CN113116972A

CN113116972A - Application of goldhair hedyotis herb extract in preparing anti-oxidation and anti-tumor medicines

Info

Publication number: CN113116972A
Application number: CN202110367921.1A
Authority: CN
Inventors: 季红; 张超; 张静; 李山诺; 陈美鸿; 谭娅玲; 陈乐贤
Original assignee: Guangzhou Medical University
Current assignee: Guangzhou Medical University
Priority date: 2021-04-06
Filing date: 2021-04-06
Publication date: 2021-07-16
Anticipated expiration: 2041-04-06
Also published as: CN113116972B

Abstract

The invention discloses an application of a goldhair hedyotis herb extract in preparing anti-oxidation and anti-tumor medicaments. The goldhair hedyotis herb extract is prepared by the following method: crushing the goldhair hedyotis herb, carrying out continuous reflux extraction by using an ethanol water solution, and concentrating an extracting solution to obtain an ethanol crude extract; and (2) dispersing the ethanol crude extract in water, sequentially and respectively extracting with petroleum ether, ethyl acetate and n-butanol, separating the extract, concentrating to constant weight, and respectively obtaining a petroleum ether extract, an ethyl acetate extract and an n-butanol extract. The goldhair hedyotis herb extract has better antioxidation and anti-tumor effects.

Description

Application of goldhair hedyotis herb extract in preparing anti-oxidation and anti-tumor medicines

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a goldhair hedyotis herb extract in preparation of anti-oxidation and anti-tumor medicines.

Background

In recent years, cancer has been second to the second leading cause of death of cardiovascular disease, despite significant advances in early screening and treatment. The incidence and mortality of cancer also presents a rapidly increasing momentum. The existing chemotherapy drugs can cause various serious toxic and side effects while playing a therapeutic role. Meanwhile, the easy generation of drug resistance also influences the effect of the chemotherapeutic drugs to a great extent. Therefore, the search for novel antitumor drugs with high efficiency and low toxicity has been an important field of drug research.

Chinese herbal medicines and natural medicines have been the important source for medicine discovery and development from ancient times to present. More and more antitumor drugs derived from Chinese herbal medicines attract great attention of medicinal chemists. The search for new antitumor drugs from natural drugs also becomes an important way for research and development of antitumor drugs. The Chinese acremon is broad, the types of medicinal plants, animals and minerals are various, and various traditional Chinese medicinal materials, national medicines and folk medicines play an important role in the aspects of prevention, health care and disease treatment. The abundant natural medicine resources, the long history of the traditional Chinese medicine and the profound traditional Chinese medicine theory enable us to have the unique advantages of discovering new anti-tumor medicines from the traditional Chinese medicines.

The goldhair hedyotis herb is a perennial herb, is widely distributed in subtropical and tropical regions of China, is mainly distributed in places such as Jiangxi, Anhui, Jiangsu, Zhejiang, Fujian, Guangdong and Guangxi, has the functions of clearing heat, removing dampness, promoting urination, calming the liver, strengthening teeth, improving eyesight, promoting blood circulation and relaxing muscles and tendons, and is used for treating jaundice, edema, chyluria, dysentery, diarrhea, traumatic injury, innominate toxic swelling, mastitis and the like. The goldhair hedyotis herb is mainly used as one of the formulas of Chinese patent medicine enteritis peaches in clinic and is used for treating acute and chronic gastroenteritis, diarrhea, bacillary dysentery and infantile dyspepsia. At present, pharmacological action and substance basis research of the goldhair hedyotis herb for playing drug effect are few, so that the goldhair hedyotis herb is deeply researched and developed and has important significance in preparing antioxidant and antitumor drugs.

Disclosure of Invention

The invention aims to provide a preparation method of a goldhair hedyotis herb extract and application of the goldhair hedyotis herb extract in preparing anti-oxidation and anti-tumor medicaments.

The first purpose of the invention is to provide the application of the goldhair hedyotis herb extract in preparing anti-oxidation or anti-tumor medicines.

Preferably, the goldhair hedyotis herb extract is an ethanol crude extract, a petroleum ether extract, an ethyl acetate extract or an n-butanol extract, and is prepared by the following method:

crushing the goldhair hedyotis herb, carrying out continuous reflux extraction by using an ethanol water solution, and concentrating an extracting solution to obtain an ethanol crude extract;

and (3) dispersing the ethanol crude extract in water, sequentially and respectively extracting with petroleum ether, ethyl acetate and n-butanol, separating the extract liquid, concentrating to constant weight, and respectively obtaining a petroleum ether extract, an ethyl acetate extract and an n-butanol extract.

Preferably, the ethanol water solution is 50-95% of ethanol water solution by volume fraction.

Preferably, the ethanol aqueous solution is 80% by volume of ethanol aqueous solution.

Preferably, the continuous reflux extraction is performed at least 3 times, each time for 1-2 h.

Preferably, the concentration is a concentration under reduced pressure.

Preferably, the application is the application of the ethyl acetate extract of the goldhair hedyotis herb in preparing the antioxidant drugs.

Preferably, the application is the application of the petroleum ether extract of the goldhair hedyotis herb in preparing the anti-tumor medicine.

Preferably, the anti-tumor drug is a drug for resisting breast cancer, colon cancer, non-small cell lung cancer, cervical cancer and oral epidermoid carcinoma.

Preferably, the antitumor drug is a drug having at least one function selected from the following (1) to (5):

(1) inhibiting tumor cell proliferation;

(2) inhibiting tumor cell migration;

(3) inhibiting tumor cell invasion;

(4) inducing apoptosis of tumor cells;

(5) increase the generation of active oxygen in tumor cells.

Compared with the prior art, the invention has the following advantages and effects:

(1) the goldhair hedyotis herb extract has better antioxidation, especially the ethyl acetate extract has the strongest antioxidation, can be applied to preparing antioxidant drugs, and has great potential for clinical application.

(2) The goldhair hedyotis herb extract has a good anti-tumor effect, particularly the petroleum ether extract has strong activity of resisting various cancers such as breast cancer, colon cancer, non-small cell lung cancer, cervical cancer, oral epidermoid carcinoma and the like, has a prominent inhibiting effect on proliferation, migration and invasion of tumor cells, can remarkably induce apoptosis of the tumor cells, can play a role in promoting apoptosis by increasing the level of Reactive Oxygen Species (ROS) in the tumor cells, can be applied to preparation of anti-tumor drugs, and provides new resources and ideas for research and development of the anti-tumor drugs.

(3) The preparation method of the extract is simple and easy to operate.

Drawings

FIG. 1 shows the effect of petroleum ether extract on MDA-MB-231(a, b) and HCT-116(c, d) cell migration ((a, b))^***p<0.001)；

Figure 2 is the effect of petroleum ether extracts on MDA-MB-231(a, b) and HCT-116(c, d) cell invasion ([ p ] p <0.05, [ p ] p <0.01, [ p ] p < 0.001);

FIG. 3 is a graph of the effect of petroleum ether extract on MDA-MB-231(a, b) and HCT-116(c, d) apoptosis ((a, b))^*p<0.05,^****p<0.0001)；

FIG. 4 is a graph of the effect of petroleum ether extract on the apoptotic morphology of MDA-MB-231(a) and HCT-116 (b);

FIG. 5 is a graph showing the effect of petroleum ether extract on intracellular reactive oxygen species production of MDA-MB-231(a, b) and HCT-116(c, d) ((^**p<0.01，***p<0.001)。

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: preparation method of herba Hedyotidis Chrysotrichae extract

Pulverizing dried whole herba Hedyotidis Chrysotrichae (1.0Kg), extracting with 80% (v/v) ethanol water solution under reflux for 3 times (2 hr each time), mixing extractive solutions, and concentrating under reduced pressure to dry to obtain ethanol crude extract total extract (2.0 g, Creude). Dispersing the total extract (100.0g) in water, sequentially extracting with equal amount of petroleum ether, ethyl acetate and n-butanol respectively, separating the extractive solutions, and concentrating under reduced pressure to constant weight to obtain petroleum ether extract (PE fraction, 2.8g), ethyl acetate extract (EtOAc fraction,3.58g) and n-butanol extract (BuOH fraction, 3.24g), respectively.

Example 2: determination of antioxidant Activity of Epimedium xanthioides extract

1, 1-diphenyl-2- [ (2,4,6) -trinitro group]Phenylhydrazine (DPPH) (3.94mg) was dissolved in absolute ethanol to prepare a 0.4mmol/L solution of DPPH in ethanol. The ethanol crude extract, the petroleum ether extract, the ethyl acetate extract and the n-butanol extract are respectively prepared into ethanol solutions with different concentration gradients (the concentration of the ethanol crude extract is 50, 100, 150, 200, 250 and 300 mu g/mL, the concentration of the petroleum ether extract is 100, 150, 200, 250, 300 and 350 mu g/mL, the concentration of the ethyl acetate extract is 100, 200, 300, 400, 500 and 600 mu g/mL, and the concentration of the n-butanol extract is 100, 200, 300, 400, 500 and 600 mu g/mL). Mixing 1.5mL of each ethanol solution with different concentration gradients with the 0.4mmol/L DPPH ethanol solution at room temperature, reacting in dark for 30min, and measuring the absorbance value of each mixed solution at 517nm with an ultraviolet spectrophotometer. Vitamin C (Vc) is used as a positive control. The percentage inhibition of free radical scavenging activity is calculated as: percentage (%) inhibition of DPPH radical scavenging activity [ < 1- (A) >_i-A_j)/A₀]X 100% where A_iTo test the absorbance of the groups, A_jAbsorbance of blank group, A₀Absorbance of the control group. Each group was run in triplicate and the IC of each sample was calculated from the concentration dependence of the concentration of the sample solution on the percent inhibition of DPPH free radical scavenging activity₅₀Values (table 1).

TABLE 1 free radical scavenging Activity of the extract of goldhair hedyotis herb

As can be seen from Table 1, the crude ethanol extract of the goldhair hedyotis herb has a certain antioxidant activity (IC)₅₀214.67 μ g/mL), IC of petroleum ether extract, ethyl acetate extract and n-butanol extract₅₀The values are 510.44, 98.67 and 176.00 mug/mL respectively, and the antioxidant activity of the ethyl acetate extract is strongest.

Example 3: determination of inhibitory Activity of Epimedium xanthioides extract on tumor cell proliferation

MTT method is adopted to determine the inhibition effect of the ethanol crude extract, the petroleum ether extract, the ethyl acetate extract and the n-butanol extract on the proliferation of 5 human tumor cell lines (human breast cancer cell MDA-MB-231, colon cancer cell HCT-116, lung cancer cell A549, cervical cancer cell Hela and oral epidermoid carcinoma cell KB). DMSO was used as a blank control, and 5-fluorouracil (5-FU) was used as a positive control.

The above 5 tumor cells were cultured at a ratio of 3X 10³The density of individual cells/well was seeded in 96-well plates at 5% CO₂Incubated at 37 ℃ for 24 hours in the presence. Then, the cells were treated with DMSO solutions of the above extracts (ethanol crude extract, petroleum ether extract, ethyl acetate extract, and n-butanol extract) at concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400. mu.g/mL for 48h, respectively. Subsequently, a concentration of 10. mu.L was added to each well5.0mg/mL MTT solution and incubated for 4 h. The medium was discarded from the wells, 100. mu.L DMSO was added to each well, and the wells were shaken on a shaker for 10 minutes. The absorbance of each well was measured using a microplate reader at wavelengths of 490nm and 540 nm. The formula is adopted: (1-A) having a cell growth inhibition ratio%_t/A_c) X 100% calculation of the cell inhibition, where A_tIs the absorbance of the drug group, A_cAbsorbance of the control group. Each group was run in triplicate and IC was calculated for each extract based on the concentration-dependent curve of inhibition of cell proliferation using GraphPad Prism 7 software₅₀The results are shown in Table 2.

TABLE 2 inhibitory Effect of the extract of Hedyotis chrysotricha on tumor cell proliferation

The results show that the crude ethanol extract, the petroleum ether extract, the ethyl acetate extract and the n-butanol extract of the goldhair hedyotis herb all show inhibition effects of different degrees on the proliferation of 5 tumor cells, and the anti-proliferation effects of the petroleum ether extract, the ethyl acetate extract and the n-butanol extract are obviously stronger than those of the crude ethanol extract. Wherein the petroleum ether extract has the strongest inhibition effect on the proliferation of two tumor cells of human breast cancer cells MDA-MB-231 and colon cancer cells HCT-116.

Example 4: inhibition of tumor cell migration by petroleum ether extracts

MDA-MB-231 cells and HCT-116 cells were plated at 5X 10⁵The density of each cell/well is inoculated in a 6-well plate, after 12 hours of culture until the cells are fused, a monolayer of cells are damaged by a sterile microtube head, and then the cells are washed three times by PBS to remove the exfoliated cells. Subsequently, 2 mL/well of serum-free medium was added to the cells and starved overnight. Cells were treated with petroleum ether extracts at different concentrations (0, 1.5, 3 and 6. mu.g/mL) for 0h, 24h and 48h, respectively, and the migration of peripheral cells to the central scratch area was observed by microscopy at different time points. Measuring the width of the scratch by adopting Image J software according to a formula: percent wound healing ═ S (S)_t/S_c) X 100% calculated injuryPercent oral healing, three replicates per group, and statistical analysis of the data using GraphPad Prism 7 software, the results are shown in figure 1.

The results show that the petroleum ether extract can obviously inhibit the migration of human breast cancer cell MDA-MB-231 (shown in figures 1a and b) and colon cancer cell HCT-116 (shown in figures 1c and d), and can effectively inhibit the migration of tumor cells at a low concentration of 1.5 mu g/mL.

Example 5: inhibition of tumor cell invasion by petroleum ether extract

Detection was performed using a Transwell cell invasion assay. The matrigel was diluted with serum-free medium at a ratio of 1:4, and the diluted matrigel (45. mu.L/chamber) was added to the upper chamber of the Transwell chamber and left at 37 ℃ for 2 hours to convert the matrigel to a gel. Prepared with serum-free DMEM medium at a concentration of 3X 10⁵Individual cells/mL of human breast cancer cells MDA-MB-231 and colon cancer cells HCT-116 cell suspensions. 200. mu.L of the above cell suspension was added to the upper chamber of the Transwell chamber, and 600. mu.L of DMEM medium containing FBS, penicillin and streptomycin was added to the lower chamber. The cells were then treated with 0, 3, 6. mu.g/mL petroleum ether extracts for 48h at 37 ℃. After discarding the medium from each well, the cells were fixed with methanol for 15min, then stained with 0.1% crystal violet for 0.5h, and washed 3 times with PBS. The non-migrated cells were physically removed from the upper surface with a cotton swab, and the cells invading through the membrane to the lower surface were observed under an inverted microscope. Randomly select 5 fields (10 times magnification), take images and calculate the number of invading cells, repeat the experiment three times per group, perform statistical analysis using GraphPad Prism 7 software, and the results are shown in fig. 2.

As shown in figure 2, compared with cells not treated by the petroleum ether extract, the drug composition has obvious inhibition effect on invasion of human breast cancer cells MDA-MB-231 and colon cancer cells HCT-116 at the concentrations of 3 mug/mL and 6 mug/mL, and is in a dose-dependent relationship, which indicates that the petroleum ether extract has obvious inhibition effect on invasion of two tumor cells.

Example 6: action of petroleum ether extract for inducing tumor cell apoptosis

Human breast cancer cell MDA-MB-231 and colon cancer cell HCT-116 were mixed at 2.5X 10⁵Individual cell or cellThe density of wells was seeded in 6-well plates and after 12h of culture, the cells were treated with petroleum ether extract at a concentration of 6. mu.g/mL for 48h, respectively. Cells were collected and washed twice with 1mL ice-cold PBS. After centrifugation, the cells were resuspended in buffer, 5 μ L FITC and 5 μ L PI were added, incubated for 10min at room temperature in the dark, and apoptosis was detected by flow cytometry with triplicate experiments per group and statistical analysis was performed using GraphPad Prism 7 software, with results shown in fig. 3 and 4.

The results show that the proportion of apoptotic cells in both cell populations was significantly increased after treating human breast cancer cells MDA-MB-231 and colon cancer cells HCT-11648 h with petroleum ether extract at 6. mu.g/mL (FIG. 3). Morphological experiments show that cells show green fluorescence after being stained by Annexin V-FITC and represent the early stage of apoptosis. Cells stained with Annexin V-FITC and PI at the same time showed red fluorescence internally and green fluorescence externally, representing the late stage of apoptosis (FIG. 4). Compared with the control group, the petroleum ether extract treatment shows higher fluorescence intensity, which indicates that the petroleum ether extract treatment can remarkably induce human breast cancer MDA-MB-231 cells and colon cancer HCT-116 cells to die.

Example 7: use of extract of goldhair hedyotis herb for increasing Reactive Oxygen Species (ROS) production in tumor cells

Human breast cancer cell MDA-MB-231 and colon cancer cell HCT-116 were mixed at 2.5X 10⁵Individual cells/well density were seeded in 6-well plates at 5% CO₂After incubation at 37 ℃ for 12h in the presence of the buffer, the cells were treated with petroleum ether extracts at concentrations of 0, 10, 20, 40. mu.g/mL for 48 h. 1mL of fluorescent probe DCFH-DA (diluted 1:1000 in serum-free medium) was added to each group of cells, and the cells were incubated at 37 ℃ in the dark for 20min and then washed 3 times with 1mL of serum-free medium. Images were collected using an inverted fluorescence microscope. After imaging, cells were centrifuged (2000r, 4 ℃, 3min), washed with serum-free medium, and fluorescence intensity analyzed using a fluorescence microplate reader (excitation 488nm, emission 525nm) and GraphPad Prism 7 software. The results of the experiment are shown in FIG. 5.

As can be seen from fig. 5a and 5c, the number of green fluorescence spots and fluorescence intensity exhibited under the microscope were significantly increased after the petroleum ether extract was treated with both cells compared to the control group. Statistical analysis showed that the petroleum ether extract significantly enhanced the fluorescence intensity of the green fluorescent spots appearing in both cells at concentrations of 10, 20 and 40 μ g/mL (fig. 5b and 5 d). These results indicate that petroleum ether extracts significantly increased ROS production in human breast cancer cells MDA-MB-231 and colon cancer cells HCT-116. It is concluded that petroleum ether extract may exert its effect of inducing apoptosis of tumor cells by increasing intracellular ROS production.

In conclusion, the goldhair hedyotis herb extract has antioxidant and antitumor effects, wherein the ethyl acetate extract has the strongest antioxidant activity, and the petroleum ether extract has the strongest antitumor effect, and particularly has the most prominent effect on cervical cancer cells and colon cancer cells. The petroleum ether extract has obvious effects of resisting proliferation, migration and invasion of tumor cells, can induce apoptosis of the tumor cells, and can possibly play a role of promoting apoptosis by increasing the generation of intracellular ROS.

Further description of the properties of the extract of goldhair hedyotis herb of the present invention:

the literature reports that the alkaloid of the goldhair hedyotis herb extract has cytotoxic effect on a tumor cell HL-60; the sterol has cytotoxic and anti-cell migration effects on human liver cancer cell SK-HEP-1, but does not promote apoptosis and has no cell cycle blocking effect.

In this experiment, we identified the goldhair hedyotis herb extract by a color reaction, in which the petroleum ether extract was a chromogenic agent for two alkaloids: neither the modified bismuth potassium iodide (Dragendorff) reagent nor the iodine-potassium iodide (Wagner) reagent developed color, indicating that no alkaloid component was contained therein. Meanwhile, petroleum ether extract and ethyl acetate extract were also negative for trichloroacetic acid reaction (Rosen-Heimer reaction) and acetic anhydride-concentrated sulfuric acid reaction (Liebermann-Burchard reaction), indicating that they also do not contain triterpenes and steroids.

Claims

1. Application of herba Hedyotidis Chrysotrichae extract in preparing antioxidant or antitumor medicine is provided.

2. The use of claim 1, wherein the goldhair hedyotis herb extract is an ethanol crude extract, a petroleum ether extract, an ethyl acetate extract or an n-butanol extract, and is prepared by the following method:

3. The use according to claim 2, wherein the aqueous ethanol solution is 50-95% by volume aqueous ethanol solution.

4. The use according to claim 3, wherein the aqueous ethanol solution is an 80% by volume aqueous ethanol solution.

5. The use according to claim 2, wherein said continuous reflux extraction is carried out at least 3 times, each time for 1-2 h.

6. Use according to claim 2, wherein the concentration is a reduced pressure concentration.

7. The use according to claim 2, wherein the ethyl acetate extract of goldhair hedyotis herb is used in the preparation of an antioxidant medicament.

8. The use according to claim 2, characterized by the use of a petroleum ether extract of goldhair hedyotis herb for the preparation of an antitumor medicament.

9. The use of claim 8, wherein the anti-tumor drug is a drug against breast cancer, colon cancer, non-small cell lung cancer, cervical cancer, oral epidermoid carcinoma.

10. The use according to claim 8, wherein the antitumor agent is an agent having at least one function selected from the group consisting of the following (1) to (5):

(1) inhibiting tumor cell proliferation;

(2) inhibiting tumor cell migration;

(3) inhibiting tumor cell invasion;

(4) inducing apoptosis of tumor cells;

(5) increase the generation of active oxygen in tumor cells.