoxide (DMSO) and penicillin-streptomycin mixture (Beijing Dingguo Changsheng Biotechnology); Huafengdan (Guizhou Wansheng Pharmaceutical Co., Ltd.).

[0023] 2. Experimental Methods

[0024] U251, U87, and A172 cells in logarithmic growth phase were seeded into 96-well plates at a density of 5 × 10³ cells / well, with 190 μl of DMEM medium containing 5% FBS per well. After 8–12 h of culture, 10 μl of a solution containing different concentrations of Huafengdan (a traditional Chinese medicine) was added to each well, with five replicates for each concentration. A saline control group was also included. After culturing the cells at 37°C for 24 h and 48 h, 20 μl of 5% MTT solution was added to each well, and after 4 h of further culture, the 96-well plates were inverted and the medium was discarded. 150 μl of DMSO was added to each well, and the mixture was thoroughly mixed on a shaker. The absorbance of each well was measured using a microplate reader at 490 nm to calculate the inhibition rate. The results are shown in the figure below. Figure 1 .

[0025] The results showed that the Miao medicine Huafengdan significantly inhibited the growth of three types of glioma cells: U251, U87, and A172. Figure 1 Furthermore, the effects of the active ingredients in Liao's Huafengdan on growth inhibition are time- and concentration-dependent. Based on the above data, it is evident that Liao's Huafengdan has the potential to be developed into an anti-glioma drug. In addition, studies have shown that preparing the active ingredients of Liao's Huafengdan into tablets, injections, capsules, oral liquids, or drop pills according to the above experimental data also exhibits the same growth-inhibiting effect.

[0026] Example 2

[0027] Flow cytometry analysis of the effect of Liao's Huafengdan on apoptosis of glioma cells U87

[0028] 1. Experimental materials

[0029] ① Preparation of sample solutions: Grind Huafengdan pills into powder, dissolve and filter with DMSO, and prepare sample solutions with concentrations of 25μg / ml, 50μg / ml and 100μg / ml respectively.

[0030] ② Cell line: Glioma cells U87 were provided by the Guizhou Provincial Natural Products Research Center Laboratory and cultured in DMEM medium containing 10% FBS in a 37℃, 5% CO2 incubator.

[0031] ③EDTA trypsin: (Hycone); FITC Annexin V Apoptosis Detection Kite I (BD Bioscence); Flow cytometer (BD Bioscence, USA); the rest are the same as above.

[0032] 2. Experimental Methods

[0033] U87 cells in logarithmic growth phase were collected and added to small dishes at a density of 8 × 10⁵ cells / dish, with 3 ml of cell suspension per dish. Five dishes were prepared at five different concentrations and incubated for 24 hours. When the growth density reached 70-80%, 2 ml of physiological saline was added for washing. The washing solution was then aspirated. 3 ml of the required experimental drugs were added to each dish according to the specified concentration, and 3 ml of culture medium was added to the control wells. The dishes were incubated for 48 hours, and cell morphology changes were observed under a microscope. The culture medium was collected into 15 ml centrifuge tubes, washed twice with 2 ml of physiological saline, and 300 μL of EDTA-free trypsin was added. The cells were then incubated at 37°C for 20 minutes to allow adherent cells to stop adhering. Fresh culture medium was added to stop the digestion. The cells were transferred to 15 ml centrifuge tubes and centrifuged. After centrifugation, the supernatant was discarded, and the cells were gently washed once with 2 ml of physiological saline. After centrifugation, the supernatant was discarded again. After resuspending cells in PBS buffer, transfer them to 1.5 ml EP tubes, add Annexin V-PI / FITC reagent, mix well, and protect from light. Flow cytometry was used to detect cell apoptosis. Flowjo software was used to analyze the flow cytometry data, and the apoptosis analysis results were exported. Results are shown below. Figure 2 .

[0034] Figure 2 The results showed that Liao's Huafengdan could induce apoptosis in U87 cells in a significant concentration-dependent manner. Data were analyzed using Excel and GraphPad Prism9 statistical software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, with P < 0.05 considered statistically significant.

[0035] Example 3

[0036] In vivo anti-glioma activity assay of Liao's Huafengdan

[0037] 1. Experimental materials

[0038] ①The animals were 30 male BALB / c Nude mice around 5 weeks old.

[0039] ② Cell line: U87 cells were cultured in DMEM medium containing 100% FBS in a 37℃, 5% CO2 incubator.

[0040] ③ Preparation of sample solutions: Grind Huafengdan into powder and prepare sample solutions with physiological saline at concentrations of 1040 mg / ml, 520 mg / ml and 260 mg / ml respectively.

[0041] ④ BALB / c Nude male mice (Guizhou Huijiu Biotechnology Co., Ltd.);

[0042] 2. Experimental Methods

[0043] U87 cells were cultured, and U87 cells in the logarithmic growth phase were prepared into a cell suspension with a concentration of 1×10⁷ cells / ml. 0.1 ml of the cell suspension was subcutaneously injected into the right anterior axilla of mice. The appearance of a tumor at the injection site after 2-3 days of routine feeding indicated successful construction of glioma-bearing mice. Mice were divided into four groups of six each: a control group (gavage with physiological saline); a low-dose group (260 mg / ml), a medium-dose group (520 mg / ml), and a high-dose group (1040 mg / ml). Each mouse in the control and treatment groups received 0.4 ml of the drug via gavage. The administration was once daily for 12 days. Twelve days after gavage, the mice were dissected, and vital organs and subcutaneous tumors were collected for pathological examination and tumor weight measurement. The statistical results of tumor weight and volume are shown below. Figure 3 .

[0044] Figure 3 The results showed that Liao's Huafengdan significantly inhibited the growth of glioma cells U87 in vivo. Statistical analysis was performed using Excel and GraphPad Prism9 software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, with P < 0.05 considered statistically significant.

[0045] Example 4

[0046] In vivo toxicity test of Liao's Huafeng Dan:

[0047] 1. Experimental materials

[0048] Xylene, anhydrous ethanol, hematoxylin, eosin, neutral resin.

[0049] 2. Experimental Methods

[0050] Paraffin sections were prepared from heart, liver, spleen, lung, kidney, and tumor tissues, and the following procedures were performed:

[0051] (1) Dewaxing and rehydration: The baked paraffin slices were immersed in xylene (I, II) for 10 min each, then in anhydrous ethanol for 10 min, 95% ethanol for 10 min, and 75% ethanol for 10 min, and finally rinsed in distilled water for 5 min.

[0052] (2) Staining: Immerse the sections in hematoxylin staining solution for 10 min, rinse with running water for 5 min, shake off the surface water, then differentiate the tissue with hydrochloric acid alcohol differentiation solution for 20 s, rinse with running water for 5 min, and finally immerse in eosin staining solution for 2 min.

[0053] (3) Dehydration and clearing: Dehydration and clearing were carried out in the following order: 3 min of 80% ethanol, 3 min of 85% ethanol, 3 min of 90% ethanol, 3 min of 95% ethanol, 3 min of anhydrous ethanol (I, II) each, and 5 min of xylene (I, II, III) each.

[0054] (4) Finally, mount the slide with neutral resin (be careful to avoid air bubbles during the process). After the slide has air-dried, observe it under a microscope and take pictures to record the results (×200).

[0055] Figure 4 The results showed no significant differences in the structure and morphology of organs and tissues between the treatment group and the control group, indicating that Huafengdan is non-toxic in vivo. Furthermore, compared with the control group, the glioma cells in the tumor tissue of the treatment group were more loosely arranged with larger intercellular spaces, indicating that Huafengdan can affect the morphology and structure of tumor tissue.

[0056] The results above indicate that Liao's Huafengdan has potential value in the prevention and treatment of glioma.

Claims

1. Application of Liao's Huafengdan in the preparation of drugs for treating glioma.

Images