A method for extracting and separating β-caryophyllene from meihan grass

Abstract

The present invention discloses a method for extracting and separating β-caryophyllene from *Meehania urticifolia*, belonging to the technical field of bioseparation engineering. The method uses freeze-drying technology combined with liquid nitrogen grinding to obtain *Meehania urticifolia* powder. Ethanol is used as an extractant and combined with ultrasonic-assisted extraction, and then concentrated to obtain a crude extract of β-caryophyllene. Macroporous resin is added to the crude extract for static adsorption, and the macroporous resin saturated with adsorption is loaded into a column for elution. The eluate containing the target compound is collected, and the eluate is concentrated and then loaded onto a C 18 spherical silica gel column, and methanol is used to elute the silica gel column, and the fractions containing the target compound are collected. This method overcomes the problem of difficult separation after liquid-liquid extraction using organic solvents in conventional methods, and at the same time has the advantages of simple and easy process, green and pollution-free, good separation and purification effect, etc. This method is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of biological separation engineering, and particularly relates to a method for extracting and separating β-caryophyllene from Herba Menthae. Background Art

[0002] Meehania (Root of Farges Meehania), first recorded in Flora of China, is also known as nettle-leaved dragonfly grass and sesame flower. It is a perennial herbaceous plant of the genus Farges in the family Lamiaceae. Meehania contains a variety of volatile oils.

[0003] β-Caryophyllene is a bicyclic sesquiterpenoid compound that is widely found in essential oils extracted from various plants. Its molecular formula is C 15 H 24 , whose structural formula is:

[0004]

[0005] β-Caryophyllene plays an important role in food flavoring and fragrance applications and has great potential for treating major human diseases such as cancer, cardiovascular and cerebrovascular diseases, and AIDS. Furthermore, β-caryophyllene's application in bioenergy has also garnered widespread attention. Sesquiterpenoids such as β-caryophyllene can reduce fuel viscosity and increase the cetane number of biofuels, enabling them to meet the requirements of aviation fuel. They are considered one of the most promising high-energy-density aviation fuels of the future.

[0006] Currently, the main extraction and separation methods for sesquiterpenoids include direct pressing, solvent extraction, steam distillation, and supercritical extraction. These methods generally suffer from low extraction efficiency, complex operations, and high solvent usage. Furthermore, it is difficult to isolate the target compound as a single component from the volatile oil. Therefore, developing an efficient, economical, and simple extraction and separation method that can effectively isolate the single component β-caryophyllene while reducing the use of organic solvents has become a pressing issue in this field. Summary of the Invention

[0007] The present invention aims to provide a method for extracting and separating β-caryophyllene from Herba Menthae.

[0008] Specifically, the present invention first provides a method for extracting and isolating β-caryophyllene from Herba Menthae, which is carried out according to the following steps:

[0009] (1) Weigh the freeze-dried Herba Menthae, grind it with liquid nitrogen, sieve it, and place it in a glass container. Add ethanol to extract it at room temperature, and then ultrasonically filter and concentrate it to obtain the crude ethanol extract.

[0010] (2) The crude extract is added to the pretreated macroporous resin for static adsorption to obtain an adsorption-saturated macroporous resin.

[0011] (3) Add the adsorption-saturated macroporous resin into a glass chromatography column and elute it with an organic solvent to obtain an eluate.

[0012] (4) The eluate is concentrated, the concentrate is loaded onto a C18 column, and eluted using a mobile phase to obtain a β-caryophyllene fraction.

[0013] Wherein, in step (1), the raw material freeze-drying time is 12-24 hours, preferably 24 hours, and the grinding mesh size is 40-60 mesh, preferably 60 mesh.

[0014] Wherein, the solid-liquid ratio (g / mL) of the extraction in step (1) is 1:20, the extraction time is 6-8 hours, preferably 8 hours, and the ultrasonic-assisted extraction is 1-2 hours, preferably 2 hours; the parameters of the ultrasonic-assisted extraction are: frequency 20-50kHz, preferably 50kHz, temperature: 20-30°C.

[0015] Wherein, the macroporous adsorption resin in step (2) is a non-polar macroporous resin, preferably one of HPD-100, ADS-5, and D101, and more preferably HPD-100 macroporous resin.

[0016] The method for pretreating the macroporous resin in step (2) is as follows: soaking the macroporous resin in anhydrous ethanol for 24 hours, filtering off the ethanol, rinsing with distilled water until neutral, then soaking it in 4% hydrochloric acid solution for 4 hours, filtering off the acid, and rinsing with distilled water until neutral, then soaking it in 4% sodium hydroxide solution for 4 hours, filtering off the alkali solution, and rinsing with distilled water until neutral, and placing it in distilled water for later use.

[0017] Wherein, in the static adsorption treatment in step (2), the system temperature is 20°C-30°C, preferably 25°C, the pH system is 2.0-6.0, preferably 2.0, and the adsorption time is 3h-5h, preferably 3h.

[0018] Wherein, the macroporous resin eluent in step (3) is alcohol, ketone or alkane, preferably alcohol, more preferably anhydrous ethanol.

[0019] In the macroporous resin elution process in step (3), the amount of eluent used is 6-7 times the volume of the resin column, preferably 7:1 (eluent volume: resin column volume).

[0020] Wherein, in the C18 spherical column adsorption elution process in step (4), the sample loading volume is 3-6 mL, preferably 5 mL.

[0021] Wherein, in the C18 spherical column adsorption elution process in step (4), the elution mobile phase is preferably alcohol, more preferably methanol.

[0022] Wherein, in the C18 spherical column adsorption elution process in step (4), the elution flow rate is 0.5-2.0 mL / min, preferably 1.0 mL / min.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a method for treating the raw material of Herba Menthae using freeze-drying technology and liquid nitrogen grinding process, thereby effectively ensuring the retention of volatile components in Herba Menthae. In addition, ethanol is selected as the extraction agent for extraction at room temperature, and the process is safe and non-toxic. Ultrasound-assisted extraction is then used to ensure the efficient extraction of the target compound.

[0025] The present invention uses a preferred non-polar macroporous resin to adsorb the target compound in the crude extract. Due to similar polarity, β-caryophyllene outcompetes polar impurities during the resin adsorption process, facilitating product enrichment. Furthermore, by selecting a suitable eluent, the target compound is selectively and maximally eluted, achieving a secondary purification effect and effectively ensuring the isolation of the target compound.

[0026] The present invention concentrates the macroporous resin eluate and then applies it to a C18 column for further purification to ensure the purity of the target compound. The fractionated liquid of the product is concentrated to increase the concentration.

[0027] The invention has mild process conditions, simple operation, low solvent consumption and recoverability, and the macroporous resin and C18 column can be reused, thereby overcoming the problems of easy loss and environmental pollution in the process of β-caryophyllene extraction and separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the static adsorption rate of the three macroporous resins;

[0029] Figure 2 This is the liquid phase detection diagram of a single component of β-caryophyllene after separation and purification by macroporous resin-C18 column;

[0030] Figure 3 This is the GC-MS detection diagram of a single component of β-caryophyllene after separation and purification by macroporous resin-C18 column. DETAILED DESCRIPTION

[0031] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention.

[0032] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions were used. All reagents or instruments without manufacturer specified are conventional products that can be purchased through regular channels.

[0033] The adsorption rate and desorption rate of macroporous resin are calculated according to the following formula:

[0034]

[0035] Where: C1, β-caryophyllene concentration in sample solution, mg / mL; C2, β-caryophyllene concentration in adsorption solution, mg / mL; C3, β-caryophyllene concentration in desorption solution, mg / mL; V, volume of adsorption solution, mL; V1, volume of desorption solution, mL.

[0036] Example 1:

[0037] To prepare a crude extract of β-caryophyllene, a certain amount of Herba Menthae was frozen at -80°C for 2 hours, then freeze-dried for 24 hours, dried, placed in a mortar, added with liquid nitrogen and ground through a 60-mesh sieve to obtain solid powder of Herba Menthae. 20 g of the solid powder was weighed and placed in a glass container, 400 mL of anhydrous ethanol was added and extracted for 8 hours, followed by ultrasonic-assisted extraction at 50 kHz and 20°C for 2 hours, filtered and concentrated (concentration factor of 20) to obtain a crude extract with a concentration of 0.725 mg / mL.

[0038] For pretreatment of macroporous resin, first soak HPD-100, ADS-5, and D101 resins in 95% ethanol solution for 24 hours. The amount of ethanol solution is twice that of the resin. Then filter out the solution and rinse with distilled water until the effluent is clear and has no alcohol taste. Then add 3 times the volume of 4% hydrochloric acid solution and soak for 4 hours. After the acid is drained, rinse with distilled water until it is neutral. Finally, soak with 3 times the volume of 4% sodium hydroxide solution for 4 hours. After the alkali solution is drained, rinse with distilled water until it is neutral and set aside.

[0039] Weigh 2 g of each of the three pretreated macroporous resins and place them in a conical flask. Add 50 mL of β-caryophyllene crude extract to each flask, place it in a constant temperature shaker, adjust the system temperature to 25°C and pH to 2.0, and shake at 150 r / min for 5 hours. Take samples to determine the β-caryophyllene content in the residual liquid and calculate the adsorption rate.

[0040] The adsorption rate results are as follows Figure 1 As shown, the results show that the HPD-100 macroporous resin has the highest adsorption rate, reaching 88.57%.

[0041] 8 g of adsorption-saturated HPD-100 macroporous resin was loaded into the column, anhydrous ethanol was used as the eluent, the elution flow rate was set to 1.0 mL / min, the eluent dosage was 6 BV, the β-caryophyllene content in the eluate was determined, and the elution rate was calculated to be 65.5%.

[0042] After the eluate was concentrated, 3 mL was loaded onto a C18 column with methanol as the mobile phase at a flow rate of 0.5 mL / min. Fractions containing the target compound were collected in one tube. The purity of the target compound was 99.4% as determined by HPLC. Figure 2 As shown in the GC-MS test results, Figure 3 shown.

[0043] Example 2:

[0044] To prepare a crude extract of β-caryophyllene, a certain amount of Herba Menthae was frozen at -80°C for 2 hours, then freeze-dried for 24 hours, placed in a mortar after drying, added with liquid nitrogen and ground through a 50-mesh sieve to obtain solid powder of Herba Menthae. 20 g of the solid powder was weighed and placed in a glass container, 400 mL of anhydrous ethanol was added and extracted for 7 hours, followed by ultrasonic-assisted extraction at 40 kHz and 25°C for 2 hours, filtered and concentrated (concentration factor of 20) to obtain a crude extract with a concentration of 0.689 mg / mL.

[0045] The macroporous resin pretreatment method is the same as that in Example 1.

[0046] Weigh 2 g of pretreated ADS-5 macroporous resin and place it in a conical flask. Add 50 mL of crude extract and place it in a constant temperature shaker. Adjust the system temperature to 25°C and pH to 6.0. Oscillate at 150 r / min for 5 hours. Take a sample to determine the β-caryophyllene content in the residual liquid. The adsorption rate is 74.56%.

[0047] 8 g of adsorption-saturated ADS-5 macroporous resin was loaded onto the column, and n-hexane was used as the eluent. The elution flow rate was set to 1.5 mL / min, the eluent dosage was 6 BV, and the β-caryophyllene content in the eluate was determined. The elution rate was calculated to be 60.3%.

[0048] After the eluate was concentrated, 4 mL was loaded onto a C18 column with methanol as the mobile phase at a flow rate of 1 mL / min. Fractions containing the target compound were collected, and the purity was 99.5% according to HPLC.

[0049] Example 3:

[0050] To prepare a crude extract of β-caryophyllene, a certain amount of Herba Menthae was frozen at -80°C for 2 hours, then freeze-dried for 5 hours, placed in a mortar after drying, added with liquid nitrogen and ground through a 40-mesh sieve to obtain solid powder of Herba Menthae. 20 g of the solid powder was weighed and placed in a glass container, 400 mL of anhydrous ethanol was added and extracted for 6 hours, followed by ultrasonic-assisted extraction at 30 kHz and 30°C for 1 hour. After filtration, it was concentrated (concentration factor of 20) to obtain a crude extract with a concentration of 0.652 mg / mL.

[0051] The macroporous resin pretreatment method is the same as that in Example 1.

[0052] Weigh 2 g of pretreated D101 macroporous resin and place it in a conical flask. Add 50 mL of crude extract and place it in a constant temperature shaker. Adjust the system temperature to 30°C and pH to 2.0. Oscillate at 150 r / min for 3 hours. Take a sample to determine the β-caryophyllene content in the residual liquid. The adsorption rate is 83.09%.

[0053] 8 g of adsorption-saturated D101 macroporous resin was loaded into the column, acetone was used as the eluent, the elution flow rate was set to 2.0 mL / min, the eluent dosage was 6 BV, the β-caryophyllene content in the eluate was determined, and the elution rate was calculated to be 55.5%.

[0054] After the eluate was concentrated, 5 mL was loaded onto a C18 column with methanol as the mobile phase at a flow rate of 1.5 mL / min. Fractions containing the target compound were collected, and the purity was 95.5% according to HPLC.

[0055] Example 4:

[0056] The method for preparing the crude β-caryophyllene extract is the same as that in Example 1.

[0057] The macroporous resin pretreatment method is the same as that in Example 1.

[0058] Weigh 2 g of pretreated HPD-100 macroporous resin and place it in a conical flask. Add 50 mL of crude extract and place it in a constant temperature shaker. Adjust the system temperature to 30°C and pH to 6.0. Oscillate at 150 r / min for 5 hours. Take a sample to determine the β-caryophyllene content in the residual liquid. The adsorption rate is 76.34%.

[0059] 8 g of adsorption-saturated HPD-100 macroporous resin was loaded into the column, anhydrous ethanol was used as the eluent, the elution flow rate was set to 2.5 mL / min, the eluent dosage was 7 BV, the β-caryophyllene content in the eluate was determined, and the elution rate was calculated to be 50.3%.

[0060] After the eluate was concentrated, 6 mL was loaded onto a C18 column with methanol as the mobile phase at a flow rate of 2 mL / min. Each 1 mL fraction was collected in one tube. Fractions containing the target compound were collected, and the purity was 90.6% according to HPLC analysis.

[0061] Example 5:

[0062] The method for preparing the crude β-caryophyllene extract is the same as that in Example 1.

[0063] The macroporous resin pretreatment method is the same as that in Example 1.

[0064] Weigh 2 g of pretreated HPD-100 macroporous resin and place it in a conical flask. Add 50 mL of crude extract and place it in a constant temperature shaker. Adjust the system temperature to 20°C and pH to 4.0. Oscillate at 150 r / min for 4 hours. Take a sample to determine the β-caryophyllene content in the residual liquid. The adsorption rate is 84.57%.

[0065] 8 g of adsorption-saturated HPD-100 macroporous resin was loaded into the column, 90% ethanol was used as the eluent, the elution flow rate was set to 1.5 mL / min, the eluent dosage was 7 BV, the β-caryophyllene content in the eluate was determined, and the elution rate was calculated to be 65.3%.

[0066] After the eluate was concentrated, 5 mL was loaded onto a C18 column with anhydrous ethanol as the mobile phase at a flow rate of 1 mL / min. Each 1 mL fraction was collected in one tube. Fractions containing the target compound were collected, and the purity was 96.8% according to HPLC analysis.

[0067] The above embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineers and technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for extracting and separating β-caryophyllene from smilax glabra, characterized in that Follow the steps below: (1) Weigh freeze-dried Herba Menthae, grind with liquid nitrogen, sieve and place in a glass container, add ethanol to extract at room temperature, perform ultrasound-assisted extraction, filter and concentrate to obtain a crude ethanol extract; (2) adding the crude extract to the pretreated macroporous adsorption resin for static adsorption to obtain an adsorption-saturated macroporous adsorption resin; (3) adding the adsorption-saturated macroporous adsorption resin into a glass chromatography column and eluting it with an organic solvent to obtain an eluate; (4) concentrating the eluate, loading the concentrate onto a C18 spherical column, and eluting with a mobile phase to obtain a β-caryophyllene fraction; The macroporous adsorption resin in step (2) is a non-polar macroporous adsorption resin, specifically one of HPD-100, ADS-5, and D101; The method for pretreating the macroporous adsorption resin in step (2) is as follows: soak the macroporous adsorption resin in anhydrous ethanol for 24 hours, filter out the ethanol, rinse with distilled water until neutral, soak it in 4% hydrochloric acid solution for 4 hours, filter out the acid solution, rinse with distilled water until neutral, soak it in 4% sodium hydroxide solution for 4 hours, filter out the alkali solution, rinse with distilled water until neutral, and place it in distilled water for use; In the static adsorption treatment in step (2), the system temperature is 20°C-30°C, and the adsorption time is 3h-5h; the pH of the ADS-5 macroporous adsorption resin is 6.0, the pH of the D101 macroporous adsorption resin is 2.0, and the pH of the HPD-100 macroporous adsorption resin is 6.

0. Wherein, in step (3), the eluent of the macroporous adsorption resin is ethanol; In the macroporous adsorption resin elution process in step (3), the amount of eluent used is 6-7 times the volume of the resin column.

2. The method for extracting and separating β-caryophyllene from Herba Meyeris according to claim 1, characterized in that: In the step (1), the freeze-drying time of the raw material is 12-24 hours, and the grinding mesh size is 40-60 mesh; The solid-liquid ratio in step (1) is 1:20, expressed in g / mL; the extraction time is 6-8 hours, and the ultrasonic-assisted extraction is 1-2 hours; the parameters of the ultrasonic-assisted extraction are: frequency 20-50 kHz, temperature: 20-30°C.

3. The method for extracting and separating β-caryophyllene from Herba Meyeris according to claim 1, characterized in that: In the C18 spherical column adsorption elution process in step (4), the sample loading volume is 3-6 mL. Wherein, in the C18 spherical column adsorption elution process in step (4), the elution mobile phase is alcohol, Wherein, in the C18 spherical column adsorption elution process in step (4), the elution flow rate is 0.5-2.0 mL / min.

4. The method for extracting and separating β-caryophyllene from Herba Meyeris according to claim 2, characterized in that: Wherein, in step (1), the freeze-drying time of the raw material is 24 hours, and the grinding mesh size is 60 mesh; In the step (1), the extraction time is 8 hours, and the ultrasonic-assisted extraction is 2 hours; the parameters of the ultrasonic-assisted extraction are: frequency 50 kHz.

5. The method for extracting and separating β-caryophyllene from Herba Meyeris according to claim 3, characterized in that: The macroporous adsorption resin in step (2) is a HPD-100 macroporous adsorption resin; In the static adsorption treatment in step (2), the system temperature is 25° C., the pH system is 2.0, and the adsorption time is 3 h.

6. The method for extracting and separating β-caryophyllene from Herba Meyeris according to claim 4, characterized in that: Wherein, in step (3), the eluent of the macroporous adsorption resin is anhydrous ethanol; In the macroporous adsorption resin elution process in step (3), the amount of eluent used is calculated as a ratio of eluent volume to resin column volume of 7:

1.

7. The method for extracting and separating β-caryophyllene from Herba Meyeris according to claim 5, characterized in that: Wherein, in the C18 spherical column adsorption elution process in step (4), the sample loading volume is 5 mL; Wherein, in the C18 spherical column adsorption elution process in step (4), the elution mobile phase is methanol; Wherein, in the C18 spherical column adsorption elution process in step (4), the elution flow rate is 1.0 mL / min.

Images