Euonymus alatus fruit and rosa multiflora fruit extract composition and application thereof in preparation of anti-colorectal cancer drugs

Abstract

The invention relates to a euonymus alatus fruit and rose fruit extract composition. The composition is prepared from euonymus alatus fruit water extract and rose fruit alcohol extract composition according to the mass ratio of 1: 2. According to the composition, the euonymus ilex fruit water extract and the rose fruit alcohol extract generate a synergistic effect at the ratio, proliferation, invasion and migration of colorectal cells can be remarkably inhibited, and the composition can be used for preparing medicines for treating colorectal cancer. The composition is derived from fruits of greening ornamental plants, is easy to obtain, is simple in extraction method, and has a good development prospect.

Description

Technical Field

[0001] The present invention relates to a composition of Euonymus japonicus Thunb. fruit extract and Rosa canina fruit extract, and more specifically to a composition composed of an aqueous extract of Euonymus japonicus Thunb. fruit and an alcohol extract of Rosa canina fruit in a mass ratio of 1:2 and its application in the preparation of anti-colorectal cancer drugs. Background Art

[0002] Colorectal cancer (CRC) is a common digestive system tumor in clinical practice and the third most common type of cancer in the world, accounting for about 10% of global new cancer cases, ranking third among all cancers globally, and second in terms of mortality among all cancers. In the early stage, colorectal cancer patients often have no obvious symptoms. As the cancer progresses, half of the patients may develop liver metastases. The mortality rate of metastatic colorectal cancer patients is high, and the 5-year survival rate is only 27.7%. However, the 5-year survival rate of patients treated in the early stage of cancer can also reach 90%. The standard treatment methods for colorectal cancer have always been surgery, chemotherapy, and radiotherapy, which can be combined for the treatment of patients. However, due to the non-specificity of chemotherapy drugs and their cytotoxicity to any cells, serious side effects are caused. In addition, chemotherapy drugs are prone to drug resistance, leading to tumor recurrence. Therefore, finding more alternative and effective treatment methods is crucial for improving the prognosis of colorectal cancer patients.

[0003] Euonymus japonicus Thunb. is a shrub plant of the genus Euonymus in the Celastraceae family, named for its evergreen characteristics throughout the four seasons. Euonymus japonicus Thunb. is native to southern Japan and is now distributed throughout China. It is a particularly common plant in the northern green belts. The flowering period of Euonymus japonicus Thunb. is from June to July, with small white or light green flowers, axillary, and cymose inflorescences. The fruit ripening period is from September to October, with capsule fruits, spherical. The young fruits are green. When mature, there is an orange-red aril on the outside and a red fruit inside. In late autumn, its aril slowly cracks, and as the maturity increases, the red fruit slowly shows outside. The roots, stems, and leaves of Euonymus japonicus Thunb. have medicinal value, can regulate menstruation and relieve pain, and are used for irregular menstruation, dysmenorrhea, traumatic injuries, fractures, and painful urination. Euonymus japonicus Thunb. has strong resistance to a variety of toxic gases, strong anti-smoke and dust absorption functions, and can purify the air. It is an ideal greening tree species in polluted areas. However, there has been no report on the research of its fruits.

[0004] Rosa sp. is a genus of plants under the Rosaceae family in the class Dicotyledoneae. Most of them are upright, spreading or climbing shrubs. There are 91 species of Rosa plants native to China, accounting for about 41% of the total number in the world. It is one of the main distribution areas of wild roses, distributed in East China, Central South and other places, such as Shandong, Henan, Jiangsu, Anhui, Xinjiang and other places. The fragrance of roses is pleasant and can be used for extracting precious aromatic oils. Rose hips are a substance that can be used both as medicine and food, containing a variety of bioactive ingredients and nutrients. Modern pharmacological research has found that rose hips have effects such as anti-inflammatory, antibacterial, antioxidant, promoting digestion, immune regulation, protecting the liver and cardiovascular system, treating obesity and diabetes, etc., and also have inhibitory effects on various tumor cells such as cervical cancer, breast cancer, ovarian cancer, bladder cancer, etc., but its effect on colorectal cancer cells has not been studied yet.

[0005] Both Euonymus japonicus and Rosa are widely distributed in China, especially in the northern regions. Both plants bear fruits in autumn. In addition to having good ornamental value, in-depth research and development of their medicinal value will bring higher economic added value to plant resources. Therefore, through a large number of activity screening experiments and research on formula ratios, the inventor of the present invention obtained the extract composition of Euonymus japonicus fruits and Rosa fruits with the best ratio of the present invention, and proved its positive effect on inhibiting the proliferation, invasion and migration of colorectal cancer cells, thus completing the present invention. The present invention provides an extract composition of Euonymus japonicus fruits and Rosa fruits, which has significant in vitro anti-colorectal cancer activity and can be used for preparing drugs for anti-colorectal cancer. Summary of the Invention

[0006] In view of the above situation, in order to overcome the deficiencies of the prior art, the purpose of the present invention is to improve the economic added value of Euonymus japonicus and Rosa, expand their application fields, and at the same time provide a new drug for treating colorectal cancer.

[0007] In order to achieve the above purpose, the present invention provides an extract composition of Euonymus japonicus fruits and Rosa fruits and its application in preparing drugs for anti-colorectal cancer.

[0008] The technical solution solved by the present invention is to provide an extract composition of Euonymus japonicus fruits and Rosa fruits, which is composed of an aqueous extract of Euonymus japonicus fruits and an ethanol extract of Rosa fruits in a mass ratio of 1:2;

[0009] The preparation method of the aqueous extract of Euonymus japonicus fruits is as follows:

[0010] Take Euonymus japonicus Thunb. fruits, dry them in the sun, crush them, place them in a round-bottomed flask, add 10 times the amount of water, heat them in an electric heating mantle until slightly boiling, keep boiling for 3 h, filter, add 6 times the amount of water to the filter residue, heat them in an electric heating mantle until slightly boiling, keep boiling for 2 h, filter, combine the two filtrates, concentrate, add ethanol until the ethanol content is 75%, let stand for 24 h, filter to obtain the filtrate, place it in a rotary evaporator to recover ethanol until there is no alcohol smell, concentrate, and dry under vacuum to obtain the water extract of Euonymus japonicus Thunb. fruits;

[0011] The preparation method of the ethanol extract of Rosa canina L. fruits is as follows:

[0012] Take Rosa canina L. fruits, dry them in the sun, crush them, place them in a round-bottomed flask, add 8 times the amount of 70% ethanol, heat them in an electric heating mantle for continuous reflux extraction for 3 h, filter, add 6 times the amount of 70% ethanol to the filter residue, heat them in an electric heating mantle for continuous reflux extraction for 2 h, filter, combine the two filtrates, place them in a rotary evaporator to recover ethanol until there is no alcohol smell, concentrate, and dry under vacuum to obtain the ethanol extract of Rosa canina L. fruits;

[0013] Weigh the water extract of Euonymus japonicus Thunb. fruits and the ethanol extract of Rosa canina L. fruits according to the mass ratio of 1:2, mix them evenly to obtain an extract composition of Euonymus japonicus Thunb. fruits and Rosa canina L. fruits.

[0014] The present invention has the following advantages compared with the prior art:

[0015] 1. The extract composition of the present invention is all derived from natural plant fruits. The extraction method is simple, easy to realize industrial production, and has low cost, which is of great significance for improving the economic added value of plants, increasing the planting rate, and creating higher economic benefits.

[0016] 2. The inventor of the present invention uniquely combines the water extract of Euonymus japonicus Thunb. fruits and the ethanol extract of Rosa canina L. fruits, conducts a large number of activity screening experiments, optimizes the proportion of the extracts, and finally finds that when the mass ratio of the water extract of Euonymus japonicus Thunb. fruits to the ethanol extract of Rosa canina L. fruits is 1:2, an obvious synergistic effect can be produced, significantly inhibiting the proliferation, invasion and migration of colorectal cancer cells, and can be used for the preparation of anti-colorectal cancer drugs. Description of the Drawings

[0017] Figure 1 It shows the effect of the extract composition of Euonymus japonicus Thunb. fruits and Rosa canina L. fruits on the migration ability of colorectal cancer RKO cells (A: Representative image of the cell scratch experiment; B: Quantitative analysis of the cell migration rate). Compared with the blank control group, *P<0.05; compared with the water extract group of Euonymus japonicus Thunb. fruits, #P<0.05; compared with the ethanol extract group of Rosa canina L. fruits, ^P<0.05.

[0018] Figure 2Effect of the extract composition of Euonymus japonicus Thunb. fruit and Rosa multiflora Thunb. fruit on the invasion ability of colorectal cancer RKO cells (A: Representative image of Transwell assay; B: Quantitative analysis of cell invasion rate). Compared with the blank control group, *P<0.05; compared with the aqueous extract group of Euonymus japonicus Thunb. fruit, #P<0.05; compared with the alcohol extract group of Rosa multiflora Thunb. fruit, ^P<0.05. Detailed implementation mode

[0019] The present invention will be described in detail below with reference to specific embodiments.

[0020] Example 1 Preparation of the extract composition of Euonymus japonicus Thunb. fruit and Rosa multiflora Thunb. fruit

[0021] Take Euonymus japonicus Thunb. fruit, dry in the sun, crush, place in a round-bottomed flask, add 10 times the amount of water, heat in an electric heating mantle until slightly boiling, keep for 3 h, filter, add 6 times the amount of water to the filter residue, heat in an electric heating mantle until slightly boiling, keep for 2 h, filter, combine the two filtrates, concentrate, add ethanol until the alcohol content is 75%, stand for 24 h, filter to obtain the filtrate, place in a rotary evaporator to recover ethanol until there is no alcohol smell, concentrate, and vacuum dry to obtain the aqueous extract of Euonymus japonicus Thunb. fruit;

[0022] Take Rosa multiflora Thunb. fruit, dry in the sun, crush, place in a round-bottomed flask, add 8 times the amount of 70% ethanol, heat in an electric heating mantle for continuous reflux extraction for 3 h, filter, add 6 times the amount of 70% ethanol to the filter residue, heat in an electric heating mantle for continuous reflux extraction for 2 h, filter, combine the two filtrates, place in a rotary evaporator to recover ethanol until there is no alcohol smell, concentrate, and vacuum dry to obtain the alcohol extract of Rosa multiflora Thunb. fruit;

[0023] Weigh the aqueous extract of Euonymus japonicus Thunb. fruit and the alcohol extract of Rosa multiflora Thunb. fruit according to the mass ratio of 1:2, mix evenly, and obtain an extract composition of Euonymus japonicus Thunb. fruit and Rosa multiflora Thunb. fruit.

[0024] The anti-colorectal cancer effect of the present invention is further elaborated below through experimental examples.

[0025] Experimental example 1 Effect of the extract composition of Euonymus japonicus Thunb. fruit and Rosa multiflora Thunb. fruit on the viability of colorectal cancer RKO cells

[0026] 1. Experimental materials

[0027] Prepare the aqueous extract of Euonymus japonicus Thunb. fruit and the alcohol extract of Rosa multiflora Thunb. fruit respectively according to Example 1, dissolve them with dimethyl sulfoxide (DMSO), and prepare solutions with concentrations of 0.5, 1, 2, 4, 8, 16, and 32 μg / mL; mix the aqueous extract of Euonymus japonicus Thunb. fruit and the alcohol extract of Rosa multiflora Thunb. fruit according to the ratios of 1:1, 1:2, and 2:1 respectively to obtain extract compositions with different ratios.

[0028] Colorectal cancer cells RKO, penicillin-streptomycin solution, DMEM medium, PBS buffer, MTT, and DMSO were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; fetal bovine serum was purchased from Sangon Biotech (Shanghai) Engineering Co., Ltd.

[0029] 2. Test methods

[0030] The cell viability was detected by MTT assay. 3 Cells (cells / well) were seeded in a 96-well plate and cultured at 37°C in a 5% CO2 incubator for 24 hours. Then, 100 μL of various concentrations of holly euonymus fruit water extract, rose hip alcohol extract, or their combination (mass ratios of 1:1, 1:2, and 2:1, respectively) were added to each well. After 24 hours of culture, 20 μL of 0.5% MTT solution was added to each well. Culture was continued for 4 hours before termination. The culture medium was carefully aspirated, and 150 μL of DMSO was added to each well. The plates were shaken at low speed for 10 minutes to fully dissolve the crystals. The optical density (OD) at 490 nm was measured using a full-wavelength microplate reader. DMEM medium was used as a control, and five replicates were set up per well. Relative cell viability (%) was calculated using DMEM medium as a control.

[0031] 3. Test results

[0032] Table 1 shows the effects of different concentrations of holly euonymus fruit water extract, rosehip alcohol extract, or their combined solutions on RKO cell viability. The results showed that either holly euonymus fruit water extract or rosehip alcohol extract alone inhibited RKO cell viability to varying degrees, with the inhibitory effect increasing with increasing concentration, demonstrating a dose-dependent relationship. Mixed holistic extracts of holly euonymus fruit water extract and rosehip alcohol extract at mass ratios of 1:1, 1:2, and 2:1, respectively, significantly increased the inhibitory effect on RKO cell viability compared to either holly euonymus fruit water extract or rosehip alcohol extract alone, suggesting a possible synergistic effect between the two.

[0033] Table 1 Effects of holly euonymus fruit water extract, rose hip alcohol extract and their combination on RKO cell viability (%, x ± SD)

[0034]

[0035] CalcuSyn software was used to calculate the half-maximal inhibitory fraction (IC50) of each drug. Combination analysis was also performed, and the combination index (CI) was calculated. CI values < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively, demonstrating the interactive effects of different combination doses. The results are shown in Tables 2 and 3.

[0036] Table 2 IC50 (μg / ml) of aqueous extract of Euonymus japonicus Thunb. fruit, alcoholic extract of Rosa multiflora Thunb. fruit and their combination against RKO cells

[0037]

[0038] Table 3 Combination index (CI) of aqueous extract of Euonymus japonicus Thunb. fruit and alcoholic extract of Rosa multiflora Thunb. fruit combination against RKO cells

[0039]

[0040] As can be seen from Table 2, after the aqueous extract of Euonymus japonicus Thunb. fruit and the alcoholic extract of Rosa multiflora Thunb. fruit were mixed at a mass ratio of 1:1, 1:2 and 2:1, the IC50 values against RKO cells were 6.84, 3.36 and 5.47 μg / mL respectively, all of which were significantly lower than the IC50 values when using only the aqueous extract of Euonymus japonicus Thunb. fruit or the alcoholic extract of Rosa multiflora Thunb. fruit alone, and the IC50 was the lowest at 1:2. As can be seen from Table 3, when the aqueous extract of Euonymus japonicus Thunb. fruit and the alcoholic extract of Rosa multiflora Thunb. fruit were mixed at a mass ratio of 1:1, the CI values at different concentrations were 1.570, 0.607, 0.380, 0.465, 0.633, 1.149 and 2.107 respectively. It showed a synergistic effect at intermediate concentrations, but an antagonistic effect at high and low concentrations; when the aqueous extract of Euonymus japonicus Thunb. fruit and the alcoholic extract of Rosa multiflora Thunb. fruit were mixed at a mass ratio of 1:2, the CI values at different concentrations were 0.544, 0.412, 0.399, 0.346, 0.545 and 0.977 respectively, showing a synergistic effect at all concentrations; when the aqueous extract of Euonymus japonicus Thunb. fruit and the alcoholic extract of Rosa multiflora Thunb. fruit were mixed at a mass ratio of 2:1, the CI values at different concentrations were 0.936, 0.566, 0.354, 0.458, 0.714 and 1.303 respectively, showing a synergistic effect at medium and low concentrations, but an antagonistic effect at high concentrations. Based on the above results, it was determined that the best synergistic effect was produced when the aqueous extract of Euonymus japonicus Thunb. fruit and the alcoholic extract of Rosa multiflora Thunb. fruit were mixed at a mass ratio of 1:2.

[0041] 4. Conclusion

[0042] When the aqueous extract of Euonymus japonicus Thunb. fruit and the alcoholic extract of Rosa multiflora Thunb. fruit were mixed at a mass ratio of 1:2, they had a synergistic inhibitory effect on RKO cells, with an IC50 of 3.36 μg / mL, which was significantly lower than that when using only the aqueous extract of Euonymus japonicus Thunb. fruit or the alcoholic extract of Rosa multiflora Thunb. fruit alone.

[0043] Experimental Example 2 Effect of the extract combination of Euonymus japonicus Thunb. fruit and Rosa multiflora Thunb. fruit on the migration of colorectal cancer RKO cells

[0044] 1. Experimental materials

[0045] The water extract of Euonymus japonicus Thunb. fruit, the alcohol extract of Rosa canina fruit, and the composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit prepared according to Example 1 were each dissolved in DMSO to prepare a 4 μg / mL solution; penicillin-streptomycin solution, fetal bovine serum, DMEM medium, PBS buffer, paraformaldehyde, crystal violet, and RKO cells were purchased from Beyotime Biotechnology Co., Ltd.

[0046] 2. Test method

[0047] The cell migration ability was detected by scratch assay. RKO cells (4×10 5 cells / well) were seeded in 6-well plates. When the cells were nearly 80 - 90% confluent, a scratch was made on the monolayer cell culture layer with the tip of a 200 μL pipette, and a photograph was taken under an inverted microscope. The damaged monolayer cells were rinsed with PBS, and then 100 μL of the water extract of Euonymus japonicus Thunb. fruit, the alcohol extract of Rosa canina fruit, and the composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit at 4 μg / mL were added respectively. The blank control group was added with an equal volume of medium, and they were incubated in an incubator containing 5% CO2 at 37 °C for 48 h, and a photograph was taken under an inverted microscope. Image J software was used for analysis, with DMEM medium as the control. The migration rate was calculated according to the following formula:

[0048]

[0049] 3. Test results

[0050] The effects of the composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit on the migration ability of colorectal cancer RKO cells are shown in Figure 1 . As shown in the figure, compared with the blank control group, the migration ability of RKO cells in the groups of the water extract of Euonymus japonicus Thunb. fruit, the alcohol extract of Rosa canina fruit, and the composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit decreased compared with the blank control group. Among them, the groups of the alcohol extract of Rosa canina fruit and the composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit had statistical significance (P < 0.05); moreover, compared with the groups of the water extract of Euonymus japonicus Thunb. fruit and the alcohol extract of Rosa canina fruit respectively, the migration ability of RKO cells in the group of the composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit decreased significantly (P < 0.05), indicating that the composition of the water extract of Euonymus japonicus Thunb. fruit and the alcohol extract of Rosa canina fruit at a mass ratio of 1:2 had a synergistic effect and significantly inhibited the migration ability of RKO cells, thus showing anti-colorectal cancer activity.

[0051] 4. Conclusion

[0052] The composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit of the present invention can significantly inhibit the migration of colorectal cancer RKO cells and has the effect of treating colorectal cancer.

[0053] Test Example 3 Effects of the composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit on the invasion ability of colorectal cancer RKO cells

[0054] 1. Test materials

[0055] The water extract of Euonymus japonicus Thunb. fruit, the alcohol extract of Rosa canina fruit, and the extract composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit prepared according to Example 1 were separately dissolved in DMSO to prepare solutions with a concentration of 4 μg / mL; penicillin-streptomycin solution, fetal bovine serum, DMEM medium, PBS buffer, paraformaldehyde, crystal violet, and RKO cells were purchased from Beyotime Biotechnology Co., Ltd.; Matrigel was purchased from BD Company, USA.

[0056] 2. Test methods

[0057] The Transwell method was used to detect the cell invasion ability. 100 μL of Matrigel was added to a 12-well Transwell plate (8 μm, Corning, NY, USA) and incubated in an incubator with 5% CO2 at 37 °C for 6 h. HGC-27 cells (2×10 5 cells / well) were seeded in the upper chamber, and 100 μL of Composition 1, Composition 2, Composition 3, and Composition 4 with a concentration of 0.5 mg / mL were separately added. DMEM complete medium was added to the lower chamber, and the cells were cultured in an incubator with 5% CO2 at 37 °C for 24 h. The non-invaded cells were wiped off, and the cells were fixed with 4% paraformaldehyde for 20 min, washed 3 times with PBS, then stained with 0.5% crystal violet for 20 min, and the crystal violet not bound to the cells was washed off with PBS and dried overnight. Images were taken using an inverted microscope, and the formed crystal violet was dissolved with 33% acetic acid, and the absorbance was measured at a wavelength of 570 nm using a full-wavelength microplate reader. The absorbance represents the amount of cells invading the lower chamber. Using DMEM medium as a control, the cell invasion rate relative to the control group was calculated.

[0058] 3. Test results

[0059] The effects of the extract composition of Euonymus japonicus Thunb. fruit and Rosa canina fruit on the invasion ability of colorectal cancer RKO cells are shown in Figure 2 . As shown in the figure, although the invasion ability of RKO cells in the water extract group of Euonymus japonicus Thunb. fruit and the alcohol extract group of Rosa canina fruit decreased, there was no significant difference compared with the blank control group (P>0.05). However, the invasion ability of RKO cells in the extract composition group of Euonymus japonicus Thunb. fruit and Rosa canina fruit decreased significantly, and there were significant differences compared with the other three groups (P>0.05), suggesting that the composition of the water extract of Euonymus japonicus Thunb. fruit and the alcohol extract of Rosa canina fruit at a mass ratio of 1:2 produced a synergistic effect, significantly inhibiting the invasion ability of RKO cells, thus showing anti-colorectal cancer activity.

[0060] 4. Conclusion

[0061] The extract composition of Euonymus japonicus Thunb. fruits and Rosa multiflora Thunb. fruits of the present invention can significantly inhibit the migration of colorectal cancer RKO cells and has the effect of treating colorectal cancer.

Claims

1. A composition of euonymus japonicus fruit and rose hip extracts, characterized in that, The described Euonymus japonicus Thunb. fruit and Rosa canina fruit extract composition is composed of a water extract of Euonymus japonicus Thunb. fruit and an alcohol extract of Rosa canina fruit in a mass ratio of 1:2; Among them, the preparation method of the water extract of Euonymus japonicus Thunb. fruit is as follows: Take Euonymus japonicus Thunb. fruit, dry it in the sun, crush it, place it in a round-bottomed flask, add 10 times the amount of water, heat it in an electric heating mantle until slightly boiling, keep it for 3 h, filter, add 6 times the amount of water to the filter residue, heat it in an electric heating mantle until slightly boiling, keep it for 2 h, filter, combine the two filtrates, concentrate, add ethanol until the alcohol content is 75%, let it stand for 24 h, filter to obtain the filtrate, place it in a rotary evaporator to recover ethanol until there is no alcohol smell, concentrate, and vacuum dry to obtain the water extract of Euonymus japonicus Thunb. fruit; Among them, the preparation method of the alcohol extract of Rosa canina fruit is as follows: Take Rosa canina fruit, dry it in the sun, crush it, place it in a round-bottomed flask, add 8 times the amount of 70% ethanol, heat it in an electric heating mantle for continuous reflux extraction for 3 h, filter, add 6 times the amount of 70% ethanol to the filter residue, heat it in an electric heating mantle for continuous reflux extraction for 2 h, filter, combine the two filtrates, place it in a rotary evaporator to recover ethanol until there is no alcohol smell, concentrate, and vacuum dry to obtain the alcohol extract of Rosa canina fruit; Weigh the water extract of Euonymus japonicus Thunb. fruit and the alcohol extract of Rosa canina fruit according to the mass ratio of 1:2, mix them evenly to obtain a Euonymus japonicus Thunb. fruit and Rosa canina fruit extract composition.

2. Use of an extract composition of Euonymus japonicus Thunb. fruits and Rosa multiflora Thunb. fruits according to claim 1, characterized in that, The composition can significantly inhibit the proliferation, invasion and migration of colorectal cancer cells and can be used to prepare anti-colorectal cancer drugs.

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