Liquorice seed oil as well as preparation method and application thereof

Abstract

The invention provides liquorice seed oil as well as a preparation method and application thereof, and relates to the field of vegetable oil extraction. The preparation method of the licorice seed oil comprises the following steps: crushing licorice seeds to obtain licorice seed powder; extracting the licorice seed powder with n-hexane, and concentrating under reduced pressure to obtain crude oil; and refining the crude oil to obtain the licorice seed oil. The invention further provides application of the liquorice seed oil in preparation of anti-tumor or anti-cancer drugs, preparation of health-care foods or drugs with anti-inflammatory activity and preparation of health-care foods or drugs with anti-oxidation activity. The method is of great significance to utilization and popularization of liquorice seed resources.

Description

Technical Field

[0001] The invention relates to the field of plant oil extraction, and in particular to liquorice seed oil and a preparation method and application thereof. Background Art

[0002] Licorice belongs to the Leguminosae family and is a traditional Chinese medicine with a long history of clinical use. The medicinal parts of licorice are the roots and rhizomes, which have the functions of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, moistening the lungs and relieving cough, relieving acute pain and harmonizing various medicines. It is known as the "king of all medicines". The roots and stems of licorice are rich in a variety of active ingredients (such as triterpenes, flavonoids, polysaccharides, organic acids, etc.), and have a wide range of pharmacological activities (such as anti-inflammatory, antiviral, antibacterial, anti-tumor, antioxidant, etc.). At present, the research on the chemical composition and pharmacological activity of licorice is relatively in-depth, but there is little research on licorice seeds.

[0003] There are few methods for extracting licorice seed oil in the prior art, and no research has been conducted on its possible effective ingredients and activities. Therefore, the value of licorice seeds is still unclear and needs to be developed and studied. Summary of the invention

[0004] Therefore, the purpose of the present invention is to provide a method for preparing liquorice seed oil with biological activity, and to study its corresponding activity and use.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing liquorice seed oil, comprising the following steps:

[0007] (1) crushing liquorice seeds to obtain liquorice seed powder;

[0008] (2) extracting the licorice seed powder with n-hexane and concentrating under reduced pressure to obtain a crude oil;

[0009] (3) refining the crude oil to obtain liquorice seed oil.

[0010] Furthermore, in step (2), the licorice seed powder is loaded into a Soxhlet extractor, extracted with n-hexane by reflux for 2 to 3 times, the n-hexane extracts are combined, and concentrated under reduced pressure to obtain a crude oil.

[0011] Furthermore, in step (3), the crude oil is passed through a neutral alumina column, eluted with n-hexane, the eluate is collected, and concentrated under reduced pressure to obtain licorice seed oil.

[0012] Furthermore, in step (1), the particle size of the licorice seed powder is 20 to 70 meshes; preferably, the particle size of the licorice seed powder is 30 to 60 meshes.

[0013] Furthermore, in step (2), the sample amount of the licorice seed powder is 50-200 g, the amount of n-hexane is 500-1000 mL; the temperature of a single Soxhlet extraction is 30-80° C., and the time is 30-120

[0014] min; concentrated under reduced pressure to 1% of the original volume; preferably, the sample amount of the licorice seed powder is 200 g, and the amount of n-hexane is 1000 mL; the temperature of a single Soxhlet extraction is 80° C., and the time is 120 min.

[0015] Furthermore, in step (3), the neutral alumina column selects a filler with a particle size of 80 to 120 mesh, and the usage amount is 30 to 60 g; the inner diameter of the column tube of the neutral alumina column is 10 to 20 mm, and the column height after filling is 5 to 10 cm; the column loading method is: dry loading; the loading amount of crude oil is 3 to 8 mL; the amount of n-hexane is 500 to 100 mL; the n-hexane flow rate is 1 to 2 mL / min; preferably, the usage amount of the neutral alumina column filler is 50 g; the inner diameter of the column tube of the neutral alumina column is 15 mm, and the column height after filling is 5 cm; the loading amount of crude oil is 5 mL; the amount of n-hexane is 500 mL; the n-hexane flow rate is 1 mL / min.

[0016] In a second aspect, the present invention provides liquorice seed oil obtained by the preparation method.

[0017] In a third aspect, the present invention provides a licorice seed oil, wherein the acid value of the licorice seed oil is 0.066±0.003 mg / g; the peroxide value is 7.267±0.023 g / 100 g; the iodine value is 37.231±0.016 g / 100 g; and the saponification value is 186.823±1.818 mg / g.

[0018] Furthermore, the trace elements in the licorice seed oil include: V 0.025μg / g, Cr 0.299μg / g, Mn0.369μg / g, Fe 5.520μg / g, Co 0.025μg / g, Ni 0.191μg / g, Cu 0.316μg / g, Zn 6.434μg / g, As0.024μg / g, Se 0.012μg / g, and Mo 0.015μg / g.

[0019] In a fourth aspect, the present invention provides the licorice seed oil obtained by the preparation method or the use of the licorice seed oil in the preparation of anti-tumor or anti-cancer drugs.

[0020] Furthermore, the anti-tumor or anti-cancer drug is an anti-lung adenocarcinoma drug or an anti-cervical cancer drug.

[0021] In a fifth aspect, the present invention provides licorice seed oil obtained by the preparation method or use of the licorice seed oil in the preparation of health food or medicine with anti-inflammatory activity.

[0022] In a sixth aspect, the present invention provides licorice seed oil obtained by the preparation method or use of the licorice seed oil in the preparation of health food or medicine with antioxidant activity.

[0023] The technical solution of the present invention has the following advantages:

[0024] The preparation method of licorice seed oil provided by the invention comprises the following steps: firstly extracting oil from licorice seed powder by using n-hexane as an extraction solvent (such as using a Soxhlet extraction method) to obtain a crude oil, and then further refining the obtained crude oil (such as passing through a neutral alumina column and eluting with n-hexane to further remove impurities such as waxy substances and pigments in the crude oil) to improve the purity of the licorice seed oil.

[0025] The licorice seed oil prepared by the preparation method has a linoleic acid content of ≥43%, an oleic acid content of ≥4%, an acid value of 0.066 mg / g, a peroxide value of 7.267 g / 100 g, an iodine value of 37.231 g / 100 g, a saponification value of 186.823 mg / g, contains a large amount of trace elements required by the human body (V 0.025 μg / g, Cr 0.299 μg / g, Mn 0.369 μg / g, Fe 5.520 μg / g, Co 0.025 μg / g, Ni 0.191 μg / g, Cu 0.316 μg / g, Zn 6.434 μg / g, As 0.024 μg / g, Se 0.012 μg / g, Mo 0.015 μg / g), and a large amount of flavonoids (up to 2.41 mg / g).

[0026] The present invention provides a preparation method of licorice seed oil and studies its biological activity, which is beneficial to the utilization and promotion of licorice seed resources. In the process of studying the biological activity of the prepared licorice seed oil, the inventor unexpectedly found that:

[0027] The licorice seed oil obtained by the preparation method of the present invention has certain inhibitory activity on lung adenocarcinoma cell line A549 and cervical cancer cell line Hela at a concentration of 100 mg / mL, and the inhibition rates are 39% and 44% respectively, so a new use of licorice seed oil in the preparation of anti-tumor or anti-cancer drugs (such as anti-lung adenocarcinoma drugs or anti-cervical cancer drugs) is provided;

[0028] The licorice seed oil obtained by the preparation method of the present invention has strong anti-inflammatory activity, and its inhibition rate on xylene-induced ear swelling of mice is as high as 47.45%, thus providing a new use of licorice seed oil in the preparation of health food or medicine with anti-inflammatory activity;

[0029] The licorice seed oil obtained by the preparation method of the invention has a strong scavenging function for DPPH free radicals, and its scavenging rate for DPPH free radicals is as high as 94.8%, thus providing a new use of licorice seed oil in preparing health food or medicine with antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the rutin standard curve established when determining the total flavonoid content in liquorice seed oil in Example 4 of the present invention. DETAILED DESCRIPTION

[0031] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0032] Instruments and reagents:

[0033] Licorice seeds (Ural licorice seeds collected from Ningxia Yanchi licorice planting base in July 2023), other chemical reagents are all domestic chemical pure reagents; CCK-8 (Shanghai Beibo Biotechnology Co., Ltd.); DMEM high glucose medium (Thermo Fisher (Suzhou) Instrument Co., Ltd.); EDTA (pancreatin) (gibco); Foetal Bovine Serum (Biological Industries); phosphate buffered saline; 96-well cell culture plate; multifunctional microplate reader; nitric acid (super pure); mixed standard solution (containing: Sb, As, Be, Cd, Ca, Cr, Co, Cu, Fe, Pb, Li, Mg, Mn, Mo, Ni, Se, Sr, Tl, Ti, V, Zn, 100 μg / mL); Al single element standard (100 μg / mL); Ba single element standard (100 μg / mL); mixed internal standard solution (containing: Sc, Ge, Rh, In, Tb, Lu, 100 μg / mL); ultrapure water (resistivity 18.2 mΩ·cm -1 ); inductively coupled plasma mass spectrometer (ICP-MS 78000); MARS microwave digestion system; Milli-Q pure water treatment system; electronic analytical balance; all glassware used was soaked in dilute nitric acid for 24 h; rutin; dexamethasone acetate tablets (Zhejiang Xianju Pharmaceutical Co., Ltd.); Tween-80 (Beijing Bio-Toda Technology Co., Ltd.).

[0034] Experimental animals: SPF grade ICR mice, male, weighing 23-30 g. Feeding environment conditions: room temperature (20±2)°C, relative humidity 50%-60%, free access to ordinary feed and water, and experiments were conducted after 3 days of adaptive feeding.

[0035] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the art can be used. The raw materials or instruments used are all conventional products that can be obtained commercially, including but not limited to the raw materials or instruments used in the examples of this application.

[0036] Example 1 Preparation of Licorice Seed Oil

[0037] This embodiment provides a method for preparing liquorice seed oil, and the specific steps are as follows:

[0038] (1) crushing licorice seeds to obtain licorice seed powder (particle size 40 mesh);

[0039] (2) Wrap 200 g of licorice seed powder with filter paper, load it into a Soxhlet extractor, and extract it with n-hexane reflux for 3 times. The amount of n-hexane used for a single Soxhlet extraction is 1000 mL, the extraction temperature is 80° C., and the extraction time is 120 min. After the extraction is completed, the n-hexane extracts are combined and concentrated under reduced pressure to 1% of the original volume to obtain a crude oil;

[0040] (3) The crude oil was passed through a neutral alumina column (column tube inner diameter of 15 mm, filler particle size of 80-120 mesh, usage amount of 50 g, column height of 5 cm after filling, dry loading, loading amount of 5 mL), eluted with 500 mL of n-hexane, flow rate of 1 mL / min, the eluate was collected, and concentrated under reduced pressure to obtain licorice seed oil, which was light orange-yellow.

[0041] Example 2 Determination of physical and chemical properties

[0042] 1. Sample preparation

[0043] Anhydrous sodium sulfate was dried at 100° C. for 2 h before use, and 2 g of anhydrous sodium sulfate was added to every 10 g of licorice seed oil (prepared in Example 1), and the mixture was stirred and adsorbed for dehydration, and then filtered with filter paper, and the clarified liquid oil after filtration was taken as a sample.

[0044] 2. Determination of acid value of licorice seed oil

[0045] Accurately weigh 20g of the sample and dissolve it in 50mL of ether-isopropionic acid mixed solvent in a conical flask. Add 4 drops of phenolphthalein solution, mix the solution and sample and shake well. Then use 0.1mol / L potassium hydroxide standard solution, titrate while shaking vigorously until the solvent turns slightly red and does not fade within 15 seconds. This is the end point of the titration. Stop the titration and record the milliliters of potassium hydroxide solution consumed. Determine the same sample three times and calculate the acid value according to the following formula.

[0046] X1=[(V-V0)×c×56.1)] / m

[0047] In the formula: X1─acid value, in milligrams per gram (mg / g); V─volume of potassium hydroxide standard solution used, in milliliters (mL); V0─volume of potassium hydroxide standard solution consumed in blank determination, in milliliters (mL); c─concentration of potassium hydroxide standard solution used, in moles per liter (mol / L); m─mass of the sample, in grams (g); 56.1─molar mass of potassium oxychloride (g / mol).

[0048] 3. Determination of peroxide value of licorice seed oil

[0049] Accurately weigh 2g of sample and put it in a conical flask, add 50mL of acetic acid-isooctane mixed solution, cover the stopper and shake vigorously until the sample is completely dissolved. Add 0.5mL of saturated potassium iodide solution, cover the stopper to react, the reaction time is 1min, shake the conical flask at least three times during the reaction to make it react completely, and then immediately add 30mL of distilled water. Titrate with 0.1mol / L sodium thiosulfate standard solution until the yellow color of iodine disappears, add 0.5mL of starch solution, and titrate with 0.01mol / L sodium thiosulfate standard solution, accompanied by strong shaking during the period, and add titration solution drop by drop until the blue color disappears, which is the measurement end point. The same sample is measured three times, and a blank experiment is performed, and the peroxide value is calculated according to the following formula.

[0050] P=[1000(V-V0)c] / m

[0051] In the formula: V─the volume of sodium thiosulfate standard solution consumed in the determination, in milliliters (mL); V0─the volume of sodium thiosulfate standard solution consumed in the blank test, in milliliters (mL); c─the concentration of sodium thiosulfate standard solution, in moles per liter (mol / L); m─the mass of the sample, in grams (g).

[0052] 4. Determination of iodine value of licorice seed oil

[0053] Accurately weigh 1.5g of the sample into a 500mL conical flask, add 20mL of cyclohexane-glacial acetic acid solution to dissolve, accurately add 25mL of Wei's reagent, cover the stopper, shake well and place the conical flask in a dark place to react for 1h. After reaching the specified reaction time, first add 20mL of potassium iodide solution, then add 150mL of water, and titrate with 0.1mol / L sodium thiosulfate standard solution until the yellow color of iodine disappears. Finally, add 0.5mL of starch solution and titrate with 0.1mol / L sodium thiosulfate standard solution. While titrating, shake the conical flask vigorously until the blue color just disappears. Measure the same sample three times, and do a blank test, and calculate the iodine value according to the following formula.

[0054] W1=[12.69×c×(V1-V2)] / m

[0055] In the formula: W1─ iodine value of the sample, expressed as the number of grams of iodine absorbed by every 100g of oil (g / 100g); c─ concentration of sodium thiosulfate standard solution, in moles per liter (mol / L); V1─ volume of sodium thiosulfate standard solution consumed in blank test, in milliliters (mL); V2─ volume of sodium thiosulfate standard solution consumed in sample test, in milliliters (mL); m─ mass of the sample, in grams (g).

[0056] 5. Determination of saponification value of licorice seed oil

[0057] Accurately weigh 20g of the sample into a conical flask, add 25mL of potassium hydroxide-ethanol to dissolve it with a pipette, connect the reflux condenser to the conical flask, put the conical flask in a heating device, heat it in a 95℃ water bath and maintain boiling for 1h, shaking it from time to time. Add 0.5mL of phenolphthalein indicator to the hot solution, and titrate it with a standard hydrochloric acid solution until the pink color of the indicator disappears. Perform three measurements on the same sample. At the same time, do a blank test with 25mL of potassium hydroxide-ethanol solution without adding a sample, and calculate the saponification value according to the following formula.

[0058] I S =[(V0-V1)×c×56.1] / m

[0059] Where: I S ─Saponification value (in terms of KOH), in milligrams per gram (mg / g); V0─the volume of the hydrochloric acid standard solution consumed in the blank test, in milliliters (mL); V1─the volume of the hydrochloric acid standard solution consumed in the test, in milliliters (mL); c─the concentration of the hydrochloric acid standard solution used, in moles per liter (mol / L); m─the mass of the test sample, in grams (g).

[0060] The results of the determination of the physical and chemical properties of the licorice seed oil prepared in Example 1 of the present invention are shown in Table 1.

[0061] Table 1 Physical and chemical properties of licorice seed oil

[0062] project index Acid value (KOH) / (mg / g) 0.066±0.003 Peroxide value (g / 100g) 7.267±0.023 Iodine value (g / 100g) 37.231±0.016 Saponification value (KOH) / (mg / g) 186.823±1.818

[0063] Example 3 Trace element content determination

[0064] 1. Sample processing and sample pretreatment

[0065] 0.3 g of the licorice seed oil prepared in Example 1 was accurately weighed and placed in a polytetrafluoroethylene (PTFE) digestion tank.

[0066] Add 10mL nitric acid, place the sample on an acid remover at 110℃ for pre-digestion for 30min, cover and seal, and digest according to the working conditions shown in Table 2. After digestion is completed, place the sample on an acid remover plate, set the acid remover at 130℃ until the solution remains 0.5mL, cool the digestion solution to below 60℃, take out the digestion tank, let it cool, transfer the digestion solution to a 25mL volumetric flask, wash the digestion tank three times with a small amount of water, combine the washing solution in the volumetric flask, make up to volume with water, and shake well to obtain the sample test solution.

[0067] Prepare blank control solution according to the above method.

[0068] Table 2 Microwave digestion conditions

[0069] Power(W) Time (min) Temperature(℃) Holding time (min) 800 10 120 5 800 5 150 5 1600 5 180 20

[0070] 2. Solution preparation

[0071] (1) Preparation of standard working stock solution

[0072] Weigh 0.1 g of the mixed standard solution of the mixed elements, and the single element standards of Al and Ba, make the volume to 10 mL with 2% nitric acid, and dilute to a mixed standard stock solution (1000 ng / mL) containing different metal elements.

[0073] (2) Preparation of a series of standard stock solutions

[0074] Weigh an appropriate amount of the above standard working stock solution and dilute it with 2% nitric acid to make a standard series mixed solution containing 0.2ng, 1ng, 5ng, 10ng, 50ng, 100ng, and 500ng of each element per 1mL.

[0075] (3) Preparation of internal standard working solution

[0076] Weigh an appropriate amount of the mixed internal standard solution and dilute it to 50 ng / mL with 2% nitric acid.

[0077] 3. Linear range investigation

[0078] The ICP-MS instrument conditions are shown in Table 3.

[0079] Table 3 ICP-MS instrument conditions

[0080] project Working conditions RF Power 1.50KW Cooling gas flow (argon) 15L / min Auxiliary gas flow (argon) 1.0L / min Nebulizer gas flow rate (argon) 1.0L / min Repetitions 3 Pool mode KED Peristaltic pump speed 0.3rps

[0081] Under the above instrument conditions, a series of standard working solutions with increasing concentrations were measured in sequence, and the ratio of the response intensity of the measured element to the internal standard element was used as the ordinate and the mass concentration was used as the abscissa to draw a standard curve and obtain the regression equation. The regression equation and correlation coefficient of each element are shown in Table 4.

[0082] Table 4 Regression equation, correlation coefficient (R)

[0083] element Regression equation R V y=0.9989x+0.7856 0.9998 Cr y=0.9982x+1.2271 0.9997 Mn y=0.9984x+1.1245 0.9998 Fe y=0.9985x+1.0445 0.9999 Co y=0.9983x+1.1886 0.9998 Ni y=0.998x+1.4199 0.9998 Cu y=0.9974x+1.8173 0.9997 Zn y=0.998x+1.4088 0.9997 As y=0.9988x+0.8514 0.9997 Se y=0.9988x+0.8512 0.9998 Mo y=0.9998x+0.1126 0.9999

[0084] The prepared sample test solution was tested under the above instrument conditions, and the contents of various trace elements were determined using the drawn standard curve. The test results of the contents of 11 trace elements in the licorice seed oil prepared in Example 1 of the present invention are shown in Table 5.

[0085] Table 5 Contents of 11 trace elements in licorice seed oil

[0086] Element Type Content of each element (μg / g) V 0.025 Cr 0.299 Mn 0.369 Fe 5.520 Co 0.025 Ni 0.191 Cu 0.316 Zn 6.434 As 0.024 Se 0.012 Mo 0.015

[0087] As shown in Table 5, the licorice seed oil prepared in Example 1 of the present invention is rich in trace elements essential to the human body. There are 11 essential trace elements that have been confirmed to be related to human health and life, namely vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, arsenic, selenium, and molybdenum.

[0088] Example 4 Determination of total flavonoid content

[0089] 1. Sample processing and standard curve drawing

[0090] (1) Extract preparation

[0091] Weigh 2 g of the licorice seed oil prepared in Example 1 into a centrifuge tube, add 3 mL of methanol (60%) and 3 mL of n-hexane, vortex for 2 min, centrifuge at 3800 r / min for 8 min, separate the upper oil phase and the lower clear liquid, take out the lower clear liquid, extract the upper oil phase twice with methanol, combine the three extracts and test.

[0092] (2) Drawing of standard curve

[0093] Weigh 0.0100g of rutin standard, add 70% ethanol to dissolve in a 100mL brown volumetric flask, dilute to the scale, and shake well to obtain a 0.10mg / mL rutin standard solution. Dilute with distilled water to standard stock solutions with concentrations of 0.0, 0.02, 0.04, 0.06, 0.08, and 0.10mg / mL, respectively. Take 1.0mL of the above standard solution, add 0.7mL of 5% sodium nitrite solution, shake well and let stand for 6min, then add 0.7mL of 10% aluminum nitrate solution, shake well and let stand for 6min, then add 5mL of 4% sodium hydroxide solution, shake well and let stand for 15min. Use 0.0mL of reagent as blank and measure its absorbance at 510nm. All experiments were measured in parallel three times. The rutin standard curve is as follows Figure 1 As shown (standard curve: y = 1.4149x-0.0039, R 2 =0.9956).

[0094] 2. Determination of flavonoid content

[0095] Take 1mL of polar accompaniment (referring to the extract of step 1), add 0.7mL of 5% sodium nitrite solution, shake and place for 6min, then add 0.7mL of 10% aluminum nitrate solution, shake and place for 6min, then add 5mL of 4% sodium hydroxide solution, shake and place for 15min. Take 0.0mL of reagent as blank and measure its absorbance value at 510nm. Calculate the total flavonoid content of the sample according to the regression equation.

[0096] The total flavonoids content in licorice seed oil was calculated according to the following formula.

[0097] M=a×c×V / W

[0098] Wherein: M is the total flavonoid content in licorice seed oil (mg / g), a is the dilution factor, c is the flavonoid content (mg / mL), V is the volume of the extract (mL), and W is the mass of licorice seed oil (g).

[0099] The total flavonoid content in the licorice seed oil prepared in Example 1 of the present invention was measured and shown in Table 6.

[0100] Table 6 Content of total flavonoids in licorice seed oil

[0101]

[0102] As shown in Table 6, the licorice seed oil prepared in Example 1 of the present invention contains a large amount of flavonoids, and the content thereof is as high as 2.41 mg / g.

[0103] Example 5 In vitro antitumor activity test

[0104] The in vitro antitumor activity of the licorice seed oil obtained in Example 1 was tested by CCK-8 method, mainly studying its in vitro inhibitory activity on lung adenocarcinoma cell line (A549) and cervical cancer cell line (Hela). The specific test process is described by taking the test process of lung adenocarcinoma cell line (A549) as an example.

[0105] 1. Preparation of test sample concentration

[0106] The licorice seed oil described above was serially diluted with DMEM to five different concentrations (100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL and 6.25 mg / mL, respectively) for cell experiments.

[0107] The experiment will set up 3 groups, A (blank group): with DMEM and CCK-8; B (control group): with DMEM, CCK-8 and cells; C (experimental group): with DMEM, CCK-8, cells and different concentrations of licorice seed oil solution.

[0108] 2. Cultivation of lung adenocarcinoma cell line (A549) and inhibitory activity test

[0109] Lung cancer A549 cells (1×10 6 cells / mL) were inoculated into 96-well plates at 100 μL per well and cultured in a 37°C incubator (95% air, 5% carbon dioxide) for 12 h; the culture medium was removed from each well, 100 μL DEME was added to each well of group A and group B, 100 μL of different concentrations of licorice seed oil solution was added to each well of group C (each concentration was repeated 5 times), and cultured at 37°C for 24 h; in addition to the culture medium, 10 μL CCK-8 was added to each well of group A, group B and group C, and cultured at 37°C for another 1 h in the dark; the absorbance (OD value) at 450 nm was measured with an enzyme marker. The OD value detected in each group was used to calculate the cell survival rate = [(A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 )]×100%.

[0110] The culture and inhibitory activity testing process of the cervical cancer cell line (Hela) is consistent with that of the lung adenocarcinoma cell line (A549).

[0111] The inhibition rate of the licorice seed oil prepared in Example 1 of the present invention on the lung adenocarcinoma cell line (A549) and the cervical cancer cell line (Hela) is shown in Table 7.

[0112] Table 7 Effects of licorice seed oil on two tumor cell lines

[0113]

[0114] As shown in Table 7, the licorice seed oil prepared in Example 1 of the present invention has a certain inhibition rate on lung adenocarcinoma cell lines and cervical cancer cell lines at a concentration of 100 mg / mL, which are 39% and 44%, respectively, which proves that licorice seed oil has certain anti-tumor / anti-cancer activity and has certain potential in the preparation of anti-tumor or anti-cancer drugs, such as being used as a raw material or excipient.

[0115] Example 6 Anti-inflammatory Activity Test

[0116] Xylene-induced ear swelling experiment in mice:

[0117] After the experimental mice were adaptively raised for 3 days, 50 mice were randomly divided into 5 groups, namely a blank control group (2% Tween-80, 10 mice), a positive control group (dexamethasone 5 mg / kg, 10 mice), a high-dose group of licorice seed oil (3 g / kg, 10 mice), a medium-dose group of licorice seed oil (0.75 g / kg, 10 mice), and a low-dose group of licorice seed oil (0.1875 g / kg, 10 mice). The licorice seed oil was prepared by Example 1.

[0118] The drug was administered by gavage once a day at a volume of 10 mL / kg for 3 consecutive days. One hour after the last administration, 40 μL of xylene was evenly smeared on the front and back of the right ear of the mouse. The mouse was killed by cervical dislocation 30 minutes after inflammation. The two ears were cut off along the ear roots, and round ear pieces of equal area were punched in the same part of the two ears with a 6 mm diameter puncher. The ear swelling degree and swelling inhibition rate were calculated according to the formula "ear swelling degree (mg) = right ear (mg) - left ear (mg), swelling inhibition rate (%) = (blank group - drug group) / blank group × 100%". The experimental results are shown in Table 8.

[0119] Table 8 Effect of licorice seed oil on xylene-induced ear swelling in mice

[0120] Group Ear swelling (mg) Swelling inhibition rate (%) Blank group 6.28±1.70 - Positive control group <![CDATA[3.53±0.38 * ]]> 43.79 High dose group <![CDATA[3.30±0.4 * ]]> 47.45 Medium dose group 4.92±2.28 21.66 Low dose group 5.48±1.90 12.74

[0121] Note: Compared with the blank group *P<0.05, **P<0.01

[0122] As shown in Table 8, at a high dose (3 g / kg), the licorice seed oil prepared in Example 1 of the present invention has a relatively stronger anti-inflammatory activity than the positive drug, and has great potential in the preparation of health foods or medicines with anti-inflammatory activity, such as being used as a raw material, additive or excipient.

[0123] Example 7 Antioxidant Activity Test

[0124] 1. Preparation of DPPH stock solution

[0125] Weigh 0.0394 g of DPPH and dilute it to a 100 mL volumetric flask with anhydrous ethanol solution to obtain a DPPH mother solution with a concentration of 1 mmoL / L. Shake well and refrigerate at 4°C and keep away from light.

[0126] 2. Preparation of DPPH application solution

[0127] Dilute the DPPH stock solution to a concentration of 0.2 mmoL / L with anhydrous ethanol, shake thoroughly to prepare the DPPH application solution, and store in a dark place.

[0128] 3. Absorbance measurement

[0129] (1) 2 mL of different concentrations (10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL) of licorice seed oil-ethanol solution (licorice seed oil prepared in Example 1) were taken and added to 10 10 mL test tubes in sequence, and then 2 mL of DPPH-ethanol solution was added to each test tube. After being fully mixed and allowed to stand for 30 min, the absorbance values ​​(A) were measured at a wavelength of 517 nm. i ), and each concentration was repeated three times.

[0130] (2) Take 2 mL of ethanol to replace the sample solution and measure its absorbance value (A0) at the same wavelength. Repeat the measurement three times.

[0131] (3) Take 2 mL of ethanol instead of DPPH solution and measure its absorbance value (A j ), and the assay was performed three times in parallel.

[0132] 4. DPPH clearance calculation

[0133] The DPPH clearance rate was calculated according to the following formula.

[0134] DPPH clearance rate (%) = [1 – (A i –A j ) / A0]×100%

[0135] The DPPH scavenging rates of the licorice seed oil prepared in Example 1 at different concentrations are shown in Table 9.

[0136] Table 9 Scavenging rate of DPPH by licorice seed oil at different concentrations

[0137]

[0138]

[0139] As shown in Table 9, the licorice seed oil prepared in Example 1 of the present invention has a DPPH free radical scavenging rate of ≥94% at a concentration of 75 mg / mL, has good antioxidant activity, and has great potential in the preparation of health foods or drugs with antioxidant activity, such as being used as a raw material, additive or auxiliary material.

[0140] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing liquorice seed oil, It is characterized in that The following steps are involved: (1) crushing liquorice seeds to obtain liquorice seed powder; (2) extracting the licorice seed powder with n-hexane and concentrating under reduced pressure to obtain a crude oil; (3) refining the crude oil to obtain liquorice seed oil.

2. The preparation method of liquorice seed oil according to claim 1, It is characterized in that In step (2), the licorice seed powder is placed in a Soxhlet extractor, extracted with n-hexane under reflux for 2 to 3 times, the n-hexane extracts are combined, and concentrated under reduced pressure to obtain a crude oil; In step (3), the crude oil is passed through a neutral alumina column, eluted with n-hexane, the eluate is collected, and concentrated under reduced pressure to obtain licorice seed oil.

3. The preparation method of liquorice seed oil according to claim 2, It is characterized in that In step (1), the particle size of the licorice seed powder is 20 to 70 meshes; preferably, the particle size of the licorice seed powder is 30 to 60 meshes; In step (2), the sample amount of the licorice seed powder is 50-200 g, the amount of n-hexane is 500-1000 mL; the temperature of a single Soxhlet extraction is 30-80° C., and the time is 30-120 min; Concentrate under reduced pressure to 1% of the original volume; preferably, the sample amount of the licorice seed powder is 200g, and the amount of n-hexane is 1000mL; the temperature of a single Soxhlet extraction is 80°C and the time is 120min; In step (3), the neutral alumina column selects a filler with a particle size of 80 to 120 mesh, and the usage amount is 30 to 60 g; the inner diameter of the column tube of the neutral alumina column is 10 to 20 mm, and the column height after filling is 5 to 10 cm; the column loading method is: dry loading; the loading amount of crude oil is 3 to 8 mL; the amount of n-hexane is 500 to 100 mL; the n-hexane flow rate is 1 to 2 mL / min; preferably, the usage amount of the neutral alumina column filler is 50 g; the inner diameter of the column tube of the neutral alumina column is 15 mm, and the column height after filling is 5 cm; the loading amount of crude oil is 5 mL; the amount of n-hexane is 500 mL; the n-hexane flow rate is 1 mL / min.

4. Licorice seed oil obtained by the preparation method according to any one of claims 1 to 3.

5. A liquorice seed oil, It is characterized in that The acid value of the licorice seed oil is 0.066±0.003 mg / g; the peroxide value is 7.267±0.023 g / 100 g; the iodine value is 37.231±0.016 g / 100 g; and the saponification value is 186.823±1.818 mg / g.

6. Licorice seed oil according to claim 5, It is characterized in that The trace elements in the licorice seed oil include: V 0.025 μg / g, Cr 0.299 μg / g, Mn 0.369 μg / g, Fe 5.520 μg / g, Co 0.025 μg / g, Ni 0.191 μg / g, Cu 0.316 μg / g, Zn 6.434 μg / g, As 0.024 μg / g, Se 0.012 μg / g, and Mo 0.015 μg / g.

7. Use of the licorice seed oil obtained by the preparation method according to any one of claims 1 to 3 or the licorice seed oil according to any one of claims 4 to 6 in the preparation of anti-tumor or anti-cancer drugs.

8. The use according to claim 7, It is characterized in that The anti-tumor or anti-cancer drug is an anti-lung adenocarcinoma drug or an anti-cervical cancer drug.

9. Use of the licorice seed oil obtained by the preparation method according to any one of claims 1 to 3 or the licorice seed oil according to any one of claims 4 to 6 in the preparation of health food or medicine with anti-inflammatory activity.

10. Use of the licorice seed oil obtained by the preparation method according to any one of claims 1 to 3 or the licorice seed oil according to any one of claims 4 to 6 in the preparation of health food or medicine with antioxidant activity.

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