Extraction method of antiviral oral liquid volatile oil

Abstract

The invention belongs to the technical field of volatile oil extraction, and particularly relates to an extraction method of antiviral oral liquid volatile oil, which comprises the following steps: weighing medicinal materials according to a prescription proportion, crushing the medicinal materials, mixing, adding into a volatile oil extraction tank, adding ionic liquid, adding distilled water, heating, and separating water and volatile oil to obtain the antiviral oral liquid volatile oil. The method for extracting the volatile oil of the antiviral oral liquid has the beneficial effects that the volatile oil is extracted by adopting an ionic liquid assisted steam distillation extraction method, and compared with a traditional extraction method, the cost is reduced, the operation steps are simplified, and the extraction rate of the volatile oil is improved; by controlling the type and concentration of the ionic liquid and controlling the liquid-material ratio, the heating temperature and the heating time, the extraction rate of the volatile oil is improved, and the activity of antiviral components in the volatile oil is ensured; the feasibility in actual operation is considered when the extraction conditions are optimized, and the method is easier to industrially extract the antiviral oral liquid volatile oil on a large scale.

Description

Technical Field

[0001] The invention belongs to the technical field of volatile oil extraction, and in particular relates to a method for extracting volatile oil from an antiviral oral liquid. Background Art

[0002] The most commonly used and traditional extraction techniques for Chinese herbal volatile oils are steam distillation and organic solvent extraction. Although steam distillation is easy to operate and requires simple equipment, the extraction rate of volatile oils is low. Traditional organic solvent extraction methods have problems such as organic solvent residues and environmental pollution; and this extraction method usually extracts fat-soluble components in Chinese herbal medicines, such as resins and waxes in the herbs, resulting in more impurities in the volatile oils; in addition, organic solvent extraction methods are not easy to industrially extract Chinese herbal volatile oils on a large scale, and are only used in laboratories to extract a small amount of volatile oils.

[0003] In recent years, new technologies for extracting essential oils include supercritical fluid extraction, enzyme method, ultrasound-assisted extraction (UAE), etc. Supercritical fluid CO2 extraction requires expensive professional equipment and high purity CO2, which leads to relatively few applications of this method in the extraction of volatile oils from traditional Chinese medicines; the harsh process conditions of enzyme-assisted extraction limit the further widespread application of this method.

[0004] Therefore, the existing volatile oil extraction methods have the problems of low extraction rate, difficulty in large-scale industrial extraction, and demanding process conditions. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for extracting volatile oil from antiviral oral liquid.

[0006] The present invention relates to a method for extracting volatile oil from an antiviral oral liquid, the steps of which are as follows: Weigh the medicinal materials according to the prescription ratio, grind and mix the medicinal materials, add them to the volatile oil extraction tank, add ionic liquid, add distilled water and heat, separate the water and volatile oil, and obtain the volatile oil of the antiviral oral liquid.

[0007] Preferably, the medicinal materials include Forsythia suspensa, Patchouli, Acorus calamus and Curcuma aromatica, and the mass ratio of the Forsythia suspensa, Patchouli, Acorus calamus and Curcuma aromatica is 1.5-2:1-1.5:0.8-1:0.8-1.

[0008] Preferably, the ionic liquid is at least one of 1-butyl-3-methylimidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium tetrafluoroborate and N-ethylpyridinium tetrafluoroborate.

[0009] Preferably, the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate.

[0010] Preferably, the concentration of the ionic liquid is 0.9% to 10%.

[0011] Preferably, the concentration of the ionic liquid is 0.9%.

[0012] Preferably, the mass ratio of the medicinal material to distilled water is 1:3-8.

[0013] Preferably, the mass ratio of the medicinal material to distilled water is 1:3.

[0014] Preferably, the heating temperature is 70°C to 100°C, and the heating time is 60 to 90 minutes.

[0015] Preferably, the heating temperature is 70° C. and the heating time is 75 min.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are: (1) The method for extracting volatile oil from the antiviral oral liquid of the present invention uses an ionic liquid-assisted steam distillation extraction method to extract volatile oil. Compared with the traditional extraction method, the extraction time is reduced from 2.5 to 3 hours to 1.25 hours (75 minutes), which reduces the cost, simplifies the operation steps, and improves the extraction rate of volatile oil; (2) The method for extracting volatile oil from the antiviral oral liquid of the present invention improves the extraction rate of volatile oil by controlling the type and concentration of the ionic liquid, and simultaneously controls the liquid-to-solid ratio, heating temperature and heating time, while ensuring the activity of the antiviral components in the volatile oil; (3) The method for extracting volatile oil from the antiviral oral liquid of the present invention further considers the feasibility in actual operation when optimizing the extraction conditions. This method is easier to carry out large-scale industrial extraction of volatile oil from the antiviral oral liquid. DETAILED DESCRIPTION

[0017] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with embodiments.

[0018] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0019] Example 1 The ionic liquid-assisted steam distillation extraction method of the present invention: According to the prescription ratio, weigh 46.43g of Forsythia suspensa, 28.57g of Patchouli, 25g of Acorus calamus and 25g of Curcuma aromatica, crush each medicinal material and mix them, add them to a volatile oil extraction tank, add 3.375ml of 1-butyl-3-methylimidazolium tetrafluoroborate, then add 371.625ml of distilled water, heat at 70℃ for 75min, separate water and volatile oil, and obtain 2.74g of volatile oil of antiviral oral solution.

[0020] Comparative Example 1 Ionic liquid ultrasonic-assisted steam distillation extraction method: According to the prescription ratio, 46.43g of Forsythia suspensa, 28.57g of Patchouli, 25g of Acorus calamus and 25g of Curcuma aromatica were weighed, each of the medicinal materials was crushed and mixed, and added to a reaction container, and then 3.375ml of 1-butyl-3-methylimidazole tetrafluoroborate was added. The reaction container was then placed in an ultrasonic cleaner and ultrasonicated at 40°C for 30min. The mixture was added to a volatile oil extraction tank, and then 371.625ml of distilled water was added. The mixture was heated at 70°C for 75min, and water and volatile oil were separated to obtain 2.76g of volatile oil of the antiviral oral liquid.

[0021] Comparative Example 2 Ultrasonic-assisted steam distillation extraction method: According to the prescription ratio, 46.43g of Forsythia suspensa, 28.57g of Patchouli, 25g of Acorus calamus and 25g of Curcuma aromatica were weighed, each of the medicinal materials was crushed and mixed, and then added to a reaction container. The reaction container was placed in an ultrasonic cleaner and ultrasonicated at 40°C for 30 minutes. The mixture was added to a volatile oil extraction tank, and then 371.625ml of distilled water was added. The mixture was heated at 70°C for 75 minutes to separate water and volatile oil to obtain 2.15g of volatile oil of the antiviral oral solution.

[0022] Comparative Example 3 Steam distillation extraction method: According to the prescription ratio, weigh 46.43g of Forsythia suspensa, 28.57g of Patchouli, 25g of Acorus calamus and 25g of Curcuma aromatica, crush each medicinal material and mix them, add them to the volatile oil extraction tank, then add 371.625ml of distilled water, heat at 70℃ for 75min, separate the water and volatile oil, and obtain 2.1g of volatile oil of antiviral oral solution.

[0023] Test example 1. The volatile oil extraction rates of Example 1 and Comparative Examples 1-3 were calculated according to formula (1). The results are shown in Table 1.

[0024] Volatile oil extraction rate = extracted volatile oil mass (g) / medicinal material mass (g) × 100% (1) Table 1. Results of volatile oil extraction rate by different extraction methods It can be seen from the data in Table 1 above that the volatile oil extraction rates of Example 1 and Comparative Example 1 are the highest, and the difference between the two is not much. Considering the simplification of steps and cost saving, the ionic liquid-assisted steam distillation extraction method of Example 1 is preferred to extract the volatile oil of the antiviral oral liquid.

[0025] 2. Single factor experiment: (1) Effect of different ionic liquids on volatile oil extraction rate: According to the prescription ratio, 11 groups of each of 46.43g of Forsythia suspensa, 28.57g of Patchouli, 25g of Acorus calamus, and 25g of Curcuma were weighed, crushed, mixed, and placed in a 1L round-bottom flask. 1-ethyl-3-methyl imidazolium bromide [EMim] Br, 1-n-butyl-3-methyl imidazolium tetrafluoroborate [BMim] BF4, 1-hexyl-3-methyl imidazolium tetrafluoroborate [HMim] BF4, 1-octyl-3-methyl imidazolium tetrafluoroborate [OMim] BF4, 1-butyl-3-methyl imidazolium hexafluorophosphate [BMim] PF6, 1-hexyl-3-methyl imidazolium hexafluorophosphate [HMim] PF6, 1-ethyl 10 ml each of 3-methylimidazolium chloride [C2Mim]Cl, 1-butyl-3-methylimidazolium tetrafluoroborate [C4Mim]BF4, 1-hexyl-3-methylimidazolium tetrafluoroborate [C6Mim]BF4, 1-octyl-3-methylimidazolium bromide [C8Mim]Br, and N-ethylpyridinium tetrafluoroborate [EPy]BF4 were added with distilled water to the scale line and heated in a water bath at 70°C for 1 h. The volatile oil was separated according to Method A of the volatile oil determination method under Item 2204 of the 2020 edition of the Pharmacopoeia of the People's Republic of China (Volume 4). The weight of the volatile oil was determined and the volatile oil extraction rate was calculated according to Formula (1). The test results are shown in Table 2.

[0026] Table 2. Volatile oil extraction results of different ionic liquids From the data in Table 2 above, it can be seen that there are significant differences in the effects of the 11 ionic liquids on the volatile oil extraction rate in the antiviral oral solution. The volatile oil extraction rates corresponding to [BMim] PF6, [EPy] BF4, and [C4Mim] BF4 are higher. After comprehensively considering the raw material cost, [C4Mim] BF4 is preferred for subsequent process research.

[0027] (2) Effect of different temperatures on volatile oil extraction rate: According to the prescription ratio, 5 groups of each of 46.43 g of Forsythia suspensa, 28.57 g of Patchouli, 25 g of Acorus calamus, and 25 g of Curcuma were weighed, crushed, mixed, and placed in a 1L round-bottom flask. 10 ml of ionic liquid [C4Mim] BF4 was added, and distilled water was added to the scale line. The mixture was heated in a water bath at 60°C, 70°C, 80°C, 90°C, and 100°C for 1 h, respectively. The volatile oil was separated according to Method A of the volatile oil determination method under Item 2204 of the General Rules of the 2020 Edition of the Pharmacopoeia of the People's Republic of China (Volume 4). The weight of the volatile oil was determined, and the volatile oil extraction rate was calculated according to Formula (1). The test results are shown in Table 3.

[0028] Table 3. Volatile oil extraction results at different temperatures From the data in Table 3 above, it can be seen that the volatile oil extraction rate is basically stable when the temperature is above 70°C. From the perspective of energy saving, 70°C is selected as the heating temperature for volatile oil extraction.

[0029] (3) Effect of different time on volatile oil extraction rate: According to the prescription ratio, 6 groups of each of 46.43 g of Forsythia suspensa, 28.57 g of Patchouli, 25 g of Acorus calamus, and 25 g of Curcuma were weighed, crushed and mixed, placed in a 1L round-bottom flask, 10 ml of ionic liquid [C4Mim] BF4 was added, distilled water was added to the scale line, and heated in a water bath at 70°C for 30 min, 45 min, 1 h, 1.5 h, 2 h, and 3 h, respectively. The volatile oil was separated according to Method A of the volatile oil determination method under Item 2204 of the General Rules of the 2020 Edition of the Pharmacopoeia of the People's Republic of China (Volume 4), the weight of the volatile oil was determined, and the volatile oil extraction rate was calculated according to Formula (1). The test results are shown in Table 4.

[0030] Table 4. Results of volatile oil extraction rate at different times From the data in Table 4 above, it can be seen that when the heating time is 1h, the volatile oil extraction rate is the highest. As the extraction time increases, the volatile oil extraction rate decreases. The reason may be that as the heating time increases, the volatile oil and distilled water produce emulsification, resulting in increased loss of volatile oil.

[0031] (4) Effect of different ionic liquid concentrations on volatile oil extraction rate: According to the prescription ratio, 6 groups of each of 46.43g of Forsythia suspensa, 28.57g of Patchouli, 25g of Acorus calamus, and 25g of Curcuma were weighed, crushed and mixed, and placed in a 1L round-bottom flask. 1ml, 5ml, 10ml, 25ml, 50ml, and 100ml of ionic liquid [C4Mim] BF4 were added respectively, and distilled water was added to the scale line. The concentrations of the ionic liquids were 0.1%, 0.5%, 1%, 2.5%, 5%, and 10%, respectively. The mixture was heated in a water bath at 70℃ for 1h. The volatile oil was separated according to Method A of the volatile oil determination method under Item 2204 of the General Rules of the 2020 Edition of the Pharmacopoeia of the People's Republic of China (Volume 4). The weight of the volatile oil was determined, and the volatile oil extraction rate was calculated according to Formula (1). The test results are shown in Table 5.

[0032] Table 5. Volatile oil extraction results at different ionic liquid concentrations From the data in Table 5 above, it can be seen that increasing the concentration of ionic liquid will enhance the ability to extract volatile oils. However, since the ionic liquid itself has a high viscosity, too high an ionic liquid concentration will increase the viscosity of the solvent, which is not conducive to the precipitation of volatile oils, resulting in a stable volatile oil extraction rate. Considering that the yield difference between 1%, 2.5%, and 5% concentrations of ionic liquids is small, 1% is selected as the optimal ionic liquid concentration from the perspective of reducing costs.

[0033] (5) Effect of different liquid-to-solid ratios (mass ratio of distilled water to medicinal materials) on the extraction rate of volatile oils: According to the prescription ratio, 5 groups of each of 46.43 g of Forsythia suspensa, 28.57 g of Patchouli, 25 g of Acorus calamus, and 25 g of Curcuma were weighed, crushed, mixed, and placed in a 1L round-bottom flask. 10 ml of deionized liquid [C4Mim] BF4 was added, and distilled water in an amount of 1, 2, 5, 6, and 8 times the weight of the medicinal materials was added, respectively. The mixture was heated in a water bath at 70°C for 1 h. The volatile oil was separated according to Method A of the volatile oil determination method under Item 2204 of the General Rules of the 2020 Edition of the Pharmacopoeia of the People's Republic of China (Volume 4). The weight of the volatile oil was determined, and the volatile oil extraction rate was calculated according to Formula (1). The test results are shown in Table 6.

[0034] Table 6. Volatile oil extraction results at different liquid-to-solid ratios It can be seen from the data in Table 6 above that when the liquid-to-solid ratio reaches 5, the volatile oil extraction rate changes little. From the perspective of reducing costs, the optimal liquid-to-solid ratio is 5.

[0035] 3. Response surface experiment: On the basis of the above single factor experiment, the extraction temperature was confirmed to be 70°C, and the three factors of heating time, ionic liquid concentration and liquid-to-solid ratio that had a greater impact on the volatile oil extraction rate were selected. The volatile oil extraction rate was taken as the response value, and the three-factor three-level Box-Behnken response surface experiment was designed using the Design-expert13 software. The experimental design and results are shown in Tables 7, 8, and 9.

[0036] Table 7. Box-Behnken experimental factor levels and coding table Table 8. Box-Behnken test results (n=3) Table 9. Box-Behnken test variance analysis results The multivariate quadratic model equation obtained by regression fitting of each factor is: Y=2.41+0.0738A+0.0650B+0.1063C+0.0525AB+0.0100AC+0.0525BC-0.1100A2-0.0825B2-0.1050C2.

[0037] Design-expert13 software was used to optimize and predict the levels of each factor, and the optimal process conditions for this experiment were obtained: the maximum predicted Y value (volatile oil extraction rate) was 2.27%. Considering the convenience of actual production operations, the optimal extraction process conditions for volatile oil were revised to: heating time of 75 min, ionic liquid concentration of 0.9%, and liquid-to-solid ratio of 3. Three parallel experiments were carried out under this condition, and the average volatile oil yield was 2.21%, which was 2.71% relative deviation from the predicted Y value, indicating that the process conditions have good stability and high accuracy.

[0038] 4. Evaluation of in vitro anti-influenza virus activity: 70 Sprague-Dawley rats without specific pathogens were randomly divided into a blank group, experimental drug high, medium and low dose groups and control drug high, medium and low dose groups; the blank group was given normal saline by gavage; the experimental drug high, medium and low dose groups were given the antiviral volatile oil of Example 1 by gavage at doses of 7.5 mg / g, 5 mg / g and 2.5 mg / g, respectively; the control drug high, medium and low dose groups were given antiviral volatile oil (volatile oil prepared according to the preparation method of antiviral oral liquid in Part I of the Pharmacopoeia of the People's Republic of China, 2020 edition) by gavage at doses of 7.5 mg / g, 5 mg / g and 2.5 mg / g, respectively; the blank group, experimental drug high, medium and low dose groups and control drug high, medium and low dose groups were all given 3 times / d, and blood was collected under anesthesia after 7 consecutive days of administration to prepare drug-containing serum.

[0039] MDCK cells that have grown into a monolayer were placed in a 96-well plate and the supernatant was discarded. Except for the normal cell control group, the virus control group, the experimental drug high, medium and low dose groups, and the control drug high, medium and low dose groups were inoculated with 100ul influenza A (H1N1) pdm09 subtype virus solution (100TCID 50 / 0.10ml), cultured at 37℃ for 120min, discarded the virus supernatant, and set up 6 duplicate wells for each group; added 0.2ml of MEM culture medium containing 10% fetal bovine serum to the normal cell group, added 0.2ml of MEM culture medium containing 10% fetal bovine serum to the virus control group, added 0.2ml of 2.5% concentration of serum containing experimental drugs to the experimental drug high, medium and low dose groups, added 0.2ml of 2.5% concentration of serum containing control drugs to the control drug high, medium and low dose groups, cultured at 37℃, observed cytopathic effect (CPE) continuously, terminated the culture when more than 75% of the virus control group had lesion wells, used MTT staining method, and measured the optical density (OD value) of each well with a microplate reader.

[0040] The results were recorded and the inhibition rate of each group of drugs on the virus was calculated using the statistical software SPSS18.0. Formula (2) is as follows: Virus inhibition rate = (OD value of drug group - OD value of virus control group) / (OD value of normal cells - OD value of virus control group) × 100% (2) Table 10. Virus inhibition rate results of each group It can be seen from the data in Table 10 above that both the experimental drug high-dose group and the control drug high-dose group have inhibitory effects on the virus, among which the inhibitory effects of the experimental drug high-dose group and the control drug high-dose group are significantly stronger than those of the control drug high-dose group and the control drug high-dose group, proving that the extraction method of the volatile oil of the antiviral oral liquid can further ensure the activity of the antiviral components in the volatile oil.

[0041] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes for application in other fields. However, any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for extracting volatile oil from an antiviral oral liquid, characterized in that: Here are the steps: Weigh the medicinal materials according to the prescription ratio, grind and mix the medicinal materials, add them to the volatile oil extraction tank, add ionic liquid, add distilled water and heat, separate the water and volatile oil, and obtain the volatile oil of the antiviral oral liquid.

2. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The medicinal materials include forsythia, patchouli, calamus and curcuma, and the mass ratio of the forsythia, patchouli, calamus and curcuma is 1.5-2:1-1.5:0.8-1:0.8-1.

3. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The ionic liquid is at least one of 1-butyl-3-methylimidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium tetrafluoroborate and N-ethylpyridinium tetrafluoroborate.

4. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate.

5. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The concentration of the ionic liquid is 0.9% to 10%.

6. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The concentration of the ionic liquid is 0.9%.

7. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The mass ratio of the medicinal material to distilled water is 1:3-8.

8. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The mass ratio of the medicinal material to distilled water is 1:

3.

9. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The heating temperature is 70°C to 100°C, and the heating time is 60 to 90 minutes.

10. The method for extracting volatile oil from antiviral oral liquid according to claim 1, characterized in that: The heating temperature was 70°C and the heating time was 75 min.

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