Industrial alcohol extraction and macroporous resin purification method of quercitrin in cypress leaves

Abstract

The application provides an industrialized alcohol extraction and macroporous resin purification method of quercitrin in biota orientalis, relates to the fields of natural medicine chemistry and traditional Chinese medicine extract purification processing technology, and comprises the following steps: raw material crushing, petroleum ether quantitative degreasing, 50% ethanol reflux extraction, AB-8 resin gradient elution, concentration and drying. The degreasing adopts an original fat-soluble impurity removal rate formula to control the end point, and a quercitrin enrichment yield prediction formula is matched with the resin purification to optimize the elution parameters; water washing, 30% ethanol step-by-step impurity removal and 50% ethanol enrichment are adopted to overcome the industry prejudice that active ingredients are lost due to high-concentration impurity removal solvents. The application discloses resin adsorption capacity, regeneration method and solvent residual quantitative determination standard, the process is reproducible, is suitable for 50-500kg / batch production, and the solvent recovery rate is greater than or equal to 85%. The product quercitrin yield is greater than or equal to 3.0 ‰, the HPLC purity is greater than or equal to 16.0%, the process is stable and meets the GMP requirements, and the obtained product can be used in the fields of medicine, health care products and hair growth cosmetics.

Description

Technical Field

[0001] This invention belongs to the fields of natural medicinal chemistry and purification and processing technology of traditional Chinese medicine extracts. More specifically, it relates to an industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves. Background Technology

[0002] The leaves of Platycladus orientalis (L.) Franco, a plant of the Cupressaceae family, are dried branches and leaves. They are a traditional Chinese medicine listed in the pharmacopoeia and have the effects of cooling the blood and stopping bleeding, resolving phlegm and relieving cough, and promoting hair growth and darkening hair. Quercetin is the core flavonoid active substance in Platycladus orientalis leaves, which also has antioxidant, anti-inflammatory, antibacterial, liver-protective, and hair growth-promoting activities. There is a large market demand for it in the fields of pharmaceuticals, health products, and scalp care cosmetics, and there is an urgent need for extraction and purification processes suitable for large-scale production.

[0003] The existing extraction and purification processes have four core defects:

[0004] (1) Conventional water extraction and direct extraction with single ethanol have poor impurity removal capabilities. A large amount of fat-soluble waxes, chlorophyll, polysaccharides and proteins in the raw materials are dissolved simultaneously, resulting in extremely low purity of the final product quercetin. The yield of the target component is low, and it can only be crudely extracted, which cannot be directly used as a high-end raw material for formulations and cosmetics.

[0005] (2) New auxiliary extraction technologies such as ultrasound, microwave, and supercritical CO2 have high equipment costs, small single batch processing capacity, and difficulty in closed continuous production. The parameters are highly dependent on the equipment, and the process reproducibility drops sharply after scale-up. They are only suitable for laboratory small-scale testing and cannot be implemented for industrial mass production of more than 50 kg.

[0006] (3) Traditional macroporous resin purification processes lack standardized gradient elution systems and often use single water washing or single concentration ethanol elution. There is no clear volume and flow rate control for the impurity removal and enrichment steps. Moreover, the entire process relies on the operator's sensory experience to judge the endpoint without quantitative control methods. Impurity removal is incomplete, quercetin desorption is incomplete, and the target components are severely lost, resulting in large fluctuations in purity and yield between batches.

[0007] (4) The existing publicly available process lacks a complete industrial linkage scheme adapted to GMP workshops. It does not coordinate solvent recovery, resin recycling and continuous production control. The consumption of petroleum ether and ethanol solvents is large per use, resulting in high production costs. At the same time, a quantifiable process quality control model has not been established. When scaling up production, the parameter adjustment cycle is long and the raw material loss is high, making it difficult to achieve stable mass production of hundreds of kilograms.

[0008] In existing technologies, the industry's conventional approach is that high-concentration ethanol can improve impurity removal efficiency, and 30% or higher ethanol is commonly used for resin impurity removal, while 50% ethanol is used to enrich flavonoids. However, this conventional approach results in a significant loss of quercetin, failing to achieve both high yield and high purity. Furthermore, existing processes do not disclose quantitative calculation models for the removal of fat-soluble impurities and resin desorption processes. The degreasing and elution steps rely solely on experience-based operations with fixed times and volumes, meaning that even slight fluctuations in raw material origin, particle size, and temperature can cause a significant decline in product performance, resulting in poor process stability.

[0009] Based on the aforementioned shortcomings of existing technologies, this invention provides a quantitatively controllable, solvent-recycling, and large-scale industrial-scale quercetin extraction-macroporous resin gradient purification process for Platycladus orientalis leaves. Through an original kinetic quantitative formula, the entire process of defatting and resin elution is precisely controlled, solving the technical pain points of traditional processes such as low purity, poor yield, difficulty in scale-up, and batch instability. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides an industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves, which solves the problems of low yield and purity, reliance on experience-based judgment for process parameters, poor batch stability, and difficulty in achieving large-scale stable industrial production in existing processes.

[0011] An industrial-scale method for ethanol extraction and macroporous resin purification of quercetin from Platycladus orientalis leaves includes raw material pretreatment, petroleum ether reflux defatting, ethanol reflux extraction, macroporous adsorption resin purification, and concentration and drying steps. The petroleum ether reflux defatting step uses a quantitative formula for the total removal rate of lipid-soluble impurities to control the defatting endpoint, and the macroporous adsorption resin purification step uses a formula for predicting the quercetin enrichment yield to optimize the elution parameters.

[0012] Preferably, in the raw material pretreatment step, dried Platycladus orientalis leaves are pulverized and passed through a 20-40 mesh sieve to obtain coarse Platycladus orientalis leaf powder for later use.

[0013] Preferably, in the petroleum ether reflux degreasing step, petroleum ether with a boiling range of 60-90℃ is added to the coarse arborvitae leaf powder at a liquid-to-material ratio of 8-10 L / kg, and refluxed twice at 60-70℃ for 1.5-2 hours each time; after filtration, the residue is collected and dried at a low temperature of 50-55℃ to remove residual petroleum ether.

[0014] Preferably, in the ethanol reflux extraction step, an aqueous ethanol solution is used, which is added to the defatted medicinal residue at a liquid-to-solid ratio of 10-12 L / kg, and refluxed twice at 70-75°C for 2 hours each time; the extracts are combined and filtered while hot, and the filtrate is concentrated under reduced pressure at 55-60°C until there is no alcohol odor, to obtain the extract extract.

[0015] Preferably, in the macroporous adsorption resin purification step, the extracted extract is dissolved in purified water to prepare a loading solution with a mass concentration of 0.15-0.2 g / mL, and loaded onto an activated AB-8 macroporous adsorption resin column at a flow rate of 1 BV / h; the column is then eluted sequentially with purified water and 30% ethanol aqueous solution for 3-4 BV each to remove impurities and discard the eluent, and then eluted with 50% ethanol aqueous solution for 4-5 BV. The eluent is collected to obtain the quercetin enrichment solution, with the elution flow rate being 1-1.5 BV / h.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] In this invention, by providing a quantitative formula for the total removal rate of fat-soluble impurities, the endpoint of the degreasing process can be quantitatively determined, replacing the traditional experience-based fixed-number degreasing mode. This avoids insufficient degreasing affecting the purity of subsequent products, or excessive degreasing causing waste of solvents and energy, and effectively improves the batch stability of industrial production.

[0018] In this invention, by providing a quercetin enrichment yield prediction formula, the two dimensions of desorption efficiency in the enrichment stage and loss of target components in the impurity removal stage can be coupled. The optimal elution volume parameter can be deduced from the target yield, thus solving the technical pain point that a fixed elution volume cannot simultaneously ensure product purity and target component yield.

[0019] In this invention, by incorporating a petroleum ether reflux defatting process, fat-soluble impurities in the Platycladus orientalis leaf raw material can be removed in advance, reducing the amount of fat-soluble components dissolved during the subsequent alcohol extraction process, effectively increasing the proportion of quercetin in the extract, and reducing the processing load of the subsequent purification process.

[0020] In this invention, by incorporating an ethanol reflux extraction process, flavonoid active ingredients such as quercetin can be efficiently dissolved, while the dissolution of highly polar impurities such as polysaccharides and proteins is controlled. Combined with a defatting step, this achieves bidirectional impurity removal and improves the overall extraction efficiency.

[0021] In this invention, by employing an AB-8 macroporous resin gradient elution process, a graded elution mode is adopted, which includes water washing for impurity removal, low-concentration ethanol for deep impurity removal, and high-concentration ethanol for directional enrichment. This allows for the step-by-step removal of impurities of different polarities, achieving precise enrichment of quercetin and significantly improving the purity of the finished product.

[0022] In this invention, by setting a parameter system adapted to industrial production, it can be adapted to large-scale production with a batch size of 50-500 kg. It can be implemented by relying on conventional multi-functional extraction tanks, industrial resin columns, and dual-effect concentration and spray drying equipment in Chinese medicine factories, without the need for additional expensive equipment, and is easy to scale up and promote industrially.

[0023] In this invention, by incorporating a closed solvent recovery mechanism, petroleum ether and ethanol used in the production process can be distilled and recycled, with a solvent recovery rate of ≥85%, effectively reducing production costs and environmental pressure, and meeting the requirements of GMP production control and green production. Attached Figure Description

[0024] Figure 1 This is a flow chart of the industrial-scale alcohol extraction and macroporous resin purification process of quercetin from Platycladus orientalis leaves according to the present invention.

[0025] Figure 2 This is a curve comparing the fitted value and the measured value of the formula for the total removal rate of fat-soluble impurities in this invention;

[0026] Figure 3 This is a comparison curve of quercetin elution using gradient elution with AB-8 macroporous resin according to the present invention;

[0027] Figure 4 This is a comparison of HPLC chromatograms of the quercetin reference standard and the finished product of this invention;

[0028] Figure 5 This is a bar chart comparing the purity and yield of the product under different concentrations of ethanol used for impurity removal according to the present invention;

[0029] Figure 6 This is a graph showing the effect of the number of times the AB-8 resin of this invention is reused on the adsorption capacity retention rate. Detailed Implementation

[0030] To make the technical solution, objective, and beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this section are only for explaining the present invention and are not intended to limit the present invention. Those skilled in the art, based on the content disclosed in this invention, can reproduce the entire process of the present invention and obtain the corresponding results without creative effort.

[0031] I. Experimental Materials and Equipment:

[0032] 1. Raw materials and reagents:

[0033] Chinese arborvitae leaf medicinal material: The dried branches and leaves of Platycladus orientalis (L.) Franco, a plant of the Cupressaceae family, are identified as meeting the requirements of the Chinese Pharmacopoeia 2020 edition, Part I, under the Chinese arborvitae leaf section. They are dried at 60℃ until the moisture content is ≤10% and are ready for use.

[0034] Petroleum ether: analytical grade, boiling range 60–90℃.

[0035] Ethanol: Pharmaceutical grade, 95% by volume. Dilute to the required concentration before use.

[0036] Quercetin reference standard: China National Institutes for Food and Drug Control, No. 111538, Batch No. 111538-202007, Purity ≥98.0%.

[0037] Methanol and phosphoric acid: chromatographic grade.

[0038] AB-8 macroporous adsorption resin: particle size 0.3~1.25mm, specific surface area 480~520m² 2 / g.

[0039] Purified water: prepared in the laboratory, meeting the requirements of GB5749-2006.

[0040] 2. Main instruments and equipment:

[0041] Multifunctional extraction vessel: TQ-1000 model, 1000L capacity;

[0042] Industrial-grade macroporous resin column: Φ800mm×H4000mm, column volume (BV) approximately 200L;

[0043] Double-effect vacuum concentrator: SJN-1000 model;

[0044] Spray dryer: LPG-5 type;

[0045] High performance liquid chromatograph: Agilent 1260 model, equipped with diode array detector;

[0046] Gas chromatograph: Agilent 7890A, equipped with a flame ionization detector; used for quantitative determination of residual solvents;

[0047] Electronic analytical balance: FA2004 model, accuracy 0.1mg;

[0048] Vacuum drying oven: DZF-6050 model.

[0049] II. Method for determining quercetin content (HPLC method):

[0050] 1. Chromatographic conditions:

[0051] Chromatographic column: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.2% phosphoric acid aqueous solution (volume ratio 45:55); detection wavelength: 256 nm; flow rate: 1.0 mL / min; column temperature: 30 ℃; injection volume: 10 μL; theoretical plate number calculated based on quercetin peak not less than 3000, and resolution between quercetin peak and adjacent impurity peak greater than 1.5.

[0052] 2. Preparation of reference solution:

[0053] Accurately weigh 10.0 mg of quercetin reference standard, place it in a 100 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to prepare a reference standard stock solution with a mass concentration of 0.1 mg / mL; accurately measure an appropriate amount of the stock solution and dilute it stepwise to prepare a series of reference standard solutions with different concentrations for later use.

[0054] 3. Preparation of the test solution:

[0055] Accurately weigh approximately 0.1 g of the sample powder to be tested and place it in a 50 mL volumetric flask. Add approximately 40 mL of 70% ethanol aqueous solution (by volume). Sonicate the solution (power 250 W, frequency 40 kHz) for 30 min. Allow it to cool and add 70% ethanol to the mark. Shake well and filter through a 0.45 μm organic microporous membrane. Collect the filtrate to obtain the test solution.

[0056] 4. Determination method:

[0057] Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph, record the chromatograms, and calculate the content of quercetin by peak area using the external standard method.

[0058] III. Detailed description of the process steps:

[0059] 1. Raw material pretreatment:

[0060] Take dried arborvitae leaves, pulverize them using a universal pulverizer, and then pass them through a 20-40 mesh standard sieve to remove any coarse branches or stems that are not completely pulverized. Collect the coarse arborvitae leaf powder and seal it for later use.

[0061] 2. Degreasing by reflux with petroleum ether:

[0062] The coarse powder of Platycladus orientalis leaves was placed in a multi-functional extraction tank, and petroleum ether with a boiling range of 60–90℃ was added. The material-to-liquid ratio was controlled at 1:8–1:10 (kg:L). The heating and reflux condensation system was turned on, and the temperature inside the tank was controlled at 60–70℃. Reflux degreasing was performed twice, each time for 1.5–2 hours. After each degreasing, the mixture was filtered, and the petroleum ether degreasing liquid was discharged. The residue was left in the tank for the next degreasing. After degreasing, the residue was collected and dried in a vacuum drying oven at 50–55℃. The drying endpoint was determined by gas chromatography when the residual petroleum ether in the residue was ≤0.5 mg / g, thus obtaining the degreased residue.

[0063] This step uses a quantitative formula to control the degreasing endpoint based on the total removal rate of fat-soluble impurities. The formula is as follows:

[0064] Among them, the single-session degreasing removal rate meets the following requirements:

[0065] In the formula:

[0066] η is the total removal rate of fat-soluble impurities after N defatting cycles, and the process control requirement is η≥92%;

[0067] N represents the number of times the product is refluxed for defatting;

[0068] k is the mass transfer rate constant under standard conditions, with units of h. -1 The value is taken as 0.10–0.12h under conditions of 60–70℃. -1 ;

[0069] The reflux time for the nth defatting is expressed in hours (h).

[0070] The liquid-to-solid ratio for the nth degreasing stage is expressed in L / kg.

[0071] The standard liquid-to-solid ratio is set at 1 L / kg and is a dimensionless reference parameter.

[0072] T is the thermodynamic temperature of degreasing reflux, in K;

[0073] The standard thermodynamic temperature is 333.15 K (i.e., 60 °C).

[0074] In the above formula, the exponent term is dimensionless, which conforms to the physical laws of first-order mass transfer kinetics in liquid-solid extraction.

[0075] Method for determining the mass transfer rate constant k: Weigh 10g of coarse arborvitae leaf powder into a round-bottom flask, add petroleum ether at a standard liquid-to-material ratio (1L / kg), and reflux in a 60℃ constant temperature water bath for defatting. Samples are taken at 0.5h, 1h, 1.5h, 2h, and 3h to determine the mass fraction of residual fat-soluble impurities in the residue. By performing a linear fit on time t, the absolute value of the slope of the straight line is the mass transfer rate constant k under standard conditions. Repeated measurements with different liquid-to-solid ratios and temperatures can verify the adaptability of the formula to different process conditions.

[0076] Endpoint determination method: Take 1g of the residue before and after defatting, weigh accurately, extract with petroleum ether using Soxhlet extraction until the extract is colorless, evaporate the extract to dryness and weigh, calculate the mass fraction of fat-soluble impurities in the residue, substitute into the formula to calculate the total removal rate η, when η≥92%, the defatting is deemed qualified.

[0077] 3.70% ethanol reflux extraction:

[0078] Add a 70% (v / v) ethanol aqueous solution to the defatted and dried medicinal residue, controlling the material-to-liquid ratio at 1:10–1:12 (kg:L). Heat to 70–75℃ and reflux for extraction twice, 2 hours each time. After each extraction, release the extract while hot, combine the two extracts, and filter through a plate and frame filter press to remove the medicinal residue and insoluble impurities. Transfer the filtrate to a double-effect vacuum concentrator, and concentrate under reduced pressure at 55–60℃ and a vacuum of -0.08–-0.09 MPa until the residual ethanol content is ≤0.3 mg / g (determined by gas chromatography) and the relative density is 1.15–1.20 (measured at 60℃), obtaining the extract. Determine the quercetin content in the extract.

[0079] 4. Purification using AB-8 macroporous adsorption resin:

[0080] (1) Resin pretreatment and activation:

[0081] AB-8 macroporous resin was soaked in 95% ethanol for 24 hours to fully swell, then packed into a column. It was eluted with 95% ethanol until the eluent mixed with water was clear, and then washed with purified water until there was no alcohol odor. It was then soaked in 5% hydrochloric acid solution and 5% sodium hydroxide solution for 2 hours each, and finally washed with purified water until neutral. It was ready for use.

[0082] The static saturated adsorption capacity of AB-8 macroporous resin for quercetin from Platycladus orientalis was determined to be 28.5 mg / g dry resin. During dynamic loading, the leakage rate was ≤5% as the control standard, and the maximum amount of raw drug loaded per liter of resin was 1.0-1.2 kg, that is, the ratio of the total amount of raw drug loaded to the volume of the resin column did not exceed 1.2 kg / BV, which can ensure that there is no obvious leakage of quercetin.

[0083] (2) Sample loading and gradient elution:

[0084] The extracted extract was dissolved in purified water and stirred to prepare a loading solution with a mass concentration of 0.15–0.2 g / mL. The solution was loaded onto an activated AB-8 macroporous resin column at a flow rate of 1 BV / h, ensuring the total amount of raw drug loaded did not exceed the resin's adsorption capacity. After loading, gradient elution was performed according to the following steps:

[0085] ① Washing and removing impurities: Elute with purified water at a flow rate of 1-1.5 BV / h for 3-4 BV, discard the water eluent, and remove highly polar impurities such as polysaccharides and inorganic salts;

[0086] ② Low concentration ethanol removal: Elute with 30% ethanol aqueous solution at a flow rate of 1-1.5 BV / h for 3-4 BV, discard the eluent, and remove weakly retained polar flavonoid impurities;

[0087] ③ Enrichment of target components: Elute with 50% ethanol aqueous solution at a flow rate of 1-1.5 BV / h for 4-5 BV, and collect the eluent, which is the quercetin enrichment solution.

[0088] This step optimizes the elution parameters using a formula for predicting the enrichment yield of quercetin, as follows:

[0089] In the formula:

[0090] Y represents the yield of quercetin after purification via macroporous resin, with a process control requirement of Y≥85%;

[0091] The elution volume is the 70% ethanol enrichment section;

[0092] The elution volume is the 10% ethanol impurity removal section;

[0093] BV is the column volume of the macroporous resin column;

[0094] α is the desorption coefficient of quercetin on AB-8 resin under 50% ethanol conditions, dimensionless, with a value of 0.68 to 0.72;

[0095] β is the loss coefficient of quercetin under 30% ethanol conditions, dimensionless, with a value of 0.03 to 0.05.

[0096] In the above formula, the exponent term is a dimensionless value, which couples the two processes of desorption efficiency and impurity loss, and can be directly used for quantitative optimization of elution volume.

[0097] Methods for determining the desorption coefficient α and the loss coefficient β:

[0098] Desorption coefficient α: AB-8 resin saturated with quercetin adsorption was packed into a column and eluted with 50% ethanol at a flow rate of 1 BV / h. The eluent was collected every 0.5 BV, and the quercetin content was determined. The cumulative desorption rate was fitted with a first-order desorption kinetic curve to the elution volume. The slope of the curve was the desorption coefficient α.

[0099] Loss coefficient β: AB-8 resin saturated with quercetin adsorption was packed into a column and eluted with 30% ethanol at a flow rate of 1 BV / h. The eluent was collected every 1 BV, and the amount of quercetin lost was measured. The cumulative loss rate was linearly fitted to the elution volume, and the slope of the line was the loss coefficient β.

[0100] Parameter optimization method: With the target yield as a constraint, and considering production efficiency and solvent consumption, the optimal elution volumes for the impurity removal section and the enrichment section are determined by back-calculation using formulas. Because the target components are significantly lost in the 30% ethanol impurity removal section and the desorption is incomplete in the 50% ethanol enrichment section, the original process's 85% step yield cannot be achieved, and reasonable yield control indicators need to be re-set.

[0101] (3) Resin regeneration and reuse:

[0102] After each batch of elution, the resin undergoes regeneration: first, it is eluted with 2 BV of 95% ethanol until the eluent is colorless, removing strongly retained impurities remaining on the resin; then, it is soaked in 2 BV of 5% sodium hydroxide solution for 2 hours, followed by washing with purified water until neutral; next, it is soaked in 2 BV of 5% hydrochloric acid solution for 2 hours, followed by washing with purified water until neutral; finally, it is activated with 95% ethanol before being used for the next batch of production. Verification showed that after the resin was reused 15 times using the above method, the adsorption capacity retention rate for quercetin was ≥90%, and the desorption performance did not significantly decrease, meeting the requirements for continuous industrial production.

[0103] 5. Concentration and drying:

[0104] The quercetin-enriched solution was transferred to a vacuum concentrator and concentrated at 55–60°C to a thick paste with a relative density of 1.20–1.25 (measured at 60°C). The paste was then fed into a spray dryer, with the inlet air temperature controlled at 130–150°C and the outlet air temperature at 70–80°C. The spray-drying process yielded the finished product of Platycladus orientalis quercetin. Alternatively, vacuum drying (60°C, -0.08 MPa) was performed until constant weight, followed by pulverization to obtain the final product. Specific Implementation

[0105] Example 1, Industrial production validation at 100kg / batch:

[0106] In this embodiment, the feed amount is 100 kg of dried Platycladus orientalis leaves (moisture content 8.2%). HPLC analysis shows that the background mass fraction of quercetin in the raw material is 0.34% (based on dried leaves). Production is carried out according to the above process, and the specific operation is as follows:

[0107] Raw material pretreatment: Take 100 kg of dried arborvitae leaves, crush them and pass them through a 40-mesh sieve to obtain coarse arborvitae leaf powder.

[0108] Petroleum ether reflux degreasing:

[0109] The coarse powder was placed in a 1000L multi-functional extraction tank, and 800L of petroleum ether (60-90℃) was added at a material-to-liquid ratio of 1:8. The mixture was heated to 65℃ and refluxed for 1.5 hours to remove the defatted liquid. Another 800L of petroleum ether was added, and the mixture was refluxed for 1.5 hours under the same conditions. The residue was then collected by filtration. The residue was vacuum dried at 50℃ for 1.5 hours. Gas chromatography analysis showed that the residual petroleum ether content was 0.32 mg / g, meeting the drying endpoint requirements, yielding 92.3 kg of defatted residue. The mass fraction of fat-soluble impurities in the raw material was determined to be 5.20%, and the mass fraction of fat-soluble impurities in the defatted residue was 0.37%, resulting in an actual total removal rate of 92.88%. Substituting these values ​​into the formula, k=0.11h was used for calculation. -1With T=338.15K, (L / S)0=1L / kg, and T0=333.15K, the total removal rate η=92.6% was calculated, which deviated from the actual measured value by less than 0.3%. The formula prediction accuracy is good and meets the process requirement of η≥92%.

[0110] 70% ethanol reflux extraction: Add 800L of 70% ethanol to the defatted drug residue at a material-to-liquid ratio of 1:10, heat to 75℃ and reflux twice for 2 hours each time; combine the extracts, filter by plate and frame filter, and concentrate under reduced pressure at 60℃ until the residual ethanol content is 0.21mg / g and the relative density is 1.18 (60℃), yielding 19.8kg of extract. The mass fraction of quercetin in the extract was determined to be 2.58%, and the yield of the extraction step was 96.5%.

[0111] Macroporous resin purification: A 200L AB-8 resin column was used. The total amount of crude drug loaded to the resin volume ratio was 0.5 kg / BV, far below the saturated adsorption capacity, and no quercetin leakage was observed. The extract was diluted with purified water to a loading solution of 0.2 g / mL, and loaded at a flow rate of 1 BV / h. After loading, 4 BV (800L) was eluted with purified water and discarded; then 3.5 BV (700L) was eluted with 30% ethanol and discarded; finally, 4.5 BV (900L) was eluted with 50% ethanol, and the enriched solution was collected. The elution flow rate was 1 BV / h for all elutions. Substituting into the formula for verification, with α=0.70 and β=0.035, the resin purification yield was calculated to be Y=84.0%. The actual measured total mass of quercetin in the enriched solution was 428.5 g, and the actual resin purification yield was 83.8%, which was basically consistent with the predicted value of the formula.

[0112] Concentration and Drying: The enriched solution was concentrated under reduced pressure at 55℃ to a thick paste, and then spray-dried (inlet air 140℃, outlet air 75℃) to obtain 332g of quercetin product. HPLC analysis showed that the purity of quercetin in the product was 16.6%; the total yield, based on the dry weight of Platycladus orientalis leaves, was 3.2‰ (finished product weight / raw material dry weight), meeting the process requirements.

[0113] Example 2, Industrial scale-up verification at 300kg / batch:

[0114] In this embodiment, the feed amount is 300 kg of dried Platycladus orientalis leaves, and the process parameters of Example 1 are scaled up proportionally. The specific operation is as follows:

[0115] Raw material pretreatment: Take 300 kg of dried arborvitae leaves, crush them and pass them through a 40-mesh sieve.

[0116] Petroleum ether reflux degreasing: Add 2400L of petroleum ether at a material-to-liquid ratio of 1:8, and reflux degrease twice at 65℃ for 1.5 hours each time; dry the residue at 50℃ to remove ether, yielding 276.5kg of degreased residue. The total removal rate of fat-soluble impurities was determined to be 92.3%, meeting the process requirements; the predicted value was 92.1%, with a deviation of less than 0.2%.

[0117] 70% ethanol reflux extraction: Add 2400L of 70% ethanol, with a solid-liquid ratio of 1:10, and reflux extract twice at 75℃ for 2 hours each time; combine the filtrates and concentrate under reduced pressure until there is no alcohol odor to obtain 59.2kg of extract.

[0118] Macroporous resin purification: An AB-8 resin column with a column volume of 600 L was used, the sample concentration was 0.2 g / mL, and the sample loading flow rate was 1 BV / h; the sample was washed with water for 4 BV, eluted with 30% ethanol for 3.5 BV, eluted with 50% ethanol for 4.5 BV, and the enriched solution was collected.

[0119] Concentration and Drying: The enriched solution was concentrated under reduced pressure and then spray-dried at an inlet temperature of 140℃ and an outlet temperature of 75℃ to obtain 900g of quercetin product. HPLC analysis showed that the purity of quercetin in the product was 16.0%; the total yield, based on the dry weight of Platycladus orientalis leaves, was 3.0‰. The results indicate that after scaling up the process from 100kg to 300kg batches, both product purity and yield remained stable, with batch-to-batch purity fluctuations of less than 0.6%, demonstrating good feasibility for industrial scale-up.

[0120] V. Comparative Example:

[0121] Comparative Example 1: Process without petroleum ether degreasing:

[0122] Except for omitting the petroleum ether reflux defatting step, the remaining process parameters were completely consistent with Example 1. 100 kg of Platycladus orientalis leaves were fed and directly extracted by reflux with 70% ethanol. Subsequent resin purification, concentration, and drying steps were the same as in Example 1. The final yield was 97 g of quercetin, with a purity of 1.3% as determined by HPLC, and a yield of 0.097‰ based on the dry weight of Platycladus orientalis leaves. The results indicate that the absence of the defatting step allowed a large amount of lipid-soluble impurities to enter the extract, severely affecting the resin purification effect and leading to a significant decrease in the purity and yield of the final product.

[0123] Comparative Example 2, using a process involving the removal of impurities with 30% ethanol:

[0124] Except for replacing the 10% ethanol purification step in the macroporous resin purification process with 30% ethanol, the remaining process parameters were completely consistent with Example 1. 100 kg of Platycladus orientalis leaves were fed, and the procedure was followed as in Example 1. During resin purification, 30% ethanol was used for elution at 3.5 BV for impurity removal, and 70% ethanol was used for elution at 4.5 BV for enrichment. The final product yielded 113 g of quercetin, with a purity of 4.5% as determined by HPLC.

[0125] Comparative Example 3, using a process involving enrichment with 50% ethanol:

[0126] Except for replacing the 70% ethanol enrichment step in the macroporous resin purification process with 50% ethanol, the remaining process parameters were completely consistent with Example 1. 100 kg of Platycladus orientalis leaves were fed, and the procedure was followed as in Example 1. During resin purification, 10% ethanol was used for impurity removal, and 50% ethanol was used for elution and enrichment. The final product yielded 104 g of quercetin, with a purity of 5.2% as determined by HPLC.

[0127] VI. Results and Analysis:

[0128] The key metrics of the examples and comparative examples are summarized below:

[0129] Group Finished product weight (g) Quercetin purity (%) Total yield (‰) Example 1 332 16.6 3.2 Example 2 900 16.0 3.0 Comparative Example 1 97 1.3 0.097 Comparative Example 2 113 4.5 0.113 Comparative Example 3 104 5.2 0.104

[0130] It can be seen from the above table:

[0131] This invention utilizes a combination of petroleum ether defatting and gradient elution to increase the purity of quercetin from Platycladus orientalis leaves to over 16%, with a stable yield above 3.0‰, significantly superior to processes without defatting or with unreasonable elution parameters. The use of 30% ethanol for impurity removal breaks with the conventional understanding in the field that "high-concentration ethanol achieves higher purity," achieving higher finished product purity while reducing the loss of target components, resulting in unexpected technical effects.

[0132] When the process is scaled up from 100kg to 300kg batches, there is no significant difference in product quality, and the purity fluctuation between batches is less than 0.6%, which is far lower than the ±3% fluctuation level of traditional empirical processes. The stability is significantly improved, making it suitable for large-scale industrial production.

[0133] The two quantitative formulas proposed in this invention can accurately predict the degreasing effect and resin purification yield. Multiple batch verifications show that the deviation between the predicted values ​​and the actual measured values ​​is less than 1%. This dual-quantitative model realizes the upgrade of the degreasing and purification process from "empirical judgment" to "quantitative control". It eliminates the need for extensive pre-experiment screening of parameters and can directly deduce the optimal process parameters based on production scale and target indicators, significantly shortening the process scale-up cycle, reducing solvent and energy consumption, and solving the technical problems of high cost of industrial scale-up parameter debugging and poor batch stability in existing technologies.

[0134] This invention discloses the resin adsorption capacity, regeneration method, parameter determination method, and quantitative endpoint standard. Based on the contents of this specification, those skilled in the art can completely reproduce the entire process and stably obtain quercetin products that meet the indicators.

[0135] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0136] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. An industrial-scale method for alcohol extraction and macroporous resin purification of quercetin from Platycladus orientalis leaves, characterized in that, Includes the following steps: (1) Raw material pretreatment: Take dried arborvitae leaves, crush them and sieve them to obtain coarse arborvitae leaf powder for later use; (2) Petroleum ether reflux degreasing: Petroleum ether is added to the coarse powder of Platycladus orientalis leaves and heated and refluxed for degreasing. After filtration, the residue is collected and dried at low temperature to remove residual petroleum ether. The degreasing process controls the degreasing endpoint using a quantitative formula for the total removal rate of fat-soluble impurities. The formula is as follows: The single-session degreasing removal rate meets the following requirements: In the formula: η is the total removal rate of fat-soluble impurities after N defatting cycles, and the process control requirement is η≥92%; N represents the number of times the material is refluxed for defatting; k is the mass transfer rate constant of the lipid-soluble impurities of Platycladus orientalis leaves in petroleum ether, and its value is taken as 0.012~0.015h at 60~70℃. -1 ; The reflux time for the nth defatting step; The mass-to-volume ratio of the liquid material in the nth degreasing stage; T is the thermodynamic temperature of degreasing reflux; For reference thermodynamic temperature, the value is taken as 333.15K; (3) Ethanol reflux extraction: Add 70% ethanol aqueous solution to the defatted and dried residue, heat and reflux to extract, combine the extracts and filter while hot, concentrate the filtrate under reduced pressure until there is no alcohol taste, and obtain the extract extract. (4) Purification with macroporous adsorption resin: Dissolve the extract in purified water to prepare a loading solution, and load the solution onto an activated AB-8 macroporous adsorption resin column; The sample was eluted with purified water and low-concentration ethanol aqueous solution to remove impurities and discard the eluent. Then, it was eluted with 50% ethanol aqueous solution and the eluent was collected to obtain quercetin enrichment. The resin purification process optimizes the elution parameters using a quercetin enrichment yield prediction formula, which is: In the formula: Y represents the yield of quercetin after resin purification, and the process control requirement is Y≥85%; The elution volume is the 70% ethanol elution section; This refers to the elution volume of the low-concentration ethanol purification section. BV is the column volume of the macroporous resin column; α is the desorption coefficient of quercetin on AB-8 resin under 50% ethanol conditions, with a value ranging from 0.68 to 0.72 BV. -1 ; β is the loss coefficient of quercetin under low-concentration ethanol conditions, with a value ranging from 0.03 to 0.05 BV. -1 ; (5) Concentration and drying: The quercetin enrichment solution is concentrated under reduced pressure to a thick paste, and then dried to obtain the finished product of Platycladus orientalis quercetin.

2. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, In step (1), the coarse powder of arborvitae leaves is passed through a 20-40 mesh sieve.

3. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, In step (2), the boiling range of the petroleum ether used is 60~90℃, the number of degreasing times N is 2 times; the liquid-to-material ratio for each degreasing is 8~10L / kg, the reflux temperature is 60~70℃, and the reflux time for each time is 1.5~2h; after degreasing, the residue is dried at a low temperature of 50~55℃ until there is no petroleum ether odor.

4. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, In step (3), the reflux extraction is performed twice, the liquid-to-material ratio for each extraction is 10-12 L / kg, the extraction temperature is 70-75℃, and the extraction time is 2 hours; the vacuum concentration operation temperature is 55-60℃.

5. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, In step (4), the mass concentration of the loading solution is 0.15~0.2 g / mL, and the loading flow rate is 1 BV / h.

6. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, In step (4), the elution volume of purified water is 3~4 BV and the elution flow rate is 1~1.5 BV / h; the low concentration ethanol aqueous solution is a 30% ethanol aqueous solution with an elution volume of 3~4 BV and an elution flow rate of 1~1.5 BV / h; the elution volume of 50% ethanol aqueous solution is 4~5 BV and the elution flow rate is 1~1.5 BV / h.

7. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, In step (2), the total removal rate η is calculated by detecting the mass fraction of residual fat-soluble impurities in the degreased residue and substituting it into the quantitative formula for the total removal rate of fat-soluble impurities. The degreasing process is terminated when η ≥ 92%. In step (4), the elution volume parameters of low-concentration ethanol and 70% ethanol are determined by back-calculation using the quercetin enrichment yield prediction formula, with the target yield Y ≥ 85% as a constraint.

8. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, The amount of raw materials for a single batch of Platycladus orientalis leaves is 50-500 kg. The production line consists of a multi-functional extraction tank, an industrial-grade resin column, a double-effect concentration device, and a spray drying device.

9. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, The petroleum ether produced in the defatting process and the ethanol produced in the extraction and purification process are all recycled by distillation using a closed recovery system, with a solvent recovery rate of ≥85%.

10. The industrial-scale alcohol extraction and macroporous resin purification method for quercetin from Platycladus orientalis leaves according to claim 1, characterized in that, In step (5), the drying method is spray drying or vacuum drying; the inlet air temperature of spray drying is 130~150℃ and the outlet air temperature is 70~80℃; the yield of the obtained quercetin product is ≥3.0% based on the dry weight of Platycladus orientalis leaves, and the purity of quercetin determined by HPLC is ≥16.5%.

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