Extractive from fructus penaeae with antitumor activity, its preparation method and application

Abstract

The application discloses an extract of penawau fruits with antitumor activity, and adopts the optimal supercritical CO2 fluid low-temperature extraction technology to extract effective components of the penawau fruits, so that the heat-intolerant active components can be reserved, and the anti-tumor screening experiment result shows that the penawau fruit extract obtained by the application has good anti-proliferation activity on multiple myeloma (MM) cell strain ARP-1 cells, human esophageal cancer cell strain Ec109, human cervical cancer cell strain Hela, human hepatoma cell strain SMMC-7721 and lung adenocarcinoma cell strain A549, especially has good inhibition activity on the MM cell strain ARP1, A549 and Ec109, and can be developed into a potential antitumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of anti-tumor, and particularly relates to an extract of fruits of Fissistigma polyanthum with anti-tumor activity and a preparation method thereof. BACKGROUND

[0002] Fissistigma polyanthum is a small evergreen tree with height of about 10 meters. The trunk is straight, and the bark is grayish brown; the young branches are thick and densely covered with rusty soft hair. The leaves are alternate, leathery, and obliquely lanceolate or elliptic, 8-25 cm long and 3-5 cm wide, with pointed or blunt apex and wedge-shaped base, and entire or sparsely serrate on upper part, and covered with soft hair on upper surface and rusty soft hair on lower surface; the leaf stalk is 2.5-3 cm long and covered with short hair. The conic inflorescence is terminal and covered with rusty soft hair; the flower is small and white; there are 5 sepals and 5 petals; and there are 5 stamens. The drupe is nearly spherical, with inclined base and 5-8 mm in diameter, and the persistent calyx is present. The flowering period is from May to June, and the fruiting period is in August.

[0003] The fruits of Fissistigma polyanthum are used as medicine, and the fruits are collected in autumn when they are mature and dried. Fissistigma polyanthum grows in mountain slopes, streamsides, forest edges, broad-leaved mixed wood or shrubs at an altitude of less than 1500 m, and is distributed in Zhejiang, Jiangxi, Fujian, Hubei, Hunan, Guangdong, Guangxi, Guizhou and Yunnan.

[0004] There is no document reporting the anti-tumor effect of the fruits of Fissistigma polyanthum. The present application studies the anti-tumor activity of the fruits of Fissistigma polyanthum, and develops new clinical uses thereof. SUMMARY

[0005] The present application aims to study the anti-tumor activity of the products of different extraction methods of the fruits of Fissistigma polyanthum, and develop new anti-tumor uses thereof.

[0006] Technical scheme: In order to achieve the above object, the technical scheme adopted by the present application is as follows:

[0007] An extract of the fruits of Fissistigma polyanthum with anti-tumor activity is prepared by the following method.

[0008] The fruits of Fissistigma polyanthum are taken, crushed, and then extracted by supercritical CO2 fluid to obtain the extract.

[0009] As a preferred scheme, the supercritical CO2 fluid extraction conditions of the extract of the fruits of Fissistigma polyanthum with anti-tumor activity are as follows: the flow rate of carbon dioxide is controlled to be 20-30 L / h, the extraction pressure is 20-40 MPa, the extraction temperature is 40-65℃, and the extraction time is 1-3 hours; the pressure of the separation kettle I is 2-6 MPa, and the temperature is 30-45℃; and the pressure of the separation kettle II is 3-6 MPa, and the temperature is 30-40℃.

[0010] As a preferred solution, the extract of the fruits of Penaeum zeylanicum with anti-tumor activity is obtained by supercritical CO2 fluid extraction under the following conditions: controlling the flow of carbon dioxide at 20 L / h, extraction pressure at 30 MPa, extraction temperature at 55℃, and extraction time at 2 hours; the pressure of separation kettle I is 5 MPa and the temperature is 45℃; the pressure of separation kettle II is 5 MPa and the temperature is 40℃.

[0011] The application of the extract of the fruits of Penaeum zeylanicum in the preparation of anti-tumor drugs, especially in the preparation of anti-myeloma, esophageal cancer, cervical cancer, liver cancer and lung cancer drugs.

[0012] A pharmaceutical preparation for treating tumors, which is prepared by mixing the extract of the fruits of Penaeum zeylanicum with a pharmaceutically acceptable carrier. The dosage form of the pharmaceutical preparation includes capsules, tablets, granules, pills, oral liquids or injection liquids.

[0013] An anti-tumor drug, which comprises the extract of the fruits of Penaeum zeylanicum prepared by the present application.

[0014] Advantages: Compared with the prior art, the present application has the following advantages:

[0015] The present application adopts the optimal supercritical CO2 fluid low-temperature extraction technology to extract the effective components of the fruits of Penaeum zeylanicum, so that the heat-intolerant active components can be preserved. The anti-tumor screening experiment results show that the extract of the fruits of Penaeum zeylanicum obtained by the present application has good anti-proliferation activity on the MM cell strain ARP-1 cells, human esophageal cancer cell strain Ec109, human cervical cancer cell strain Hela, human liver cancer cell strain SMMC-7721 and lung adenocarcinoma cell strain A549, especially has good inhibitory activity on the MM cell strain ARP1, A549 and Ec109, and can be developed as a potential anti-tumor drug.

[0016] The extract of the fruits of Penaeum zeylanicum is a natural product, has low adverse reactions and small toxic and side effects, can be prepared into various dosage forms for convenient clinical use, and develops a new clinical use of the fruits of Penaeum zeylanicum. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 IC of the inhibitory effect of the supercritical CO2 fluid extract of the fruits of Penaeum zeylanicum on different tumor cells 50 Bar chart.

[0018] Figure 2 IC of the inhibitory effect of the ethanol extract of the fruits of Penaeum zeylanicum on different tumor cells 50 Bar chart. DETAILED DESCRIPTION

[0019] The present application is further illustrated by the following examples, but the present application is not limited thereto.

[0020] Example 1 Screening of supercritical CO2 extraction process

[0021] In order to further screen out the best extraction process, the present application investigates important factors such as extraction temperature, extraction time, extraction pressure, and carbon dioxide flow rate.

[0022] 1. Effect of extraction pressure on extraction yield

[0023] The present application screens the effect of different extraction pressures on the extraction yield of Boro Suber fruit under the conditions of 50℃, 2 hours of extraction, and a carbon dioxide flow rate of 30L / h, and the specific experimental results are shown in Table 1.

[0024] Table 1 Effect of different extraction pressures on the extraction yield of Boro Suber fruit

[0025] Extraction pressure (MPa) Extraction yield (%) 15 8.5 20 18.4 25 21.2 30 25.4 35 25.5 40 25.7

[0026] As shown by the experimental results in Table 1, the extraction yield of Boro Suber fruit increases with the increase of extraction pressure from 15MPa to 40MPa, and when the pressure reaches 30MPa, the extraction yield basically reaches the highest. Considering the safety and reliability of the equipment, the extraction pressure is selected to be 30MPa.

[0027] 2. Effect of extraction temperature on extraction yield

[0028] The present application screens the effect of different extraction temperatures on the extraction yield of Boro Suber fruit under the conditions of 30MPa, 2 hours of extraction, and a carbon dioxide flow rate of 30L / h, and the specific experimental results are shown in Table 2.

[0029] Table 2 Effect of different extraction temperatures on extraction yield

[0030] Extraction temperature (°C) Extraction yield (%) 45 19.1 50 25.7 55 27.5 60 26.3 65 25.0

[0031] As shown by the experimental results in Table 2, when the temperature reaches 55℃, the extraction yield is the highest, and when the temperature continues to rise, the extraction yield has a decreasing trend, which indicates that high temperature has a certain effect on the heat-sensitive products of Boro Suber fruit. Therefore, the preferred extraction temperature is 55℃.

[0032] 3. Effect of extraction time on extraction yield

[0033] The present application screens the effect of different extraction times on the extraction yield of Boro Suber fruit under the conditions of 30MPa, 55℃, and a carbon dioxide flow rate of 30L / h, and the specific experimental results are shown in Table 3.

[0034] Table 3 Effect of different extraction times on extraction yield

[0035] Extraction time (hours) Extraction yield (%) 0.5 10.4 1 16.3 2 28.0 3 28.5

[0036] As shown by the experimental results in Table 3, the extraction yield gradually increases with the extension of time, and reaches a high value after the extraction time is 2 hours, and therefore, the extraction time is preferably 2 hours in consideration of the economic cost and extraction efficiency.

[0037] 4, Influence of carbon dioxide flow on extraction yield

[0038] The present application screens the influence of different carbon dioxide flows on the extraction yield of the fruit of Broussonetia papyrifera under the conditions of an extraction pressure of 30 MPa, an extraction temperature of 55 DEG C and an extraction time of 2 hours, and the specific experimental results are shown in Table 4.

[0039] Table 4 Influence of different extraction times on extraction yield

[0040] Different carbon dioxide flow (L / h) Extraction yield (%) 10 26 20 28 30 28 40 27

[0041] As shown by the experimental results in Table 4, the extraction yield gradually increases and then decreases with the increase of the carbon dioxide flow, and therefore, the carbon dioxide flow is selected as 20 L / h in consideration of the economic cost.

[0042] Through the screening experiments of the above factors, the optimal process of supercritical CO2 extraction of the fruit of Broussonetia papyrifera provided by the present application is that the extraction pressure is 30 MPa, the extraction temperature is 55 DEG C, the extraction time is 2 hours, and the carbon dioxide flow is selected as 20 L / h.

[0043] The present application obtains the optimal supercritical CO2 extraction process through screening, and compared with the traditional ethanol extraction, the supercritical CO2 extraction has the advantage of high extraction efficiency for active ingredients with poor thermal stability.

[0044] Example 2

[0045] Preparation of the extract of the fruit of Broussonetia papyrifera: the fruit of Broussonetia papyrifera is taken, crushed and passed through a 40-mesh sieve, and then placed in a supercritical extraction device (Nantong Huaan HA221-40-48 type supercritical extraction device) for extraction, and the specific conditions of the supercritical CO2 fluid extraction are that the carbon dioxide flow is controlled as 20 L / h, the extraction pressure is 30 MPa, the extraction temperature is 55 DEG C, and the extraction time is 2 hours; the pressure of separation kettle I is 5 MPa, and the temperature is 45 DEG C; the pressure of separation kettle II is 5 MPa, and the temperature is 40 DEG C, and the supercritical CO2 fluid extract of the fruit of Broussonetia papyrifera is obtained.

[0046] Example 3

[0047] Preparation of the extract of the fruit of Broussonetia papyrifera: the fruit of Broussonetia papyrifera is taken, crushed and passed through a 40-mesh sieve, and then placed in a supercritical extraction device (Nantong Huaan HA221-40-48 type supercritical extraction device) for extraction, and the specific conditions of the supercritical CO2 fluid extraction are that the carbon dioxide flow is controlled as 20 L / h, the extraction pressure is 30 MPa, the extraction temperature is 55 DEG C, and the extraction time is 2 hours; the pressure of separation kettle I is 5 MPa, and the temperature is 45 DEG C; the pressure of separation kettle II is 5 MPa, and the temperature is 40 DEG C, and the supercritical CO2 fluid extract of the fruit of Broussonetia papyrifera is obtained.

[0048] Experiment of anti-tumor activity in vitro of Example 4

[0049] 1. Experimental materials

[0050] Tumor cell lines: multiple myeloma (MM) cell line ARP-1 cells, human esophageal cancer cell line Ec109, human cervical cancer cell line Hela, human hepatocarcinoma cell line SMMC-7721, lung adenocarcinoma cell line A549.

[0051] Test drugs: the supercritical CO2 fluid extract of the fruits of Piliocalyx benguetensis prepared in Example 2 and the ethanol extract of the fruits of Piliocalyx benguetensis prepared in Example 3.

[0052] 96-well plates were purchased from Costar Company; RPMI1640 medium was purchased from Gibco Company; DMEM medium was purchased from Gibco Company; thiazole blue MTT was purchased from Amresco Company. Newborn bovine serum was purchased from Hangzhou Sijiqing Company.

[0053] The experimental instruments used in the following examples are as follows:

[0054] Experimental instrument: BIO-RAD Bole 680 enzyme marker.

[0055] 2. Experimental method

[0056] In the present application, the multiple myeloma MM cell line ARP-1, the human esophageal cancer cell line Ec109, the human cervical cancer Hela, the human hepatocarcinoma SMMC-7721 and the lung adenocarcinoma cell line A549 were digested with trypsin, counted and prepared into cell suspensions with a concentration of 5x10 4 cells / ml.

[0057] 100 μl of the cell suspension was added to each well of the 96-well plate, 3 replicate wells were set for each group, the test drugs were diluted with incomplete medium to the required different gradient concentrations, 100 μL of the corresponding drug-containing medium was added to each well, a negative control group without the addition of drugs was set up, and incubation was carried out in a 37℃, 5% CO2 incubator for 48 h; MTT reagent (5 mg / ml) was freshly prepared and added to each well, and incubation was continued in a 37℃, 5% CO2 incubator for 4 h; centrifugation was carried out in a centrifuge, 150 μL of DMSO was added to each well; then the OD of each well at 570 nm wavelength was determined by an enzyme marker, and after data processing, the growth curve of the cells was drawn with the concentrations of each group as the horizontal coordinate and the cell survival rate as the vertical coordinate, and the IC 50 values of the test drugs of each group were calculated by the two-point method (Reed and Muench method). The specific experimental results are shown in Table 5, Figure 1 and Figure 2 Extraction pressure (MPa) Extraction yield (%) Extraction temperature (°C) Extraction yield (%) Extraction time (hours) Extraction yield (%) Different carbon dioxide flow (L / h) Extraction yield (%)

[0058] ​Table 1 shows the inhibition (IC 50 values)

[0059]

[0060] The experimental results of Table 1 above show that the supercritical CO2 fluid extract has good anti-proliferation activity on the multiple myeloma (MM) cell line ARP-1 cells, human esophageal cancer cell line Ec109, human cervical cancer cell line Hela, human hepatoma cell line SMMC-7721, and lung adenocarcinoma cell line A549, especially on the MM cell line ARP1, A549 and Ec109, and has the potential to be developed as an anti-tumor drug.

Claims

1. An extract of *Prunus armeniaca* fruit with antitumor activity, characterized in that, The extract was prepared by the following method: Take the fruit of the *Philodendron chinense*, crush it, and then extract it using supercritical CO2 fluid to obtain the extract; The supercritical CO2 fluid extraction conditions are as follows: the carbon dioxide flow rate is controlled at 20~30 L / h, the extraction pressure is 20~40 MPa, the extraction temperature is 40~65℃, and the extraction time is 1~3 hours; the pressure of separation vessel I is 2~6 MPa, and the temperature is 30~45℃; the pressure of separation vessel II is 3~6 MPa, and the temperature is 30~40℃.

2. The extract of *Prunus armeniaca* fruit with antitumor activity according to claim 1, characterized in that, The supercritical CO2 fluid extraction conditions were as follows: CO2 flow rate was controlled at 20 L / h, extraction pressure at 30 MPa, extraction temperature at 55℃, and extraction time at 2 hours; the pressure of separation vessel I was 5 MPa and the temperature was 45℃; the pressure of separation vessel II was 5 MPa and the temperature was 40℃.

3. The use of the extract of the fruit of *Prunus armeniaca* as described in claim 1 or 2 in the preparation of a medicament for treating myeloma, esophageal cancer, cervical cancer, liver cancer, or lung cancer.

4. An antitumor pharmaceutical preparation, characterized in that, It is prepared by combining the extract of the fruit of the *Prunus armeniaca* as described in claim 1 with a pharmaceutically acceptable carrier.

5. The pharmaceutical preparation according to claim 4, characterized in that, The dosage forms of the pharmaceutical preparations include capsules, tablets, granules, pills, oral liquids, or injections.

6. An antitumor drug, characterized in that, Including the extract of the fruit of the *Philodendron* as described in claim 1 or 2.

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