CN118615282B - Radix clematidis alkaloid compound and application thereof - Google Patents

Abstract

The invention discloses the application of clematis alkaloid compounds as anti-tumor drugs. The alkaloid compounds are obtained from Clematis root and rhizome as raw materials, extracted with alcohol and water, concentrated, extracted with ethyl acetate, separated by silica gel column chromatography, and purified by HPLC. Cell experiments show that the compounds of the application have good inhibitory effects on human leukemia cells and liver cancer cells.

Description

Clematis alkaloid compounds and uses thereof

Technical Field

The invention relates to the fields of natural drug chemistry and pharmacology, and in particular to the use of alkaloid compounds in the traditional Chinese medicine Clematis chinensis.

Background Art

Clematis is a traditional Chinese medicinal material. It is the dried root and rhizome of Clematis chinensis Osbeck, Clematis hexapetala Pall. or Clematis manshurica Rupr. of the Ranunculaceae family. Previous studies have shown that the main chemical components of Clematis include pentacyclic triterpenoid saponins, flavonoids, coumarins, macrocyclic glycosides, alkaloids, etc. In recent years, the pharmacological effects of Clematis such as antibacterial and anti-inflammatory, dehumidifying and analgesic, and anti-tumor have attracted widespread attention. Clematis has different degrees of inhibitory effects on various cancers, such as significantly inhibiting cancer cells such as gastric cancer, ovarian cancer, breast cancer, and cervical cancer, which has guiding significance for anti-tumor treatment.

Acute myeloid leukemia (AML) is a blood system malignancy characterized by excessive proliferation, abnormal differentiation or inhibited apoptosis of hematopoietic progenitor cells or stem cells. Chemotherapy is currently the main treatment for AML, but its clinical application is limited by high medical costs, adverse reactions and drug resistance. Therefore, it is of great clinical significance to explore new and safe anti-cancer solutions. In recent years, Chinese herbal medicine has been valued by many biological and medical researchers for its obvious anti-cancer effects and high safety.

Li Junyan et al. (Study on the inhibitory effect of Clematis polysaccharide on the growth of tongue squamous cell carcinoma cells, Biotechnology Communications) found that the inhibitory effect of Clematis polysaccharide on tongue squamous cell carcinoma cell Tca-8113 cells increased with the increase of concentration, and the inhibition rate of Clematis polysaccharide on Tca-8113 cells stabilized at a certain level after 72 hours of action. This suggests that Clematis polysaccharide has an inhibitory effect on the growth of tongue squamous cell carcinoma Tca-8113 cells and is time- and dose-dependent. Chen Yuru et al. (Study on the inhibitory effect of Clematis saponins on the growth of HL-60 cells, Journal of Luzhou Medical College) used Clematis saponins as the research object and arsenic as the positive control to explore its effect on the growth of acute myeloid leukemia HL-60 cell line. The cell growth inhibition test of HL-60 cells was carried out by CCK-8 method and compared with the effect of arsenic trioxide; the changes in cell morphology were observed by inverted microscope.

The results showed that Clematis saponins had a significant growth inhibitory effect on leukemia HL-60 cells.

Hai-Bing Jiang et al. ("Phenolic glycosides and indole alkaloids isolatedfrom the roots and rhizomes of Clematis chinensis and their anti-inflammatory activity", Phytochemistry, Vol. 215) isolated three new phenolic glycosides and three new indole alkaloids from the aqueous extracts of the roots and rhizomes of Clematis chinensis and tested their anti-inflammatory activity.

The present application separates, purifies and detects the anti-cancer activity of alkaloid compounds in Clematis chinensis, and finds that the alkaloid compounds have good anti-leukemia activity.

Summary of the invention

Clematis alkaloid compounds and their use as anti-tumor drugs, the alkaloid compounds are obtained from Clematis root and rhizome as raw materials, extracted with alcohol and water, concentrated, extracted with ethyl acetate, separated by silica gel column chromatography, and purified by HPLC. Cell experiments show that the compounds of the present application have a good inhibitory effect on human leukemia cells HL-60 and liver cancer hepG2 cells. The present application obtains compounds with obvious inhibitory effects on leukemia and liver cancer cell lines by screening the main active ingredients in Clematis, providing more candidate compounds for the new drug development of anti-leukemia and liver cancer drugs.

The present invention first provides the use of the compound clematisindolin A, 6-hydroxy-1H-indole-3-carboxylic acid methyl ester, 1H-indole-3-carboxylic acid methyl ester, and 1H-indole-3-carboxaldehyde in the preparation of an anticancer pharmaceutical composition, wherein the chemical structure of the compound clematisindolin A is as follows:

.

Furthermore, the compound of the present invention can be used as the only active ingredient in an anticancer drug. Furthermore, the pharmaceutical composition of the present application can also contain other anticancer active ingredients, and the cancer is leukemia or liver cancer.

The anticancer drug of the present invention is in the form of an oral agent or an injection.

Preferably, the anticancer drug of the present invention can be prepared into common dosage forms by adding the compound of the present invention to a pharmaceutically acceptable excipient, such as granules, tablets, capsules, injections, etc., which are known to those skilled in the art.

Preferably, the active ingredient of the anti-leukemia drug of the present invention is clematisindolin A.

Furthermore, the cancer described in the present invention is leukemia or liver cancer. Experimental verification shows that the compound clematisindolin A has good inhibitory activity against leukemia and liver cancer cells, and the inhibitory activity is better than other alkaloid compounds in Clematis chinensis.

The alkaloid compounds of the present application are isolated by referring to the method reported by Hai-Bing Jiang et al. ("Phenolic glycosides and indole alkaloids isolated from the roots and rhizomes of Clematis chinensis and their anti-inflammatory activity", Phytochemistry, Vol. 215).

Beneficial effects: The present invention verifies for the first time the inhibitory effect of the monomeric compounds of the Clematis chinensis indole alkaloids on leukemia and liver cancer cells, and can be used to prepare drugs against leukemia and liver cancer, establishes the material basis for the anti-cancer activity of red peony root, and provides more candidate compounds for the research and development of new anti-cancer drugs.

DETAILED DESCRIPTION

The compounds to be tested: clematisindolin A, 6-hydroxy-1H-indole-3-carboxylic acid methyl ester, 1H-indole-3-carboxylic acid methyl ester, 1H-indole-3-carboxaldehyde, which are isolated from the traditional Chinese medicine Clematis chinensis. Other test materials and reagents are known materials that can be purchased commercially.

Example 1 Preparation of Compounds

40 kg of dried roots and rhizomes of Clematis chinensis were taken and boiled and stirred with 70% EtOH-H2O (3×120L) for 3 times (3 hours each time), and then the extract was concentrated under reduced pressure to obtain a concentrated extract, which was suspended in water and extracted with ethyl acetate. The ethyl acetate extract (318 g) was chromatographed on a silica gel column, using petroleum ether-ethyl acetate gradient elution (50:1, 10:1, 3:1), and then eluted with 95% ethanol and 55% ethanol to obtain fractions 1-5 in sequence. Fraction 3 (25 g) and fraction 4 (31 g) were eluted and separated using a silica gel column (petroleum ether: EtOAc), respectively, to obtain 10 parts (fractions 3.1-3.10) and (fractions 4.1-4.10), respectively. Fractions 3.1-3.10 and fractions 4.1-4.10 eluted from the silica gel column were separated by semi-preparative HPLC. The compounds clematisindolin A (18 mg), methyl 6-hydroxy-1H-indole-3-carboxylate (32 mg), methyl 1H-indole-3-carboxylate (39 mg), and 1H-indole-3-carboxaldehyde (30 mg) were obtained.

Example 2 Detection of the inhibitory activity of compounds on leukemia cell proliferation

Test reagents and preparation:

Experimental drugs and reagents: homemade compounds, positive control drug 5-Fu, DMSO to the required concentration (DMSO concentration ≤ 1‰), sterilized and stored at 4°C. MTT (methyl tetrazolyl blue) reagent was purchased from Sigma. Human leukemia HL-60 cells were purchased from Pronosai Biotechnology. 10% SDS reagent, RPMI-1640 (GiBCo, USA) culture medium containing 20% fetal bovine serum (FBS), other reagents are commercially available analytical grade. Subculture was carried out in an incubator at 37°C, 5% CO2, and saturated humidity, and the cells were used for experiments when they were in the logarithmic growth phase.

Experimental method: The experiment was carried out in a 96-well plate, and the system contained 1×10 ⁵ cells and the whole culture medium of the target compound at different concentrations. The total volume of each well was 100 μL, and each group had 8 replicates. The drug color control wells (without cells) and the culture wells containing cells and drugs were set up. After culturing for 44 hours, MTT (5 mg/mL, 10 μL) was added to each well, and the culture was continued for 4 hours. Then 100 μL of 10% SDS was added to terminate the reaction, and the reaction was incubated at 37°C overnight. The absorbance A value of each well at 570 nm was detected by enzyme-linked immunosorbent assay. The tumor growth inhibition rate was calculated according to the following formula: inhibition rate % = (A control - A test) / A control * 100%. The IC50 value for tumor cells was calculated.

The same operation as above was used to detect the inhibitory activity of the compound on human liver cancer cell Hep G2. The results are shown in Table 1.

Table 1: In vitro test results of inhibiting tumor cell growth

Table 1 Anti-tumor cell activity assay shows that the Clematis alkaloid compounds have a good inhibitory effect on the proliferation of leukemia HL-60 cells. Clematis alkaloid compounds have a significant inhibitory effect on leukemia HL-60 cells, and clematisindolin A and 1H-indole-3-carboxylic acid methyl ester also have significant inhibitory activity on liver cancer cells Hep G2. Among them, the IC50 value of clematisindolin A is lower than that of the clinical anticancer drug 5-fluorouracil (5-FU). Clematisindolin A is the compound with the most potential anti-leukemia activity in the series of compounds.

Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (2)

1. The application of the clematis root alkaloid compound in preparing a pharmaceutical composition for treating cancers, wherein the cancers are leukemia, the clematis root alkaloid compound is clematisindolin A, the clematisindolin A is used as the only active ingredient of an anti-leukemia drug, and the clematisindolin A chemical structural formula is as follows:

。

2. The use according to claim 1, wherein the medicament is in the form of granules, tablets, capsules or injection.