Extraction method and application of desensitized and attenuated fingered citron essential oil

Abstract

The invention relates to the technical field of separation and extraction of fingered citron essential oil, in particular to an extraction method and application of desensitized and attenuated fingered citron essential oil. The extraction method of the desensitized and attenuated fingered citron essential oil comprises the following steps that fingered citron is pretreated, then essential oil is extracted through a steam distillation method, and pretreatment comprises at least one of microwave treatment, ultrasonic treatment and alkali liquor treatment. According to the method, the content of the sensitizing substances in the fingered citron essential oil can be effectively reduced, particularly, the alkali liquor pretreatment has the best removing effect on the sensitizing substances, and a theoretical basis is provided for the reduction technology of the sensitizing substances of the fingered citron essential oil and application and development of the fingered citron essential oil.

Description

Technical Field

[0001] The invention relates to the technical field of bergamot essential oil extraction, and in particular to an extraction method and application of desensitized and attenuated bergamot essential oil. Background Art

[0002] Buddha's hand, also known as five-finger citrus, longevity citrus, and citron, is a plant of the genus Citrus in the Rutaceae family.

[0003] The dried fruit of L. var. sarcodactylis Swingle has the effects of soothing the liver and regulating qi, soothing the stomach and relieving pain, and removing dampness and phlegm. The bergamot mainly contains chemical components such as flavonoids, volatile oils, coumarins, polysaccharides, etc., and is a Chinese medicine with both medicinal and edible properties. Bergamot contains a variety of active substances, among which essential oil is the main component of the bergamot peel and has pharmacological activity. On the one hand, the volatile essential oil of bergamot has certain physiological activity and has good clinical efficacy. It can improve depression and anxiety, relieve emotions, and also has antitussive and antiasthmatic effects and antioxidant effects. On the other hand, the bergamot essential oil has a fresh and fragrant smell. As a precious natural fragrance, it has good application value in industries such as food, cosmetics, and medicines.

[0004] Allergic reaction is a series of overreactions made by the immune system when the human body comes into contact with specific allergens. This phenomenon is considered to be a disorder of the immune system, that is, allergic reaction is an abnormal, pathological, and harmful immune reaction to the human body, also known as hypersensitivity or allergic reaction. When the human body comes into contact with allergens (such as insect venom, food and drugs, injections through skin contact, etc.), it is likely to cause an allergic reaction. When cosmetics or skin care products are applied to the surface of the human skin, an abnormal immune response is produced to the specific chemicals in them, causing disorders in the body's physiological functions, and even the risk of tissue damage, triggering a series of mild or severe clinical symptoms.

[0005] The terpenoids such as limonene, geraniol and linalool contained in citrus essential oils make the essential oils have certain phototoxicity and can cause damage to the skin. Therefore, it is particularly important to study the key technologies for reducing the allergenic components of citrus bergamot essential oils and develop safe and reliable bergamot desensitizing essential oil products. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for extracting desensitized and attenuated bergamot essential oil and its application, so as to effectively reduce the content of allergenic substances in the extracted bergamot essential oil.

[0007] In a first aspect, the present invention provides a method for extracting desensitized and attenuated bergamot essential oil, comprising the following steps:

[0008] The bergamot is pre-treated and then essential oil is extracted by steam distillation. The pre-treatment includes at least one of microwave treatment, ultrasonic treatment and alkali solution treatment.

[0009] Optionally, the pre-treatment includes alkali solution treatment.

[0010] Optionally, the power of the microwave treatment is 450-550 W, the temperature of the microwave treatment is 70-80° C., and the duration of the microwave treatment is 40-50 min.

[0011] Optionally, the power of the ultrasonic treatment is 250-350 W, the temperature of the ultrasonic treatment is 45-55° C., and the duration of the ultrasonic treatment is 35-45 min.

[0012] Optionally, the alkali solution is selected from sodium hydroxide solution.

[0013] Optionally, the concentration of the alkali solution is 0.5wt%-1.5wt%.

[0014] Optionally, the alkali solution treatment includes: using the alkali solution as the reflux medium and boiling and refluxing for 0.5-1.5 hours.

[0015] Optionally, the material ratio of the bergamot to the alkali solution is 50-80g:500-600mL.

[0016] Optionally, the extraction duration is 3-4 hours.

[0017] In a second aspect, the present invention also provides bergamot essential oil extracted according to the method as described above.

[0018] The beneficial effects of the present invention include:

[0019] The application of bergamot essential oil can cause allergic phenomena of varying degrees on the skin of guinea pigs, such as redness, swelling, spots, thickening of the stratum corneum, edema, infiltration of inflammatory cells such as lymphocytes and eosinophils, congestion and bleeding of the deep dermis, necrosis and degeneration of connective tissue, and increased immunoglobulin. The present invention can reduce the content of allergenic substances in bergamot essential oil, especially the alkaline solution pretreatment has the best removal effect on allergenic substances. The present invention provides a theoretical basis for the reduction technology of allergenic substances in bergamot essential oil and the application and development of bergamot essential oil.

[0020] The present invention reduces the allergenic components in bergamot essential oil by different pre-treatment methods such as microwave treatment, ultrasonic treatment and alkali solution treatment, and the reduction effects are different. Among them, chemical pre-treatment has the best effect on reducing the allergenic components of bergamot essential oil, and can significantly reduce the contents of three allergenic components, limonene, linalool and geraniol. The chemical pre-treatment is simple to operate, efficient, and the cost of the chemical reagents used is low. The reduced bergamot essential oil can be developed into a safe and reliable bergamot essential oil product. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 1 is the GC-MS total ion current detection result of different pre-treated bergamot essential oils, where A is the bergamot essential oil group without pre-treatment, B is the bergamot essential oil group with ultrasonic pre-treatment, C is the bergamot essential oil group with microwave pre-treatment, and D is the bergamot essential oil group with alkali pre-treatment. 1 is limonene, 2 is linalool, and 3 is geraniol.

[0022] Figure 2 The figure is the test result of the effect of bergamot essential oil on guinea pig skin allergy, wherein A is the normal control group, B is the group without pretreatment 0.025mL / pig, C is the group with ultrasonic pretreatment 0.025mL / pig, D is the group with microwave pretreatment 0.025mL / pig, E is the group with alkali solution pretreatment 0.025mL / pig, F is the positive control group, G is the group without pretreatment 0.05mL / pig, H is the group with ultrasonic pretreatment 0.05mL / pig, I is the group with alkali solution pretreatment 0.05mL / pig, and J is the group with microwave pretreatment 0.05mL / pig;

[0023] Figure 3 Figure 1 is the test result of the effect of bergamot essential oil on the pathology of guinea pig skin tissue sections (×100), wherein A is the normal control group, B is the positive control group, C is the group without pretreatment 0.025mL, D is the group without pretreatment 0.05mL, E is the group with ultrasonic pretreatment 0.025mL, F is the group with ultrasonic pretreatment 0.05mL, G is the group with alkali solution pretreatment 0.025mL, H is the group with alkali solution pretreatment 0.05mL, I is the group with microwave pretreatment 0.025mL, and J is the group with microwave pretreatment 0.05mL;

[0024] Figure 4 Effects of bergamot essential oil on the levels of IgE (A), IgA (B), and IgG (C) in guinea pig serum n=8) Test result graph, among which, compared with the normal control group: *p<0.05, **p<0.01, compared with the untreated bergamot essential oil 0.025mL group: #p<0.05, ##p<0.01; compared with the untreated bergamot essential oil 0.05mL group: &p<0.05, &&p<0.01;

[0025] Figure 5 Effects of bergamot essential oil on the levels of IgE (A), IgA (B), and IgG (C) in guinea pig skin n=8) Test result graph, wherein, compared with the normal control group: *p<0.05, **p<0.01, compared with the unpretreated bergamot essential oil 0.025mL group: #p<0.05, ##p<0.01; compared with the unpretreated bergamot essential oil 0.05mL group: &p<0.05, &&p<0.01. DETAILED DESCRIPTION

[0026] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0027] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0028] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0029] 1. Experimental Materials

[0030] 1.1 Experimental animals

[0031] 80 healthy guinea pigs, ordinary grade, with a body mass of 280-350g, were provided by Guizhou Yikeda Biotechnology Co., Ltd., license number SCXK(Guizhou)2021-0001, and were kept in the animal room of Chongqing Institute of Traditional Chinese Medicine. They were fed with a mixture of granular feed (consisting of feed containing 10wt% protein and rabbit feed in a mass ratio of 4:1); at the same time, each guinea pig was supplemented with two leaves of romaine lettuce every day; the drinking water was a 250mL bottle of purified water with 4 vitamin C tablets added. After one week of adaptive feeding, the experiment began; the animal experiment ethics approval unit was the Animal Institute of Chongqing Institute of Traditional Chinese Medicine, with approval number YHS2024-07.

[0032] 1.2 Experimental drugs and reagents

[0033] Xylene (Shandong Keyuan Biochemical Co., Ltd., batch number C15720136), 4% paraformaldehyde universal tissue fixative (biosharp, batch number 23333866), distilled water, physiological saline (Shanghai Yuanye Biotechnology Co., Ltd., batch number JR23137A), sodium hydroxide (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number C2110062), hydrochloric acid (Chongqing Chuandong Chemical Co., Ltd., batch number 20220501), anhydrous sodium sulfate (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number 30538), anhydrous ethanol (AR grade) (Sichuan Xilong Scientific Co., Ltd., batch number: 2201121), xylene (AR grade) (Sichuan Xilong Scientific Co., Ltd., batch number: 2201111), hematoxylin stain (Wuhan Sewell Biotechnology Co., Ltd., batch number: CR2109146) eosin stain (Wuhan Sewell Biotechnology Co., Ltd., batch number: CR2011064), immunoglobulin A kit (batch number: E20240422-400234Q), immunoglobulin E kit (batch number: E20240422-400336Q), immunoglobulin G kit (batch number:

[0034] E20240422-400335Q), protein quantification (batch number: E20240422-KM0488), isoflurane (Shenzhen Ruiwode Life Science Co., Ltd., batch number: 2024031201). The bergamot samples used in the experiment were all produced in the Mankangshe bergamot planting base in Babian Village, Mohei Town, Ning'er County, Pu'er City, Yunnan Province, and were identified as the dried fruits of Citrus.medica L.var.Sarcodactylis Swingle, a plant of the Rutaceae family, by researcher Qu Xianyou of the Institute of Chinese Materia Medica and Pharmacognosy of Chongqing Academy of Traditional Chinese Medicine.

[0035] 1.3 Experimental instruments

[0036] RWBZ-08S microwave vacuum dryer (Nanjing Su Enrui Drying Equipment Co., Ltd.), electric heating jacket PTHW type (Shanghai Yu Kang Science and Education Instrument Equipment Co., Ltd.), solid ultrasonic cleaning machine (Guangdong Good Ultrasonic Co., Ltd., VGT-2120QT) small animal anesthesia machine (Shenzhen Ruiwode Life Science Technology Co., Ltd., R500IE), electric constant temperature incubator (Wuhan Yiheng Sujing Scientific Instrument Co., Ltd.), 988 plate washer (Tianshi), RT-6100 microplate reader (Rayto), TG16W micro high-speed centrifuge (Changsha Xiangzhi Centrifuge Instrument Co., Ltd.), TGL16M desktop high-speed refrigerated centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.), HT-111B constant temperature shaker, AE1204 model one millionth electronic analytical balance (Shanghai Liangping Instrument Co., Ltd.) (2) Swiss PRECISA electronic analytical balance XB220A (Hai Liangping Instrument Co., Ltd.), LECO Pegasus BT4D (LECO, St. Joseph, MI, USA) Agilent 8890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA), double-stage jet modulator and split / non-split, LECO Pegasus BT 4D mass spectrometer (LECO, St. Joseph, MI, USA), rotary slicer (Leica-2016, Germany), JT-12S automatic tissue dehydrator (Wuhan Junjie Electronics Co., Ltd.), BMJ-A embedding machine (Changzhou Suburb Zhongwei Electronic Instrument Factory), RS36 automatic staining machine (Changzhou Paisjie Medical Equipment Co., Ltd.), PHY-Ⅲ pathological tissue bleaching and drying instrument (Changzhou Zhongwei Electronic Instrument Co., Ltd.), Pannoramic 250 digital slide scanner (3DHISTECH, Hungary)

[0037] (II) Experimental methods

[0038] 2.1 Preparation of bergamot essential oil

[0039] Comparative Example 1, extraction of bergamot essential oil without pretreatment, specific steps:

[0040] S1. Take fresh bergamot fruit and chop it into small pieces of about 0.5 cm × 0.5 cm;

[0041] S2. Weigh 180g of fruit particles and place them in a 1000mL round-bottom flask, then add 360mL of water, and perform steam distillation for 3.5 hours according to the volatile oil extraction method A in the 2020 edition of the "Chinese Pharmacopoeia", collect the oil-water mixture, and then add an appropriate amount of desiccant anhydrous sodium sulfate. Shake continuously. The organic phase should become clearer and clearer with the addition of desiccant. After it becomes transparent, the desiccant should be loose and not lumpy when shaken. Then let it stand for 2 hours, centrifuge, and store the upper layer of oil obtained by centrifugation in a 4°C refrigerator for later use.

[0042] Embodiment 1:

[0043] S1. Take fresh bergamot fruit and chop it into small pieces (also called granules) of about 0.5 cm × 0.5 cm;

[0044] S2. Place the fruit particles (i.e. the small pieces) in a microwave vacuum dryer, and microwave treat them for 45 minutes at a power of 500 W and a temperature of 75°C to obtain a dried bergamot sample. Weigh 180 g of the dried bergamot sample and place it in a 1000 mL round-bottom flask. Add 360 mL of water and use steam distillation to extract the bergamot essential oil. After steam distillation for 3.5 hours according to the volatile oil extraction method A in the 2020 edition of the "Chinese Pharmacopoeia", collect the oil-water mixture, add an appropriate amount of desiccant anhydrous sodium sulfate, and shake continuously. The organic phase should become clearer and clearer with the addition of the desiccant. After it becomes transparent, the desiccant should be loose and not lumpy when shaken. Then let it stand for 2 hours and centrifuge. The upper layer of oil obtained by centrifugation should be stored in a refrigerator at 4°C for later use.

[0045] That is, the difference between this embodiment and comparative example 1 is that the bergamot fruit is pre-treated with microwaves.

[0046] Embodiment 2:

[0047] S1. Take fresh bergamot fruit and chop it into small pieces of about 0.5 cm × 0.5 cm;

[0048] S2. 50 g of fruit particles were placed in a 250 mL conical flask, 100 mL of water was added, and ultrasonic treatment was performed at 300 W and 50 ° C for 40 min to obtain a bergamot sample;

[0049] Weigh 180g of bergamot sample and place it in a 1000mL round-bottom flask, then add 360mL of water, and use steam distillation to extract bergamot essential oil. After steam distillation for 3.5 hours according to the volatile oil extraction method A in the 2020 edition of the "Chinese Pharmacopoeia", collect the oil-water mixture, add an appropriate amount of desiccant anhydrous sodium sulfate, and shake continuously. The organic phase should become clearer and clearer with the addition of desiccant. After it becomes transparent, the desiccant should be loose and not agglomerated when shaken. Then let it stand for 2 hours, centrifuge, and store the upper oil obtained by centrifugation in a 4°C refrigerator for later use.

[0050] Embodiment 3:

[0051] S1. Take fresh bergamot fruit and chop it into small pieces of about 0.5 cm × 0.5 cm;

[0052] S2. Weigh 70 g of fruit particles into a 1000 mL round-bottom flask, add 560 mL of 1 wt% NaOH solution, boil and reflux for 1 h, filter while hot, adjust the pH of the filtrate to neutral (measured with a test paper) with 5% hydrochloric acid solution, combine the neutral filtrate and the filter residue to obtain a mixture;

[0053] Weigh 180g of the mixture and place it in a 1000mL round-bottom flask, then add 360mL of water, and use steam distillation to extract bergamot essential oil. After steam distillation for 3.5 hours according to the volatile oil extraction method A in the 2020 edition of the "Chinese Pharmacopoeia", collect the oil-water mixture, add an appropriate amount of desiccant anhydrous sodium sulfate, and shake continuously. The organic phase should become clearer and clearer with the addition of desiccant. After it becomes transparent, the desiccant should be loose and not agglomerated when shaken. Then let it stand for 2 hours, centrifuge, and store the upper layer of oil obtained by centrifugation in a 4°C refrigerator for later use.

[0054] 2.2 Determination of allergenic components in bergamot essential oil by GC×GC-TOF MS

[0055] 2.2.1GC Chromatographic Conditions

[0056] Chromatographic column DB-Heavy Wax (30m×250μm×0.5μm); high-purity helium as carrier gas with a constant flow rate of 1.0mL / min; initial temperature was 40°C, maintained for 3min, increased to 200°C at a rate of 6°C / min, and finally increased to 250°C at a rate of 10°C / min and maintained for 5min.

[0057] 2.2.2 Mass spectrometry conditions

[0058] LECO Pegasus BT 4D mass spectrometer detector (LECO, St. Joseph, MI, USA); mass spectrometer transmission line temperature was 250°C; ion source temperature was 250°C; acquisition rate was 10spectra / s, electron bombardment source was 70eV, detector voltage was 2011V; mass spectrometer scanning range was m / z 35-550.

[0059] 2.2.3 Solution preparation

[0060] 2.2.3.1. Dissolve the 2-octanol standard reference substance in 50% ethanol aqueous solution to prepare a single standard solution with a concentration of 100 mg / L and store it in a refrigerator at 4°C.

[0061] 2.2.3.2. Take an appropriate amount of 1000 mg / L normal alkane standard reference substance, dilute it step by step with n-hexane to prepare a single standard solution with a concentration of 1 mg / L, and store it in a refrigerator at 4°C.

[0062] 2.2.4 Extraction of volatile substances

[0063] Accurately pipette 10 μL of the samples obtained in Comparative Example 1 and Examples 1-3 into 20 mL headspace injection bottles, and add 10 μL of the single-label solution into the samples;

[0064] The samples were incubated at 50°C for 10 min;

[0065] The SPME extraction tip was aged at 270°C for 10 min;

[0066] The aged SPME extraction tip was transferred to the incubation chamber and adsorbed at 50°C for 20 min.

[0067] After the adsorption, the SPME extraction head was transferred to the GC injection port and desorbed at 250 °C for 5 min;

[0068] After injection, the SPME extraction tip was aged at 270 °C for 10 min;

[0069] Take 10 μL of n-alkane solution into a 20 mL headspace injection vial, incubate at 50°C for 10 min, and then inject.

[0070] 2.3 Animal guinea pig experiment

[0071] 2.3.1 Guinea pig grouping and drug administration

[0072] A total of 80 guinea pigs were randomly divided into 10 groups, with 8 in each group, half male and half female, including normal control group, positive control xylene group, low-dose and high-dose bergamot essential oil groups without pretreatment, low-dose and high-dose bergamot essential oil groups with microwave heating pretreatment, low-dose and high-dose bergamot essential oil groups with ultrasound pretreatment, and low-dose and high-dose bergamot essential oil groups with alkali solution pretreatment.

[0073] The normal control group was given 0.05 mL / mouse of distilled water; the positive control group was given 0.05 mL / mouse of xylene; the low-dose and high-dose groups of bergamot essential oil without pretreatment, the low-dose and high-dose groups of bergamot essential oil treated with microwave, the low-dose and high-dose groups of bergamot treated with ultrasound, and the low-dose and high-dose groups of bergamot essential oil treated with chemical treatment were given 0.025 mL / mouse and 0.05 mL / mouse of the corresponding extracted essential oil, respectively, that is, each low-dose group was given 0.025 mL / mouse of the corresponding extracted essential oil, and each high-dose group was given 0.05 mL / mouse of the corresponding extracted essential oil.

[0074] The drug was administered through transdermal application. The experiment was divided into two stages: induction and stimulation. First, during the induction stage, the guinea pigs were shaved on the back with an electric and manual shaver before the experiment. The shaved area was 2 cm × 2 cm.

[0075] Apply xylene and the test substance to the hairless area on the back of the guinea pig; after administration, first cover the administration site with gauze, then cover the hairless area with a layer of transparent tape to prevent the tape from contacting the skin, seal and fix for 6 hours, remove the covering, and clean the remaining test sample;

[0076] Before each administration, the hair at the corresponding location of the administration area of ​​each group of animals was removed again;

[0077] The drug was applied on the 1st, 8th, and 15th day of the experiment, for a total of 3 times;

[0078] Then, a challenge was given on the 14th day after the last administration in the induction period; the administration method, dosage and volume were the same as those in the induction period.

[0079] 2.3.2 General observations on guinea pig skin sensitization

[0080] After removing the covering, observe the sensitization reaction of erythema, edema and hemorrhage on the skin of guinea pigs in each group, and take photos of the sensitized parts of guinea pigs at 0h, 24h, 48h and 72h.

[0081] The sensitization rate was calculated according to the allergic reaction degree scoring standard, and the sensitization of the guinea pig skin in each group was evaluated according to the sensitization rate. The evaluation standards are shown in Table 1 and Table 2. Among them, the sensitization rate formula was calculated according to the formula sensitization rate = number of guinea pigs with allergic reactions / total number of guinea pigs. The results are shown in Figure 2 And as shown in Table 4.

[0082] Table 1 Evaluation criteria for the degree of skin allergic reaction in guinea pigs

[0083]

[0084] Table 2 Evaluation criteria for guinea pig skin sensitization

[0085] Sensitization rate / % Sensitization 0-10 Non-allergenic 11-30 Mild allergenicity 31-70 Moderate sensitization 71-90 Severe allergenicity 91-100 Extremely allergenic

[0086] 2.3.3 Sample collection

[0087] 2.3.3.1 Blood sample collection

[0088] One hour after the last administration, the guinea pigs in each group were weighed; after the guinea pigs were anesthetized with isoflurane, the abdominal cavity was opened, and blood was collected from the abdominal aorta. The blood was collected in a blood collection tube without anticoagulant, placed on ice for a period of time, centrifuged at 4000r / min and 4℃ for 10min, the supernatant was collected, packaged, and temporarily stored in a 20℃ refrigerator.

[0089] 2.3.3.2 Collection of skin tissue specimens

[0090] After blood was collected from the abdominal aorta, the sensitized skin was quickly removed, the surrounding excess tissue was removed, and after washing with physiological saline, the water was absorbed with filter paper and weighed with a precision balance. The skin index was calculated according to the format of skin index = skin mass / guinea pig body mass. The results are shown in Table 5;

[0091] A guinea pig skin tissue sample was fixed in 4% paraformaldehyde for examination; another skin tissue was taken and temporarily stored in a -20°C refrigerator.

[0092] 2.3.4 Observation of guinea pig skin tissue pathology

[0093] The skin tissue was placed in 4% paraformaldehyde solution and stained with HE. According to the pathological examination SOP procedure, it was dehydrated with gradient ethanol, transparentized with xylene, embedded in paraffin, sectioned, stained with hematoxylin-eosin (HE), sealed with neutral gum, and finally observed under a microscope for specific lesions. The results are shown in Figure 1. Figure 3 shown.

[0094] 2.3.5 Detection of IgA, IgG, and IgE levels in guinea pig serum and skin tissue

[0095] According to the operation method of the enzyme-linked immunosorbent assay kit, the levels of immunoglobulins IgA, IgG and IgE in guinea pig serum and skin tissue homogenate supernatant were detected. The results are as follows Figure 4 and Figure 5 shown.

[0096] 2.3.6 Data processing

[0097] Graphpad Prism software was used for data analysis. ANOVA was used to compare the means of multiple samples, and t-test was used to compare the differences between multiple groups. P < 0.05 indicated that the difference was statistically significant.

[0098] 3 Results

[0099] 3.1 Determination of volatile allergenic components in bergamot essential oil

[0100] Solid phase microextraction combined with GC-Q-TOF-MS was used to inject samples according to the analytical conditions in Section 2.2 to obtain the total ion current of bergamot essential oil. The target compound was identified by the mass spectrum database (NIST14) provided by the GC-MS workstation, and the relative percentage content of each chemical component was calculated according to the peak area normalization method. The results are shown in the figure. Figure 1 And as shown in Table 3.

[0101] Depend on Figure 1It can be seen that the substances corresponding to the retention times of 13.82min, 21.53min and 27.38min are limonene, linalool and geraniol, respectively.

[0102] 3.2 Effects of different pretreatment methods on the allergenic components of bergamot essential oil

[0103] Table 3 Detection results of allergenic components and their contents in bergamot essential oil with different pretreatment methods

[0104]

[0105] From Table 3 and Figure 1 It can be seen that compared with the essential oil group without pretreatment, the content of limonene in the essential oil group prepared by ultrasonic pretreatment changed the most, from 15.3060% to 2.9565%; geraniol was second, from 6.0674% to 1.7031%; the content of linalool did not change much. The content of geraniol in the essential oil group prepared by microwave pretreatment changed the most, from 6.0674% to 0.0001%, while the contents of limonene and linalool did not change much. The contents of the three substances in the essential oil group prepared by alkali solution pretreatment were all reduced, and the content of limonene and geraniol changed from 15.3060 to 8.9033% and from 6.0674% to 0.0001, respectively. The results suggest that the essential oil prepared by alkali solution pretreatment has the greatest effect on the content changes of the three allergenic components of limonene, linalool and geraniol, and has the best reduction effect.

[0106] 3.3 General observation on the sensitization of bergamot essential oil to guinea pig skin

[0107] Table 4 Effect of bergamot essential oil on skin allergic reaction in guinea pigs ( n=8)

[0108]

[0109] Depend on Figure 2 As shown in Table 4, no local and systemic allergic reactions were observed on the skin of guinea pigs in the normal control group. Compared with the normal control group, after administration of the positive control xylene group, the guinea pigs showed obvious abnormal reactions such as erythema, edema and writhing. The allergic condition was further observed, and the erythema and edema gradually subsided, and the skin returned to normal after about 72 hours. After 24 hours of administration, the guinea pigs in the untreated bergamot essential oil group, the microwave pre-treated bergamot essential oil group, and the ultrasound pre-treated bergamot essential oil group all showed erythema, edema and other reactions on their skin. The abnormal reaction of the alkali pre-treated bergamot essential oil group was not obvious, with only a few subtle erythema. After 48 hours of administration, the erythema began to gradually subside, and the allergic phenomenon further subsided after 72 hours.

[0110] In addition, compared with the 0.25 mL / unit dose group of essential oil without pretreatment, the sensitization rates of the microwave, ultrasound, and alkali pretreatment essential oil groups with the same dosage were all reduced, among which the alkali pretreatment essential oil group had the lowest sensitization rate. Compared with the 0.50 mL / unit dose group of essential oil without pretreatment, the sensitization rates of the microwave and ultrasound pretreatment essential oil groups with the same dosage increased, while the sensitization rate of the alkali pretreatment essential oil group decreased. Compared with the 0.25 mL / unit dose group of essential oil with the same pretreatment, the sensitization rates of the high-dose groups of ultrasound and microwave pretreatment increased; the chemical 0.50 mL / unit dose group had little change.

[0111] 3.4 Effect of bergamot essential oil on guinea pig skin index

[0112] Table 5 Effects of different pre-treatment bergamot essential oils on body weight (day 31) and skin index of guinea pigs ( n=8)

[0113] Group Dosage (mL / piece) Guinea pig body weight / g Skin index / % Normal control group 0.05 356.70±1.67 0.0014±0.0000 Positive control group 0.05 429.10±16.56 0.0027±0.0003 No pretreatment low dose group 0.025 381.90±14.99 <![CDATA[0.2108±0.0863 *# ]]> No pretreatment high dose group 0.05 410.30±27.73 0.0785±0.0396 Ultrasonic pretreatment low dose group 0.025 359.80±28.93 0.1203±0.0533 Ultrasonic pretreatment high dose group 0.05 435.10±14.37 0.1071±0.0421 Microwave pretreatment low dose group 0.025 423.90±14.94 0.0760±0.0373 Microwave pretreatment high dose group 0.05 408.10±25.33 0.1150±0.0481 Alkaline solution pretreatment low dose group 0.025 423.20±19.72 0.0658±0.0331 Alkaline solution pretreatment high dose group 0.05 432.20±16.75 0.1044±0.0406

[0114] Note: Compared with the normal control group: * p<0.05, ** p<0.01; compared with the positive control group: # p<0.05, ## p<0.01.

[0115] As shown in Table 5, compared with the normal control group, the 0.025 mL group of bergamot essential oil without pretreatment significantly increased the skin index of guinea pigs ( * p<0.05). Compared with the untreated chemical essential oil group, the skin index of bergamot essential oil prepared by ultrasound pretreatment, microwave pretreatment and alkali solution pretreatment had no significant difference.

[0116] 3.5 HE staining pathological results

[0117] Depend on Figure 3The results show that the skin tissue structure is intact, the positive control group, ultrasound pretreatment 0.025mL group and alkali pretreatment 0.05mL group have hyperkeratosis and the stratum corneum is significantly thickened; the positive control group, no pretreatment 0.025mL group, no pretreatment 0.05mL group, ultrasound pretreatment 0.025mL group, ultrasound pretreatment 0.05mL group, alkali pretreatment 0.025mL group, alkali pretreatment 0.05mL group, microwave pretreatment 0.025mL group and microwave pretreatment 0.05mL group have different degrees of thickening of the epidermis, and the number of stratum spinosum cells increases; the epidermis of the no pretreatment 0.05mL group is necrotic. Necrotic cells disintegrated and fragmented. In the positive control group, the 0.025mL group without pretreatment, the 0.05mL group without pretreatment, the 0.025mL group with ultrasound pretreatment, the 0.05mL group with ultrasound pretreatment, the 0.05mL group with alkali solution pretreatment, the 0.025mL group with microwave pretreatment and the 0.05mL group with microwave pretreatment, the collagen fibers in the dermis were necrotic, and the necrotic collagen fibers were dissolved and broken. The necrotic areas in the 0.05mL group without pretreatment and the 0.025mL group with microwave pretreatment were infiltrated with a small amount of inflammatory cells, mainly neutrophils. The structures of the appendages such as hair follicles and sebaceous glands were intact. No other obvious pathological changes were found.

[0118] 3.6 Effect of bergamot essential oil on IgE, IgA and IgG levels in guinea pig serum

[0119] 3.6.1 Effect of bergamot essential oil on IgE levels in guinea pig serum

[0120] Depend on Figure 4 A shows that compared with the normal control group, the IgE level in the guinea pig serum of the 0.025mL and 0.05mL groups of bergamot essential oil without pretreatment and ultrasound pretreatment was significantly increased (p < 0.01). The IgE level in the guinea pig serum of the 0.025mL group of alkali pretreatment was significantly increased (p < 0.05). There was no significant difference in the IgE level in the serum of the 0.025mL and 0.05mL groups of microwave pretreatment, the 0.05mL group of alkali pretreatment and the positive control group.

[0121] Depend on Figure 4 A shows that compared with the untreated bergamot essential oil 0.025mL group, the IgE level in the guinea pig serum of the bergamot essential oil 0.025mL and 0.05mL groups pretreated with microwave was significantly reduced (p < 0.01), and there was no significant difference in the IgE level in the guinea pig serum of the bergamot essential oil 0.025mL and 0.05mL groups pretreated with ultrasound and alkali solution.

[0122] Depend on Figure 4A shows that compared with the untreated bergamot essential oil 0.05mL group, the IgE level in the guinea pig serum of the bergamot essential oil 0.025mL and 0.05mL groups pretreated with microwave was significantly reduced (p < 0.01), and the IgE in the guinea pig serum of the bergamot essential oil 0.025mL and 0.05mL groups pretreated with alkali solution was significantly reduced (p < 0.05), and there was no significant difference in IgE in the guinea pig serum between the ultrasonically treated bergamot essential oil 0.025mL and 0.05mL groups and the untreated bergamot essential oil 0.025mL group.

[0123] 3.6.2 Effect of bergamot essential oil on IgA levels in guinea pig serum

[0124] Depend on Figure 4 B shows that compared with the normal control group, the IgA level in the guinea pig serum of the 0.025mL and 0.05mL bergamot essential oil groups without pretreatment and ultrasound pretreatment was significantly increased (p < 0.01). There was no significant difference in the IgA level in the guinea pig serum of the 0.025mL and 0.05mL bergamot essential oil groups pretreated with microwave and alkali solution and the positive control group.

[0125] Depend on Figure 4 B shows that compared with the untreated bergamot essential oil 0.025mL group, the IgA level in the guinea pig serum of the microwave pre-treated bergamot essential oil 0.025mL group was significantly reduced (p < 0.05). The IgA level in the guinea pig serum of the microwave pre-treated bergamot essential oil 0.05mL and alkali pre-treated bergamot essential oil 0.025mL groups was significantly reduced (p < 0.01). There was no significant difference in the IgA level in the guinea pig serum of the ultrasonic pre-treated bergamot essential oil 0.025mL and alkali pre-treated bergamot essential oil 0.05mL groups.

[0126] Depend on Figure 4 B shows that compared with the untreated bergamot essential oil 0.05mL group, the bergamot essential oil 0.05mL pre-treated with ultrasound and microwave and the bergamot essential oil 0.025mL pre-treated with alkali solution had significantly lower IgA levels in guinea pig serum (p < 0.01), and the bergamot essential oil 0.025mL pre-treated with microwave had significantly lower IgA levels in guinea pig serum (p < 0.05). There was no significant difference in IgA levels in guinea pig serum without the pre-treatment of bergamot essential oil 0.025mL. Figure 4 B.

[0127] 3.6.3 Effect of bergamot essential oil on IgG levels in guinea pig serum

[0128] Depend on Figure 4C shows that compared with the normal control group, the IgG level in the guinea pig serum of the 0.025mL and 0.05mL bergamot essential oil groups without pretreatment and ultrasound pretreatment and the positive control group was significantly increased (p < 0.01). The IgG level in the guinea pig serum of the 0.025mL and 0.05mL bergamot essential oil groups pretreated with alkali solution was significantly increased (p < 0.05). There was no significant difference in the IgG level in the guinea pig serum of the 0.025mL and 0.05mL bergamot essential oil groups pretreated with microwave and alkali solution.

[0129] Depend on Figure 4 C shows that compared with the untreated bergamot essential oil 0.025mL group, the IgG level in the guinea pig serum of the microwave pre-treated bergamot essential oil 0.025mL group was significantly reduced (p < 0.01). The IgG level in the guinea pig serum of the microwave pre-treated bergamot essential oil 0.05mL group was significantly reduced (p < 0.05), and there was no significant difference in the IgG level in the guinea pig serum of the ultrasonic pre-treated and alkali pre-treated bergamot essential oil 0.025mL and 0.05mL groups.

[0130] Depend on Figure 4 C shows that compared with the group without pretreatment of bergamot essential oil 0.05mL, the IgG level in guinea pig serum in the groups with microwave pretreatment of bergamot essential oil 0.025mL and 0.05mL and the groups with alkali pretreatment of bergamot essential oil 0.025mL and 0.05mL was significantly reduced (p < 0.01), and there was no significant difference in the IgG level in guinea pig serum in the groups with ultrasound pretreatment of bergamot essential oil 0.025mL and 0.05mL and the group without pretreatment of bergamot essential oil 0.025mL.

[0131] 3.7 Effect of bergamot essential oil on IgE, IgA, and IgG levels in guinea pig skin

[0132] 3.7.1 Effect of bergamot essential oil on IgE levels in guinea pig skin

[0133] Depend on Figure 5 A shows that compared with the normal control group, the IgE level in the guinea pig skin of the 0.05mL untreated bergamot essential oil and the 0.025mL and 0.05mL ultrasonic pre-treated bergamot essential oil groups was significantly increased (p < 0.01), the IgE level in the guinea pig skin of the 0.025mL bergamot essential oil pre-treated group and the positive control group was significantly increased (p < 0.05), and there was no significant difference in the IgE level in the guinea pig skin of the 0.025mL untreated bergamot essential oil, the 0.05mL bergamot essential oil pre-treated group and the 0.025mL and 0.05mL microwave pre-treated bergamot essential oil groups.

[0134] Depend on Figure 5A shows that compared with the group without pretreatment of bergamot essential oil 0.025mL, the IgE level in the guinea pig skin of the groups pretreated with bergamot essential oil 0.025mL and 0.05mL by ultrasound was significantly increased (p < 0.01), and there was no significant difference in the IgE level in the guinea pig skin of the groups pretreated with bergamot essential oil 0.025mL and 0.05mL by microwave and 0.025mL and 0.05mL by alkali solution.

[0135] Depend on Figure 5 A shows that compared with the untreated bergamot essential oil 0.05mL group, the guinea pig skin IgE levels in the ultrasound pre-treated bergamot essential oil 0.025mL and 0.05mL groups were significantly increased (p < 0.05), and there was no significant difference in the guinea pig skin IgE levels in the microwave pre-treated bergamot essential oil 0.025mL and 0.05mL and alkali pre-treated bergamot essential oil 0.025mL and 0.05mL groups and the untreated bergamot essential oil 0.025mL group. Figure 5 A.

[0136] 3.7.2 Effect of bergamot essential oil on IgA levels in guinea pig skin

[0137] Depend on Figure 5 B shows that compared with the normal control group, the IgA level in the guinea pig skin of the untreated bergamot essential oil 0.025mL and 0.05mL group, the ultrasound pre-treated bergamot essential oil 0.025mL and 0.05mL group, the alkali solution pre-treated bergamot essential oil 0.025mL and 0.05mL group and the positive control group was significantly increased (p < 0.01), the IgA level in the guinea pig skin of the microwave pre-treated bergamot essential oil 0.025mL group was significantly increased (p < 0.05), and there was no significant difference in the IgA level in the guinea pig skin of the microwave pre-treated bergamot essential oil 0.05mL group.

[0138] Depend on Figure 5 B shows that compared with the untreated bergamot essential oil 0.025mL group, there was no significant difference in the IgA level in guinea pig skin among the bergamot essential oil 0.025mL and 0.05mL groups pretreated with ultrasound, the bergamot essential oil 0.025mL and 0.05mL groups pretreated with microwave, and the bergamot essential oil 0.025mL and 0.05mL groups pretreated with alkali solution.

[0139] Depend on Figure 5 B shows that compared with the 0.05mL bergamot essential oil group without pretreatment, there was no significant difference in the IgA level in guinea pig skin among the 0.025mL and 0.05mL bergamot essential oil groups with ultrasound pretreatment, the 0.025mL and 0.05mL bergamot essential oil groups with microwave pretreatment, the 0.025mL and 0.05mL bergamot essential oil groups with alkali solution pretreatment, and the 0.025mL bergamot essential oil group without pretreatment.

[0140] 3.7.3 Effect of bergamot essential oil on IgG levels in guinea pig skin

[0141] Depend on Figure 5 C shows that compared with the normal control group, the IgG level in the guinea pig skin of the groups without pretreatment of bergamot essential oil 0.05mL, ultrasound pretreatment of bergamot essential oil 0.025mL and 0.05mL and alkali solution pretreatment of bergamot essential oil 0.05mL was significantly increased (p < 0.01), the IgG level in the guinea pig skin of the group without pretreatment of bergamot essential oil 0.025mL and the positive control group was significantly increased (p < 0.05), and there was no significant difference in the IgG level in the guinea pig skin of the groups with alkali solution pretreatment of bergamot essential oil 0.025mL and microwave pretreatment of bergamot essential oil 0.05mL and 0.025mL.

[0142] Depend on Figure 5 C shows that compared with the untreated bergamot essential oil 0.025mL group, there was no significant difference in the IgG level in guinea pig skin among the bergamot essential oil 0.025mL and 0.05mL groups pretreated with ultrasound, the bergamot essential oil 0.025mL and 0.05mL groups pretreated with microwave, and the bergamot essential oil 0.025mL and 0.05mL groups pretreated with alkali solution.

[0143] Depend on Figure 5 C shows that compared with the group without pretreatment of bergamot essential oil 0.05mL, the IgG level in the guinea pig skin of the microwave pretreated bergamot essential oil 0.025mL and 0.05mL groups was significantly reduced (p < 0.01), and the IgG level in the guinea pig skin of the alkali pretreated bergamot essential oil 0.025mL group was significantly reduced (p < 0.05), and there was no significant difference in the IgG level in the guinea pig skin between the ultrasound pretreated bergamot essential oil 0.025mL and 0.05mL groups and the unpretreated bergamot essential oil 0.025mL group and the alkali pretreated 0.05mL group.

[0144] 3.8 Comparison of essential oil allergy reduction technologies

[0145] In summary, the present invention carried out GC-MS detection on untreated bergamot essential oil, microwave pre-treated bergamot essential oil, ultrasonic pre-treated bergamot essential oil and alkali solution pre-treated bergamot essential oil, and the results showed that compared with the untreated bergamot essential oil group, the bergamot essential oil pre-treated with alkali solution had the lowest allergenicity, and the bergamot essential oils pre-treated with different methods had different effects on reducing the allergic substances in the bergamot essential oil, indicating that the bergamot essential oil pre-treated with alkali solution had the best allergenicity reducing effect.

[0146] The present invention carries out pathological sectioning on untreated bergamot essential oil, microwave pre-treated bergamot essential oil, ultrasound pre-treated bergamot essential oil and alkali solution pre-treated bergamot essential oil, and the results show that among the three pre-treatment methods, the bergamot essential oil pre-treated with ultrasound has the strongest sensitizing effect, while the bergamot essential oil pre-treated with alkali solution has the lowest sensitizing effect.

[0147] The invention detects the levels of IgE, IgA and IgG immunoglobulins on untreated bergamot essential oil, microwave pre-treated bergamot essential oil, ultrasonic pre-treated bergamot essential oil and alkali pre-treated bergamot essential oil. The results show that compared with a normal control group, there are no significant differences in the levels of IgE, IgA and IgG in the serum and skin of guinea pigs in the microwave pre-treated bergamot essential oil group, and the levels of IgE, IgA and IgG in the serum and skin of guinea pigs in the ultrasonic and alkali pre-treated bergamot essential oil groups are increased to varying degrees; compared with a bergamot essential oil group not pre-treated, the levels of IgE, IgA and IgG in the serum and skin of guinea pigs in the microwave pre-treated bergamot essential oil group are reduced to varying degrees, the levels of IgE, IgA and IgG in the serum and skin of guinea pigs in the alkali pre-treated bergamot essential oil group are partially reduced to varying degrees, and the IgE level in the skin of guinea pigs in the ultrasonic pre-treated bergamot essential oil group is significantly increased.

[0148] Obviously, the immunoglobulin levels of the guinea pig serum and skin of the ten groups of guinea pigs in the present invention were found to be lower in the guinea pig immunoglobulin level of the bergamot essential oil group pretreated with microwave than those pretreated with alkali solution and ultrasound, indicating that microwave pretreatment has a better effect on reducing allergenic substances.

[0149] The present invention observes the allergy experiment of bergamot essential oil on guinea pig skin by transdermal application, and the results show that the inducing effect of bergamot essential oil on guinea pig skin symptoms such as erythema, edema, scab, ulceration, etc. is visible to the naked eye. Guinea pig skin pathological sections show pathological changes such as thickening of the epidermis, necrosis of the epidermis, necrosis of collagen fibers in the dermis, and infiltration of inflammatory cells in the guinea pig skin, among which the 0.05mL group of ultrasonic pre-treatment bergamot essential oil has the strongest sensitization. The bergamot essential oil application causes skin allergies of varying degrees, among which the 0.05mL group of ultrasonic pre-treatment bergamot essential oil has the strongest sensitization. The bergamot essential oil application causes skin allergies of varying degrees, and the ultrasonic pre-treatment bergamot essential oil transdermal application causes the strongest allergic reaction.

[0150] In summary, the application of bergamot essential oil can cause different degrees of allergic phenomena on the skin of guinea pigs, causing redness, spots, thickening of the stratum corneum, edema, infiltration of inflammatory cells such as lymphocytes and eosinophils, deep cortical congestion, hemorrhage, connective tissue necrosis, degeneration and increased immunoglobulin, etc. Among the above different pre-treatment methods for reducing the allergenic substances of bergamot essential oil, the alkali solution pre-treatment effect is the best, and the ultrasonic pre-treatment effect is the worst. The present invention provides a theoretical basis for the reduction technology of bergamot essential oil allergenic substances and the application and development of bergamot essential oil.

[0151] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for extracting desensitized and attenuated bergamot essential oil, characterized in that: The following steps are involved: The bergamot is pre-treated and then essential oil is extracted by steam distillation. The pre-treatment includes at least one of microwave treatment, ultrasonic treatment and alkali solution treatment.

2. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, wherein: The pre-treatment includes alkali solution treatment.

3. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, wherein: The power of the microwave treatment is 450-550 W, the temperature of the microwave treatment is 70-80° C., and the duration of the microwave treatment is 40-50 min.

4. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, wherein: The power of the ultrasonic treatment is 250-350 W, the temperature of the ultrasonic treatment is 45-55° C., and the duration of the ultrasonic treatment is 35-45 min.

5. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, characterized in that, The alkali solution is selected from sodium hydroxide solution.

6. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, characterized in that: The concentration of the alkali solution is 0.5wt%-1.5wt%.

7. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, characterized in that: The alkali solution treatment comprises: using the alkali solution as a reflux medium and boiling and refluxing for 0.5-1.5 hours.

8. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, characterized in that: The material ratio of the bergamot to the alkali solution is 50-80g:500-600mL.

9. The method for extracting the desensitized and attenuated bergamot essential oil as claimed in claim 1, characterized in that: The extraction time is 3-4 hours.

10. The bergamot essential oil obtained according to the method described in any one of claims 1 to 9.

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