Agilawood extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of natural medicines and large health products, and particularly relates to an agilawood extract as well as a preparation method and application thereof. The preparation method of the agilawood volatile oil and the agilawood extract powder provided by the invention comprises the following steps: soaking agilawood, and performing CO2 supercritical extraction to obtain the agilawood volatile oil and agilawood dregs; performing reflux extraction, concentration and drying on the agilawood dregs to obtain agilawood extract powder; according to the preparation method provided by the invention, the extraction rate of the agilawood volatile oil is high, and the agilawood dregs are extracted through an ethanol reflux extraction method, so that medicinal components such as agilawood tetrol and the like in the agilawood dregs can be better extracted, and the utilization rate of the agilawood medicinal material is greatly improved. Moreover, according to the obtained agilawood pharmacodynamic component, an experiment on the influence of nasal administration on an insomnia rat shows that the obtained agilawood pharmacodynamic component has an influence on the expression level of neurotransmitters and amino acids in the brain of the rat, and a reference is provided for the study of a nasal preparation of the traditional Chinese medicine agilawood on the anti-insomnia effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicines, and particularly relates to an agarwood volatile oil, an agarwood extract powder, and a preparation method and application thereof. Background Art

[0002] Agarwood is the resin-containing wood of Aquilaria sinensis (Lour.) Gilg of the Thymelaeaceae family. As a traditional Chinese medicine in China, agarwood has a pungent taste and a slightly warm nature, and has pharmacological effects such as sedation and hypnosis, antidepressant, anti-inflammatory, antibacterial, and anti-tumor. The active ingredients of agarwood are mainly volatile oils, including sesquiterpenoids, chromones, aromatic and fatty acid compounds, etc.

[0003] At present, steam distillation, solvent extraction, and supercritical CO2 fluid extraction methods are mainly used to extract agarwood volatile oil. Although the steam distillation method is simple and convenient and easy to implement in industrial production, its process temperature is relatively high, which is likely to cause the volatilization loss of low-boiling components, accelerate the isomerization reaction of unstable components, and the overall extraction rate is relatively low, and the effective components in agarwood medicinal materials cannot be effectively utilized. When extracting agarwood volatile oil by solvent extraction, the extraction rate is often only a few per thousand, resulting in the waste of precious agarwood medicinal materials, and the organic reagents are toxic and harmful, which will damage the human body and pollute the environment. The supercritical CO2 fluid extraction method is a highly efficient new separation technology. Its principle is to use CO2 in a supercritical state as a solvent for extraction, which can be extracted and separated under near-ambient temperature conditions, and CO2 is odorless, non-toxic, safe, low in price, high in purity, easy to prepare, and can be recycled repeatedly, effectively reducing costs. Although the supercritical CO2 fluid extraction method can meet the requirements of environmental protection and safety, the current supercritical CO2 fluid extraction process for agarwood still has the problem of poor yield (the extraction rate is below 1.65%). Summary of the Invention

[0004] The purpose of the present invention is to provide an agarwood volatile oil, an agarwood extract powder, and a preparation method and application thereof. The preparation method provided by the present invention has a relatively high extraction rate for agarwood volatile oil.

[0005] In order to achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method of an agarwood extract, comprising the following steps:

[0007] After impregnating the agarwood, perform CO2 supercritical extraction to obtain the agarwood volatile oil and agarwood medicinal residues; the solvent for impregnation is anhydrous ethanol.

[0008] Preferably, the particle size of the agarwood is less than 0.85 mm;

[0009] The solid-liquid ratio of the impregnation is 1 g: 0.6 - 2.1 mL, and the time is 1 - 24 h.

[0010] Preferably, the extraction pressure of the CO2 supercritical extraction is 20 - 35 MPa, the extraction temperature is 40 - 50 °C, and the extraction time is 0.5 - 5 h.

[0011] Preferably, the separation pressure of the CO2 supercritical extraction is 4 - 6 MPa, and the separation temperature is 45 - 55 °C.

[0012] The present invention also provides a combined preparation method of agarwood volatile oil - agarwood extract powder, comprising the following steps:

[0013] Performing reflux extraction on the agarwood medicinal residues to obtain an agarwood medicinal residue extract; the precipitated medicinal residues are prepared by the preparation method described in the above technical solution;

[0014] Concentrating the agarwood medicinal residue extract to obtain agarwood extract;

[0015] Drying the agarwood extract to obtain the agarwood extract powder.

[0016] Preferably, the solid-liquid mass ratio of the reflux extraction is 1:5 - 10, the extraction time is 1 - 5 h, and the extraction times are 1 - 5 times; the solvent for the reflux extraction is an ethanol aqueous solution; the volume concentration of ethanol in the ethanol aqueous solution is 1% - 99%.

[0017] Preferably, the concentration is vacuum concentration; the temperature of the vacuum concentration is 30 - 60 °C;

[0018] The drying is vacuum drying; the temperature of the vacuum drying is 40 - 100 °C.

[0019] The present invention also provides the agarwood volatile oil prepared by the preparation method described in the above technical solution.

[0020] The present invention also provides the agarwood extract powder prepared by the preparation method described in the above technical solution.

[0021] The present invention also provides the application of the agarwood volatile oil described in the above technical solution and / or the agarwood extract powder described in the above technical solution in the preparation of a preparation for treating insomnia.

[0022] The present invention provides a method for preparing agarwood volatile oil, comprising the following steps: After impregnating the agarwood, perform CO2 supercritical extraction to obtain the agarwood volatile oil and agarwood medicinal residues; the solvent for impregnation is anhydrous ethanol. The present invention first impregnates the agarwood medicinal materials with anhydrous ethanol, which helps to extract the volatile oil better. Then, the CO2 supercritical extraction method is used to extract the agarwood volatile oil. By contacting CO2 in the supercritical state with the agarwood medicinal materials to be separated, the agarwood volatile oil is selectively extracted, effectively preventing the oxidation and escape of thermosensitive substances. Moreover, anhydrous ethanol can act as an entrainer during the extraction process, which helps to extract the substances with high boiling points, low volatility, and easy pyrolysis in agarwood at a temperature far lower than their boiling points. This preparation method has good selectivity, high extraction efficiency, low energy consumption, improves production efficiency, reduces preparation costs, and can achieve the maximum retention of effective substances in agarwood. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 Physical diagram of the agarwood volatile oil (AEO) in the agarwood medicinal components prepared in Example 1;

[0025] Figure 2 Physical diagram of the agarwood extract powder (EA) in the agarwood medicinal components prepared in Example 1;

[0026] Figure 3 Physical comparison diagram of β-CD, the physical mixture of β-CD and the agarwood volatile oil obtained by the preparation method in Example 1, and the agarwood volatile oil β-cyclodextrin inclusion complex (Agarwood-β-CD) in Example 7;

[0027] Figure 4 Scanning electron microscope comparison diagram of β-CD, the physical mixture of β-CD and the agarwood volatile oil obtained by the preparation method in Example 1, and the agarwood volatile oil β-cyclodextrin inclusion complex (Agarwood-β-CD) in Example 7. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention provides a method for preparing an agarwood extract, comprising the following steps:

[0029] After impregnating the agarwood, perform CO2 supercritical extraction to obtain the agarwood volatile oil and agarwood medicinal residues; the solvent for impregnation is anhydrous ethanol.

[0030] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0031] In the present invention, the particle size of the aloeswood is preferably less than 0.85 mm, more preferably 0.1 - 0.85 mm, and most preferably 0.42 - 0.85 mm.

[0032] In the present invention, when the particle size of the aloeswood is not within the above range, it is also preferably included to successively pulverize and screen the aloeswood; the mesh number of the sieve for screening is preferably 20 - 60 meshes, more preferably 40 - 60 meshes; the present invention has no other limitations on the processes of pulverizing and screening, and the processes well-known to those skilled in the art can be adopted.

[0033] In the present invention, the solid-liquid ratio of the impregnation is preferably 1 g : 0.6 - 2.1 mL, more preferably 1 g : 1 - 2.1 mL, and most preferably 1 g : 1.2 - 1.8 mL; the time is preferably 1 - 24 h, more preferably 5 - 24 h, and most preferably 12 - 24 h; the impregnation is preferably sealed cold impregnation; the addition method of absolute ethanol in the sealed cold impregnation is preferably pumped in by a supercritical fluid extractor pump or added by spraying.

[0034] In the present invention, the extraction pressure of the CO2 supercritical extraction is preferably 20 - 35 MPa, more preferably 25 - 35 MPa; the extraction temperature is preferably 40 - 50 °C, more preferably 45 - 50 °C; the extraction time is preferably 0.5 - 5 h, more preferably 2 - 4 h; the separation pressure is preferably 4 - 6 MPa, more preferably 5 - 6 MPa; the separation temperature is preferably 45 - 55 °C, more preferably 50 - 55 °C.

[0035] In the present invention, after obtaining the aloeswood volatile oil, it is also preferably included to successively centrifuge and concentrate; the rotation speed of the centrifuge is preferably 3000 - 10000 r / min, more preferably 5000 - 10000 r / min, and most preferably 8000 - 10000 r / min; the time is preferably 5 - 15 min, more preferably 10 - 15 min; the concentration is preferably to concentrate the supernatant obtained by the centrifugation; the concentration is preferably vacuum concentration; the temperature of the vacuum concentration is preferably 30 - 50 °C, more preferably 30 - 40 °C; the present invention has no special limitations on the process of the vacuum concentration, and it is concentrated in a manner well-known to those skilled in the art until no liquid drips from the receiver under the condenser tube.

[0036] The present invention first impregnates the Aquilariae Lignum Resinatum medicinal material with absolute ethanol, which helps to better extract the volatile oil. Then, the supercritical CO2 extraction method is used to extract the volatile oil of Aquilariae Lignum Resinatum. By contacting CO2 in the supercritical state with the Aquilariae Lignum Resinatum medicinal material to be separated, the volatile oil of Aquilariae Lignum Resinatum is selectively extracted, effectively preventing the oxidation and volatilization of heat-sensitive substances. Moreover, during the extraction process, absolute ethanol can act as an entrainer, which helps to extract the substances with high boiling points, low volatility, and easy pyrolysis in Aquilariae Lignum Resinatum at a temperature far lower than their boiling points. This preparation method has good selectivity, high extraction efficiency, low energy consumption, improves production efficiency, reduces the preparation cost, and can achieve the maximum retention of effective substances in Aquilariae Lignum Resinatum.

[0037] The present invention also provides a combined preparation method of volatile oil of Aquilariae Lignum Resinatum - Aquilariae Lignum Resinatum extract powder, comprising the following steps:

[0038] Performing reflux extraction on the residue of Aquilariae Lignum Resinatum medicinal material to obtain an extraction solution of the residue of Aquilariae Lignum Resinatum medicinal material; concentrating the extraction solution of the residue of Aquilariae Lignum Resinatum medicinal material to obtain an Aquilariae Lignum Resinatum extract; the residue of Aquilariae Lignum Resinatum medicinal material is prepared by the preparation method described in the above technical solution;

[0039] Drying the Aquilariae Lignum Resinatum extract to obtain the Aquilariae Lignum Resinatum extract powder.

[0040] The present invention performs reflux extraction on the residue of Aquilariae Lignum Resinatum medicinal material to obtain an extraction solution of the residue of Aquilariae Lignum Resinatum medicinal material; the residue of Aquilariae Lignum Resinatum medicinal material is prepared by the preparation method described in the above technical solution.

[0041] In the present invention, the solid-liquid mass ratio of the reflux extraction is preferably 1:5 - 10, more preferably 1:8 - 10, and most preferably 1:10; the extraction time is preferably 1 - 5 h, more preferably 1 - 3 h, and most preferably 2 - 3 h; the number of extraction times is preferably 1 - 5 times, more preferably 2 - 3 times, and most preferably 2 times; the solvent for the reflux extraction is preferably an ethanol aqueous solution; the volume concentration of ethanol in the ethanol aqueous solution is preferably 1% - 99%, more preferably 10% - 90%, and most preferably 25% - 75%.

[0042] After obtaining the extraction solution of the residue of Aquilariae Lignum Resinatum medicinal material, the present invention concentrates the extraction solution of the residue of Aquilariae Lignum Resinatum medicinal material to obtain an Aquilariae Lignum Resinatum extract.

[0043] In the present invention, the concentration is preferably vacuum concentration; the temperature of the vacuum concentration is preferably 30 - 60 °C, more preferably 40 - 60 °C, and most preferably 45 - 60 °C.

[0044] After obtaining the Aquilariae Lignum Resinatum extract, the present invention dries the Aquilariae Lignum Resinatum extract to obtain the Aquilariae Lignum Resinatum extract powder.

[0045] In the present invention, the drying is preferably vacuum drying; the temperature of the vacuum drying is preferably 40 to 100 °C, more preferably 50 to 90 °C, and most preferably 60 to 80 °C; the present invention has no special limitation on the time of the vacuum drying, and it can be dried until the product is of constant weight.

[0046] The method for jointly preparing the agarwood volatile oil-agarwood extract powder provided by the present invention has a high extraction rate of the agarwood volatile oil, and by using the ethanol reflux extraction method to extract the agarwood medicinal residues, the active ingredient agalloquilol in the agarwood medicinal residues can be better extracted, and the obtained agarwood extract powder is rich in agalloquilol, thereby greatly improving the utilization rate of the agarwood medicinal materials.

[0047] The present invention also provides the agarwood volatile oil prepared by the preparation method described in the above technical solution.

[0048] The present invention also provides the agarwood extract powder prepared by the preparation method described in the above technical solution.

[0049] The present invention also provides the application of the agarwood volatile oil and / or the agarwood extract powder described in the above technical solution in the preparation of a preparation for treating insomnia.

[0050] In the present invention, the application form of the agarwood volatile oil and / or the agarwood extract powder in the preparation of a preparation for treating insomnia is preferably a traditional Chinese medicine preparation; the agarwood volatile oil is preferably an agarwood volatile oil β-cyclodextrin clathrate.

[0051] In the present invention, the application form of the agarwood volatile oil and / or the agarwood extract powder in the preparation of a preparation for treating insomnia is preferably a nasal mucosa administration preparation.

[0052] The present invention applies the agarwood volatile oil and / or the agarwood extract powder to the preparation of a preparation for treating insomnia. By simulating agarwood incense through the nasal mucosa administration method, the active substances of agarwood can bypass the blood-brain barrier and directly enter the brain, achieving brain targeting in the drug administration route, which is beneficial to the treatment of insomnia and provides a reference for the development of nasal preparations of traditional Chinese medicine agarwood in the treatment of insomnia.

[0053] In order to further illustrate the present invention, the agarwood volatile oil, the agarwood extract powder, and their preparation methods and applications provided by the present invention will be described in detail below with reference to the attached tables, drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] Preparation of agarwood volatile oil:

[0056] The Aquilariae Lignum Resinatum medicinal material was crushed and passed through a 60-mesh sieve. 100 g of the crushed Aquilariae Lignum Resinatum powder was sealed and cold-soaked with 1.8 times the amount of anhydrous ethanol entrainer for 1 h, and then CO2 supercritical extraction was carried out. The extraction parameters were an extraction pressure of 35 MPa, a separation pressure of 6 MPa, an extraction temperature of 45 °C, a separation temperature of 50 °C, and an extraction time of 1 h, to obtain the crude extract of Aquilariae Lignum Resinatum volatile oil and the dried medicinal residue of Aquilariae Lignum Resinatum after supercritical fluid extraction;

[0057] The crude extract of Aquilariae Lignum Resinatum volatile oil was centrifuged at 8000 r / min for 10 min, and the supernatant was taken. The supernatant was concentrated under reduced pressure at 30 °C to obtain Aquilariae Lignum Resinatum volatile oil.

[0058] Preparation of Aquilariae Lignum Resinatum extract powder:

[0059] The above-mentioned Aquilariae Lignum Resinatum medicinal residue was reflux-extracted twice with 10 times the amount of 65% ethanol, with a single reflux extraction time of 1 h, to obtain the Aquilariae Lignum Resinatum medicinal residue extract;

[0060] The Aquilariae Lignum Resinatum medicinal residue extract was filtered under reduced pressure while it was hot, and the filtrate was taken. After being concentrated under reduced pressure at 45 °C, it was vacuum-dried at 45 °C to obtain Aquilariae Lignum Resinatum extract powder.

[0061] The above-mentioned Aquilariae Lignum Resinatum volatile oil and Aquilariae Lignum Resinatum extract powder were combined to obtain the medicinal components of Aquilariae Lignum Resinatum.

[0062] The physical picture of the Aquilariae Lignum Resinatum volatile oil prepared in Example 1 is as Figure 1 shown; the physical picture of the Aquilariae Lignum Resinatum extract powder prepared in Example 1 is as Figure 2 shown.

[0063] Example 2

[0064] Preparation of Aquilariae Lignum Resinatum volatile oil:

[0065] The Aquilariae Lignum Resinatum medicinal material was crushed and passed through a 60-mesh sieve. 100 g of the crushed Aquilariae Lignum Resinatum powder was sealed and cold-soaked with 1.8 times the amount of anhydrous ethanol entrainer for 1 h, and then CO2 supercritical extraction was carried out. The extraction parameters were an extraction pressure of 25 MPa, a separation pressure of 6 MPa, an extraction temperature of 50 °C, a separation temperature of 50 °C, and an extraction time of 3 h, to obtain the crude extract of Aquilariae Lignum Resinatum volatile oil and the dried medicinal residue of Aquilariae Lignum Resinatum after supercritical fluid extraction;

[0066] The crude extract of Aquilariae Lignum Resinatum volatile oil was centrifuged at 8000 r / min for 10 min, and the supernatant was taken. The supernatant was concentrated under reduced pressure at 30 °C to obtain Aquilariae Lignum Resinatum volatile oil.

[0067] Preparation of Aquilariae Lignum Resinatum extract powder:

[0068] The above-mentioned Aquilariae Lignum Resinatum medicinal residue was reflux-extracted in 10 times the amount of 50% ethanol for 3 h, with a reflux extraction time of 1 h, to obtain the Aquilariae Lignum Resinatum medicinal residue extract;

[0069] The agarwood residue extract was filtered under reduced pressure while hot, the filtrate was concentrated under reduced pressure at 45°C, and then vacuum dried at 45°C to obtain agarwood extract powder.

[0070] The agarwood volatile oil is combined with agarwood extract powder to obtain the agarwood medicinal component.

[0071] Example 3

[0072] Preparation of agarwood volatile oil:

[0073] The agarwood medicinal material was crushed through a 40-mesh sieve, 100 g of the crushed agarwood powder was weighed and sealed and cold-soaked for 1 hour with 0.6 times the amount of anhydrous ethanol entrainer, and CO2 supercritical extraction was performed. The extraction parameters were extraction pressure 20 MPa, separation pressure 4 MPa, extraction temperature 40°C, separation temperature 45°C and extraction time 0.5 h, and a crude extract of agarwood volatile oil and agarwood dry residue after supercritical fluid extraction were obtained;

[0074] The crude extract of agarwood volatile oil was centrifuged at 3000 r / min for 5 min, the supernatant was collected, and the supernatant was concentrated under reduced pressure at 50° C. to obtain agarwood volatile oil.

[0075] Preparation of agarwood extract powder:

[0076] Reflux extraction of the agarwood residue in 5 times the amount of 50% ethanol for 1 hour, reflux extraction 5 times, to obtain an agarwood residue extract;

[0077] The agarwood residue extract was filtered under reduced pressure while hot, the filtrate was concentrated under reduced pressure at 30°C, and then vacuum dried at 40°C to obtain agarwood extract powder.

[0078] The agarwood volatile oil is combined with agarwood extract powder to obtain the agarwood medicinal component.

[0079] Example 4

[0080] Preparation of agarwood volatile oil:

[0081] The agarwood medicinal material was crushed through a 40-mesh sieve, 100 g of the crushed agarwood powder was weighed and sealed and cold-soaked in 1.35 times the amount of anhydrous ethanol entrainer for 12 hours, and CO2 supercritical extraction was performed. The extraction parameters were extraction pressure 30 MPa, separation pressure 5 MPa, extraction temperature 45°C, separation temperature 50°C and extraction time 2.5 hours to obtain a crude extract of agarwood volatile oil and agarwood dry residue after supercritical fluid extraction;

[0082] The crude extract of agarwood volatile oil was centrifuged at 6500 r / min for 10 min, the supernatant was collected, and the supernatant was concentrated under reduced pressure at 30° C. to obtain agarwood volatile oil.

[0083] Preparation of agarwood extract powder:

[0084] Reflux extract the agarwood residue in 8 times the amount of 75% ethanol for 3 h, with reflux extraction once, to obtain the agarwood residue extract;

[0085] Filter the agarwood residue extract while it is hot under reduced pressure, take the filtrate, concentrate it under reduced pressure at 45 °C, and then dry it under vacuum at 70 °C to obtain the agarwood extract powder.

[0086] Combine the agarwood volatile oil and the agarwood extract powder to obtain the agarwood medicinal efficacy component.

[0087] Example 5

[0088] Preparation of agarwood volatile oil:

[0089] Crush the agarwood medicinal material through a 60-mesh sieve, weigh 100 g of the crushed agarwood powder, seal and cold soak it with 2.1 times the amount of absolute ethanol entrainer for 1 h, and carry out CO2 supercritical extraction. The extraction parameters are extraction pressure 30 MPa, separation pressure 6 MPa, extraction temperature 50 °C, separation temperature 55 °C, and extraction time 5 h, to obtain the crude agarwood volatile oil extract and the dried agarwood residue after supercritical fluid extraction;

[0090] Centrifuge the crude agarwood volatile oil extract at 10000 r / min for 15 min, take the supernatant, and concentrate the supernatant under reduced pressure at 30 °C to obtain the agarwood volatile oil.

[0091] Preparation of agarwood extract powder:

[0092] Reflux extract the agarwood residue in 10 times the amount of absolute ethanol for 3 h, with reflux extraction once, to obtain the agarwood residue extract;

[0093] Filter the agarwood residue extract while it is hot under reduced pressure, take the filtrate, concentrate it under reduced pressure at 60 °C, and then dry it under vacuum at 100 °C to obtain the agarwood extract powder.

[0094] Combine the agarwood volatile oil and the agarwood extract powder to obtain the agarwood medicinal efficacy component.

[0095] Example 6

[0096] Preparation of agarwood volatile oil:

[0097] Crush the agarwood medicinal material through a 40-mesh sieve, weigh 100 g of the crushed agarwood powder, seal and cold soak it with 2.1 times the amount of absolute ethanol entrainer for 24 h, and carry out CO2 supercritical extraction. The extraction parameters are extraction pressure 25 MPa, separation pressure 4 MPa, extraction temperature 45 °C, separation temperature 45 °C, and extraction time 2 h, to obtain the crude agarwood volatile oil extract and the dried agarwood residue after supercritical fluid extraction;

[0098] Centrifuge the crude extract of agarwood volatile oil at 3000 r / min for 15 min, take the supernatant, and concentrate the supernatant under reduced pressure at 30 °C to obtain agarwood volatile oil.

[0099] Preparation of agarwood extract powder:

[0100] Reflux extract the agarwood residue in 10 times the amount of 75% ethanol for 5 h, reflux extract once to obtain the agarwood residue extract;

[0101] Filter the agarwood residue extract while it is hot under reduced pressure, take the filtrate, concentrate it under reduced pressure at 60 °C, and then vacuum dry it at 100 °C to obtain agarwood extract powder.

[0102] Combine the agarwood volatile oil and the agarwood extract powder to obtain the agarwood medicinal component.

[0103] Example 7

[0104] Preparation of agarwood volatile oil β-cyclodextrin inclusion compound:

[0105] Using the saturated aqueous solution method, weigh 1.5 g of β-cyclodextrin (β-CD) accurately, place it in a 100 mL conical flask, add 40 mL of pure water, heat to dissolve β-CD, and prepare a β-CD saturated aqueous solution. In a magnetic stirrer, add 0.25 g of agarwood volatile oil (prepared according to Example 1) (dissolved in an equal amount of anhydrous ethanol) drop by drop, the temperature is 40 °C, the inclusion time is 2 h, after cooling to room temperature, refrigerate it at 4 °C in the refrigerator for 24 h, filter it, wash it 3 times with 10 mL of anhydrous ethanol and distilled water respectively, and dry it in an oven at 45 °C for 5 h to obtain the agarwood volatile oil β-cyclodextrin inclusion compound.

[0106] Preparation of purified agarwood extract powder:

[0107] Taking the purity of agalloquil as the index, the purification process was selected as the agarwood extract (prepared according to Example 1), passing through AB-8 macroporous resin, loading the column wetly, with a diameter-height ratio of 1:5, a sample loading amount (0.1 mol / mL crude drug amount, 60 mL), a sample loading speed of 2 BV / h, a impurity removal solvent (0.1 mol / mL NaCl: 1 BV, 2 BV / h), and an elution solvent (65% ethanol, 6 BV, 2 BV / h). The results showed that the purity of agalloquil reached 23.82 ± 3.04%, and the extract yield was 62.93 ± 4.81%.

[0108] Figure 3 It is a comparison physical map of β-CD, the physical mixture of β-CD and the agarwood volatile oil obtained by the preparation method of Example 1, and the agarwood volatile oil β-cyclodextrin inclusion compound (Agarwood-β-CD) in Example 7;

[0109] Figure 4 The scanning electron microscope comparison diagram of β-CD in Example 7, the physical mixture of β-CD and agarwood volatile oil prepared according to the method of Example 1, and the β-cyclodextrin inclusion complex of agarwood volatile oil (Agarwood-β-CD). Figure 3 and Figure 4 It can be seen that the inclusion of β-CD to agarwood volatile oil cannot be achieved through simple physical mixing. Figure 4 It can be seen that β-cyclodextrin is blocky and has a firm texture. Under the same magnification, the volume of β-cyclodextrin is larger than that of the inclusion compound. The physical mixture of β-CD and agarwood volatile oil has basically no regular clumps and a loose texture. The β-CD inclusion compound of agarwood volatile oil is a small flaky crystal with clear edges. The particle size and morphology are completely different from those of β-CD and the physical mixture of β-CD and agarwood volatile oil, indicating that the agarwood volatile oil in the agarwood volatile oil β-cyclodextrin inclusion compound (Agarwood-β-CD) is included by cyclodextrin.

[0110] Comparative Example 1

[0111] Preparation of agarwood volatile oil:

[0112] The agarwood medicinal material was crushed through a 20-mesh sieve, and 100 g of the crushed agarwood powder was weighed and subjected to CO2 supercritical extraction. The extraction parameters were extraction pressure 20 MPa, separation pressure 4 MPa, extraction temperature 40°C, separation temperature 45°C, and extraction time 0.5 h to obtain a crude extract of agarwood volatile oil and agarwood dry residue after supercritical fluid extraction;

[0113] The crude extract of agarwood volatile oil was centrifuged at 3000 r / min for 5 min, the supernatant was collected, and the supernatant was concentrated under reduced pressure at 50° C. to obtain agarwood volatile oil.

[0114] Preparation of agarwood extract powder:

[0115] The agarwood residues were subjected to reflux extraction in 5 times the amount of pure water for 5 hours, and the reflux extraction was repeated once to obtain an agarwood residue extract;

[0116] The agarwood residue extract was filtered under reduced pressure while hot, the filtrate was concentrated under reduced pressure at 30°C, and then vacuum dried at 40°C to obtain agarwood extract powder.

[0117] The agarwood volatile oil is combined with agarwood extract powder to obtain the agarwood medicinal component.

[0118] Comparative Example 2

[0119] Preparation of agarwood volatile oil:

[0120] Crush the Aquilariae Lignum Resinatum medicinal material through a 40-mesh sieve, weigh 100 g of the crushed Aquilariae Lignum Resinatum powder, perform CO2 supercritical extraction, with the extraction parameters being an extraction pressure of 1 MPa, a separation pressure of 4 MPa, an extraction temperature of 40 °C, a separation temperature of 45 °C, and an extraction time of 1 h, to obtain the crude extract of Aquilariae Lignum Resinatum volatile oil and the dried medicinal residue of Aquilariae Lignum Resinatum after supercritical fluid extraction;

[0121] Centrifuge the crude extract of Aquilariae Lignum Resinatum volatile oil at 4000 r / min for 8 min, take the supernatant, and concentrate the supernatant under reduced pressure at 30 °C to obtain Aquilariae Lignum Resinatum volatile oil.

[0122] Preparation of the Aquilariae Lignum Resinatum extract powder:

[0123] Reflux extract the above-mentioned Aquilariae Lignum Resinatum medicinal residue in 5 times the amount of 15% ethanol for 5 h, with 1 reflux extraction, to obtain the extract of Aquilariae Lignum Resinatum medicinal residue;

[0124] Filter the extract of Aquilariae Lignum Resinatum medicinal residue while it is hot under reduced pressure, take the filtrate, concentrate it under reduced pressure at 30 °C, and then dry it under vacuum at 40 °C to obtain the Aquilariae Lignum Resinatum extract powder.

[0125] Combine the above-mentioned Aquilariae Lignum Resinatum volatile oil and the Aquilariae Lignum Resinatum extract powder to obtain the effective component of Aquilariae Lignum Resinatum.

[0126] Test Example 1

[0127] Measure and calculate the extraction rate of Aquilariae Lignum Resinatum volatile oil and the yield of Aquilariae Lignum Resinatum extract powder in the above-mentioned examples and comparative examples. Among them, the extraction rate of Aquilariae Lignum Resinatum volatile oil (%) = the amount of Aquilariae Lignum Resinatum volatile oil (g) / the amount of the cut crude drug (g) * 100%, and the yield of Aquilariae Lignum Resinatum extract (%) = the amount of extract obtained from the cut crude drug (g) / the amount of the cut crude drug (g) * 100%. The results are shown in Table 1.

[0128] Table 1 Data of the extraction rate of Aquilariae Lignum Resinatum volatile oil and the yield of Aquilariae Lignum Resinatum extract powder in the examples and comparative examples

[0129]

[0130] As can be seen from Table 1, through impregnation with absolute ethanol and CO2 supercritical extraction, after optimizing the extraction process, the extraction amount of Aquilariae Lignum Resinatum volatile oil has increased significantly; through ethanol reflux extraction of the Aquilariae Lignum Resinatum medicinal residue, after optimizing the process, the yield of Aquilariae Lignum Resinatum extract powder has increased significantly; the preparation method provided by the present invention has a high extraction rate of Aquilariae Lignum Resinatum volatile oil and a high yield of Aquilariae Lignum Resinatum extract powder, and can improve the utilization rate of Aquilariae Lignum Resinatum medicinal material.

[0131] Test Example 2

[0132] A single-factor investigation was conducted on the preparation method of agarwood volatile oil. The contour lines and response surface plots were drawn using Design-Expert 8.0.6 software to obtain the interactive effects among various factors. When the extraction temperature remained constant, the extraction rate of volatile oil first gradually increased and then showed a gentle downward trend as the extraction pressure increased; when the extraction pressure remained constant, the extraction rate of volatile oil first increased and then decreased with the increase of extraction temperature. When the extraction pressure remained constant, the effect of separation temperature on the extraction rate of volatile oil was to increase first and then decrease; when the separation temperature remained constant, the extraction rate of volatile oil first gradually increased with the increase of extraction pressure and then showed a downward trend. When the separation temperature remained constant, the extraction rate of volatile oil increased with the increase of extraction temperature; when the extraction temperature remained constant, the extraction rate of volatile oil first gradually increased and then showed a gentle downward trend with the increase of separation temperature.

[0133] A single-factor investigation was conducted on the preparation method of agarwood extract powder. The contour lines and response surface plots were drawn using Design-Expert 8.0.6 software to obtain the interactive effects among various factors. When the solvent volume remained constant, the extract yield first gradually increased and then decreased as the solvent concentration increased; when the solvent concentration remained constant, the extract yield gradually increased with the increase of solvent volume. When the solvent volume remained constant, the effect of extraction time on the extract yield was not significant; when the extraction time remained constant, the extract yield gradually increased with the increase of solvent volume. When the solvent concentration remained constant, the extract yield slowly increased with the increase of extraction time; when the extraction time remained constant, the extract yield first increased and then decreased with the increase of solvent concentration.

[0134] Test Example 3

[0135] Pharmacodynamic study on the anti-insomnia effect of agarwood pharmacodynamic components

[0136] 1. Preparation and treatment of drugs

[0137] Agarwood pharmacodynamic components (prepared through Example 1, the agarwood volatile oil in the agarwood pharmacodynamic components is denoted as AEO, and the agarwood extract powder in the agarwood pharmacodynamic components is denoted as EA). The volatile oil and extract powder were administered jointly (dissolved by ultrasonic treatment with a 4% ethanol-saline solution), and the dosage for rats was converted. Low, medium, and high dose groups were set; the agarwood volatile oil β-cyclodextrin inclusion complex (Agarwood-β-CD) prepared in Example 7 was mixed with the purified agarwood extract powder (EA) prepared in Example 7, and after dissolving with normal saline, it was set as the β-cyclodextrin inclusion complex group. After the preparation of each group of drugs, they were aliquoted and stored at 4°C for standby.

[0138] 2. Experimental animals

[0139] Forty-two male Sprague-Dawley rats, SPF grade, weighing 180-220 g, were purchased from the Experimental Animal Center of Zhejiang Chinese Medical University (Experimental Animal Production License No., Shanghai Slack Experimental Animal Co., Ltd., SCXK (Shanghai) 2017-0005; Experimental Animal Qualification Certificate No., SYXK (Zhejiang) 2018-0012).

[0140] 3. Materials and Reagents

[0141] 4-Chloro-DL-phenylalanine (PCPA, aladdin, batch number: J2022233); Sodium pentobarbital (provided by the Experimental Animal Center of Zhejiang Chinese Medical University); Diazepam (Shandong Xinyi Pharmaceutical Co., Ltd., national drug approval number H37023039); Rat norepinephrine ELISA kit (Jiangsu Enzyme Immuno Biotechnology Co., Ltd., product number: MM-20302R1); Rat 5-hydroxytryptamine ELISA kit (Jiangsu Enzyme Immuno Biotechnology Co., Ltd., product number: MM-0442R1); Rat γ-aminobutyric acid ELISA kit (Jiangsu Enzyme Immuno Biotechnology Co., Ltd., product number: MM-0441R1); Rat glutamate ELISA kit (Jiangsu Enzyme Immuno Biotechnology Co., Ltd., product number: MM-0601R1); BCA protein concentration assay kit (Beyotime, product number: LotNo.082820201207).

[0142] 4. Experimental Groups

[0143] After referring to the scope of application of Aquilariae Lignum in the 2020 edition of the Chinese Pharmacopoeia and the results of preliminary pre-experiments, the low, medium, and high doses were determined. Forty-two SD rats were randomly divided into 7 groups, with 6 rats in each group. The grouping is as follows:

[0144] A. Normal control group: No treatment

[0145] B. Model group: Normal saline

[0146] C. Diazepam group: Diazepam (DZP, 0.92 mg / kg)

[0147] D. Low-dose group of Aquilariae Lignum pharmacodynamic components: A mixture of AEO (12.43 mg / kg) and EA (3.63 mg / kg)

[0148] E. Medium-dose group of Aquilariae Lignum pharmacodynamic components: A mixture of AEO (24.85 mg / kg) and EA (7.28 mg / kg)

[0149] F. High-dose group of Aquilariae Lignum pharmacodynamic components: A mixture of AEO (49.70 mg / kg) and EA (14.55 mg / kg)

[0150] Group of G.β - cyclodextrin inclusion complex: A mixture of Agarwood - β - CD (79.52 mg / kg) and EA (14.55 mg / kg).

[0151] 5. Experimental methods

[0152] Establishment of insomnia rat model: Intraperitoneally inject PCPA (450 mg / kg) from 9:00 - 10:00 am every day to make a rat insomnia model. PCPA is made into a suspension with weakly alkaline saline (pH: 7 - 8) at a ratio of 1 mL / 100 g for 2 consecutive days. Randomly select 6 SD rats as the normal control group without any treatment for 9 consecutive days. Except for the normal control group, the other groups are modeled for two days. From the 3rd day, the model group: Administer normal saline (50 μL / rat) by nasal mucosa; the diazepam group: Administer diazepam aqueous solution (gavage at 0.92 mg / kg, 100 g / mL); the low - dose group, medium - dose group, high - dose group, and β - cyclodextrin inclusion complex group: All are administered by nasal mucosa (50 μL / rat) for 7 consecutive days. After 7 days, relevant behavioral experiments are carried out. After the experiment, anesthetize each group of rats and remove the hypothalamus tissue on ice. Process it according to the ELISA kit instructions, and detect the contents of 5 - HT, NE, GABA, and Glu in the hypothalamus tissue with an enzyme - labeled instrument. The experimental data are expressed as (x ± s). Statistical analysis is performed using SPSS 20.0 mathematical software. Normal distribution, variance analysis, and t - test are carried out on the experimental results. p < 0.05 is considered to have a significant difference, and p < 0.01 is considered to have a highly significant difference. Animals only perform the same behavioral experiment once, and each behavioral experiment needs to be completed. GraphPad Prism 5.01.336 software is used for graphing.

[0153] 6. Results of the test on the synergistic effect of pentobarbital sodium in inducing sleep in rats

[0154] (1) Effect of subthreshold dose of pentobarbital sodium on the sleep - onset rate of rats

[0155] After the last administration of each group, intraperitoneally inject a subthreshold dose of pentobarbital sodium (30 mg / kg), and observe and record whether the animals fall asleep.

[0156] The results of the hypnotic effect of subthreshold pentobarbital sodium on the sleep - onset rate of rats are shown in Table 2. Compared with the model group, the sleep - onset rate of the blank control group is 100%, with a statistical difference (p < 0.01); the sleep - onset rate of the model group is 0.0%; the sleep - onset rates of the diazepam group and the medium - dose group are both 83.3%, with a statistically significant difference (p < 0.01); the low - dose, high - dose, and β - cyclodextrin inclusion complex groups are all 66.7%, with a statistically significant difference compared with the model group (p < 0.05). The results show that all the drug - administered groups have a hypnotic effect, the efficacy of the medium - dose group is more significant, and the efficacy of the low - dose, high - dose, and β - cyclodextrin inclusion complex groups is equivalent.

[0157] Table 2 Effects of Synergistic Subthreshold Dose of Sodium Pentobarbital on the Sleep Onset Rate of Rats

[0158] Sleep onset rate Mean value (%) SD Normal control group 100.00 0.00 Model group 0.00 0.00 Diazepam group 83.33 0.41 Low-dose group 66.67 0.52 Medium-dose group 83.33 0.41 High-dose group 66.67 0.52 β-cyclodextrin inclusion complex group 66.67 0.52

[0159] (2) Effects of Synergistic Suprathreshold Dose of Sodium Pentobarbital on the Sleep Latency and Sleep Duration of Rats

[0160] After the last administration in each group, a suprathreshold dose of sodium pentobarbital (45 mg / kg) was intraperitoneally injected, and the sleep conditions of the animals were observed and recorded. If the disappearance of the righting reflex was greater than 1 min, the rat entered the sleep state, and the sleep duration was the time from the disappearance of the righting reflex to recovery, and the sleep latency was determined as the time from injection to the disappearance of the righting reflex.

[0161] The effects of synergistic suprathreshold sodium pentobarbital on sleep latency and sleep time are shown in Table 3 and Table 4. The results showed that compared with the model group, all the drug administration groups could shorten the sleep latency time of rats and prolong the sleep time. Compared with the model group, there were statistically significant differences in the effects of the positive drug group, medium-dose group, and β-cyclodextrin inclusion complex group on sleep latency; however, the high-dose effect was better than the low-dose effect but weaker than the medium-dose effect, and the dose relationship showed a "peak" shape; the effect of the β-cyclodextrin inclusion complex group was better than that of the high-dose group. The effects on sleep time showed a dose-dependent relationship in the low-dose, medium-dose, and high-dose groups, and the β-cyclodextrin inclusion complex group was also extremely significant.

[0162] Table 3 Effects of Synergistic Suprathreshold Dose of Sodium Pentobarbital on the Sleep Latency of Rats

[0163]

[0164] Table 4 Effects of Synergistic Suprathreshold Dose of Sodium Pentobarbital on the Sleep Duration of Rats

[0165]

[0166]

[0167] (3) Results of Open Field Test

[0168] The results of the effects of the drug on the distance and frequency of rats in each group entering the central area in the open field test are shown in Tables 5 and 6, and the results of the effects of the drug on the total movement distance and the number of animal stools in the open field test are shown in Tables 7 and 8. Compared with the model group, there were statistically significant differences in the distance (%) and frequency (times) of the blank group entering the central area (p < 0.001); there was an improving trend in the low-dose group of the drug-administered group, but there was no statistical significance compared with the model group. For the distance of entering the central area, the medium dose > the β-cyclodextrin inclusion complex group > the high-dose group; for the frequency of entering the central area, there was a dose-dependent relationship, the β-cyclodextrin inclusion complex group > the high-dose group > the medium-dose group. The results of the total movement distance in the open field showed that there were statistical differences between each drug-administered group and the model group (p < 0.05); after drug administration, it could reduce the number of stools of insomnia animals, and there was a dose-dependent relationship, and the β-cyclodextrin inclusion complex group was better than the high-dose group. The positive control group of diazepam had fewer activity times and total distances during the entire open field test. The reason might be that diazepam had a sedative and hypnotic effect, resulting in a decrease in the number of spontaneous activities of animals.

[0169] Table 5 Effects of the open field test on the distance of rats entering the central area

[0170]

[0171] Table 6 Effects of the open field test on the frequency of rats entering the central area

[0172]

[0173]

[0174] Table 7 Effects of the open field test on the total movement distance of rats

[0175]

[0176] Table 8 Effects of the open field test on the number of animal stools of rats

[0177]

[0178] (4) Results of the tail suspension test

[0179] The effects of the drug on the immobile time of rats in each group in the tail suspension test are shown in Table 9. After the model group was injected with PCPA, the immobile time of animals was prolonged, indicating its anxiety state to a certain extent; both diazepam and the drug-administered group could reduce the immobile time of animals, and there were statistical differences compared with the model group; the drug-administered group could reduce the immobile time of animals in the tail suspension test, indicating that the effective components of Aquilaria sinensis could alleviate the anxiety of insomnia rats, and the curative effect relationship was medium dose > β-cyclodextrin inclusion complex group > high dose group > low dose group.

[0180] (5) Results of the elevated plus maze test

[0181] Comparison results of the effects of the drug on the number of entries into the open arms (Table 10) and residence time (Table 11), the number of entries into the closed arms (Table 12) and residence time (Table 13) of rats in the elevated plus-maze test, and the comparison results of the effects on the total distance of entry of each group are shown in Table 14. The drug-administered group can increase the number of entries into the open arms; it can also prolong the residence time in the open arms, showing a dose-dependent relationship. Compared with the model group, there was a statistically significant decrease in the number of entries of the drug-administered group into the closed arms (p < 0.05), but there was no significant difference between groups; each group had no obvious effect on the time of rats entering the closed arms. The low-dose group and the medium-dose group had a tendency to reduce the total movement distance but there was no statistical significance; compared with the model group, there were statistical differences in the total distance of the high-dose group and the β-cyclodextrin inclusion complex group entering the elevated plus-maze (p < 0.05). Comprehensive description of each index shows that the effective components of Aquilariae Lignum Resinatum can, to a certain extent, alleviate the anxiety of insomnia rats. Among them, the positive drug diazepam group had fewer activity times and total distance during the entire elevated plus-maze test, which was consistent with the results of the open field test.

[0182] Table 9 Effects on the immobile time of rats in the tail suspension test

[0183]

[0184] Table 10 Effects on the number of entries of rats into the open arms in the elevated plus-maze test

[0185]

[0186] Table 11 Effects on the residence time of rats in the open arms in the elevated plus-maze test

[0187]

[0188] Table 12 Effects on the number of entries of rats into the closed arms in the elevated plus-maze test

[0189]

[0190]

[0191] Table 13 Effects on the residence time of rats in the closed arms in the elevated plus-maze test

[0192]

[0193] Table 14 Effects on the total distance of rats in the elevated plus-maze test

[0194]

[0195] (6) Effects on monoamine neurotransmitters and amino acids

[0196] Effects of the drug on the norepinephrine (Table 15) and serotonin content (Table 16) in the hypothalamus of rats. After injecting PCPA, the NE in the hypothalamus of insomnia rats increased compared with the blank control group. Compared with the model group, there were significant differences in the positive drug and each administration group, indicating an improvement effect on the NE level. The β-cyclodextrin inclusion complex group was better, with the medium dose being equivalent to the high dose, and the low dose being the second best; PCPA would affect the expression of 5-HT in the hypothalamus of rats, and nasal mucosa administration of the effective components of Aquilaria sinensis had a certain improvement effect on the 5-HT level in the hypothalamus of animals. The medium dose had better efficacy, followed by the high dose and the β-cyclodextrin inclusion complex group, and the low dose and the diazepam group had an increase.

[0197] Table 15 Effects of the drug on norepinephrine in the hypothalamus of rats

[0198]

[0199]

[0200] Table 16 Effects of the drug on serotonin content in the hypothalamus of rats

[0201]

[0202] (7) Results of the ratio of glutamate and γ-aminobutyric acid in the hypothalamus of rats

[0203] The effects of the drug on the ratio of glutamate and γ-aminobutyric acid in the hypothalamus of rats are shown in Table 17. After injecting PCPA, insomnia would affect the secretion of GABA and GLU in its hypothalamus, manifested as an abnormal GABA / GLU ratio. The GABA / GLU ratio of insomnia rats was higher than that of normal rats, and the diazepam group was significantly improved. The Aquilaria sinensis administration group could improve the expression levels of GABA and GLU, showing a dose-dependent relationship.

[0204] Table 17 Effects of the drug on the ratio of glutamate and γ-aminobutyric acid in the hypothalamus of rats

[0205]

[0206] The effective components of Aquilaria sinensis prepared in Examples 2 to 6 have the same performance as those in Example 1.

[0207] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing an agarwood extract, characterized in that, It includes the following steps: After impregnating the agarwood, carry out CO2 supercritical extraction to obtain the agarwood volatile oil and agarwood medicinal residues; the solvent for impregnation is absolute ethanol.

2. The preparation method according to claim 1, wherein The particle size of the agarwood is less than 0.85 mm; The solid-liquid ratio for impregnation is 1 g: 0.6 - 2.1 mL, and the time is 1 - 24 h.

3. The preparation method according to claim 1, characterized in that, The extraction pressure for the CO2 supercritical extraction is 20 - 35 MPa, the extraction temperature is 40 - 50 °C, and the extraction time is 0.5 - 5 h.

4. The preparation method according to claim 1 or 3, characterized in that, The separation pressure for the CO2 supercritical extraction is 4 - 6 MPa, and the separation temperature is 45 - 55 °C.

5. A combined preparation method of agarwood volatile oil-agarwood extract powder, characterized in that, It includes the following steps: Carry out reflux extraction on the agarwood medicinal residues to obtain an agarwood medicinal residue extract; the precipitated medicinal residues are prepared by the preparation method according to any one of claims 1 - 4; Concentrate the agarwood medicinal residue extract to obtain an agarwood extract; Dry the agarwood extract to obtain the agarwood extract powder.

6. The preparation method according to claim 5, wherein, The solid-liquid mass ratio for the reflux extraction is 1: 5 - 10, the extraction time is 1 - 5 h, and the number of extraction times is 1 - 5 times; the solvent for the reflux extraction is an ethanol aqueous solution; the volume concentration of ethanol in the ethanol aqueous solution is 1% - 99%.

7. The preparation method according to claim 5, characterized in that, The concentration is vacuum concentration; the temperature for the vacuum concentration is 30 - 60 °C; The drying is vacuum drying; the temperature for the vacuum drying is 40 - 100 °C.

8. The agarwood volatile oil prepared by the preparation method according to any one of claims 1 - 4.

9. The agarwood extract powder prepared by the preparation method according to any one of claims 5 - 7.

10. The use of the agarwood volatile oil according to claim 8 and / or the agarwood extract powder according to claim 9 in the preparation of a preparation for treating insomnia.

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