Multi-stage extraction method of active ingredients of cacumen biotae

Abstract

The invention provides a multi-stage extraction method of active ingredients of cacumen biotae, and belongs to the technical field of plant extracts. The preparation method comprises the following steps: squeezing fresh cacumen biotae for the first time, adding enzyme and sterile water into primary leaf residues obtained after the first-time squeezing, separating hydrolysate from the leaf residues after enzymolysis, and squeezing for the second time; mixing the two-time squeezed liquid and the hydrolysate, concentrating, adding ammonium sulfate for precipitation, centrifuging, and drying the precipitate to obtain total protein; after centrifugation, carrying out electrodialysis desalination on supernate, and freeze-drying to obtain total sugar; performing supercritical extraction on the leaf residues subjected to secondary squeezing to obtain total tannin; extracting three-time leaf residues after extraction with ethanol, concentrating an extracting solution, separating with silica gel, eluting with petroleum ether-n-butyl alcohol and petroleum ether-ethanol respectively, and performing reduced pressure distillation to obtain volatile oil and total flavonoids respectively. According to the method, multiple active ingredients in the cacumen biotae can be obtained at the same time in a multi-stage extraction mode, the content is high, and the comprehensive utilization rate of cacumen biotae resources is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extracts, and in particular relates to a multi-stage extraction method for active components of Platycladus orientalis leaves. Background Art

[0002] Platycladus orientalis is an evergreen tree of the genus Platycladus in the Cupressaceae family. It is one of the tree species with widespread resource distribution in my country. It is cold-resistant, drought-resistant, and long-lived, and is widely used in landscaping. Platycladus orientalis leaves, also known as cypress leaves, cypress leaves, and cypress leaves, come from the branches and leaves of Platycladus orientalis, a plant of the Cupressaceae family. They are mostly harvested in summer and autumn and dried in the shade. Platycladus orientalis leaves, as a traditional Chinese medicinal material, refer to the dried branches and leaves of Platycladus orientalis. The main chemical components of Platycladus orientalis leaves are volatile oils, flavonoids, polysaccharides, tannins, etc. In addition, there are proteins, fats, vitamins, organic acids, amino acids, pigments, minerals and other components. Modern medicine believes that Platycladus orientalis leaves have multiple functions such as hemostasis, anti-inflammatory, hair growth and nourishment, anti-oxidation, anti-tumor, and antibacterial. Chen Xingfen et al. found that the volatile oil in Platycladus orientalis leaves has a significant inhibitory effect on Aspergillus niger, Penicillium and Rhizopus (Chen Xingfen, Shan Chengying, Ma Shihong, et al. Experimental study on the inhibition of fungi by volatile oil from Platycladus orientalis leaves [J]. Food Research and Development, 2012, 33(06): 198-201.); Zhou Yufan et al. found that the volatile oil from Platycladus orientalis leaves can effectively inhibit the chelation of ferrous ions with ferrous ions, which proves that it has antioxidant capacity (Zhou Yufan, Wang Jihua, Liu Zhilong, et al. Antibacterial and antioxidant activities of Platycladus orientalis leaf essential oil (English) )[J]. Natural Product Research and Development, 2011, 23(04): 620-624+646. DOI: 10.16333 / j.1001-6880.2011.04.041.); Lei Meng found that Platycladus orientalis leaf volatile oil can induce the expression of β-catenin protein in mouse hair follicle cells, thereby promoting hair follicle regeneration (Lei Meng. Mechanism of Platycladus orientalis leaf volatile oil inducing hair growth in C57BL / 6 mice[D]. Northwest University, 2018.). Li Yuanhui et al. determined the ability of Platycladus orientalis leaf polysaccharides to scavenge superoxide anion free radicals. When the polysaccharide concentration was 0.5 g / L, its scavenging rate was as high as 72.97%, indicating that the polysaccharide had antioxidant capacity (Li Yuanhui, Wen Quantai, Zhang Zhaoping, et al. Study on the extraction and antioxidant activity of Platycladus orientalis leaf polysaccharides [J]. Chinese Journal of Traditional Chinese Medicine and Pharmacy, 2016, 23(01): 40-42+47.); Miao Hui studied the anti-tumor activity of Platycladus orientalis leaf polysaccharides on the H22 mouse subcutaneous transplanted tumor model. The results showed that Platycladus orientalis leaf polysaccharides can achieve anti-tumor effects by improving the body's immunity and antioxidant capacity (Miao Hui. Study on the chemical composition and pharmacological activity of Platycladus orientalis leaves [D]. Jilin Agricultural University, 2018.). Liang Tong et al. found that the total flavonoids of Platycladus orientalis leaves at 5.0-45.0 mg / L can significantly inhibit the release of β-glucuronidase and play a role in controlling inflammation (Liang Tong, Qin Yanmei, Liang Nianci, et al. Anti-inflammatory effect and mechanism of total flavonoids of Platycladus orientalis [J]. Chinese Pharmacological Bulletin, 2003, (12): 1407-1410.). It can be seen that the efficacy of Platycladus orientalis leaves is closely related to its chemical components, and obtaining these active ingredients has important application value.

[0003] At present, most of the existing technologies dry and crush Platycladus orientalis leaves into powder for extraction. Chinese patent CN104523767B discloses "a method for extracting flavonoids from Platycladus orientalis leaves", which involves drying, crushing and sieving Platycladus orientalis leaves to obtain Platycladus orientalis leaf powder and then starting water extraction to finally obtain flavonoids. Chinese patent CN115093452A discloses "a method for preparing Platycladus orientalis leaf extract", which involves crushing the Platycladus orientalis leaf raw material into 40-60 meshes, performing steam distillation and macroporous resin separation, and finally obtaining Platycladus orientalis leaf essential oil and high-purity quercetin and myricetin. Some of the active ingredients in Platycladus orientalis leaves will be lost as the water evaporates during the drying process, causing certain losses.

[0004] In addition, the existing technology mainly extracts a single component or a few components. Chinese patent CN118109241A discloses "a method for extracting essential oil from Platycladus orientalis leaves", and the essential oil is obtained by ultrasound-assisted distillation process after freeze-thawing and high-temperature treatment of Platycladus orientalis leaf powder. Chinese patent CN118340694A discloses "a method for preparing and purifying flavonoids from Platycladus orientalis leaves and its application", and the total flavonoids from Platycladus orientalis leaves are obtained by microwave-assisted extraction and supercritical fluid separation and purification using a low eutectic solvent. Chinese patent CN106823454A opens "a continuous extraction method for volatile oil and flavonoids in Platycladus orientalis leaves", and supercritical CO2 extraction technology is used to simultaneously obtain Platycladus orientalis leaf essential oil and flavonoids under different conditions. In the process of extracting the target component, the existing technology treats other effective ingredients as waste residue, which causes great waste. In summary, it is urgent to develop an efficient, low-loss extraction method that can simultaneously obtain various active resources in Platycladus orientalis leaves. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a multi-stage extraction method for active ingredients in Platycladus orientalis, which adopts fresh squeezing and enzymatic hydrolysis to quickly and gently break the wall, and obtain the active components in Platycladus orientalis leaves step by step by peeling off the silk without destroying the active ingredients, so as to deeply explore the application value of Platycladus orientalis leaves.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a multi-stage extraction method of active ingredients from Platycladus orientalis leaves, comprising the following steps:

[0008] 1) squeezing fresh Platycladus orientalis leaves once to obtain a squeezed liquid and a leaf residue, mixing the leaf residue with water, stirring, enzymatically hydrolyzing, and centrifuging to obtain a hydrolyzate and leaf residue;

[0009] 2) squeezing the leaf residue to obtain a secondary squeezed liquid and secondary leaf residue, and mixing the secondary squeezed liquid, the hydrolyzate, and the primary squeezed liquid to obtain a Platycladus orientalis leaf extract;

[0010] 3) After the Platycladus orientalis leaf extract is concentrated by vacuum distillation, an inorganic salt is added, the extract is allowed to stand for precipitation, and the extract is centrifuged to obtain a precipitate and a supernatant, and the precipitate is dried to obtain Platycladus orientalis leaf protein, and the supernatant is the Platycladus orientalis leaf deproteinized liquid;

[0011] 4) desalting the deproteinized solution of Platycladus orientalis leaves by electrodialysis, and freeze-drying the desalted sample to obtain Platycladus orientalis leaf total polysaccharides;

[0012] 5) extracting the secondary leaf residue with supercritical carbon dioxide to obtain an extract and tertiary leaf residue, and distilling the extract under reduced pressure to obtain total tannins from Platycladus orientalis leaves;

[0013] 6) Mix the leaf residues from the three times with ethanol, stir, let stand, and filter to separate to obtain ethanol solution and leaf residue, concentrate the ethanol solution by distillation under reduced pressure, and then perform silica gel column chromatography, first elute with petroleum ether-n-butanol, collect the eluate, and distill under reduced pressure to obtain Platycladus orientalis leaf volatile oil; then elute with petroleum ether-ethanol, collect the eluate, and distill under reduced pressure to obtain Platycladus orientalis leaf total flavonoids.

[0014] Preferably, in step 1), the fresh Platycladus orientalis leaves are cut before the first pressing, and the cutting length is 3 to 5 cm; the time of the first pressing is 3 to 30 minutes, and the temperature of the first pressing is 20 to 35° C.;

[0015] The primary squeezed liquid is mixed with a preservative and an antioxidant and then sealed and stored. The preservative is any one of benzoate, sorbate, and pyrosulfite, and the amount of the preservative added is 0.05 to 0.5 g / L; the antioxidant is isoascorbic acid or L-ascorbic acid, and the amount of the antioxidant added is 0.01 to 0.2 g / L.

[0016] Preferably, in step 1), the mass ratio of the primary leaf residue to water is 1:2-5; the enzymatic hydrolysis uses cellulase and hemicellulase, the added amount of the cellulase is 10-50 U / g primary leaf residue; the added amount of the hemicellulose is 5-30 U / g primary leaf residue;

[0017] The stirring speed is 100-400 rpm; the enzymatic hydrolysis temperature is 30-50° C., and the enzymatic hydrolysis time is 12-20 h; the centrifugal speed is 5000-9000 rpm, and the centrifugal time is 5-25 min.

[0018] Preferably, the time of the secondary pressing in step 2) is 5 to 25 minutes, and the temperature of the secondary pressing is 20 to 45°C.

[0019] Preferably, in step 3), the multiple of the concentration of the Platycladus orientalis leaf extract by vacuum distillation is 3 to 8; the inorganic salt is ammonium sulfate, and the final concentration of the ammonium sulfate in the Platycladus orientalis leaf extract after vacuum distillation concentration is 65% to 80%;

[0020] The standing temperature is 2-6°C, and the standing time is 12-30h; the centrifugal speed is 6000-12000rpm, the centrifugal temperature is 2-12°C, and the centrifugal time is 10-30min; the drying temperature is 35-50°C, and the drying time is 8-24h.

[0021] Preferably, the operating voltage of the electrodialysis method in step 4) is 100-220V, the operating temperature is 20-45°C, and the injection flow rate is 5-35L / h; the pH value of the deproteinized solution of Platycladus orientalis leaves is 5.0-8.0;

[0022] The freeze drying is vacuum freeze drying, the temperature of the vacuum freeze drying is -90 to -70°C, the vacuum degree of the vacuum freeze drying is 3 to 12 Pa, and the time of the vacuum freeze drying is 20 to 52 hours.

[0023] Preferably, in step 5), the time of the supercritical carbon dioxide extraction is 1 to 6 hours, the temperature of the supercritical carbon dioxide extraction is 30 to 50° C., and the pressure of the supercritical carbon dioxide extraction is 15 to 55 MPa; the carrying agent for the supercritical carbon dioxide extraction is an ethanol solution, the volume fraction of the ethanol solution is 40% to 60%, and the amount of the carrying agent is 1 to 6 mL / g of secondary leaf residue.

[0024] Preferably, in step 6), the mass volume ratio of the tertiary leaf residue and ethanol is 1 g: 10-30 mL; the stirring speed is 80-220 rpm, and the stirring time is 10-20 h; the standing time is 0.5-3 h; the ethanol liquid is concentrated by vacuum distillation by 6-12 times; the volume ratio of petroleum ether and n-butanol in the petroleum ether-n-butanol is 25-50:1, and the elution volume of the petroleum ether-n-butanol is 3-12 BV; the volume ratio of petroleum ether and ethanol in the petroleum ether-ethanol is 4-9:1, and the elution volume of the petroleum ether-ethanol is 12-22 BV.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention is different from previous studies, which aim to extract one or two main active ingredients such as volatile oil and / or flavonoids from Platycladus orientalis leaves, and the remaining ingredients are treated as waste residue or waste liquid. The present invention adopts a graded extraction method to obtain the main active ingredients in Platycladus orientalis leaves, including total protein, total sugar, total tannin, volatile oil and total flavonoids, respectively, through enzymatic hydrolysis and mild means of breaking the wall, and fully treats the Platycladus orientalis leaves to avoid the waste of effective ingredients, thereby improving the comprehensive utilization rate of Platycladus orientalis leaf resources.

[0027] The present invention adopts a fresh squeeze method to avoid the inactivation and denaturation of some components as the water evaporates during the drying process. The contents of total protein, total sugar, total tannin, volatile oil and total flavonoids extracted by the present invention are respectively higher than 80%, 90%, 85%, 82% and 88%. These active ingredients can be directly applied to the fields of daily chemicals and agriculture, or further separated and purified to obtain monomers for application in the fields of medicine and cosmetics, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a process flow diagram of the multi-stage extraction method of active ingredients from Platycladus orientalis leaves of the present invention;

[0029] Figure 2 It is a structural schematic diagram of the screw press of the present invention;

[0030] Figure 3 This is a diagram of the experimental device of the supercritical extraction instrument of the present invention;

[0031] Figure 4 This is a standard curve diagram for determining the total protein content in the embodiment of the present invention;

[0032] Figure 5 This is a standard curve diagram for determining the total sugar content in the embodiment of the present invention;

[0033] Figure 6 This is a standard curve diagram for determining the total tannin content in the embodiment of the present invention;

[0034] Figure 7 This is a standard curve diagram for determining the total flavonoid content in the embodiment of the present invention. DETAILED DESCRIPTION

[0035] The present invention provides a multi-stage extraction method of active ingredients from Platycladus orientalis leaves, comprising the following steps:

[0036] 1) squeezing fresh Platycladus orientalis leaves once to obtain a squeezed liquid and a leaf residue, mixing the leaf residue with water, stirring, enzymatically hydrolyzing, and centrifuging to obtain a hydrolyzate and leaf residue;

[0037] 2) squeezing the leaf residue to obtain a secondary squeezed liquid and secondary leaf residue, and mixing the secondary squeezed liquid, the hydrolyzate, and the primary squeezed liquid to obtain a Platycladus orientalis leaf extract;

[0038] 3) After the Platycladus orientalis leaf extract is concentrated by vacuum distillation, an inorganic salt is added, the extract is allowed to stand for precipitation, and the extract is centrifuged to obtain a precipitate and a supernatant, and the precipitate is dried to obtain Platycladus orientalis leaf protein, and the supernatant is the Platycladus orientalis leaf deproteinized liquid;

[0039] 4) desalting the deproteinized solution of Platycladus orientalis leaves by electrodialysis, and freeze-drying the desalted sample to obtain Platycladus orientalis leaf total polysaccharides;

[0040] 5) extracting the secondary leaf residue with supercritical carbon dioxide to obtain an extract and tertiary leaf residue, and distilling the extract under reduced pressure to obtain total tannins from Platycladus orientalis leaves;

[0041] 6) Mix the leaf residues from the three times with ethanol, stir, let stand, filter and separate to obtain ethanol solution and leaf residue, concentrate the ethanol solution by vacuum distillation, and then perform silica gel column chromatography, first elute with petroleum ether-n-butanol, collect the eluate, and obtain Platycladus orientalis leaf volatile oil by vacuum distillation; then elute with petroleum ether-ethanol, collect the eluate, and obtain Platycladus orientalis leaf total flavonoids by vacuum distillation. The flow chart of the multi-stage extraction method of active ingredients in Platycladus orientalis leaves is as follows: Figure 1 shown.

[0042] In the present invention, fresh Platycladus orientalis leaves are squeezed once to obtain a squeezed liquid and a leaf residue, and the leaf residue is mixed with water, stirred, and enzymatically hydrolyzed and centrifuged to obtain a hydrolyzed liquid and leaf residue. The fresh Platycladus orientalis leaves are cut before the first squeezing, and the length of the cut is preferably 3 to 5 cm, and more preferably 4 cm; the squeezing is preferably carried out in a screw press (structure diagram as shown in FIG. Figure 2 The process is carried out in a vacuum evaporator (as shown in the figure), the power of the screw press is preferably 5-15kw, and more preferably 8-12kw; the time of the primary pressing is preferably 3-30min, and more preferably 5-25min; the temperature of the primary pressing is preferably 20-35°C, and more preferably 25-30°C; the primary pressing liquid is mixed with a preservative and an antioxidant and then sealed and stored, the preservative is preferably any one of benzoate, sorbate, and pyrosulfite, and the amount of the preservative added is preferably 0.05-0.5g / L, and more preferably 0.1-0.4g / L; the antioxidant is preferably isoascorbic acid or L-ascorbic acid, and the amount of the antioxidant added is preferably 0.01-0.2g / L, and more preferably 0.05-0.15g / L; the temperature of the sealed storage is preferably 2-6°C, and more preferably 3-5°C. Compared with the pretreatment method of drying and then crushing the fresh Platycladus orientalis leaves for solvent extraction, the loss of active ingredients can be reduced by 10% to 30% by the rapid pressing extraction of fresh Platycladus orientalis leaves. By strictly controlling the pressing time and temperature, it is possible to avoid excessive heat generated during the spiral pressing process, which may lead to inactivation and denaturation of heat-sensitive active ingredients such as proteins in the Platycladus orientalis leaves. At the same time, adding preservatives and antioxidants at appropriate concentrations can prevent the primary pressing liquid from being contaminated by microorganisms, and the active ingredients from being oxidized or decomposed by microorganisms.

[0043] The mass ratio of the primary leaf residue and water is preferably 1:2-5, and more preferably 1:3-4; the enzymatic hydrolysis preferably uses cellulase and hemicellulase, and the added amount of the cellulase is preferably 10-50U / g primary leaf residue, and more preferably 15-45U / g primary leaf residue; the added amount of hemicellulose is preferably 5-30U / g primary leaf residue, and more preferably 10-25U / g primary leaf residue; the stirring speed is preferably 100-400rpm, and more preferably 150-350rmp; the enzymatic hydrolysis temperature is preferably 30-50°C, and more preferably 35-45°C; the enzymatic hydrolysis time is preferably 12-20h, and more preferably 14-18h; the centrifugal speed is preferably 5000-9000rpm, and more preferably 6000-8000rpm; the centrifugal time is preferably 5-25min, and more preferably 10-20min. One-time pressing usually does not break the cell wall sufficiently. Adding cellulase and hemicellulase to fully hydrolyze under mild conditions can effectively destroy the plant cell wall and release the active ingredients completely. On the other hand, the monosaccharides and oligosaccharides produced after the hydrolysis of cellulose and hemicellulose can also be further collected and extracted as active ingredients. At the same time, controlling the enzymatic hydrolysis temperature within the appropriate temperature range can not only avoid the temperature being too low to reduce the enzyme activity and thus affect the enzymatic hydrolysis efficiency, but also avoid the temperature being too high to cause the enzyme and product to lose activity.

[0044] In the present invention, the leaf residue is squeezed to obtain a secondary squeezed liquid and a secondary leaf residue, and the secondary squeezed liquid, the hydrolyzate, and the primary squeezed liquid are mixed to obtain a Platycladus orientalis leaf extract. The secondary squeeze time is preferably 5 to 25 minutes, more preferably 10 to 20 minutes; the secondary squeeze temperature is preferably 20 to 45°C, more preferably 25 to 40°C.

[0045] In the present invention, the Platycladus orientalis leaf extract is concentrated by vacuum distillation, and then an inorganic salt is added, and the extract is allowed to stand for precipitation, and centrifuged to obtain a precipitate and a supernatant. The precipitate is dried to obtain Platycladus orientalis leaf protein, and the supernatant is Platycladus orientalis leaf deproteinized liquid. The multiple of vacuum distillation concentration of the Platycladus orientalis leaf extract is preferably 3 to 8, and more preferably 4 to 7; the inorganic salt is preferably ammonium sulfate, and the final concentration of ammonium sulfate in the Platycladus orientalis leaf extract after vacuum distillation concentration is preferably 65% ​​to 80%, and more preferably 70 to 75%; the temperature when adding the inorganic salt is preferably 15 to 40°C, and more preferably 20 to 35°C; the standing temperature is preferably 2 to 6°C, and more preferably 3 to 5°C; the standing time is preferably 12 to 30h, and more preferably 15 to 25h; the centrifugal speed is preferably 7000 to 11000rpm, and the centrifugal temperature is preferably 2 to 12°C, and more preferably 4 to 10°C; the centrifugal time is preferably 10 to 30min, and more preferably 15 to 25min; the drying temperature is preferably 35 to 50°C, and more preferably 40 to 45°C; the drying time is preferably 8 to 24h, and more preferably 10 to 14h. The protein in the water extract of Platycladus orientalis leaves will gradually precipitate out due to the salting-out effect as the concentration of the precipitant ammonium sulfate in the water increases. When the ammonium sulfate is saturated, all the protein will precipitate out. Compared with the acid-base precipitation method or the organic solvent precipitation method, ammonium sulfate is mild and stable, and the salting-out process will not destroy the protein structure and activity, and the Platycladus orientalis leaf protein content can be obtained to be ≥80%.

[0046] In the present invention, the deproteinized solution of Platycladus orientalis leaves is desalted by electrodialysis, and the desalted sample is freeze-dried to obtain the total polysaccharides of Platycladus orientalis leaves. The operating voltage of the electrodialysis method is preferably 100-220V, more preferably 120-200V, and the operating temperature is preferably 20-45°C, more preferably 25-40°C; the injection flow rate is preferably 5-35L / h, more preferably 10-30L / h; the pH value of the deproteinized solution of Platycladus orientalis leaves is preferably 5.0-8.0, more preferably 6.0-7.0; the ammonium sulfate introduced by the precipitated protein can be quickly removed by electrodialysis, and the end point of desalination can be determined according to the concentration or conductivity of the ammonium salt in the sample. The freeze-drying is vacuum freeze-drying, and the temperature of the vacuum freeze-drying is preferably -90--70°C, more preferably -85--75°C; the vacuum degree of the vacuum freeze-drying is preferably 3-12Pa, and the time of the vacuum freeze-drying is 20-52h. The content of total sugars in Platycladus orientalis leaves can be obtained to be ≥90%.

[0047] In the present invention, the secondary leaf residue is extracted with supercritical carbon dioxide to obtain an extract and tertiary leaf residue, and the extract is distilled under reduced pressure to obtain total tannins from Platycladus orientalis leaves. Figure 3As shown, the time of supercritical carbon dioxide extraction is preferably 1 to 6 hours, and more preferably 2 to 5 hours; the temperature of supercritical carbon dioxide extraction is preferably 30 to 50°C, and more preferably 35 to 45°C; the pressure of supercritical carbon dioxide extraction is preferably 15 to 55 MPa, and more preferably 20 to 50 MPa; the carrier of supercritical carbon dioxide extraction is preferably an ethanol solution, and the volume fraction of the ethanol solution is preferably 40% to 60%, and more preferably 45 to 55%; the amount of the carrier is preferably 1 to 6 mL / g secondary leaf residue, and more preferably 2 to 5 mL / g secondary leaf residue. Supercritical carbon dioxide extraction is relatively simple to operate, efficient, and mild in conditions, which can avoid the denaturation of chemically unstable tannin compounds during the extraction process; and has high selectivity, and the impurity content in the product is less. Compared with other organic solvents, the ethanol-water system is more environmentally friendly as a carrier. The content of total tannins in Platycladus orientalis leaves directly separated is ≥85%.

[0048] In the present invention, the leaf residues of three times are mixed with ethanol, stirred, allowed to stand, filtered and separated to obtain ethanol solution and leaf residues, the ethanol solution is concentrated by distillation under reduced pressure, and then subjected to silica gel column chromatography, firstly eluted with petroleum ether-n-butanol, the eluate is collected, and the volatile oil of Platycladus orientalis leaves is obtained after distillation under reduced pressure; then eluted with petroleum ether-ethanol, the eluate is collected, and the total flavonoids of Platycladus orientalis leaves are obtained after distillation under reduced pressure. The mass volume ratio of the tertiary leaf residue and ethanol is preferably 1g:10-30mL, and more preferably 1g:15-25mL; the stirring speed is preferably 80-220rpm, and more preferably 100-200rpm; the stirring time is preferably 10-20h, and more preferably 12-18h; the standing time is preferably 0.5-3h, and more preferably 1-2h; the multiple of the ethanol solution concentrated by vacuum distillation is preferably 6-12 times, and more preferably 7-11 times; the silica gel column chromatography method is specifically: the ethanol solution concentrated by vacuum distillation is mixed with silica gel to obtain sample-mixed silica gel, and the sample-mixed silica gel and separation silica gel are sequentially added from top to bottom into the separation column for elution; the mesh number of the silica gel is preferably 60-100 meshes, The preferred size is 80-100 mesh; the mass ratio of the solid content of the ethanol solution after vacuum distillation and concentration to the silica gel is preferably 1:1-2, and more preferably 1:1.2-1.8; the mass ratio of the sample mixing silica gel to the separation silica gel is preferably 1:2-6, and more preferably 1:3-5; the volume ratio of petroleum ether and n-butanol in the petroleum ether-n-butanol is preferably 25-50:1, and more preferably 30-45:1; the elution volume of the petroleum ether-n-butanol is preferably 3-12 BV, and more preferably 4-10 BV; the volume ratio of petroleum ether and ethanol in the petroleum ether-ethanol is preferably 4-9:1, and more preferably 5-8:1; the elution volume of the petroleum ether-ethanol is preferably 12-22 BV, and more preferably 14-20 BV. Silica gel has the characteristics of high separation efficiency and strong selectivity. The polarity of volatile oils and flavonoids is quite different. By selecting silica gel with suitable particle size and reasonable solvent combination, a variety of different types of compounds can be obtained through one separation. The content of volatile oil in the Platycladus orientalis leaves that can be obtained is ≥82%, and the content of total flavonoids in the Platycladus orientalis leaves that can be obtained is ≥88%.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Materials and instruments used in this invention:

[0051] Fresh Platycladus orientalis leaves are produced in Shandong.

[0052] Coomassie Brilliant Blue G-250, bovine serum albumin, casein, cellulase, and hemicellulase were purchased from Solarbio.

[0053] Rutin standard was purchased from Chengdu Purifa Technology Development Co., Ltd.

[0054] 60-100 mesh silica gel was purchased from Qingdao Bangkai High-tech Materials Co., Ltd.

[0055] SHYZ-200 / SHYZ-300 screw press (Shandong Shanheng Environmental Protection Technology Co., Ltd.), CR22GIII centrifuge (HITACHI), DSAD-IV electrodialysis membrane separator (Jiangsu Ritai Environmental Protection Engineering Co., Ltd.), SYnergyMX multifunctional microplate reader (BioTek, USA), Cleverchem380 water quality analyzer (Dechem-Tech, Germany), BT85 vacuum freeze dryer (MillRock, USA), supercritical extraction device (Guizhou Aerospace Wujiang Electromechanical Company).

[0056] Example 1

[0057] This embodiment is a multi-stage extraction method of active ingredients from Platycladus orientalis leaves, which consists of the following steps:

[0058] Step 1: Take 5 kg of fresh Platycladus orientalis leaves in summer, wash and cut into small sections with a length of 3 cm, slowly put the cut Platycladus orientalis leaves into the SHYZ-200 screw press with a power of 5.5 kW, press for 5 minutes, and the pressing temperature is 22°C. Then collect 1.4 kg of primary press liquid and 3.55 kg of primary leaf residue respectively, add 0.1 g / L sodium metabisulfite and 0.05 g / L L-ascorbic acid to the primary press liquid, and then seal and store at 4°C.

[0059] Step 2: Add sterile water 3 times the mass of the leaf residue, 20U / g of cellulase and 25U / g of hemicellulase to the primary leaf residue, control the stirring speed at 200rpm, the hydrolysis temperature at 40℃, hydrolyze for 16h, centrifuge at 8000rpm for 10min to separate the hydrolyzate and leaf residue, and obtain 10.7kg of hydrolyzate and 3.5kg of leaf residue. Press the leaf residue for a second time for 8min at a temperature of 35℃, then collect 0.6kg of secondary pressed liquid and 2.8kg of secondary leaf residue, respectively, mix the primary pressed liquid, hydrolyzate and secondary pressed liquid to obtain a total of 12.7kg of Platycladus orientalis leaf extract.

[0060] Step 3: Concentrate the Platycladus orientalis leaf extract by 4 times to 3.18 kg by reduced pressure distillation, slowly add ammonium sulfate with a final concentration of 70% to the concentrated Platycladus orientalis leaf extract at 20°C, let it stand at 4°C for 18 hours, then centrifuge at 9000 rpm at 4°C for 15 minutes, use the supernatant after centrifugation as the Platycladus orientalis leaf deproteinized liquid, dry the centrifugal precipitate at 40°C for 10 hours, and obtain 47g of Platycladus orientalis leaf total protein.

[0061] Step 4: Desalt the deproteinized solution of Platycladus orientalis leaves using an electrodialysis membrane separator, with an operating voltage of 200 V, an operating temperature of 26° C., a pH value of the deproteinized solution of Platycladus orientalis leaves of 5.4, and a sample flow rate of 10 L / h. After desalting, the ammonium salt concentration of the sample was determined by a water quality analyzer using a salicylic acid method to be 95 mg / L. The desalted sample was pre-frozen and freeze-dried for 30 hours using a vacuum freeze dryer at a freeze drying temperature of -80° C. and a vacuum degree of 5 Pa to obtain 105.1 g of total sugar from Platycladus orientalis leaves.

[0062] Step 5: Use a supercritical carbon dioxide extraction device (instrument such as Figure 7 The secondary leaf residue sample was extracted with the extraction time of 5h, the extraction temperature of 45°C and the extraction pressure of 30MPa; the carrier was ethanol-water with an ethanol content of 45%, and the carrier dosage ratio was 3mL / g secondary leaf residue sample. After the extract was distilled under reduced pressure to remove the solvent, 9.5g of total tannins from Platycladus orientalis leaves were obtained.

[0063] Step 6: Add 15 times of ethanol (mL / g) to the three leaf residues, stir at a speed of 200 rpm, stir for 16 hours and then stand for 1 hour, filter and separate the ethanol solution, concentrate the ethanol solution by vacuum distillation to 8 times, add 60-100 mesh silica gel with a total solid content of 1.5 times that of the ethanol solution, mix evenly and evaporate the solvent as the sample mixing silica gel, add 3 times the amount of sample mixing silica gel to the separation column, and then add the sample mixing silica gel again, use 40:1 petroleum ether (60-90℃) / n-butanol as eluent, elute 6BV, and obtain 47.9g of Platycladus orientalis leaf volatile oil after vacuum distillation of the eluent; continue eluting with 8:1 petroleum ether (60-90℃) / ethanol as eluent, elute 15BV, and obtain 115.7g of Platycladus orientalis leaf total flavonoids after vacuum distillation of the eluent.

[0064] Example 2

[0065] This embodiment is a multi-stage extraction method of active ingredients from Platycladus orientalis leaves, which consists of the following steps:

[0066] Step 1: Take 10 kg of fresh Platycladus orientalis leaves in summer, wash and cut into small sections with a length of 5 cm, slowly put the cut Platycladus orientalis leaves into the SHYZ-200 screw press with a power of 5.5 kW, press for 15 min, and the pressing temperature is 25°C. Then collect 3.1 kg of primary press liquid and 6.8 kg of primary leaf residue respectively, add 0.3 g / L potassium sorbate and 0.08 g / L isoascorbic acid to the primary press liquid, and then seal and store at 4°C.

[0067] Step 2: Add sterile water 4 times the mass of the leaf residue, 25U / g of cellulase and 20U / g of hemicellulase to the primary leaf residue, control the stirring speed at 250rpm, the hydrolysis temperature at 38℃, hydrolyze for 18h, centrifuge at 7000rpm for 15min to separate the hydrolyzate and leaf residue, and obtain 27.3kg of hydrolyzate and 6.6kg of leaf residue. Press the leaf residue for the second time for 15min at a temperature of 30℃, then collect 1.3kg of secondary pressed liquid and 5.2kg of secondary leaf residue respectively, mix the primary pressed liquid, hydrolyzate and secondary pressed liquid to obtain 31.7kg of Platycladus orientalis leaf extract in total.

[0068] Step 3: Concentrate the Platycladus orientalis leaf extract 5 times to 6.3 kg by reduced pressure distillation, slowly add ammonium sulfate with a final concentration of 72% to the concentrated Platycladus orientalis leaf extract at 25°C, let it stand at 4°C for 20 hours, then centrifuge at 8000 rpm at 10°C for 20 minutes, use the supernatant after centrifugation as the Platycladus orientalis leaf deproteinized liquid, dry the centrifugal precipitate at 45°C for 14 hours, and obtain 128.5 g of Platycladus orientalis leaf total protein.

[0069] Step 4: Desalting the deproteinized solution of Platycladus orientalis leaves by using an electrodialysis membrane separator, the operating voltage is 180V, the operating temperature is 30°C, the pH value of the deproteinized solution of Platycladus orientalis leaves is 5.2, the sampling flow rate is 15L / h, and the ammonium salt concentration of the sample is 78mg / L after desalting by using a water quality analyzer by the salicylic acid method. The desalted sample is pre-frozen and freeze-dried for 40h using a vacuum freeze dryer at a freeze drying temperature of -80°C and a vacuum degree of 6Pa to obtain 247.5g of total sugar from Platycladus orientalis leaves.

[0070] Step 5: The secondary leaf residue sample was extracted with a supercritical carbon dioxide extraction device, with an extraction time of 4 hours, an extraction temperature of 40°C, and an extraction pressure of 20 MPa; the carrier was ethanol-water with an ethanol content of 48%, and the carrier dosage ratio was 2 mL / g of secondary leaf residue sample. After the extract was distilled under reduced pressure to remove the solvent, 21.2 g of total tannins from Platycladus orientalis leaves were obtained.

[0071] Step 6: Add 20 times of ethanol (mL / g) to the three leaf residues, stir at a speed of 150 rpm, stir for 16 hours and then stand for 1 hour, filter and separate the ethanol solution, concentrate the ethanol solution by vacuum distillation to 10 times, add 60-100 mesh silica gel with a total solid content of 1.6 times that of the ethanol solution, mix evenly and evaporate the solvent as the sample mixing silica gel, add 3.5 times the amount of sample mixing silica gel to the separation column, and then add the sample mixing silica gel again, use 38:1 petroleum ether (60-90℃) / n-butanol as the eluent, elute 8BV, and obtain 112.5g of Platycladus orientalis leaf volatile oil after vacuum distillation of the eluent; continue eluting with 7:1 petroleum ether (60-90℃) / ethanol as the eluent, elute 16BV, and obtain 242.3g of Platycladus orientalis leaf total flavonoids after vacuum distillation of the eluent.

[0072] Example 3

[0073] This embodiment is a multi-stage extraction method of active ingredients from Platycladus orientalis leaves, which consists of the following steps:

[0074] Step 1: Take 3 kg of fresh Platycladus orientalis leaves in autumn, wash and cut into small sections of 4 cm in length, slowly put the cut Platycladus orientalis leaves into the SHYZ-200 screw press with a power of 5.5 kW, pressing for 8 min and a pressing temperature of 30°C, then collect 0.78 kg of primary pressed liquid and 2.08 kg of primary leaf residue respectively, add 0.2 g / L potassium benzoate and 0.12 g / L L-ascorbic acid to the primary pressed liquid, and then seal and store at 4°C.

[0075] Step 2: Add sterile water 5 times the mass of the leaf residue, 40U / g of cellulase and 30U / g of hemicellulase to the primary leaf residue, control the stirring speed at 300rpm, the hydrolysis temperature at 45℃, hydrolyze for 18h, centrifuge at 6000rpm for 20min to separate the hydrolyzate and leaf residue, and obtain 9.7kg of hydrolyzate and 2.68kg of leaf residue. Press the leaf residue for the second time for 10min at a temperature of 40℃, then collect 0.25kg of secondary pressed liquid and 2.37kg of secondary leaf residue respectively, mix the primary pressed liquid, hydrolyzate and secondary pressed liquid to obtain a total of 10.73kg of Platycladus orientalis leaf extract.

[0076] Step 3: Concentrate the Platycladus orientalis leaf extract by 6 times to 1.79 kg by reduced pressure distillation, slowly add ammonium sulfate with a final concentration of 77% to the concentrated Platycladus orientalis leaf extract at 35°C, let it stand at 4°C for 24 hours, then centrifuge at 8000 rpm at 4°C for 25 minutes, use the supernatant after centrifugation as the Platycladus orientalis leaf deproteinized liquid, dry the centrifugal precipitate at 45°C for 8 hours, and obtain 39g of Platycladus orientalis leaf total protein.

[0077] Step 4: Desalting the deproteinized solution of Platycladus orientalis leaves by using an electrodialysis membrane separator, the operating voltage is 220V, the operating temperature is 32°C, the pH value of the deproteinized solution of Platycladus orientalis leaves is 5.8, the sampling flow rate is 12L / h, and the ammonium salt concentration of the sample is 54mg / L after desalting by using a water quality analyzer with a salicylic acid method. The desalted sample is pre-frozen and freeze-dried for 24h using a vacuum freeze dryer at a freeze drying temperature of -80°C and a vacuum degree of 8Pa to obtain 84.4g of total sugars from Platycladus orientalis leaves.

[0078] Step 5: The secondary leaf residue sample was extracted with a supercritical carbon dioxide extraction device, with an extraction time of 2.5 h, an extraction temperature of 40°C, and an extraction pressure of 40 MPa; the carrier was ethanol-water with an ethanol content of 48%, and the carrier dosage ratio was 3.5 mL / g of secondary leaf residue sample. After the extract was distilled under reduced pressure to remove the solvent, 5.4 g of total tannins from Platycladus orientalis leaves were obtained.

[0079] Step 6: Add 25 times of ethanol (mL / g) to the three leaf residues, stir at a speed of 150 rpm, stir for 12 hours, then let stand for 1 hour, filter and separate the ethanol solution, concentrate the ethanol solution by vacuum distillation to 10 times, add 60-100 mesh silica gel with a total solid content of 1.8 times that of the ethanol solution, mix evenly and evaporate the solvent as the sample mixing silica gel, add 4 times the amount of sample mixing silica gel to the separation column, and then add the sample mixing silica gel again, use 35:1 petroleum ether (60-90℃) / n-butanol as eluent, elute 7BV, and obtain 32.1g of Platycladus orientalis leaf volatile oil after vacuum distillation of the eluent; continue eluting with 7:1 petroleum ether (60-90℃) / ethanol as eluent, elute 13BV, and obtain 55.9g of Platycladus orientalis leaf total flavonoids after vacuum distillation of the eluent.

[0080] Example 4

[0081] This embodiment is a multi-stage extraction method of active ingredients from Platycladus orientalis leaves, which consists of the following steps:

[0082] Step 1: Take 20 kg of fresh Platycladus orientalis leaves in autumn, wash and cut into small sections with a length of 5 cm, slowly put the cut Platycladus orientalis leaves into the SHYZ-300 screw press with a power of 10 kW, pressing for 25 min, and a pressing temperature of 28°C. Then collect 5.9 kg of primary pressed liquid and 13.8 kg of primary leaf residue respectively, add 0.2 g / L sodium benzoate and 0.1 g / L L-ascorbic acid to the primary pressed liquid, and then seal and store at 4°C.

[0083] Step 2: Add sterile water (2 times the mass of the leaf residue), 15U / g of cellulase and 12U / g of hemicellulase to the primary leaf residue, control the stirring speed at 200rpm, the hydrolysis temperature at 42°C, hydrolyze for 14h, centrifuge at 8000rpm for 10min to separate the hydrolyzate and leaf residue, and obtain 26.2kg of hydrolyzate and 14.2kg of leaf residue. Press the leaf residue for a second time for 20min at a temperature of 37°C, then collect 1.25kg of secondary pressed liquid and 12.6kg of secondary leaf residue, respectively, mix the primary pressed liquid, hydrolyzate and secondary pressed liquid, and obtain a total of 33.35kg of Platycladus orientalis leaf extract.

[0084] Step 3: Concentrate the Platycladus orientalis leaf extract by 4 times to 8.3 kg by reduced pressure distillation, slowly add ammonium sulfate with a final concentration of 75% to the concentrated Platycladus orientalis leaf extract at 30°C, let it stand at 4°C for 22 hours, then centrifuge at 10,000 rpm at 6°C for 15 minutes, use the supernatant after centrifugation as the Platycladus orientalis leaf deproteinized liquid, and dry the centrifugal precipitate at 45°C for 19 hours to obtain 268.9 g of Platycladus orientalis leaf total protein.

[0085] Step 4: Desalting the deproteinized solution of Platycladus orientalis leaves by using an electrodialysis membrane separator, the operating voltage is 220V, the operating temperature is 28°C, the pH value of the deproteinized solution of Platycladus orientalis leaves is 6.1, the sampling flow rate is 20L / h, and the ammonium salt concentration of the sample is 43mg / L after desalting by using a water quality analyzer by the salicylic acid method. The desalted sample is pre-frozen and freeze-dried for 48h in a vacuum freeze dryer at a freeze drying temperature of -80°C and a vacuum degree of 10Pa to obtain 517.4g of total sugars from Platycladus orientalis leaves.

[0086] Step 5: The secondary leaf residue sample was extracted with a supercritical carbon dioxide extraction device, with an extraction time of 3 hours, an extraction temperature of 35°C, and an extraction pressure of 40 MPa; the carrier was ethanol-water with an ethanol content of 55%, and the carrier dosage ratio was 5 mL / g of secondary leaf residue sample. After the extract was distilled under reduced pressure to remove the solvent, 30.8 g of total tannins from Platycladus orientalis leaves were obtained.

[0087] Step 6: Add 15 times of ethanol (mL / g) to the three leaf residues, stir at a speed of 100 rpm, stir for 12 hours and then stand for 2 hours, filter and separate the ethanol solution, concentrate the ethanol solution by vacuum distillation to 8 times, add 60-100 mesh silica gel with a total solid content of 1.8 times that of the ethanol solution, mix evenly and evaporate the solvent as the sample mixing silica gel, add 5 times the amount of sample mixing silica gel to the separation column, and then add the sample mixing silica gel again, use 30:1 petroleum ether (60-90℃) / n-butanol as eluent, elute 10BV, and obtain 197.1g of Platycladus orientalis leaf volatile oil after vacuum distillation of the eluent; continue eluting with 5:1 petroleum ether (60-90℃) / ethanol as eluent, elute 20BV, and obtain 413g of Platycladus orientalis leaf total flavonoids after vacuum distillation of the eluent.

[0088] Example 5

[0089] This embodiment is a multi-stage extraction method of active ingredients from Platycladus orientalis leaves, which consists of the following steps:

[0090] Step 1: Take 40 kg of fresh Platycladus orientalis leaves in autumn, wash and cut into small sections with a length of 5 cm, slowly put the cut Platycladus orientalis leaves into the SHYZ-300 screw press with a power of 10 kW, press for 30 minutes, and a pressing temperature of 24°C. Then collect 13.5 kg of primary press liquid and 24.8 kg of primary leaf residue respectively, add 0.4 g / L potassium benzoate and 0.2 g / L isoascorbic acid to the primary press liquid, and then seal and store at 4°C.

[0091] Step 2: Add sterile water (2 times the mass of the leaf residue), 45U / g of cellulase and 30U / g of hemicellulase to the primary leaf residue, control the stirring speed at 350rpm, and the hydrolysis temperature at 45°C. Hydrolyze for 18h, centrifuge at 8000rpm for 20min to separate the hydrolyzate and leaf residue, and obtain 45.4kg of hydrolyzate and 25.4kg of leaf residue. Press the leaf residue for a second time for 20min at a temperature of 26°C, then collect 2.15kg of secondary pressed liquid and 22.1kg of secondary leaf residue, respectively, mix the primary pressed liquid, hydrolyzate and secondary pressed liquid, and obtain a total of 61.05kg of Platycladus orientalis leaf extract.

[0092] Step 3: Concentrate the Platycladus orientalis leaf extract 5 times to 12.2 kg by reduced pressure distillation, slowly add ammonium sulfate with a final concentration of 73% to the concentrated Platycladus orientalis leaf extract at 25°C, let it stand at 4°C for 18 hours, then centrifuge at 9000 rpm at 4°C for 20 minutes, use the supernatant after centrifugation as the Platycladus orientalis leaf deproteinized liquid, dry the centrifugal precipitate at 40°C for 23 hours, and obtain 498.1g of Platycladus orientalis leaf total protein.

[0093] Step 4: Desalt the deproteinized solution of Platycladus orientalis leaves using an electrodialysis membrane separator, with an operating voltage of 220V, an operating temperature of 25°C, a pH value of 6.3, and a sample flow rate of 30L / h. After desalting, the ammonium salt concentration of the sample was determined by a water quality analyzer using a salicylic acid method to be 88mg / L. The desalted sample was pre-frozen and freeze-dried for 48h using a vacuum freeze dryer at a freeze drying temperature of -80°C and a vacuum degree of 5Pa to obtain 1047.2g of total sugar from Platycladus orientalis leaves.

[0094] Step 5: The secondary leaf residue sample was extracted with a supercritical carbon dioxide extraction device, the extraction time was 2h, the extraction temperature was 45°C, and the extraction pressure was 50MPa; the carrier was ethanol-water, the ethanol content was 50%, and the carrier dosage ratio was 4mL / g secondary leaf residue sample. After the extract was distilled under reduced pressure to remove the solvent, 63.9g of total tannins from Platycladus orientalis leaves were obtained.

[0095] Step 6: Add 18 times of ethanol (mL / g) to the three leaf residues, stir at a speed of 150 rpm, stir for 18 hours, then let stand for 1 hour, filter and separate the ethanol solution, concentrate the ethanol solution by vacuum distillation to 9 times, add 60-100 mesh silica gel with a total solid content of 1.5 times that of the ethanol solution, mix evenly and evaporate the solvent as the sample mixing silica gel, add 3 times the amount of sample mixing silica gel to the separation column, and then add the sample mixing silica gel again, use 35:1 petroleum ether (60-90℃) / n-butanol as eluent, elute 5BV, and obtain 415.4g of Platycladus orientalis leaf volatile oil after vacuum distillation of the eluent; continue eluting with 5.5:1 petroleum ether (60-90℃) / ethanol as eluent, elute 14BV, and obtain 853g of Platycladus orientalis leaf total flavonoids after vacuum distillation of the eluent.

[0096] Experimental Example 1

[0097] The detection method of total protein refers to the Coomassie Brilliant Blue staining method, as follows:

[0098] Reagent preparation: Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of 95% ethanol, add 100 mL of 85% phosphoric acid, dilute to 1000 mL with distilled water, and filter with filter paper. The final reagent contains 0.01% (W / V) Coomassie Brilliant Blue G-250, 4.7% (W / V) ethanol, and 8.5% (W / V) phosphoric acid.

[0099] Preparation of standard protein solution: Crystallized bovine serum albumin was prepared into 1 mg / mL protein solution with 0.15 mol NaCl.

[0100] Preparation of standard curve and sample testing: Add 5.0mL of Coomassie Brilliant Blue reagent to 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, and 0.8mL of standard protein solution, respectively, and shake evenly. After 5 minutes, measure the absorbance at 595nm, and draw a standard curve with absorbance as the ordinate and protein concentration as the abscissa. The standard curve is as shown in the figure. Figure 4 As shown. For the unknown protein sample concentration, the total protein of Platycladus orientalis leaves of Examples 1 to 5 was taken as a sample, and its measured value was within the linear range of the standard curve. According to the measured absorbance value at 595 nm, the protein concentration of the unknown sample was calculated and converted into protein content.

[0101] Experimental results: as shown in Table 1.

[0102] Table 1 Total protein content of Platycladus orientalis leaves obtained in different treatment groups

[0103]

[0104]

[0105] As can be seen from Table 1, under the method of the present invention, the total protein content obtained after salting out precipitation is higher than 80%, and can reach up to 85.9%. When the total protein content extracted in Example 3 is the highest, the temperature for adding ammonium sulfate is the highest, which is 35°C; the final concentration of ammonium sulfate added is also the highest, which is 77%; and the salting out time is the longest, which is 24h. It can be seen that the higher the temperature when adding ammonium sulfate during the salting out process, the higher the proportion of ammonium sulfate added, the higher the degree of salt saturation, and the better the salting out effect. In addition, the longer the salting out time, the more complete the protein precipitation, and the higher the content of total protein finally obtained.

[0106] Experimental Example 2

[0107] The detection method of total sugar refers to the phenol-sulfuric acid method, as follows:

[0108] Preparation of reference solution: Weigh 1000 mg of anhydrous glucose dried to constant weight at 105°C, place in 50 mL, and add water to dissolve to volume.

[0109] Preparation of standard curve: Measure 0, 0.5, 1, 2, 5, and 10 mL of the reference solution into a 25 mL stoppered test tube, fill to 10 mL with distilled water, add 1.0 mL of 5% phenol solution, shake well, add 9 mL of concentrated sulfuric acid, shake well, boil in a boiling water bath for 3 to 5 minutes, take out and place at room temperature. Measure the absorbance at a wavelength of 485 nm with the reagent blank as a reference. Draw a standard curve with absorbance as the ordinate and glucose concentration as the abscissa. The standard curve is as follows: Figure 5 shown.

[0110] Preparation of sample solution and sample testing: Weigh the total sugar of Platycladus orientalis leaves of Examples 1 to 5 as a sample, so that its measured value is within the linear range of the standard curve, place it in a 50mL volumetric flask, add an appropriate amount of hot water and ultrasonically vibrate to dissolve it, and then make up to volume to obtain the sample solution. Take 2mL of the sample solution in a 25mL stoppered test tube, fill it up to 10mL with distilled water, add 1.0mL of 5% phenol solution, shake well, quickly add 9mL of concentrated sulfuric acid, and shake well. Boil in a boiling water bath for 2 to 3 minutes, take it out and place it at room temperature, which is the sample solution to be tested. The absorbance of the sample solution to be tested is measured at 485nm with the reagent blank as a reference, so as to calculate the sample concentration and convert it into sugar content.

[0111] Experimental results: as shown in Table 2.

[0112] Table 2 Total sugar content of Platycladus orientalis leaves obtained in different treatment groups

[0113]

[0114]

[0115] As shown in Table 2, under the method of the present invention, the content of total sugar obtained after desalting is higher than 90%, and can reach up to 93.1%. When the total sugar content extracted in Example 5 is the highest, the amount of cellulase and hemicellulase added is the highest, which are 45U / g and 30U / g respectively; it can be seen that within a certain range, the higher the amount of cellulase and hemicellulase added, the more complete the hydrolysis of plant structural polysaccharides, and the higher the content of total sugar obtained.

[0116] Experimental Example 3

[0117] The detection method of total tannins refers to the phosphomolybdic acid-casein colorimetric method, as follows:

[0118] Preparation of phosphomolybdic tungstic acid test solution: Take 100g of sodium tungstate and 25g of sodium molybdate, add 700mL of water to dissolve, add 100mL of hydrochloric acid and 50mL of phosphoric acid, heat and reflux for 10h, cool, then add 150g of lithium sulfate, 50mL of water and 0.2mL of bromine, boil to remove the residual bromine (about 15min), cool, dilute to 1000mL with water, filter, and the phosphomolybdic tungstic acid test solution is obtained.

[0119] Preparation of reference solution: Weigh 50 mg of gallic acid sample, place in a 100 mL brown volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0120] Preparation of standard curve: Take 1mL, 2mL, 3mL, 4mL, and 5mL of reference solution, add 1mL of phosphomolybdic acid test solution to each, dilute to 10mL with 29% sodium carbonate solution, and shake well. Use the corresponding reagent as blank, measure the absorbance at a wavelength of 760nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa. The standard curve is as follows: Figure 6 shown.

[0121] Preparation of sample solution: Take the total tannins from Platycladus orientalis leaves prepared in Examples 1 to 5 as a sample, place it in a 250 mL brown volumetric flask, add 150 mL of water, leave it overnight, ultrasonically treat it for 10 min, cool it, dilute it to the scale with water, shake it well, let it stand, filter it, discard 50 mL of the initial filtrate, measure 20 mL of the subsequent filtrate, place it in a 100 mL brown volumetric flask, dilute it to the scale with water, shake it well, and obtain the sample solution.

[0122] Determination of total phenols: Weigh 2 mL of sample solution, refer to the standard curve preparation method, add 1 mL of phosphomolybdic acid test solution, add water to 10 mL, determine the absorbance in the same way, read the amount of gallic acid in the sample solution from the standard curve, and calculate the total phenol content.

[0123] Determination of non-adsorbed polyphenols: Take 25 mL of the sample solution, add it to a 100 mL stoppered conical flask containing 0.6 g of casein, seal it, and place it in a 30°C shaking water bath for 1 hour. Take it out and shake it well, filter it, discard the initial filtrate, measure 2 mL of the subsequent filtrate, and refer to the standard curve preparation method. Start by adding 1 mL of phosphomolybdic acid test solution and add water to 10 mL. Determine the absorbance in the same way, read the amount of gallic acid in the sample solution from the standard curve, and calculate the amount of non-adsorbed polyphenols.

[0124] Tannin content: Tannin content is the difference between total phenol content and non-adsorbed polyphenol content.

[0125] Experimental results: as shown in Table 3.

[0126] Table 3 Total tannin content of Platycladus orientalis leaves obtained in different treatment groups

[0127] Treatment Group Total tannin content Example 1 87.5％ Example 2 85.4％ Example 3 88.1％ Example 4 87.6％ Example 5 85.7％

[0128] It can be seen from Table 3 that, under the method of the present invention, the content of total tannins obtained by supercritical extraction is higher than 85%, and can reach up to 88.1%. Within a certain range, appropriately increasing the extraction pressure can increase the content of total tannins, but the pressure should not be too high, which will affect the safety of the equipment and the stability of the extraction, and may also destroy the target product with an unstable chemical structure. The extraction pressure is most suitably controlled at 30-40MPa.

[0129] Experimental Example 4

[0130] The volatile oil from the Platycladus orientalis leaves of Examples 1 to 5 was taken as a sample, and the detection method of the volatile oil was carried out by steam distillation with reference to the volatile oil determination method A of the 2020 edition of the Pharmacopoeia-2204.

[0131] Experimental results: as shown in Table 4.

[0132] Table 4 Volatile oil content of Platycladus orientalis leaves obtained in different treatment groups

[0133] Treatment Group Volatile oil content Example 1 84.6％ Example 2 85.7％ Example 3 82.8％ Example 4 86.4％ Example 5 83.7％

[0134] As shown in Table 4, under the method of the present invention, the total volatile oil content obtained by silica gel separation is higher than 82%, and can reach 86.4% at the highest. When the total volatile oil content extracted in Example 4 is the highest, the ratio of eluent petroleum ether (60-90°C) / n-butanol is 30:1; the elution volume is 10BV. It can be seen that within a certain range, the greater the polarity of the mixed eluent, the easier it is for the target product volatile oil to be eluted, the larger the elution volume, the more sufficient the separation effect, and the higher the final volatile oil content.

[0135] Experimental Example 5

[0136] The detection method of total flavonoids refers to the phenol-sulfuric acid method, as follows:

[0137] Preparation of reference solution: Weigh 10 mg of rutin standard and dissolve it in ethanol in a 10 mL volumetric flask to make a reference solution with a concentration of 1.0 mg / mL.

[0138] Preparation of standard curve: weigh 0, 1, 2, 4, 6, 8 mL of rutin reference solution, dilute to 10 mL with ethanol, take 1 mL of the solution to be tested in a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution, mix well and let stand for 6 min, add 0.5 mL of 10% aluminum nitrate solution, mix well and let stand for 6 min, add 4 mL of 4% sodium hydroxide solution, mix well and let stand for 15 min, dilute to volume with ethanol, and measure its absorbance at 510 nm. Draw a standard curve with absorbance as the ordinate and rutin concentration as the abscissa. The standard curve is as follows: Figure 7 shown.

[0139] Preparation of sample solution and sample test: Take an appropriate amount of total flavonoids of Platycladus orientalis leaves in Examples 1 to 5 as a sample, so that its measured value is within the linear range of the standard curve, add 100 mL of ethanol until it is completely dissolved, which is the sample solution. Take 1 mL of the sample solution in a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution in sequence, mix and let stand for 6 minutes, add 0.5 mL of 10% aluminum nitrate solution, mix and let stand for 6 minutes, add 4 mL of 4% sodium hydroxide solution, mix and let stand for 15 minutes, make up to volume with ethanol, and measure the absorbance of the sample solution to be tested at 510 nm with the reagent blank as a reference, so as to calculate the sample concentration converted into flavonoid content.

[0140] Experimental results: as shown in Table 5.

[0141] Table 5 Total flavonoids content of Platycladus orientalis leaves obtained in different treatment groups

[0142]

[0143]

[0144] As shown in Table 5, under the method of the present invention, the total flavonoid content obtained by silica gel separation is higher than 88%, and can reach up to 92.7%. When the total flavonoid content extracted in Example 4 is the highest, the ratio of petroleum ether (60-90°C) / ethanol of the eluent is 5:1; the elution volume is 20BV. Similar to the separation of volatile oils, within a certain range, the greater the polarity of the mixed eluent, the easier it is for the target product total flavonoids to be eluted, the larger the elution volume, the more complete the separation effect, and the higher the final total flavonoid content.

[0145] It can be seen from the above embodiments and experimental examples that the present invention adopts a graded extraction method to obtain the main active ingredients in Platycladus orientalis leaves by breaking the wall through mild enzymatic hydrolysis, and the contents of total protein, total sugar, total tannin, volatile oil and total flavonoids obtained by extraction are respectively higher than 80%, 90%, 85%, 82% and 88%, which greatly improves the comprehensive utilization rate of Platycladus orientalis leaf resources.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A multi-stage extraction method of active ingredients from Platycladus orientalis leaves, characterized in that: The following steps are involved: 1) squeezing fresh Platycladus orientalis leaves once to obtain a squeezed liquid and a leaf residue, mixing the leaf residue with water, stirring, enzymatically hydrolyzing, and centrifuging to obtain a hydrolyzate and leaf residue; 2) squeezing the leaf residue to obtain a secondary squeezed liquid and secondary leaf residue, and mixing the secondary squeezed liquid, the hydrolyzate, and the primary squeezed liquid to obtain a Platycladus orientalis leaf extract; 3) After the Platycladus orientalis leaf extract is concentrated by vacuum distillation, an inorganic salt is added, the extract is allowed to stand for precipitation, and the extract is centrifuged to obtain a precipitate and a supernatant, and the precipitate is dried to obtain Platycladus orientalis leaf protein, and the supernatant is the Platycladus orientalis leaf deproteinized liquid; 4) desalting the deproteinized solution of Platycladus orientalis leaves by electrodialysis, and freeze-drying the desalted sample to obtain Platycladus orientalis leaf total polysaccharides; 5) extracting the secondary leaf residue with supercritical carbon dioxide to obtain an extract and tertiary leaf residue, and distilling the extract under reduced pressure to obtain total tannins from Platycladus orientalis leaves; 6) Mix the leaf residues from the three times with ethanol, stir, let stand, and filter to separate to obtain ethanol solution and leaf residue, concentrate the ethanol solution by distillation under reduced pressure, and then perform silica gel column chromatography, first elute with petroleum ether-n-butanol, collect the eluate, and distill under reduced pressure to obtain Platycladus orientalis leaf volatile oil; then elute with petroleum ether-ethanol, collect the eluate, and distill under reduced pressure to obtain Platycladus orientalis leaf total flavonoids.

2. The multi-stage extraction method according to claim 1, characterized in that: Step 1) the fresh Platycladus orientalis leaves are cut before the first pressing, the cutting length is 3 to 5 cm; the time of the first pressing is 3 to 30 minutes, and the temperature of the first pressing is 20 to 35° C.; The primary squeezed liquid is mixed with a preservative and an antioxidant and then sealed and stored. The preservative is any one of benzoate, sorbate, and pyrosulfite, and the amount of the preservative added is 0.05 to 0.5 g / L; the antioxidant is isoascorbic acid or L-ascorbic acid, and the amount of the antioxidant added is 0.01 to 0.2 g / L.

3. The multi-stage extraction method according to claim 1, characterized in that: Step 1) The mass ratio of the primary leaf residue to water is 1:2-5; the enzymatic hydrolysis uses cellulase and hemicellulase, the added amount of the cellulase is 10-50 U / g primary leaf residue; the added amount of the hemicellulose is 5-30 U / g primary leaf residue; The stirring speed is 100-400 rpm; the enzymatic hydrolysis temperature is 30-50° C., and the enzymatic hydrolysis time is 12-20 h; the centrifugal speed is 5000-9000 rpm, and the centrifugal time is 5-25 min.

4. The multi-stage extraction method according to claim 1, characterized in that: Step 2) The time of the secondary pressing is 5 to 25 minutes, and the temperature of the secondary pressing is 20 to 45°C.

5. The multi-stage extraction method according to claim 1, characterized in that: Step 3) the Platycladus orientalis leaf extract is concentrated by vacuum distillation at a multiple of 3 to 8; the inorganic salt is ammonium sulfate, and the final concentration of the ammonium sulfate in the Platycladus orientalis leaf extract after vacuum distillation and concentration is 65% to 80%; The standing temperature is 2-6°C, and the standing time is 12-30h; the centrifugal speed is 6000-12000rpm, the centrifugal temperature is 2-12°C, and the centrifugal time is 10-30min; the drying temperature is 35-50°C, and the drying time is 8-24h.

6. The multi-stage extraction method according to claim 1, characterized in that: Step 4) The operating voltage of the electrodialysis method is 100-220V, the operating temperature is 20-45°C, and the injection flow rate is 5-35L / h; the pH value of the deproteinized solution of Platycladus orientalis leaves is 5.0-8.0; The freeze drying is vacuum freeze drying, the temperature of the vacuum freeze drying is -90 to -70°C, the vacuum degree of the vacuum freeze drying is 3 to 12 Pa, and the time of the vacuum freeze drying is 20 to 52 hours.

7. The multi-stage extraction method according to claim 1, characterized in that: Step 5) The time of the supercritical carbon dioxide extraction is 1 to 6 hours, the temperature of the supercritical carbon dioxide extraction is 30 to 50° C., and the pressure of the supercritical carbon dioxide extraction is 15 to 55 MPa; the carrying agent of the supercritical carbon dioxide extraction is an ethanol solution, the volume fraction of the ethanol solution is 40% to 60%, and the amount of the carrying agent is 1 to 6 mL / g of secondary leaf residue.

8. The multi-stage extraction method according to claim 1, characterized in that: Step 6) The mass volume ratio of the tertiary leaf residue and ethanol is 1g:10-30mL; the stirring speed is 80-220rpm, and the stirring time is 10-20h; the standing time is 0.5-3h; the ethanol solution is concentrated by vacuum distillation by 6-12 times; the volume ratio of petroleum ether and n-butanol in the petroleum ether-n-butanol is 25-50:1, and the elution volume of the petroleum ether-n-butanol is 3-12BV; the volume ratio of petroleum ether and ethanol in the petroleum ether-ethanol is 4-9:1, and the elution volume of the petroleum ether-ethanol is 12-22BV.

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