Stepped preparation method of indocalamus leaf essential oil flavone polysaccharide

Abstract

The invention discloses a gradient preparation method of indocalamus leaf essential oil flavone polysaccharide, which is characterized in that specific steps and a specific process are matched for multi-step gradual low-temperature graded extraction and separation to ensure that plant essential oil which is free of organic solvent residue, green and environment-friendly and high in flavor reduction degree is finally obtained; only three times of simple ultrasonic oscillation are added in the essential oil extraction process, so that the water phase after essential oil extraction is rich in active ingredients such as polysaccharide and flavone, crude polysaccharide and crude flavone can be obtained through further graded extraction and separation, and the utilization rate of raw materials is remarkably improved; the method provided by the invention has small damage to essential oil, polysaccharide and flavone, and the essential oil, polysaccharide and flavone in the raw materials can be reduced to the greatest extent.

Description

Technical Field

[0001] The invention relates to the field of preparation of essential oil flavonoid polysaccharides, and in particular to a step-by-step preparation method of indocalamus leaf essential oil flavonoid polysaccharides. Background Art

[0002] Indocalamus refers to the plant of the genus Indocalamus in the subfamily Bambusoideae of the Graminales family. Its leaves are broad and have a special aroma. Since ancient times, people in the Jiangnan region have used it as a packaging material for food such as rice dumplings, tea, and rice cakes. Indocalamus leaves, also known as Indocalamus leaves, are not only used as food packaging materials, but are also a traditional Chinese medicine. The Compendium of Materia Medica records that "Indocalamus leaves are sweet, cold, and non-toxic", "treat vomiting blood in men and women", "unblock urination, benefit lung qi, throat paralysis, and eliminate carbuncle", etc. The Ben Jing Feng Yuan records that "Indocalamus grows from small bamboos and its leaves are the largest. It can be used as a hat, which can unblock urination and benefit lung qi", and "decoction can treat stomach heat hiccups, and its nature is slightly milder than that of Perilla frutescens". Modern pharmacological studies have shown that Indocalamus leaves are rich in nutritional active ingredients such as flavonoids, phenolic acids, lignans, terpenes, polysaccharides, volatile oils, and mineral elements, and have antibacterial, antiseptic, antioxidant, anticancer, and antiviral functions. Among them, essential oil, polysaccharides and flavonoids are the three main active ingredients of indocalamus leaves.

[0003] Existing active ingredient extraction technologies often only extract one of the essential oils, polysaccharides, and flavonoids, and when extracting one of the ingredients, it will cause damage to other ingredients or adversely affect the extraction and separation efficiency of other ingredients, resulting in a huge waste of resources. For example, when extracting essential oils with water, the temperature will reach 100°C during high-temperature steaming to extract essential oils. At this time, the sugar chain of polysaccharides will be destroyed, causing problems such as loss of activity; if conventional 90°C hot water is used for polysaccharide extraction, essential oils cannot be extracted. If low-temperature acid-base extraction of polysaccharides is used, not only will essential oils not be extracted from the remaining raw materials, but the extraction rate of flavonoids will also be reduced. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a step-by-step preparation method of flavonoid polysaccharide from indocalamus leaf essential oil.

[0005] The invention adopts low-temperature step extraction technology to extract three active components of essential oil, polysaccharide and flavonoids from indocalamus leaves at one time. The reduction degree of the extract is high, and the invention is a green and efficient active component preparation technology.

[0006] A stepwise preparation method of indigo leaf essential oil flavonoid polysaccharide, using fresh indigo leaf as raw material, comprises the following steps:

[0007] (1) adding water to the raw materials to make pulp to obtain a slurry;

[0008] (2) adding water to the slurry, grinding at a temperature greater than 0° C. and less than or equal to 20° C. until the particle size is less than or equal to 500 meshes, to obtain a grinding liquid;

[0009] (3) subjecting the grinding liquid to ultrasonic oscillation, and then centrifuging at 8000 rpm to 12000 rpm and a temperature greater than 0° C. and less than or equal to 20° C. to separate and obtain a first liquid A1 and a first solid B1;

[0010] (4) adding water to the first solid B1, and ball-milling the solid at a temperature greater than 0° C. and less than or equal to 20° C. until the particle size is less than or equal to 3000 mesh, to obtain a ball-milling liquid;

[0011] (5) subjecting the ball milling liquid to ultrasonic oscillation, and then centrifuging at 8000 rpm to 12000 rpm and a temperature greater than 0° C. and less than or equal to 20° C. to separate and obtain a second liquid A2 and a second solid B2;

[0012] (6) adding water to the second solid B2, stirring for 3 min-10 min, then ultrasonically oscillating, and then centrifuging at 8000 rpm-12000 rpm and a temperature greater than 0° C. and less than or equal to 20° C. to separate and obtain a third liquid A3 and a third solid B3;

[0013] (7) combining the first liquid A1, the second liquid A2 and the third liquid A3 to obtain an extract, adding an inorganic salt to the extract, slowly stirring at a speed of 30 r / min-240 r / min at a temperature greater than 0° C. and less than or equal to 20° C., standing to separate the essential oil and the first aqueous phase after oil-water separation, and obtaining the essential oil;

[0014] (8) concentrating the first aqueous phase to within 5 times the weight of the raw material under a pressure of 1 kPa-1.2 kPa and a temperature of 25° C.-30° C. to obtain aqueous solution C;

[0015] (9) adding petroleum ether to aqueous solution C, mixing evenly, standing to separate layers, and separating to obtain a second aqueous phase and petroleum ether containing a pigment;

[0016] (10) adding n-butanol to the second aqueous phase, mixing evenly and standing to separate, separating to obtain a third aqueous phase and a first n-butanol phase, repeatedly adding n-butanol to the third aqueous phase, mixing evenly and standing to separate, separating to obtain a precipitate, a fourth aqueous phase and a second n-butanol phase, and combining the first n-butanol phase and the second n-butanol phase to obtain a third n-butanol phase;

[0017] (11) freeze-drying the precipitate obtained in step (10) to obtain crude polysaccharide;

[0018] (12) Concentrating and removing the solvent from the third n-butanol phase obtained in step (10) to obtain a concentrate and crude flavonoids.

[0019] The invention adopts the above-mentioned specific steps and specific processes in combination to gradually extract and separate at low temperature in multiple steps, so as to ensure that the plant essential oil with no organic solvent residue, green and environmental protection and high flavor restoration is finally obtained; only by adding simple three ultrasonic oscillations in the essential oil extraction process, the aqueous phase after the plant essential oil is extracted can be rich in active ingredients such as polysaccharides and flavonoids, and further graded extraction and separation can obtain crude polysaccharides and crude flavonoids, which significantly improves the utilization rate of raw materials; the method of the invention has little destructiveness to plant essential oils, polysaccharides and flavonoids, and can restore the essential oils, polysaccharides and flavonoids in the raw materials to the maximum extent.

[0020] In order to achieve better technical effects, the following optimization is performed:

[0021] In step (1), the weight ratio of the raw material to water is 1:1-3. The slurry has a particle size of at least 16 mesh; it is further preferably processed into a slurry with a smaller particle size, such as a particle size of 50 mesh.

[0022] In step (2), the weight ratio of the slurry to water is 1:0.5-5. The grinding is performed by a colloid mill. More preferably, the grinding is performed by a colloid mill for multiple cycles (most preferably more than three times).

[0023] In step (3), the ultrasonic oscillation conditions are: ultrasonic oscillation for 5 minutes to 10 minutes at a frequency of 20KHz to 30KHz and a vibration-off ratio of 5s:1s.

[0024] In step (4), the weight ratio of the first solid B1 to water is 1:0.5-5. The ball milling uses zirconium oxide ball milling beads to avoid metal residue. Further preferably, water is added to the first solid B1, and the ball milling is performed at a temperature greater than 0°C and less than or equal to 20°C until the particle size is less than or equal to 5000 mesh to obtain a ball milling liquid.

[0025] In step (5), the ultrasonic oscillation conditions are: ultrasonic oscillation for 5 minutes to 10 minutes at a frequency of 40KHz to 50KHz and a vibration-off ratio of 5s:1s.

[0026] In step (6), the ultrasonic oscillation conditions are: ultrasonic oscillation for 20min-30min at a frequency of 40KHz-50KHz and a vibration-off ratio of 5s:1s. The weight ratio of the second solid B2 to water is 1:0.5-5. The stirring can be carried out using a beater.

[0027] The frequency of ultrasonic oscillation in the present invention is preferably selected into two different frequencies according to the fineness of the materials in each step, because at low frequency, the cavitation intensity is large, the cavitation bubbles are large, but the impact surface is small, and at high frequency, the cavitation intensity is small, the cavitation bubbles are small, but the contact surface is large. The ultrasonic oscillations of different frequencies in specific steps adopted in the present invention are mainly to enrich the highly active polysaccharides and flavonoids extracted from the raw materials in the first aqueous phase after the essential oil is extracted, especially to extract flavonoids from the material without affecting the composition and yield of the essential oil.

[0028] In step (7), the amount of the inorganic salt added is 3%-8% of the weight of the extract, preferably 5%. The inorganic salt is selected from sodium chloride, potassium chloride, etc. The slow stirring time is generally 12 hours to 48 hours. The slow stirring can be magnetic stirring. The water separated after the oil-water separation can be recycled and used for essential oil extraction of the same plant.

[0029] In step (7), at a separation temperature greater than 0°C and less than or equal to 20°C and a stirring rate of 30rpm-240rpm, a certain amount of essential oil with rich component types can be obtained. More preferably, at a separation temperature greater than 0°C and less than or equal to 10°C and a stirring rate of 30rpm-90rpm, essential oil with rich component types can be obtained with a high yield. Even more preferably, at a separation temperature greater than 0°C and less than or equal to 8°C (most preferably 2°C-4°C) and a stirring rate of 30rpm-60rpm, essential oil with rich component types can be obtained with a high yield.

[0030] In step (9), petroleum ether is added to aqueous solution C three times, each time the amount of petroleum ether added is 5%-10% of the weight of aqueous solution C, mixed evenly, allowed to stand for stratification, the aqueous phase and the petroleum ether containing the pigment are separated, and the aqueous phases obtained by the three separations are combined to obtain a second aqueous phase.

[0031] In step (10), n-butanol in an amount of 2 to 4 times the weight of the second aqueous phase is added to the second aqueous phase; and n-butanol in an amount of 2 to 4 times the weight of the third aqueous phase is repeatedly added to the third aqueous phase.

[0032] In step (11), preferably: add a first low eutectic solvent (DES) accounting for 20 to 22 times the mass of the precipitate to the precipitate obtained in step (10), ultrasonically extract for 10 min to 15 min, add ethanol to the extract, mix well and then let it stand for precipitation, freeze-dry the precipitate to obtain crude polysaccharide; the purity of the crude polysaccharide can be improved by secondary extraction of the precipitate obtained in step (10) with the first low eutectic solvent. The first low eutectic solvent is preferably composed of water, choline chloride and malonic acid or water, choline chloride and succinic acid, wherein the molar ratio of choline chloride to malonic acid or succinic acid is 1:2, and the weight percentage of water is 10%. Further preferably, the first low eutectic solvent is prepared by hot stirring. The conditions of the hot stirring are preferably: 75°C-85°C for uniform mixing and stirring. Preferably, ethanol accounting for 4 to 6 times the volume of the extract is added to the extract. The first low eutectic solvent and ethanol separated in the standing precipitation step can be recycled and reused. For example, vacuum concentration can be used to distill out ethanol, and the remainder is the first low eutectic solvent.

[0033] In step (12), preferably: the third n-butanol phase obtained in step (10) is concentrated and desolventized to obtain a concentrate, a second low eutectic solvent accounting for 10 to 15 times the volume of the concentrate is added to the concentrate, ultrasonic extraction is performed for 10 to 20 minutes, a macroporous resin AB-8 accounting for more than 10% of the weight of the extract is added to the extract, stirring and adsorption is performed, vacuum filtration is performed, the macroporous resin AB-8 is eluted with ethanol, and the eluent is concentrated to obtain crude flavonoids; the third n-butanol phase concentrate is extracted with the second low eutectic solvent to improve the purity of the crude flavonoids; since the extract extracted with the second low eutectic solvent is difficult to separate using conventional separation methods, the present invention uses macroporous resin AB-8 to well separate the crude flavonoids and the second low eutectic solvent. The second low eutectic solvent is composed of water, choline chloride and propylene glycol or water, choline chloride and malonic acid, wherein the molar ratio of choline chloride to propylene glycol or malonic acid is 1:2, and the weight percentage of water is 15%. Further preferably, the second low eutectic solvent is prepared by hot stirring. The hot stirring condition is preferably: 75°C-85°C for uniform mixing and stirring. Preferably: the macroporous resin AB-8 is eluted with ethanol in an amount of 5 to 8 times the volume of the macroporous resin AB-8. The second low eutectic solvent separated in the vacuum filtration step and the ethanol obtained by concentrating the eluent can be recycled and reused.

[0034] In the essential oil extraction stage of the present invention, the operating temperatures of grinding, ball milling, centrifugation, etc. are all controlled below 20° C., thereby avoiding the loss of essential oil components during the operation process.

[0035] In the present invention, the water is preferably purified water.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The essential oil extraction process is carried out at a low temperature throughout, which will not cause the oxidation of the essential oil components and the loss of low-boiling point components.

[0038] The essential oil extraction process is a fully physical processing process, without any organic solvents, and there is no solvent residue or loss caused by desolventization.

[0039] The essential oil extraction process is a full essential oil preparation process, without the polarity selection problem of CO2 and organic solvents, and the obtained essential oil flavor composition has a high degree of restoration.

[0040] The close combination of the steps of the method of the present invention can simultaneously extract the three components of essential oil, polysaccharide and flavonoids.

[0041] All operations are performed at relatively low temperatures, such as room temperature, to avoid the loss of activity of active ingredients caused by high temperature; short-time ultrasound and limited temperature rise can both extract the active ingredients and avoid the loss of activity of the active ingredients.

[0042] The crude polysaccharides and crude flavonoids of the crude extracts have higher extraction rates and higher purity than those obtained by traditional extraction methods.

[0043] The water and extraction solvent used can be recycled and reused, avoiding waste of resources and environmental pollution, and significantly reducing production costs.

[0044] Each step in the method of the present invention is simple to operate, and adopts process parameters that are friendly to the environment and equipment, which is conducive to industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The present invention is a process flow chart of the preparation method.

[0046] Figure 2 GCMS chromatograms of Indocalamus leaf essential oil obtained by four methods.

[0047] Figure 3 This is a comparison chart of the component quantities of Indocalamus leaf essential oil obtained by four methods.

[0048] Figure 4 The relative contents of the essential oil components of Indocalamus leaf obtained by four methods.

[0049] Figure 5 This is the particle size distribution diagram of the material after ball milling in the present invention.

[0050] Figure 6 This is a particle size distribution diagram of the liquid solute after the ball-milled material is centrifuged in the present invention.

[0051] Figure 5 , Figure 6In the table, Results means results, Diam. means diameter, Intensity means intensity, Width means width, Z-Average means the average particle size obtained by intensity distribution, PdI means aggregation index, Intercept means the intercept of the particle size distribution curve on the horizontal axis, Result quality means result quality, Good means good, peak means peak, Size Distribution by Intensity means size distribution by intensity, Size means size, Record means record, and Refer to quality report means reference to quality report.

[0052] Figure 7 This is a graph showing the relationship between material size and essential oil yield (based on dry raw material) obtained using different methods.

[0053] Figure 8 This is a graph showing the effect of secondary extraction of crude polysaccharides using different DES extractants on the extraction rate and purity.

[0054] Fig. 9 Comparison chart of the yield, purity and maximum molecular weight of crude polysaccharides obtained by different extraction methods.

[0055] Fig.10 This is a comparison chart of the monosaccharide composition of crude polysaccharides extracted by three methods.

[0056] Fig.11 This is a graph showing the effect of crude polysaccharides extracted by different methods on the DPPH free radical scavenging rate.

[0057] Fig.12 ABTS analysis of crude polysaccharides extracted by different methods + Effect diagram of free radical scavenging rate.

[0058] Fig.13 This is a graph showing the effect of secondary extraction of crude flavonoids using different DES extractants on the extraction rate and purity.

[0059] Fig.14 This is a comparison chart of the extraction rate and purity of crude flavonoids obtained by different extraction methods.

[0060] Fig.15 This is an analysis chart of the component composition of crude flavonoids obtained by different extraction methods (1-3 represent the analysis results of three repetitions of the same method), where the scale on the right is the result after data normalization, the bluer the color, the higher the content, and the redder the color, the lower the content, comparing the content of the same substance in crude flavonoids obtained by the three methods.

[0061] Fig.16 The figure is a comparison of the free radical scavenging ability of crude flavonoids obtained by different extraction methods. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the technical solutions of the present invention.

[0063] The content of crude polysaccharide was determined by phenol-sulfuric acid method at a wavelength of 490 nm.

[0064] The molecular weight of crude polysaccharide was determined by high performance gel filtration chromatography-differential multi-angle laser light scattering.

[0065] The extraction rate is the percentage of the weight of the extraction target to the weight of the raw material (calculated on the basis of the weight of the dry raw material, the moisture content of fresh Indocalamus japonica leaves is 60%).

[0066] The purity of crude polysaccharide is the weight percentage of polysaccharide in the crude polysaccharide.

[0067] The purity of crude flavonoids is the weight percentage of flavonoids in the crude flavonoids.

[0068] Determination of flavonoid content: Using rutin as the standard sample, the content was determined by aluminum nitrate complex spectrophotometry at a wavelength of 510 nm.

[0069] Determination of flavonoids in crude extracts (crude flavonoids): The crude extracts were freeze-dried, ground with a grinder, dissolved in methanol, vortexed, filtered, and identified by ultra-performance liquid chromatography-mass spectrometry (UPLCMS), and the relative content was determined using the relative peak area of ​​the ion fragment peaks.

[0070] Embodiment 1:

[0071] (1) 10 kg of fresh Indocalamus japonica leaves were collected, washed, added with 10 kg of purified water, and beaten with a pulper to obtain a slurry with a fineness of 16 meshes;

[0072] (2) Add 10 kg of pure water to the 20 kg slurry, put it into the colloid mill, control the temperature at 15°C-20°C, and grind it in cycles at three levels of grinding disc gap: high, medium and low. Grind it three times in total, each time for 30 minutes, to obtain a grinding liquid with a fineness of 500 mesh;

[0073] (3) The grinding liquid was first subjected to ultrasonic oscillation at a frequency of 20 kHz and a vibration-stop ratio of 5s:1s for 5 minutes, and then added to a low-temperature centrifuge and centrifuged at 10,000 rpm and a temperature of 10° C. for 5 minutes to separate and obtain a first liquid A1 and 8.4 kg of a first solid B1;

[0074] (4) Add 7 kg of pure water to the first solid B1, stir and mix, then add to a low-temperature ball mill, use zirconium oxide grinding beads, and ball mill for 45 minutes at a temperature of 10° C. to a ball milling liquid with a fineness of at least 3000 mesh;

[0075] (5) The ball-milled liquid was first subjected to ultrasonic oscillation at a frequency of 40 kHz and a vibration-stop ratio of 5 s:1 s for 10 min, and then added to a low-temperature centrifuge and centrifuged at 10,000 rpm and 10° C. to obtain a second liquid A2 and 7.3 kg of a second solid B2;

[0076] (6) Add 10 kg of pure water to the second solid B2, stir repeatedly for 3 min with a beater, and then ultrasonically oscillate for 30 min at a frequency of 40 kHz and a vibration-stop ratio of 5 s:1 s, and then centrifuge at 10,000 rpm and 10° C. with a low-temperature centrifuge to obtain a third liquid A3 and a third solid B3;

[0077] (7) The first liquid A1, the second liquid A2 and the third liquid A3 were combined to obtain 40 kg of extract, which was added to a flat-bottomed narrow-mouthed separatory bottle, and 2 kg of sodium chloride was added. The mixture was slowly stirred at a speed of 45 r / min at a temperature of 2° C. for 48 hours. After standing for oil-water separation, the essential oil and the first aqueous phase were separated to obtain 26.14 g of Indocalamus leaf essential oil. The essential oil yield (i.e., the extraction rate) was 2.61 mg / g based on the fresh raw material, and the yield converted to dry raw material based on the moisture content of the raw material was 6.53 mg / g.

[0078] (8) The first aqueous phase was concentrated to within 5 times the weight of the raw material under the conditions of 1 kPa pressure and 30°C to obtain aqueous solution C.

[0079] (9) adding petroleum ether to aqueous solution C three times, with the amount of petroleum ether added each time accounting for 5% of the weight of aqueous solution C; mixing evenly, standing to separate the layers, separating the aqueous phase and the petroleum ether containing the pigment (i.e., the petroleum ether pigment extract), and combining the aqueous phases obtained by the three separations to obtain a second aqueous phase.

[0080] (10) adding n-butanol in an amount twice the weight of the second aqueous phase to the second aqueous phase, mixing evenly and allowing to stand to separate, separating to obtain a third aqueous phase and a first n-butanol phase, repeatedly adding n-butanol in an amount twice the weight of the third aqueous phase to the third aqueous phase, mixing evenly and allowing to stand to separate, separating to obtain a precipitate, a fourth aqueous phase (i.e., aqueous phase) and a second n-butanol phase, combining the first and second n-butanol phases to obtain a third n-butanol phase (i.e., organic phase).

[0081] (11) Add 20 times the mass of the precipitate to the first low eutectic solvent (composed of water, choline chloride and malonic acid, wherein the molar ratio of choline chloride to malonic acid is 1:2, and the weight percentage of water is 10%; mixed and stirred at 80°C to be uniformly prepared.), ultrasonic extraction for 10 minutes, add 4 times the volume of ethanol to the extract, mix evenly and let stand to precipitate, freeze-dry the precipitate to obtain a crude polysaccharide (i.e., crude polysaccharide K). The separated solvent: the first low eutectic solvent and ethanol are recycled, vacuum concentration is used to distill out the ethanol, and the remainder is the first low eutectic solvent.

[0082] (12) The third n-butanol phase is concentrated and desolventized to obtain a concentrate, and a second low eutectic solvent (composed of water, choline chloride and propylene glycol, wherein the molar ratio of choline chloride to propylene glycol is 1:2, and the weight percentage of water is 15%; mixed and stirred at 80°C) is added to the concentrate in an amount 10 times the volume of the concentrate, and ultrasonic extraction is performed for 10 minutes. A macroporous resin AB-8 accounting for 10% of the weight of the extract is added to the extract, and the mixture is stirred for adsorption, and vacuum filtration is performed to separate and obtain a second EDS solvent and macroporous resin AB-8. The macroporous resin AB-8 is eluted with ethanol accounting for 5 times the volume of the macroporous resin AB-8, and the eluate is concentrated to obtain crude flavonoids (i.e., crude flavonoids L). The second EDS solvent separated in the vacuum filtration step and the ethanol obtained by concentrating the eluate are recycled.

[0083] Comparative Example 1: Steam distillation

[0084] Weigh 10 kg of fresh Indocalamus japonicus leaves, wash them, add 10 kg of pure water, heat to boiling, condense and reflux to extract, and obtain 29.1 g of Indocalamus japonicus leaf water-extracted essential oil. The essential oil yield is 2.91 mg / g.

[0085] Comparative Example 2: Supercritical CO2 Extraction

[0086] 10 kg of fresh Indocalamus japonicus leaves were weighed and extracted with supercritical CO2 fluid at 35°C to separate 162.74 g of Indocalamus japonicus leaf supercritical extraction essential oil. The yield of essential oil was 16.27 mg / g.

[0087] Comparative Example 3: Dynamic Headspace Method

[0088] The dynamic headspace method is to continuously blow nitrogen through the sample, carry the volatile essential oil components to the adsorption tube through nitrogen, and be adsorbed by the adsorption material, and concentrate them at -100℃, and then quickly heat and inject them for GCMS analysis. This method has a high recognition rate for plant aromatic components and a good composition reduction degree. The essential oil obtained by this method was used to detect the composition of the essential oil in the leaves of Indocalamus japonica.

[0089] Weigh 4 g of fresh Indocalamus japonicus leaves, continuously blow nitrogen through the Indocalamus japonicus leaf sample, carry the volatile essential oil components to the adsorption tube through the nitrogen and be adsorbed by the adsorption material (Tetax material, the component is 2,6-dibenzofuran porous polymer resin), and concentrate it at -100°C to obtain the dynamic headspace essential oil of Indocalamus japonicus leaves.

[0090] Embodiment 2:

[0091] Except for “step (3) directly adding the grinding liquid into a low-temperature centrifuge, centrifuging at 10000 rpm and 10°C for 5 minutes to separate and obtain the first liquid A1 and 8.5 kg of the first solid B1;

[0092] Step (5) directly adding the ball milling liquid into a low-temperature centrifuge, and centrifuging at 10000 rpm and 10° C. to obtain a second liquid A2 and 7.5 kg of a second solid B2;

[0093] Step (6) Add 10 kg of pure water directly to the second solid B2, stir repeatedly for 3 min with a beater, and then centrifuge at 10000 rpm and 10° C. in a low-temperature centrifuge to obtain a third liquid A3 and a third solid B3; ", and the remaining operations are the same as those in Example 1, and 26.14 g of indocalamus leaf essential oil, 20.8 g of indocalamus leaf crude polysaccharide, and 11.6 g of indocalamus leaf crude flavonoids are obtained respectively.

[0094] The yield of essential oil from indocalamus leaves (i.e., extraction rate) is 2.61 mg / g based on fresh raw material, and the yield converted into dry raw material based on the moisture content of the raw material is 6.53 mg / g.

[0095] The yield of crude polysaccharide from Indocalamus japonicus leaves converted into dry raw material based on the moisture content of the raw material was 0.52%±0.05% (n=3).

[0096] The yield of crude flavonoids from Indocalamus leaves converted into dry raw material based on the moisture content of the raw material was 0.29%±0.09% (n=3).

[0097] It can be seen that adding ultrasonic oscillation in the specific steps of the method of the present invention, namely step (3), step (5) and step (6), has no obvious effect on the extraction of essential oils. It is mainly to enrich the first aqueous phase after the essential oils are extracted with polysaccharides and flavonoids extracted from the raw materials, and further fractional extraction and separation can obtain a considerable amount of crude polysaccharides and crude flavonoids with high activity, especially flavonoids can be better extracted from the material without affecting the composition and yield of the essential oils. The reason may be that the essential oils exist in the glands, while flavonoids and polysaccharides exist in the cells.

[0098] Comparison of the essential oil obtained by the present invention with other methods:

[0099] The essential oils were extracted and compared using steam distillation, supercritical extraction, dynamic headspace method and the method of the present invention.

[0100] Take 10 μL of the essential oil of Indocalamus leaf in Example 1, 10 μL of the essential oil of Indocalamus leaf water extraction in Comparative Example 1, 10 μL of the essential oil of Indocalamus leaf supercritical extraction in Comparative Example 2, and 10 μL of the essential oil of Indocalamus leaf in Example 2, respectively, add 2 mL of n-hexane for oscillation and dissolution, pass through an organic filter membrane, and directly inject the essential oil of Indocalamus leaf dynamic headspace adsorption in Comparative Example 3, and analyze it by GCMS. The results are shown in Table 1. Figure 2 , Figure 3 and Figure 4 .

[0101] GCMS conditions: chromatographic column DB-HeavyWAX (30m×0.25mm, 0.25μm), carrier gas (high purity helium) flow rate 1.0mL / min, injection port temperature 250℃, heating program: initial temperature 40℃, hold for 5min, then heat to 280℃ at a rate of 4℃ / min, hold for 5min. Mass spectrometer interface temperature 280℃, ion source temperature 230℃, mass scan range m / z 35-550, qualitative library NIST2020.

[0102] The components of the indocalamus leaf essential oil obtained in Example 1 of the present invention are consistent with those of the indocalamus leaf essential oil obtained in Example 2. By comparing the components of the conventional water-extracted essential oil, the dynamic headspace essential oil, and the supercritical extraction essential oil, it can be found that the indocalamus leaf essential oil obtained in Example 1 of the present invention and the indocalamus leaf essential oil obtained in Example 2 are closest to the essential oil components obtained by the dynamic headspace method, the types are basically the same, and the low-boiling point and high-boiling point components are relatively uniform; the extremely low-boiling point components in the water-extracted essential oil obtained by the steam distillation method are also lost in large quantities, and the high-boiling point components are basically absent; the essential oil components obtained by supercritical carbon dioxide have serious losses of low-boiling point components, which are mainly concentrated in the high-boiling point components.

[0103] from Figure 3 It can be seen that the composition of the essential oil obtained in Example 2 of the present invention is similar to that obtained by the dynamic headspace method, in which phenols and alkanes are more abundant than those by the dynamic headspace method, which is related to the extraction principle. The dynamic headspace method relies on nitrogen purge to capture, and phenols and alkanes are difficult to be blown out at low temperatures, so there are fewer related components. Figure 4 It can also be seen from the relative content graph that the composition of essential oils obtained by the method of the present invention is basically consistent with that by the dynamic headspace method. The contents of alkane substances that contribute less to flavor are relatively low in both methods; while the contents of alkane substances that contribute less to flavor in the essential oils obtained by steam distillation and supercritical extraction are significantly higher than those in the method of the present invention. It can be seen that compared with the essential oils obtained by steam distillation and supercritical extraction, the composition reduction degree of the essential oil of Indocalamus leaf obtained by the method of the present invention is the highest.

[0104] Table 1 Comparison of essential oil components obtained by four different methods

[0105]

[0106]

[0107]

[0108] Example 3: Effect of crushed particle size on essential oil extraction rate

[0109] The present invention finds that the grinding degree of the material is closely related to the extraction efficiency of the essential oil. In Example 2, after the fresh indocalamus leaves are pulped, colloid milled and ball milled, the particle size distribution of the ball milling liquid material is shown in FIG. Figure 5 ,from Figure 5 It can be seen that after the beating, colloid milling and ball milling of the present invention, the material reaches a fineness of 5000 mesh (about 2.6 μm), the average particle size of the material is 0.3011 μm, and the distribution range is 20 nm-3 μm. At this time, the yield of essential oil becomes high. The material is centrifuged and the second liquid A2 contains a small amount of impurities and essential oil components. The particle size distribution of the solute in the second liquid A2 is shown in FIG. Figure 6 , the average particle size of the solute becomes 3.054nm, the distribution range is 1nm-3μm, and it is mainly distributed in the range of 1nm-3nm and 10nm-40nm. In Example 1, after the fresh Indocalamus japonica leaves are pulped, colloid milled, and ball milled, the particle size distribution of the ball milling liquid material and the particle size distribution of the second liquid A2 solute are the same as those in Example 2.

[0110] The slurry obtained by the operation in step (1) of Example 2 was added to a low-temperature centrifuge and centrifuged at 10000 rpm and 10°C to obtain a liquid and a solid. The liquid was added to a flat-bottomed narrow-mouthed separation bottle, and 2 kg of sodium chloride was added. The mixture was slowly stirred at a speed of 45 r / min at a temperature of 2°C for 48 hours. After standing for oil and water separation, 0.04 g of Indocalamus leaf essential oil was obtained. The essential oil yield based on fresh raw material was 0.004 mg / g (the yield converted to dry raw material was 0.01 mg / g), recorded as: 1-shear only.

[0111] The first liquid A1 obtained by the operations in steps (1), (2) and (3) of Example 2 was added to a flat-bottomed narrow-mouthed separatory flask, and 2 kg of sodium chloride was added. The mixture was slowly stirred at a speed of 45 r / min at a temperature of 2° C. for 48 hours. After standing for oil-water separation, 5.21 g of Indocalamus leaf essential oil was obtained. The essential oil yield based on fresh raw material was 0.521 mg / g (the yield converted to dry raw material was 1.3 mg / g), recorded as: 2-colloid mill.

[0112] Except that the ball milling time in step (4) of Example 2 was replaced by 5 min to obtain a ball milling liquid with an average particle size of 4.5 μm, the remaining operations were the same as in Example 1 to obtain 7.22 g of indocalamus leaf essential oil, with an essential oil yield of 0.722 mg / g based on fresh raw material (1.81 mg / g based on dry raw material), recorded as: 3-ball milling 1.

[0113] Except that the ball milling time in step (4) of Example 2 was replaced with 10 min to obtain a ball milling solution with an average particle size of 0.9 μm, the remaining operations were the same as in Example 1 to obtain 12.48 g of indocalamus leaf essential oil. The essential oil yield based on fresh raw material was 1.248 mg / g (the yield converted to dry raw material was 3.12 mg / g), recorded as: 4-ball milling 2.

[0114] According to the operation of Example 2, a ball milling liquid with an average particle size of 0.3 μm was obtained, and 26.14 g of Indocalamus leaf essential oil was obtained. The essential oil yield based on fresh raw materials was 2.614 mg / g (converted to a dry raw material yield of 6.53 mg / g), recorded as: 5-ball mill 3.

[0115] The material size obtained by different grinding processes and the essential oil yield can be seen in Figure 7 ,from Figure 7 It can be seen that the essential oil yield is significantly positively correlated with the fineness of the material. The fineness of the material obtained under the process conditions of the present invention can obtain a relatively high essential oil yield while ensuring a high degree of reduction of the essential oil components.

[0116] Example 4

[0117] 1. First low eutectic solvent screening study

[0118] Choline chloride was used as a hydrogen bond acceptor, urea, acetic acid, propionic acid and other 12 reagents were used as hydrogen bond donors, water was added and mixed at 80°C, the molar ratio of hydrogen bond acceptor to hydrogen bond donor was 1:2, the weight percentage of water was 10%, and the first low eutectic solvent was used for the secondary extraction of crude polysaccharides. The rest of the operations were performed according to Example 1. The research results are shown in Figure 8 The study found that choline chloride + malonic acid and choline chloride + succinic acid had the highest extraction rates for crude polysaccharides, reaching 0.68% and 0.69% respectively, and the purity of crude polysaccharides extracted by choline chloride + malonic acid reached 63.49%, which was higher than the purity of 60.00% of crude polysaccharides extracted by choline chloride + succinic acid. Although the purity of crude polysaccharide extracts of some first low eutectic solvents was higher than that of choline chloride + malonic acid, the extraction rate was not ideal and was significantly lower than that of choline chloride + malonic acid (p < 0.05). Taking all factors into consideration, the first low eutectic solvent preferably used choline chloride + malonic acid (molar ratio 1:2) + 10% (weight percentage) water or choline chloride + succinic acid (molar ratio 1:2) + 10% (weight percentage) water.

[0119] 2. Comparison of the polysaccharide extraction rate of the present invention with conventional hot water extraction and ultrasonic water extraction

[0120] Ultrasonic water extraction method (recorded as: ultrasonic extraction or ultrasonic extraction): weigh 10g of fresh Indocalamus leaf raw material, add 200g of distilled water, ultrasonically treat for 45min, repeat the extraction three times (add 200g of distilled water each time), combine the three filtrates and concentrate to 200mL, add 95% ethanol aqueous solution with a mass percentage concentration of 4 times the volume of the concentrate for alcohol precipitation, centrifuge to obtain the precipitate, freeze-dry to obtain crude polysaccharide.

[0121] Hot water extraction method (recorded as: hot water extraction or hot water extraction): weigh 10g of fresh Indocalamus leaf raw material, add 200g of distilled water, extract in a 90°C water bath for 2 hours, repeat the extraction three times (add 200g of distilled water each time), combine the three filtrates and concentrate to 200mL, add 95% ethanol aqueous solution with a mass percentage concentration of 4 times the volume of the concentrate for alcohol precipitation, centrifuge to obtain the precipitate, freeze-dry to obtain crude polysaccharide.

[0122] Method 1 of the present invention: According to steps (1) to (10) of Example 1, the precipitate obtained in step (10) is freeze-dried to obtain crude polysaccharide (i.e., crude extract after n-butanol precipitation).

[0123] Method 2 of the present invention (referred to as the present invention): According to steps (1) to (11) of Example 1, a crude polysaccharide is obtained (the crude extract after precipitation with n-butanol is purified by secondary extraction with the first low eutectic solvent).

[0124] The extraction rate, purity and maximum molecular weight of crude polysaccharides in each method are shown in Fig. 9 The results showed that the crude polysaccharide extraction rates of the methods 1 and 2 of the present invention were much higher than those of hot water extraction and ultrasonic extraction (comparison between the method 1 of the present invention and hot water extraction, p < 0.01; comparison between the method 1 of the present invention and ultrasonic extraction, p < 0.01; comparison between the method 2 of the present invention and hot water extraction, p < 0.01; comparison between the method 2 of the present invention and ultrasonic extraction, p < 0.05), and the maximum molecular weight of the crude polysaccharide of the method 2 of the present invention reached 6.3×10 3 KD (KDa), the maximum molecular weight of crude polysaccharide in method 1 of the present invention reaches 6.25×10 3 KD, which is much higher than the maximum molecular weight of crude polysaccharide extracted by hot water (1.0×10 3 The maximum molecular weight of crude polysaccharide extracted by KD and ultrasound is 1.7×10 3KD, indicating that the method of the present invention has the least destructive effect on polysaccharide extraction. The purity of crude polysaccharides obtained by the methods 1 and 2 of the present invention is significantly higher than that of conventional hot water extraction and ultrasonic extraction (comparison between the method 1 of the present invention and hot water extraction, p < 0.01; comparison between the method 1 of the present invention and ultrasonic extraction, p < 0.05; comparison between the method 2 of the present invention and hot water extraction, p < 0.001; comparison between the method 2 of the present invention and ultrasonic extraction, p < 0.001), among which the purity of crude polysaccharides obtained by the method 2 of the present invention is the highest, reaching 63.49%.

[0125] The HPLC monosaccharide compositions of crude polysaccharides extracted from Indocalamus chinensis leaves by different methods after derivatization were compared and analyzed. Fig.10 , showing that: the HPLC monosaccharide composition of crude polysaccharide after derivatization by method 2 of the present invention is not significantly different from the HPLC monosaccharide composition of crude polysaccharide after derivatization by conventional hot water extraction and the HPLC monosaccharide composition of crude polysaccharide after derivatization by ultrasonic extraction. The HPLC monosaccharide composition of crude polysaccharide after derivatization by method 2 of the present invention is basically the same as the HPLC monosaccharide composition of crude polysaccharide after derivatization by method 1 of the present invention. The maximum molecular weight of crude polysaccharide obtained by the present invention is much greater than that of crude polysaccharide obtained by hot water extraction and ultrasonic extraction, indicating that the method of the present invention has the least destructive effect on polysaccharide. From the perspective of monosaccharide composition, the monosaccharide composition of crude polysaccharides obtained by different extraction methods is mainly composed of three monosaccharides: Gal (galactose), Glc (glucose) and Ara (arabinose), and the difference is not large. However, the GalUA (galacturonic acid) content in the crude polysaccharides obtained by the method of the present invention is significantly higher than that in the crude polysaccharides obtained by the other two methods (hot water extraction and ultrasonic extraction) (compared with hot water extraction, p < 0.05; compared with ultrasonic extraction, p < 0.05), the proportion of GalUA in the crude polysaccharides of the present invention is 4.74%, while the proportions of GalUA in the crude polysaccharides obtained by ultrasonic extraction and hot water extraction are 1.18% and 0.82%, respectively.

[0126] 3. Activity analysis of crude polysaccharides extracted by this method

[0127] Vc and crude polysaccharides obtained by different extraction methods were prepared into solutions of different concentrations with water, and vitamin C (Vc) was used as a control. The DPPH free radical scavenging and ABTS activities of the crude polysaccharides were measured by spectrophotometry. + The free radical scavenging ability was determined and the results are shown in Fig.11 and Fig.12 , showing that the crude polysaccharide obtained by the present invention has higher activity than the crude polysaccharide extracted by conventional hot water and ultrasonic extraction (DPPH free radical scavenging ability: concentration 0.75mg / mL-1.5mg / mL, the present invention is compared with hot water extraction, p < 0.05, the present invention is compared with ultrasonic extraction, p < 0.05; ABTS +Free radical scavenging ability: at a concentration of 0.5 mg / mL or above, the present invention is compared with hot water extraction, p < 0.05; the present invention is compared with ultrasonic extraction, p < 0.05).

[0128] 4. Selection of a low eutectic solvent for secondary extraction of flavonoids from the third n-butanol phase concentrate

[0129] Choline chloride is used as a hydrogen bond acceptor, urea, acetic acid, propionic acid and other 12 reagents are used as hydrogen bond donors, water is added and mixed at 80°C, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:2, and the weight percentage of water is 15%, forming a second low eutectic solvent for secondary extraction of crude flavonoids from the third n-butanol phase concentrate. Except for step (12) of "concentrating and desolventizing the third n-butanol phase to obtain a concentrate, adding a second low eutectic solvent in an amount 10 times the volume of the concentrate to the concentrate, ultrasonically extracting for 10 minutes, and centrifuging to separate the extract", the remaining operations are the same as in Example 1, and the various extracts obtained using different second low eutectic solvents are tested for flavonoid extraction rate and purity. The research results are shown in Fig.13 The study found that choline chloride + malonic acid and choline chloride + propylene glycol had the highest extraction rates, reaching 0.53% and 0.55% respectively, which were significantly higher than other low eutectic solvents; the purity of crude flavonoids extracted by choline chloride + propylene glycol reached 42.29%, and the purity of crude flavonoids extracted by choline chloride + malonic acid reached 41.7%, which was significantly higher than other low eutectic solvents. Taking all factors into consideration, the second low eutectic solvent preferably uses choline chloride + propylene glycol (molar ratio 1:2) + 15% (weight percentage) water or choline chloride + malonic acid (molar ratio 1:2) + 15% (weight percentage) water.

[0130] 5. Comparison of crude flavonoid extraction rate and purity obtained by different methods

[0131] Method 3 of the present invention (referred to as: present invention 3): According to step (1) to step (10) of Example 1, a third n-butanol phase is obtained, and the third n-butanol phase obtained in step (10) is concentrated and desolventized to obtain a concentrate, i.e., crude flavonoids.

[0132] Method 4 of the present invention (referred to as: present invention 4): According to steps (1) to (10) of Example 1, a third n-butanol phase is obtained, and crude flavonoids are obtained according to step (12) of Example 1.

[0133] Methanol, ethanol, and 75% by mass ethanol aqueous solution: Using fresh Indocalamus japonicus leaves as raw materials, these three solvents were used as extractants, and ultrasonic extraction was carried out three times with a solid-liquid ratio of 10:1 by weight. The extracts were combined to recover the solvent, and crude flavonoids extracted with methanol, crude flavonoids extracted with ethanol, and crude flavonoids extracted with 75% by mass ethanol aqueous solution were obtained, respectively.

[0134] The extraction rate and purity of crude flavonoids by each method are shown in Fig.14 , it can be seen from the figure that the crude flavonoids obtained by the method 4 of the present invention do not have an advantage in the extraction rate. The method 4 of the present invention is similar to the ultrasonic extraction of methanol, which is lower than the extraction rate of ethanol, 75% ethanol aqueous solution and the method 3 of the present invention; however, the purity of flavonoids in the crude flavonoids of the method 4 of the present invention is significantly improved, which is significantly higher than the method 3 of the present invention (p < 0.01), the ultrasonic extraction of methanol (p < 0.001), the ultrasonic extraction of ethanol (p < 0.001), and the ultrasonic extraction of 75% ethanol aqueous solution (p < 0.001). The crude flavonoids obtained by the method 3 of the present invention do not have an advantage in the extraction rate. The method 3 of the present invention is similar to the ultrasonic extraction of ethanol, which is higher than the extraction rate of the ultrasonic extraction of methanol and the method 4 of the present invention, and slightly lower than the extraction rate of the 75% ethanol aqueous solution, but the purity of flavonoids in the crude flavonoids of the method 3 of the present invention is also significantly improved, which is significantly higher than the ultrasonic extraction of methanol (p < 0.05), the ultrasonic extraction of ethanol (p < 0.05), and the ultrasonic extraction of 75% ethanol aqueous solution (p < 0.05). The extraction rate of 75% ethanol aqueous solution is the highest, which can reach 0.69%, but the purity is the lowest, which is only 9.89%. The extraction rate of the intermediate crude extract (method 3 of the present invention) can reach 0.64%, which is slightly lower than the extraction rate of 75% ethanol water, but the purity can reach 26.55%, which is much higher than the conventional extraction method. The extraction rate of the secondary extraction of flavonoids (method 4 of the present invention) of the present invention is 0.55%, which is lower than that of method 3 of the present invention, but the purity is increased to 62.29%, and the effect is significant.

[0135] 6. Differences in components of crude flavonoids obtained by different methods

[0136] UPLCMS was used to compare the differences in flavonoid composition among the crude flavonoids obtained by method 4 of the present invention, crude flavonoids extracted with methanol, and crude flavonoids extracted with 75% ethanol aqueous solution. Fig.15 , the main flavonoid composition is listed in the figure. It is observed that the total content of flavonoid glycosides in the crude flavonoids obtained by the method of the present invention is higher than the total content of flavonoid glycosides in the crude flavonoids extracted by methanol, and is similar to the total content of flavonoid glycosides in the crude flavonoids extracted by 75% ethanol, but the content of various flavonoid glycosides is also different. Among the flavonoid glycosides in the crude flavonoids extracted by the present invention, apigenin-7-O-neohesperidin, apigenin-8C-xylose-6C-glucoside, orientin, etc. have the highest content, and the relative content exceeds 10%; the relative content of flavonoid glycosides in the crude flavonoids extracted by methanol exceeds 10%, and the content of non-glycoside flavonoids such as vitexin in the crude flavonoids extracted by methanol is relatively high, which may be caused by the hydrolysis of glycoside flavonoids during the extraction process.

[0137] 7. Differences in the activity of crude flavonoids obtained by different extraction methods

[0138] The crude flavonoids obtained by method 4 of the present invention, the crude flavonoids extracted by methanol, and the crude flavonoids extracted by 75% ethanol aqueous solution were respectively prepared with water into 1 mg / mL concentration of flavonoid solutions, and the scavenging abilities of DPPH, superoxide anion and hydroxyl free radical were compared. The results are shown in Table 1. Fig.16 .from Fig.16 It can be seen that the crude flavonoids obtained by the present invention have significantly higher activity than the other two (crude flavonoids extracted with methanol and crude flavonoids extracted with 75% ethanol aqueous solution) at the same concentration (DPPH scavenging ability: p<0.01 for the crude flavonoids extracted by the present invention compared with the crude flavonoids extracted with methanol, and p<0.05 for the crude flavonoids extracted by the present invention compared with the crude flavonoids extracted with 75% ethanol aqueous solution; hydroxyl radical scavenging ability: p<0.01 for the crude flavonoids extracted by the present invention compared with the crude flavonoids extracted with methanol, and p<0.05 for the crude flavonoids extracted by the present invention compared with the crude flavonoids extracted with 75% ethanol aqueous solution); mass spectrometry analysis found that in addition to flavonoids, the crude flavonoids also contain some active ingredients such as phenolic acid and phenylpropanoids, as well as impurities such as protein, sugar, cellulose, and pigment, which will affect the activity.

[0139] The change of parameters in the preparation method of the present invention, such as the change of the amount of water added, does not affect the preparation of essential oils, crude polysaccharides, and crude flavonoids, so any combination of parameters in the preparation method of the present invention can achieve the preparation of essential oils, crude polysaccharides, and crude flavonoids. They are not listed one by one here.

Claims

1. A stepwise preparation method of flavonoid polysaccharide from indocalamus leaf essential oil, characterized in that: The method uses fresh indigo bamboo leaves as raw materials, and comprises the following steps: (1) adding water to the raw materials to make pulp to obtain a slurry; (2) adding water to the slurry, grinding at a temperature greater than 0° C. and less than or equal to 20° C. until the particle size is less than or equal to 500 meshes, to obtain a grinding liquid; (3) subjecting the grinding liquid to ultrasonic oscillation, and then centrifuging at 8000 rpm to 12000 rpm and a temperature greater than 0° C. and less than or equal to 20° C. to separate and obtain a first liquid A1 and a first solid B1; (4) adding water to the first solid B1, and ball-milling the solid at a temperature greater than 0° C. and less than or equal to 20° C. until the particle size is less than or equal to 3000 mesh, to obtain a ball-milling liquid; (5) subjecting the ball milling liquid to ultrasonic oscillation, and then centrifuging at 8000 rpm to 12000 rpm and a temperature greater than 0° C. and less than or equal to 20° C. to separate and obtain a second liquid A2 and a second solid B2; (6) adding water to the second solid B2, stirring for 3 min-10 min, then ultrasonically oscillating, and then centrifuging at 8000 rpm-12000 rpm and a temperature greater than 0° C. and less than or equal to 20° C. to separate and obtain a third liquid A3 and a third solid B3; (7) combining the first liquid A1, the second liquid A2 and the third liquid A3 to obtain an extract, adding an inorganic salt to the extract, slowly stirring at a speed of 30 r / min-240 r / min at a temperature greater than 0° C. and less than or equal to 20° C., standing to separate the essential oil and the first aqueous phase after oil-water separation, and obtaining the essential oil; (8) concentrating the first aqueous phase to within 5 times the weight of the raw material under a pressure of 1 kPa-1.2 kPa and a temperature of 25° C.-30° C. to obtain aqueous solution C; (9) adding petroleum ether to aqueous solution C, mixing evenly, standing to separate layers, and separating to obtain a second aqueous phase and petroleum ether containing a pigment; (10) adding n-butanol to the second aqueous phase, mixing evenly and standing to separate, separating to obtain a third aqueous phase and a first n-butanol phase, repeatedly adding n-butanol to the third aqueous phase, mixing evenly and standing to separate, separating to obtain a precipitate, a fourth aqueous phase and a second n-butanol phase, and combining the first n-butanol phase and the second n-butanol phase to obtain a third n-butanol phase; (11) freeze-drying the precipitate obtained in step (10) to obtain crude polysaccharide; (12) Concentrating and removing the solvent from the third n-butanol phase obtained in step (10) to obtain a concentrate and crude flavonoids.

2. The preparation method according to claim 1, characterized in that: In step (1), the weight ratio of the raw material to water is 1:1-3; Alternatively, in step (2), the weight ratio of the slurry to water is 1:0.5-5; Alternatively, in step (4), the weight ratio of the first solid B1 to water is 1:0.5-5; Alternatively, in step (6), the weight ratio of the second solid B2 to water is 1:0.5-5.

3. The preparation method according to claim 1, characterized in that: In step (3), the ultrasonic oscillation conditions are: ultrasonic oscillation for 5 min to 10 min at a frequency of 20 KHz to 30 KHz and an oscillation-off ratio of 5 s:1 s; Alternatively, in step (5), the ultrasonic oscillation conditions are: ultrasonic oscillation for 5 min to 10 min at a frequency of 40 KHz to 50 KHz and an oscillation-off ratio of 5 s:1 s; Alternatively, in step (6), the ultrasonic oscillation conditions are: ultrasonic oscillation for 20 minutes to 30 minutes at a frequency of 40KHz to 50KHz and an oscillation-off ratio of 5s:1s.

4. The preparation method according to claim 1, characterized in that: In step (7), the weight of the inorganic salt is 3%-8% of the weight of the extract; the inorganic salt is selected from sodium chloride and potassium chloride.

5. The preparation method according to claim 1, characterized in that: In step (9), petroleum ether is added to aqueous solution C three times, each time the amount of petroleum ether added is 5%-10% of the weight of aqueous solution C, mixed evenly, allowed to stand for stratification, the aqueous phase and the petroleum ether containing the pigment are separated, and the aqueous phases obtained by the three separations are combined to obtain a second aqueous phase.

6. The preparation method according to claim 1, characterized in that: In step (10), n-butanol in an amount of 2 to 4 times the weight of the second aqueous phase is added to the second aqueous phase; and n-butanol in an amount of 2 to 4 times the weight of the third aqueous phase is repeatedly added to the third aqueous phase.

7. The preparation method according to claim 1, characterized in that: In step (11), a first low eutectic solvent in an amount of 20 to 22 times the mass of the precipitate is added to the precipitate obtained in step (10), ultrasonic extraction is performed for 10 to 20 minutes, ethanol is added to the extract, the mixture is mixed evenly and then allowed to stand for precipitation, and the precipitate is freeze-dried to obtain a crude polysaccharide.

8. The preparation method according to claim 7, characterized in that: In step (11), the first deep eutectic solvent consists of water, choline chloride and malonic acid or consists of water, choline chloride and succinic acid, wherein the molar ratio of choline chloride to malonic acid or succinic acid is 1:2, and the weight percentage of water is 10%.

9. The preparation method according to claim 1, characterized in that: In step (12), the third n-butanol phase obtained in step (10) is concentrated and desolventized to obtain a concentrate, and a second low eutectic solvent in an amount of 10 times the weight of the concentrate is added to the concentrate, and ultrasonic extraction is performed for 10 min-15 min, and a macroporous resin AB-8 in an amount of more than 10% by weight of the extract is added to the extract, and the mixture is stirred for adsorption, vacuum filtered, and the macroporous resin AB-8 is eluted with ethanol, and the eluate is concentrated to obtain crude flavonoids.

10. The preparation method according to claim 9, characterized in that: In step (12), the second low eutectic solvent consists of water, choline chloride and propylene glycol or consists of water, choline chloride and malonic acid, wherein the molar ratio of choline chloride to propylene glycol or malonic acid is 1:2, and the weight percentage of water is 15%.

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