CN115671181B - Atomization application method of terpene component derived from fingered citron in relieving cough and verification method thereof - Google Patents
Atomization application method of terpene component derived from fingered citron in relieving cough and verification method thereof Download PDFInfo
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Abstract
The application provides an atomization application method of terpene components derived from fingered citron in relieving cough and a verification method thereof. The atomization application method of the terpene component derived from the fingered citron in relieving cough comprises the following steps of: obtaining fingered citron; distilling fingered citron to obtain a terpene-containing precursor, wherein the terpene-containing precursor mainly contains D-limonene and gamma-terpinene, the content of the D-limonene is not lower than 200mg/g, and the content of the gamma-terpinene is not lower than 50mg/g; and preparing the terpene-containing precursor at a low temperature to obtain a terpene-containing atomized liquid, wherein the terpene-containing atomized liquid is used for atomizing to obtain a terpene-containing aerosol. The atomization application method of the terpene components from the fingered citron in relieving cough can provide scientific basis for further excavating the action mechanism of the terpene components in the fingered citron for treating cough, and can provide a new thinking method for the drug administration mode of the fingered citron for relieving cough.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicine biological effect evaluation, in particular to an atomization application method of terpene components derived from fingered citron in relieving cough and a verification method thereof.
Background
Cough is a common and frequently encountered disease in clinic, and is clinically manifested by persistent cough, itching throat, little or no phlegm, usually induced by inhalation of pungent smell and cold air, allergen contact and exercise, and is effective in oral administration of antihistamine and glucocorticoid. The cough is generally considered to belong to airway inflammation, the pathogenesis has seasonal characteristics, repeated attacks, lingering and difficult to heal, the life of a patient is seriously influenced, the traditional Chinese medicine has long-term clinical experience in treating allergic cough, and the traditional Chinese medicine has definite curative effect and small adverse reaction.
The fingered citron is the fruit of the fingered citron of the Rutaceae, and the extract of the fingered citron has the effects of resisting inflammation, resisting tumor, regulating blood sugar, resisting depression, resisting oxidation and the like, and the report that the fingered citron extract is orally taken for treating respiratory diseases is also available in the past, but the mechanism of the fingered citron is not clear, so that the curative effect of the fingered citron on relieving cough is necessarily researched by an advanced and scientific method, a scientific basis is provided for further excavating the action mechanism of the fingered citron for treating cough, and a novel thinking method is provided for the medicament taking mode of the fingered citron for relieving cough.
Disclosure of Invention
The application aims to overcome the defects in the prior art, and provides an atomization application method and a verification method of terpene components derived from fingered citron in relieving cough, so that scientific basis can be provided for further excavating the action mechanism of the terpene components in the fingered citron for treating cough, and a new thinking method can be provided for the drug administration mode of the fingered citron for relieving cough.
The aim of the application is realized by the following technical scheme:
an atomization application method of terpene components derived from fingered citron in relieving cough comprises the following steps:
obtaining fingered citron;
distilling fingered citron to obtain a terpene-containing precursor;
the terpene-containing precursor is pretreated to obtain terpene-containing atomized essential oil, wherein the terpene-containing atomized essential oil is used for low-temperature direct thermal atomization to obtain terpene-containing aerosol, the low-temperature direct thermal atomization temperature is 80-150 ℃, the terpene-containing essential oil mainly contains D-limonene and gamma-terpinene, the content of the D-limonene is not lower than 200mg/g, and the content of the gamma-terpinene is not lower than 50mg/g.
In one embodiment, the fingered citron is obtained, specifically, fresh fruits with the picking time of 7-11 months are cut into slices, and the slices are dried at a low temperature or in the shade until the moisture is less than or equal to 15%.
In one embodiment, the distillation of fingered citron comprises the steps of:
weighing the fingered citron with the mass of m g; adding n milliliters of water into the fingered citron for soaking treatment, wherein n: m is 8-12, m is more than 0;
and carrying out distillation and extraction operation on the fingered citron after the soaking treatment and water.
In one embodiment, before the step of adding water with a volume of n milliliters to the fingered citron for soaking treatment, and after the step of weighing m grams of the fingered citron, the step of distilling the fingered citron further comprises the following steps:
and crushing the fingered citron.
In one embodiment, the grinding operation of the fingered citron is specifically: pulverizing fructus Citri Sarcodactylis into coarse powder.
In one embodiment, the soaking time of soaking treatment by adding n milliliters of water into the fingered citron is 45-75 minutes.
In one embodiment, the temperature of the operation of distilling and extracting the fingered citron after the soaking treatment and the water is 80-100 ℃.
In one embodiment, the time for distillation and extraction of the soaked fingered citron and water is 3-5 hours.
In one embodiment, the pre-treating the terpene containing precursor is to remove water from the terpene containing precursor with a desiccant.
In one embodiment, the D-limonene and gamma terpinene in the terpene containing precursor are obtained by GC-MS analysis.
A method for verifying the application of terpenes derived from bergamot in the atomization of cough, comprising the steps of:
verifying at the animal level terpene components derived from fingered citron: atomizing the terpene-containing atomized liquid in any embodiment, and introducing the generated terpene-containing aerosol into a cough model mouse induced by ammonia water;
verifying at the cellular level terpene components derived from fingered citron: atomizing the terpene-containing atomized liquid of any embodiment, and respectively introducing the generated terpene-containing aerosol into an LPS-induced MH-S cell inflammation model.
Compared with the prior art, the application has at least the following advantages:
according to the application method of the terpene components in the fingered citron in the cough relieving, the fingered citron is obtained and subjected to experimental analysis, so that the usage method of the fingered citron is to obtain the terpene-containing essential oil by carrying out distillation operation and pretreatment on the fingered citron, the content of D-limonene in the obtained terpene-containing essential oil is controlled to be not lower than 200mg/g and the content of gamma-terpinene is controlled to be not lower than 50mg/g, the terpene-containing essential oil is directly subjected to low-temperature direct thermal atomization at 80-150 ℃ to obtain the terpene-containing aerosol which can be directly subjected to atomization utilization to achieve the effect of better cough relieving, namely, the conclusion that the terpene-containing essential oil with the content of D-limonene is not lower than 200mg/g and the content of gamma-terpinene can be atomized at low temperature to achieve the effect of better cough relieving by means of atomization, the specific and feasible atomization mode of the fingered citron applied to the cough relieving is provided, namely, under the condition that the cough relieving mechanism of the fingered citron is not required to be further subjected to the cough relieving by the novel method of the fingered citron, and the cough relieving method is further provided according to the application of the application.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method of applying terpenes derived from fingered citron to atomization in relieving cough according to an embodiment of the present application;
FIG. 2 is a flow chart of a method of validating the use of a bergamot-derived terpene component in the atomization of a cough according to one embodiment of the present application;
FIG. 3 is a bar graph of inflammatory factor mRNA expression levels in MH-S cell inflammation models at various concentrations of bergamot terpene components;
FIG. 4 is a bar graph of inflammatory factor expression levels in MH-S cell inflammation models at various concentrations of bergamot terpene components;
FIG. 5 is a bar graph of the level of epithelial-to-mesenchymal transition of BEAS-2B cells at various concentrations of bergamot terpene component;
FIG. 6 is a GC-MS chromatogram of a bergamot terpene component;
FIG. 7 is a GC chromatogram for the identification of bergamot terpene components.
Detailed Description
In order that the application may be readily understood, a more complete description of the application will be rendered by reference to the appended drawings. The drawings illustrate preferred embodiments of the application. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It will be understood that when an element is referred to as being "fixed to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like are used herein for illustrative purposes only and are not meant to be the only embodiment.
Unless defined otherwise, 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 application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The application provides an atomization application method of terpene components derived from fingered citron in relieving cough. The atomization application method of the terpene component derived from the fingered citron in relieving cough comprises the following steps of: obtaining fingered citron; distilling fingered citron to obtain a terpene-containing precursor; the terpene-containing precursor is pretreated to obtain terpene-containing atomized essential oil, wherein the terpene-containing atomized essential oil is used for low-temperature direct thermal atomization to obtain terpene-containing aerosol, the low-temperature direct thermal atomization temperature is 80-150 ℃, the terpene-containing essential oil mainly contains D-limonene and gamma-terpinene, the content of the D-limonene is not lower than 200mg/g, and the content of the gamma-terpinene is not lower than 50mg/g.
According to the application method of the terpene components derived from the fingered citron in relieving cough, the fingered citron is obtained and subjected to experimental analysis, so that the application method of the fingered citron is that the fingered citron is subjected to distillation operation and pretreatment to obtain the terpene-containing essential oil, when the content of D-limonene in the obtained terpene-containing essential oil is not lower than 200mg/g and the content of gamma-terpinene is not lower than 50mg/g, the terpene-containing essential oil is directly subjected to low-temperature direct-heating atomization at the temperature of 80-150 ℃ to obtain the conclusion that the terpene-containing aerosol can be directly subjected to atomization utilization to achieve the effect of better cough, namely, the conclusion that the terpene-containing essential oil with the content of D-limonene is not lower than 200mg/g and the content of gamma-terpinene can be atomized at the low temperature to achieve the effect of better cough by the atomization utilization, the specific and feasible atomization mode of the fingered citron applied to the cough is provided, namely, under the condition that the concept of the cough of the fingered citron is not met, the novel application method of the fingered citron is provided for relieving cough by the medical science and the application method according to the application.
The present methods mainly include antihistamines, glucocorticoids, leukotriene receptor antagonists and Th2 cytokine inhibitors, which are used for relieving cough, and have obvious side effects and certain limitations although the effects are still good. Traditional Chinese medicine is used for integrally treating diseases through multiple paths and multiple targets, is deeply favored by people because of small side effects, fingered citron is commonly used for treating hypochondriac pain, chest distress, abdominal distending pain, eructation, nausea and long-term expectoration in clinic, has reported that the fingered citron has a certain anti-inflammatory effect, but the mechanism is not clear, and the existing technology of atomizing and utilizing the fingered citron extract is not clear, such as patent CN113243550A, wherein the fingered citron extract is obtained by distilling and extracting the fingered citron for three times, but the efficacy of relieving cough is not related to the fingered citron, namely, the drug taking mode of the fingered citron for relieving cough is not related to, and solvents such as glycerol and propylene glycol are used, and the inhalation of the glycerol and the propylene glycol is harmful, and the atomizing temperature of an atomizing system is increased. In addition, the existing technology for adding the extracting solution of the fingered citron to assist other substances in achieving atomization and cough relieving is disclosed in the patents CN114432401A, CN114522198A, CN114191509A and CN113730479A, wherein the disclosed decocted extract of the fingered citron is used for atomization and cough relieving and is added in an auxiliary manner according to the general curative effect of the fingered citron and is used for playing the role of assisting in cough relieving, and the research on the cough relieving curative effect of the fingered citron is not involved, namely the fingered citron is used by utilizing the existing cough relieving effect of the fingered citron, scientific basis cannot be provided for further excavating the action mechanism of the fingered citron for treating cough, and a new thinking method cannot be provided for the drug administration mode of the fingered citron for relieving cough; in addition, the use of solvents such as water, propylene glycol or glycerol, which are all involved in the above patents, but substances insoluble in solvents such as water, propylene glycol and glycerol, such as D-limonene and gamma-terpinene, exist in fingered citron, and D-limonene and gamma-terpinene have a great influence on the cough relieving effect of fingered citron, if the contents of D-limonene and gamma-terpinene in aerosol containing D-limonene and gamma-terpinene formed by atomization are varied in the case of uneven dispersion of D-limonene and gamma-terpinene, the cough relieving effect of fingered citron is easily affected, and the problem is not considered in the prior art, the application breaks through the prior art and researches, verifies the effect of terpene components in fingered citron on cough relieving in an in vivo model, and forms a novel concept of atomized administration of terpene components in fingered citron for relieving cough, and provides a new administration method for the fingered citron for relieving cough.
It should be noted that, for the people who need to perform cough relieving treatment, more oral medicines are difficult to perform treatment, the experiment of the application obtains that the fingered citron is distilled to obtain the terpene-containing precursor, the content of D-limonene of the terpene-containing precursor is not less than 200mg/g, and the content of gamma-terpinene is not less than 50mg/g, and the application has better cough relieving effect when being used for treating cough by atomization administration, and provides convenience for people who are not suitable for oral medicines and do need to relieve cough.
In particular, the application uses terpene-containing essential oil, wherein the content of D-limonene is not lower than 200mg/g and the content of gamma-terpinene is not lower than 50mg/g, so that the terpene-containing aerosol is obtained by adopting low-temperature direct thermal atomization, namely, the aerosol can be formed by atomization at low temperature in a direct heating mode to realize atomization utilization, thus achieving better cough relieving effect, and if the essential oil or other extracts of fingered citron are added into an atomization solvent, such as water, propylene glycol or glycerol, on the one hand, the content of D-limonene and gamma-terpinene in the fingered citron is difficult to ensure, on the other hand, the atomization temperature is increased, the atomization of the atomized liquid containing the fingered citron and the solvent can be realized only at a higher temperature, such as 200-270 ℃, the consumption of energy is increased, or the atomization of the atomized liquid containing the fingered citron and the solvent can be realized in an ultrasonic atomization mode, and the accumulated water is caused to cause the influence on health after inhalation.
In order to better understand the atomization application method of the terpene component derived from fingered citron in relieving cough, the following further explains the atomization application method of the terpene component derived from fingered citron in relieving cough:
an embodiment of the method for atomizing a bergamot-derived terpene component for use in relieving cough comprises the steps of:
s100, obtaining fingered citron. It can be understood that the fingered citron is orally taken when being used for relieving cough, but the cough relieving mechanism of the fingered citron is not clear, so as to provide scientific basis for further excavating the action mechanism of the fingered citron for treating cough, and provide a new thought method for the drug administration mode of the fingered citron for relieving cough.
And S200, performing distillation operation on fingered citron to obtain a terpene-containing precursor. It can be understood that the distillation operation is carried out on the fingered citron, so that the extraction of terpene components in the fingered citron can be better realized, and further, a material basis is better provided for demonstrating the curative effect of terpene components in the fingered citron for treating cough.
S300, preprocessing a terpene-containing precursor to obtain terpene-containing atomized essential oil, wherein the terpene-containing atomized essential oil is used for low-temperature direct thermal atomization to obtain terpene-containing aerosol, the low-temperature direct thermal atomization temperature is 80-150 ℃, the terpene-containing essential oil mainly contains D-limonene and gamma-terpinene, the content of the D-limonene is not lower than 200mg/g, and the content of the gamma-terpinene is not lower than 50mg/g. It can be understood that the pretreatment of the terpene-containing precursor comprises the steps of drying and impurity removal of the terpene-containing precursor obtained by distillation operation, wherein the impurity removal is mainly to only reserve terpene components in the terpene-containing precursor, namely, the terpene-containing atomized essential oil is better obtained, so that the content of D-limonene is not lower than 200mg/g and the content of gamma-terpinene is not lower than 50mg/g, thereby better ensuring the accuracy of the curative effect of the terpene component in the fingered citron for treating cough, and when the terpene-containing essential oil mainly contains the D-limonene with the content of not lower than 200mg/g and the content of not lower than 50mg/g gamma-terpinene, the terpene-containing essential oil is directly atomized at a low temperature of 80-150 ℃ to obtain a conclusion of a better cough effect by atomization, namely, the method can be further provided for realizing a better cough-relieving mechanism by the fingered citron by directly heating the terpene-containing essential oil, namely, the novel method for realizing the cough-relieving cough by using the aerosol at a low temperature is further provided, and the cough can be further provided for realizing a better cough-relieving mechanism by the fingered citron cough by the fingered citron.
According to the application method of the terpene components derived from the fingered citron in relieving cough, the fingered citron is obtained and subjected to experimental analysis, so that the application method of the fingered citron is that the fingered citron is subjected to distillation operation and pretreatment to obtain the terpene-containing essential oil, when the content of D-limonene in the obtained terpene-containing essential oil is not lower than 200mg/g and the content of gamma-terpinene is not lower than 50mg/g, the terpene-containing essential oil is directly subjected to low-temperature direct-heating atomization at the temperature of 80-150 ℃ to obtain the conclusion that the terpene-containing aerosol can be directly subjected to atomization utilization to achieve the effect of better cough, namely, the conclusion that the terpene-containing essential oil with the content of D-limonene is not lower than 200mg/g and the content of gamma-terpinene can be atomized at the low temperature to achieve the effect of better cough by the atomization utilization, the specific and feasible atomization mode of the fingered citron applied to the cough is provided, namely, under the condition that the concept of the cough of the fingered citron is not met, the novel application method of the fingered citron is provided for relieving cough by the medical science and the application method according to the application.
In one embodiment, the fingered citron is obtained, particularly fresh fruits with the picking time of 7 months to 11 months are cut into slices, and the fresh fruits are dried or dried in the shade until the moisture content is less than or equal to 15 percent under the condition of shading, so that the content of terpene components in the fingered citron is better ensured, particularly, the content of D-limonene containing terpene atomized essential oil in the fingered citron after distillation operation and pretreatment is not less than 200mg/g and the content of gamma-terpinene is not less than 50mg/g, further, the conclusion that the content of D-limonene is not less than 200mg/g and the content of gamma-terpinene is not less than 50mg/g is better obtained, and the effect that the aerosol formed by atomizing the terpene-containing essential oil for atomization is used for realizing better cough relieving can be realized under the low temperature in a direct heating mode is more clearly given.
In one embodiment, the fingered citron producing area is Guangdong, guangxi, zhejian, fujian or Sichuan area, and the fingered citron is picked for 9 months to 10 months in autumn, so that the content of D-limonene containing terpene atomized essential oil in the fingered citron after distillation operation and pretreatment is not lower than 200mg/g and the content of gamma-terpinene is not lower than 50mg/g.
In one embodiment, fingered citron is weighed to a mass of m grams. Further, adding water with the volume of n milliliters into the fingered citron for soaking treatment, wherein n: m is 8-12, m is more than 0. Further, the soaked fingered citron and water are distilled and extracted together. It will be appreciated that the number of components,
in one embodiment, the distillation of fingered citron before the step of adding n milliliters of water to the fingered citron for soaking treatment, and after the step of weighing m grams of fingered citron, further comprises the steps of: and (5) crushing the fingered citron.
In one embodiment, the grinding operation of the fingered citron is specifically: pulverizing fructus Citri Sarcodactylis into coarse powder. It will be understood that the coarse powder is one that passes all through the No. two screen, but is mixed with no more than 40% of the powder that passes through the No. four screen.
In one embodiment, the soaking time of soaking treatment by adding n milliliters of water into fingered citron is 45-75 minutes.
In one embodiment, the time for distillation and extraction of the soaked fingered citron and water is 3-5 hours.
In one embodiment, the soaked fingered citron and water are distilled and extracted together at the temperature of 80-100 ℃.
In one embodiment, the fingered citron is subjected to a distillation operation, specifically: weighing a certain mass of fingered citron medicinal material, crushing the fingered citron medicinal material into coarse powder (sieving with a No. 1 sieve and 10 meshes), adding purified water with the volume of 8-12 times, soaking for 45-75 min, extracting the coarse powder with a volatile oil extractor for 3-5 h, drying the volatile oil with anhydrous sodium sulfate to obtain a terpene-containing precursor, wherein the terpene-containing precursor comprises the total terpene components of the fingered citron, so that the effective extraction of the terpene components in the fingered citron, especially the full extraction of D-limonene and gamma-terpinene, is better realized, the content of D-limonene is better than 200mg/g and the content of gamma-terpinene is better than 50mg/g, the conclusion that the terpene-containing essential oil can be atomized to form aerosol for atomization at low temperature in a direct heating mode to achieve a better cough relieving effect is better, and a specific and feasible atomization medicament application mode of the fingered citron is better provided.
In one embodiment, the terpene containing precursor is pre-treated to remove water from the terpene containing precursor with a desiccant.
In one embodiment, the desiccant is anhydrous sodium sulfate.
In one embodiment, the terpene containing precursor is pre-treated to remove water from the terpene containing precursor with a desiccant, and to remove organic acids and esters of the terpene containing precursor. Generally, conventional methods for removing organic acids and esters are employed.
In one embodiment, the D-limonene and gamma terpinene in the terpene containing precursor are obtained by GC-MS analysis.
The application also provides a verification method for the atomization application of the terpene component derived from the fingered citron in relieving cough. The verification method for the atomization application of the terpene component derived from the fingered citron in relieving cough comprises the following steps of: verifying at the animal level terpene components derived from fingered citron: atomizing the terpene-containing atomized liquid in any embodiment, and introducing the generated terpene-containing aerosol into a cough model mouse induced by ammonia water; verifying at the cellular level terpene components derived from fingered citron: atomizing the terpene-containing atomized liquid in any embodiment, and respectively introducing the generated terpene-containing aerosol into an LPS-induced MH-S cell inflammation model.
According to the verification method for the atomization application of the terpene components derived from the fingered citron in relieving cough, the terpene components derived from the fingered citron are verified at the animal level and the terpene components derived from the fingered citron are verified at the cell level, and the effect of the terpene components derived from the fingered citron on relieving cough is verified when the terpene components derived from the fingered citron are atomized and administrated, so that the verification of the curative effect of the terpene components derived from the fingered citron for atomizing and administrating the terpene components derived from the fingered citron for relieving cough is relatively visual and relatively scientifically obtained, and the accuracy and the confidence of the verification of the curative effect of the terpene components derived from the fingered citron for atomizing and administrating the terpene components derived from the fingered citron for relieving cough are improved.
In order to better understand the verification method for the atomization application of the bergamot-derived terpene component in cough according to the present application, the verification method for the atomization application of the bergamot-derived terpene component in cough according to the present application is further explained as follows:
the verification method for the atomization application of the terpene component derived from the fingered citron in relieving cough according to one embodiment comprises the following steps:
s10, verifying terpene components from fingered citron at animal level: atomizing the terpene-containing atomized liquid in any embodiment, and introducing the generated terpene-containing aerosol into a cough model mouse induced by ammonia water. It can be understood that the terpene components from fingered citron are verified on animal level by means of atomization administration, the effect of the terpene components from fingered citron on relieving cough during atomization administration is intuitively and accurately obtained, and further the effective verification of the curative effect of the terpene components from fingered citron for relieving cough during atomization administration is well realized.
S20, verifying terpene components from fingered citron at a cellular level: atomizing the terpene-containing atomized liquid in any embodiment, and respectively introducing the generated terpene-containing aerosol into an LPS-induced MH-S cell inflammation model. It can be understood that after verifying the terpene components derived from fingered citron at animal level, further combining with verifying the terpene components derived from fingered citron at cellular level, namely, the generated terpene-containing aerosol is respectively introduced into an MH-S cell inflammation model induced by LPS, the influence of the terpene components derived from fingered citron on the expression of mRNA of inflammatory factors IL-6, IL-1 beta and TNF-alpha during atomization administration is scientifically obtained, and further, the effective verification of the curative effect of the terpene components derived from fingered citron for relieving cough during atomization administration is scientifically realized.
According to the verification method for the atomization application of the terpene components derived from the fingered citron in relieving cough, the terpene components derived from the fingered citron are verified at the animal level and the terpene components derived from the fingered citron are verified at the cell level, and the effect of the terpene components derived from the fingered citron on relieving cough is verified when the terpene components derived from the fingered citron are atomized and administrated, so that the verification of the curative effect of the terpene components derived from the fingered citron for atomizing and administrating the terpene components derived from the fingered citron for relieving cough is relatively visual and relatively scientifically obtained, and the accuracy and the confidence of the verification of the curative effect of the terpene components derived from the fingered citron for atomizing and administrating the terpene components derived from the fingered citron for relieving cough are improved.
It should be noted that, the report of cough stopping is not about the terpene components of fingered citron, wherein the content of D-limonene is not less than 200mg/g and the content of gamma-terpinene is not less than 50mg/g, so that the terpene components of fingered citron are used for directly acting on uncomfortable parts to achieve the cough stopping effect in an atomized administration mode, namely, the application mode of fingered citron for stopping cough is improved, and the verification method of the atomization application of the terpene components of fingered citron in stopping cough proves that the terpene components of fingered citron are used for directly acting on uncomfortable parts to achieve the cough stopping effect in an atomized administration mode, so that the cough stopping effect is better, and a scientific basis is provided for further excavating the action mechanism of fingered citron for treating cough.
In one embodiment, the method of validating the application of the bergamot-derived terpenes to the atomization in the cough, at the cellular level, further comprises the steps of: atomizing the terpene-containing atomized liquid according to any of claims 1 to 9 and separately passing the resulting terpene-containing aerosol into a bed-2B cell model. It can be understood that the generated terpene-containing aerosol is further led into the BEAS-2B cell model respectively, the influence of terpene components from fingered citron on the epithelial-mesenchymal transition of BEAS-2B cells during atomization administration is obtained more scientifically, and the effective verification of the curative effect of the terpene components from fingered citron for relieving cough during atomization administration is realized more scientifically.
Compared with the prior art, the application has at least the following advantages:
according to the application method of the terpene components in the fingered citron in the cough relieving, the fingered citron is obtained and subjected to experimental analysis, so that the usage method of the fingered citron is to obtain the terpene-containing essential oil by carrying out distillation operation and pretreatment on the fingered citron, the content of D-limonene in the obtained terpene-containing essential oil is controlled to be not lower than 200mg/g and the content of gamma-terpinene is controlled to be not lower than 50mg/g, the terpene-containing essential oil is directly subjected to low-temperature direct thermal atomization at 80-150 ℃ to obtain the terpene-containing aerosol which can be directly subjected to atomization utilization to achieve the effect of better cough relieving, namely, the conclusion that the terpene-containing essential oil with the content of D-limonene is not lower than 200mg/g and the content of gamma-terpinene can be atomized at low temperature to achieve the effect of better cough relieving by means of atomization, the specific and feasible atomization mode of the fingered citron applied to the cough relieving is provided, namely, under the condition that the cough relieving mechanism of the fingered citron is not required to be further subjected to the cough relieving by the novel method of the fingered citron, and the cough relieving method is further provided according to the application of the application.
Specific examples are set forth below, and all references to percentages are by weight. It should be noted that the following examples are not exhaustive of all possible scenarios, and that the materials used in the examples described below are commercially available unless otherwise specified.
Example 1
Weighing 100 g of fingered citron medicinal material, crushing into coarse powder (sieving with a No. 1 sieve and 10 meshes), picking up the fingered citron in the production place of Guangdong in the month No. 7 and the month No. 2, cutting fresh fruits into slices, drying at low temperature or drying in the shade until the water content is 15%, soaking the fingered citron coarse powder in 8 times of pure water (1 g=8 ml) for 45min, collecting the extracted terpene components by using a steam distillation device with the distillation temperature of 80 ℃, extracting for 5h each time, and sealing and storing the freshly prepared fingered citron terpene components in a refrigerator at the temperature of 4 ℃ for further experiments.
Example 2
Weighing 100 g of fingered citron medicinal material, crushing into coarse powder (sieving with a No. 1 sieve and 10 meshes), picking up the fingered citron with a production place of Guangxi, carrying out Gregorian calendar No. 8 months and 10, cutting fresh fruits into slices, drying at low temperature or drying in shade to water of 12%, placing the fingered citron coarse powder into 10 times of pure water (1 g=10 ml) to soak for 50min, collecting the extracted terpene components by using a steam distillation device with the distillation temperature of 90 ℃, extracting for 4.5h each time, and sealing and storing the freshly prepared fingered citron terpene components in a refrigerator with the temperature of 4 ℃ for further experiments.
Example 3
Weighing 100 g of fingered citron medicinal material, crushing into coarse powder (sieving with a No. 1 sieve and 10 meshes), picking up the fingered citron with a production place of Fujian and a Gregorian calendar of No. 9 and 20 months, cutting fresh fruits into slices, drying at a low temperature or drying in the shade until the water content is 13%, soaking the fingered citron coarse powder in 11 times of pure water (1 g=11 ml) for 60min, collecting the extracted terpene components by using a steam distillation device with the distillation temperature of 95 ℃ for 4h each time, and sealing and storing the freshly prepared fingered citron terpene components in a refrigerator at 4 ℃ for further experiments.
Example 4
Weighing 100 g of fingered citron medicinal material, crushing into coarse powder (sieving with a No. 1 sieve and 10 meshes), picking up the fingered citron in a place of Sichuan with a Gregorian calendar of No. 28, cutting fresh fruits into slices, drying at a low temperature or drying in the shade to water content of 10%, soaking the fingered citron coarse powder in 12 times of pure water (1 g=12 ml) for 75min, collecting the extracted terpene components by using a steam distillation device with a distillation temperature of 100 ℃, extracting for 3h each time, and sealing and storing the freshly prepared fingered citron terpene components in a refrigerator with a temperature of 4 ℃ for further experiments.
The test samples containing the bergamot terpene components of examples 1-4 were tested at different concentrations.
1. Verifying at the animal level terpene components derived from fingered citron:
animals were randomly divided into 5 groups (5 mice per group): ammonia water model (model, M) and examples 1-4 were used for atomizing and administering the composition, and the bergamot terpene components were heated by a low-temperature heating atomizer at 80℃to 120 ℃. Mice were fed adaptively for one week, model group mice were subjected to a 1mL ammonia solution aspiration using cotton balls, mice were allowed to inhale for 2min in a closed container to construct a mouse cough model, and the number of times of cough was recorded. Examples 1-4 nebulized dosing groups were administered in a presaturated environment after molding and inhaled for 2min in different drug concentration environments, the number of coughs was recorded, and the average cough frequency was calculated and the results are shown in table 1:
table 1: mouse cough frequency of atomized administration by 80 ℃ low-temperature heating atomizer
| Test examples | Cough frequency (2 min) |
| Model set (without atomization administration) | 135±9 |
| EXAMPLE 1 nebulized administration set | 80±39* |
| EXAMPLE 2 nebulized administration set | 72±21** |
| EXAMPLE 3 nebulized administration set | 65±14** |
| EXAMPLE 4 nebulized administration set | 64±12** |
Table 2: mouse cough frequency of 90 ℃ low-temperature heating atomizer atomized administration
| Test examples | Cough frequency (2 min) |
| Model set (without atomization administration) | 135±9 |
| EXAMPLE 1 nebulized administration set | 78±39* |
| EXAMPLE 2 nebulized administration set | 70±21** |
| EXAMPLE 3 nebulized administration set | 64±14** |
| EXAMPLE 4 nebulized administration set | 64±12** |
Table 3: mouse cough frequency of 100 ℃ low-temperature heating atomizer atomized administration
| Test examples | Cough frequency (2 min) |
| Model set (without atomization administration) | 135±9 |
| EXAMPLE 1 nebulized administration set | 78±39* |
| EXAMPLE 2 nebulized administration set | 70±21** |
| EXAMPLE 3 nebulized administration set | 64±14** |
| EXAMPLE 4 nebulized administration set | 63±12** |
Table 4: mouse cough frequency of atomized administration by 120 ℃ low-temperature heating atomizer
| Test examples | Cough frequency (2 min) |
| Model set (without atomization administration) | 135±9 |
| EXAMPLE 1 nebulized administration set | 76±39* |
| EXAMPLE 2 nebulized administration set | 70±21** |
| EXAMPLE 3 nebulized administration set | 62±14** |
| EXAMPLE 4 nebulized administration set | 62±12** |
Note that: p <0.05, P <0.01 compared to model group.
As can be seen from tables 1 to 4, the number of cough in mice (P <0.05, P < 0.01) was significantly reduced by the administration of the aerosol in each example, compared with the model group, indicating that the terpene component extracted from fingered citron had a significant cough suppressing effect under the conditions set forth in this example.
2. The terpene components derived from fingered citron (performed with the fingered citron terpene components of example 2) were validated at the cellular level:
a) Cell culture and grouping: MH-S cells were grown in 1640 medium containing 10% FBS and 1% PS at 37deg.C in 5% CO 2 Is cultured in a cell culture incubator. Cells were divided into normal, model (LPS stimulated, concentration 0.1. Mu.g/mL), and bergamot terpene component administration groups.
b) Fluorescent real-time quantitative PCR (polymerase chain reaction) detection of protein expression changes of IL-6 and IL-1 beta of cells: RNA extracted from cells was inverted to cDNA, and quantitative cDNA (2. Mu.L), primers (2. Mu.L in total for the front and rear primers, see tables 2-4 of the section), SYBR (10. Mu.L) and enzyme-free water (6. Mu.L) were taken and assayed for the relative expression of the relevant factor mRNA.
c) The method comprises the steps of planting MH-S cells in a six-hole plate when the cell fusion degree reaches about 70%, adding LPS (final concentration is 0.1 mug/mL), carrying out drug administration and culture for 24 hours, setting different drug administration concentrations, dividing into five groups of control, model, LPS-1, LPS-10 and LPS-100 mug/mL (final concentration), collecting supernatant after culture for 24 hours, respectively detecting the expression quantity of IL-6 protein, centrifuging the cell supernatant for 20 minutes by 1,000Xg, and taking the supernatant for later detection of the Elisa.
The test data are recorded as shown in fig. 2 to 4, wherein C is the control group. M is a model group.
FIG. 3 is a bar graph of inflammatory factor mRNA expression levels in MH-S cell inflammation models at various concentrations of bergamot terpene components, wherein (a) inflammatory factor IL-6mRNA expression levels are determined by qRT-PCR; (b) Determining the expression level of inflammatory factor IL-1 beta mRNA by qRT-PCR; (c) Determining the expression level of inflammatory factor TNF- α mRNA by qRT-PCR, P <0.05, P <0.01 and P <0.001 compared to the model group; compared with the control group, the expression of inflammatory factor mRNA can be obviously reduced by the bergamot terpene component, wherein the expression of inflammatory factor mRNA can be obviously reduced by the bergamot terpene component, and the expression of inflammatory factor mRNA can be obviously reduced by the bergamot terpene component.
FIG. 4 is a bar graph of inflammatory factor expression levels in MH-S cell inflammation models at various concentrations of bergamot terpene components, (a) detection of inflammatory factor IL-6 expression levels by Elisa; (b) Detecting the expression level of inflammatory factor IL-1 beta by Elisa; (c) Detecting the expression level of inflammatory factor TNF- α by Elisa, P <0.05, P <0.01 and P <0.001 compared to the model group; compared with the control group #P <0.05, #P <0.01 and #P <0.001, the bergamot terpene component can obviously reduce the expression of inflammatory factor proteins.
FIG. 5 is a bar graph of BEAS-2B cell epithelial mesenchymal transition levels at various concentrations of bergamot terpene components, (a) determining the expression level of α -SMA mRNA by qRT-PCR; (b) determining the expression level of COL1A1 mRNA by qRT-PCR; (c) determining the expression level of Vimentin mRNA by qRT-PCR; (d) Determining the expression level of E-cadherein mRNA by qRT-PCR; (e) Determining the level of N-cadherein mRNA expression by qRT-PCR, P <0.05, P <0.01 and P <0.001 compared to the model group; compared with the control group, the composition of the bergamot terpenes is proved to be capable of obviously improving the epithelial-mesenchymal transition by the composition of the bergamot terpenes, wherein the composition of the bergamot terpenes is proved to be less than 0.05, the composition of the bergamot terpenes is proved to be more than 0.01 and the composition of the bergamot terpenes is proved to be more than 0.001. 3. The bergamot terpene components were analyzed by GC-MS (with the bergamot terpene components of example 2).
Fig. 6 is a GC-MS chromatogram of the bergamot terpene-based component, fig. 7 is a GC chromatogram for the bergamot terpene-based component identification, and fig. 5 is a component table of the bergamot terpene-based component.
Table 5: bergamot terpene class ingredient list
As can be seen from fig. 6, 7 and table 5, the extracted total terpene components of fingered citron mainly include terpene components such as D-limonene and gamma-terpinene.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (3)
1. A method for preparing terpene components from fingered citron for relieving cough, which is characterized by comprising the following steps:
the method comprises the steps of obtaining fingered citron, picking fresh fruits with the picking time of 7-11 months, cutting the fresh fruits into slices, and drying the fresh fruits at a low temperature or drying the fresh fruits in shade until the water content is 10-13%;
weighing m grams of fingered citron, crushing the fingered citron into coarse powder, and adding n milliliters of water into the fingered citron for soaking treatment, wherein n: m is 11-12, m is more than 0, and the soaking time is 60-75 min;
carrying out distillation and extraction operation on the fingered citron after the soaking treatment and water to obtain a terpene-containing precursor, wherein the temperature is 95-100 ℃ and the time is 3-4 hours;
the terpene-containing precursor is pretreated to obtain terpene-containing atomized essential oil, wherein the terpene-containing atomized essential oil is used for low-temperature direct thermal atomization to obtain terpene-containing aerosol, the low-temperature direct thermal atomization temperature is 80-120 ℃, the terpene-containing essential oil mainly contains D-limonene and gamma-terpinene, the content of the D-limonene is not less than 200mg/g, and the content of the gamma-terpinene is not less than 50mg/g.
2. The method of claim 1, wherein the pre-treating the terpene containing precursor with a desiccant removes water from the terpene containing precursor.
3. The method for preparing terpene components derived from fingered citron for relieving cough according to claim 1 or 2, wherein D-limonene and gamma-terpinene in the terpene containing precursor are obtained by GC-MS analysis.
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