Method for simultaneously preparing essential oil and total flavonoids of Yingghe citrus

Abstract

The invention particularly relates to a method for simultaneously preparing essential oil and total flavonoids of Yingghe citrus. Citrus essential oil is extracted from Yinghe citrus by adopting a reduced pressure distillation method, and by-products are citrus residues and a water extract of the citrus essential oil; extracting the citrus pulp with ethanol water to obtain a flavone extracting solution; combining with a water extract and a concentrated solution of the byproduct citrus essential oil, adsorbing through a resin column, and collecting effluent; washing the resin column with water and collecting water eluate; adding low-concentration ethanol to continuously wash the resin column, collecting eluent, combining the eluent with the water eluent and the effluent, and concentrating and drying to obtain a citrus flavone product I; and adding high-concentration ethanol into the resin column eluted by the low-concentration ethanol for elution, collecting the eluent, concentrating and recovering the ethanol, and drying the concentrated solution to obtain a citrus flavone product II. The method for analyzing the citrus flavone product II through HPLC is established, the accuracy is high, essential oil and flavone products are prepared through a one-step method, the utilization rate of citrus is greatly increased, the technology is green and environmentally friendly, and the market prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant active ingredient extraction, and specifically relates to a method for simultaneously preparing Yinhewan orange essential oil and total flavonoids. BACKGROUND

[0002] Yinhewan is located in the southeast corner of Huzhuapu Town, Gushi County, Xinyang, and is close to the Guan River, a tributary of the Huaihe River. Orange trees have been planted for nearly 300 years, and Yinhewan is known as the "Central Plains Orange Village". Yinhewan oranges are unique due to their special geographical location and excellent geographical environment.

[0003] During the processing of oranges, the main by-product is peel residue (remaining material after juice removal), which accounts for 20-40% of the total mass of oranges. However, the utilization rate of orange peel is currently low, and most of it is discarded as waste, not only resulting in low comprehensive utilization of resources, but also causing environmental pollution, except for a small part used to make food, extract essential oils, pectin, and flavonoids.

[0004] Orange essential oil is mainly distributed in oil sacs (oil cells) in the outer pericarp. Oil sacs are small round dots visible to the naked eye on the surface of the orange pericarp, and they are organs that secrete and store essential oils. Lightly squeezing the pericarp, the aromatic oil mist sprayed is from here. The secondary distribution location is flowers and leaves, but the essential oil used for extraction is mainly from the pericarp. Flavonoids in oranges are mainly divided into two categories: flavonoid glycosides and polymethoxyflavones (PMFs), and their distribution locations are different. Oranges also contain an important component, flavonoids, which include two main categories: flavonoid glycosides and polymethyl flavones; flavonoid glycosides include hesperidin and naringin, and are mainly distributed in the pericarp (white layer of the mesocarp) and the pulp (sarcocarp), especially the white layer, which contains the highest content. This is why the white part of the inner layer of the orange pericarp tastes bitter, because it is rich in bitter flavonoids such as naringin and hesperidin. Polymethoxyflavones (PMFs), such as nobiletin and tangeretin, are mainly distributed in the pericarp (exocarp). They are usually coexisting with essential oils in oil sacs or on the surface of the pericarp. Orange essential oil is used in the food industry to improve the flavor and quality of products, and also has medicinal value. Flavonoids also have the effects of expanding coronary blood vessels, reducing capillary permeability, antioxidant, anti-inflammatory, anti-allergic, antibacterial, and antiviral effects, but the existing extraction process has low raw material utilization rate and high cost.

[0005] Patent CN 105213560A discloses a method for efficiently extracting flavonoids from orange peel. The orange peel is crushed and extracted with ethanol, then filtered and extracted after ultrasonic extraction, to obtain flavonoid substances. This process uses organic solvents, which causes VOC pollution, and the essential oils and other components in the orange peel are not extracted, resulting in low utilization rate of raw materials.

[0006] Patent CN 114874853 A discloses a citrus-based essential oil extraction method, including the following steps: using a cold press machine to cold press citrus peels to obtain crude citrus essential oil; using a washing and filtering integrated device to filter and wash the crude citrus essential oil to obtain filtrate and filter residue, mixing and standing until oil and water are layered to obtain refined citrus essential oil; and refining the essential oil to remove terpene to improve stability. The generated waste residue and waste liquid are not fully utilized, causing great waste of resources.

[0007] If a process for simultaneously extracting citrus essential oil and total flavonoids from citrus can be developed, the added value of Yinghe citrus industry can be greatly improved. In addition, the existing technology for detecting citrus flavonoids is not clear or the operation is complex, and a simple, accurate and effective detection method is urgently needed. SUMMARY

[0008] In view of the problem of waste of effective components in the existing technology, the present application provides an integrated process: a method for simultaneously preparing Yinghe citrus essential oil and total flavonoids. The utilization rate of raw materials is improved, and various effective components in citrus peels, such as essential oil and flavonoids, are effectively extracted. The process is green and environmentally friendly, and has strong practicality.

[0009] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0010] A method for simultaneously preparing Yinghe citrus essential oil and total flavonoids, comprising the following steps:

[0011] (1) crushing Yinghe citrus (whole fruit) and mixing with water, and extracting by vacuum distillation, and separating the distillate to obtain citrus essential oil, and the by-products are citrus residue and water extract of citrus essential oil;

[0012] (2) mixing the by-product citrus residue with an ethanol water extraction solution, extracting, filtering to obtain a flavonoid extract solution; concentrating and recovering ethanol, and the concentrated solution is reserved;

[0013] (3) combining the water extract of the by-product citrus essential oil and the concentrated solution of step (2), and adsorbing through a resin column to collect the effluent; washing the resin column with water to collect the water eluate;

[0014] (4) adding low-concentration ethanol with a concentration of 10-30% (v / v) to the washed resin column, eluting, collecting the eluate, and combining it with the water eluate and effluent of step (3), and concentrating and drying to obtain a citrus flavonoid product I with hesperidin as the main component;

[0015] (5) adding high-concentration ethanol with a concentration of 50-90% (v / v) to the resin column eluted with low-concentration ethanol, eluting, recovering ethanol by vacuum concentration, and drying the concentrated solution to obtain a citrus flavonoid product II with hesperidin, nobiletin and tangeretin as the main components.

[0016] Further, the volume ratio of the Yinhewan citrus to water in step (1) is 1:10-14; the vacuum degree of the reduced pressure distillation is -0.06 MPa to -0.09 MPa, the temperature is 40-60°C, and the time is 0.5-3 h.

[0017] Further, the volume concentration of ethanol in the ethanol water extraction solution in step (2) is 50-95% (v / v), the material ratio of the citrus residue to the ethanol water extraction solution is 1:10-30, and the reflux extraction time is 0.5-3 h;

[0018] or the microwave extraction time is 5-10 min, and the microwave extraction power is 500-700 W;

[0019] or the ultrasonic extraction time is 20-60 min, and the ultrasonic power is 100-300 W.

[0020] Further, in step (3), the resin column is a single or multiple resin column in series, the resin type is macroporous adsorption resin, and the resin is D101; the resin to citrus (the mass of the fruit, i.e., the mass of the medicinal material) dosage ratio is 1:1-10:1 (w / w), and the resin column height to diameter ratio is 5:1-30:1; the adsorption temperature is 20-40°C.

[0021] Further, in step (3), the adsorption flow rate is 0.5-1 BV / h; and the water flushing resin column flow rate is 1-3 BV.

[0022] Further, in steps (4) and (5), the low concentration ethanol and high concentration ethanol flushing resin column flow rates are each independently 1-5 BV, the eluent flow rate is 1-2 BV / h; the elution process is isocratic elution or gradient elution; and the elution temperature is 25-60°C.

[0023] Further, in step (1), the main component of the citrus essential oil is D-limonene, and the content is not less than 80%; in step (4), the content of hesperidin in the citrus flavonoid product I is 1-10%; and in step (5), the content of hesperidin, nobiletin, and tangeretin in the obtained citrus flavonoid product II is 20-50%.

[0024] Further, a method for determining the contents of hesperidin, nobiletin, and tangeretin prepared by the above method using HPLC, the determination method comprising the following steps:

[0025] 1) standard control solution preparation: precisely weighing hesperidin, nobiletin, and tangeretin standard control substances, adding 50% methanol to prepare a hesperidin, nobiletin, and tangeretin mixed control solution, and reserving;

[0026] 2) Preparation of sample solution: accurately weigh the citrus flavonoids product I and citrus flavonoids product II to be tested, respectively, and add 50% methanol by volume to ultrasonically dissolve, add methanol to make up the weight, take the supernatant through a 0.45 μm microporous filter membrane, and reserve for use;

[0027] 3) Chromatographic conditions: the chromatographic column is 4.6*250mm, 3 μm, Omega Polar 100-5-C18 chromatographic column, the mobile phase is acetonitrile (A)-water (B), the flow rate is 0.6 mL·min -1 ; the column temperature is 35℃; the detection wavelength is 330nm, and analysis is carried out;

[0028] 4) Record the peak area, and calculate the contents of hesperidin, nobiletin and tangeretin in the citrus flavonoids product I and the citrus flavonoids product II according to the standard curve obtained from the control sample solution prepared in step a).

[0029] Further, the gradient elution program of the mobile phase in step 3) is as follows:

[0030] Time / min Acetonitrile (A) % Water (B) % 0-5 23 77 5-10 23→25 77→75 10-25 25→48 75→52 25-30 48→68 52→32 30-40 68→75 32→25 .

[0031] Further, the concentrations of the hesperidin, nobiletin and tangeretin control sample solutions in step 1) are 87.0 μg / mL, 22.5 μg / mL and 12.0 μg / mL, respectively; and the concentrations of the sample citrus flavonoids product I and the citrus flavonoids product II in step 2) are independently 10-200 μg / mL.

[0032] The beneficial effects of the present application are as follows:

[0033] 1. The present application provides a method for simultaneously preparing Yinhai citrus essential oil and total flavonoids, which utilizes citrus resources through multiple processes, and extracts essential oil and total flavonoids simultaneously, thereby being highly efficient.

[0034] 2. The present application uses reduced pressure distillation to prepare essential oil, which improves the extraction efficiency of essential oil while not damaging heat-sensitive components in the essential oil. The boiling point is reduced by adjustable vacuum degree, and the essential oil is obtained by passing through an oil-water separation system, and the waste residue and waste liquid after extraction of essential oil are extracted again, and flavonoids are obtained by chromatographic column separation, and crude flavonoids and pure flavonoids are effectively separated.

[0035] 3, the relative content of D-limonene in the essential oil obtained by the application is 84.83%, the main component of the citrus flavone product I is hesperidin, and the content is 12.7mg / g, the main components of the citrus flavone product II are hesperidin, nobiletin and tangeretin, and the contents are 16.9mg / g, 120mg / g and 67mg / g respectively; The content of the essential oil and flavone product prepared by the method of the application is high, and the subsequent development of flavone health care products is facilitated.

[0036] 4, the application simultaneously determines three main components (hesperidin, nobiletin and tangeretin) in the total flavone extracted from Yinghe citrus by HPLC, the sample pretreatment of this method is simple, the cost is low, the separation degree between each other is good, and baseline separation is achieved with other components, the recovery rate of the three total flavone substances is between 98.6% and 101.85%, the detection method of the total flavone (hesperidin, nobiletin and tangeretin) extracted from Yinghe citrus has high reliability and small systematic error; The RSD% of the precision, stability and repeatability of the test method is between 1.09 and 1.51, which also proves that the detection method of the application has high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creating any creative labor.

[0038] Figure 1 The process flow chart for simultaneously preparing Yinghe citrus essential oil and total flavone of the application.

[0039] Figure 2 The high performance liquid chromatogram of the mixed reference substance (A) and the flavone product II (B) of example 1; wherein, Figure 2 A in the above formula (1) represents the high performance liquid chromatogram of the mixed reference substance; Figure 2 B in the above formula (2) represents the high performance liquid chromatogram of the sample.

[0040] Figure 3 The linear relationship diagram of hesperidin.

[0041] Figure 4 The linear relationship diagram of nobiletin.

[0042] Figure 5 The linear relationship diagram of tangeretin.

[0043] Figure 6 The high performance liquid chromatogram of the determination of the citrus flavone product I.

[0044] Figure 7High performance liquid chromatogram determined for citrus flavonoid product II. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort belong to the scope of protection of the present application.

[0046] The medicinal material used in the present application is the fruit of Citrus reticulata Blanco in 2024, which is identified as the mature fruit of Citrus reticulata Blanco and its cultivated varieties.

[0047] 1. Materials and reagents

[0048] Table 1. Materials and reagents

[0049] Name Name Batch No. / Specification Manufacturer Yinghe citrus peel — Henan Beigujiangnan Agricultural Technology Co., Ltd. Hesperidin B20182 / Reference substance Shanghai Yuan Ye Biological Technology Co., Ltd. Nobiletin B20199 / Reference substance Shanghai Yuan Ye Biological Technology Co., Ltd. Tangeritin B20646 / Reference substance Shanghai Yuan Ye Biological Technology Co., Ltd. Methanol Analytical pure Zhengzhou Panyi Chemical Reagent Factory Methanol Chromatographic pure Fisher Company Acetonitrile Chromatographic pure Fisher Company Anhydrous ethanol Analytical pure Zhengzhou Panyi Chemical Reagent Factory 95% ethanol Analytical pure Zhengzhou Panyi Chemical Reagent Factory Ash-free filter paper 16946859 Shanghai Weixi Biological Technology Co., Ltd.

[0050] 2. Experimental instruments

[0051] Table 2. Experimental instruments

[0052] Name Model Manufacturer High-performance liquid chromatograph Waters 2996 Waters Technology Co., Ltd. Gas chromatography-mass spectrometer TRACE 1310-ISQ 7000 Thermo Fisher Scientific Ultrasonic cleaner KQ-500DE Kunshan Ultrasonic Instrument Co., Ltd. High-speed traditional Chinese medicine grinder LG-01 Rui'an Bai Xin Pharmaceutical Machinery Co., Ltd. Electric heating constant-temperature water bath DKZW-type Beijing Yongguangming Medical Instrument Co., Ltd. Temperature-regulating electric heating jacket ZNHW-type Shanghai Yingdi Instrument and Equipment Co., Ltd. Rotary evaporator RE-205-type Shanghai Kan Kun Instrument and Equipment Co., Ltd. Pure water machine UPT-60-type Sichuan Youpu Super Pure Technology Co., Ltd. Refrigerator BCD-555WKM Hefei Midea Refrigerator Co., Ltd. Electronic balance (0.0001 g) BS124S-type Sartorius Gmbh Electronic balance (0.001 g) Secura 613-type Sartorius Gmbh Electric heating air-drying oven 101-3B Zhejiang Lichen Instrument Technology Co., Ltd. Juicer QMZ-PBJ-02-type Foshan Hansenray Electrical Appliance Co., Ltd.

[0053] Example 1

[0054] The present embodiment provides a method for simultaneously preparing Citrus reticulata Blanco essential oil and total flavonoids, and the process flow is as shown in Figure 1 The preparation steps are as follows:

[0055] (1) 50 g of fresh Citrus reticulata Blanco was used as raw material, crushed by a juicer (all were reserved for extraction), and then loaded into a single-neck flask containing 12 times the amount of water. A volatile oil tester, a condenser tube and a vacuum device were connected, and the vacuum degree was adjusted to liquid boiling by a vacuum system cock valve at a certain temperature. The vacuum degree of the reduced pressure distillation was-0.08 MPa, the temperature was 45℃, the time was 1 h, and the reflux boiling was maintained for a certain time until the oil amount in the extractor no longer increased, i.e. the extraction was stopped. The lower distillation water was removed by a separatory funnel to obtain an oil-like essential oil crude extract, which was sealed and stored in a 4℃ refrigerator for detection;

[0056] (2) The water extract of the extracted Citrus reticulata Blanco was filtered and concentrated for standby use; the remaining Citrus reticulata Blanco residue was added with 20 times the volume of 50% ethanol aqueous solution, and reflux extracted in an electric heating jacket for 2 h. The extract was concentrated under reduced pressure, ethanol was recovered, and the concentrated solution was standby used. An appropriate amount of the concentrated solution was added with water to make the extract liquid clear, and the water extract and the clear liquid were combined for separation and purification on a resin column;

[0057] (3) Weigh 100 g of pretreated D101 macroporous adsorption resin, and wet fill a glass column with an inner diameter of 4 cm and a column height of 30 cm. Citrus extract liquid flows through the resin column for adsorption, the flow rate is 0.5 BV / h, the temperature of the resin column is 30 DEG C, and the effluent is collected. 2 BV of pure water is added to the adsorbed resin column at a flow rate of 1 BV / h, and the water eluent is collected. Then, 20% ethanol aqueous solution (v / v) is used to elute the resin column, the elution temperature is 30 DEG C, the flow rate is 1 BV / h, and the eluent amount is 4 BV. The eluent, the effluent, and the water eluent are combined, concentrated under reduced pressure, and dried under normal pressure to obtain citrus flavone product I for detection. Finally, 80% ethanol aqueous solution (v / v) is used to elute the resin, the elution temperature is 30 DEG C, the flow rate is 1 BV / h, and the eluent amount is 5 BV. The eluent is collected, concentrated under reduced pressure to recover ethanol, and dried to obtain yellow citrus flavone product II for detection.

[0058] 2. GC-MC determination of essential oil components

[0059] (1) Preparation of sample solution for injection

[0060] The determination method of the essential oil of the present application adopts GC-MS analysis method, 50 μL of essential oil product is taken, dissolved in anhydrous ethanol, and diluted to 10 mL in a volumetric flask. After shaking, anhydrous sodium sulfate is added, and the solution is filtered through a 0.22 μm organic microporous filter. The filtrate is taken into an injection vial to obtain the sample for injection.

[0061] (2) Gas chromatography conditions

[0062] Programmed temperature rising: the initial column temperature is 10 DEG C, which is raised to 160 DEG C at a speed of 10 DEG C / min and maintained for 5 min, and then raised to 280 DEG C at a speed of 10 DEG C / min and maintained for 5 min; the carrier gas used is high-purity helium; the column flow rate is 1.0 mL / min; the injection amount is 5.0 μL, and no split is performed;

[0063] (3) Mass spectrometry conditions

[0064] EI ion source, electron energy 70 eV, scanning range 33-350 amu, ion source temperature 220 DEG C, and transfer line temperature 280 DEG C. 5.0 μL of the test sample solution is injected into the GC-MS instrument, and run for 50 min to record the chromatogram. The chemical components of the citrus essential oil are analyzed by gas chromatography-mass spectrometry (GC-MS) method to obtain the total ion current chromatogram.

[0065] (4) Experimental results and analysis

[0066] The essential oil obtained by extraction is detected by GC-MS according to the GC-MS analysis methods of items (2) and (3), and compared with the standard NIST spectral library to obtain the chemical components and their normalized contents in the essential oil as shown in Table 3.

[0067] Table 3 GC-MS analysis results of citrus essential oil

[0068]

[0069] As can be seen from Table 3, the citrus essential oil mainly contains D-limonene and γ-terpinene, and the content of D-limonene is the highest, accounting for 84.83%, which can be used for the development of essential oil health care products in subsequent.

[0070] 3. Determination of citrus flavone content by HPLC method

[0071] (1) Preparation of test sample

[0072] Preparation of reference solution: accurately weigh a certain amount of hesperidin, nobiletin and tangeretin reference substances, and add chromatographic methanol to prepare a mixed reference solution of hesperidin, nobiletin and tangeretin with concentrations of 87.0 μg / mL, 22.5 μg / mL and 12.0 μg / mL respectively, and reserve.

[0073] Preparation of sample solution: accurately weigh about 20 mg of each of the citrus flavone products I and II (passed through a 60-mesh sieve) to be tested in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh, ultrasonically dissolve for 0.5 h, weigh, add methanol to make up the weight, shake well, stand, and then pass the supernatant through a 0.45 μm microporous filter membrane to obtain the sample solution.

[0074] (2) Content determination

[0075] The determination method of total flavones of the present application adopts high performance liquid chromatography, and the chromatographic column is OmegaPolar100-5-C18 chromatographic column (4.6x250mm, 3μm); mobile phase: acetonitrile (A)-water (B), gradient elution (0-5min, 23% A; 5-10min, 23% A→25% A; 10-25min, 25% A→48% A; 25-30min, 48% A→68% A; 30-40min, 68% A→75% A); flow rate: 0.6mL·min-1; column temperature: 35℃; detection wavelength: 330nm; running time: 40min.

[0076] Under this chromatographic condition, hesperidin, nobiletin and tangeretin are well separated from each other and baseline separated from other components, as shown in Figure 2 .

[0077] (3) Linear relationship investigation

[0078] Accurately pipette 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 mL of the mixed control sample stock solution prepared in (1) into 10 mL volumetric flasks, dilute to the mark with methanol, and mix well. Pipette appropriate amounts of the above series of solutions into 10 mL volumetric flasks, and dilute to the mark with methanol. Analyze the samples by HPLC under the conditions in (2). Regress the mass concentration (X, μg / mL) of the three components on the peak area (Y) to obtain the linear equation. The results are shown in Table 4. Figure 3 , Figure 4 , Figure 5 Table 4. Linear relationship test

[0079] Table 4. Linear relationship test

[0080] Compound Regression equation r Linear range (μg / mL) Hesperidin Y = 430889.4839X - 6983.1404 0.9997 17.4～348.0 Nobiletin Y = 5128242.4795X - 28148.9438 0.9999 4.5～90.0 Tangeritin Y = 5237661.9162X - 20872.5779 0.9998 2.4～48.0

[0081] (4) Precision test

[0082] Accurately pipette the same sample solution, and continuously inject the sample 6 times under the conditions in (2). The RSD of the peak area is shown in Table 5. The results show that the precision of the instrument is good.

[0083] (5) Stability test

[0084] Accurately pipette the same sample solution, and continuously inject the sample 6 times under the conditions in (2). The RSD of the peak area is shown in Table 5. The results show that the precision of the instrument is good.

[0085] (6) Reproducibility test

[0086] Accurately weigh the same batch of citrus flavone product II, and prepare a sample solution. Inject the sample under the conditions in (2), and record the peak area. The average content of the three components (hesperidin, nobiletin and tangeretin) is 16.88 mg / g, 120.62 mg / g and 67.32 mg / g, respectively, and the RSD is less than 3.0%, indicating that the reproducibility of the method is good. The RSD of the content is shown in Table 5.

[0087] Table 5. Precision, stability and reproducibility test results of the three components in citrus flavone product II

[0088] Compound Precision RSD (%) Stability RSD (%) Repeatability RSD (%) Hesperidin 1.13 1.34 1.51 Nobiletin 1.25 1.47 1.46 Tangeritin 1.43 1.36 1.09

[0089] (7) Recovery test

[0090] Take about 0.02 g of the known content of citrus flavonoid product II powder (hesperidin content of 16.88 mg / g, nobiletin of 120.62 mg / g, and tangeretin of 67.32 mg / g), 6 portions in total, accurately weigh and place in a 25 mL conical flask with a stopper, accurately transfer 10.0 mL of methanol into each, and then accurately add 0.5 mL of the control solution with a concentration of 870.0 μg / mL of hesperidin, 5.0 mL of the control solution with a concentration of 450.0 μg / mL of nobiletin, and 5.0 mL of the control solution with a concentration of 240.0 μg / mL of tangeretin, respectively. Prepare the sample solution according to the preparation method under the conditions of item (1). Perform the determination according to the conditions under item (2), and calculate the average recovery rate and RSD of each component to be determined, as shown in Table 6. The average recovery rate of the three components to be determined is 98.67% to 101.85%, and the RSD is less than 3.0%, indicating that the accuracy of the method is good.

[0091] Table 6: Recovery rate test of three components in citrus flavonoid product II (n = 6)

[0092]

[0093]

[0094] (8) Sample determination

[0095] Sample content determination: accurately weigh 3 portions of the citrus flavonoid products I and II to be determined, prepare the sample solution according to item (1), and analyze and determine according to the conditions under item (2), as shown in Table 7. Figure 6 Figure 7 The content is calculated, and the results are shown in Table 7.

[0096] Table 7: Determination results of hesperidin, nobiletin, and tangeretin contents in citrus flavonoid products

[0097] Sample Hesperidin content (mg / g) Nobiletin content (mg / g) Tangeritin content (mg / g) Citrus flavonoid product I-1 12.76 0 0 Citrus flavonoid product I-2 12.77 0 0 Citrus flavonoid product I-3 12.76 0 0 Citrus flavonoid product II-1 16.93 120.50 67.50 Citrus flavonoid product II-2 16.91 120.17 67.40 Citrus flavonoid product II-3 16.91 120.08 67.46

[0098] As shown in Table 7, the content of total flavonoids (hesperidin, nobiletin, and tangeretin) in citrus flavonoid product II can reach 204.62 mg / g (i.e., 20.46%), which can be used for the development of flavonoid health care products in the future.

[0099] Example 2

[0100] The present embodiment provides a method for simultaneously preparing Yinhai citrus essential oil and total flavonoids, and the preparation steps are as follows:

[0101] ​(1) Take fresh Yinhelu orange (whole fruit) 50g as raw material, crush by juicer, then put into a single neck flask containing 12 times amount of water, connect volatile oil tester, condenser and vacuum device, adjust vacuum degree to liquid boiling by vacuum system cock valve at certain temperature. The vacuum degree of reduced pressure distillation is -0.08 MPa, the temperature is 60℃, the time is about 2h, until the oil amount in the extractor does not increase, stop extraction, remove the lower distillation water with separatory funnel to obtain oil-like essential oil crude extract, seal and store in 4℃ refrigerator, detect the prepared essential oil by GC-MS method in example 1;

[0102] (2) Filter the water extract of extracted orange, concentrate, and reserve; add 30 times volume (i.e. 30 times of the original medicinal material 50g) of 70% ethanol aqueous solution to the remaining orange residue, reflux extract in an electric heating jacket for 2h, concentrate the extract under reduced pressure, recover ethanol, and reserve the concentrated solution, take an appropriate amount of concentrated solution, add water to make the extract liquid clear, combine the water extract and the clear liquid, and then separate and purify on a resin column;

[0103] (3) Take pretreated D101 macroporous adsorption resin 100g, wet fill into a glass column with inner diameter of 4cm and column height of 20cm. Orange extract flows through the resin column for adsorption, the flow rate is 0.5BV / h, the temperature of the resin column is 40℃, and the effluent is collected. Add pure water to the adsorbed resin column at a flow rate of 0.5BV / h to remove impurities, the amount of water is 1BV, and the water eluate is collected. Then elute the resin column with 20% ethanol aqueous solution (v / v), the elution temperature is 30℃, the flow rate is 5BV / h, and the amount of eluent is 4BV. Combine the eluate, effluent and water eluate, concentrate under reduced pressure, and dry under normal pressure to obtain orange flavone product I for detection. Finally, elute the resin with 90% ethanol aqueous solution (v / v), the elution temperature is 30℃, the flow rate is 1BV / h, and the amount of eluent is 5BV. Collect the eluate, concentrate under reduced pressure to recover ethanol, and dry to obtain yellow orange flavone product II for detection.

[0104] Example 3

[0105] The present embodiment provides a method for simultaneously preparing Yinhelu orange essential oil and total flavones, and the preparation steps are as follows:

[0106] (1) Take fresh Yinhelu orange 50g as raw material, crush by juicer, then put into a single neck flask containing 14 times amount of water, connect volatile oil tester, condenser and vacuum device, adjust vacuum degree to liquid boiling by vacuum system cock valve at certain temperature. The vacuum degree of reduced pressure distillation is -0.090 MPa, the temperature is 60℃, the time is about 2h, until the oil amount in the extractor does not increase, stop extraction, remove the lower distillation water with separatory funnel to obtain oil-like essential oil crude extract, seal and store in 4℃ refrigerator, detect the prepared essential oil by GC-MS method in example 1;

[0107] (2) Filter and concentrate the aqueous extract of citrus and set aside; add 20 times the volume (20 times the original 50g of medicinal material) of 95% ethanol aqueous solution to the remaining citrus residue and extract it by ultrasonic extraction in an ultrasonic water bath with a power of 300W for 40min, concentrate the extract under reduced pressure, recover the ethanol, and set aside the concentrate. Take an appropriate amount of the concentrate, add water to make the extract clear, combine the aqueous extract and the clear extract, and then separate and purify them by resin column.

[0108] (3) Weigh 100g of pretreated D101 macroporous adsorption resin and pack it into a glass column with an inner diameter of 3cm and a column height of 25cm using a wet method. Citrus extract is passed through the resin column for adsorption at a flow rate of 0.5 BV / h and a column temperature of 20℃. The eluent is collected. Pure water is added to the adsorbed resin column at a flow rate of 3 BV / h to remove impurities, and the eluent is collected. Then, the resin column is eluted with a 20% ethanol aqueous solution (v / v) at a elution temperature of 65℃, a flow rate of 2 BV / h, and an eluent volume of 4 BV. The eluent, eluent, and water eluent are combined, concentrated under reduced pressure, and dried at normal pressure to obtain citrus flavonoid product I, which is then tested. Finally, the resin is eluted with a 50% ethanol aqueous solution (v / v) at a elution temperature of 30℃, a flow rate of 1 BV / h, and an eluent volume of 5 BV. The eluent was collected, concentrated under reduced pressure to recover ethanol, and dried to obtain yellow citrus flavonoid product II, which was then tested.

[0109] Example 4

[0110] This embodiment provides a method for determining total flavonoids in Yinghe tangerines using HPLC, which is basically the same as the method for determining total flavonoids in Yinghe tangerines in Example 1. The difference lies in the comparison of the purification effects of different types of macroporous adsorption resins on total flavonoids in Yinghe tangerines in this embodiment:

[0111] Resins: Four common macroporous adsorption resins were selected: D101 (non-polar), HPD417 (weakly polar), ADS-21 (medium polar), and AB-8 (weakly polar).

[0112] Sample: Crude extract of flavonoids from Yinghe citrus prepared according to steps (1) and (2) of Example 2 (concentration uniformly 0.5 mg / mL based on hesperidin).

[0113] Method: Accurately weigh the pretreated resins and load them into the column (column bed volume 10 mL). Load 50 mL of the crude extract at a flow rate of 1 BV / h, and collect the effluent. Elute with 2 BV of water at a flow rate of 1 BV / h, and collect the water eluate (this part is combined with the effluent, corresponding to the precursor of product I). Then, sequentially elute with 4 BV of 30% ethanol and 4 BV of 80% ethanol at a flow rate of 1 BV / h, and collect the eluate of the two concentrations respectively (the 30% ethanol eluate corresponds to product I, and the 80% ethanol eluate corresponds to product II). Combine the effluent, water eluate, and 30% ethanol eluate, and concentrate and dry to obtain citrus flavone product I; concentrate and dry the 80% ethanol eluate to obtain citrus flavone product II. Determine the total flavone concentration (HPLC method, based on the total amount of hesperidin, nobiletin, and tangeretin) of each product.

[0114] Evaluation index: Calculate the adsorption rate, 80% ethanol elution rate (product II yield), and product II purity of each resin.

[0115] Total adsorption rate (%) = (1 - effluent flavone amount / sample flavone amount) x 100%

[0116] 80% ethanol elution rate (%) = (80% ethanol eluate flavone amount / total adsorbed flavone) x 100%

[0117] The results are shown in Table 8 below.

[0118] Table 8: Resin type and product purity and elution rate:

[0119]

[0120] *Purity is based on the weight percentage of total flavones in the dried solid (HPLC method).

[0121] As can be seen from Table 8, D101 is a non-polar resin made of styrene-divinylbenzene polymerization. Its skeleton is purely benzene ring and alkyl chain, and almost does not contain any polar functional groups. The main target of product II is polymethoxylated flavones (PMFs), such as nobiletin and tangeretin. These molecules are characterized by strong hydrophobicity, and they almost do not have hydrophilic groups (such as hydroxyl groups) on their structure, but are covered by multiple hydrophobic methoxyl groups (-OCH3). The non-polar characteristics of D101 resin and the most valuable strong hydrophobic active ingredients in citrus (polymethoxylated flavones PMFs) produce the strongest and most specific "hydrophobic interaction" and "π-π stacking interaction".

[0122] The surface micro-chemical environment of HPD417 is more suitable for the adsorption of polymethoxyflavones (strong hydrophobicity) such as nobiletin and tangeretin, and the hydrophobic interaction and π-π stacking effect are stronger, so the selective adsorption of the target product is better, and the purity after elution is higher. The surface of AB-8 may contain slightly more polar groups, so it adsorbs more strongly to hesperidin (containing a sugar group) which is slightly more polar, but this may also make it more difficult to elute (slightly lower desorption rate), thereby affecting the yield and purity of high-purity product II.

[0123] Comparative Examples 1-3

[0124] Comparative Examples 1-3 each provide a method for detecting hesperidin, nobiletin and tangeretin in citrus flavone products by HPLC, which differs from Example 1 in that different mobile phases and gradient elution are used, and the rest of the process is the same as in Example 1. The scheme is shown in Table 9:

[0125] Table 9: Mobile phase and elution gradient of Comparative Examples 1-3

[0126]

[0127]

[0128] Evaluation index:

[0129] Total running time (min): the time from sample injection to the end of the last peak.

[0130] Resolution (Rs): focus on the resolution (Rs1) of hesperidin and the adjacent impurity peak and the resolution (Rs2) between nobiletin and tangeretin, and Rs>1.5 is considered baseline separation.

[0131] Theoretical plate number (N): calculated based on the hesperidin peak to evaluate column efficiency and peak shape. The higher the N value, the sharper the peak and the stronger the separation ability.

[0132] Peak symmetry factor (As): evaluate peak shape, ideal value is 1.0, acceptable range is 0.9-1.2.

[0133] Column pressure (psi): record the initial pressure and maximum pressure.

[0134] The results are shown in Table 10:

[0135] Table 10: Resolution, theoretical plate number and column pressure of examples and comparative examples

[0136] Evaluation index Example 4 Comparative example 1 Comparative example 2 Comparative example 3 Total running time (min) 40 45 45 40 Rs1 (hesperidin separation degree) >2.0 >1.8 >1.9 <1.2 (not baseline separated) Rs2 (nobiletin separation degree) >2.5 >2.0 >2.3 ~1.0 (co-melting) Theoretical plate number (N) >15000 ～12000 ～14000 ～10000 Peak symmetry factor (As) 0.95-1.05 0.85-1.15 0.90-1.10 1.20-1.40 (tailing) System column pressure (psi) ～2200 ～3200 ～2300 ～2100

[0137] Comparative Example 3 has the same mobile phase as Example 4, but the elution gradient is a single linear gradient, and separation fails. The nobiletin and tangeretin peaks are severely overlapped, and hesperidin is not completely separated from the adjacent impurities. It is proved that a simple linear gradient cannot solve the separation problem of structurally similar compounds in this complex matrix.

[0138] Comparative Example 1 has the mobile phase of methanol-acid water (containing 0.1% glacial acetic acid), and although it can achieve basic separation, it has obvious disadvantages: ① the longest analysis time; ② the column pressure is significantly higher (because the viscosity of methanol is greater than that of acetonitrile), which is large to the system and the chromatographic column; ③ the peak shape and column efficiency are slightly inferior to the acetonitrile system.

[0139] Comparative Example 2 has the mobile phase of acetonitrile-acid water, which adds acid to suppress peak tailing on the basis of the present application, but uses a simpler linear gradient. The performance is close to Example 1 of the present application, but the addition of the acid modifier may bring two potential problems: ① negative impact on the service life of the chromatographic column; ② additional additives increase the instability and transfer difficulty of the method. In contrast, the "acetonitrile-water" binary simple system of the present application is more green, stable and durable.

[0140] Example 4 has the best overall performance. Within a reasonable analysis time, baseline separation (Rs>1.5) of all target compounds is achieved, and the peak shape is sharp and symmetrical (high column efficiency), and the system pressure is low. Its multi-order linear gradient program is the result of careful optimization.

[0141] In summary, the multi-order gradient elution system of "acetonitrile-water" used in Example 1 of the present application has significant and unexpected advantages in separation efficiency, analysis speed, system pressure, peak shape quality and method robustness compared with various alternative solutions. This method successfully solves the technical problem of simultaneously analyzing hesperidin (glycoside) with large polarity difference and nobiletin and tangeretin (aglycones) with weak polarity in a single chromatographic system, fully demonstrating its creativity.

[0142] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for simultaneously preparing Yinghe citrus essential oil and total flavonoids, characterized in that, Includes the following steps: (1) Yinghe tangerines were crushed and mixed with water, and extracted by vacuum distillation. The distillate was separated into oil and water to obtain tangerine essential oil. The by-products were tangerine residue and an aqueous extract of tangerine essential oil. (2) The by-product citrus pomace was mixed with the ethanol-water extraction solution for extraction. After filtration, the flavonoid extract was obtained. The ethanol was recovered by concentration and the concentrate was kept for later use. (3) Combine the aqueous extract of the by-product citrus essential oil and the concentrate from step (2), adsorb them through a resin column, and collect the eluent; rinse the resin column with water and collect the water eluent. (4) Add 10-30% (v / v) low-concentration ethanol to the resin column after water washing, elute, collect the eluent, and combine it with the water eluent and effluent from step (3), concentrate and dry to obtain citrus flavonoid product I with hesperidin as the main component. (5) Add 50-90% (v / v) high-concentration ethanol to the resin column after elution with low-concentration ethanol for elution, collect the eluent, concentrate under reduced pressure to recover ethanol, and dry the concentrate to obtain citrus flavonoid product II with hesperidin, norihesperidin and tangeretin as the main components.

2. The method for simultaneously preparing Yinghe citrus essential oil and total flavonoids according to claim 1, characterized in that: In step (1), the volume ratio of Yinghe tangerines to water is 1:10-14; the vacuum degree of vacuum distillation is -0.06MPa to -0.09MPa, the temperature is 40-60℃, and the time is 0.5-3h.

3. The method for simultaneously preparing Yinghe citrus essential oil and total flavonoids according to claim 1 or 2, characterized in that: In step (2), the volume concentration of ethanol in the ethanol-water extraction solution is 50-95% (v / v); the material ratio of citrus pomace to ethanol-water extraction solution is 1:10-30, and the extraction is carried out by reflux for 0.5-3 hours. Alternatively, microwave extraction can be performed for 5–10 minutes at a power of 500–700W. Alternatively, extract using ultrasound for 20–60 minutes at an ultrasound power of 100–300W.

4. The method for simultaneously preparing Yinghe citrus essential oil and total flavonoids according to claim 1, characterized in that: In step (3), the resin column is a single column or multiple resin columns connected in series. The resin type is macroporous adsorption resin D101. The mass ratio of resin to citrus is 1:1 to 10:1 (w / w), the height-to-diameter ratio of the resin column is 5:1 to 30:1, and the adsorption temperature is 20-40℃.

5. The method for simultaneously preparing Yinghe citrus essential oil and total flavonoids according to claim 1, characterized in that: In step (3), the adsorption flow rate is 0.5-1 BV / h; the flow rate of water rinsing the resin column is 1-3 BV.

6. The method for simultaneously preparing Yinghe citrus essential oil and total flavonoids according to claim 1, characterized in that: In steps (4) and (5), the flow rates of low-concentration ethanol and high-concentration ethanol rinsing the resin column are each 1-5 BV, and the eluent flow rate is 1-2 BV / h; the elution process is isocratic elution or gradient elution; the elution temperature is 25-60℃.

7. The method for simultaneously preparing Yinghe citrus essential oil and total flavonoids according to claim 1, characterized in that: The main component of the citrus essential oil in step (1) is D-limonene with a content of not less than 80%. The content of citrus flavonoid product I in step (4) is 1-10% based on hesperidin. The content of citrus flavonoid product II obtained in step (5) is 20-50% based on hesperidin, nosocomialin, and tangeretin.

8. A method for determining the content of hesperidin, nobiletin, and tangeretin prepared by any one of the methods described in claims 1-7 using HPLC, characterized in that: The determination method includes the following steps: 1) Preparation of standard reference solution: Accurately weigh the hesperidin, nonocitretin and tangeretin standard references, add 50% methanol to prepare a mixed reference solution of hesperidin, nonocitretin and tangeretin, for later use; 2) Preparation of sample solution: Accurately weigh the citrus flavonoid product I and citrus flavonoid product II to be tested, add 50% methanol (v / v) and dissolve by ultrasonication. Take the supernatant and filter it through a 0.45μm microporous membrane for later use. 3) Chromatographic conditions: The chromatographic column was 4.6 × 250 mm, 3 μm. An Omega Polar 100-5-C18 column was used, with acetonitrile (A)-water (B) as the mobile phase, and a flow rate of 0.6 mL / min. -1 The column temperature was 35℃; the detection wavelength was 330nm for analysis. 4) Record the peak area and calculate the contents of hesperidin, nonotrimonin and citrus flavonoid product I and citrus flavonoid product II based on the standard curve obtained from the reference solution prepared in step a).

9. The method for determining total flavonoids in Yinghe citrus by HPLC according to claim 8, characterized in that: The gradient elution procedure for the mobile phase in step 3) is as follows: 。 10. The method for determining total flavonoids in Yinghe citrus by HPLC according to claim 8, characterized in that: In step 1), the concentrations of the reference solutions of hesperidin, nobiletin, and citrus peel were 87.0 μg / mL, 22.5 μg / mL, and 12.0 μg / mL, respectively; in step 2), the concentrations of citrus flavonoid product I and citrus flavonoid product II were each independently 10-200 μg / mL.

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