Method for preparing high-purity 3-acetyl-11-keto-beta-masticinic acid through high-speed countercurrent chromatography separation

Abstract

The invention relates to a method for preparing high-purity 3-acetyl-11-keto-beta-boswellic acid through high-speed countercurrent chromatography separation, which comprises the following steps: by taking commercially available frankincense extract as a raw material, fully shaking a solvent system, standing for phase separation, and separately collecting an upper phase and a lower phase; dissolving the frankincense extract in the upper phase, performing separation and purification by using high-speed counter-current chromatography to obtain a mixed solution of 3-acetyl-11-keto-beta-masticinic acid and the upper phase, removing the organic solvent in the upper phase to obtain a turbid liquid containing 3-acetyl-11-keto-beta-masticinic acid, and centrifuging to remove an aqueous solution to obtain the frankincense extract. And filtering to obtain the 3-acetyl-11-keto-beta-masticinic acid. The purity of the 3-acetyl-11-keto-beta-masticinic acid prepared by the method disclosed by the invention is up to 98% or above; reagents of a solvent system can be recycled and reused, and good energy-saving and environment-friendly functions are achieved.

Description

Technical Field

[0001] This invention belongs to the field of high-efficiency separation and purification technology of effective components of traditional Chinese medicine, and specifically relates to a method for high-purity 3-acetyl-11-keto-β-boswellic acid by high-speed countercurrent chromatography separation. Background Technology

[0002] Frankincense is the resin exuded from the bark of the Boswellia carterii Birdw. and Boswellia bhaurdajiana Birdw., both belonging to the Burseraceae family. Frankincense is pungent and warming, possessing the effects of promoting blood circulation, relieving pain, reducing swelling, and promoting tissue regeneration. It also has anti-inflammatory, antioxidant, and hypoglycemic pharmacological effects. It is mainly used to treat chest pain, stomach pain, dysmenorrhea, amenorrhea, postpartum blood stasis, abdominal masses, rheumatic pain, muscle spasms, and traumatic injuries. In terms of formulations, domestic frankincense-based medicines are mainly compound preparations; single-ingredient frankincense preparations such as tablets and topical ointments are only found in overseas markets. Clinically, it is widely used for dysmenorrhea, amenorrhea, stomach pain, rheumatic pain, traumatic injuries, carbuncles, and intestinal abscesses, making it a plant-based medicine with broad development prospects.

[0003] Current literature reports that the main chemical components isolated from frankincense are pentacyclic triterpenes and macrocyclic diterpenes, among which frankincense acids and macrocyclic diterpenes are the effective components for their anti-inflammatory and anti-tumor effects. The main method for separating and purifying these active components from frankincense is column chromatography, such as silica gel column chromatography. However, this method has drawbacks: long separation cycle, low recovery rate, and unsatisfactory separation effect. Furthermore, prolonged contact with the silica gel packing material during silica gel column chromatography can cause structural changes in these components.

[0004] Some literature reports the use of high-speed countercurrent chromatography to separate and prepare frankincense monomers, but the solvent system used—petroleum ether, ethyl acetate, methanol, and water—is time-consuming, and the solvent cannot be recycled, which is not conducive to large-scale production; the purity of the separated monomers is only 97%. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for high-purity 3-acetyl-11-keto-β-boswellic acid separation and preparation by high-speed countercurrent chromatography. The 3-acetyl-11-keto-β-boswellic acid prepared by this method has a purity of over 98%. The solvent system reagents can be recycled and reused, which has good energy-saving and environmental protection functions.

[0006] This invention provides a method for preparing high-purity 3-acetyl-11-keto-β-boswellic acid by high-speed countercurrent chromatography, comprising the following steps:

[0007] Commercially available frankincense extract was used as the raw material. The solvent system was thoroughly shaken, allowed to stand and separate into two phases, and the upper and lower phases were collected separately. The frankincense extract was dissolved in the upper phase and purified by high-speed countercurrent chromatography to obtain a mixture of 3-acetyl-11-keto-β-boswellic acid and the upper phase. The organic solvent in the upper phase was removed to obtain a suspension containing 3-acetyl-11-keto-β-boswellic acid. The aqueous solution was removed by centrifugation to obtain 3-acetyl-11-keto-β-boswellic acid. The solvent system was prepared by mixing n-heptane, dichloromethane, ethanol and water in a volume ratio of 3-6:0.5-2:3-5:1-3.

[0008] Preferably, the commercially available frankincense extract contains 60% 3-acetyl-11-keto-β-boswellic acid.

[0009] Preferably, the separately collected upper and lower phases are pre-treated with ultrasonic degassing.

[0010] Preferably, the high-speed countercurrent chromatography adopts an operating mode that combines forward rotation with the lower phase as the mobile phase and forward rotation with the upper phase as the mobile phase.

[0011] Preferably, the chromatographic conditions for the high-speed countercurrent chromatography are: rotation speed of 900 rpm; column temperature of 25℃; flow rate of mobile phase of 5 mL / min; and detection wavelength of detector of 254 nm.

[0012] Preferably, the process conditions for removing organic solvents from the upper phase are: rotary evaporation vacuum drying at 55°C and -0.085 MPa.

[0013] The molecular formula of the 3-acetyl-11-keto-β-boswellic acid is C 32 H 48 O5; The structural formula is shown below:

[0014]

[0015] Beneficial effects

[0016] This invention utilizes high-speed countercurrent chromatography to separate and purify high-purity 3-acetyl-11-keto-β-boswellic acid from commercially available frankincense extract, achieving a purity of over 98%. The solvent system and reagents are recyclable, demonstrating excellent environmental protection properties. This invention is highly efficient, simple to operate, stable, allows for large-scale production, and has low overall cost, making it highly valuable for widespread application. Attached Figure Description

[0017] Figure 1 High-speed countercurrent chromatography (HSC) chromatogram of frankincense extract containing 60% 3-acetyl-11-keto-β-boswellic acid.

[0018] Figure 2 The HPLC chromatogram of 3-acetyl-11-keto-β-boswellic acid was obtained by high-speed countercurrent chromatography separation. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0020] The reagents and instruments used in the examples are as follows:

[0021] Reagents: n-Heptane, dichloromethane, and ethanol were all analytical grade reagents produced by Sinopharm Chemical Reagent Co., Ltd.; water was deionized water; and frankincense extract was a commercially available product.

[0022] Instrument: The high-speed countercurrent chromatograph is a TBE-300C model manufactured by Shanghai Tongtian Biotechnology Co., Ltd.

[0023] Example 1

[0024] A solvent system was prepared by mixing n-heptane, dichloromethane, ethanol, and water in a volume ratio of 5:1:3.5:1.5. The mixture was added to a separatory funnel, shaken thoroughly, and allowed to stand for phase separation, yielding a two-phase mixture. The upper and lower solvent phases were collected separately and subjected to ultrasonic degassing in an ultrasonic oscillator. A frankincense extract containing 60% 3-acetyl-11-keto-β-boswellic acid was dissolved in the upper phase and purified using high-speed countercurrent chromatography (HSCLC). The chromatographic conditions were set as follows: rotation speed 900 rpm; column temperature 25℃; mobile phase flow rate 5 mL / min; and detector wavelength 254 nm.

[0025] Taking the completion of sample injection as 0 min, the mobile phase was changed from forward to forward contact with the lower phase for 0-65 min, and from forward to reverse contact with the upper phase for 65-120 min. The mixture of 3-acetyl-11-keto-β-boswellic acid and the upper phase was collected from 95-110 min to obtain a mixture of 3-acetyl-11-keto-β-boswellic acid and the upper phase. The mixture was then placed in a rotary evaporator and subjected to rotary evaporation and vacuum drying at a water bath temperature of 55℃ and a vacuum pressure of -0.085MPa to remove the organic solvent from the upper phase, resulting in a suspension containing 3-acetyl-11-keto-β-boswellic acid. The aqueous solution was removed by centrifugation to obtain high-purity 3-acetyl-11-keto-β-boswellic acid.

[0026] The purity of 3-acetyl-11-keto-β-boswellic acid obtained in this example was analyzed by HPLC, and the results showed that the purity of 3-acetyl-11-keto-β-boswellic acid was 98.88%.

[0027] Example 2

[0028] A solvent system was prepared by mixing n-heptane, dichloromethane, ethanol, and water in a volume ratio of 5:1:3.5:2. The mixture was added to a separatory funnel, shaken thoroughly, and allowed to stand for phase separation to obtain a two-phase mixture. The upper and lower phase solvents were collected separately and subjected to ultrasonic degassing in an ultrasonic oscillator. A frankincense extract containing 60% 3-acetyl-11-keto-β-boswellic acid was dissolved in the upper phase and purified using high-speed countercurrent chromatography (HSCLC). The chromatographic conditions were set as follows: rotation speed 900 rpm; column temperature 25℃; mobile phase flow rate 5 mL / min; and detector wavelength 254 nm.

[0029] Taking the completion of sample injection as 0 min, the mobile phase was changed from forward to forward contact with the lower phase for 0-105 min; the mobile phase was changed from forward to reverse contact with the upper phase for 105-160 min. The mixture of 3-acetyl-11-keto-β-boswellic acid and the upper phase was collected for 135-150 min. The mixture of 3-acetyl-11-keto-β-boswellic acid and the upper phase was then placed in a rotary evaporator and dried under vacuum conditions of 55℃ water bath and -0.085MPa to remove the organic solvent in the upper phase, resulting in a suspension containing 3-acetyl-11-keto-β-boswellic acid. The aqueous solution was removed by centrifugation to obtain high-purity 3-acetyl-11-keto-β-boswellic acid.

[0030] The purity of 3-acetyl-11-keto-β-boswellic acid obtained in this example was analyzed by HPLC, and the results showed that the purity of 3-acetyl-11-keto-β-boswellic acid was 99.19%.

Claims

1. A method for preparing high-purity 3-acetyl-11-keto-β-boswellic acid by high-speed countercurrent chromatography, comprising the following steps: Commercially available frankincense extract was used as the raw material. The solvent system was thoroughly shaken, allowed to stand for phase separation, and the upper and lower phases were collected separately. The frankincense extract was dissolved in the upper phase and purified by high-speed countercurrent chromatography to obtain a mixture of 3-acetyl-11-keto-β-boswellic acid and the upper phase. The organic solvent in the upper phase was removed to obtain a suspension containing 3-acetyl-11-keto-β-boswellic acid. The aqueous solution was removed by centrifugation to obtain 3-acetyl-11-keto-β-boswellic acid. The solvent system is prepared by mixing n-heptane, dichloromethane, ethanol, and water in a volume ratio of 3-6:0.5-2:3-5:1-3.

2. The method according to claim 1, characterized in that: The commercially available frankincense extract contains 60% 3-acetyl-11-keto-β-boswellic acid.

3. The method according to claim 1, characterized in that: The upper and lower phases collected separately are pre-treated with ultrasonic degassing.

4. The method according to claim 1, characterized in that: The high-speed countercurrent chromatography employs an operating mode that combines forward rotation with the lower phase as the mobile phase and forward rotation with the upper phase as the mobile phase.

5. The method according to claim 1, characterized in that: The chromatographic conditions for the high-speed countercurrent chromatography are as follows: rotation speed of 900 rpm; column temperature of 25℃; flow rate of mobile phase of 5 mL / min; and detection wavelength of detector of 254 nm.

6. The method according to claim 1, characterized in that: The process conditions for removing organic solvents from the upper phase are: rotary evaporation vacuum drying at 55°C and -0.085 MPa.

Images