Method for separating antioxidant components from schizonepeta fordii essential oil

Abstract

The invention belongs to the technical field of compound separation and preparation, and particularly relates to a method for separating antioxidant components from schizonepeta fordii essential oil. According to the present invention, n-hexane-ethyl acetate-methanol-water with a volume ratio of 19: 1: 19: (0.99-1.01) is adopted as the two-phase solvent system in the high-speed counter-current chromatography, such that a variety of the antioxidant components, such as 1-octylene-3-ol, alpha-terpineol, piperonone, methyl cyclohexenone, menthone and pulegone, can be effectively separated from the schizonepeta leaf essential oil.

Description

Technical Field

[0001] The invention belongs to the technical field of compound separation, and particularly relates to a method for separating antioxidant components from schizonepeta tenuifolia essential oil. Background Art

[0002] Nepeta tenuifolia is a perennial herbaceous plant of the Lamiaceae family. As the main drug in traditional Chinese medicine and Jingfang granules, it is widely planted in China, Japan and Africa. The stems and leaves of Nepeta tenuifolia are not only used as herbs, but also as ingredients in food, herbal tea, beneficial drinks, and medicinal diets. In terms of pharmacological activity, Nepeta tenuifolia has antibacterial, antioxidant, antipyretic and immunomodulatory effects.

[0003] Natural products play an important role in the treatment of various diseases, and the extraction methods of natural products are relatively complex, including extraction, separation, identification and activity determination. As a traditional Chinese medicine resource, schizonepeta essential oil contains a variety of antioxidant components, which have multiple pharmacological effects on the human body, such as anti-inflammatory, antioxidant, and antibacterial. However, the content of antioxidant components in essential oils is low and the composition is complex. Therefore, the antioxidant components separated from schizonepeta essential oil by conventional gas chromatography, liquid chromatography or gas chromatography-mass spectrometry are relatively single. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for separating antioxidant components from schizonepeta essential oil, which can separate multiple antioxidant components from schizonepeta essential oil.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows.

[0006] A method for separating antioxidant active ingredients from schizonepeta tenuifolia essential oil comprises the following steps:

[0007] Select n-hexane-ethyl acetate-methanol-water to form a two-phase solvent system, pour n-hexane-ethyl acetate-methanol-water in a volume ratio of 19:1:19:0.99-1.01 into a separatory funnel in sequence, mix thoroughly, and let stand until stratification to obtain an upper phase and a lower phase, the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase, and the stationary phase and the mobile phase constitute a two-phase solvent system for high-speed countercurrent chromatography;

[0008] At 18℃~25℃, pump the stationary phase into the main unit at a flow rate of 10mL / min~30mL / min. After the liquid flows out of the outlet, start the centrifuge and pump the mobile phase at a speed of 800r / min~900r / min and a flow rate of 1mL / mi~3mL / min. After the outflowing liquid is separated into upper and lower phases, record the volume of the upper phase and calculate the retention rate of the stationary phase.

[0009] The split-leaf schizonepeta essential oil is mixed with the upper phase and then loaded onto the sample, fractions are collected and tested, fractions containing the same antioxidant components are combined, distilled under reduced pressure, and recrystallized with ethanol to obtain antioxidant active components.

[0010] The invention adopts high-speed countercurrent chromatography, uses n-hexane-ethyl acetate-methanol-water with a volume ratio of 19:1:19:0.99-1.01 as a two-phase system, and can effectively separate multiple antioxidant activities in schizonepeta essential oil at a flow rate of 10 mL / min-30 mL / min for the stationary phase and 1 mL / mi-3 mL / min for the mobile phase.

[0011] In another preferred embodiment, the antioxidant active ingredients are 1-octen-3-ol, α-terpineol, piperonene, methylcyclohexenone, menthone and pulegone.

[0012] In another preferred embodiment, the volume ratio of the Nepeta tenuifolia essential oil to the upper phase is 800 μL-1000 μL:15 mL-25 mL.

[0013] In another preferred embodiment, thin layer chromatography is used for detection, and the specific process is as follows:

[0014] The collected fractions are spotted on a detection plate and placed in a developing agent. After being taken out and blown dry, they are sprayed with a color developer and heated to obtain a color band. The ratio shift value is calculated based on the color band, and fractions with the same ratio shift value are combined.

[0015] In another preferred embodiment, the developing solvent is a mixture of n-hexane and ethyl acetate in a volume ratio of 17:2.99-3.01;

[0016] The color developer is prepared by mixing vanillin, anhydrous ethanol and concentrated sulfuric acid in a mass volume ratio of 2.5 g to 2.52 g: 45 mL: 5 mL; wherein the mass percentage of the concentrated sulfuric acid is not less than 70%.

[0017] In another preferred embodiment, the heating temperature is 60°C to 80°C.

[0018] In another preferred embodiment, the essential oil of Nepeta tenuifolia is prepared by steam distillation.

[0019] In another preferred embodiment, the conditions in the steam distillation method are as follows:

[0020] The mass volume ratio of Schizonepeta tenuifolia to water is 100g-150g:800mL-1500mL; and the distillation time is 3h-5h.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention utilizes high-speed countercurrent chromatography to realize simultaneous elution and pushing, and separates antioxidant components in the split-leaf essential oil in sections. The upper phase and the lower phase are both weakly polar, and n-hexane and water with weak polarity are used as a basic solvent system. Ethyl acetate and methanol, solvents with polarity between that of n-hexane and water, are added to the upper phase and the lower phase to adjust the polarity of the two-phase solvent system, so as to obtain a two-phase solvent system of n-hexane-ethyl acetate-methanol-water with a volume ratio of 19:1:19:0.99-1.01. The antioxidant components in the split-leaf essential oil can be effectively separated, and thin-layer chromatography and bioautographic technology are combined to quickly determine the antioxidant components in the split-leaf schizonepeta essential oil, which are 1-octen-3-ol, α-terpineol, piperonene, methylcyclohexenone, menthone and pulegone. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a picture of the essential oil of Schizonepeta tenuifolia extracted in the embodiment of the present invention.

[0024] Figure 2 Graph showing the total ion current of the Schizonepeta tenuifolia essential oil in an embodiment of the present invention.

[0025] Figure 3 It is a high-speed countercurrent chromatogram of the Schizonepeta tenuifolia essential oil in the embodiment of the present invention.

[0026] Figure 4 1 are thin layer chromatographic analysis graphs of different flow fractions in the embodiments of the present invention; wherein A is a thin layer chromatographic analysis graph of 1 to 10 flow fractions, B is a thin layer chromatographic analysis graph of 11 to 20 flow fractions, C is a thin layer chromatographic analysis graph of 21 to 30 flow fractions, D is a thin layer chromatographic analysis graph of 31 to 40 flow fractions, E is a thin layer chromatographic analysis graph of 41 to 50 flow fractions, and F is a thin layer chromatographic analysis graph of 51 to 60 flow fractions.

[0027] Figure 5 1 and 1 are bioautographic analysis diagrams of different fractions in the examples of the present invention; a is the result after color development using vanillin sulfate, and b is the result after color development using DPPH free radical solution. DETAILED DESCRIPTION

[0028] The technical solutions in the present invention will be described clearly and completely below in conjunction with the specific implementation of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0030] 1. Experimental materials and instruments

[0031] 1.1 Experimental materials

[0032] The medicinal material of Schizonepeta tenuifolia is produced in Anguo, Hebei, and the batch number is: 23062502.

[0033] 1.2 Experimental Reagents

[0034] Table 1 Experimental reagents

[0035]

[0036] 1.3 Experimental instruments

[0037] Table 2 Experimental instruments

[0038] name model factory Gas Chromatography-Mass Spectrometry 7890A-5975C Agilent Technologies High Speed ​​Countercurrent Chromatography TBE300A Shanghai Tongtian Biotechnology Co., Ltd. Electronic balance AL204 Mettler-Toledo Instruments (Shanghai) Co., Ltd. Rotary evaporator RE-2000A Shanghai Yarong Biochemical Instrument Factory Heating plate LC-DB-XAB Junteng Electronic Instrument Co., Ltd.

[0039] 2 Experimental steps and conditions

[0040] 2.1 Qualitative analysis of chemical components of Schizonepeta tenuifolia essential oil

[0041] 1) Extraction of Nepeta tenuifolia essential oil

[0042] Take 150g of Nepeta tenuifolia medicinal material and put it into a 2000mL distillation bottle. After adding water to 1000mL, connect the condenser and the water separator, use steam to distill for 3h, and use steam distillation to collect essential oil. The extracted essential oil is placed in a -4℃ refrigerator for standby use. Finally, the oil yield of Nepeta tenuifolia essential oil is calculated using formula (1):

[0043]

[0044] Where: W is the oil yield of Nepeta tenuifolia essential oil, unit is %; M 1 is the mass of Nepeta tenuifolia, in g; M 2 is the mass of Nepeta tenuifolia essential oil, in g.

[0045] 2) Analysis of essential oils by gas chromatography-mass spectrometry

[0046] The essential oil of Nepeta tenuifolia was mixed with n-hexane at a volume ratio of 1:100, and the essential oil was analyzed by gas chromatography-mass spectrometry.

[0047] Chromatographic conditions: Chromatographic column: HP-5MS (30m×0.25mm×0.25μm) quartz capillary column; Temperature program: initial temperature 60℃ maintained for 3min, increased to 220℃ at 4℃ / min; carrier gas is helium (He), flow rate is 0.8mL / min; Injection mode: split, split ratio is 50:1, injection volume is 0.6μL.

[0048] Mass spectrometry conditions: electron impact (EI) ion source; electron energy 70 eV; transfer line temperature 290 ° C, ion source temperature 230 ° C, quadrupole temperature 150 ° C; sampling frequency 50 Hz, sampling mode: full scan; mass scanning range m / z 40amu ~ 550amu. The experimental results were qualitatively analyzed by MassHunter workstation. Under the same heating program, the C 7 -C 30 Normal alkanes were used as references to compare sample retention times, and the NIST14 spectral library was used to search for chemical compositions of unknown substances.

[0049] 2.2 Separation of essential oils

[0050] 1) Screening of two-phase solvent system

[0051] The distribution coefficient K value of the target components menthone and pulegone in a two-phase solvent system with different compositions and proportions was determined by GC-MS, and the two-phase solvent system was selected according to the K value. The essential oil of schizonepeta tenuifolia was dissolved in the two-phase solvent system, and the upper phase and the lower phase were respectively subjected to GC-MS detection. The distribution coefficient K value was calculated according to formula (2), and the calculation formula of the K value was as follows:

[0052]

[0053] In the formula, A upper is the peak area of ​​the component in the upper phase, A lower is the peak area of ​​the component in the lower phase.

[0054] The specific composition of the two-phase solvent system is shown in Table 3.

[0055] Table 3 Different two-phase solvent systems

[0056]

[0057]

[0058] 2) Separation of antioxidant components in essential oils by high-speed countercurrent chromatography

[0059] Pour the screened solvent system into a separatory funnel at room temperature, mix thoroughly, and let stand to separate (total volume 1000mL), then take the upper phase as the stationary phase and the lower phase as the mobile phase. Ultrasonicate the separated upper and lower phases for 30 minutes, and pour them into the upper phase bottle and lower phase bottle of high-speed countercurrent chromatography respectively. Pump the stationary phase (upper phase) at a flow rate of 20mL / min and wait for the liquid to flow out. Turn on the centrifuge and pump the mobile phase (lower phase) at a speed of 900r / min and a flow rate of 2mL / min. Wait for the outflowing liquid to separate into upper and lower phases, record the volume of the upper phase, and calculate the stationary phase retention rate using formula (3). Dissolve 800μL of essential oil in 20mL of the upper phase, inject with a syringe, record on the workstation, receive the fractions, and after the equipment runs for 300min, stop the host rotation, collect the fractions, and save the data.

[0060]

[0061] Where: R f is the retention rate, in %; V c is the coil volume in mL; V s is a fixed volume in mL.

[0062] 2.3 Screening of antioxidant activity

[0063] 1) TLC-HSCC fraction merging

[0064] n-hexane and ethyl acetate are mixed in a volume ratio of 17:3 to obtain a developing agent, and the developing agent is poured into a developing tank; vanillin, anhydrous ethanol, and concentrated sulfuric acid are mixed in a mass volume ratio of 2.5 g:45 mL:5 mL to obtain a color developer, wherein the mass percentage of concentrated sulfuric acid is 98%.

[0065] Take a silica gel plate, draw a line about 1 cm from the bottom with a pencil, and collect fractions about 1 cm apart on the line with a capillary tube with an inner diameter of about 1 mm for spotting. After spotting, put it into the developing agent, blow dry after development, spray the color developer on it, and bake it at 60°C on a hot plate until the color band appears. Calculate the ratio shift value, and merge the fractions with the same ratio shift value.

[0066] 2) Analysis of antioxidant activity by thin layer chromatography

[0067] The combined fractions were subjected to rotary evaporation and the obtained samples were spotted on two identical silica gel plates. After spotting, the plates were placed in a developing agent for development. After development, the plates were taken out and blown dry. One silica gel plate was sprayed with a color developer and baked on a hot plate to observe the separation phenomenon. The other silica gel plate was dipped in a DPPH free radical solution, placed in a dark place for reaction, and observed whether the color faded.

[0068] 3 Results

[0069] 3.1 Results of qualitative analysis of chemical components of Schizonepeta tenuifolia essential oil

[0070] Take 5kg of Nepeta tenuifolia medicinal materials for steam distillation and extract about 16.5g of essential oil. The oil yield is about 0.33%. The extracted Nepeta tenuifolia essential oil is light yellow and slightly fragrant. Figure 1 shown.

[0071] The total ion current of GC-MS analysis of Nepeta tenuifolia essential oil is shown in Figure 2 As shown. The chemical composition of the essential oil extracted by steam distillation was analyzed by GC-MS. After comparison with the NIST standard spectral library and combined with the retention index comparison, a total of 31 peaks were detected, and 30 compounds were identified, accounting for 99.35% of the total essential oil compounds. These compounds include monoterpene hydrocarbons (3.63%), oxygenated monoterpenes (91.14%), sesquiterpene hydrocarbons (1.90%), oxygenated sesquiterpenes (0.56%), etc. Among them, the compound with the highest content is menthone (47.16%), followed by pulegone (39.97%). Table 4 is a qualitative table of the essential oil of Schizonepeta tenuifolia extracted by steam distillation.

[0072] Table 4 Qualitative properties of Nepeta tenuifolia essential oil extracted by steam distillation

[0073]

[0074]

[0075]

[0076]

[0077] Note: R t is the retention time, RI a Calculate the retention value, RI b The retention time of the n-alkane standard was used to calculate the retention value of the compound.

[0078] 3.2 Analysis results of essential oil separation by high-speed countercurrent chromatography

[0079] 1) Analysis of solvent system screening results

[0080] The invention takes menthone and pulegone as separation targets, realizes simultaneous elution and pushing by high-speed countercurrent chromatography, realizes segmented separation of antioxidant components in split leaf essential oil, adopts upper phase and lower phase with weak polarity, takes weak polarity n-hexane / n-heptane and water as basic solvent system, adds solvent with polarity between n-hexane and water into the upper phase and lower phase to adjust polarity of two-phase solvent system, finally selects n-hexane-ethyl acetate-methanol-water and n-heptane-ethyl acetate-methanol-water solvent system through K value, and the measured distribution coefficients of solvent systems with different volume ratios of menthone and pulegone are shown in Table 5. In n-heptane-ethyl acetate-methanol-water, the distribution coefficients of n-heptane-ethyl acetate-methanol-water are 0.0447 W / v / v, and the distribution coefficients of n-heptane-ethyl acetate-methanol-water are 0.033 W / v / v. In the water system, as the ratio of n-heptane to methanol continued to increase, the distribution coefficients of menthone and pulegone decreased accordingly. However, considering that the boiling point of n-heptane is high, it is not conducive to the separation and purification of the target compound in the later stage. Therefore, the n-hexane-ethyl acetate-methanol-water system was screened. When the volume ratio of n-hexane-ethyl acetate-methanol-water system increased from 4:1:4:1 to 9:1:9:1, the distribution coefficient of menthone in the two phases decreased from 3.514 to 1.894, and the distribution coefficient K of pulegone in the two phases decreased from 2.148 to 1.170; further increasing the volume ratio of n-hexane and methanol in the solvent system can obtain a more ideal distribution coefficient. Therefore, n-hexane-ethyl acetate-methanol-water has a more suitable K value of 0.5-2 when the volume ratio is 19:1:19:1, and it is selected as the optimal solvent system for the separation of menthone and pulegone in schizonepeta essential oil.

[0081] Table 5 Solvent system screening results

[0082]

[0083] 2) High-speed countercurrent chromatography

[0084] After the upper phase fills the coil, the lower phase is pumped in, and stratification occurs. The upper phase volume is 79 mL, and the stationary phase retention rate is calculated to be 71.79%. The sample is added to the high-speed countercurrent chromatography and separated by high-speed countercurrent chromatography at a speed of 900 r / min and a flow rate of 2 mL / min. The high-speed countercurrent chromatogram is obtained as shown below. Figure 3 , which showed four major peaks, and a total of 58 fractions were collected.

[0085] 3.3 Antioxidant activity screening analysis results

[0086] 1) Thin layer chromatography analysis of fractions

[0087] The flow fractions were spotted on the silica gel plate. Figure 4As shown. The results show that different compounds of the schizonepeta essential oil are developed in the same developing solvent to obtain different ratio values. By calculating the ratio value, the fractions with the same ratio value are merged, and the merged fractions are subjected to GC-MS detection. The results of the merger and its chemical composition are shown in Table 6.

[0088] Table 6 Flow fraction merging results

[0089]

[0090] Note: - indicates that no chemical components were detected.

[0091] 2) Thin layer chromatography-bioautography analysis

[0092] The combined fractions were spotted on two identical silica gel plates, and then developed with a developing agent, followed by color development with vanillin sulfate and DPPH free radical solution, respectively. The results were as follows: Figure 5 The results showed that after dipping in DPPH solution, 4 tubes of the fractions showed DPPH free radical solution discoloration spots. The 4 tubes of the fractions were analyzed by GC-MS, and the antioxidant active ingredients were speculated to be 1-octen-3-ol, α-terpineol, piperonene, methylcyclohexenone, menthone and pulegone.

[0093] The invention uses schizonepeta as a raw material, adopts a steam distillation method to extract schizonepeta essential oil, and obtains that the main components of the schizonepeta essential oil are 47.16% of menthone and 39.97% of pulegone by GC-MS analysis; menthone and pulegone with high purity are successfully separated by a high-speed countercurrent chromatography method, and antioxidant active components in the schizonepeta essential oil are quickly screened out by combining a thin-layer chromatography-bioautography technology, which are 1-octen-3-ol, α-terpineol, piperonene, methylcyclohexenone, menthone and pulegone.

[0094] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0095] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for separating antioxidant components from Nepeta tenuifolia essential oil, characterized in that: The following steps are involved: n-hexane-ethyl acetate-methanol-water are mixed in a volume ratio of 19:1:19:0.99-1.01, and allowed to stand until separation to obtain an upper phase and a lower phase, wherein the upper phase is used as a stationary phase and the lower phase is used as a mobile phase, and the stationary phase and the mobile phase constitute a two-phase solvent system for high-speed countercurrent chromatography; At 18°C ​​to 25°C, the stationary phase is pumped in at a flow rate of 10mL / min to 30mL / min, and the mobile phase is pumped in at a flow rate of 1mL / min to 3mL / min. After the mobile phase is equilibrated, the split-leaf schizonepeta essential oil is mixed with the upper phase and then loaded onto the sample. The fractions are collected and tested, and the fractions containing the same antioxidant components are combined, distilled under reduced pressure, and recrystallized with ethanol to obtain the antioxidant components.

2. The method for separating antioxidant components from Nepeta tenuifolia essential oil according to claim 1, characterized in that: The antioxidant ingredients are 1-octen-3-ol, α-terpineol, piperonene, methylcyclohexenone, menthone and pulegone.

3. The method for separating antioxidant components from Nepeta tenuifolia essential oil according to claim 1, characterized in that: The volume ratio of the schizonepeta tenuifolia essential oil to the upper phase is 800 μL to 1000 μL: 15 mL to 25 mL.

4. The method for separating antioxidant components from Nepeta tenuifolia essential oil according to claim 1, characterized in that: Thin layer chromatography was used for detection, and the specific process is as follows: The collected fractions are spotted on a detection plate, placed in a developing agent, blown dry, sprayed with a color developer, and heated to obtain a color band. The ratio shift value is calculated based on the color band, and fractions with the same ratio shift value are combined.

5. The method for separating antioxidant components from Nepeta tenuifolia essential oil according to claim 4, characterized in that: The developing solvent is a mixture of n-hexane and ethyl acetate in a volume ratio of 17:2.99-3.01; The color developer is prepared by mixing vanillin, anhydrous ethanol and concentrated sulfuric acid in a mass volume ratio of 2.5 g to 2.52 g: 45 mL: 5 mL.

6. The method for separating antioxidant components from Nepeta tenuifolia essential oil according to claim 4, characterized in that: The heating temperature is 60°C to 80°C.

7. The method for separating antioxidant components from Nepeta tenuifolia essential oil according to claim 1, characterized in that: The essential oil of Nepeta tenuifolia was prepared by steam distillation.

8. The method for separating antioxidant components from Nepeta tenuifolia essential oil according to claim 7, characterized in that: The conditions in the steam distillation method are specifically as follows: The mass volume ratio of Schizonepeta tenuifolia to water is 100g-150g:800mL-1500mL; and the distillation time is 3h-5h.

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