Application of frankincense processed product volatile oil

Abstract

The invention provides application of frankincense processed product volatile oil in preparation of a medicine. The medicine is used for resisting apoptosis or promoting cell proliferation. The frankincense processed product volatile oil can activate a cAMP signal channel and promote the expression of PKA so as to influence a downstream signal channel, inhibit the phosphorylation of pro-apoptotic protein Bad and promote the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, thereby effectively inhibiting the apoptosis. Therefore, when the frankincense processed product volatile oil is prepared into the medicine, cell apoptosis can be effectively inhibited, and then cell proliferation is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular, the present application relates to the use of volatile oil of frankincense processed products. BACKGROUND

[0002] Modern pharmacological studies show that volatile oil of frankincense processed products has various effects, such as analgesia, penetration promotion, bacteriostasis, anti-inflammatory, etc. The perception of nature and taste of traditional Chinese medicine is related to the biological mechanism of signal transduction, such as human bitter taste receptor TAS2Rs and mammalian olfactory receptor (OR). The main function of olfactory receptor is to identify various odors, and it also has ectopic expression outside the nasal cavity. OR is not only the olfactory receptor expressed in pure olfactory neurons (OSN), but also a general chemical receptor involved in the whole body physiology and pathophysiology process.

[0003] Most ectopic ORs are involved in intercellular recognition, migration, proliferation, apoptosis, exocytosis, etc. Ectopic ORs not only play a role in the human body, but also play a similar role in other mammals. Studies have shown that methylisoeugenol as a weak Olfr73 agonist in mice can trigger a signal cascade independent of the classical pathway mediated by Gnao activation, leading to Cl - efflux, coexist with the classical cAMP signal pathway in olfactory neurons, and can be triggered by the same OR in a ligand-selective manner. The classical olfactory signal transduction pathway is that under the stimulation of odorants, OR activates the heterotrimeric Gs protein, triggers protein dissociation into α, β, γ subunits, Gnαs activates ACIII, promotes ATP conversion to generate the second messenger cAMP, and then opens Ca 2+ and Na + conducting cyclic nucleotide-gated (CNG) channels. Ca 2+ activated Cl - (CACL) channels open, leading to enhanced OSN depolarization through Cl - efflux. cAMP pathway activation often triggers a cascade reaction, such as opening Ca 2+ and Na + conducting cyclic nucleotide-gated (CNG) channels, Ca 2+ activated Cl - (CACL) channels open leading to Cl - efflux, inhibition of Bad phosphorylation, etc.

[0004] Therefore, in view of the unique pharmacological activity of volatile oil of frankincense processed products and the complexity of the key target points predicted by olfactory receptor network pharmacology, the interaction between the two needs to be studied. SUMMARY

[0005] The present application aims to solve at least one of the technical problems existing in the prior art at least to some extent.

[0006] The present application is based on the following findings of the inventors:

[0007] The volatile components of the processed products of Boswellia sacra have a multi-target and multi-path combined effect on olfactory receptors, mainly involving GO functions such as G protein function, neurotransmitter activity, and synaptic composition, and regulating neural-receptor signaling pathways, calcium signaling pathways, and cAMP signaling pathways. According to the C-T-P network analysis, the main volatile active components of the processed products of Boswellia sacra include geranyl acetate, neryl acetate, octyl acetate, linalool, and octyl formate. Among them, octyl acetate is a representative component of the volatile oil of Boswellia sacra and its processed products; linalool is the key component that causes the odor difference among Boswellia sacra, vinegar Boswellia sacra, and rush-fried Boswellia sacra. Due to the differences in the relative content of these core volatile components in the volatile oil, the effects of the volatile oils of the three on olfactory receptors and cells may be different.

[0008] Therefore, in order to further explore the influence mechanism of the volatile oil of the processed products of Boswellia sacra on cell signal transduction, the inventors carried out cell experiments. The experimental results show that the volatile oil of the processed products of Boswellia sacra may regulate cell signal transduction through the G protein-mediated cAMP signaling pathway. Specifically, by detecting the expression of key proteins GnαS, PKA, and AC III in the cAMP signaling pathway and the level of intracellular cAMP, the mechanism of action of the volatile oil of the processed products of Boswellia sacra is revealed: promoting the dissociation of Gs protein into its alpha subtype (GnαS), activating AC protein into AC III, and then promoting the conversion of ATP into second messenger cAMP and the expression of PKA, followed by inhibiting the phosphorylation of apoptosis protein Bad. This series of changes affects the downstream cell apoptosis signaling pathway, promotes the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, and thus inhibits cell apoptosis. Further experimental verification shows that the volatile oil of the processed products of Boswellia sacra can regulate the key proteins of the downstream cell apoptosis function of the cAMP signaling pathway. Specifically, it inhibits the phosphorylation of pro-apoptotic protein Bad, while promoting the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, and thus effectively inhibits cell apoptosis. These findings provide important molecular mechanism basis for understanding the cell protection effect of the volatile oil of the processed products of Boswellia sacra.

[0009] Based on this, in a first aspect of the present application, the present application provides a use of a volatile oil of a processed product of Olibanum in the preparation of a medicament for resisting cell apoptosis or promoting cell proliferation. The inventors have found through experiments that the volatile oil of the processed product of Olibanum can activate the cAMP signal pathway, promote the expression of PKA, and then affect the downstream signal pathway, inhibit the phosphorylation of the pro-apoptotic protein Bad, and promote the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl, thereby effectively inhibiting cell apoptosis. Therefore, the volatile oil of the processed product of Olibanum prepared into a medicament can effectively inhibit cell apoptosis, and then promote cell proliferation.

[0010] In a second aspect of the present application, the present application provides a use of a volatile oil of a processed product of Olibanum in the preparation of a product for at least one of the following: activating the cAMP signal pathway; regulating the expression of GnαS; regulating the expression of PKA; inhibiting the phosphorylation of Bad; promoting the expression of Bcl-2 and Bcl-xl; promoting the expression of ACIII; promoting the expression of NMDAR; and inhibiting the expression of AMPAR. The inventors have found through experiments that the volatile oil of the processed product of Olibanum can regulate the expression of GnαS, activate the AC protein to generate ACIII, activate the cAMP signal pathway, thereby regulate the expression of PKA, and then affect the downstream signal pathway, promote the expression of NMDAR and the anti-apoptotic proteins Bcl-2 and Bcl-xl, inhibit the expression of AMPAR, and promote the phosphorylation of the pro-apoptotic protein Bad, thereby inhibiting cell apoptosis.

[0011] In a third aspect of the present application, the present application provides a method for promoting cell proliferation or resisting cell apoptosis in vitro. According to an embodiment of the present application, the method comprises: co-culturing a volatile oil of a processed product of Olibanum with the cells. As described above, the volatile oil of the processed product of Olibanum can inhibit cell apoptosis, and therefore culturing the volatile oil of the processed product of Olibanum with the cells can promote cell proliferation or resist cell apoptosis.

[0012] In a fourth aspect of the present application, the present application provides a method for increasing the expression of cAMP in cells in vitro. According to an embodiment of the present application, the method comprises: co-culturing a volatile oil of a processed product of Olibanum with the cells. As described above, the volatile oil of the processed product of Olibanum can regulate the expression of GnαS, activate the AC protein to generate ACIII, and then promote the conversion of ATP to the second signal molecule cAMP. Therefore, the method of the present application can increase the expression of cAMP in cells.

[0013] Additional aspects and advantages of the present application will be partially given in the following description, partially will become apparent from the following description, or will be understood by practicing the present application. BRIEF DESCRIPTION OF DRAWINGS

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0015] Figure 1 The results are from the cytotoxicity test according to Example 2 of the present invention; wherein, olibanum is frankincense volatile oil, vinegar olibanum is vinegar-flavored frankincense volatile oil, and Rush-olibanum is frankincense volatile oil stir-fried with rush pith.

[0016] Figure 2 The graph shows the concentration results of the volatile oils of frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith according to Example 3 of the present invention on the proliferation-promoting effect on RAW 264.7 cells, where A is frankincense volatile oil, B is vinegar-processed frankincense volatile oil, and C is frankincense stir-fried with rush pith volatile oil.

[0017] Figure 3 The graph shows the results of intracellular cAMP level measurement in RAW 264.7 cells of each experimental group according to Example 4 of the present invention. Statistical analysis shows that compared with the control group, * represents P<0.05; ** represents P<0.01; and *** represents P<0.001.

[0018] Figure 4 The graph shows the results of intracellular ACⅢ expression levels in RAW 264.7 cells in each experimental group according to Example 5 of the present invention. In the graph, A is the cell immunofluorescence result (scale bar: 20 μm), single staining: ACⅢ (red) / DAPI (blue); B is the ACⅢ quantitative measurement result.

[0019] Figure 5 The figures show the intracellular NMDAR and AMPAR expression levels of RAW 264.7 cells in each experimental group according to Example 5 of the present invention. A represents the results of cellular immunofluorescence (scale bar: 20 μm), double staining: AMPAR (green) / DAPI (blue), NMDAR (red) / DAPI (blue); B represents the quantitative determination results of NMDAR and AMPAR.

[0020] Figure 6 This refers to the expression of GnαS and PKA, key proteins of the cAMP signaling pathway, in RAW 264.7 cells of each experimental group according to Example 6 of the present invention. Wherein, # represents P<0.05, ## represents P<0.01, ### represents P<0.001 (compared to the frankincense group); * represents P<0.05, ** represents P<0.01, *** represents P<0.001 (compared to the control group);

[0021] Figure 7 The expression of intracellular apoptosis-related proteins p-Bad / Bad, Bcl-2, and Bcl-xl in RAW 264.7 cells in each experimental group according to Example 6 of the present invention;

[0022] Figure 8 This refers to the expression of key genes Gnαs, Adcy1, and Prkca mRNA in the intracellular cAMP signaling pathway of RAW 264.7 cells in each experimental group according to Example 7 of the present invention.

[0023] Figure 9 This refers to the expression of intracellular apoptosis-related genes Bad, Bcl-2, and Bcl-xlmRNA in RAW 264.7 cells of each experimental group according to Example 7 of the present invention.

[0024] Figure 10 This refers to the expression of intracellular signal transduction-related genes Grin1 and Gria1 mRNA in RAW 264.7 cells of each experimental group according to Example 7 of the present invention. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below, and are intended to explain the present invention, but should not be construed as limiting the present invention.

[0026] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0027] For the sake of brevity, this article only discloses some specific numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the invention, are intended to cover non-exclusive inclusion.

[0029] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0030] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0031] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0032] This invention proposes the use of frankincense processed volatile oil in drug preparation, the use in product preparation, a method for promoting cell proliferation or inhibiting apoptosis in vitro, and a method for increasing intracellular cAMP expression in vitro. These will be described in detail below.

[0033] use

[0034] In a first aspect, the present invention proposes the use of frankincense processed product volatile oil in the preparation of a pharmaceutical product for anti-apoptosis or cell proliferation promotion. The inventors have experimentally discovered that frankincense processed product volatile oil can activate the cAMP signaling pathway, promote PKA expression, and thereby affect downstream signaling pathways, inhibiting the phosphorylation of the pro-apoptotic protein Bad and promoting the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, thus effectively inhibiting apoptosis. Therefore, preparing frankincense processed product volatile oil into a pharmaceutical product can effectively inhibit apoptosis and thus promote cell proliferation.

[0035] In some embodiments of the present invention, the cells contain at least one of the following proteins: Bad, p-Bad, Bcl-2, Bcl-xl, GnαS, PKA, AMPAR, NMDAR, and ACⅢ. Therefore, the volatile oil from frankincense processed products can regulate the expression of the above proteins, thereby achieving anti-apoptosis.

[0036] In some embodiments of the present invention, the cells contain at least one of the following genes: Bad, Bcl-2, Bcl-xl, Gnαs, Adcy1, Prkca, Grin1, and Gria1. Therefore, the volatile oil from frankincense processed products can regulate the expression of the above-mentioned genes, thereby achieving anti-apoptosis.

[0037] In this paper, the term "regulation" is a two-way process that includes both upregulation (promotion) and downregulation (inhibition), depending on the type of regulatory mechanism and the cellular environment.

[0038] It should be noted that this invention does not specifically limit the cell type and is applicable to any mammalian cell capable of expressing the following proteins or encoding corresponding genes: Proteins: Bad, p-Bad, Bcl-2, Bcl-xl, GnαS, PKA, AMPAR, NMDAR, ACⅢ; Genes: Bad, Bcl-2, Bcl-xl, Gnαs, Adcy1, Prkca, Grin1, Gria1. It is worth noting that apoptosis inhibition of different cell types can target different diseases. For example, the cells of this invention can be inflammatory cells. On the one hand, regulating the anti-apoptotic effect of inflammatory cells can reduce excessive apoptosis of inflammatory cells, thereby alleviating the damage of the inflammatory response to the cardiovascular system; on the other hand, the anti-apoptotic effect of inflammatory cells helps reduce neuroinflammation, protect nerve cells, and delay the progression of neurodegenerative diseases; furthermore, the anti-apoptotic effect of inflammatory cells can regulate bone metabolism, reduce bone resorption, increase bone density, and prevent and / or treat bone diseases. Therefore, in some embodiments of this invention, the drug can also be used to prevent and / or treat at least one of cardiovascular diseases, neurodegenerative diseases, and bone diseases.

[0039] In this document, the term "treatment" refers to the administration of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a disease in individuals susceptible to the disease but not yet diagnosed with it; (b) suppression of a disease, such as inhibiting disease progression; or (c) alleviating a disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of recombinant immune cells, pharmaceutical compositions, or drugs to an individual to treat, cure, alleviate, improve, reduce, or suppress a disease in that individual, including but not limited to administration of drugs containing the inhibitory chimeric antigen receptor, recombinant immune cells, or pharmaceutical compositions described herein to an individual in need.

[0040] In some embodiments of the present invention, the volatile oil of the frankincense processed product includes at least one of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense volatile oil stir-fried with rush pith. Therefore, the aforementioned volatile oil can effectively inhibit cell apoptosis and promote cell proliferation.

[0041] In some embodiments of the present invention, the volatile oil of frankincense stir-fried with rush pith is obtained by the following method: frankincense is subjected to a first stir-frying treatment; rush pith is added, and a second stir-frying treatment is performed to obtain frankincense stir-fried with rush pith; the frankincense stir-fried with rush pith is pulverized sequentially to obtain coarse powder; the coarse powder is mixed with water to obtain a mixture; and the mixture is subjected to volatile oil extraction treatment to obtain frankincense stir-fried with rush pith volatile oil. Therefore, the frankincense stir-fried with rush pith volatile oil prepared by the method of the present invention can effectively inhibit cell apoptosis and thus effectively promote cell proliferation.

[0042] In some embodiments of the present invention, the particle size of the frankincense is 4 to 7 mm. For example, it can be 4 mm, 5 mm, 6 mm, 7 mm, etc., or it can be any range of the above values.

[0043] In some embodiments of the present invention, the mass ratio of frankincense to rush pith is 10:(0.3 to 0.5). For example, it can be 10:0.3, 10:0.3.5, 10:0.4, 10:0.45, 10:0.5, etc., or it can be any range of the above values.

[0044] In some embodiments of the present invention, the temperatures of the first and second stir-frying processes are 75–85°C, respectively. For example, these temperatures can be 75°C, 77°C, 80°C, 82°C, 85°C, or any range of the above values.

[0045] In some embodiments of the present invention, the first frying time is 30 to 90 seconds. For example, it can be 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, or any range of the above values.

[0046] In some embodiments of the present invention, the second frying time is 4-6 minutes. For example, it can be 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, etc., or it can be any range of the above values.

[0047] In some embodiments of the present invention, the rush pith is added in two batches.

[0048] In some embodiments of the present invention, the mass ratio of the two batches of rush pith is 1:1.

[0049] In some embodiments of the present invention, the frankincense volatile oil is obtained by extracting volatile oil from frankincense.

[0050] In some embodiments of the present invention, the volatile oil of frankincense is obtained by extracting volatile oil from frankincense.

[0051] In some embodiments of the present invention, the extraction of volatile oil is carried out according to Method A of Volatile Oil Extraction Method in General Chapter 2204 of Part IV of the Chinese Pharmacopoeia 2020.

[0052] In a second aspect, the present invention proposes the use of frankincense processed product volatile oil in the preparation of products, said products being used for at least one of the following: activating the cAMP signaling pathway; regulating GnαS expression; regulating PKA expression; inhibiting Bad phosphorylation; promoting Bcl-2 and Bcl-xl expression; promoting ACⅢ expression; promoting NMDAR expression; and inhibiting AMPAR expression. The inventors have experimentally discovered that frankincense processed product volatile oil can regulate GnαS expression, activate the conversion of AC protein into ACⅢ, activate the cAMP signaling pathway, thereby regulating PKA expression, and further affecting downstream signaling pathways, promoting the expression of NMDAR, anti-apoptotic proteins Bcl-2 and Bcl-xl, inhibiting AMPAR expression, and inhibiting the phosphorylation of the pro-apoptotic protein Bad, thereby inhibiting cell apoptosis.

[0053] In some embodiments of the present invention, the frankincense processed product volatile oil includes at least one of frankincense volatile oil, vinegar-processed frankincense volatile oil, and rush pith-processed frankincense volatile oil.

[0054] In some embodiments of the present invention, the product can also be used to regulate the expression of the following gene mRNAs: Gnαs, Adcy1, Prkca, Bad, Bcl-2, Bcl-xl, Grin1, and Gria1. In some embodiments of the present invention, the product can inhibit the expression of Gnαs mRNA, inhibit the expression of Adcy1 mRNA, inhibit the expression of Prkca mRNA, inhibit the expression of Bad mRNA, promote the expression of Bcl-2 mRNA, promote the expression of Bcl-xl mRNA, promote the expression of Grin1 mRNA, and inhibit the expression of Gria1 mRNA. Therefore, it can effectively inhibit apoptosis and thus promote cell proliferation.

[0055] method

[0056] In a third aspect, the present invention provides a method for promoting cell proliferation or inhibiting apoptosis in vitro. According to an embodiment of the present invention, the method includes: co-culturing the cells with the volatile oil from frankincense processed products. As mentioned above, the volatile oil from frankincense processed products can inhibit apoptosis; therefore, culturing cells with the volatile oil from frankincense processed products can promote cell proliferation or inhibit apoptosis.

[0057] In some embodiments of the present invention, the volatile oil of the frankincense processed product includes at least one of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense volatile oil stir-fried with rush pith. Therefore, it can effectively promote cell proliferation or inhibit cell apoptosis.

[0058] In some embodiments of the present invention, the volatile oil of the frankincense processed product is frankincense volatile oil, and the concentration of the frankincense volatile oil in the co-culture treatment is less than 0.8 μg / μL. Therefore, it can effectively promote cell proliferation or inhibit cell apoptosis.

[0059] In some embodiments of the present invention, the volatile oil of the frankincense product is acetic frankincense volatile oil, and the concentration of the acetic frankincense volatile oil is less than 0.5 μg / μL during the co-culture treatment. Therefore, it can effectively promote cell proliferation or inhibit apoptosis.

[0060] In some embodiments of the present invention, the volatile oil of the frankincense processed product is the volatile oil of frankincense stir-fried with rush pith, and in the co-culture treatment, the concentration of the volatile oil of frankincense stir-fried with rush pith is less than 1.6 μg / μL. Therefore, it can effectively promote cell proliferation or inhibit apoptosis.

[0061] In some embodiments of the present invention, the cells contain at least one of the following proteins: Bad, p-Bad, Bcl-2, Bcl-xl, GnαS, PKA, AMPAR, NMDAR, and ACⅢ.

[0062] In some embodiments of the present invention, the cell contains at least one of the following genes: Bad, Bcl-2, Bcl-xl, Gnαs, Adcy1, Prkca, Grin1, Gria1.

[0063] In a fourth aspect, the present invention provides a method for enhancing intracellular cAMP expression in vitro. According to an embodiment of the present invention, the method includes: co-culturing frankincense processing product volatile oil with the cells. As mentioned above, frankincense processing product volatile oil can activate the conversion of AC protein to ACⅢ by regulating GnαS expression, thereby promoting the conversion of ATP to the second signal molecule cAMP. Therefore, the method of the present invention can enhance intracellular cAMP expression.

[0064] In some embodiments of the present invention, the frankincense processed product volatile oil includes at least one of frankincense volatile oil, vinegar-processed frankincense volatile oil, and rush pith-processed frankincense volatile oil.

[0065] In some embodiments of the present invention, the volatile oil of the frankincense processed product is frankincense volatile oil, and the concentration of the frankincense volatile oil is less than 0.2 μg / μL. This effectively enhances the expression of intracellular cAMP.

[0066] In some embodiments of the present invention, the volatile oil of the frankincense processed product is acetic frankincense volatile oil, and the concentration of the acetic frankincense volatile oil is less than 0.3 μg / μL. This effectively enhances the expression of intracellular cAMP.

[0067] In some embodiments of the present invention, the volatile oil of the frankincense processed product is frankincense volatile oil stir-fried with rush pith, and the concentration of the frankincense volatile oil stir-fried with rush pith is less than 0.2 μg / μL. Therefore, it can effectively increase the expression of cAMP in cells.

[0068] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0069] Example 1: Preparation of volatile oils from frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith

[0070] 1. Preparation of frankincense volatile oil and vinegar-processed frankincense volatile oil: Weigh about 50g each of vinegar-processed frankincense and frankincense, crush them through a No. 2 sieve, accurately weigh them, place them in a 500mL round-bottom flask, add 250mL of water, and extract the volatile oil according to Method A of Volatile Oil Extraction in General Chapter 2204 of Part IV of the Chinese Pharmacopoeia 2020. Collect the volatile oil in a brown volumetric flask and store it at 4℃.

[0071] 2. Preparation of volatile oil from stir-fried rush pith and frankincense:

[0072] (1) Preparation of stir-fried frankincense with rush pith: Select medium-sized frankincense (4-7mm) and put it into an 80℃ pot. Add 2g of rush pith to every 50g of frankincense, divide it into two portions, and add it at the 2nd minute and 4th minute of stir-frying respectively. Stir-fry continuously until the 6th minute to obtain stir-fried frankincense with rush pith.

[0073] (2) Preparation of volatile oil from stir-fried frankincense from rush pith: Weigh about 50g of stir-fried frankincense from rush pith, crush it through a No. 2 sieve, weigh it accurately, place it in a 500mL round-bottom flask, add 250mL of water, and extract the volatile oil according to Method A of Volatile Oil Extraction Method 2204 in General Chapter 4 of the 2020 edition of the Chinese Pharmacopoeia. Collect the volatile oil in a brown volumetric flask and store it at 4℃.

[0074] Example 2: Cytotoxicity of volatile oils from frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith

[0075] The three volatile oils were first dissolved in a certain amount of DMSO. Then, the DMSO containing frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense stir-fried with rush pith were diluted with DMEM medium to make the final concentrations of frankincense volatile oil in DMEM medium 1.2255 μg / μL, 2.4510 μg / μL, 4.9020 μg / μL, and 9.8040 μg / μL, the final concentrations of vinegar-processed frankincense volatile oil 1.1355 μg / μL, 2.2710 μg / μL, 4.5420 μg / μL, and 9.0840 μg / μL, and the final concentrations of frankincense stir-fried with rush pith 1.1575 μg / μL, 2.3150 μg / μL, 4.6300 μg / μL, and 9.2600 μg / μL. Different concentrations of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense stir-fried with rush pith were co-cultured with RAW 264.7 cells for 24 h. Cell viability was calculated, and the relative viability of the drug-treated groups to the DMSO solvent group (DMSO group cell viability was 100%) and EC5 values ​​of each drug-treated group were calculated. 50 value.

[0076] The test results are as follows Figure 1 As shown, this indicates that: α = 0.05, EC50 of the Juncus effusus-fried frankincense group is... 50 The EC values ​​were 5.828 μg / μL to 6.708 μg / μL; for the frankincense group... 50 The EC values ​​for the frankincense group ranged from 6.792 μg / μL to 8.657 μg / μL. 50 The concentrations ranged from 5.315 μg / μL to 7.495 μg / μL. The EC values ​​of frankincense volatile oil and frankincense were... 50 The values ​​were all higher than those of the volatile oil from stir-fried rush pith and frankincense. Specifically, the EC value of the volatile oil from stir-fried rush pith and frankincense was... 50 The lowest value indicates the smallest concentration range with cytotoxicity to RAW 264.7 cells, suggesting that the cytotoxicity of the volatile oil from stir-fried frankincense from rush pith is lower than that of vinegar-processed frankincense and frankincense volatile oil.

[0077] Example 3: Screening of cell proliferation-promoting concentrations of volatile oils from frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith.

[0078] Frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense stir-fried with rush pith were first dissolved in DMSO, and then diluted to different concentrations using DMEM medium. After co-culturing different concentrations of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense stir-fried with rush pith with RAW 264.7 cells for 24 h, cell viability was calculated, and the relative viability of the drug-treated groups relative to the DMSO solvent group was calculated (the cell viability of the DMSO group was 100%).

[0079] Experimental results are as follows Figure 2As shown, the results indicated that when CCK-8 was used to screen three concentrations of volatile oils to promote proliferation, frankincense volatile oil had the best proliferation-promoting effect, especially when the frankincense volatile oil concentration c < 0.3947 μg / μL. Figure 2 A) Frankincense volatile oil had a significant proliferative effect on RAW 264.7 cells (P<0.001); the concentration of frankincense volatile oil in vinegar was c<0.1192μg / μL ( Figure 2 B), the volatile oil of frankincense cooked with vinegar significantly promoted the proliferation of RAW 264.7 cells (P<0.001); the concentration of volatile oil of frankincense stir-fried with rush pith was c<0.7814μg / μL ( Figure 2 C) The volatile oil from stir-fried rush pith and frankincense significantly promoted the proliferation of RAW 264.7 cells (P<0.001).

[0080] Example 4: Results of intracellular cAMP level measurement in RAW 264.7 cells

[0081] To investigate whether frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith had any effect on cAMP production, the inventors co-cultured different concentrations of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense stir-fried with rush pith volatile oil with RAW 264.7 cells for 24 hours. The drug concentrations that showed the most significant proliferative effect on RAW 264.7 cells were selected, and each group was dissolved in a certain amount of DMSO. Then, the DMSO containing frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense stir-fried with rush pith were diluted with DMEM medium to make the final concentrations of frankincense volatile oil in DMEM medium 0.0493 μg / μL, 0.0987 μg / μL, and 0.1973 μg / μL, the final concentrations of vinegar-processed frankincense volatile oil 0.0596 μg / μL, 0.1192 μg / μL, and 0.2384 μg / μL, and the final concentrations of frankincense stir-fried with rush pith volatile oil 0.0488 μg / μL, 0.0977 μg / μL, and 0.1955 μg / μL.

[0082] The intracellular cAMP expression level of cultured RAW 264.7 cells was detected by ELISA, and the results are as follows: Figure 3 As shown, the concentration of volatile oil that promotes RAW 264.7 cell proliferation does not necessarily promote intracellular cAMP expression, but appropriate concentrations of volatile oil can significantly promote cAMP production. Specifically, the promoting effect was most pronounced when the concentration of frankincense volatile oil was 0.0493 μg / μL; the promoting effect was also most pronounced when the concentration of frankincense volatile oil (processed with vinegar) was 0.1192 μg / μL; and the promoting effect was most pronounced when the concentration of frankincense volatile oil (processed with rush pith) was 0.0488 μg / μL. Among the three volatile oils, the promoting effect of frankincense volatile oil (processed with vinegar) was the most significant.

[0083] Example 5: Effects of volatile oils from frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith on the expression levels of AMPAR, NMDAR, and ACⅢ proteins.

[0084] To investigate the effects of three volatile oils on the expression of key proteins AMPAR and NMDAR, as well as membrane protein ACⅢ, in the downstream signal transduction cascade of the cAMP signaling pathway, RAW 264.7 cells were cultured for 24 h with the optimal proliferation-promoting concentration of each volatile oil. The expression levels of each protein in the cells were then measured, and the average values ​​were taken. Specifically, the final concentration of frankincense volatile oil in DMEM medium was 0.0493 μg / μL, the final concentration of vinegar-processed frankincense volatile oil in DMEM medium was 0.1192 μg / μL, and the final concentration of stir-fried frankincense volatile oil from rush pith in DMEM medium was 0.0488 μg / μL.

[0085] Given that ACⅢ is a membrane protein with low expression levels, immunofluorescence (IF) was used to semi-quantitatively determine ACⅢ protein expression, which is more intuitive than Western blot. Experimental results are shown below. Figure 4 As shown, the ACⅢ expression levels in the vinegar-processed frankincense group and the frankincense stir-fried with rush pith group were significantly higher than those in the control group and the frankincense group (P>0.01), indicating that the volatile oils of vinegar-processed frankincense and frankincense stir-fried with rush pith can activate ACⅢ in the cAMP pathway.

[0086] The expression of NMDAR and AMPAR proteins was semi-quantitatively determined using cellular immunofluorescence assay. The experimental results are as follows: Figure 5 As shown, the volatile oils of frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith can all promote NMDAR expression. Among them, the volatile oil of vinegar-processed frankincense has a highly significant ability to promote NMDAR protein expression (P>0.0001). The AMPAR expression level in the three treatment groups was lower than that in the control group, indicating that the three volatile oils have an inhibitory effect on AMPAR protein expression.

[0087] Example 6: Effects of volatile oils from frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith on GnαS, PKA proteins, and apoptosis-related proteins.

[0088] To investigate the effects of three volatile oils on the expression of key upstream proteins GnαS and PKA in the cAMP signaling pathway, and key downstream proteins involved in the apoptosis cascade, P-Bad / Bad, Bcl-2, and Bcl-xl, RAW 264.7 cells were cultured for 24 h with the optimal concentration of each volatile oil used for cell proliferation. The expression levels of each protein in the cells were then measured, and the average values ​​were taken. Specifically, the final concentration of frankincense volatile oil in DMEM medium was 0.0493 μg / μL, the final concentration of vinegar-processed frankincense volatile oil in DMEM medium was 0.1192 μg / μL, and the final concentration of stir-fried frankincense volatile oil from rush pith in DMEM medium was 0.0488 μg / μL.

[0089] After the culture was completed, the expression levels of GnαS and PKA, key upstream proteins of the cAMP signaling pathway, were measured using Western blotting in RAW 264.7 cells. The results are as follows: Figure 6 As shown in the figure, the volatile oil of frankincense processed with vinegar promoted the expression of GnαS protein in RAW 264.7 cells, and the volatile oil of frankincense processed with rush pith promoted the expression of PKA protein in RAW 264.7 cells. These results indicate that the classic cAMP signaling pathway, regulated by olfactory receptors (ORs) in olfactory neurons (OSNs), also exists in mouse somatic RAW 264.7 cells and is regulated by the volatile oil components of frankincense and its processed products. The volatile oil of frankincense processed with rush pith promoted the dissociation of Gs protein into the α isoform and simultaneously promoted PKA production; however, the PKA expression level in the vinegar-processed frankincense group was significantly lower than that in the rush pith-processed frankincense group.

[0090] P-Bad / Bad, Bcl-2, and Bcl-xl are key proteins downstream of the cAMP signaling pathway involved in the apoptosis cascade. The inventors used Western blotting to detect the expression levels of these apoptosis-related proteins, such as... Figure 7 As shown in the figure, the p-Bad / Bad values ​​of the vinegar-processed frankincense group and the frankincense-stirred with rush pith group were lower than those of the control group and the frankincense group, indicating that the volatile oils of vinegar-processed frankincense and frankincense-stirred with rush pith can inhibit the phosphorylation of the pro-apoptotic protein Bad. Compared with the control group, the expression levels of the anti-apoptotic proteins Bcl-2 and Bcl-xl were higher in the vinegar-processed frankincense group and the frankincense-stirred with rush pith group. The volatile oils of vinegar-processed frankincense and frankincense-stirred with rush pith can inhibit the phosphorylation of the pro-apoptotic protein Bad, while promoting the expression of the anti-apoptotic proteins Bcl-xl and Bcl-2, thereby inhibiting RAW264.7 cell apoptosis. Among them, the volatile oil of frankincense-stirred with rush pith has better anti-RAW 264.7 cell apoptosis ability, which may be one of the reasons why the volatile oil of frankincense-stirred with rush pith has less cytotoxicity to RAW 264.7 cells.

[0091] Example 7: Effects of volatile oils from frankincense, vinegar-processed frankincense, and frankincense stir-fried with rush pith on the mRNA expression levels of related genes.

[0092] To investigate the effects of three volatile oils on the mRNA expression levels of related genes, RAW 264.7 cells were cultured for 24 h using the optimal concentration of each volatile oil to promote cell proliferation. The expression levels of various proteins in the cells were then measured, and the average values ​​were taken. Specifically, the final concentrations of frankincense volatile oil, vinegar-processed frankincense volatile oil, and stir-fried rush pith frankincense volatile oil in DMEM medium were 0.0493 μg / μL, 0.1192 μg / μL, and 0.0488 μg / μL.

[0093] The expression levels of Gnαs, Adcy1, and Prkca mRNA, corresponding to key upstream proteins in the cAMP signaling pathway, were measured using RT-qPCR. Figure 8 As shown in the figure, compared with the control group, the expression levels of GnαS, Adcy1, and Prkca, the corresponding genes of key upstream proteins GnαS, ACⅢ, and PKA in the cAMP signaling pathway, were significantly reduced in the drug-treated group. The expression level of Adcy1, the gene corresponding to ACⅢ, was extremely low, indicating that the classic cAMP signaling pathway, regulated by olfactory receptors (ORs) in olfactory neurons (OSNs) at the gene level, also exists in mouse somatic RAW 264.7 cells and is regulated by the volatile oil components of frankincense and its processed products. Compared with the frankincense group, the expression levels of GnαS protein and genes were higher in the frankincense-processed group, indicating that more α-isoforms of Gs protein dissociated. The gene expression levels of Adcy1 and Prkca in the frankincense-processed group were higher than those in the frankincense group and the vinegar-processed frankincense group, indicating that the volatile oil of frankincense-processed group is more effective in stimulating the cAMP signaling pathway than that of vinegar-processed frankincense.

[0094] The expression levels of apoptosis-related genes Bad, Bcl-2, and Bcl-xl mRNA were measured using RT-qPCR, such as... Figure 9 As shown in the figure, compared with the control group, the expression level of bad mRNA corresponding to the pro-apoptotic protein Bad was significantly reduced in the treatment group, and the expression level of Bcl-2 and Bcl-xl mRNA corresponding to the anti-apoptotic proteins Bcl-2 and Bcl-xl was higher in the treatment group. Compared with the frankincense group, the expression levels of anti-apoptotic genes Bcl-2 and Bcl-xl mRNA were higher in the Juncus effusus-fried frankincense group and the vinegar-processed frankincense group. Compared with the vinegar-processed frankincense group, the expression level of bad mRNA corresponding to the pro-apoptotic protein Bad was lower in the Juncus effusus-fried frankincense group, while the expression levels of anti-apoptotic genes Bcl-2 and Bcl-xl mRNA were higher. This indicates that the volatile oil of Juncus effusus-fried frankincense may have a better anti-apoptotic function in RAW 264.7 cells, and is superior to that of vinegar-processed frankincense.

[0095] The expression levels of Grin1 and Gria1 mRNA, genes involved in signal transduction, were measured using RT-qPCR. Figure 10 As shown in the figure. The results indicate that the volatile oil of frankincense in vinegar has a strong upregulatory effect on the Grin1 gene, and the volatile oil of frankincense in vinegar and frankincense stir-fried with rush pith has a very significant inhibitory effect on the Grin1 gene (P>0.001).

[0096] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of frankincense volatile oil in the preparation of pharmaceuticals, wherein the pharmaceuticals are used to inhibit apoptosis or promote cell proliferation.

2. The use according to claim 1, characterized in that, The cells contain at least one of the following proteins: Bad, p-Bad, Bcl-2, Bcl-xl, GnαS, PKA, AMPAR, NMDAR, ACⅢ; Optionally, the cell contains at least one of the following genes: Bad, Bcl-2, Bcl-xl, Gnαs, Adcy1, Prkca, Grin1, Gria1.

3. The use according to claim 2, characterized in that, The frankincense processed product volatile oil includes at least one of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense volatile oil stir-fried with rush pith.

4. The use according to claim 3, characterized in that, The volatile oil from stir-fried rush pith and frankincense was obtained by the following method: The frankincense undergoes a first stir-frying process. Add rush pith and stir-fry for a second time to obtain rush pith stir-fried with frankincense; The stir-fried rush pith and frankincense were pulverized to obtain coarse powder; The coarse powder is mixed with water to obtain a mixture; The mixture was subjected to volatile oil extraction treatment to obtain volatile oil from stir-fried frankincense with rush pith.

5. The use according to claim 4, characterized in that, The particle size of the frankincense is 4-7 mm; Optionally, the mass ratio of the frankincense to the rush pith is 10:(0.3-0.5); Optionally, the temperatures of the first and second stir-frying processes are 75–85°C, respectively. Optionally, the first stir-frying process takes 30 to 90 seconds; Optionally, the second stir-frying process takes 4 to 6 minutes; Optionally, the rush pith is added in two batches; Optionally, the mass ratio of the two batches of rush pith is 1:

1.

6. Use of frankincense processed volatile oil in the preparation of a product, wherein the product is used in at least one of the following: Activate the cAMP signaling pathway; Regulates the expression of GnαS; Regulate PKA expression; Inhibits phosphorylation of Bad; Promotes the expression of Bcl-2 and Bcl-xl; Promotes the expression of ACⅢ; Promotes NMDAR expression; Inhibit AMPAR expression.

7. A method for promoting cell proliferation or inhibiting apoptosis in vitro, characterized in that, include: The volatile oil from the processed frankincense product was co-cultured with the cells.

8. The method according to claim 7, characterized in that, The frankincense processed product volatile oil includes at least one of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense volatile oil stir-fried with rush pith; Optionally, the volatile oil of the frankincense product is frankincense volatile oil, and in the co-cultivation treatment, the concentration of the frankincense volatile oil is less than 0.8 μg / μL; Optionally, the volatile oil of the frankincense product is acetosaccharin volatile oil, and in the co-cultivation treatment, the concentration of the acetosaccharin volatile oil is less than 0.5 μg / μL; Optionally, the volatile oil of the frankincense product is the volatile oil of frankincense stir-fried with rush pith, and in the co-culture treatment, the concentration of the volatile oil of frankincense stir-fried with rush pith is less than 1.6 μg / μL; Optionally, the cells contain at least one of the following proteins: Bad, p-Bad, Bcl-2, Bcl-xl, GnαS, PKA, AMPAR, NMDAR, ACⅢ; Optionally, the cell contains at least one of the following genes: Bad, Bcl-2, Bcl-xl, Gnαs, Adcy1, Prkca, Grin1, Gria1.

9. A method for increasing intracellular cAMP expression in vitro, characterized in that, include: The volatile oil from the processed frankincense product was co-cultured with the cells.

10. The method according to claim 9, characterized in that, The frankincense processed product volatile oil includes at least one of frankincense volatile oil, vinegar-processed frankincense volatile oil, and frankincense volatile oil stir-fried with rush pith; Optionally, the volatile oil of the frankincense product is frankincense volatile oil, and in the co-cultivation treatment, the concentration of the frankincense volatile oil is less than 0.2 μg / μL; Optionally, the volatile oil of the frankincense product is acetic frankincense volatile oil, and in the co-cultivation treatment, the concentration of the acetic frankincense volatile oil is less than 0.3 μg / μL; Optionally, the volatile oil of the frankincense product is the volatile oil of frankincense stir-fried with rush pith, and in the co-culture treatment, the concentration of the volatile oil of frankincense stir-fried with rush pith is less than 0.2 μg / μL.

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