Extraction method and application of lysimachia foenum-graecum volatile
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN POLYTECHNIC
- Filing Date
- 2024-11-25
- Publication Date
- 2026-05-26
Smart Images
Figure CN122081007A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bio-extraction technology, and in particular to a method for extracting volatile oil from *Gynostemma pentaphyllum* and its application. Background Technology
[0002] Lavender volatile oil is extracted from lavender (Lysimachia fenum-graecum Hance), a plant belonging to the genus Lysimachia in the Primulaceae family. Also known as lavender or herb, it is mainly produced in Guangxi, Guangdong, and Yunnan provinces in my country. It is a valuable raw material for the fragrance industry and is widely used to flavor high-end cigarettes and alcohol. Lavender also has a pungent, sweet, and warm nature, and has the effects of clearing heat, promoting qi circulation, relieving pain, and expelling parasites. It is used to treat colds, headaches, toothaches, sore throats, chest tightness, and abdominal distension, thus having high medicinal value.
[0003] Currently, there are many methods for extracting plant essential oils, and the conditions for different methods vary considerably, resulting in different extraction rates. Furthermore, research has found that the extraction method significantly impacts the aroma, composition, and activity of essential oils. The traditional method for extracting plant essential oils is steam distillation. This method is simple, inexpensive, and widely used, suitable for extracting highly volatile components that are not easily destroyed by high temperatures and do not react with water. However, it is not suitable for extracting heat-labile components. However, steam distillation has drawbacks such as long extraction time, high energy consumption, and a tendency to lose volatile, heat-sensitive, and polar molecules from the essential oil, leading to lower quality essential oils. Summary of the Invention
[0004] This invention provides a method for extracting volatile oil from *Gynura divaricata* and its application, solving the problem of low extraction quality of volatile oil from *Gynura divaricata* in the prior art. Specifically, it is achieved through the following techniques.
[0005] In a first aspect, the present invention provides a method for extracting volatile oil from *Gynura divaricata*, comprising the following steps:
[0006] The *Lingxiang* micro powder and the enzymatic decomposition system were added to the bacterial culture mixture for fermentation to obtain *Lingxiang* fermentation broth.
[0007] The fermented broth of the fragrant grass was sequentially subjected to ultrasonic extraction and microwave extraction, and then freeze-dried into a fine fermented powder.
[0008] The fermented powder was extracted using supercritical CO2 fluid extraction to obtain a paste.
[0009] The paste was subjected to solvent extraction to obtain a solvent extract; the solvent extract was then separated and purified to obtain volatile oil of *Gynura divaricata*.
[0010] The microbial mixture includes Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689, and Aspergillus niger CICC41796; the amount of microorganisms added to the microbial mixture is 0.01-0.1 g / L based on the total volume of the fermentation system.
[0011] The enzymatic degradation system includes flavin mononucleotide (FMN), lipoic acid (prosthetic group L), pyrophosphate (prosthetic group TPP), and pyridoxine phosphate (prosthetic group P-CH2NH2); the amount of the enzymatic degradation system added is 0.2-0.5 g / L based on the total volume of the fermentation system.
[0012] The *Aspergillus oryzae* CICC40108, *Penicillium citrinum* CICC2689, and *Aspergillus niger* CICC41796 used in this invention can all produce various cellulases, including cellulase, β-glucanase, and xylanase. These enzymes can decompose cellulose into cellulose monomers or oligosaccharides with lower polymerization degrees, disrupt the cell wall structure, and promote the release of volatile oil from *Gynostemma pentaphyllum*. The four raw materials in the enzymatic decomposition system of this invention are all coenzymes, capable of hydrolyzing or degrading the components of the cell wall (cellulose, hemicellulose, and pectin), disrupting the cell wall structure, and dissolving, suspending, or gelling the intracellular components in the solvent, thus promoting the release of volatile oil from *Gynostemma pentaphyllum*. This invention, by combining cellulose-decomposing bacteria with an enzymatic reaction system to extract volatile oil from *Gynostemma pentaphyllum*, fully utilizes the synergistic effect of both, significantly improving the extraction efficiency of volatile oil from *Gynostemma pentaphyllum*.
[0013] Further, the mass ratio of Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689, and Aspergillus niger CICC41796 is (0.4-0.45):(0.42-0.52):(0.03-0.18).
[0014] Further, the composition consists of flavin mononucleotide (FMN), lipoic acid (prosthetic group L), pyrophosphate (prosthetic group TPP), and pyridoxine phosphate (prosthetic group P-CH2NH2), with a mass ratio of (0.3-0.38):(0.1-0.15):(0.4-0.45):(0.02-0.2).
[0015] Furthermore, the mass ratio of the *Lysimachia christinae* micro powder to the bacterial strain is 100:(0.1-0.3).
[0016] Optionally, the fermentation conditions include: a fermentation temperature of 25-35℃ and a fermentation time of 120-144 h.
[0017] Optionally, the microbial mixture is prepared by inoculating Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689 and Aspergillus niger CICC41796 into a fermentation medium and then activating them.
[0018] Optionally, the activation treatment conditions include: adjusting the pH of the fermentation medium to neutral, the culture temperature to 28-30℃, and the culture time to 60-80 h.
[0019] Optionally, the ultrasonic extraction process is as follows: extraction at a temperature of 33-42℃ and an ultrasonic power of 60-180 W for 45-60 min; the microwave extraction process is as follows: extraction at a microwave power of 200-600 W for 60-120 s.
[0020] Secondly, the present invention also provides a volatile oil of *Gynura divaricata*, which is obtained by the above-described extraction method.
[0021] Thirdly, the present invention also provides the application of volatile oil of *Gynura divaricata* in the preparation of mosquito repellent dispersion.
[0022] Optionally, the mass percentage of geranium volatile oil in the mosquito repellent dispersion is 4-12%.
[0023] The volatile oil of *Lingcao Xiang* obtained by this invention has a strong scavenging ability against DPPH free radicals, thus exhibiting strong antioxidant properties. When the obtained *Lingcao Xiang* volatile oil is applied to mosquito repellent treatment, it has a good mosquito repellent effect.
[0024] Compared with the prior art, the advantages of the present invention are:
[0025] 1. The *Aspergillus oryzae* CICC40108, *Penicillium citrinum* CICC2689, and *Aspergillus niger* CICC41796 used in this invention can all produce various cellulases, including cellulase, β-glucanase, and xylanase. These enzymes can decompose cellulose into cellulose monomers or oligosaccharides with lower polymerization degrees, disrupt the cell wall structure, and promote the release of volatile oil from *Gynostemma pentaphyllum*. The enzymatic decomposition system in this invention can hydrolyze or degrade the components of the cell wall (cellulose, hemicellulose, and pectin), disrupting the cell wall structure and dissolving, suspending, or gelling the intracellular components in the solvent, thus promoting the release of volatile oil from *Gynostemma pentaphyllum*. This invention, by combining cellulose-decomposing bacteria with an enzymatic reaction system to extract volatile oil from *Gynostemma pentaphyllum*, fully utilizes the synergistic effect of both, significantly improving the extraction efficiency of volatile oil from *Gynostemma pentaphyllum*.
[0026] 2. The volatile oil of *Lingcao Xiang* obtained by this invention has a strong scavenging ability against DPPH free radicals, thus exhibiting strong antioxidant properties. The obtained *Lingcao Xiang* volatile oil was mixed with D-limonene to prepare a mosquito repellent dispersion. The experimental results showed that the volatile oil of *Lingcao Xiang* had a good mosquito repellent effect. Attached Figure Description
[0027] Figure 1 The infrared spectrum of the vanilla essential oil prepared in Example 3 is shown. In the figure, 2951.28 cm⁻¹... -1 The infrared peak at 1655.95 cm⁻¹ is the out-of-plane antisymmetric stretching vibration peak of CH. -1 The stretching vibration peak belongs to the C=O group of the aldehyde group, at 1372.55 cm⁻¹. -1 1450.9 cm -1 The peak at 876.31 cm⁻¹ belongs to the in-plane stretching vibration peak of the C=C group in olefins. -1 The nearby characteristic peaks indicate that it contains a cyclic structure, which is consistent with the standard infrared spectrum of D-limonene, indicating that lingonberry essential oil contains D-limonene.
[0028] Figure 2 The image shows the gas chromatography-mass spectrometry (GC-MS) spectrum of the vanilla essential oil prepared in Example 3. In the spectrum, D-limonene is present at a retention time of 6.558, and its peak area reaches 80%, indicating that the main component of the prepared vanilla essential oil is D-limonene.
[0029] Figure 3-5 Infrared spectra of the essential oils of *Gynura divaricata* prepared in Comparative Examples 1-3.
[0030] Figure 6-8 The images shown are gas chromatography-mass spectra of the essential oil of *Lysimachia christinae* prepared in Comparative Example 4.
[0031] Figure 9 The gas chromatogram of the essential oil of *Lysimachia christinae* prepared in Comparative Example 5 is shown.
[0032] Figure 10-12 The image shows a gas chromatography-mass spectrometry (GC-MS) image of the lingxiang essential oil prepared in Comparative Example 6. Detailed Implementation
[0033] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0034] In some embodiments of the present invention, the method for extracting the volatile oil of *Gynostemma pentaphyllum* includes the following steps:
[0035] S1. Add the vanilla powder and enzymatic decomposition system to the bacterial culture mixture for fermentation to obtain vanilla fermentation broth.
[0036] Among them, the ginseng powder is obtained by ball milling, freeze drying and sieving of ginseng. First, the dried ginseng is ball milled with liquid nitrogen using a nano planetary ball mill to obtain fine powder with a particle size of 10-40 μm.
[0037] In some embodiments of the present invention, the grinding parameters of the nano-planetary ball mill are as follows: electromagnetic frequency 10 Hz, liquid nitrogen filling, agate grinding jar 200 ml, revolution speed 600 rpm, rotation speed 800 rpm, and running time 10 min.
[0038] The fine powder obtained by freeze-drying the ball-milled powder and then sieving it to obtain a 200-300 mesh powder is called lingxiang micro powder.
[0039] In some embodiments of the present invention, the freeze-drying conditions are: -60~-50℃, 2-10 Pa, 48-72 h.
[0040] The microbial mixture includes Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689, and Aspergillus niger CICC41796.
[0041] The enzymatic degradation system includes flavin mononucleotide (FMN), lipoic acid (prosthetic group L), pyrophosphate (prosthetic group TPP), and pyridoxine phosphate (prosthetic group P-CH2NH2).
[0042] S2. The above-mentioned fermented broth of *Lingxiangcao* is successively subjected to ultrasonic extraction and microwave extraction, and then freeze-dried into a fine fermented powder.
[0043] The paste was obtained by supercritical CO2 fluid extraction.
[0044] The paste was subjected to solvent extraction to obtain a solvent extract, which was then separated and purified to obtain the volatile oil of *Gynura divaricata*.
[0045] The *Aspergillus oryzae* CICC40108, *Penicillium citrinum* CICC2689, and *Aspergillus niger* CICC41796 mentioned in this invention can all produce a variety of cellulases, including cellulase, β-glucanase, and xylanase. These enzymes can decompose cellulose into cellulose monomers or oligosaccharides with lower polymerization degree, disrupt the cell wall structure, and promote the release of volatile oil from *Gynura divaricata*.
[0046] The enzymatic decomposition system in this invention can hydrolyze or degrade the components of the cell wall (cellulose, hemicellulose, and pectin), disrupting the cell wall structure and allowing intracellular components to dissolve, suspend, or gel in the solvent, thus promoting the release of volatile oil from *Gynostemma pentaphyllum*. This invention, by combining cellulose-decomposing bacteria with an enzymatic reaction system to extract volatile oil from *Gynostemma pentaphyllum*, fully leverages the synergistic effect of both, significantly improving the extraction efficiency of volatile oil from *Gynostemma pentaphyllum*.
[0047] In some embodiments of the present invention, the mass ratio of Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689, and Aspergillus niger CICC41796 is (0.4-0.45):(0.42-0.52):(0.03-0.18).
[0048] In some embodiments of the present invention, the amount of microorganisms added to the microbial mixture is 0.01-0.1 g / L, based on the total volume of the fermentation system.
[0049] In some embodiments of the present invention, the mass ratio of flavin mononucleotide (FMN), lipoic acid (prosthetic group L), pyrophosphate (prosthetic group TPP), and pyridoxine phosphate (prosthetic group P-CH2NH2) is (0.3-0.38):(0.1-0.15):(0.4-0.45):(0.02-0.2).
[0050] In some embodiments of the present invention, the amount of the enzymatic decomposition system added is 0.2-0.5 g / L, based on the total volume of the fermentation system.
[0051] In some embodiments of the present invention, the mass ratio of the *Lysimachia christinae* micro powder to the bacterial strain is 100:(0.1-0.3).
[0052] In some embodiments of the present invention, the fermentation conditions include: a fermentation temperature of 25-35°C and a fermentation time of 120-144 h.
[0053] In some embodiments of the present invention, the above-mentioned bacterial mixture is prepared by inoculating Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689 and Aspergillus niger CICC41796 into a fermentation medium and then activating them.
[0054] In some embodiments of the present invention, the conditions for the activation treatment include: adjusting the pH of the fermentation medium to neutral, the culture temperature to 28-30°C, and the culture time to 60-80 h.
[0055] Optionally, the pH of the 0014 PDA medium can be adjusted to neutral using a citrate-disodium hydrogen phosphate buffer solution.
[0056] In some embodiments of the present invention, the above-mentioned ultrasonic extraction process is as follows: extraction is performed at a temperature of 33-42°C and an ultrasonic power of 60-180W for 45-60 minutes.
[0057] In some embodiments of the present invention, the microwave extraction process is as follows: extraction is performed for 60-120 seconds at a microwave power of 200-600 W.
[0058] In some embodiments of the present invention, the extraction process of the supercritical CO2 fluid extraction method is as follows: the fermented powder is soaked and swollen in n-hexane, and then extracted with supercritical CO2 fluid. After the supercritical CO2 fluid extraction is completed, the n-hexane is removed by rotary evaporation to obtain a paste.
[0059] The extraction conditions for supercritical CO2 fluid extraction are as follows: extraction pressure of 15-25 MPa, material-to-liquid ratio of 1:6-18 g / mL, extraction time of 60-240 min, and temperature of 36-48℃.
[0060] Specifically, the obtained paste is extracted sequentially with different solvents to separate the aqueous layer and collect the solvent extracts.
[0061] More specifically, the sample was extracted three times each with chloroform, ethyl acetate and n-butanol, and the extracts from the three extractions were combined.
[0062] Specifically, the solvent extract is separated and purified to obtain the volatile oil of *Gynostemma pentaphyllum*. The specific steps include: First, the solvent extract is mixed with Sevag reagent in an equal proportion, stirred at room temperature for 30 min, centrifuged at low temperature, and then introduced into a separatory funnel to collect the supernatant solution.
[0063] The Sevag reagent consists of chloroform and methanol in a volume ratio of 10:(1-1.5).
[0064] Then, the collected supernatant solution was centrifuged to remove the precipitate, and this process was repeated until no precipitate formed at the interface between the upper and lower layers. Four times the volume of anhydrous ethanol was added to the above solution, and the mixture was allowed to stand for 24 hours. After rotary evaporation under reduced pressure, the solution was dispersed in distilled water and dialyzed against running water for 72 hours. The supernatant was collected and frozen at low temperature to separate the ice crystals, yielding purified vanilla volatile oil.
[0065] The conditions for low-temperature centrifugation were: temperature -3 to 2℃, rotation speed 8000 rpm / min, and centrifugation time 10 min.
[0066] The present invention uses the above method to extract and prepare the volatile oil of *Lysimachia christinae* into a mosquito repellent dispersion for practical application.
[0067] Optionally, the mass concentration of geranium volatile oil in the mosquito repellent dispersion is 4-12%.
[0068] The volatile oil of *Lingcao Xiang* obtained by this invention has a strong scavenging ability against DPPH free radicals, thus exhibiting strong antioxidant properties. When the obtained *Lingcao Xiang* volatile oil is applied to mosquito repellent treatment, it has a good mosquito repellent effect.
[0069] The following description is based on specific embodiments and comparative examples.
[0070] Example 1
[0071] The extraction method of volatile oil from *Gynura divaricata* in this embodiment includes the following steps:
[0072] (1) Prepare 100 ml of 0014 PDA medium with 0.1 mol / L citric acid and 0.1 mol / L disodium hydrogen phosphate buffer solution, and adjust the pH value to 6.6-7.4; add 1 g of vanilla powder, 2 mg of inoculum and 0.03 g of enzymatic decomposition system and mix well before fermentation. In the fermentation system, the concentration of vanilla powder (i.e., the material-to-liquid ratio) is 10 g / L, the concentration of inoculum (i.e., the material-to-liquid ratio) is 0.02 g / L, and the concentration of enzymatic decomposition system (i.e., the material-to-liquid ratio) is 0.3 g / L.
[0073] The fungal strains used in this embodiment are a mixture of Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689, and Aspergillus niger, with an activity of (4000-50000 IU) and a mass ratio of 0.42:0.45:0.13.
[0074] The fermentation conditions used in this embodiment are: fermentation temperature 25℃, culture time 80 h.
[0075] In the enzymatic degradation system used in this embodiment, the mass ratio of flavin mononucleotide, lipoic acid, pyrophosphate, and pyridoxine phosphate is 0.3:0.1:0.4:0.2.
[0076] (2) Take 50 ml of the above fermentation mixture and perform ultrasonic-microwave combined extraction.
[0077] The ultrasonic extraction process was as follows: extraction was performed at 35°C and 60 W ultrasonic power for 60 min.
[0078] The microwave extraction process involves extracting for 60 seconds at a microwave power of 200 W. The filtrate from the extracted solution is then freeze-dried to obtain the fermented powder of *Gynura divaricata*.
[0079] (3) The prepared storax fermented fine powder was subjected to supercritical CO2 fluid extraction. The extraction process was as follows: 1 g of the above storax fermented fine powder was swollen with 30 ml of n-hexane and then extracted with supercritical CO2 fluid.
[0080] The extraction conditions for supercritical CO2 fluid extraction are as follows: extraction pressure of 22 MPa, material-to-liquid ratio of 1:18 g / mL, extraction time of 60 min, and temperature of 36℃.
[0081] After supercritical CO2 fluid extraction, hexane is removed by rotary evaporation to obtain a paste.
[0082] (4) The obtained paste was sequentially extracted three times each with chloroform, ethyl acetate and n-butanol, and the three extracts were combined.
[0083] The solvent extract was mixed with Sevag's reagent (chloroform:methanol = 10:1), stirred at room temperature for 30 min, centrifuged at low temperature, and the mixture was transferred to a separatory funnel to collect the supernatant. The precipitate was removed by centrifugation, and the process was repeated until no precipitate formed at the interface.
[0084] Add 4 times the volume of anhydrous ethanol to the above solution and let it stand for 24 h.
[0085] After vacuum rotary evaporation, the extract was dispersed in distilled water. After dialyzing with running water for 72 h, the supernatant was collected and subjected to low-temperature refrigerated centrifugation (temperature -2℃, speed 8000 rpm / min, centrifugation time 10 min) to obtain purified vanilla volatile oil.
[0086] The extraction yield of the dye from the stem of Saposhnikovia divaricata obtained in this embodiment was determined to be 17.42 mg / kg.
[0087] Example 2
[0088] The extraction method of volatile oil from *Gynura divaricata* in this embodiment includes the following steps:
[0089] (1) Prepare 100 ml of 0014 PDA medium with 0.1 mol / L citric acid and 0.1 mol / L disodium hydrogen phosphate buffer solution, and adjust the pH value to 6.6-7.4; add 1.11 g of vanilla powder, 1.8 mg of inoculum and 0.04 g of enzymatic decomposition system, mix well and ferment. In the fermentation system, the concentration of vanilla powder (i.e., the material-to-liquid ratio) is 11.1 g / L, the concentration of inoculum (i.e., the material-to-liquid ratio) is 0.018 g / L, and the concentration of enzymatic decomposition system (i.e., the material-to-liquid ratio) is 0.4 g / L.
[0090] The bacterial strains and their activity levels used in this embodiment are the same as in Example 1, and the mass ratio of the three bacterial strains is 0.45:0.45:0.1.
[0091] The fermentation conditions used in this embodiment are: fermentation temperature 30℃ and fermentation time 60 h.
[0092] In the enzymatic degradation system used in this embodiment, the mass ratio of flavin mononucleotide, lipoic acid, pyrophosphate, and pyridoxine phosphate is 0.38:0.15:0.42:0.05.
[0093] (2) Take 50 ml of the above fermentation mixture and perform ultrasonic-microwave combined extraction.
[0094] The ultrasonic extraction process was as follows: extraction was performed at 35°C and 80 W ultrasonic power for 60 min.
[0095] The microwave extraction process involves extracting for 60 seconds at a microwave power of 400 W. The filtrate from the extracted solution is then freeze-dried to obtain the fermented powder of *Gynura divaricata*.
[0096] (3) The prepared fragrant grass fermentation powder was subjected to supercritical CO2 fluid extraction. The extraction process was as follows: 1g of the above fermentation powder was swollen with 25ml of n-hexane and then extracted with supercritical CO2 fluid.
[0097] The extraction conditions for supercritical CO2 fluid extraction are as follows: extraction pressure of 15 MPa, material-to-liquid ratio of 1:12 g / mL, extraction time of 120 min, and temperature of 40℃.
[0098] After supercritical CO2 fluid extraction, hexane is removed by rotary evaporation to obtain a paste.
[0099] (4) This step is the same as step (1) in Example 1, and purified vanilla volatile oil is obtained.
[0100] The extraction yield of the dye from the stem of Saposhnikovia divaricata obtained in this embodiment was determined to be 19.96 mg / kg.
[0101] Example 3
[0102] The extraction method of volatile oil from *Gynura divaricata* in this embodiment includes the following steps:
[0103] (1) Prepare 100 ml of 0014 PDA medium using 0.1 mol / L citric acid and 0.1 mol / L disodium hydrogen phosphate buffer solution, adjust the pH to 6.6-7.4, add 1.25 g of vanilla powder, 2.5 mg of inoculum, and 0.05 g of enzymatic decomposition system, mix well, and then ferment. In the fermentation system, the concentration of vanilla powder (i.e., the material-to-liquid ratio) is 12.5 g / L, the concentration of inoculum (i.e., the material-to-liquid ratio) is 0.025 g / L, and the concentration of enzymatic decomposition system (i.e., the material-to-liquid ratio) is 0.5 g / L.
[0104] The bacterial strains and their activity levels used in this embodiment are the same as in Example 1, and the mass ratio of the three bacterial strains is 0.4:0.5:0.1.
[0105] The fermentation conditions used in this embodiment are: fermentation temperature of 28℃ and fermentation time of 70 h.
[0106] In the enzymatic degradation system used in this embodiment, the mass ratio of flavin mononucleotide, lipoic acid, pyrophosphate, and pyridoxine phosphate is 0.3:0.1:0.4:0.2, which is the same as in Example 1.
[0107] (2) Take 50 ml of the above fermentation mixture and perform ultrasonic-microwave combined extraction.
[0108] The ultrasonic extraction process was as follows: extraction was performed at 35°C and 80 W ultrasonic power for 60 min.
[0109] The microwave extraction process involves extracting for 60 seconds at a microwave power of 400 W. The filtrate from the extracted solution is then freeze-dried to obtain the fermented powder of *Gynura divaricata*.
[0110] (3) The prepared storax fermented fine powder was subjected to supercritical CO2 fluid extraction. The extraction process was as follows: 1 g of the above storax fermented fine powder was swollen with 25 ml of n-hexane and then extracted with supercritical CO2 fluid.
[0111] The extraction conditions for supercritical CO2 fluid extraction are as follows: extraction pressure of 20 MPa, material-to-liquid ratio of 1:10 g / mL, extraction time of 120 min, and temperature of 40℃.
[0112] After supercritical CO2 fluid extraction, hexane is removed by rotary evaporation to obtain a paste.
[0113] (4) This step is the same as step (1) in Example 1, and purified vanilla volatile oil is obtained.
[0114] The extraction yield of volatile oil from *Lysimachia christinae* obtained in this embodiment was determined to be 23.45 mg / kg.
[0115] Figure 1 The infrared spectrum of the vanilla essential oil prepared in Example 3 is shown. In the figure, 2951.28 cm⁻¹... -1 The infrared peak at 1655.95 cm⁻¹ is the out-of-plane antisymmetric stretching vibration peak of CH. -1 The stretching vibration peak belongs to the C=O group of the aldehyde group, at 1372.55 cm⁻¹. -1 1450.9 cm -1 The peak at 876.31 cm⁻¹ belongs to the in-plane stretching vibration peak of the C=C group in olefins. -1 The nearby characteristic peaks indicate that it contains a cyclic structure, which is consistent with the standard infrared spectrum of D-limonene, indicating that lingonberry essential oil contains D-limonene.
[0116] Figure 2 Table 1 below shows the gas chromatography-mass spectrometry (GC-MS) chromatogram of the *Lysimachia christinae* essential oil prepared in this embodiment. In the figure, D-limonene is present at a retention time of 6.558, and its peak area reaches 80%, indicating that the main component of the prepared *Lysimachia christinae* essential oil is D-limonene.
[0117] Table 1
[0118]
[0119] Comparative Examples 1-3: Effects of Fermentation Strains on the Extraction Efficiency of Vanilla frutescens Volatile Oil
[0120] Comparative Examples 1-3 prepared vanilla extract volatile oil using essentially the same steps as in Example 3, the difference being the change in the amount of mold used, or the substitution of other mold strains, as shown in Table 2 below. The infrared spectra of the vanilla extract volatile oils from Comparative Examples 1-3 are as follows: Figure 3-5 As shown.
[0121] Table 2
[0122]
[0123] As can be seen from Table 2, the extraction efficiency of volatile oil from *Gynostemma pentaphyllum* was significantly improved by using a combination of microbial strains.
[0124] Depend on Figure 1 and Figure 3-5 As can be seen, the three substances in Example 3 and Comparative Examples 1-3 share common characteristic peak positions, indicating that the extracts of Example 3 and Comparative Examples 1-3 mainly contain the same types of compounds. Among the common peak positions are 2951.28, 2923.67, 2922.61, and 2920.00 cm⁻¹. -1 The infrared peaks at these locations are out-of-plane antisymmetric stretching vibration peaks of CH; 1655.95, 1641.63, 1674.69 cm⁻¹. -1 Peaks associated with the stretching vibration of the aldehyde group (C=O): 1450.9, 1435.92, 1454.29, 1446.94 cm⁻¹ -1 The peaks at these locations belong to the in-plane stretching vibration peaks of the C=C group in olefins; 876.31, 888.57, and 837.14 cm⁻¹. -1 The nearby transmission peaks indicate the presence of a ring-like structure. This is consistent with... Figure 1 The characteristic peaks of D-limonene in the infrared spectral analysis were basically consistent. The characteristic peak of the cyclic structure at 876.31 in Example 3 was significantly stronger than that in Comparative Examples 1-3, which is consistent with the extraction rate results.
[0125] Comparative Examples 4-6: Effect of Fermentation Strains on the Extraction Efficiency of Vanilla Root Volatile Oil
[0126] Comparative Examples 4-6 prepared vanilla volatile oil using essentially the same steps as in Example 3, except that they did not contain a certain component of the enzymatic decomposition system, as shown in Table 3 below.
[0127] Table 3
[0128]
[0129] As can be seen from Table 2, the extraction efficiency of volatile oil from *Gynostemma pentaphyllum* was significantly improved by using a combination of microbial strains.
[0130] Figure 6-8 Tables 4 and 4-6 show the gas chromatography-mass spectrometry (GC-MS) analysis results of the volatile oil of *Gnaphalium affine* from Comparative Example 4. Figure 9 The gas chromatogram of the vanilla essential oil prepared in Comparative Example 5 is shown. The mass spectrum of the vanilla volatile oil in Comparative Example 5 is compared with that in Comparative Example 4. Figure 8 The mass spectra are the same. Figure 10-12 The image shows a gas chromatography-mass spectrometry (GC-MS) image of the lingxiang essential oil prepared in Comparative Example 6.
[0131] Table 4
[0132]
[0133] Table 5
[0134]
[0135] Table 6
[0136]
[0137] Depend on Figure 6 It can be seen that the volatile oil of *Gnaphalium affine* prepared in Comparative Example 4 showed a very strong peak at a retention time of 6.44, with a molecular weight of 136, which is consistent with... Figure 1 The limonene content is the same, differing only in relative content, with D-limonene content at 49%.
[0138] Depend on Figure 9 It can be seen that the volatile oil of *Gnaphalium affine* prepared in Comparative Example 5 showed a very strong peak at a retention time of 6.39, with a molecular weight of 136, which is consistent with... Figure 1 The limonene content is the same, differing only in relative content; the D-limonene content is 45%.
[0139] Depend on Figure 10 It can be seen that the volatile oil of *Gnaphalium affine* prepared in Comparative Example 6 showed a very strong peak at a retention time of 6.173, with a molecular weight of 136, which is consistent with... Figure 1 The limonene content is the same, differing only in relative content, with D-limonene content being 20%.
[0140] Comparative Examples 8-9: The Synergistic Effect of Enzymatic Decomposition System and Microbial Mixture on Extraction Efficiency / Component Ratio in Volatile Oil. Comparative Examples 8-9 prepared volatile oil of *Gnaphalium affine* using essentially the same steps as in Example 3. The difference was that in Comparative Example 8, only the enzymatic decomposition system was added for fermentation in step (1), while in Comparative Example 9, only the microbial mixture was added. The extraction rate results are shown in Table 7 below.
[0141] Table 7
[0142]
[0143] As shown in Table 1, the extraction efficiency of volatile oil from *Gnaphalium affine* decreased significantly when an enzymatic decomposition system or a mixed microbial culture was lacking.
[0144] Experimental Example 1: Determination of the scavenging ability of safflower volatile oil essential oil against DPPH free radicals
[0145] 1. Measurement method:
[0146] The essential oil of *Gynura divaricata* obtained in Example 3 was diluted with anhydrous ethanol to obtain *Gynura divaricata* essential oils with a mass concentration of 10-50 mg / mL. The mass concentrations of *Gynura divaricata* essential oils at different concentrations are shown in Table 8.
[0147] Table 8. Volatile oil of *Gynura divaricata* at different concentrations
[0148]
[0149] Add 6.0 mL of 0.6 mmol / L DPPH solution and shake well. After reacting for 30 min, 60 min, and 90 min, measure the absorbance at 517 nm. Use anhydrous ethanol instead of DPPH solution as the sample background and DMSO instead of vanilla extract as the blank control to calculate the scavenging rate.
[0150]
[0151] In the formula: C l Here, A0 represents the clearance rate, and A is the absorbance of the blank control. i A represents the absorbance of the experimental group. j The absorbance is the background absorbance of the sample.
[0152] 2. Measurement Results
[0153] The scavenging ability of vanilla volatile oil against DPPH free radicals is shown in Table 9.
[0154] Table 9. DPPH radical scavenging rate of different concentrations of vanilla volatile oil dilutions
[0155]
[0156] Table 5 shows that the DPPH radical scavenging rate of each concentration of vanilla styrax volatile oil dilution increased rapidly from 0 to 60 min. From 60 to 90 min, the trend of DPPH radical scavenging rate of each concentration of vanilla styrax volatile oil dilution gradually leveled off. Meanwhile, the DPPH solution remained basically stable within the prepared 90 min period. Therefore, 60 min was selected as the reaction time for vanilla styrax volatile oil to scavenge DPPH radicals, resulting in the best DPPH radical scavenging rate.
[0157] Furthermore, with the addition of different concentrations of vanilla scabra volatile oil, the scavenging rate of DPPH free radicals gradually increased, and the color of the DPPH free radical solution changed from dark purple to light purple, eventually turning yellow. This indicates that the ability of vanilla scabra volatile oil to scavenge DPPH free radicals increases with increasing concentration. At a concentration of 50 mg / mL, the scavenging rate of vanilla scabra volatile oil for DPPH free radicals essentially reached saturation.
[0158] At a concentration of 50 mg / mL, the volatile oil of *Gynura divaricata* exhibited a scavenging rate of up to 83.19% against DPPH free radicals. Furthermore, at a lower concentration of 20 mg / mL, the scavenging capacity of *Gynura divaricata* volatile oil against DPPH free radicals also reached 10.97%. Therefore, *Gynura divaricata* volatile oil possesses extremely strong scavenging ability against DPPH free radicals, thus demonstrating its properties.
[0159] Application example: Preparation of mosquito repellent dispersion using the volatile oil of *Gynura divaricata*.
[0160] The volatile oil of *Lysimachia christinae* obtained in Example 3 was used to prepare a mosquito repellent dispersion.
[0161] In this application example, the mosquito repellent dispersions consist of sage extract, D-limonene, an essential oil emulsifier, and deionized water. The sage extract volatile oil concentrations were 4%, 6%, 8%, 10%, and 12% by mass; the D-limonene concentration was 1.5% by mass; the essential oil emulsifier concentration was 0.5% by mass; and the deionized water concentrations were 95%, 92%, 90%, 88%, and 86% by mass. Different concentrations of mosquito repellent dispersions were prepared. A control group without sage extract volatile oil was used. The formulations of the mosquito repellent dispersions are shown in Table 10.
[0162] Table 10 Formulation of mosquito repellent dispersible liquid
[0163]
[0164] The mosquito repellency effect was evaluated using the mosquito bite prevention rate as the test index. The formula for mosquito repellency (repellency) effect was based on the calculation formula for repellency rate of repellents in GB / T13917.9-2009 "Indoor Efficacy Tests and Evaluation of Sanitary Insecticides for Pesticide Registration", and the test method was optimized by simulating the indoor environment. The mosquito repellency effect is shown in Table 11 below.
[0165] Table 11 Results of mosquito repellent effect test
[0166]
[0167] Table 4 shows that the mosquito-repellent effect of the mosquito-repellent dispersion gradually increases with the increase of the concentration of *Gynostemma pentaphyllum* volatile oil, and the mosquito-repellent rate is significantly improved. Specifically, when the concentration of *Gynostemma pentaphyllum* volatile oil exceeds 10%, the mosquito-repellent rate can reach over 90%. The results indicate that *Gynostemma pentaphyllum* volatile oil has a good mosquito-repellent effect.
[0168] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A method for extracting volatile oil from *Gynura divaricata*, characterized in that, Includes the following steps: The *Lingxiang* micro powder and the enzymatic decomposition system were added to the bacterial culture mixture for fermentation to obtain *Lingxiang* fermentation broth. The fermented broth of the fragrant grass was sequentially subjected to ultrasonic extraction and microwave extraction, and then freeze-dried into a fine fermented powder. The fermented powder was extracted using supercritical CO2 fluid extraction to obtain a paste. The paste was subjected to solvent extraction to obtain a solvent extract, which was then separated and purified to obtain the volatile oil of *Gynura divaricata*. The microbial mixture includes Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689, and Aspergillus niger CICC41796; the amount of microorganisms added to the microbial mixture is 0.01-0.1 g / L based on the total volume of the fermentation system. The enzymatic decomposition system includes flavin mononucleotide, lipoic acid, pyrophosphate, and pyridoxine phosphate; the amount of the enzymatic decomposition system added is 0.2-0.5 g / L based on the total volume of the fermentation system.
2. The method for extracting the volatile oil of *Gynura divaricata* according to claim 1, characterized in that, The mass ratio of Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689, and Aspergillus niger CICC41796 is (0.4-0.45):(0.42-0.52):(0.03-0.18).
3. The method for extracting the volatile oil of *Gynura divaricata* according to claim 1, characterized in that, The mass ratio of flavin mononucleotide, lipoic acid, pyrophosphate, and pyridoxine phosphate is (0.3-0.38):(0.1-0.15):(0.4-0.45):(0.02-0.2).
4. The method for extracting the volatile oil of *Gynura divaricata* according to claim 1, characterized in that, The mass ratio of the *Lingxiang* micro powder to the bacterial strain is 100:(0.1-0.3).
5. The method for extracting the volatile oil of *Gynura divaricata* according to claim 1, characterized in that, The fermentation conditions include: a fermentation temperature of 25-35℃ and a fermentation time of 120-144 h.
6. The method for extracting the volatile oil of *Gynura divaricata* according to claim 1, characterized in that, The microbial mixture was prepared by inoculating Aspergillus oryzae CICC40108, Penicillium citrinum CICC2689 and Aspergillus niger CICC41796 into a fermentation medium and then activating the mixture.
7. The method for extracting the volatile oil of *Gynura divaricata* according to claim 6, characterized in that, The activation treatment conditions include: adjusting the pH of the fermentation medium to neutral, the culture temperature to 28-30℃, and the culture time to 60-80 h.
8. The method for extracting the volatile oil of *Gynura divaricata* according to claim 1, characterized in that, The ultrasonic extraction process is as follows: extraction is performed at a temperature of 33-42℃ and an ultrasonic power of 60-180 W for 45-60 min; the microwave extraction process is as follows: extraction is performed at a microwave power of 200-600 W for 60-120 s.
9. A volatile oil of *Gnaphalium affine* prepared by the extraction method according to any one of claims 1 to 8.
10. The use of the vanilla volatile oil according to claim 9 in the preparation of a mosquito repellent dispersion.