CN106038737B - A method of coupling ultrasonic wave and two-phase system to extract flavonoids from Sorbus elm - Google Patents

Abstract

The invention relates to a method for extracting Sorbus elm flavonoids by coupling ultrasonic wave and two-phase system, which is characterized in that: the specific steps of the method are as follows: Step 1) extraction and separation of flavonoids from Sorbus elm leaves, using propanol-sulfuric acid ammonium two-phase aqueous phase to obtain the upper phase liquid and the lower phase liquid; step 2) lower the temperature of the lower phase liquid in step 1), crystallize, filter, and recover ammonium sulfate crystals; step 3) use the upper phase liquid in step 1) with Anhydrous ethanol purified, concentrated and dried finished product. The method has the advantages of rapid phase separation, integrated extraction and separation process, improved concentration and purity of flavonoids in the upper phase; reduced material consumption, low toxicity, easy recycling of ethanol and ammonium sulfate; The recovery rate is high, the production method is simple, easy to master, and is not affected by the environment; the cost is low, and it is suitable for industrial production.

Description

A method of coupling ultrasonic wave and two-phase system to extract flavonoids from Sorbus elm

technical field

The invention belongs to the field of separation and purification of effective components of natural products, and relates to a method for extracting the flavonoids of Sorbus elm by coupling ultrasonic wave and two-phase system.

Background technique

Sorbus alnifolia ( Sorbus alnifolia ) is a plant of the genus Sorbus in the family Rosaceae. It is distributed in Heilongjiang, Jilin, Liaoning, Henan, Shaanxi, etc., and is distributed on the north and south slopes of Qinling Mountains in Shaanxi. Tianshan Sorbus elm contains rich flavonoids, anthocyanins, bisphenols, cyanogenic glycosides and other components, and has strong anti-oxidation, anti-radiation, anti-cancer, cough and asthma effects in pharmacological effects. However, there is no report on the active ingredients of Sorbus elm.

Aqueous Two Phase Extraction ( Aqueous Two Phase Extraction, ATPE ) is a new type of separation technology, due to its advantages of high efficiency, mildness, easy operation, easy scale-up, and ability to effectively maintain the activity of heat-sensitive components, etc. It is widely used in natural product separation, biological extraction, pharmaceutical, food chemical industry, separation of metal ions and other fields. However, the traditional polymer-salt aqueous two-phase system has disadvantages such as high cost, difficult recovery, and difficulty in subsequent treatment due to the use of polymers with high viscosity. With the continuous deepening of the research on the two-phase water technology, the ordinary organic two-phase water-phase system that replaces the polymer with inorganic salts has the advantages of low price, low toxicity, simple system, easy recycling of solvents, and easy follow-up treatment. The system components are relatively polar, especially suitable for the extraction and purification of polar substances, making the industrialization of two-phase aqueous phase possible.

At present, the traditional extraction methods of flavonoids include water decoction, organic solvent reflux, Soxhlet extraction, infiltration method, etc. These methods have the disadvantages of low extraction efficiency, time-consuming, energy-consuming, and easy decomposition of heat-sensitive components. In recent years, new methods such as enzymatic extraction and microwave method have emerged. The enzymatic method has mild conditions and high extraction rate, but takes a long time and the extraction efficiency is not high. The microwave method is widely used in the extraction of active ingredients due to its strong ability to break the walls of plant cells, high efficiency, energy saving, and time saving, but it has great damage to heat-sensitive ingredients.

Ultrasound is an elastic wave that generates and transmits powerful energy. Ultrasonic wave technology can greatly improve the extraction rate and extraction speed, because the ultrasonic wave has a series of mechanical, thermal, electromagnetic and chemical ultrasonic effects, cavitation effects, thermal effects and chemical effects. Among them, the cavitation effect of the ultrasonic wave promotes the solvent to penetrate into the cells to a greater extent. When the vibration is in a sparse state, the ultrasonic wave penetrates deeper than the electromagnetic wave in the plant tissue cells, and the residence time is longer, which improves the material transmission speed; and it Secondary effects such as mechanical effects, mechanical vibration, emulsification, diffusion, crushing and chemical effects can rupture the cell wall and accelerate the diffusion and release of active ingredients in plants. The biological activity of the soaked active ingredients remains unchanged at the moment of being crushed, and the crushing speed and extraction rate are improved at the same time. Ultrasound can produce solubilization and is widely used in the enhanced extraction of active ingredients. It has the advantages of low cost, simple equipment, easy operation, and high extraction rate.

Contents of the invention

The purpose of the present invention is to address the deficiencies in the prior art, to provide a method for coupling extraction of Sorbus elm flavonoids by ultrasonic wave and two-phase system, which not only promotes the intersection, penetration and fusion of extraction and separation techniques, but also effectively improves It not only improves the efficiency of extraction and separation, but also avoids the use of toxic solvents and the decomposition of heat-sensitive components, and can separate the flavonoids in the crude extract from impurities such as hydrophilic polysaccharides and proteins, so as to achieve the purpose of purifying flavonoids and separating impurities . The method is low in cost and simple in operation; at the same time, it also makes up for the lack of research on the active components of Sorbus chinensis.

The technical scheme of the present invention: a method for coupling extraction of Sorbus elm flavonoids by ultrasonic wave and two-phase system, characterized in that: the specific steps of the method are as follows:

Step 1) extraction and separation of flavonoids from the leaves of Sorbus chinensis, using propanol-ammonium sulfate two-phase aqueous phase to obtain upper phase liquid and lower phase liquid;

Step 2) cooling the lower phase liquid in step 1), crystallizing, filtering, and recovering ammonium sulfate crystals;

Step 3) Purify, concentrate and dry the upper phase liquid in step 1) with absolute ethanol.

Said step 1) extraction and separation of flavonoids from Sorbus chinensis leaves: dry fresh Sorbus elm leaves and crush them; first use 4.50-6.00g of ammonium sulfate, 6mL-7mL of propanol, and 10mL of water to form a stable dihydrate phase system, add 0.5-0.7g of Sorbus elm leaves to the two-phase system, conduct ultrasonic extraction at 300W and 50°C for 20-40min, suction filter, remove Sorbus slag, and let the filtrate stand for 1- 3h, stratify into upper phase liquid and lower phase liquid, separate upper phase liquid and lower phase liquid while hot to obtain upper phase liquid and lower phase liquid.

Step 3) Purify, concentrate, and dry the upper phase liquid in step 1) with absolute ethanol to obtain a finished product: add 3-5 times the volume of absolute ethanol to the upper phase liquid to precipitate to remove residual impurities of ammonium sulfate, and recover ethanol , the supernatant liquid is concentrated and vacuum-dried at 60°C-70°C to obtain the finished product of flavonoids from the leaves of Sorbus elm.

The upper phase liquid and the lower phase liquid in the step 1) are cooled to room temperature, and the upper phase liquid and the lower phase liquid are respectively measured by using the Al(NO ₃ ) ₃ -NaNO ₂ colorimetric method with rutin as the standard sample, The distribution coefficient of the flavonoids in the upper phase and the lower phase is greater than 12, and the recovery rate is between 81.4-91.4%.

The alcohol-water ratio of the propanol and water is 0.5-0.7.

Said step 1) Extraction and separation of flavonoids from Sorbus chinensis leaves: dry fresh Sorbus elm leaves and crush them; first use 5.25 g of ammonium sulfate, 6 mL of propanol, and 10 mL of water to form a stable two-phase system. Add 0.5 g of Sorbus elm leaves to the two-phase system, perform ultrasonic extraction for 40 minutes at a power of 300W and a temperature of 50°C, filter with suction to remove Sorbus elm residues, let the filtrate stand for 2 hours, and separate into upper phase liquid and lower phase. Separate the upper phase liquid and the lower phase liquid while hot to obtain the upper phase liquid and the lower phase liquid.

In the present invention, ultrasonic waves are coupled with a two-phase system to extract the flavonoids of Sorbus elm, which has obvious advantages:

(1) Rapid phase separation

In the experiment, it was observed that the flavonoids of Sorbus chinensis were extracted by coupling ultrasonic wave with propanol-ammonium sulfate two-phase system, and Sorbus chinensis leaves, propanol, and ammonium sulfate were added in proportion, and after ultrasonic extraction, it took about 10 minutes to complete Phase separation (no centrifugation required) and no emulsified layer shortens the test period.

(2) Process integration

Ultrasound and propanol-ammonium sulfate two-phase extraction and separation not only achieve the purpose of extraction and purification, but also concentrate the feed liquid. The performance is that impurities (such as hydrophilic sugar, protein, etc.) enter the lower phase, flavonoids are enriched in the upper phase, and the upper phase is reduced compared with the original treatment solution, and the concentration and purity of total flavonoids are both increased.

The distribution coefficient K of Sorbus flavonoids in the system was 16.218, and the extraction rate Y was as high as 91.4%.

(3) Reduce material consumption

The method of the invention uses propanol as the organic solvent of the two-phase water, has low toxicity, and the propanol and ammonium sulfate are easy to recycle, thereby greatly reducing the loss of raw materials.

The method of the invention has a high recovery rate of the Sorbus elm flavonoids, a simple production method, easy to grasp, not affected by the environment, low cost and suitable for industrialized production.

The present invention will be described in further detail below through specific examples, but not as a limitation of the present invention.

Detailed ways

Example 1

A method for coupling extraction of Sorbus elm flavonoids by ultrasonic wave and two-phase system, the specific steps are as follows:

Step 1) extraction and separation of flavonoids from the leaves of Sorbus chinensis, using propanol-ammonium sulfate two-phase aqueous phase to obtain upper phase liquid and lower phase liquid;

Step 2) cooling the lower phase liquid in step 1), crystallizing, filtering, and recovering ammonium sulfate crystals;

Step 3) Purify, concentrate and dry the upper phase liquid in step 1) with absolute ethanol.

Example 2

A method for coupling extraction of Sorbus elm flavonoids by ultrasonic wave and two-phase system, the specific steps are as follows:

Step 1) Extraction and separation of flavonoids from Sorbus chinensis leaves, and use propanol-ammonium sulfate two-phase aqueous phase to obtain the upper and lower phase liquids: dry the fresh Sorbus chinensis leaves and crush them; first use ammonium sulfate 4.50- 6.00g, propanol 6mL-7mL, water 10mL to form a stable two-phase system, add 0.5-0.7g of Sorbus elm leaves to the two-phase system, and perform ultrasonic extraction at 300W and 50°C for 20- 40min, filter with suction, remove Sorbus elm residue, let the filtrate stand for 1-3h, separate into upper phase liquid and lower phase liquid, separate upper phase liquid and lower phase liquid while hot, and obtain upper phase liquid and lower phase liquid .

Step 2) cooling the lower phase liquid in step 1), crystallizing, filtering, and recovering ammonium sulfate crystals;

Step 3) Purify, concentrate and dry the upper phase liquid in step 1) with absolute ethanol: add 3-5 times the volume of absolute ethanol to the upper phase liquid for precipitation to remove residual impurities of ammonium sulfate, recover ethanol, and put After the phase liquid is concentrated, vacuum-dry at 60°C-70°C to obtain the finished product of the flavonoids from the leaves of Sorbus elm.

In the two-phase aqueous phase enrichment technology of flavonoids, the widely used macromolecular polymers in the enrichment phase are not only difficult to separate from flavonoids, but also the price of macromolecular polymers is generally relatively high. cost.

The propanol-ammonium sulfate two-phase aqueous phase is used, and the propanol is used as the organic solvent of the two-phase aqueous phase. The propanol and the ammonium sulfate can be recycled, and the cost is low and the operation is simple. With the method of the present invention, the flavonoids of Sorbus elm leaves are enriched and separated in the propanol phase of the upper phase, and the upper phase is precipitated with absolute ethanol to remove residual impurities such as ammonium sulfate, then the ethanol is recovered, concentrated, and vacuum-dried to obtain water The finished product of flavonoids from the leaves of Sorbus elm; the ammonium sulfate crystals filtered by the cooling and crystallization of the lower phase liquid are recycled.

Example 3

A method for coupling extraction of Sorbus elm flavonoids by ultrasonic wave and two-phase system, the specific steps are as follows:

Step 1) Extraction and separation of flavonoids from Sorbus chinensis leaves, and use propanol-ammonium sulfate two-phase aqueous phase to obtain the upper and lower phase liquids: dry the fresh Sorbus chinensis leaves and crush them; first use 5.25g of ammonium sulfate , propanol 6mL, and water 10mL to form a stable two-phase system. Add 0.5 g of Sorbus elm leaves to the two-phase system, perform ultrasonic extraction for 40 minutes at a power of 300W and a temperature of 50°C, and filter to remove the Sorbus slag, the filtrate was left standing for 2 hours, stratified into an upper phase liquid and a lower phase liquid, and the upper phase liquid and the lower phase liquid were separated while hot to obtain the upper phase liquid and the lower phase liquid. After cooling the upper phase liquid and the lower phase liquid to room temperature, take the volumes of the upper phase liquid and the lower phase liquid, which are 6mL and 9.26mL respectively, and the ratio (volume ratio of the upper phase liquid and the lower phase liquid) is 0.6522, with rutin as the standard The upper phase liquid and the lower phase liquid were measured respectively by Al(NO ₃ ) ₃ -NaNO ₂ colorimetric method, the flavonoid concentration of the upper phase liquid was 7.039 mg/mL, and the flavonoid concentration of the lower phase liquid was 0.434 mg/mL , the distribution coefficient of flavonoids in the upper phase and the lower phase was 16.218, and the yield of total flavonoids was 91.4%.

Step 2) cooling the lower phase liquid in step 1), crystallizing, filtering, and recovering ammonium sulfate crystals;

Step 3) Purify, concentrate, and dry the upper phase liquid in step 1) with absolute ethanol to obtain a finished product: Precipitate 5 times the volume of the upper phase liquid with absolute ethanol to remove residual impurities of ammonium sulfate, recover ethanol, and concentrate the upper phase liquid Afterwards, the finished product (0.04782 g) of the flavonoids from the leaves of Sorbus elm was dried under vacuum at 60°C. This embodiment is the best embodiment.

Example 4

A method for coupling extraction of Sorbus elm flavonoids by ultrasonic wave and two-phase system, the specific steps are as follows:

Step 1) Extraction and separation of flavonoids from Sorbus chinensis leaves, and use propanol-ammonium sulfate two-phase aqueous phase to obtain upper phase liquid and lower phase liquid: dry fresh Sorbus elmia leaves and crush them; first use ammonium sulfate 6g, 6mL of propanol and 10mL of water form a stable two-phase system. Add 0.6g of Sorbus elm leaves to the two-phase system, perform ultrasonic extraction at 300W and 50°C for 20min, and filter with suction to remove elm flowers Catalpa slag, the filtrate was allowed to stand for 1 hour, stratified into an upper phase liquid and a lower phase liquid, and the upper phase liquid and the lower phase liquid were separated while hot to obtain the upper phase liquid and the lower phase liquid. After cooling the upper phase liquid and the lower phase liquid to room temperature, take the upper phase liquid and the lower phase liquid volume, respectively 4.2mL and 12mL, compared (the volume ratio of the upper phase liquid and the lower phase liquid) 0.3500, with rutin as the standard The upper phase liquid and the lower phase liquid were measured respectively by Al(NO ₃ ) ₃ -NaNO ₂ colorimetric method, the flavonoid concentration of the upper phase liquid was 11.829 mg/mL, and the flavonoid concentration of the lower phase liquid was 0.945 mg/mL , the distribution coefficient of flavonoids in the upper phase and lower phase was 12.517, and the yield of total flavonoids was 81.4%.

Step 2) cooling the lower phase liquid in step 1), crystallizing, filtering, and recovering ammonium sulfate crystals;

Step 3) Purify, concentrate, and dry the upper phase liquid in step 1) with absolute ethanol to obtain a finished product: Precipitate 4 times the volume of the upper phase liquid with absolute ethanol to remove residual impurities of ammonium sulfate, recover ethanol, and concentrate the upper phase liquid Afterwards, the finished product (0.05419g) of the flavonoids from Sorbus chinensis leaves was vacuum-dried at 65°C.

Example 5

A method for coupling extraction of Sorbus elm flavonoids by ultrasonic wave and two-phase system, the specific steps are as follows:

Step 1) Extraction and separation of flavonoids from Sorbus chinensis leaves, and use propanol-ammonium sulfate two-phase aqueous phase to obtain the upper and lower phase liquids: dry the fresh Sorbus chinensis leaves and crush them; first use 5.25g of ammonium sulfate , 7 mL of propanol, and 10 mL of water to form a stable two-phase system. Add 0.7 g of Sorbus elm leaves to the two-phase system, perform ultrasonic extraction at 300W and 50°C for 20 minutes, and filter with suction to remove the Sorbus slag, the filtrate was left standing for 3 hours, stratified into an upper phase liquid and a lower phase liquid, and the upper phase liquid and the lower phase liquid were separated while hot to obtain the upper phase liquid and the lower phase liquid. After cooling the upper phase liquid and the lower phase liquid to room temperature, take the volume of the upper phase liquid and the lower phase liquid, which are 5.4mL and 11.5mL respectively, compared with (the volume ratio of the upper phase liquid and the lower phase liquid) 0.4696. The standard sample was determined by Al(NO ₃ ) ₃ -NaNO ₂ colorimetric method on the upper phase liquid and the lower phase liquid respectively. mL, the partition coefficient of flavonoids in the upper phase and the lower phase was 15.300, and the yield of total flavonoids was 81.4%.

Step 2) cooling the lower phase liquid in step 1), crystallizing, filtering, and recovering ammonium sulfate crystals;

Step 3) Purify, concentrate, and dry the upper phase liquid in step 1) with absolute ethanol to obtain a finished product: Precipitate 3 times the volume of the upper phase liquid with absolute ethanol to remove residual impurities of ammonium sulfate, recover ethanol, and concentrate the upper phase liquid Afterwards, the finished product (0.06205 g) of the flavonoids from the leaves of Sorbus elm was dried under vacuum at 70°C.

The present invention is based on the discovery that the flavonoid content in the leaves of Sorbus elm is relatively high. Table 1 shows the partition coefficients of flavonoids from Sorbus elm leaves in propanol-ammonium sulfate two-phase aqueous phase. The principle of the extraction and separation technology of the two-phase system is: the flavonoids from the leaves of Sorbus elm are selectively distributed in the two-phase system, showing a certain distribution coefficient. The flavonoids of Sorbus elm leaves have affinity for propanol, which makes it easier for the flavonoids of Sorbus elm leaves to distribute in the upper phase rich in propanol, while some impurities such as sugar and protein are distributed in the lower phase. The partition coefficient of lutein was greater than 12 in the experimental range, and the recovery rate was between 81.4-91.4%.

Table 1 Partition behavior of flavonoids from leaves of Sorbus elm in propanol-ammonium sulfate two-phase aqueous phase

In the table: R=V _t /V _b ; K=C _t /C _b ; Y=RK/(RK+1)

In the formula: R---comparison; V _t upper phase volume; V _b lower phase volume; K—distribution coefficient of flavonoids from Sorbus chinensis leaves in two aqueous phases; C _t ---upper phase liquid Shui elm The mass concentration of flavonoids from Sorbus chinensis leaves, mg/mL; C _b --- the mass concentration of flavonoids from Sorbus chinensis leaves in the lower phase, mg/mL; Y—extraction rate (yield).

Therefore, through the above-mentioned ultrasonic wave and propanol-ammonium sulfate two-phase system coupling extraction, separation, and absolute ethanol precipitation, the flavonoids of Sorbus chinensis leaves were extracted, concentrated and purified, and finally obtained high-quality A finished product of Sorbus elm flavonoids with high yield and low cost.

The present embodiment does not have detailed process steps, which belong to known common methods in this industry, and will not be described here one by one.

Claims (2)

1. a kind of ultrasonic wave couples the method for extracting Sorbus alnifolia flavones with double-aqueous phase system, it is characterized in that: this method is specific Steps are as follows:

Step 1) Sorbus alnifolia leaf extracting flavonoids and separation obtain phase liquid and lower phase liquid using propyl alcohol-ammonium sulfate aqueous two-phase；

Lower phase liquid in step 1) is cooled down, crystallization, filtering, recycles ammonia sulfate crystal by step 2；

The finished product that upper phase liquid dehydrated alcohol in step 1) is purified, is concentrated, dried by step 3)；

The step 1) Sorbus alnifolia leaf extracting flavonoids and separation: fresh Sorbus alnifolia leaf is dried, and is crushed；First use ammonium sulfate 4.50-6.00g, propyl alcohol 6mL-7mL, water 10mL form stable double-aqueous phase system, Sorbus alnifolia are added in double-aqueous phase system Leaf 0.5-0.7g carries out ultrasonic wave extraction 20-40min at power 300W, temperature 50 C, filters, and removes Sorbus alnifolia slag, filter Liquid stands 1-3h, is layered as phase liquid and lower phase liquid, while hot separates upper phase liquid and lower phase liquid, obtains phase liquid and lower phase liquid；

The step 3) is by the upper phase liquid dehydrated alcohol purifying in step 1), concentration, dry finished product: upper phase liquid is added 3-5 After the dehydrated alcohol precipitating of times volume removes ammonium sulfate residual impurities, ethyl alcohol is recycled, it is true at 60 DEG C -70 DEG C after upper phase liquid concentration Sky is dried to obtain Sorbus alnifolia leaf flavones finished product；

The upper phase liquid of the step 1) and lower phase liquid are by being cooled to room temperature, using rutin as standard specimen, using Al (NO₃)₃-NaNO₂Than Color method is respectively measured upper phase liquid and lower phase liquid, and flavones is greater than 12 in the distribution coefficient of upper phase liquid and lower phase liquid, returns Yield is between 81.4-91.4%.

2. a kind of ultrasonic wave according to claim 1 couples the method for extracting Sorbus alnifolia flavones with double-aqueous phase system, Be characterized in: the step 1) Sorbus alnifolia leaf extracting flavonoids and separation: fresh Sorbus alnifolia leaf is dried, and is crushed；First use sulfuric acid Ammonium 5.25g, propyl alcohol 6mL, water 10mL form stable double-aqueous phase system, and Sorbus alnifolia leaf 0.5g is added in double-aqueous phase system, Ultrasonic wave extraction 40min is carried out at power 300W, temperature 50 C, is filtered, Sorbus alnifolia slag is removed, and filtrate stands 2h, layering For upper phase liquid and lower phase liquid, upper phase liquid and lower phase liquid are separated while hot, obtain phase liquid and lower phase liquid.