Anti-inflammatory volatile oil of euonymus alatus flower and preparation method of anti-inflammatory volatile oil

Abstract

The invention relates to the technical field of medicine, and particularly discloses volatile oil for extracting euonymus alatus flowers and a preparation method thereof.The preparation method comprises the steps of extraction and purification, according to the extraction step, 200 g of euonymus alatus flowers are placed in 3000 ml of ultrapure water to be soaked overnight, then heating is conducted through a constant-temperature electric jacket, and the temperature is kept at 180-200 DEG C; adding 250mL of dichloromethane into a 500mL round-bottom flask, heating in an oil bath, keeping the temperature at about 80 DEG C, and extracting for 8 hours by adopting a simultaneous distillation and extraction instrument; the chemical components of the euonymus alatus flower volatile oil are analyzed by adopting GC-MS (Gas Chromatography-Mass Spectrometer) in combination with retention indexes, and the volatile oil has the advantages that the chemical components of the euonymus alatus flower volatile oil can be analyzed by adopting the GC-MS in combination with the retention indexes; and anti-inflammatory and analgesic activity tests are carried out on the euonymus alatus by adopting a toe inflammatory swelling experiment and a mouse twisting experiment, so that a scientific basis is provided for further development and utilization of the euonymus alatus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an anti-inflammatory volatile oil of Euonymus japonicus flower and a preparation method thereof. Technical Background

[0002] Holly Euonymus belongs to the genus Euonymus of the family Celastraceae. It is a broad-leaved evergreen shrub. Holly Euonymus has strong adaptability, wide distribution and cultivation range, and strong resistance to air pollution, cold, drought, and soil barrenness. "Shennong's Herbal Classic" and "Compendium of Materia Medica" record that plants of the Celastraceae can be used to treat pain, menstrual disorders, skin swelling and bruises, rheumatoid arthritis, and detoxification. In recent years, literature reports have shown that plants of the Celastraceae have anti-inflammatory, anti-diabetic, anti-tumor, liver-protecting, antibacterial and antioxidant activities, and are effective for hyperglycemia, chronic kidney disease, rheumatoid arthritis, cor pulmonale, bronchial asthma, allergic diseases, urinary tract infections, and prostate diseases. "Chu Yi Materia Medica", "Xiang Lan Kao" and "Tujia Medicine" also record that the roots, stems and leaves of Holly Euonymus have the effect of regulating menstruation and relieving pain, and are used for irregular menstruation, dysmenorrhea, traumatic injuries, fractures and urinary pain. Based on this, this study intends to use GC-MS combined with retention index to analyze the chemical components of the volatile oil of Euonymus japonicus flowers, and use the toe inflammation and swelling experiment and the mouse writhing experiment to test its anti-inflammatory and analgesic activity, aiming to provide a scientific basis for the further development and utilization of Euonymus japonicus. Summary of the invention

[0003] The object of the present invention is to provide a method for preparing volatile oil from euonymus flower, comprising the steps of extracting volatile oil, weighing 200g of euonymus flower, placing it in a 5000mL round-bottom flask, adding 3000mL of ultrapure water, soaking it overnight, heating it with a constant temperature electric heating jacket, and keeping the temperature at 180°C to 200°C; adding 250mL of dichloromethane to the 500mL round-bottom flask, heating it with an oil bath, and keeping the temperature at about 80°C, and extracting it for 8h with a simultaneous distillation extractor. After the dichloromethane extract is separated and dried over anhydrous magnesium sulfate to remove water, it is filtered and rotary evaporated to obtain 1.84g of a light yellow oily substance, which is placed in a refrigerator at 4°C for standby use, and the volatile oil extraction rate is 0.92%.

[0004] GC-MS analysis conditions of anti-inflammatory volatile oil from Euonymus japonicus flowers:

[0005] Chromatographic conditions: Agilent HP-5MS (30m×0.25mm×0.25μm) capillary column, carrier gas: He, flow rate: 1mL / min. Initial temperature: 40°C, hold for 5min, increase temperature to 280°C at 5°C / min, hold for 5min, injection port temperature 290°C, injection volume 1μL, no splitting.

[0006] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; interface temperature 260 ° C; quadrupole temperature 150 ° C; mass scanning range m / z: 30-500 amu; acquisition mode: full scan mode. Determination of retention index (RI): C7-C 30 The normal alkane standard was subjected to GC-MS analysis under the same conditions as above, and its retention time was determined. The retention index RI of each component of the volatile oil was calculated according to the linear formula: RI = 100n + 100 [t R(x) -t R(n) ] / [t R(n+1) -t R(n) ], t R(x) is the retention time of the analyzed component, t R(n) and t R(n+1) are the retention times of normal alkanes with n and n+1 carbon atoms, respectively, and t R(n) <t R(x) <t R(n+1) .

[0007] The invention discloses an anti-inflammatory volatile oil from Euonymus japonicus flower, which mainly comprises beta-cerebrene A (28.56%), eicosane (16.18%), heneicosane (10.75%), tetracosane (3.63%), leaf alcohol (2.20%), ethyl α-linolenate (2.16%), ethyl linoleate (1.64%) and cis-myrtanol (1.43%).

[0008] An anti-inflammatory volatile oil of Euonymus japonicus flower comprises hydrocarbon compounds, the content of which accounts for 62.63% of the total volatile oil, wherein the hydrocarbon compounds mainly include (-)-cepene A (28.56%), eicosane (16.18%), heneicosane (10.75%), tetracosane (3.63%), (Z)-9-tricosene (0.94%), and farnesene (0.61%);

[0009] It also includes alcohols accounting for 14.13%, mainly leaf alcohol (2.20%), cis-myrtanol (1.43%), 2-heptanol (1.23%), myrtenol (0.79%), linalool (0.61%), and geraniol (0.58%);

[0010] It also includes esters accounting for 6.11%, mainly ethyl linolenate (2.16%), ethyl linoleate (1.64%), ethyl palmitate (1.05%), and methyl palmitate (0.39%);

[0011] It also includes aldehydes accounting for 1.24%, mainly n-nonanal (0.63%), n-hexanal (0.21%), and 1-octanal (0.13%);

[0012] It also includes carboxylic acids accounting for 1.17%, mainly palmitic acid (1.17%);

[0013] It also includes ethers accounting for 0.29%, mainly cis-rose oxide (0.20%) and trans-rose oxide (0.09%).

[0014] The beneficial effects of the present invention are as follows: the volatile oil of Euonymus japonicus flower of the present invention has stable performance, good positive drug performance, has a certain anti-inflammatory effect, and its anti-inflammatory ability is gradually enhanced with increasing dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure is the total ion current diagram of the volatile oil of Euonymus japonicus flower of the present invention. DETAILED DESCRIPTION

[0016] The specific implementation modes of the present invention are described in detail below in combination with experimental results.

[0017] A preparation method of anti-inflammatory volatile oil of euonymus flower comprises the following steps of extracting volatile oil: weighing 200g of euonymus flower, placing it in a 5000mL round-bottom flask, adding 3000mL of ultrapure water, soaking it overnight, heating it with a constant temperature electric heating jacket, and keeping the temperature at 180°C to 200°C; adding 250mL of dichloromethane to the 500mL round-bottom flask, heating it in an oil bath, and keeping the temperature at about 80°C, and extracting it for 8h with a simultaneous distillation extractor. After the dichloromethane extract is separated and dried over anhydrous magnesium sulfate to remove water, it is filtered and rotary evaporated to obtain 1.84g of a light yellow oily substance, which is placed in a refrigerator for refrigeration at 4°C for standby use, and the volatile oil extraction rate is 0.92%.

[0018] GC-MS analysis conditions of essential oils

[0019] Chromatographic conditions: Agilent HP-5MS (30m×0.25mm×0.25μm) capillary column, carrier gas: He gas, flow rate: 1mL / min. Initial temperature: 40°C, maintained for 5min, heated to 280°C at 5°C / min, maintained for 5min, injection port temperature was 290°C, injection volume was 1μL, no splitting.

[0020] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; interface temperature 260°C; quadrupole temperature 150°C; mass scanning range m / z: 30-500 amu; acquisition mode: full scan mode.

[0021] Determination of retention index (RI): C7~C 30 The normal alkane standard was subjected to GC-MS analysis under the same conditions as above, and its retention time was determined. The retention index RI of each component of the volatile oil was calculated according to the linear formula: RI = 100n + 100 [tR(x) -t R(n) ] / [t R(n+1) -t R(n) ], t R(x) is the retention time of the analyzed component, t R(n) and t R(n+1) are the retention times of normal alkanes with n and n+1 carbon atoms, respectively, and t R(n) <t R(x) <t R(n+1) .

[0022] Carrageenan-induced swelling experiment in mice

[0023] Fifty female Kunming mice were randomly divided into five groups, with 10 mice in each group, namely the model group, aspirin group, and low, medium, and high dose groups of volatile oil from euonymus flowers. According to the daily doses of 0.025, 0.050, and 0.100 g / kg·BW, the volatile oil from euonymus flowers was prepared with distilled water to prepare sample suspensions with concentrations of 1.25, 2.50, and 5.00 g / L, respectively. The mice were gavaged at a dose of 20 ml / kg·BW. The model group was gavaged with an equivalent volume of distilled water, and the aspirin group was administered at 0.25 g / kg·BW. The mice were gavaged once a day for 7 consecutive days. 30 minutes after the last administration, the thickness of the front right toe of the mouse was measured with a vernier caliper, and the average of three times was taken as the thickness of the toe before inflammation. After the measurement, 0.1 ml of 1% carrageenan solution was injected subcutaneously into the front right toe of the mouse to induce inflammation. The thickness of the front right toe was measured three times 3 hours later and the average value was taken.

[0024] Toe swelling (mm) = toe thickness after injection (mm) - toe thickness before injection (mm)

[0025] Inflammation inhibition rate (%) = (toe swelling of model group - toe swelling of drug-treated group) / toe swelling of model group × 100%

[0026] Acetic acid writhing test

[0027] The analgesic in vivo experiment used the mouse acetic acid writhing test. The animal grouping and drug administration were the same as the "carrageenan-induced inflammatory mouse foot swelling experiment". One hour after the last administration, each mouse was intraperitoneally injected with 1% glacial acetic acid. After 5 minutes, the mouse behavior stabilized, and the number of mouse writhing within 15 minutes was observed and recorded. The drug pain inhibition rate was calculated.

[0028] Pain inhibition rate (%) = (number of twists in the control group - number of twists in the drug group) / number of twists in the control group × 100%

[0029] Statistical methods

[0030] The test results were expressed as mean ± standard deviation (x ± s), and the statistical analysis method used was the inter-group t test.

[0031] Analysis of essential oil components

[0032] Total ion current diagram of volatile oil from Euonymus japonicus flower Figure 1 The components and relative contents are shown in Table 1. From the chemical components and relative contents of volatile oil from Euonymus japonicus flowers in Table 1, it can be seen that 43 chemical components were identified from the volatile oil from Euonymus japonicus flowers, accounting for 85.57% of the total content of volatile oil, and the main components were (-)-cepene A (28.56%), eicosane (16.18%), heneicosane (10.75%), tetracosane (3.63%), leaf alcohol (2.20%), α-linolenic acid ethyl ester (2.16%), linoleic acid ethyl ester (1.64%) and cis-myrtanol (1.43%). The content of hydrocarbon compounds is relatively high, accounting for 62.63% of the total volatile oil, mainly including (-)-cerebellene A (28.56%), eicosane (16.18%), heneicosane (10.75%), tetracosane (3.63%), (Z)-9-tricosene (0.94%), farnesene (0.61%), etc.; alcohols account for 14.13%, mainly including leaf alcohol (2.20%), cis-myrtanol (1.43%), 2-heptanol (1.23%), myrtenol (0.79%), linalool (0.61%). ), geraniol (0.58%), etc.; esters accounted for 6.11%, mainly ethyl linolenate (2.16%), ethyl linoleate (1.64%), ethyl palmitate (1.05%), methyl palmitate (0.39%), etc.; aldehydes accounted for 1.24%, mainly nonanal (0.63%), hexanal (0.21%), 1-octanal (0.13%); carboxylic acids accounted for 1.17%, mainly palmitic acid (1.17%); ethers accounted for 0.29%, mainly cis-rose oxide (0.20%) and trans-rose oxide (0.09%),

[0033] Table 1 is as follows: RIexp is the retention index relative to normal alkanes (C7-C30) measured on the HP-5MS chromatographic column; RIref is the literature value of the retention index of each compound.

[0034]

[0035]

[0036]

[0037]

[0038] Effects of volatile oil from Euonymus japonicus flowers on foot swelling in mice induced by carrageenan inflammation

[0039] The experiment used the degree of foot swelling before and after inflammation in mice and the rate of foot swelling inhibition as evaluation indicators of anti-inflammatory activity. The experimental results showed that the positive drug aspirin group had a very significant (P<0.01) swelling inhibition rate compared with the model group, indicating that the model was stable and the positive drug performed well. From Table 2, it can be seen that the effect of ilex euonymus flower volatile oil on mouse foot swelling can be seen that 3h after inflammation, compared with the model group, the low, medium and high experimental groups of ilex euonymus flower volatile oil all had a certain swelling inhibition rate, and showed a dose-dependent relationship. Compared with the model group, the high-dose group can significantly inhibit the degree of swelling (P<0.05). Table 2 Effect of ilex euonymus flower volatile oil on mouse foot swelling (n=10) is as follows:

[0040] Group <![CDATA[Dose / (mg·kg -1 )]]> 3-hour plantar swelling / mm Swelling inhibition rate / % Model Group - 0.26±0.13 - aspirin 5 <![CDATA[0.12±0.05 ## ]]> 53.85％ Low dose group 5 0.21±0.14 19.23％ Medium dose group 10 0.15±0.10 42.31％ High dose group 15 <![CDATA[0.14±0.10 # ]]> 46.15％

[0041] Compared with the model group: # indicates P < 0.05; compared with the aspirin group and the model group: ## indicates P < 0.01.

[0042] Experimental study on analgesic activity of volatile oil from Euonymus japonicus flowers

[0043] As shown in Table 3 of the experimental results, compared with the model group, the number of writhing times of mice in the positive drug aspirin group was significantly reduced (P<0.01). Compared with the model group, the inhibitory effect of each dose group of euonymus flower volatile oil on mouse writhing showed a downward trend with the increase of dose, and there was a dose-dependent relationship. The number of writhing times in the high-dose group was significantly reduced compared with the model group (P<0.05). Comparing the inhibition rate of mouse writhing in each drug group, the positive drug group had the best inhibition rate of writhing, reaching 52.9%, and the high-dose group also showed a certain inhibition rate of writhing.

[0044] See Table 3 for the effects of Euonymus japonicus essential oil on acetic acid writhing reaction in mice (n=10):

[0045] Group <![CDATA[Dose / (mg·kg -1 )]]> Twist times / times Torsion inhibition rate / % Model Group - 63.9±10.11 - aspirin 5 <![CDATA[30.1±10.67 ## ]]> 52.90％ Low dose group 5 54.5±12.78 14.71％ Medium dose group 10 52.7±13.59 17.53％ High dose group 15 <![CDATA[44.7±13.03 1# ]]> 30.05％

[0046] Note: Comparison between high-dose group and model group: # indicates P<0.05; Comparison between aspirin group and model group:

[0047] ## indicates P<0.01.

[0048] The volatile oil of Euonymus ilex was extracted by SED method, and the chemical components of the volatile oil of Euonymus ilex were identified and analyzed by GC-MS technology. The majority of the volatile oil components are hydrocarbons (62.63%), alcohols (14.13%) and carboxylic acid ester compounds (6.11%). Among them, the content of terpenes and their oxygen-containing derivatives reached 37.75%. The main component (-)-cepene A (28.56%) is a macrocyclic diterpene tetraene compound with a fourteen-carbon ring. It was found in Australian termites, medicinal plants and marine organisms. It is a natural pheromone and is also the first to be found in Euonymus plants. According to literature reports, (-)-cepene A has physiological activities such as anti-inflammatory and anti-tumor, and can significantly inhibit the activity of LPS-induced B cell proliferation and ConA-induced T cell proliferation. Its IC 50 The concentrations of ED and ED were 16.7 and 44.3 μM respectively, which could significantly reduce the activity of human pancreatic phospholipase A2 bile salt, activate hPLA2 activity to 5 μM, and have significant inhibitory activity against four tumor cells: A549, HT-29, KB, and P-388. 50 The results showed that the volatile oil contained a variety of active ingredients, such as α-linolenic acid ethyl ester, linoleic acid ethyl ester, palmitic acid and other fatty acids and fatty acid esters, which have the effects of lowering blood lipids, lowering cholesterol, improving immunity, protecting nerves, anti-inflammatory and anti-tumor; eucalyptol has expectorant, antitussive, decongestant and other effects, and can treat bronchitis, sinusitis and rheumatism; linalool has significant analgesic, antianxiety, sedative hypnotic, anti-inflammatory and other pharmacological activities; geraniol and trumpet alcohol have anti-inflammatory, antibacterial, antioxidant and neuroprotective effects; myrtenol and (-)-cis-myrtanol have significant anti-inflammatory, analgesic and antibacterial activities; guaiacol has anti-proliferation, autophagy promotion, insecticide, antianxiety, anti-inflammatory and blood pressure lowering biological activities.

[0049] The anti-inflammatory effect of volatile oil from Euonymus japonicus flowers was observed by using carrageenan-induced mouse paw swelling experiment. The results showed that volatile oil from Euonymus japonicus flowers had a certain anti-inflammatory effect, and its anti-inflammatory ability gradually increased with increasing doses. This may be related to the high content of anti-inflammatory active substance (-)-cepene A, terpenoids such as myrtenol, cis-myrtanol, linalool, etc., as well as linoleate and palmitate in Euonymus japonicus flowers.

[0050] In the mouse writhing experiment, the volatile oil of ilex euonymus flower can reduce the number of mouse writhing times in a dose-dependent manner. This may be related to the obvious analgesic effect of monoterpene alcohols and sesquiterpene alcohols contained in the volatile oil, such as linalool, myrtenol, geraniol, and guaiacol.

Claims

1. An anti-inflammatory volatile oil from Euonymus japonicus flower, characterized in that: The main components include -bene A (28.56%), eicosane (16.18%), heneicosane (10.75%), tetracosane (3.63%), leaf alcohol (2.20%), ethyl α-linolenate (2.16%), ethyl linoleate (1.64%) and cis-myrtanol (1.43%).

2. The anti-inflammatory volatile oil of Euonymus japonicus flower according to claim 1, characterized in that: The content of hydrocarbon compounds accounts for 62.63% of the total volatile oil, and the hydrocarbon compounds mainly include (-)-cene A (28.56%), eicosane (16.18%), heneicosane (10.75%), tetracosane (3.63%), (Z)-9-tricosene (0.94%), and farnesene (0.61%); It also includes alcohols accounting for 14.13%, mainly leaf alcohol (2.20%), cis-myrtanol (1.43%), 2-heptanol (1.23%), myrtenol (0.79%), linalool (0.61%), and geraniol (0.58%); It also includes esters accounting for 6.11%, mainly ethyl linolenate (2.16%), ethyl linoleate (1.64%), ethyl palmitate (1.05%), and methyl palmitate (0.39%); It also includes aldehydes accounting for 1.24%, mainly n-nonanal (0.63%), n-hexanal (0.21%), and 1-octanal (0.13%); It also includes carboxylic acids accounting for 1.17%, mainly palmitic acid (1.17%); It also includes ethers accounting for 0.29%, mainly cis-rose oxide (0.20%) and trans-rose oxide (0.09%).

3. A method for preparing anti-inflammatory volatile oil from Euonymus japonicus flowers, characterized in that: The invention comprises the anti-inflammatory volatile oil of Euonymus japonicus flower according to any one of claims 1 and 2: Including the extraction of essential oils, Weigh 200g of Euonymus japonicus flower, place it in a 5000mL round-bottom flask, add 3000mL of ultrapure water, soak overnight, heat with a constant temperature electric heating mantle, and keep the temperature at 180℃~200℃; add 250mL of dichloromethane to the 500mL round-bottom flask, heat in an oil bath, keep the temperature at about 80℃, and use a simultaneous distillation extractor to extract for 8h. The dichloromethane extract is separated, dried over anhydrous magnesium sulfate to remove water, filtered, and rotary evaporated to obtain 1.84g of light yellow oily substance, which is placed in a refrigerator at 4℃ for future use. The volatile oil extraction rate is 0.92%; Among them; volatile oil GC-MS analysis conditions Chromatographic conditions: Agilent HP-5MS (30m×0.25mm×0.25μm) capillary column, carrier gas: He, flow rate: 1mL / min. Initial temperature: 40℃, maintained for 5min, heated to 280℃ at 5℃ / min, maintained for 5min, injection port temperature was 290℃, injection volume was 1μL, no split; Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70eV; interface temperature 260℃; quadrupole temperature 150℃; mass scanning range m / z: 30~500amu; acquisition mode: full scan mode; retention index (rentention index) Determination of retention index (RI) [9]: C7-C30 normal alkane standards were subjected to GC-MS analysis under the same conditions as above, and their retention times were determined. The retention index RI of each component of the volatile oil was calculated according to the linear formula: RI = 100n + 100 [tR(x) - tR(n)] / [tR(n+1) - tR(n)], where tR(x) is the retention time of the component being analyzed, tR(n) and tR(n+1) are the retention times of normal alkanes with n and n+1 carbon atoms, respectively, and tR(n) <tR(x)<tR(n+1)。

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