Extraction method and application of effective components in larch

Abstract

The invention discloses an extraction method and application of effective components in larch. A preparation method of a larch extract comprises the following steps: S1, peeling and crushing; s2, continuous countercurrent extraction and concentration; s3, flocculation and centrifugation; (S4); performing microfiltration and clarification; s5, separating sugar from flavone; s6, purifying the dihydroquercetin; and S7, purifying the arabinogalactan. According to the method, larch is comprehensively developed, the green and environment-friendly concept is met, and meanwhile the production cost of an enterprise is remarkably reduced. The finished product is controllable in quality and high in stability, and the larch extract which is wide in application and good in effect can be provided for the industries of cosmetics, food and health food.

Description

Technical Field

[0001] The invention discloses a method for extracting effective components from larch and application thereof, belonging to the technical field of cosmetic raw material preparation. Background Art

[0002] Larch (Larix gmelinii (Rμpr.) Kμzen.) is a tree of the genus Larix in the Pinaceae family. It is mostly distributed in the cooler temperate regions of the northern hemisphere, especially in the northern forests of the northern hemisphere, such as Siberia, Russia, Canada, North American forests, and Northeast China. It grows widely in the upper part of the mountainous area and has a high utilization value. Due to the needs of domestic production, a large amount of larch wood is cut down every year, and there are more than 10 million tons of larch roots remaining underground. Dihydroquercetin, as an effective ingredient in larch, has a high content and can be used as a raw material for dihydroquercetin extraction. Although dihydroquercetin exists in many natural plants, the content is very low, and the extraction and preparation make it expensive and cannot be effectively fully utilized. At the same time, the content of arabinogalactan in larch roots is extremely rich, which can reach 15%-30%. It is a neutral polysaccharide composed of arabinose and galactose. In the food industry, it is often used as a sweetener, emulsifier, stabilizer, thickener and other food additives. It has physiological functions such as enhancing immunity, inhibiting tumors, directional transport, and improving intestinal flora. It is an extremely important resource for the utilization of abandoned larch. Drugs made with arabinogalactan as the main ingredient have also appeared internationally.

[0003] In view of the current situation of extracting and utilizing various bioactive components contained in the residues after logging and processing, we can recycle and reuse larch wood waste. Optimizing the extraction and purification process and developing corresponding technologies for the effective utilization of active substances in wood are important measures to utilize larch plant resources, improve the industrial structure of forest areas, achieve the reuse of waste wood chips, and reduce production costs.

[0004] Patent CN101157733A discloses a method for simultaneously extracting dihydroquercetin and arabinogalactan from larch processing residues, using ester or ether solvents for extraction and n-butanol for precipitation of arabinogalactan. Although this preparation method can obtain high-purity dihydroquercetin and arabinogalactan, the steps are complicated, a large amount of harmful organic solvents are introduced, and n-butanol is difficult to recover due to its high boiling point, which is not suitable for sustainable development.

[0005] Patent CN110408324A discloses a pretreatment steam extraction method for rosin, arabinogalactan and dihydroquercetin in larch wood powder, wherein arabinogalactan is extracted with water, and dihydroquercetin and rosin are extracted with steam. The concentrated and dried extract is recrystallized to obtain rosin and dihydroquercetin. This method uses steam at 124-140°C for extraction, which consumes huge energy and has high production costs.

[0006] Patent CN1844095 discloses a method for extracting dihydroquercetin from larch, wherein water is used as an extractant to extract the Dahurian larch, and the obtained aqueous extract is subjected to liquid-liquid extraction using methyl tert-butyl ether as an extractant, and the extract is adsorbed and decolorized using activated carbon or activated clay, and the paste after the solvent is removed is crystallized and purified using water to obtain dihydroquercetin. Methyl tert-butyl ether is a carcinogen, and the dihydroquercetin produced has residues and is harmful to the health of production personnel.

[0007] In view of the many shortcomings of the existing larch extract preparation methods, and the fact that they are not suitable for comprehensive extraction of effective components in larch, it is urgent to develop a method for rapid and large-scale preparation of effective components in larch that can be widely used in the research and development of various common foods, health foods, and cosmetics. Summary of the invention

[0008] The purpose of the present invention is to overcome the above-mentioned technical defects and provide a method for extracting larch which is simple to operate, uses less raw materials and is fast.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] A method for extracting effective ingredients from larch comprises the following steps:

[0011] S1. Peeling and crushing

[0012] Select air-dried larch roots with a diameter of more than 5 cm, remove the red root bark, and grind them with a plant grinder, and pass them through a 1-3 mm sieve. The sieved material is the grinded larch root powder;

[0013] S2. Water extraction and concentration

[0014] The ground larch root powder in S1 is put into hot water heated to a certain temperature for continuous countercurrent extraction, and after the extraction is completed, the solid and liquid are separated by squeezing to obtain an extract, and the extract is concentrated under reduced pressure using a single-effect concentrator to obtain a concentrate;

[0015] S3, Flocculation Centrifugation

[0016] The concentrated solution obtained in step S2 is cooled to a certain temperature and then the pH is adjusted, and the ZTC clarifier B component and the ZTC clarifier A component are added to the concentrated solution in sequence, and after stirring for sufficient reaction, the flocculation is removed by centrifugation;

[0017] S4, microfiltration clarification

[0018] The supernatant obtained in step S3 after flocculation is filtered through a microfiltration membrane, and when the retentate is 5% of the original volume, it is washed with deionized water, and the microfiltration permeate and the washing permeate are combined and collected to obtain a microfiltration membrane liquid;

[0019] S5, sugar and flavonoid separation

[0020] The microfiltration membrane liquid obtained in step S3 is pumped into an intermittent stirring tank, a certain amount of polyamide powder is added to the stirring tank, and after being fully stirred, it is pumped into a plate and frame filter press for filtration to obtain a first filtrate and a first filter cake, and after the filtration is completed, the first filter cake is washed with deionized water, and the first water washing liquid is obtained by filtration. The obtained first filtrate and the first water washing liquid are combined for standby use, and the filter cake is transferred to a reactor, and a urea solution is pumped into the reactor. After being fully stirred for reaction, it is pumped into a plate and frame filter press to obtain a second filtrate and a second filter cake, and the second filter cake is discharged after being treated, and after the filtration is completed, the filter cake is washed with deionized water and then filtered to obtain a second water washing liquid, and the obtained second filtrate and the second water washing liquid are combined for standby use;

[0021] S6, Dihydroquercetin Purification

[0022] The second filtrate and the second water washing liquid of step S5 are combined to obtain a liquid, a saturated lead acetate aqueous solution is added to the solution, the solution is allowed to stand for precipitation, and then the solution is washed with water to obtain a first precipitate. The first precipitate is dissolved and then subjected to an ion exchange resin column to remove lead. The effluent is collected and concentrated and dried to obtain a crude dihydroquercetin product. The crude dihydroquercetin product is mixed with a solvent, heated to dissolve, and filtered to obtain a third filtrate and a second precipitate. The third filtrate is cooled and crystallized, and the crystals are filtered and dried to obtain;

[0023] S7. Arabinogalactan purification

[0024] The first filtrate and the first washing liquid in step S5 are combined to obtain a liquid which is passed through an anion exchange resin and a cation exchange resin in sequence. After the column loading is completed, 1BV of the liquid is washed in sequence. The effluent and the washing liquid are collected and combined, desalted and concentrated by a nanofiltration membrane. After the retentate is concentrated, 95% ethanol is added to the ethanol content to 80%, and the alcohol insoluble matter is obtained by filtration. The alcohol insoluble matter is washed with a certain concentration of ethanol until the filtrate is colorless to obtain a third filter cake. The third filter cake is dried to obtain arabinogalactan.

[0025] Preferably, in step S2, the continuous countercurrent extraction time is 0.5-1.5h; the weight ratio of larch root powder to water is 1:3-1:15; the number of extractions is 2-3 times; and the extraction water temperature is 70-90°C.

[0026] Preferably, in step S2, when the single-effect concentrator is concentrated under reduced pressure, the temperature is 50-75° C. and the vacuum degree is 0.07 MPa-0.09 MPa.

[0027] Preferably, in step S2, the concentration endpoint is 0.1-1 g / ml.

[0028] Preferably, in step S3, the flocculation pH=3.0-6.0.

[0029] Preferably, in step S3, the addition amounts of the ZTC1+1Ⅱ type clarifier B component and the ZTC1+1Ⅱ type clarifier A component are 0.5%-10% and 0.05%-4% of the mass of the concentrated liquid, and the intervals for adding the flocculation component B and the flocculation component A are 1-3h, respectively.

[0030] Preferably, in step S3, the centrifugation is one of butterfly centrifugation, horizontal screw centrifugation and tubular centrifugation, and the rotation speed of the centrifugation is 5000-20000 rpm / min.

[0031] Preferably, in step S4, the filtration pore size of the microfiltration membrane is 0.1-1 mm; the filtration pore size of the microfiltration membrane is 0.1-1 mm; the microfiltration membrane element is one of a ceramic membrane, a flat membrane, a spiral membrane and a hollow fiber membrane,

[0032] Preferably, the microfiltration membrane element is a ceramic membrane.

[0033] Preferably, in step S4, the operating pressure of the microfiltration is 0.01-0.2 MPa; the microfiltration temperature is 10-45° C.; and the microfiltration membrane filtration method is cross-flow filtration.

[0034] Preferably, in step S4, the endpoint of the microfiltration water washing is that the concentration of the permeate is less than 0.2 Brix.

[0035] Preferably, in step S5, the concentration of the urea solution is 0.1-5% (w / v), and the volume of the urea is 1-20 times (v / w) the mass of the first filter cake.

[0036] Preferably, in step S6, the saturated lead acetate aqueous solution is prepared on-the-go.

[0037] Preferably, in step S6, the ion exchange resin column is a strongly acidic styrene-based cation exchange resin (hydrogen type), preferably one of a Diaion SK-103 cation exchange resin column, an Amberlite IR-120 cation exchange resin column and a D001 cation exchange resin column.

[0038] Preferably, in step S6, the drying is vacuum drying or spray drying.

[0039] Preferably, in step S7, the anion exchange resin is a chloride-type weakly basic anion exchange resin with styrene as a skeleton, and the anion exchange resin is a salt-type resin.

[0040] Preferably, in step S7, the cation exchange resin is a sodium-type weakly acidic cation exchange resin with styrene as a skeleton, and the cation exchange resin is a salt-type resin.

[0041] Preferably, in step S7, the molecular weight cut-off of the nanofiltration membrane is 100-1000D.

[0042] Preferably, in step S7, the nanofiltration operating pressure is 0.5-2.0 MPa; the microfiltration temperature is 10-45° C.; and the nanofiltration membrane filtration method is cross-flow filtration.

[0043] Preferably, in step S7, the concentration of ethanol used for washing the alcohol-insoluble matter is 70%-95%.

[0044] Preferably, in step S7, the drying is spray drying or freeze drying.

[0045] The present invention also discloses an application of an effective ingredient in larch obtained by any of the above-mentioned extraction methods, wherein the effective ingredient is applied in a non-therapeutic cosmetic, which is a skin external preparation prepared by using the effective ingredient as an active component and adding conventional pharmaceutical or cosmetic excipients or auxiliary ingredients.

[0046] Preferably, the non-therapeutic cosmetics are one or more of lotion, essence, cream, mask and gel.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The present invention provides a new method for extracting high-purity dihydroquercetin and arabinogalactan from larch roots. The purity of the prepared dihydroquercetin is greater than 95% (HPLC) and the recovery rate is greater than 91%; the content of arabinogalactan is greater than 90% and the recovery rate is greater than 92%. The obtained dihydroquercetin can be used as a high-quality plant raw material in cosmetics and sold on the market.

[0049] (2) The method and process provided by the present invention is highly operable, low-cost, pollution-free, and suitable for industrial production. It not only realizes the comprehensive utilization of larch resources, turning waste into treasure and creating considerable economic benefits, but also solves the problem of environmental pollution caused by waste liquid after the extraction of dihydroquercetin from larch.

[0050] (3) The dihydroquercetin prepared by the present invention has been tested to have multiple effects such as antioxidant, whitening and anti-wrinkle, and is expected to become a highly effective cosmetic plant raw material. DETAILED DESCRIPTION

[0051] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0053] Example 1

[0054] S1. Peeling and crushing

[0055] Select larch roots with a diameter of more than 5 cm, remove the red root bark and grind them with a plant grinder, then pass them through a 1 mm sieve. The sieved material is the grinded larch root powder.

[0056] S2. Water extraction and concentration

[0057] Accurately weigh 100 kg of larch root powder, add it to 500 L of hot water at 85°C for continuous countercurrent extraction for 90 minutes, extract the residue with 400 L of water under the same conditions for 60 minutes, squeeze and separate the solid and liquid after two extractions, and combine the liquids obtained from the two extractions to obtain the extract. Pump the extract into a single-effect concentrator, concentrate the extract at 70°C and 0.09 MPa, and concentrate the extract under reduced pressure to a volume of 300 L.

[0058] S3, Flocculation Centrifugation

[0059] The concentrated solution obtained in step S2 is cooled to a temperature of 60°C, and the pH is adjusted to 5.0 with hydrochloric acid. 12.0 L of fully swollen clarifier ZTC clarifier B component is added to the concentrated solution while stirring the feed liquid. After 1.5 hours, 6.0 L of fully swollen clarifier ZTC clarifier B component is added. After sufficient stirring, the flocculation is removed by centrifugation to obtain a clear solution.

[0060] S4, microfiltration clarification

[0061] The flocculated clear liquid obtained in step S3 is filtered through a microfiltration membrane. When the retentate is 15 L, deionized water is introduced into the storage barrel to wash out the target components in the retentate. The microfiltration permeate and the water washing permeate are combined and collected to obtain the microfiltration membrane liquid.

[0062] S5, sugar and flavonoid separation

[0063] The microfiltration membrane liquid obtained in step S3 is pumped into an intermittent stirring tank, 20 kg of polyamide powder is added to the stirring tank, and the stirring paddle is turned on to stir at a speed of 100 rpm / min for 90 minutes. After the stirring is completed, the slurry is pumped into a plate-frame filter press for filtration, and the filtrate and filter cake are collected to obtain the first filtrate and the first filter cake. After the filtration is completed, the first filter cake is washed with deionized water, and the first water washing liquid is obtained by filtration. The obtained first filtrate and the first water washing liquid are combined for standby use. After the first filter cake is unloaded, it is transferred to a reactor, and a urea solution with a volume of 60 L and a concentration of 2% is pumped into the reactor. The stirring paddle is turned on to stir at a speed of 100 rpm / min for 90 minutes to obtain a second filtrate and a second filter cake. After the second filter cake is unloaded, it is treated and discharged, and after the filtration is completed, the filter cake is washed with deionized water and then filtered to obtain a second water washing liquid. The obtained second filtrate and the second water washing liquid are combined for standby use.

[0064] S6, Dihydroquercetin Purification

[0065] The second filtrate of step S5 and the second washing liquid were combined to obtain a saturated lead acetate aqueous solution until no new precipitation was generated, stirred and allowed to stand for 30 minutes, and centrifuged to obtain a first precipitation. After the first precipitation was dissolved, it was removed from the D001 ion exchange resin column at a flow rate of 1BV / h, washed with 1BV of deionized water after the column was completed, and the effluent and washing liquid were combined and collected and concentrated and dried to obtain a crude dihydroquercetin product. The crude dihydroquercetin product was mixed with 12.0L deionized water, heated to dissolve and filtered to obtain a third filtrate and a second precipitate. The third filtrate was cooled and crystallized at a temperature of 2°C for 16h to obtain dihydroquercetin crystals. After filtering the crystals, vacuum drying was performed at a temperature of 45°C to obtain 1.091kg of white dihydroquercetin, and the purity was 97.67% by HPLC, and the recovery rate was 91.95%.

[0066] S7. Arabinogalactan purification

[0067] The supernatant liquid obtained by combining the first filtrate and the first water washing liquid in step S5 is successively passed through a series-connected 335-type weakly basic anion exchange column (chloride form) and a D110-type weakly acidic cation exchange column (sodium form). After the column loading is completed, it is washed with water for 1 BV successively. The effluent and the washing liquid are combined and passed through a nanofiltration membrane with a molecular weight cut-off of 200 D under a pressure of 1.5 MPa for desalination and concentration to 25.0 Brix. The retentate of the nanofiltration membrane is pumped into an alcohol precipitation tank, and 95% ethanol pre-cooled to 5°C is added to the alcohol precipitation tank while stirring. After adding until the alcohol content reaches 80%, it is left standing for 2 h, filtered, and the filter cake is washed with 95% ethanol at 5°C until the filtrate is colorless. The filter cake is obtained as 19.7 kg of arabinogalactan after freeze-drying. The obtained arabinogalactan is hydrolyzed and pre-column derivatized, and the arabinogalactan content is measured to be 93.11% and the recovery rate is 93.94% by HPLC.

[0068] Example 2

[0069] S1. Peeling and pulverizing

[0070] Select larch roots with a diameter of more than 5 cm, remove the red root bark, pulverize them with a plant pulverizer, and pass through a 1-mm sieve. The material passing through the sieve is the pulverized larch root powder.

[0071] S2. Water extraction and concentration

[0072] Accurately weigh 200 kg of larch root powder, feed it into 1000 L of hot water at 86°C, and perform continuous countercurrent extraction for 90 min. After the extraction is completed, the residue is extracted with 700 L of water under the same conditions for 60 min. After the two extractions are completed, the solid and liquid are separated by extrusion respectively. The liquids obtained from the two extractions are combined to obtain an extract. The extract is pumped into a single-effect concentrator, and the extract is concentrated at a temperature of 70°C and a pressure of 0.09 MPa. The extract is concentrated under reduced pressure to a volume of 600 L.

[0073] S3. Flocculation and centrifugation

[0074] Cool the concentrated liquid obtained in step S2 to a temperature of 60°C, adjust the pH of the concentrated liquid to 5.2 with hydrochloric acid, and add 25.0 L of the fully swollen clarifying agent ZTC clarifying agent B component to the concentrated liquid while stirring. After reacting for 1.5 h, add 12.50 L of the fully swollen clarifying agent ZTC clarifying agent B component, and stir well to sediment impurities such as proteins, tannins, and gums. Centrifuge to remove the flocculent mass to obtain a clear liquid.

[0075] S4. Microfiltration clarification

[0076] The flocculated clear liquid obtained in step S3 is pumped into an inorganic ceramic membrane with a filtration pore size of 0.4 μm for filtration. When the retentate is 30 L, deionized water is introduced into the storage barrel for washing to wash out the target components in the retentate. When the concentration of the permeate is less than 0.2 Brix, the washing is stopped, and the microfiltration permeate and the water-washing permeate are combined and collected to obtain the microfiltration membrane liquid.

[0077] S5, sugar and flavonoid separation

[0078] The microfiltration membrane liquid obtained in step S3 is pumped into an intermittent stirring tank, 40.2 kg of polyamide powder is added to the stirring tank, and the stirring paddle is turned on to stir at a speed of 110 rpm / min for 90 min. After the stirring is completed, the slurry is pumped into a plate-frame filter press for filtration, and the filtrate and filter cake are collected to obtain the first filtrate and the first filter cake. After the filtration is completed, the first filter cake is washed with deionized water, and the first water washing liquid is obtained by filtration. The obtained first filtrate and the first water washing liquid are combined for standby use. After the first filter cake is unloaded, it is transferred to a reactor, and a urea solution with a volume of 125.0 L and a concentration of 2% is pumped into the reactor. The stirring paddle is turned on to stir at a speed of 100 rpm / min for 60 min to obtain a second filtrate and a second filter cake. After the second filter cake is unloaded, it is treated and discharged, and after the filtration is completed, the filter cake is washed with deionized water and then filtered to obtain a second water washing liquid. The obtained second filtrate and the second water washing liquid are combined for standby use.

[0079] S6, Dihydroquercetin Purification

[0080] The second filtrate of step S5 and the second washing liquid were combined to obtain a saturated lead acetate aqueous solution until no new precipitation was generated, stirred and allowed to stand for 30 minutes, and centrifuged to obtain a first precipitation. After the first precipitation was dissolved, it was removed from the lead on a DiaionSK-103 ion exchange resin column at a flow rate of 1BV / h. After the column was completed, it was washed with 0.8BV of deionized water, and the effluent and washing liquid were collected and concentrated and dried to obtain a crude dihydroquercetin product. The crude dihydroquercetin product was mixed with 12.0L deionized water, heated to dissolve and filtered to obtain a third filtrate and a second precipitation. The third filtrate was cooled and crystallized at a temperature of 4°C for 12h to obtain dihydroquercetin crystals. After filtering the crystals, vacuum drying was performed at a temperature of 45°C to obtain 2.06kg of light yellow pure dihydroquercetin, and the purity was 97.08% by HPLC, and the recovery rate was 92.08%.

[0081] S7. Arabinogalactan purification

[0082] The liquid supernatant obtained by combining the first filtrate and the first water washing liquid in step S5 is sequentially passed through a 335 type weak alkaline anion exchange column (chloride type) and a D110 type weak acid cation exchange column (sodium type) connected in series, and then washed with water for 1BV after the column loading is completed. The effluent and the water washing liquid are combined and collected, desalted and concentrated to 25.0 Brix through a nanofiltration membrane with a molecular weight cutoff of 200D under a pressure of 1.5MPa. The nanofiltration membrane retentate is pumped into an alcohol precipitation tank, and 95% ethanol precooled to 5°C is added to the alcohol precipitation tank while stirring. After the alcohol content is 80%, it is allowed to stand for 2h, filtered, and the filter cake is washed with 95% ethanol at 5°C until the filtrate is colorless. After freeze-drying the filter cake, 39.01kg of arabinogalactan is obtained. The obtained arabinogalactan is hydrolyzed and pre-column derivatized. HPLC measures the arabinogalactan content to be 93.11%, and the recovery rate is 93.94%.

[0083] Example 3

[0084] S1. Peeling and crushing

[0085] Select larch roots with a diameter of more than 5 cm, remove the red root bark, and then grind them with a plant grinder and pass them through a 1 mm sieve. The sieved material is the grinded larch root powder.

[0086] S2. Water extraction and concentration

[0087] Accurately weigh 1000 kg of larch root powder, add it to 5000 L of hot water at 88°C for continuous countercurrent extraction for 60 minutes, extract the residue with 4000 L of water under the same conditions for 60 minutes, squeeze the solid and liquid after the two extractions, and combine the liquids obtained from the two extractions to obtain the extract. Pump the extract into a single-effect concentrator, concentrate the extract at a temperature of 70°C and a pressure of 0.095 MPa, and concentrate the extract under reduced pressure to a volume of 3000 L.

[0088] S3, Flocculation Centrifugation

[0089] The concentrated solution obtained in step S2 is cooled to a temperature of 55° C., and the pH value of the concentrated solution is adjusted to 5.2 with hydrochloric acid. 120.0 L of fully swollen clarifier ZTC clarifier B component is added thereto while stirring the concentrated solution. After fully reacting for 2.0 h, 60.0 L of fully swollen clarifier ZTC clarifier B component is added, and the solution is fully stirred to precipitate impurities such as protein, tannin and gum, and the flocculants are removed by centrifugation to obtain a clear solution.

[0090] S4, microfiltration clarification

[0091] The flocculated clear liquid obtained in step S3 is pumped into an inorganic ceramic membrane with a filtration pore size of 0.4 μm for filtration. When the retentate is 150.1 L, deionized water is introduced into the storage barrel for washing to wash out the target components in the retentate. When the concentration of the permeate is less than 0.2 Brix, the washing is stopped, and the microfiltration permeate and the water washing permeate are combined and collected to obtain the microfiltration membrane liquid.

[0092] S5, sugar and flavonoid separation

[0093] The microfiltration membrane liquid obtained in step S3 is pumped into an intermittent stirring tank, 201.0 kg of polyamide powder is added to the stirring tank, and the stirring paddle is turned on to stir at a speed of 110 rpm / min for 90 min. After the stirring is completed, the slurry is pumped into a plate-frame filter press for filtration, and the filtrate and filter cake are collected to obtain the first filtrate and the first filter cake. After the filtration is completed, the first filter cake is washed with deionized water, and the first water washing liquid is obtained by filtration. The obtained first filtrate and the first water washing liquid are combined for standby use. After the first filter cake is unloaded, it is transferred to a reactor, and a urea solution with a volume of 550 L and a concentration of 2.1% is pumped into the reactor, and the stirring paddle is turned on to stir at a speed of 100 rpm / min for 60 min to obtain a second filtrate and a second filter cake. After the second filter cake is unloaded, it is treated and discharged, and after the filtration is completed, the filter cake is washed with deionized water and then filtered to obtain a second water washing liquid. The obtained second filtrate and the second water washing liquid are combined for standby use.

[0094] S6, Dihydroquercetin Purification

[0095] The first filtrate of step S5 and the first water washing liquid were combined to obtain a saturated lead acetate aqueous solution until no new precipitation was generated, stirred and allowed to stand for 30 minutes, and centrifuged to obtain the first precipitation. After the first precipitation was dissolved, it was removed from the AmberliteIR-120 ion exchange resin column at a flow rate of 1BV / h, and washed with 0.8BV of deionized water after the column was completed. The effluent and the washing liquid were combined and concentrated and dried to obtain a crude dihydroquercetin product. The crude dihydroquercetin product was mixed with 12.0L deionized water, heated to dissolve and filtered to obtain a third filtrate and a second precipitate. The third filtrate was cooled and crystallized at a temperature of 4°C for 12h to obtain dihydroquercetin crystals. After filtering the crystals, vacuum drying was performed at a temperature of 45°C to obtain a light yellow pure dihydroquercetin product 2056.9g, and the HPLC detection purity was 96.17%, and the recovery rate was 91.50%.

[0096] S7. Arabinogalactan purification

[0097] The liquid supernatant obtained by combining the second filtrate and the second water washing liquid in step S5 is sequentially passed through a 335 type weak alkaline anion exchange column (chloride type) and a D110 type weak acid cation exchange column (sodium type) connected in series, and then washed with water for 1BV after the column loading is completed. The effluent and the water washing liquid are combined and collected, desalted and concentrated to 25.0 Brix through a nanofiltration membrane with a molecular weight cutoff of 200D under a pressure of 1.5MPa. The nanofiltration membrane retentate is pumped into an alcohol precipitation tank, and 95% ethanol precooled to 5°C is added to the alcohol precipitation tank while stirring. After the alcohol content is 80%, it is allowed to stand for 2h, filtered, and the filter cake is washed with 95% ethanol at 5°C until the filtrate is colorless. After freeze-drying the filter cake, 19.07g of arabinogalactan is obtained. The obtained arabinogalactan is hydrolyzed and pre-column derivatized. HPLC measures the arabinogalactan content to be 95.11%, and the recovery rate is 93.94%.

[0098] Comparative Example 1

[0099] Compared with Example 1, the temperature of the continuous countercurrent extraction described in step S2 is to keep the liquid surface slightly boiling, and the remaining steps are exactly the same as in Example 1. The mass of the obtained dihydroquercetin in step S6 is 0.85kg, the content of dihydroquercetin is 93.16%, the mass of the obtained arabinogalactan is 15.39kg, and the content is 85.64%. Compared with Example 1, the yields of the obtained dihydroquercetin and arabinogalactan are significantly reduced, and the purity is reduced to a certain extent. It is analyzed that the high-temperature water extraction leaches impurities such as rosin, etc., and rosin is easy to combine dihydroquercetin and arabinogalactan, causing dihydroquercetin to be difficult to be completely precipitated by lead acetate during the purification process, and arabinogalactan is more difficult to be precipitated by alcohol and left in ethanol, affecting the final yield and purity of the two.

[0100] Comparative Example 2

[0101] Compared with Example 1, the process flow minus the flocculation centrifugation of step S3, and the remaining steps are exactly the same as Example 1. The mass of dihydroquercetin obtained in step S6 is 1.01 kg, and the content of dihydroquercetin is 69.16%, and the mass of arabinogalactan obtained is 19.12 kg, and the content is 76.84%. After minus this step, the purity of dihydroquercetin and arabinogalactan is greatly reduced. Analysis shows that the macromolecular structure of the protein wraps a large amount of target components, which are precipitated together with the target components by the reagent precipitation, resulting in a decrease in the purity of both.

[0102] Comparative Example 3

[0103] Compared with Example 1, the urea in step S5 is changed to 95% ethanol, and the remaining steps are exactly the same as in Example 1. The mass of dihydroquercetin obtained in step S6 is 0.53 kg, the content of dihydroquercetin is 92.85%, and the mass of arabinogalactan obtained is 19.03 kg, and the content is 93.02%. The yield of the obtained dihydroquercetin is greatly reduced, which is inferred to be that 95% ethanol is difficult to completely react with the first filter cake, so that the dihydroquercetin is transferred to 95% ethanol, resulting in a significant decrease in the yield.

[0104] In order to better illustrate the advantages of the present invention, four efficacy experiments of dihydroquercetin provided by the present invention are given below:

[0105] Experiment 1: DPPH free radical scavenging experiment

[0106] Accurately weigh 41.67 mg of DPPH reagent, dilute to 100 ml brown volumetric flask, take 5 ml of it and dilute to 50 ml brown volumetric flask with anhydrous ethanol to obtain DPPH test solution (0.1 mmol / L). Pipette 1 ml of DPPH test solution respectively, and add 0.5 ml of 10 μg / ml concentration of dihydroquercetin prepared in Examples 1-3 and Comparative Examples 1-3 respectively as shown in Table 2. After fully mixing, let stand in the dark for 30 minutes, use anhydrous ethanol as a blank control, measure its absorbance at 517 nm, and set vitamin C as a control, and calculate the scavenging rate of DPPH by the sample according to the following formula:

[0107] DPPH clearance rate (%) = [1-(As-Ar) / A0] × 100%, where: As is the absorbance of the extract and DPPH reaction system; Ar is the absorbance of the extract-anhydrous ethanol; A0 is the absorbance of the blank control anhydrous ethanol.

[0108] Table 1. Reaction solution composition

[0109] Absorbance Extraction solution added (ml) DPPH test solution (ml) Anhydrous ethanol (ml) A0 0 1 0.5 Ar 0.5 0 1 As 0.5 1 0

[0110] The free radicals produced by mitochondria during electron transfer will attack cell membranes, proteins and even DNA, leading to cell aging and even death, and are closely related to a variety of diseases. Inhibiting free radical activity can effectively alleviate the degradation of cell structure and function under the action of excessive free radicals produced after external stimulation, thereby maintaining the normal barrier structure of various organs including the skin. The higher the clearance rate, the better the antioxidant level, and it also promotes the repair of the skin barrier.

[0111] Table 2. Effect of dihydroquercetin on scavenging DPPH free radicals

[0112] Dihydroquercetin (10.0 μg / ml) DPPH clearance rate (%) Example 1 63.66 Example 2 65.72 Example 3 64.19 Comparative Example 1 48.72 Comparative Example 2 42.95 Comparative Example 3 47.28

[0113] As can be seen from Table 2, the dihydroquercetin samples prepared in Examples 1-3 and Comparative Examples 1-3 have a DPPH free radical scavenging rate of more than 45%, which is good. The scavenging rate of Examples 1-3 is significantly higher than that of Comparative Examples 1-3, indicating that the extracts prepared by a specific process have better antioxidant effects. The DPPH free radical scavenging rate of Examples 1-3 is as high as more than 80%. In summary, it is shown that dihydroquercetin has a good DPPH free radical scavenging rate, good antioxidant capacity, and can effectively alleviate the degradation of cell structure function under the action of excessive free radicals generated after external stimulation, and has a good effect of promoting skin barrier repair.

[0114] Experiment 2: ABTS free radical scavenging experiment

[0115] ABTS free radical scavenging ability is also often used to indicate antioxidant capacity. Its principle is to react with potassium persulfate to generate a stable blue-green cationic free radical ABTS. + The antioxidant active substance reacts with the ion, and a single electron is transferred from the antioxidant molecule to the oxidant molecule, causing the blue color in the reaction system to fade. The absorbance change at 734nm can be measured under an ultraviolet spectrophotometer to evaluate its ability to scavenge free radicals.

[0116] Accurately pipette 0.2 ml of sample solution, add 7.8 ml of ABTS free radical working solution, mix well and stand at room temperature (25°C) in the dark for 10 min, measure the absorbance at 734 nm, calculate the clearance rate, prepare a standard curve with the clearance rate as y and the concentration of Trolox solution (μg / ml) as x, and measure the clearance rate of each sample at a concentration of 100 μg / ml for ABTS.

[0117] y=(A0-A1-A2) / A0*100%, where: y——scavenging rate, %; A0——absorbance of 0.2ml distilled water+7.8ml, ABTS free radical working solution; A1——absorbance of 0.2ml sample / Trolox standard solution+ABTS free radical working solution; A2——absorbance of 0.2ml sample / Trolox standard solution+ethanol solution (to eliminate color errors between different samples).

[0118] Trolox standard working solution was used as the positive control, and anhydrous ethanol was used as the blank control.

[0119] Table 3. Effect of dihydroquercetin on scavenging ABTS free radicals

[0120] Dihydroquercetin (5.0 μg / ml) ABTS free radical scavenging rate (%) Example 1 93.80 Example 2 94.14 Example 3 93.79 Comparative Example 1 85.14 Comparative Example 2 77.32 Comparative Example 3 83.28

[0121] As shown in Table 3, the dihydroquercetin samples prepared in Examples 1-3 and Comparative Examples 1-3 have a scavenging rate of ABTS free radicals of more than 75%, which is a good scavenging rate. The scavenging rate of Examples 1-3 is significantly higher than that of Comparative Examples 1-3, indicating that the extracts prepared by a specific process have better antioxidant effects. The scavenging rate of ABTS free radicals in Examples 1-3 is as high as more than 93.0%. In summary, it is shown that dihydroquercetin has a good scavenging rate of ABTS free radicals, has good antioxidant capacity, and can effectively alleviate the degradation of cell structure and function under the action of excessive free radicals generated after external stimulation.

[0122] Test 3: Tyrosinase inhibition test

[0123] The tyrosinase inhibitory activity of dihydroquercetin was determined by spectrophotometry. Take 60 μL of 100 μg / ml dihydroquercetin solution, add 40 μL (500 μg / ml) tyrosinase solution, and incubate at 25°C for 5 minutes. Then add 200 μL of -DOPA solution (0.5 mM) to start the reaction, react at 25°C for 5 minutes, and quickly measure the absorbance of the reaction solution at 475 nm after the reaction is completed.

[0124] The tyrosinase inhibition rate of peptides at different concentrations was calculated using the following formula:

[0125] Tyrosinase inhibitory activity (%) = [(AB) - (CD)] × 100 / (AB)

[0126] Where A is the absorbance value of the reaction solution without inhibitor, B is the absorbance value of the reaction solution without inhibitor and enzyme, C is the absorbance value of the reaction solution containing L-DOPA, and D is the absorbance value of the reaction solution without enzyme.

[0127] Definition of tyrosinase activity: At 25°C, the amount of enzyme required to catalyze L-DOPA to produce 1 mol dopaquinone is defined as one unit (μ) of tyrosinase activity.

[0128] Table 4. Inhibitory effect of dihydroquercetin on tyrosinase

[0129] Group P-value Significance Inhibition rate (%) BC / / / PC 0.0001 *** 51.24 Example 1 0.022 * 50.60 Example 2 0.008 * 52.06 Example 3 0.015 * 50.79 Comparative Example 1 0.029 * 35.84 Comparative Example 2 0.019 * 31.12 Comparative Example 3 0.009 * 44.38

[0130] As can be seen from Table 4, the dihydroquercetin prepared in Examples 1-3 and Comparative Examples 1-3 has a tyrosinase inhibition rate of more than 31% at a concentration of 100 μg / ml, and has a good inhibitory effect on tyrosinase. The clearance rate of Examples 1-3 is significantly higher than that of Comparative Examples 1-3, indicating that the extract prepared by a specific process has a better tyrosinase inhibition effect.

[0131] Test 4: Cellular level - anti-wrinkle efficacy test

[0132] The dihydroquercetin prepared in Examples 1-3 and Comparative Examples 1-3 was selected for testing, and the test was as follows:

[0133] Cytotoxicity assay: Adjust the concentration of HFF-1 cell suspension and add it to a 96-well plate. Incubate at 37°C and 5% CO2 for 24 hours. Prepare a series of concentration gradient samples and add 100 μl / well to a 96-well plate. Incubate at 37°C and 5% CO2 for 24 hours. 2 The culture medium was discarded, the cells were washed twice with PBS, 50 μl / well of MTT solution was added, and the cells were incubated at 37°C and 5% CO 2 Culture for 2-6 hours under the same conditions. Discard the medium containing MTT, add DMSO, 150 μl / well, shake for 5-10 minutes to mix, and measure the OD value at 570 nm. Select the concentration without obvious cytotoxicity for protein content determination experiment.

[0134] Determination of type I collagen content: Adjust the concentration of HFF-1 cell suspension and add it to a 96-well plate at 37°C and 5% CO 2 The old culture medium was discarded, and the model was induced with μVB. After irradiation, 100 μl of DMEM culture medium containing the sample was added to each well. At the same time, a culture medium blank control group and a culture medium containing TGF-β were set as positive control groups. The cells were placed at 37°C and 5% CO 2 Culture under the conditions of 24h±2h. After the culture, the content of human type I collagen was determined according to the instructions of the ELISA kit. A standard curve was made with the concentration of the standard as the horizontal axis and the OD450 value as the vertical axis. According to the OD450 value of the sample, the standard curve was inserted to obtain the content of collagen in the experimental group. Finally, the average value of 3 replicate wells in each group was selected as the final collagen result. GraphPadPrism or other statistical software was used for statistical and significance analysis, and the P value was calculated. P<0.05 indicated a significant difference, otherwise there was no significant difference.

[0135] Up-regulation rate (%) = (T / C-1) * 100%, where: T is the average collagen value of the experimental group; C is the average collagen value of the blank / solvent group.

[0136] The results of the cytotoxicity test showed that when the concentration was 150 μg / ml, the survival rate of HFF-1 cells was 99.25%, so the concentration of 150 μg / ml was selected for the test.

[0137] Table 5. Experimental results of dihydroquercetin on collagen content

[0138] Group Collagen mean (ng / ml) P-value Significance Inhibition rate (%) BC 0.18 / / / PC 0.37 0.0001 **** 106.13 Example 1 0.57 0.0003 **** 215.19 Example 2 0.58 0.0001 **** 220.13 Example 3 0.57 0.0001 **** 219.18 Comparative Example 1 0.52 0.0001 **** 192.78 Comparative Example 2 0.50 0.0001 **** 181.05 Comparative Example 3 0.53 0.0001 **** 195.90

[0139] The experimental results of dihydroquercetin on collagen content are shown in Table 5 above, NC is the experimental blank control, and PC group is the positive control. As can be seen from the table, when adding examples 1-3 within the safe concentration range, the collagen upregulation rate is higher than that of the samples of comparative examples 1-3, and the average intracellular collagen is 0.34ng / ml. Compared with the NC group, the collagen content is increased by 88.34%, which is slightly lower than the positive group, indicating that the composition has a good effect of promoting cell collagen synthesis and can play a good anti-wrinkle effect.

[0140] In summary, the present invention studies the characteristic plant larch in the field of cosmetic technology, and obtains high-purity dihydroquercetin and arabinogalactan powders through peeling and crushing, continuous countercurrent extraction and concentration, flocculation centrifugation, microfiltration clarification, sugar and flavonoid separation, dihydroquercetin purification, arabinogalactan purification and other processes, wherein dihydroquercetin has good antioxidant, anti-wrinkle and whitening effects, can effectively brighten the skin, slow down skin aging, deeply improve the skin condition, and has good effects.

[0141] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for extracting effective ingredients from larch, It is characterized in that The following steps are involved: S1. Peeling and crushing Select air-dried larch roots with a diameter of more than 5 cm, remove the red root bark, and grind them with a plant grinder, and pass them through a 1-3 mm sieve. The sieved material is the grinded larch root powder; S2. Water extraction and concentration The ground larch root powder in S1 is put into hot water heated to a certain temperature for continuous countercurrent extraction, and after the extraction is completed, the solid and liquid are separated by squeezing to obtain an extract, and the extract is concentrated under reduced pressure using a single-effect concentrator to obtain a concentrate; S3, Flocculation Centrifugation The concentrated solution obtained in step S2 is cooled to a certain temperature and then the pH is adjusted, and the ZTC clarifier B component and the ZTC clarifier A component are added to the concentrated solution in sequence, and after stirring for sufficient reaction, the flocculation is removed by centrifugation; S4, microfiltration clarification The supernatant obtained in step S3 after flocculation is filtered through a microfiltration membrane, and when the retentate is 5% of the original volume, it is washed with deionized water, and the microfiltration permeate and the washing permeate are combined and collected to obtain a microfiltration membrane liquid; S5, sugar and flavonoid separation The microfiltration membrane liquid obtained in step S3 is pumped into an intermittent stirring tank, a certain amount of polyamide powder is added to the stirring tank, and after being fully stirred, it is pumped into a plate and frame filter press for filtration to obtain a first filtrate and a first filter cake, and after the filtration is completed, the first filter cake is washed with deionized water, and the first water washing liquid is obtained by filtration. The obtained first filtrate and the first water washing liquid are combined for standby use, and the filter cake is transferred to a reactor, and a urea solution is pumped into the reactor. After being fully stirred for reaction, it is pumped into a plate and frame filter press to obtain a second filtrate and a second filter cake, and the second filter cake is discharged after being treated, and after the filtration is completed, the filter cake is washed with deionized water and then filtered to obtain a second water washing liquid, and the obtained second filtrate and the second water washing liquid are combined for standby use; S6, Dihydroquercetin Purification The second filtrate and the second water washing liquid of step S5 are combined to obtain a liquid, a saturated lead acetate aqueous solution is added to the solution, the solution is allowed to stand for precipitation, and then the solution is washed with water to obtain a first precipitate. The first precipitate is dissolved and then subjected to an ion exchange resin column to remove lead. The effluent is collected and concentrated and dried to obtain a crude dihydroquercetin product. The crude dihydroquercetin product is mixed with a solvent, heated to dissolve, and filtered to obtain a third filtrate and a second precipitate. The third filtrate is cooled and crystallized, and the crystals are filtered and dried to obtain; S7. Arabinogalactan purification The first filtrate and the first washing liquid in step S5 are combined to obtain a liquid which is passed through an anion exchange resin and a cation exchange resin in sequence. After the column loading is completed, 1BV of the liquid is washed in sequence. The effluent and the washing liquid are collected and combined, desalted and concentrated by a nanofiltration membrane. After the retentate is concentrated, 95% ethanol is added to the ethanol content to 80%, and the alcohol insoluble matter is obtained by filtration. The alcohol insoluble matter is washed with a certain concentration of ethanol until the filtrate is colorless to obtain a third filter cake. The third filter cake is dried to obtain arabinogalactan.

2. The extraction method according to claim 1, It is characterized in that In step S2, the continuous countercurrent extraction time is 0.5-1.5 hours, the weight ratio of larch root powder to water is 1:3-1:15, the number of extractions is 2-3 times; and the extraction water temperature is 70-90°C.

3. The extraction method according to claim 1, It is characterized in that In step S2, when the single-effect concentrator is concentrated under reduced pressure, the temperature is 50-75°C, the vacuum degree is 0.07MPa-0.09MPa, and the concentration end point is 0.1-1g / ml.

4. The extraction method according to claim 1, It is characterized in that In step S3, the flocculation pH is 3.0-6.0, the addition amounts of the ZTC1+1Ⅱ type clarifier B component and the ZTC1+1Ⅱ type clarifier A component are 0.5%-10% and 0.05%-4% of the mass of the concentrated liquid, respectively, the intervals for adding the flocculation component B and the component A are 1-3h, respectively, the centrifugation is one of butterfly centrifugation, horizontal screw centrifugation and tubular centrifugation, and the rotation speed of the centrifugation is 5000-20000rpm / min.

5. The extraction method according to claim 1, It is characterized in that In step S4, the filtration pore size of the microfiltration membrane is 0.1-1 mm; the microfiltration membrane element is one of a ceramic membrane, a flat membrane, a rolled membrane and a hollow fiber membrane; the operating pressure of the microfiltration is 0.01-0.2 MPa; the microfiltration temperature is 10-45°C; the microfiltration membrane filtration mode is cross-flow filtration, and the endpoint of the microfiltration water washing is that the concentration of the permeate is less than 0.2 Brix.

6. The extraction method according to claim 1, It is characterized in that In step S4 and step S5, the concentration of the urea solution is 0.1-5%, and the volume of the urea is 1-20 times the mass of the first filter cake.

7. The extraction method according to claim 1, It is characterized in that In step S6, the ion exchange resin column is a strongly acidic styrene-based cation exchange resin, preferably one of a Diaion SK-103 cation exchange resin column, an Amberlite IR-120 cation exchange resin column and a D001 cation exchange resin column, and the drying is vacuum drying or spray drying.

8. The extraction method according to claim 1, It is characterized in that In step S7, the anion exchange resin is a weakly basic anion exchange resin with styrene as the skeleton, the cation exchange resin is a weakly acidic cation exchange resin with styrene as the skeleton, the molecular weight cutoff of the nanofiltration membrane is 100-1000D, the operating pressure of the nanofiltration is 0.5-2.0MPa; the nanofiltration temperature is 10-45°C; and the filtration mode of the nanofiltration membrane is cross-flow filtration.

9. Use of effective ingredients in larch obtained by the extraction method according to any one of claims 1 to 8, It is characterized in that The active ingredient is used in non-therapeutic cosmetics, which are skin external preparations prepared with the active ingredient as an active component and conventional pharmaceutical or cosmetic excipients or auxiliary ingredients.

10. The use according to claim 9, It is characterized in that The non-therapeutic cosmetics are one or more of lotions, essences, creams, facial masks, and gels.