CN106867674B - Extraction, purification and detection method of bean curd vegetable volatile oil and application thereof - Google Patents
Extraction, purification and detection method of bean curd vegetable volatile oil and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/027—Recovery of volatiles by distillation or stripping
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/85—Verbenaceae (Verbena family)
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/022—Refining
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/025—Recovery by solvent extraction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Alternative & Traditional Medicine (AREA)
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- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Botany (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
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- General Physics & Mathematics (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses an extraction, purification and detection method of bean curd vegetable volatile oil and application thereof. The invention adopts the steam distillation and organic extraction technology to extract and purify the bean curd vegetable volatile oil for the first time, the extraction and purification method has simple process and low cost, the distilled water and the organic solvent in the preparation process can be repeatedly utilized, the volatile oil obtained by the preparation process has low water content, and simultaneously has good antioxidant, bacteriostatic and anticancer activities through test verification, has good bacteriostatic action on staphylococcus aureus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa, has good anticancer and cancer suppression effects on HepG2 (human liver cancer cells) and MCF-7 (human breast cancer cells), and is suitable for industrial production and application.
Description
Technical Field
The invention belongs to the technical field of research and development of medical products, and particularly relates to an extraction, purification and detection method of bean curd vegetable volatile oil and application thereof.
Background
Bean curd vegetable (Premna microphylla Turcz) belongs to Verbenaceae, genus Premna, and is called as herba Premnae, Premna microphylla, folium Premna microphylla and radix et rhizoma Rhei Lijiangensis, and root, stem and leaf can be used as medicine for clearing away heat and toxic materials, relieving swelling and arresting bleeding. It can be used for treating venomous snake bite, undefined lump, and traumatic hemorrhage. The ecological water-saving agent is widely distributed in China, is mainly distributed in areas of south of Yangtze river basin including east China, south China, Sichuan China, Guizhou and the like, and is suitable for hillsides, forest edges, forest thinning, irrigation clusters on two sides of brook ditches and roads below the elevation of 1000 m.
In the existing research reports, the leaves and twigs of the bean curd vegetable contain a large amount of pectin, protein and vitamins, and the contents of total sugar and reducing sugar are also high.
Volatile oils (also known as essential oils) are a generic term for water-immiscible oily liquids that can be volatilized at ambient temperature and distilled with steam. Most essential oils have a fragrant odor. The volatile oil is an important active component, and can be directly applied to crude drugs mainly containing the volatile oil clinically, and also can be applied to volatile oil refined from the crude drugs, such as eucalyptus oil, peppermint oil and the like. The volatile oil has effects of dispersing exterior syndrome, inducing resuscitation with aromatics, regulating qi-flowing, relieving pain, dispelling pathogenic wind, removing dampness, promoting blood circulation, removing blood stasis, dispelling cold, warming interior, clearing away heat and toxic materials, relieving summer-heat, removing dirt, killing parasites, and resisting bacteria, such as oleum Menthae Dementholatum for dispelling pathogenic wind, invigorating stomach, radix Angelicae sinensis oil for relieving pain, bupleuri radix oil for relieving fever, and herba Chenopodii oil for expelling intestinal parasites, and can be widely used in production of perfume, food and cosmetic.
Therefore, in order to further expand the application field of the bean curd vegetable, the research on the bean curd vegetable volatile oil is necessary. However, the prior art has not reported the bean curd vegetable volatile oil.
Disclosure of Invention
Aiming at the prior art, the invention provides a separation and extraction method of the bean curd vegetable volatile oil and application of the bean curd vegetable volatile oil.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting and purifying bean curd vegetable volatile oil comprises the following steps:
(1) cleaning fresh bean curd vegetable, air drying, pulverizing to obtain powder with particle size of 50-100 mesh, adding distilled water, stirring, and steam distilling to obtain water system containing volatile oil;
(2) adding organic solvent into the volatile oil-containing water system for extraction, discarding the separated water layer, adding dehydration drying agent into the volatile oil-containing organic solvent for dehydration, filtering and recovering the organic solvent to obtain dry and pure volatile oil.
Further, the mass ratio of the cleaned and aired tofu and the distilled water is 1: 1-3, more preferably 1: 2; the crushing particle size of the bean curd vegetable is preferably 60 meshes; the inventor finds in experimental study that the larger size of the crushed particles causes water vapor to flow out from the gaps of the raw materials, thereby reducing the extraction rate and wasting resources; the crushed particle size is too small, so that the steam cannot uniformly penetrate through the raw materials, the steam escapes from the flushed raw materials, and the extraction rate is low;
further, the steam distillation extraction time is 5-6 h; the proper extraction time can ensure that the volatile oil component is extracted from the bean curd vegetable as far as possible, and simultaneously, the resource waste caused by overlong extraction time is also avoided;
further, the organic solvent is selected from one or more of ethyl acetate, ethyl acrylate, toluene, diethyl ether and chloroform, preferably diethyl ether; the applicant finds that the diethyl ether has the best extraction effect on the volatile oil components of the bean curd vegetable and has a better extraction rate compared with other organic solvents;
further, the dehydration drying agent is selected from anhydrous sodium sulfate or anhydrous calcium sulfate, and is preferably anhydrous sodium sulfate;
the extraction and purification method of the bean curd vegetable volatile oil comprises the following optimal processes:
(1) cleaning fresh bean curd vegetable, air drying, pulverizing to obtain powder with particle size of 60 mesh, adding distilled water, stirring, and performing steam distillation for 5 hr to obtain water system containing volatile oil; wherein the mass ratio of the cleaned and aired tofu and the distilled water is 1: 2;
(2) adding ether into the volatile oil-containing water system for extraction, discarding the separated water layer, adding anhydrous sodium sulfate into the volatile oil-containing ether solution for dehydration, filtering and recovering ether to obtain dry and pure volatile oil.
The invention also discloses the bean curd vegetable volatile oil prepared by the extraction and purification method.
In addition, the invention provides a detection method of the bean curd vegetable volatile oil prepared by the extraction and purification method, which comprises the following steps of analyzing and detecting by adopting a gas chromatography-mass spectrometry (GC-MS), wherein the detection conditions are as follows: HP-5MS column (30 m.times.0.25 mm.times.0.25 μm); carrier gas: helium gas; flow rate: 1.2mL/min, solvent delay 4 min; temperature rising procedure: the initial temperature is 60 deg.C, holding for 1min, heating to 200 deg.C at 15 deg.C/min, holding for 5min, heating to 280 deg.C at 5 deg.C/min, and holding for 2 min. An ionization mode: EI, 70 eV; ion source temperature: 250 ℃; carrier gas: helium gas; column flow rate: 1.2 mL/min.
Wherein the chemical components of the helichrysum volatile oil comprise bloom C, cedrene, carene, alpha-guaifene, cryptopine and isolongifol.
The invention also discloses application of the bean curd vegetable volatile oil, wherein the bean curd vegetable volatile oil is used as an antioxidant to be applied to food, medicines, cosmetics or health products;
the invention also discloses application of the bean curd vegetable volatile oil, wherein the bean curd vegetable volatile oil is used as a bacteriostatic agent to be applied to foods, medicines, cosmetics or health-care products; further, the bean curd vegetable volatile oil is used as a bacteriostatic agent for staphylococcus aureus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa.
Finally, the invention discloses the application of the bean curd vegetable volatile oil, wherein the bean curd vegetable volatile oil is applied to a medicine as an anticancer cell agent; further, the bean curd vegetable volatile oil is used as an anticancer cell agent of HepG2 (human liver cancer cell) and MCF-7 (human breast cancer cell).
The invention has the beneficial effects that: the invention adopts the steam distillation and organic extraction technology to extract and purify the bean curd vegetable volatile oil for the first time, the extraction and purification method has simple process and low cost, the distilled water and the organic solvent in the preparation process can be repeatedly utilized, the volatile oil obtained by the preparation process has low water content, and simultaneously has good antioxidant, bacteriostatic and anticancer activities through test verification, has good bacteriostatic action on staphylococcus aureus, bacillus subtilis, escherichia coli and pseudomonas aeruginosa, has good anticancer and cancer suppression effects on HepG2 (human liver cancer cells) and MCF-7 (human breast cancer cells), and is suitable for industrial production and application.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, the tofu vegetable is rich in nutrition in the prior art, so that in order to further expand the application field of tofu vegetable, the study on the tofu vegetable volatile oil is necessary, however, no report on the tofu vegetable volatile oil is reported in the prior art.
In a typical embodiment of the present application, there is provided a method for extracting and purifying a bean curd vegetable volatile oil, comprising the steps of:
(1) cleaning fresh bean curd vegetable, air drying, pulverizing to obtain powder with particle size of 50-100 mesh, adding distilled water, stirring, and steam distilling to obtain water system containing volatile oil;
(2) adding organic solvent into the volatile oil-containing water system for extraction, discarding the separated water layer, adding dehydration drying agent into the volatile oil-containing organic solvent for dehydration, filtering and recovering the organic solvent to obtain dry and pure volatile oil.
Wherein the mass ratio of the cleaned and aired tofu and the distilled water is 1: 1-3, more preferably 1: 2; the crushing particle size of the bean curd vegetable is preferably 60 meshes; the inventor finds in experimental study that the larger size of the crushed particles causes water vapor to flow out from the gaps of the raw materials, thereby reducing the extraction rate and wasting resources; the crushed particle size is too small, so that the steam cannot uniformly penetrate through the raw materials, the steam escapes from the flushed raw materials, and the extraction rate is low;
the steam distillation extraction time is 5-6 h; the proper extraction time can ensure that the volatile oil component is extracted from the bean curd vegetable as far as possible, and simultaneously, the resource waste caused by overlong extraction time is also avoided;
the organic solvent is selected from one or more of ethyl acetate, ethyl acrylate, toluene, diethyl ether and chloroform, and diethyl ether is preferred; the applicant finds that the diethyl ether has the best extraction effect on the volatile oil components of the bean curd vegetable and has a better extraction rate compared with other organic solvents;
the dehydration drying agent is selected from anhydrous sodium sulfate or anhydrous calcium sulfate, and is preferably anhydrous sodium sulfate;
in another exemplary embodiment of the present invention, a method for extracting and purifying a bean curd vegetable volatile oil is provided, which comprises:
(1) cleaning fresh bean curd vegetable, air drying, pulverizing to obtain powder with particle size of 60 mesh, adding distilled water, stirring, and performing steam distillation for 5 hr to obtain water system containing volatile oil; wherein the mass ratio of the cleaned and aired tofu and the distilled water is 1: 2;
(2) adding ether into the volatile oil-containing water system for extraction, discarding the separated water layer, adding anhydrous sodium sulfate into the volatile oil-containing ether solution for dehydration, filtering and recovering ether to obtain dry and pure volatile oil.
In another exemplary embodiment of the present invention, a method for detecting the bean curd vegetable volatile oil prepared by the above extraction and purification method is provided, the method comprises analyzing and detecting by gas chromatography-mass spectrometry (GC-MS), and the detection conditions are as follows: HP-5MS column (30 m.times.0.25 mm.times.0.25 μm); carrier gas: helium gas; flow rate: 1.2mL/min, solvent delay 4 min; temperature rising procedure: the initial temperature is 60 deg.C, holding for 1min, heating to 200 deg.C at 15 deg.C/min, holding for 5min, heating to 280 deg.C at 5 deg.C/min, and holding for 2 min. An ionization mode: EI, 70 eV; ion source temperature: 250 ℃; carrier gas: helium gas; column flow rate: 1.2 mL/min.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
Example 1 extraction and purification method of essential oil of Helianthus tuberosus
(1) Cleaning 500g of fresh bean curd vegetable, air drying, crushing to obtain 60-mesh powder, adding 1000g of distilled water, stirring, and performing steam distillation for 5 hours to obtain a water system containing volatile oil;
(2) adding ether into the volatile oil-containing water system for extraction, discarding the separated water layer, adding anhydrous sodium sulfate into the volatile oil-containing ether solution for dehydration, filtering and recovering ether to obtain dry and pure volatile oil.
Example 2 detection method of Helianthus tuberosus volatile oil
GC-MS is adopted to detect the bean curd vegetable volatile oil obtained in the example 1, and the specific detection conditions are as follows: HP-5MS column (30 m.times.0.25 mm.times.0.25 μm); carrier gas: helium gas; flow rate: 1.2mL/min, solvent delay 4 min; temperature rising procedure: the initial temperature is 60 deg.C, holding for 1min, heating to 200 deg.C at 15 deg.C/min, holding for 5min, heating to 280 deg.C at 5 deg.C/min, and holding for 2 min. An ionization mode: EI, 70 eV; ion source temperature: 250 ℃; carrier gas: helium gas; column flow rate: 1.2 mL/min. 49 chemical components in the bean curd vegetable volatile oil are detected together, and the specific chemical components and the relative content are shown in table 1.
TABLE 1 Bean curd vegetable volatile oil chemical composition
Example 3 antioxidant Effect test of Bean curd vegetable volatile oil
1 preparation of DPPH test solution 0.08mmol/L preparation of DPPH solution: accurately weighing DPPH8.0mg, dissolving with absolute ethanol, and diluting to a constant volume of 200ml in a brown volumetric flask to obtain DPPH solution with the concentration of 0.004%, and storing in dark for later use.
2, experimental steps: taking 1.0ml of each sample solution (0.24,0.48,0.72,0.96 and 1.20mg/ml) with different concentrations, respectively, putting the sample solutions into a 10ml centrifuge tube, adding 3.0ml of DPPH solution, reacting for 30min at room temperature in a dark place, and measuring the light absorption value at the wavelength of 517nm by taking absolute ethyl alcohol as a blank. The DPPH radical clearance was calculated according to the following formula. DPPH radical scavenging (%). The formula for A0- (As-Ac)/A0X 100% is shown in the following, where the absorbance values for A0-1.0 ml of distilled water +3.0ml of DPPH solution, As-, 1.0ml of sample solution +3.0ml of DPPH solution, Ac-, 1.0ml of sample solution +3.0ml of absolute ethanol, are repeated three times to obtain the average value of the scavenging rates.
The antioxidant activity of the essential oils was determined using a DPPH basic protocol. The radical scavenging activity of the essential oil was measured by a spectrophotometer at 517 nm. A solution of DPPH in methanol was prepared immediately before the measurement. 2mL of each sample (0.100,0.150,0.200,0.250,0.300,0.350,0.400,0.450,0.500mg/mL) at various concentrations was added to 2mL of DPPH solution. The reaction mixture was shaken and allowed to stand in the shade for 30 minutes. The absorbance of the sample was measured by a spectrophotometer at 517 nm. In this assay, Butylated Hydroxytoluene (BHT) was used as a standard antioxidant to validate the assay. The experiment was repeated three times.
The results are expressed as DPPH radical scavenging rates, as shown in Table 2.
TABLE 2 antioxidant action of Helianthus tuberosus volatile oil
As can be seen from Table 2, the essential oil of Helianthus tuberosus has a good scavenging effect on free radicals.
The specific implementation example shows that the bean curd vegetable volatile oil can be used as a natural antioxidant in the industries of food, medicines and health products or cosmetics to replace a synthetic antioxidant with toxic and side effects.
Example 4 bacteriostatic action of Helianthus tuberosus volatile oil
Test strains and culture Medium
Test strains: four bacterial strains were selected for bacteriostatic testing, including gram-positive bacteria: staphylococcus aureus (ATCC 6538), bacillus subtilis (ATCC 6633), and gram-negative bacteria: escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853).
Test medium: bacteria MHB (Mueller Hinton Broth) medium.
2 test method
(1) And (3) sterilization:
and (3) performing damp-heat sterilization on a culture medium, a test tube, a gun head, distilled water and the like required by the experiment at 121 ℃ for 20min, and placing the sterilized culture medium, the test tube, the gun head, the distilled water and the like on an ultra-clean workbench for ultraviolet sterilization for 30 min.
(2) Preparing a bacterial suspension:
from the beginning to the end of the passageSelecting a ring of bacteria from the slant of the transformed bacteria, inoculating the bacteria into a corresponding MHB culture medium, and placing the MHB culture medium into a constant temperature shaking table for overnight culture at 37 ℃. 0.5mL of the above cultured test bacterial solution in logarithmic phase was aspirated, and diluted with MH broth to obtain a bacterial suspension of 0.5 McLeod standard. Diluting the bacterial suspension with MH broth at a ratio of 1: 100 to obtain 1-2X 106CFU/ml for use.
(3) Determination of MIC:
the diluted bacterial liquid is sucked and added into a 96-hole cell culture plate, each hole is 100 mu L, and 100 mu L of prepared medicines with different concentrations are added. The penultimate well was a control well for bacterial growth with no drug (medium and bacteria only) and 100. mu.L of diluted bacteria added. The last column is the positive control chloramphenicol. Placing the mixture on an oscillator for oscillation for 1min to fully and uniformly mix the solution in the holes, shaking a 96-hole plate uniformly, placing the plate in a constant temperature incubator at 37 ℃ for culturing for 16-18 hours, adding 20 mu L of 1% TTC (2,3, 5-triphenyltetrazolium chloride) solution prepared by sterile water into each hole, shaking the plate uniformly, culturing in the constant temperature incubator at 37 ℃ for 30-60 minutes, and observing the color change of each hole. And taking the concentration of the volatile oil contained in the last hole which does not show color change as the minimum inhibitory concentration MIC of the volatile oil.
Measurements of all MIC values were repeated in triplicate.
3 results of the experiment
The bacteriostatic effect is expressed by the minimum bacteriostatic concentration MIC, and the measurement result is shown in Table 3.
TABLE 3 bacteriostatic activity of essential oil of bean curd vegetable
As can be seen from Table 3, the volatile oil of Eucheuma Gelatinosum has certain inhibitory effect on 4 test bacteria, the minimum inhibitory concentration on Staphylococcus aureus is 0.270mg/mL, the minimum inhibitory concentration on Bacillus subtilis is 0.270mg/mL, the minimum inhibitory concentration on Escherichia coli is 0.150mg/mL, and the minimum inhibitory concentration on Pseudomonas aeruginosa is 0.370 mg/mL.
The specific implementation example shows that the bean curd vegetable volatile oil can be used as a natural antibacterial agent in the industries of food, medicines and health products or cosmetics to replace a synthetic antibacterial agent with toxic and side effects.
Example 5 cytotoxicity of Helianthus tuberosus oil
1 cell culture
HepG2 (human liver cancer cell) and MCF-7 (human breast cancer cell) cell lines were cultured in RPMI1640 medium containing 10% newborn calf serum and 100U/mL each of penicillin and streptomycin at 37 ℃ in a saturated humidity incubator with 5% CO 2.
2MTT method for detecting cell viability
Suspending tumor cells in logarithmic growth phase in RPMI-1640 medium containing 10% fetal bovine serum and 2mM glutamine, gently blowing the suspension with a glass pipette to form a single cell suspension, and counting the suspension with a TC10 automatic cell counter. Inoculating cells into 96-well culture plate with cell density of 5 × 103200. mu.L/mL per well. After the cells were adhered by pre-culturing in an incubator containing 5% CO2 at 37 ℃ for 24 hours, the tumor cells were treated with the previously prepared volatile oil solutions at concentrations of 0.016, 0.031, 0.063, 0.125, 0.250, 0.500, 1 and 2mg/mL, respectively. And setting negative control and blank control. Then continuously culturing for 24h, 48h and 72h respectively, and then determining by using an MTT method. mu.L of 5mg/mL MTT solution was added to each well, and after further incubation for 2h, the supernatant was aspirated. mu.L of DMSO was added to each well, the crystals were completely dissolved by shaking with a micro-shaker for 10min, and the cell proliferation inhibition rate was calculated by measuring the absorbance (A) value at a wavelength of 570nm in a Multiskan Ascent microplate reader in triplicate. The cell proliferation inhibition rate was calculated as follows.
Inhibition of cell proliferation/% ([ 1-OD)Treatment group/ODControl]×100％
3 results of the experiment
Cytotoxicity was expressed as the concentration of the sample required to inhibit 50% of cell growth, i.e. (IC50 value), and the results of measurement are shown in tables 4 and 5.
TABLE 4 cytotoxic Activity of Helianthus tuberosus oil on HepG2
TABLE 5 Bean curd volatile oil for MCF-7 cytotoxic Activity
As can be seen from tables 4-5, the bean curd vegetable volatile oil has certain inhibition effect on both HepG2 and MCF-7 cells, and the IC50 of the bean curd vegetable volatile oil is 0.315mg/mL for HepG 248 hours, and the IC50 of the bean curd vegetable volatile oil is 0.072mg/mL for 48 hours; IC50 was 0.751mg/mL for MCF-748 hr and IC50 was 0.188mg/mL for 48 hr.
The specific implementation example shows that the bean curd vegetable volatile oil can be used as a natural anticancer cell agent in the pharmaceutical industry.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (6)
1. The application of the bean curd vegetable volatile oil in preparing an anticancer cell agent is characterized in that the anticancer cell agent is applied to a medicine, and the anticancer cell agent is an anticancer cell agent of human liver cancer cells and human breast cancer cells; the bean curd vegetable volatile oil comprises bloom C, cedrene, carene, alpha-guaifene, cryptopine and isolongifol;
the extraction and purification of the bean curd vegetable volatile oil comprises the following steps:
(1) cleaning fresh bean curd vegetable, air drying, pulverizing to obtain powder with particle size of 50-100 mesh, adding distilled water, stirring, and steam distilling to obtain water system containing volatile oil;
(2) adding an organic solvent into the volatile oil-containing water system for extraction, discarding a separated water layer, adding a dehydration drying agent into the volatile oil-containing organic solvent for dehydration, and filtering and recovering the organic solvent to obtain dry and pure volatile oil;
the mass ratio of the cleaned and aired tofu vegetable to the distilled water is 1: 1-3;
the organic solvent is selected from one or more of ethyl acetate, ethyl acrylate, toluene, diethyl ether and chloroform;
the dehydration drying agent is selected from anhydrous sodium sulfate or anhydrous calcium sulfate.
2. The use of the essential oil of yucca schidigera as set forth in claim 1 for preparing an anticancer agent, wherein the yucca schidigera has a ground particle size of 60 mesh.
3. The application of the bean curd vegetable volatile oil in preparing the anticancer cell agent as claimed in claim 1, wherein the mass ratio of the cleaned and aired bean curd vegetable to the distilled water is 1: 2.
4. the use of the essential oil of gelidium amansii as claimed in claim 1, wherein the organic solvent is diethyl ether.
5. The use of the essential oil of Helianthus tuberosus L.as claimed in claim 1, wherein the dehydrating drying agent is anhydrous sodium sulfate.
6. The use of the essential oil of yucca schidigera as claimed in claim 1 in the preparation of an anticancer agent, wherein the extraction and purification of the essential oil of yucca schidigera comprises the following steps:
(1) cleaning fresh bean curd vegetable, air drying, pulverizing to obtain powder with particle size of 60 mesh, adding distilled water, stirring, and performing steam distillation for 5 hr to obtain water system containing volatile oil; wherein the mass ratio of the cleaned and aired tofu and the distilled water is 1: 2;
(2) adding ether into the volatile oil-containing water system for extraction, discarding the separated water layer, adding anhydrous sodium sulfate into the volatile oil-containing ether solution for dehydration, filtering and recovering ether to obtain dry and pure volatile oil.
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