Use of a Citrofortunella microcarpa in the preparation of an antitumor medicament
By extracting and separating axillary citronella alkaloids A, B, C, and D from *Citrus axillary*, an antitumor drug was prepared, solving the safety and side effects problems of existing tumor treatment methods and achieving effective inhibition of various tumor cells.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENYANG PHARMA UNIV
- Filing Date
- 2022-07-15
- Publication Date
- 2026-07-07
AI Technical Summary
Existing cancer treatments such as surgical resection and chemotherapy have significant harmful effects on the human body and cause serious side effects. The search for safe and effective anti-cancer drugs has become a research hotspot, and natural active substances have advantages in anti-tumor treatment.
Using *Citrus aurantium* as raw material, its roots and aerial parts were extracted with water or organic solvents, and *Citrus aurantium* alkaloids A, B, C, and D were obtained through multi-step chromatographic separation. These alkaloids were then used to prepare antitumor drugs.
The extracts and compounds of *Citrus aurantium* significantly inhibited the growth of human colorectal cancer, leukemia, gastric cancer, lung cancer, and liver cancer cells in vitro. They exhibited multi-target effects, superior to traditional drugs, and have broad application prospects.
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Figure CN117427097B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of *Citrus aurantium* in the preparation of antitumor drugs. Background Technology
[0002] Cancer, also known as malignant tumor, is an abnormal type of cell growth caused by mutations in certain proto-oncogenes and tumor suppressor genes, resulting in irreversible damage to DNA. Due to the influence of various internal and external factors, the incidence of tumors is on the rise, leading to increasing research on tumor treatment. Modern treatments for tumors include chemotherapy and surgical resection. However, surgical resection carries significant risks, including complications during the procedure and the possibility of tumor metastasis. Furthermore, surgical resection has poor efficacy in treating metastatic cancer. Chemotherapy has significant side effects and causes considerable harm to the body, including adverse reactions such as gastrointestinal problems, gastrointestinal issues, and damage to the nervous system. Therefore, finding new and safer anti-cancer drugs is crucial. Natural active substances have significant anti-tumor effects and possess advantages such as high activity, low toxicity, and good safety. Therefore, searching for anti-tumor active substances from natural products has become a major research focus.
[0003] *Melodinus axillaris* WTWang ex Tsiang et PTL, a climbing shrub belonging to the Apocynaceae family and the *Melodinus* genus, has axillary cymose inflorescences with sharply pointed flower buds. It is the only known species of *Melodinus* from the *Melodinus* section native to China, endemic to Yunnan province, and typically grows in sparse forests and damp areas at altitudes above 1000 meters. The dried berries of *Melodinus* can be used internally as a decoction to treat stomach pain and chest fullness due to diaphragm obstruction. Externally, a suitable amount can be decocted for washing or ground into powder and applied topically to treat skin toxicity, tinea, scabies, infantile malnutrition, hernia, and scrofula. In addition, *Melodinus axillaris* is also commonly used in folk medicine to treat indigestion, orchitis, pediatric hernia, abdominal pain, pediatric meningitis, fractures, and rheumatic heart disease. Summary of the Invention
[0004] The purpose of this invention is to provide an application of *Citrus aurantium* in the preparation of antitumor drugs.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] Application of a type of axillary orange in the preparation of antitumor drugs.
[0007] The axillaris species mentioned refers to the roots and / or above-ground parts of the climbing shrub Melodinus axillaris WTWang ex Tsiang et PTL, belonging to the genus Melodinus in the family Apocynaceae.
[0008] The axillary flower orange is an extract of the axillary flower orange.
[0009] The extract of *Citrus aurantium* is obtained by extracting the roots and / or aerial parts of *Citrus aurantium* from water or organic solvents.
[0010] A compound with antitumor activity, the compounds are compounds (1)-(4),
[0011]
[0012] Preparation methods of extracts and compounds with antitumor effects:
[0013] 1) After pulverizing the dried axillary flower orange herb, repeatedly soak and circulate it with 90% ethanol at room temperature for extraction, and set aside for later use;
[0014] 2) The above-obtained *Citrus aurantium* extract was suspended in water, and the pH was adjusted to 2-3 with hydrochloric acid. Then, it was extracted with petroleum ether and ethyl acetate respectively. The acid solution after extraction was then adjusted to pH 9-10 with saturated 10% ammonia solution, and then extracted with CH2Cl2 to obtain crude alkaloids. The crude alkaloids obtained by extraction were subjected to silica gel open column chromatography with gradient elution using dichloromethane-methanol (100:0→0:100) (v / v). According to the elution order and the different polarities of the components, 8 fractions (Fr.1-8) were obtained.
[0015] 3) Select the above Fr.3 fraction and perform gradient elution using an ODS open column chromatography system with a methanol-water mixture of 20:80→100:0 (v / v). Based on the elution order and the different polarities of the components, 9 fractions (Fr.3.1-Fr.3.9) were obtained.
[0016] 4) The Fr.3.6 fraction was subjected to semi-preparative high performance liquid chromatography system with C18 reversed phase column chromatography and eluted with a methanol-water mixture with a volume ratio of 67:33. Based on the different retention times and elution order, and further identified by spectroscopic methods, compounds (1), (2), (3), and (4) were obtained.
[0017] An application of the prepared compounds, wherein the compounds (1)-(4) (axillary salicylate A, axillary salicylate B, axillary salicylate C, axillary salicylate D) are used in the preparation of antitumor drugs.
[0018] An antitumor drug formulation is prepared by using one or more of the extracts of the axillary orange tree, compounds (1) to (4) as active ingredients, and combining them with a pharmaceutically acceptable carrier, excipients or pharmaceutically acceptable excipients.
[0019] The dosage forms of the drug formulations include liquid, solid, semi-solid, and gaseous dosage forms, as well as targeted formulations and sustained-release and controlled-release formulations suitable for tumor treatment.
[0020] Liquid dosage forms include compound preparations, oral liquids, tinctures, syrups, and injections; solid dosage forms include powders, granules, tablets, capsules, drop pills, films, suppositories, microcapsules, and microspheres; semi-solid dosage forms include ointments, pastes, extracts, and licks; and gaseous dosage forms include aerosols and inhalers.
[0021] The tumors mentioned include colorectal cancer, rectal cancer, leukemia, stomach cancer, lung cancer, and liver cancer, etc.
[0022] Advantages of this invention:
[0023] This invention uses *Citrus aurantium* as raw material to obtain for the first time an extract with antitumor activity and a novel compound with extremely high activity. Furthermore, it provides a new natural method for obtaining *Citrus aurantium* alkaloids A, B, C, and D. The above extracts and isolated compound monomers can effectively inhibit the growth of human colorectal cancer, leukemia, gastric cancer, lung cancer, and liver cancer cells in vitro. The obtained extracts and compound monomers can target a wide range of cancer cells and have significant effects on various cancer cells, thus achieving therapeutic effects and showing broad application prospects. Attached Figure Description
[0024] Figure 1 The compound axillary purpuricine A(1) provided in the embodiments of the present invention 1 H NMR spectrum.
[0025] Figure 2 The compound axillary purpuricine A(1) provided in the embodiments of the present invention 13 C NMR spectrum.
[0026] Figure 3 The compound axillary argentine B(2) provided in the embodiments of the present invention 1 H NMR spectrum.
[0027] Figure 4 The compound axillary argentine B(2) provided in the embodiments of the present invention 13 C NMR spectrum.
[0028] Figure 5The compound axillary purpuricine C(3) provided in the embodiments of the present invention 1 H NMR spectrum.
[0029] Figure 6 The compound axillary purpuricine C(3) provided in the embodiments of the present invention 13 C NMR spectrum.
[0030] Figure 7 The compound axillary argentine D(4) provided in the embodiments of the present invention 1 H NMR spectrum.
[0031] Figure 8 The compound axillary argentine D(4) provided in the embodiments of the present invention 13 C NMR spectrum. Detailed Implementation
[0032] The following detailed examples illustrate the application of *Citrus aurantium* extract and *Citrus aurantium* alkaloid in antitumor activity.
[0033] Example 1: Preparation of Orange Blossom Extract
[0034] After pulverizing the dried *Citrus aurantium* herb, soak it in 90% ethanol at room temperature for 4 days, and repeat the extraction process 3 times until the extract is relatively clear. This is considered complete extraction, and the extract is *Citrus aurantium* extract.
[0035] Example 2 Preparation of compounds (1)-(4)
[0036] 1) The *Citrus aurantium* extract obtained in Example 1 was suspended in water, and the pH was adjusted to 2-3 with hydrochloric acid. Then, it was extracted with petroleum ether and ethyl acetate respectively to remove the oil. The pH was then adjusted to 9-10 with saturated 10% ammonia solution, followed by extraction with CH2Cl2 to obtain crude alkaloids. The crude alkaloids were subjected to silica gel open column chromatography with gradient elution using dichloromethane-methanol (100:0, 100:2.5, 100:5, 100:10, 100:20, 100:50, 100:100, 0:100) (v / v), with a retention volume of 2 L for each gradient; a total of 8 fractions (Fr. 1-8) were obtained.
[0037] 2) Select the above Fr.3 fraction and perform gradient elution with methanol-water as the eluent (20:80; 30:70; 40:60; 50:50; 60:40; 70:30; 80:20; 90:10; 100:0) (v / v) by ODS open column chromatography. The retention volume of each gradient is 500 mL, and a total of 9 fractions (Fr.3.1-Fr.3.9) are obtained.
[0038] 3) The Fr.3.6 fraction was subjected to semi-preparative high-performance liquid chromatography (HPLC) using a C18 reversed-phase column chromatography system (Cosmosil 5C18-MS-II 10*250mm 5um). Elution was performed with a methanol-water mixture at a volume ratio of 67:33 at a flow rate of 3.5 ml / min. Based on the different retention times and peak sequences, and further identified by spectroscopic methods, compound (1) was obtained (see...). Figure 1-2 (Retention time: 30.0 min), Compound (2) (See) Figure 3-4 (Retention time: 36.0 min), Compound (3) (See) Figure 5-6 (Retention time: 54.0 min), Compound (4) (See) Figure 7-8 (Retention time: 70.0 min).
[0039] Example 3 Formulation
[0040] The present invention describes an extract obtained from *Citrus aurantium* as an active ingredient, and a monomeric compound as an active ingredient, which is then combined with a pharmaceutically acceptable carrier, excipient or pharmaceutically acceptable excipient to form a pharmaceutical formulation.
[0041] The dosage form of the pharmaceutical preparation is liquid, solid, etc.
[0042] Liquid dosage forms include oral liquids and injections; solid dosage forms include granules, tablets, and capsules.
[0043] 3.1 Preparation of oral liquid
[0044] Take 1g of axillary orange extract, dissolve it in distilled water, adjust the pH value with dilute sodium bicarbonate solution, add 0.05% potassium sorbate preservative, and then fill and sterilize.
[0045] Take 25 mg of the monomer compound succinate A(1), dissolve it in distilled water, adjust the pH value with dilute sodium bicarbonate solution, add 0.05% potassium sorbate as preservative, and then fill and sterilize.
[0046] Take 25 mg of the monomer compound succinate B(2), dissolve it in distilled water, adjust the pH value with dilute sodium bicarbonate solution, add 0.05% potassium sorbate as preservative, and then fill and sterilize.
[0047] Take 25 mg of the monomer compound succinate C(3), dissolve it in distilled water, adjust the pH value with dilute sodium bicarbonate solution, add 0.05% potassium sorbate as preservative, and then fill and sterilize.
[0048] Take 25 mg of the monomer compound succinate D(4), dissolve it in distilled water, adjust the pH value with dilute sodium bicarbonate solution, add 0.05% potassium sorbate as preservative, and then fill and sterilize.
[0049] 3.2 Preparation of Injection Solution
[0050] Take 0.2g of *Citrus aurantium* extract, solubilize with Tween-80, adjust the pH to 6.0 with saturated sodium bicarbonate solution, add mannitol and water for injection, treat with 0.2% activated carbon, pre-freeze at -25℃ for 10h, vacuum, program temperature increase at 2℃ / hr, keep at -15℃ for 10h, and finally dry at 8℃ / hr to 25℃ for 5h. Then fill and sterilize.
[0051] Take 10 mg of the monomeric compound scutellarin A(1), solubilize it with Tween-80, adjust the pH to 6.0 with saturated sodium bicarbonate solution, add mannitol and water for injection, treat with 0.2% activated carbon, pre-freeze at -25℃ for 10 h, vacuum, program the temperature to rise at 2℃ / hr, keep at -15℃ for 10 h, and finally dry at 25℃ at 8℃ / hr for 5 h. Then fill and sterilize.
[0052] Take 10 mg of the monomeric compound citric acid B(2), solubilize it with Tween-80, adjust the pH to 6.0 with saturated sodium bicarbonate solution, add mannitol and water for injection, treat with 0.2% activated carbon, pre-freeze at -25℃ for 10 h, vacuum, program the temperature to rise at 2℃ / hr, keep at -15℃ for 10 h, and finally dry at 25℃ at 8℃ / hr for 5 h. Then fill and sterilize.
[0053] Take 10 mg of the monomeric compound citric acid C(3), solubilize it with Tween-80, adjust the pH to 6.0 with saturated sodium bicarbonate solution, add mannitol and water for injection, treat with 0.2% activated carbon, pre-freeze at -25℃ for 10 h, vacuum, program the temperature to rise at 2℃ / hr, keep at -15℃ for 10 h, and finally dry at 25℃ at 8℃ / hr for 5 h. Then fill and sterilize.
[0054] Take 10 mg of the monomeric compound scutellarin D(4), solubilize it with Tween-80, adjust the pH to 6.0 with saturated sodium bicarbonate solution, add mannitol and water for injection, treat with 0.2% activated carbon, pre-freeze at -25℃ for 10 h, vacuum, program the temperature to rise at 2℃ / hr, keep at -15℃ for 10 h, and finally dry at 25℃ at 8℃ / hr for 5 h. Then fill and sterilize.
[0055] 3.3 Tablet Preparation
[0056] Take 1g of axillary orange extract, grind it into a fine powder, add an appropriate amount of starch paste as a binder and an appropriate amount of starch as a diluent, and compress it into tablets.
[0057] Take 50 mg of the monomer compound citric acid A(1), make it into a fine powder, add an appropriate amount of starch paste as a binder and an appropriate amount of starch as a diluent, and compress it into tablets.
[0058] Take 50 mg of the monomer compound citric acid B(2), make it into a fine powder, add an appropriate amount of starch paste as a binder and an appropriate amount of starch as a diluent, and compress it into tablets.
[0059] Take 50 mg of the monomer compound citric acid C(3), make it into a fine powder, add an appropriate amount of starch paste as a binder and an appropriate amount of starch as a diluent, and compress it into tablets.
[0060] Take 50 mg of the monomer compound citric acid D(4), make it into a fine powder, add an appropriate amount of starch paste as a binder and an appropriate amount of starch as a diluent, and compress it into tablets.
[0061] Example 4: Evaluation of the antitumor activity of *Citrus aurantium* var. *axillosa*
[0062] 4.1 Experimental Materials and Instruments
[0063] HCT-116 (human colorectal cancer cells), K562 (human chronic myeloid leukemia cell line), AGS (human gastric adenocarcinoma cell line), A549 (human non-small cell lung cancer cell line), HepG2 (human liver cancer cell line), DMEM medium, trypsin, fetal bovine serum (FBS), CCK-8, 5-fluorouracil, cisplatin, cell culture incubator, microplate reader, pipette, centrifuge.
[0064] 4.2 Experimental Methods
[0065] HCT-116 (human colorectal cancer cells), K562 (human chronic myeloid leukemia cell line), AGS (human gastric adenocarcinoma cell line), A549 (human non-small cell lung cancer cell line), and HepG2 (human liver cancer cell line) in logarithmic growth phase were cultured to 5 × 10⁻⁶ cells / year. 4Cells were seeded at a density of [number] cells / mL in 96-well plates. The culture medium was DMEM (RPMI-1640) containing 10-15% fetal bovine serum. The *Citrus aurantium* extract and *Citrus aurantium* alkaloid (AD) monomer compounds obtained in the above examples were dissolved in 0.1% DMSO and added to the culture medium to prepare different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL or 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM) of analytes. Cells were incubated in an incubator (37℃, 5% CO2) for 12 h and observed under a microscope. Once cell adhesion was observed, the culture medium was aspirated, and cells were administered according to their assigned groups. Positive control drugs were 5-fluorouracil and cisplatin. 100 μL of culture medium containing different concentrations (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM) of extracts and compounds (without serum) was added to each well. Three replicates were set up for each drug, creating five dosing gradients. Two blank control groups and four control groups were also included on each plate. After drug administration, incubation continued for 48 h, followed by the addition of 10 μL of CCK-8 solution. Cells were incubated for 2 h, and the absorbance (OD value) was measured at 450 nm using a microplate reader. All compound solutions, 5-fluorouracil and cisplatin solutions, and CCK-8 were prepared fresh for each use.
[0066] Cell inhibition rate = (control group - experimental group) / (control group - blank group) × 100%.
[0067] All experiments were performed in triplicate for each group, and data processing and statistical analysis were conducted using SPSS Statistics 26. Experimental results were expressed as IC50. 50 To evaluate the cellular activity of experimental compounds.
[0068] 4.3 Experimental Results and Discussion
[0069] The experimental results are shown in Table 1. Both *Citrus aurantium* extract and *Citrus aurantium* alkaloid (AD) exhibited varying degrees of inhibitory effects on the proliferation of HCT-116, K562, AGS, A549, and HepG2 cells. Among them, *Citrus aurantium* extract showed the strongest inhibitory effect on the HCT116 cell line, with an IC50 concentration of [missing value]. 50 The concentration was 9.74 ± 1.32 μg / mL, followed by the AGS cell line (IC50). 50=13.60±1.92μg / mL). For the monomeric compounds, axillary argentine (AD) showed strong activity in all five tumor cell lines, with the strongest inhibitory effect on the HCT116 cell line. In addition, compounds (3-4) generally showed better inhibitory effects than compounds (1-2) in all five cell lines, and were also better than the positive control drugs 5-fluorouracil and cisplatin. In particular, compound (4) showed the strongest inhibitory activity against cell proliferation in all five cell lines.
[0070] Table 1. In vitro antitumor activity of Citrus aurantium.
[0071]
[0072]
[0073] a Positive drug
[0074] The above-described embodiments have verified that the extract of *Citrus aurantium* and its alkaloid (AD) exhibit good inhibitory activity against various tumor cell lines, including HCT-116, K562, AGS, A549, and HepG2. This suggests that the extract and alkaloid may possess multi-target anti-tumor capabilities. In particular, compounds (3) and (4) show significantly better inhibitory activity than positive control drugs, and thus have the potential to become anti-tumor lead compounds. Therefore, it can be concluded that the present invention has good application prospects for the treatment of tumors.
Claims
1. The use of compound (1), compound (2), compound (3), or compound (4) in the preparation of antitumor drugs: 。 2. The application according to claim 1, characterized in that: Using the compound as the active ingredient, an antitumor drug formulation is formed with a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.
3. The application according to claim 1, characterized in that: The tumor is colorectal cancer, rectal cancer, leukemia, stomach cancer, lung cancer, or liver cancer.