A kind of frankincense volatile oil and its extraction method and application

Abstract

The present invention relates to the technical field of natural plant extract products, and specifically discloses a kind of frankincense volatile oil and its extraction method and application. The present invention adopts the idea of ​​composite enzymolysis-agar extraction, and in the enzymolysis process, by coordinating surfactants and metal ion solutions, combined with ultrasonic technology, most of the frankincense volatile oil in the composite enzyme is dissolved in the enzymolysis solution; further, acetone and glycerol are used as auxiliary reagents during agar extraction, acetone and β-glucanase in the composite enzyme are used in a synergistic combination, and agar is dissolved at a lower temperature and forms a semi-colloidal state by changing the agar structure, and the addition of glycerol can make the frankincense raw material and agar fully mixed, and can also synergize with agar, so that the composite enzyme and the reaction substrate that has not undergone enzymolysis reaction or enzymolysis reaction is not thorough undergo secondary enzymolysis reaction, and agar extraction is assisted by microwave heating to promote the rapid release of volatile oil in frankincense cells; through the specific extraction process provided by the present invention, the extraction efficiency and oil yield are greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of natural plant extraction products, in particular to frankincense volatile oil and an extraction method and application thereof. Background Art

[0002] Frankincense comes from the cortical resin of the small tree Boswellia carlesii and the small tree Boswellia carlesii of the olive family. The main producing areas of frankincense include Somalia, Ethiopia, southern Arabian Peninsula, Turkey, Libya, Sudan and Egypt in Africa. It is a traditional medicine widely used in India, China and Arabia.

[0003] Frankincense is yellow-white and translucent on the surface. The color deepens with long storage. It is brittle and softens when heated. The broken surface has a glassy or waxy luster. Frankincense mainly contains volatile oils, resins and gums. It has the effects of activity, swelling and pain relief. Its potential has been widely recognized, especially in anti-inflammatory, bactericidal, neuromodulatory and anti-tumor applications.

[0004] Volatile oil is one of the important components of frankincense, and studies have shown that frankincense volatile oil has a good therapeutic effect on certain diseases. However, due to the low content of volatile oil in frankincense, it is necessary to extract the volatile oil in frankincense by an extraction method. At present, the extraction method for frankincense volatile oil mostly adopts steam distillation or solvent extraction, but these methods have the problems of low oil yield, high cost, huge waste of resources. Therefore, it is of great significance to provide a method for extracting frankincense volatile oil to improve oil yield. Summary of the invention

[0005] Aiming at the problem of low oil yield in the extraction process of frankincense volatile oil in the prior art, the present invention provides frankincense volatile oil and an extraction method and application thereof.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] The first aspect of the present invention provides a method for extracting frankincense volatile oil, comprising the following steps:

[0008] Step 1, soaking the frankincense raw material in water, then adding a complex enzyme, a surfactant and a metal ion solution, adjusting the pH to 5.8-6, and performing enzymolysis at 38° C.-42° C. under ultrasonic conditions to obtain an enzymolysis solution;

[0009] Step 2, heating the enzymatic hydrolyzate to 45°C-48°C, adding agar, acetone and glycerol, letting it stand for 15min-30min, and then heating it to 93°C-95°C for microwave extraction, collecting the distillate, extracting, drying, and concentrating under reduced pressure to obtain the frankincense volatile oil;

[0010] Wherein, the complex enzyme is a complex enzyme of cellulase, galacturonase, protease and β-glucanase;

[0011] The metal ion solution is a metal ion solution containing calcium ions and magnesium ions;

[0012] The surfactant is a mixture of Tween 20 and Triton X-100.

[0013] Compared with the prior art, the preparation method of frankincense volatile oil provided by the present invention first utilizes a specific enzymolysis solution, a surfactant and a metal ion solution to enzymolyze the frankincense raw material, wherein the combined use of cellulase, galacturonase and protease in the complex enzyme is beneficial to destroying cell walls and improving the enzymolysis efficiency; further, the inventors have found through a large number of screenings that the addition of beta-glucanase is beneficial to the action with the other three enzymes, making the enzymolysis reaction more thorough; the addition of a specific surfactant can increase the contact area between the frankincense raw material and the complex enzyme, and increase the enzymolysis efficiency; the present invention also adds specific metal ions during the research process, and the addition of the surfactant is beneficial to dispersing calcium ions and magnesium ions, and the calcium and magnesium ions can react with some inorganic substances in the frankincense raw material to improve its fluidity, thereby improving the enzymatic reaction. The calcium and magnesium ions are combined with the complex enzyme, and the stability and catalytic effect of the complex enzyme are improved by adjusting the conformation of the enzyme. The calcium and magnesium ions can also react with polysaccharides, proteins, etc. in frankincense to form a complex reaction, which can not only change the molecular structure of the frankincense raw material and improve the enzymolysis efficiency, but also increase the contact area between the frankincense raw material and the complex enzyme by combining with the complex enzyme and the frankincense raw material, give full play to the interaction between the frankincense raw material and the complex enzyme, and greatly improve the extraction rate of frankincense volatile oil. In the enzymolysis process, the present invention uses an ultrasonic process as an auxiliary, and the cavitation and vibration of the ultrasonic wave are conducive to improving the destruction rate of the cell wall, and can also accelerate the frankincense volatile oil in the frankincense raw material to be fully dissolved in the solvent, and further reduce the enzymolysis temperature to improve the extraction rate of frankincense volatile oil.

[0014] The invention further processes the prepared enzymolysis solution, and adds agar, acetone and glycerol therein, wherein agar can form a semi-colloidal state in water, has extremely high stability, and forms a three-dimensional network structure, which is conducive to adsorbing frankincense volatile oil and reducing the loss rate of frankincense volatile oil; however, agar may be insoluble in water at a relatively low temperature, and the invention unexpectedly finds that further adding acetone to the enzymolysis solution can synergize with beta-glucanase in the composite enzyme, change the structure of agar, and thus promote the solubility of agar at a relatively low temperature, so that the agar can form a semi-colloidal state at a low temperature, and thus can prevent the frankincense volatile oil from volatilizing due to the excessively high temperature of dissolving agar; the addition of glycerol is conducive to making the remaining frankincense in the enzymolysis The raw materials and agar are mixed uniformly, thereby improving the compatibility of frankincense volatile oil, thereby improving the extraction rate of frankincense volatile oil; the agar and glycerol added in the enzymolysis solution can increase the contact area between the frankincense tissue and the composite enzyme, so that the composite enzyme can better penetrate into the frankincense cells, which is conducive to the composite enzyme to better perform secondary enzymolysis reaction with the reaction substrate that has not been enzymolyzed or is not completely enzymolyzed, and the frankincense volatile oil dissolved by the enzymolysis reaction can be better adsorbed in the agar in a semi-colloidal state, thereby greatly improving the extraction rate of the frankincense volatile oil; the present invention further uses a microwave extraction method in combination with agar, and microwave heating is also conducive to further promoting the rapid release of volatile oil in frankincense cells. The synergistic effect of the two means is conducive to obtaining a more efficient extraction rate in a shorter time.

[0015] The invention adopts the idea of ​​composite enzymolysis-agar extraction. In the enzymolysis process, a surfactant and a metal ion solution are matched and combined with an ultrasonic process to improve the enzymolysis efficiency, so that most of the frankincense volatile oil in the composite enzyme is dissolved in the enzymolysis solution. Furthermore, acetone and glycerine are used as auxiliary reagents during agar extraction. The acetone and the beta-glucanase in the composite enzyme are used in a coordinated manner. The agar structure is changed so that the agar can be dissolved at a relatively low temperature and form a semi-colloidal state. The addition of glycerine can not only fully mix the frankincense raw material and the agar, but also synergize with the agar to cause the composite enzyme and the reaction substrate that has not undergone an enzymolysis reaction or has not undergone a thorough enzymolysis reaction to undergo a secondary enzymolysis reaction. The dissolved frankincense volatile oil is adsorbed in the agar in the semi-colloidal state, thereby reducing the loss of the frankincense volatile oil. In addition, the agar extraction is supplemented with microwave heating to promote the rapid release of the volatile oil in the frankincense cells. The specific extraction process provided by the invention greatly improves the extraction efficiency and the oil yield.

[0016] The method for extracting frankincense volatile oil provided by the invention is simple to operate, has a short process time, and has a high oil yield of the obtained frankincense volatile oil, thus providing a new idea for the development of extracting frankincense volatile oil.

[0017] Preferably, in step 1, the volatile oil content of the frankincense raw material is 5.2%-5.8%.

[0018] Preferably, in step 1, the particle size of the frankincense raw material is 15 mesh to 20 mesh.

[0019] Too fine a grinding may cause some volatile components to evaporate and be lost. By limiting the particle size of the frankincense raw material, it is beneficial to further increase the yield of frankincense volatile oil.

[0020] Preferably, in step 1, the mass ratio of the frankincense raw material to water is 1:(6-8).

[0021] Preferably, in step 1, the temperature of the water is 55°C-60°C.

[0022] Preferably, in step 1, the soaking time is 3h-5h.

[0023] Controlling the water temperature and soaking time can make the frankincense raw material reach an optimal expansion state, improve the efficiency of subsequent enzymatic hydrolysis, and thus increase the yield of frankincense volatile oil.

[0024] Preferably, in step 1, the molar ratio of calcium ions to magnesium ions in the metal ion solution is 1:(0.5-0.7).

[0025] Preferably, in step 1, the concentration of the metal ion solution is 0.7 mol / L-1.1 mol / L.

[0026] Preferably, in step 1, the mass ratio of Tween 20 to Triton X-100 in the surfactant is 1:(1.3-1.5).

[0027] Preferably, in step 1, the mass ratio of cellulase, galacturonase, protease and β-glucanase in the complex enzyme is 1:(0.7-0.9):(0.6-0.8):(0.2-0.3).

[0028] Preferably, in step 1, the mass volume ratio of the frankincense raw material, the complex enzyme, the surfactant and the metal ion solution is 1 g: (1.3-1.5) g: (0.6-0.8) mL: (0.6-0.7) mL.

[0029] Preferably, in step 1, the enzymatic hydrolysis time is 55 min-75 min.

[0030] Preferably, in step 1, the power of the ultrasound is 250W-300W.

[0031] Preferably, in step 1, the ultrasonic time is 20 min-30 min. Preferably, in step 2, after adding agar, acetone and glycerol to the enzymatic hydrolysate, the solution is stirred at a rate of 30 rpm-40 rpm for 1 min-1.5 min.

[0032] The stirring treatment not only accelerates the dissolution of agar, but also helps to further promote the secondary enzymatic hydrolysis reaction of the complex enzyme with the unenzymatically hydrolyzed or incompletely hydrolyzed reaction substrate.

[0033] Preferably, in step 2, the mass ratio of the enzymatic hydrolysate, agar, acetone and glycerol is 1:(0.03-0.05):(0.04-0.06):(0.02-0.04).

[0034] Preferably, in step 2, the power of the microwave extraction is 300W-350W.

[0035] Preferably, in step 2, the condition for ending the microwave extraction is that no distillate flows out.

[0036] Preferably, in step 2, the organic solvent used for the extraction is petroleum ether.

[0037] The second aspect of the present invention provides a frankincense volatile oil obtained by extraction using the above-mentioned frankincense volatile oil extraction method.

[0038] The third aspect of the present invention provides the use of the above-mentioned frankincense volatile oil in the preparation of a product for inhibiting melanin deposition. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] In order to better illustrate the present invention, further examples are given below.

[0041] Example 1

[0042] This embodiment provides a method for extracting frankincense volatile oil, comprising the following steps:

[0043] Step 1, adding 10g of frankincense raw material with a volatile oil content of 5.2% to 60g of 55°C water and soaking for 3h, then adding 13g of complex enzyme, 6mL of surfactant and 6mL of metal ion solution with a concentration of 0.7mol / L, adjusting the pH to 5.8-6, and enzymolyzing at 38°C for 55min, requiring ultrasonic assistance during enzymolysis, the ultrasonic power is 250W, and the ultrasonic time is 20min, to obtain an enzymolysis solution;

[0044] The particle size of the frankincense raw material is 15 meshes, the complex enzyme is a complex enzyme of cellulase, galacturonase, protease and β-glucanase in a mass ratio of 1:0.7:0.8:0.2, the surfactant is a mixture of Tween 20 and TritonX-100 in a mass ratio of 1:1.3; the metal ion solution is a mixed metal ion solution of calcium ions and magnesium ions in a molar ratio of 1:0.5, the calcium ions can be added in the form of calcium chloride, and the magnesium ions can be added in the form of magnesium chloride;

[0045] Step 2, heating the enzymatic hydrolyzate to 45° C., adding agar, acetone and glycerol, stirring at a rate of 30 rpm for 1 min, and then standing for 15 min, heating the solution to 95° C. for microwave extraction, setting the microwave power to 300 W, collecting the distillate until no distillate is left, extracting the distillate with petroleum ether, drying, and concentrating under reduced pressure to obtain frankincense volatile oil;

[0046] Wherein, the mass ratio of the enzymatic hydrolysate, agar, acetone and glycerol is 1:0.05:0.06:0.04.

[0047] Example 2

[0048] This embodiment provides a method for extracting frankincense volatile oil, comprising the following steps:

[0049] Step 1, adding 10g of frankincense raw material with a volatile oil content of 5.8% to 80g of 60°C water and soaking for 5h, then adding 15g of complex enzyme, 8mL of surfactant and 7mL of metal ion solution with a concentration of 1.1mol / L, adjusting the pH to 5.8-6, and performing enzymolysis at 42°C for 75min, requiring ultrasonic assistance during enzymolysis, with an ultrasonic power of 300W and an ultrasonic time of 30min, to obtain an enzymolysis solution;

[0050] The particle size of the frankincense raw material is 20 meshes, the complex enzyme is a complex enzyme of cellulase, galacturonase, protease and β-glucanase in a mass ratio of 1:0.9:0.6:0.3, the surfactant is a mixture of Tween 20 and TritonX-100 in a mass ratio of 1:1.5; the metal ion solution is a mixed metal ion solution of calcium ions and magnesium ions in a molar ratio of 1:0.7, the calcium ions can be added in the form of calcium chloride, and the magnesium ions can be added in the form of magnesium chloride;

[0051] Step 2, heating the enzymatic hydrolyzate to 48° C., adding agar, acetone and glycerol, stirring at a rate of 40 rpm for 1.5 min, and then standing for 30 min, heating the enzymatic hydrolyzate to 93° C. for microwave extraction, setting the microwave power to 350 W, collecting the distillate until no distillate is left, extracting the distillate with petroleum ether, drying, and concentrating under reduced pressure to obtain frankincense volatile oil;

[0052] Wherein, the mass ratio of the enzymatic hydrolysate, agar, acetone and glycerol is 1:0.03:0.04:0.02.

[0053] Example 3

[0054] This embodiment provides a method for extracting frankincense volatile oil, comprising the following steps:

[0055] Step 1, adding 10g of frankincense raw material with a volatile oil content of 5.6% to 70g of 58°C water and soaking for 4h, then adding 14g of complex enzyme, 7mL of surfactant and 6.5mL of 1mol / L metal ion solution, adjusting the pH to 5.8-6, enzymolysis at 40°C for 65min, requiring ultrasonic assistance during enzymolysis, the ultrasonic power is 280W, and the ultrasonic time is 25min to obtain an enzymolysis solution;

[0056] The particle size of the frankincense raw material is 18 meshes, the complex enzyme is a complex enzyme of cellulase, galacturonase, protease and β-glucanase in a mass ratio of 1:0.8:0.7:0.25, the surfactant is a mixture of Tween 20 and TritonX-100 in a mass ratio of 1:1.4; the metal ion solution is a mixed metal ion solution of calcium ions and magnesium ions in a molar ratio of 1:0.6, the calcium ions can be added in the form of calcium chloride, and the magnesium ions can be added in the form of magnesium chloride;

[0057] Step 2, heating the enzymatic hydrolyzate to 47° C., adding agar, acetone and glycerol, stirring at a rate of 35 rpm for 1.3 min, letting stand for 20 min, and then heating with microwaves to 94° C. for microwave extraction, with the microwave power set to 320 W, collecting the distillate until no distillate is left, extracting the distillate with petroleum ether, drying, and concentrating under reduced pressure to obtain frankincense volatile oil;

[0058] Wherein, the mass ratio of the enzymatic hydrolysate, agar, acetone and glycerol is 1:0.04:0.05:0.03.

[0059] Comparative Example 1

[0060] This comparative example provides a method for extracting frankincense volatile oil, which is different from Example 1 in that:

[0061] In step 1, the magnesium ions are replaced with an equal amount of manganese ions;

[0062] The other components and operations were the same as in Example 1.

[0063] Comparative Example 2

[0064] This comparative example provides a method for extracting frankincense volatile oil, which is different from Example 1 in that:

[0065] In step 1, β-glucanase was replaced with an equal amount of cellulase;

[0066] The other components and operations were the same as in Example 1.

[0067] Comparative Example 3

[0068] This comparative example provides a method for extracting frankincense volatile oil, which is different from Example 1 in that:

[0069] In step 1, Tween 20 was replaced with octadecyltrimethylammonium chloride;

[0070] The other components and operations were the same as in Example 1.

[0071] Comparative Example 4

[0072] This comparative example provides a method for extracting frankincense volatile oil, which is different from Example 1 in that:

[0073] In step 2, acetone was replaced with an equal amount of ethanol;

[0074] The other components and operations were the same as in Example 1.

[0075] Comparative Example 5

[0076] This comparative example provides a method for extracting frankincense volatile oil, which is different from Example 1 in that:

[0077] In step 2, replace agar with an equal amount of pectin;

[0078] The other components and operations were the same as in Example 1.

[0079] Calculate the oil yield and transfer rate of frankincense volatile oil obtained in Examples 1-3 and Comparative Examples 1-5, wherein oil yield (%) = volatile oil content / test sample weight*100%, transfer rate (%) = oil yield / theoretical content of volatile oil contained in the raw material*100%;

[0080] The specific test results are shown in Table 1:

[0081] Table 1

[0082]

[0083] The frankincense volatile oil extracted by the present invention was subjected to a drug efficacy test. A total of 6 test groups were set up, and each group was set up with 12 wild zebrafish, specifically wild-type AB Danio rerio, wherein the first group is a control group of zebrafish embryos, the second group is a 100% inhibition group treated with benthiocarbamide, the third group is a positive control group treated with kojic acid, and the fourth to sixth groups are test groups treated with different concentrations of frankincense volatile oil; specifically, the embryos of the first group are cultured in 200 μL of water, the second group is cultured in a 30 mg / L benthiocarbamide solution, the third group is cultured in a 2.5 g / L kojic acid solution, the fourth group is cultured in an aqueous solution containing the frankincense volatile oil extracted from Example 1 with a mass concentration of 0.5‰; the fifth group is cultured in an aqueous solution containing the frankincense volatile oil extracted from Example 1 with a mass concentration of 5‰; the sixth group is cultured in an aqueous solution containing the frankincense volatile oil extracted from Example 1 with a mass concentration of 1%; the specific culture conditions are: cultured at 28°C for 48 hours; after the incubation is completed, the melanin intensity is measured by a microscope (AZ100, Nikon), and analyzed by Image J, and the melanin inhibition rate can be calculated as follows:

[0084] Melanin inhibition rate (%) = (melanin intensity of control group - melanin intensity of experimental group) / (melanin intensity of control group - melanin intensity of inhibition group)

[0085] The specific test results are shown in Table 2:

[0086] Test items Melanin inhibition rate (%) Group 1 0 Group 2 100 Group 3 50 Group 4 40.3 Group 5 85.7 Group 6 92.3

[0087] As shown in Table 2, the frankincense volatile oil extracted by the method for extracting frankincense volatile oil provided by the present invention also has the function of inhibiting melanin deposition, and its melanin inhibition rate reaches 92.3%.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting frankincense volatile oil, characterized in that: The steps include: Step 1, adding frankincense raw materials into water for soaking, then adding complex enzyme, surfactant and metal ion solution, adjusting the pH to 5.8-6, performing enzymolysis at 38° C.-42° C. under ultrasonic conditions to obtain enzymolysis solution; Step 2, heating the enzymatic hydrolyzate to 45°C-48°C, adding agar, acetone and glycerol, letting it stand for 15min-30min, and then heating it to 93°C-95°C with microwaves, performing microwave extraction, collecting the distillate, extracting, drying, and concentrating under reduced pressure to obtain frankincense volatile oil; In step 1, the temperature of the water is 55°C-60°C; In step 1, the soaking time is 3h-5h; In step 1, the molar ratio of calcium ions to magnesium ions in the metal ion solution is 1:(0.5-0.7); In step 1, the mass ratio of cellulase, galacturonase, protease and β-glucanase in the complex enzyme is 1: (0.7-0.9): (0.6-0.8): (0.2-0.3); In step 1, the concentration of the metal ion solution is 0.7 mol / L-1.1 mol / L; In step 1, the mass ratio of Tween 20 to Triton X-100 in the surfactant is 1:(1.3-1.5); In step 1, the mass volume ratio of the frankincense raw material, the complex enzyme, the surfactant and the metal ion solution is 1 g: (1.3-1.5) g: (0.6-0.8) mL: (0.6-0.7) mL; In step 2, the mass ratio of the enzymatic hydrolysate, agar, acetone and glycerol is 1: (0.03-0.05): (0.04-0.06): (0.02-0.04); In step 2, the power of the microwave extraction is 300W-350W.

2. The method for extracting frankincense volatile oil according to claim 1, characterized in that: In step 1, the volatile oil content of the frankincense raw material is 5.2%-5.8%; In step 1, the particle size of the frankincense raw material is 15 meshes to 20 meshes.

3. The method for extracting frankincense volatile oil according to claim 1, characterized in that: In step 1, the mass ratio of the frankincense raw material to water is 1:(6-8).

4. The method for extracting frankincense volatile oil according to claim 1, characterized in that: In step 1, the enzymatic hydrolysis time is 55min-75min; In step 1, the power of the ultrasound is 250W-300W; In step 1, the ultrasonic time is 20min-30min.

5. The method for extracting frankincense volatile oil according to claim 1, characterized in that: In step 2, the condition for ending the microwave extraction is: no distillate flows out.

6. A frankincense volatile oil, characterized in that: The frankincense volatile oil is extracted by the extraction method of any one of claims 1 to 5.

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