A method for extracting, separating and purifying high-antioxidant-activity polyphenols from tender leaves of highland barley

Abstract

The application discloses a method for extracting, separating and purifying high-antioxidant activity highland barley tender leaf polyphenols. The application aims to provide a simple and efficient method for extracting, separating and purifying high-antioxidant activity polyphenols from highland barley tender leaves. The method specifically comprises the following steps: (1) ultrasonic-assisted highland barley tender leaf crude polyphenol extraction; (2) alcohol precipitation impurity removal; (3) n-butanol extraction; (4) macroporous resin activation; and (5) macroporous resin purification to obtain high-antioxidant activity highland barley tender leaf polyphenols. The application can obtain high-antioxidant activity highland barley tender leaf polyphenols, and the extraction and purification process is simple, the technical requirements for enterprises are low, and the method can be popularized on a large scale. The polyphenols have high antioxidant activity and can be used for the development of health-care food.

Description

Technical Field

[0001] This invention relates to the field of food processing, and more specifically, to a method for extracting, separating, and purifying polyphenols from young barley leaves with high antioxidant activity. Background Technology

[0002] Barley (Hordeum vulgare Linn. var. nudum Hook. f.) is a variety of barley (Hordeum vulgare L.) belonging to the genus Hordeum in the family Poaceae. Because its palea and lemma separate from the caryopsis, exposing the grain, it is also known as naked barley. Farmers in Tibet also call it naked wheat, raw barley, rice barley, and rice barley. Barley originated on the Qinghai-Tibet Plateau, where farmers cultivated wild barley into barley, a practice with a cultivation history of over 3,500 years. It is the primary source of carbohydrates for the Tibetan people. Due to its strong resistance to adverse conditions, wide adaptability, and stable yield, its cultivation area has expanded to other parts of China. Currently, barley cultivation in my country is mainly distributed in the Tibet Autonomous Region, Qinghai Province, Sichuan Province, Gansu Province, and parts of the Yunnan-Guizhou Plateau. The extreme environment of the Qinghai-Tibet Plateau, characterized by high altitude, low oxygen levels, and intense sunlight, has endowed barley with exceptional resilience and a rich variety of unique nutrients, including protein, polyphenols, minerals, vitamins, and dietary fiber. The Tibetan people primarily use barley to make tsampa, which is considered the most important of the four treasures of Tibetan cuisine (tsampa, butter, tea, and beef / mutton). Despite the harsh natural environment of the Qinghai-Tibet Plateau, centenarians are not uncommon, which is likely closely related to the long-term consumption of barley, known for its rich nutritional value and significant medicinal benefits. Barley sprouts, generally referring to the fresh, tender stems and leaves of barley plants 15-30cm in height, are commonly known as barley seedlings and have high medicinal value and a long history of medicinal use. The accumulation of nutrients in barley sprouts reaches its peak when they reach 15-30cm in height, containing abundant dietary fiber, protein, minerals, chlorophyll, and polyphenols. Developing and utilizing plant polyphenols with antioxidant and other biological activities to prevent and treat various diseases caused by oxidation and free radicals has become a current research hotspot. However, problems such as insufficient resources, low content of effective components, and immature extraction and purification processes still exist. Barley, as a special type of barley, can enrich the source of phenolic substances by extracting phenols and other active substances from its young leaves. However, there is currently a lack of research on the extraction, separation, and purification of polyphenols from young barley leaves. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for extracting, separating, and purifying polyphenols from young barley leaves with high antioxidant activity. This invention is the first to use young barley leaves as raw material for polyphenol extraction, which is economical and environmentally friendly, thus increasing the economic value of young barley leaves. Utilizing an ultrasound-assisted polyphenol extraction process, compared to traditional extraction methods, ultrasonic extraction requires less solvent, has a shorter extraction time, and a higher extraction rate, while minimizing the degradation of heat-sensitive compounds. Liquid-liquid extraction with n-butanol is used to first obtain polyphenols with higher antioxidant activity, achieving a preliminary purification effect, reducing the loading capacity of macroporous resin, and improving the purification effect of macroporous resin on polyphenols, resulting in polyphenols with higher antioxidant activity. Finally, n-butanol extraction combined with macroporous resin method yielded the polyphenols from young barley leaves with the highest antioxidant activity. In vitro antioxidant results showed that the polyphenol exhibited a DPPH radical scavenging rate of 159,568 μmol TE / g, an ABTS radical scavenging rate of 163,802 μmol TE / g, and a FRAP radical scavenging rate of 193,708 μmol TE / g. This method possesses a large adsorption capacity, good selectivity, rapid adsorption rate, simple and mild desorption conditions, reusable resin, and low cost. Therefore, the method of this invention has broad market prospects.

[0004] The objective of this invention is achieved through the following solution:

[0005] This invention provides a method for extracting, separating, and purifying polyphenols from young barley leaves with high antioxidant activity, comprising the following steps:

[0006] S1. Defatted whole barley leaf powder was mixed with 70-90% methanol solution, ultrasonically treated, centrifuged, the precipitate was mixed with 70-90% methanol, ultrasonicated, centrifuged, the supernatants were combined, rotary evaporated, freeze-dried, precipitated with ethanol, centrifuged, the supernatant was rotary evaporated, and freeze-dried to obtain crude polyphenol extract.

[0007] S2. Liquid-liquid extraction: Dissolve the crude extract of phenolic substances from young barley leaves in water, and mix the crude polyphenol solution with an organic solvent using a separatory funnel. Repeat twice; with the organic solvent in the upper layer and the water layer in the lower layer, collect the extract; repeat the extraction twice, and freeze-dry the extract after rotary evaporation.

[0008] S3. Activation of macroporous resin: Soak macroporous resin in 90-95% ethanol for 12-24 hours, wash with deionized water until no white floating matter and no alcohol odor remain; soak the resin in 3-5% hydrochloric acid solution and 2-4% sodium hydroxide solution (twice the volume of the resin) for 4-6 hours, and wash with deionized water until neutral.

[0009] S4. The activated saturated wet resin is wet-packed into a chromatography column. The polyphenols are dissolved in 70-90% methanol to prepare a solution. The sample is loaded, and after washing with deionized water to remove impurities, the target substance is eluted with 70-75% ethanol. The eluent is collected, the ethanol is removed by rotary evaporation, and the sample is freeze-dried under vacuum to obtain the polyphenol extract of young barley leaves.

[0010] In one embodiment of the present invention, in step S1, the ratio of the whole powder to 70-90% methanol is 1g:20mL to 1g:25mL.

[0011] In one embodiment of the present invention, in step S1, the ultrasonic power is 480-560W, the ultrasonic time is 30-60min, and the ultrasonic temperature is 40-50℃.

[0012] In one embodiment of the present invention, in step S1, after centrifugation, the residue precipitate is mixed with 70-90% methanol and ultrasonically extracted 2-3 times.

[0013] In one embodiment of the present invention, in step S1, polysaccharide substances are removed by precipitation using three times the volume of ethanol.

[0014] In one embodiment of the present invention, in step S2, the mass-volume fraction of crude extract of phenolic substances from young barley leaves and water is 0.03-0.05 g / mL.

[0015] In one embodiment of the present invention, in step S2, the organic reagent is n-butanol, and the volume ratio of crude polyphenol solution to organic reagent is 1:1 to 1:1.5.

[0016] In one embodiment of the present invention, AB-8 macroporous resin is selected in step S2.

[0017] In one embodiment of the present invention, in step S3, the concentration of the loading solution is 2-3 mg / mL, and 4 BV of the sample is loaded at a flow rate of 2-3 BV / min. After removing impurities with 4 BV of deionized water, the target substance is eluted with 70% ethanol at a flow rate of 2.5-3 BV / min.

[0018] The high antioxidant activity barley leaf polyphenols obtained by the aforementioned method are polyphenols with high DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and oxygen free radical scavenging capacity, and can be used in the development of health foods. Therefore, the high antioxidant activity barley leaf polyphenols obtained by this invention and their use in the preparation of health foods are also within the scope of protection of this invention.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The method of the present invention is simple and easy to implement, with high polyphenol extraction rate, high antioxidant activity of purified polyphenols, and low production cost.

[0021] (2) The present invention optimizes the ultrasonic-assisted polyphenol extraction process. Compared with the traditional extraction method, ultrasonic extraction requires less solvent, has a shorter extraction time, and a higher extraction rate. It can also minimize the degradation of heat-sensitive compounds and retain highly active polyphenol compounds.

[0022] (3) This invention proposes a polyphenol purification method with the highest antioxidant activity, namely n-butanol extraction combined with macroporous resin method. This method first uses n-butanol to preliminarily purify polyphenols with high antioxidant activity, and then performs macroporous resin purification. It has a large adsorption capacity, good selectivity, fast adsorption speed, simple and mild desorption conditions, and the macroporous resin can be reused multiple times. The purified polyphenols have stronger antioxidant activity and better health care effects, and have potential application value in the production of health food. Attached Figure Description

[0023] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0024] Figure 1 A graph showing the total phenol content;

[0025] Figure 2 DPPH free radical scavenging rate graph;

[0026] Figure 3 A graph showing the ABTS free radical scavenging rate;

[0027] Figure 4 This is a graph showing the FRAP free radical scavenging rate. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0029] The methods for determining total phenolic content and antioxidant activity involved in the following examples and comparative examples are as follows:

[0030] 1. Determination of total phenol content

[0031] Determination of total phenol content using the Folin-Ciocalteu method: Add 2.5 mL of 0.2 mol / L Folin-Ciocalteu solution diluted 1:10 to a test tube, then add 500 μL of the diluted extract of the above phenolic substances, and vortex thoroughly to mix. React at room temperature for 4 min, then add 2 mL of 75 g / L sodium carbonate solution, vortex thoroughly to mix, and incubate in the dark at room temperature (22 ± 1 °C) for 2 h. Measure the absorbance of the mixture at 760 nm. Accurately prepare gallic acid solutions of 0, 16, 20, 40, and 80 mg / L for the preparation of standard curves. Calculation of results: The total phenol content in the sample was calculated based on the linear regression equation of the standard curve and the measured absorbance values, expressed in milligrams of gallic acid equivalent per 100 g sample (mg GAE / 100 g DW).

[0032] 2. Methods for determining antioxidant activity

[0033] 2.1 DPPH radical scavenging rate: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine, 60 μM) was dissolved in 80% methanol to obtain a mother liquor, which was then diluted 5-10 times with 80% methanol to adjust the absorbance at 515 nm to approximately 0.70 to obtain a reaction solution. 100 μL of polyphenol extract was reacted with 1.9 mL of DPPH solution in the dark for 2 h, and the absorbance was measured at 515 nm. A standard curve of 0-600 μM was prepared using the Trolox concentration range. The DPPH radical scavenging rate of the sample was calculated based on the standard curve and expressed as micromolar Trolox equivalents (μmol TE / g).

[0034] 2.2 ABTS radical scavenging rate: A stock solution was prepared by mixing ABTS (2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, 7 mM) with potassium persulfate (2.45 mM) at a 1:1 ratio and storing it in the dark for at least 16 hours. This stock solution was diluted 30-40 times with 80% methanol to adjust the absorbance of the stock solution at 734 nm to approximately 0.70, thus obtaining the ABTS reaction solution. 200 μL of polyphenol extract was reacted with 1.8 mL of ABTS solution in the dark for 6 minutes, and the absorbance was measured at 734 nm. A standard curve was prepared with a Trolox concentration range of 0-600 μM. The ABTS radical scavenging rate of the sample was calculated based on the standard curve and expressed as micromolar Trolox equivalents (μmol TE / g).

[0035] 2.3 FRAP radical scavenging rate: FRAP reagent: TPTZ reagent (2,4,6-tris(2-pyridyl)-s-triazine) 10mM: ferric chloride solution (20mM): sodium acetate solution (300mM) = 1:1:10. The reaction solution was prepared and preheated to 37℃. 100μL of polyphenol extract was reacted with 3mL of FRAP reagent for 4min, and the absorbance was measured at 593nm. A standard curve of 0-600μM was prepared using the trolox concentration range. The FRAP radical scavenging rate of the sample was calculated based on the standard curve and expressed as micromolar Trolox equivalents (μmol TE / g).

[0036] Example 1

[0037] This embodiment relates to a method for extracting, separating, and purifying polyphenols from young barley leaves with high antioxidant activity. The steps are as follows:

[0038] Step 1: Preparation of crude polyphenols from young barley leaves

[0039] After defatting with petroleum ether, the whole powder of young barley leaves was mixed with 80% methanol solution at a ratio of 1g:20mL. The mixture was ultrasonically sonicated at 40℃ for 30min using 480W, followed by centrifugation at 3000×g for 5min. The supernatant was collected. The precipitate was extracted twice more under the same ultrasonic conditions. The supernatants from the three extractions were combined and lyophilized under reduced pressure at 55℃ using a rotary evaporator. The lyophilized product was prepared into a 2mg / mL solution. Under magnetic stirring, three volumes of 100% ethanol were slowly added via a constant flow pump. After standing overnight, the precipitate was removed by centrifugation. The supernatant was then lyophilized again after removing the ethanol by rotary evaporation to obtain a crude extract of phenolic substances from young barley leaves.

[0040] Step 2: Activation of macroporous resin

[0041] The macroporous resin was soaked in 95% ethanol for 24 hours and then washed with deionized water until no white floating matter and no alcohol odor remained. The resin was then soaked in twice its volume of 5% hydrochloric acid solution and 2% sodium hydroxide solution for 4 hours and then washed with deionized water until neutral.

[0042] Step 3: Purification of polyphenols from young barley leaves

[0043] The pretreated AB-8 saturated wet resin was packed into a chromatography column using a wet chromatography method. The crude extract of phenolic substances from young barley leaves was loaded at a concentration of 2 mg / mL and a flow rate of 2 BV / min for 4 BV. After removing most impurities with 4 BV of deionized water, the target analytes were eluted with 70% ethanol at a flow rate of 2.5 BV / min. The collected 6 BV eluent was used to remove the ethanol using a rotary evaporator, followed by vacuum freeze-drying to obtain the purified polyphenol extract from young barley leaves.

[0044] Implementation results: Figure 1 It can be seen that the total phenol content of the polyphenol extract in Example 1 is 24780 mg GAE / 100 g DW, which is derived from... Figure 2-4 It can be seen that the DPPH radical scavenging rate is 138047 μmol TE / g, the ABTS radical scavenging rate is 143170 μmol TE / g, and the FRAP radical scavenging rate is 155100 μmol TE / g.

[0045] Example 2

[0046] This embodiment relates to a method for extracting, separating and purifying polyphenols from young barley leaves with high antioxidant activity. The difference from Embodiment 1 is that step 3 is repeated twice, and the polyphenol extract is purified twice using macroporous resin.

[0047] Implementation results: Figure 1 It can be seen that the total phenol content of the polyphenol extract in Example 2 was 24220 mg GAE / 100 g DW, which did not increase the total phenol content. Figure 2-4 It can be seen that the DPPH radical scavenging rate, ABTS radical scavenging rate, and FRAP radical scavenging rate are 110570, 124792, and 128902 μmol TE / g, respectively, which are lower than those in Example 1. Therefore, repeated macroporous resin purification is unnecessary; the purification effect can be achieved in one step, saving time and effort.

[0048] Example 3

[0049] This embodiment relates to a method for extracting, separating and purifying polyphenols from young barley leaves with high antioxidant activity. The difference from Embodiment 1 is that ultrasonic-assisted extraction was not performed in step 1, and extraction was only performed at 40°C for 30 minutes.

[0050] Implementation results: Figure 1 It can be seen that the total phenol content of the polyphenol extract in Example 3 was 12630 mg GAE / 100 g DW, which was significantly lower than that in Example 1. Figure 2-4 It was found that the DPPH radical scavenging rate, ABTS radical scavenging rate, and FRAP radical scavenging rate were 90570, 94732, and 89892 μmol TE / g, respectively, which were lower than those in Example 1. Therefore, ultrasound-assisted extraction is helpful in extracting, separating, and purifying high-antioxidant-activity barley leaf polyphenols.

[0051] Example 4

[0052] This embodiment relates to a method for extracting, separating and purifying polyphenols from young barley leaves with high antioxidant activity. The difference from Embodiment 1 is that the crude polyphenol extract in step 1 was not subjected to ethanol precipitation for impurity removal.

[0053] Implementation results: Figure 1It can be seen that the total phenolic content of the polyphenol extract in Example 4 was 16290 mg GAE / 100 g DW, which was significantly lower than that in Example 1. Figure 2-4 It was found that the DPPH radical scavenging rate, ABTS radical scavenging rate, and FRAP radical scavenging rate were 99300, 92844, and 92342 μmol TE / g, respectively, which were lower than those in Example 1. Therefore, the ethanol purification step helps to extract, separate, and purify the polyphenols from young barley leaves with high antioxidant activity.

[0054] Example 5

[0055] This embodiment relates to a method for extracting, separating and purifying polyphenols from young barley leaves with high antioxidant activity. The difference from Embodiment 1 is that steps 2 and 3 are not performed.

[0056] Implementation results: Figure 1 It can be seen that the total phenol content of the crude extract in Example 5 was 5360 mg GAE / 100 g DW, significantly lower than that in Example 1. Figure 2-4 It can be seen that the DPPH radical scavenging rate, ABTS radical scavenging rate and FRAP radical scavenging rate are 21390, 22504 and 23638 μmol TE / g, respectively, which are lower than those in Example 1.

[0057] Example 6

[0058] This comparative example relates to a method for extracting, separating, and purifying polyphenols from young barley leaves with high antioxidant activity. The steps are as follows:

[0059] Step 1: Preparation of crude polyphenols from young barley leaves

[0060] After defatting with petroleum ether, the whole powder of young barley leaves was mixed with 80% methanol solution at a ratio of 1g:20mL. The mixture was ultrasonically sonicated at 40℃ for 30min using 480W, followed by centrifugation at 3000×g for 5min. The supernatant was collected. The precipitate was extracted twice more under the same ultrasonic conditions. The supernatants from the three extractions were combined and lyophilized under reduced pressure at 55℃ using a rotary evaporator. The lyophilized product was prepared into a 2mg / mL solution. Under magnetic stirring, three volumes of 100% ethanol were slowly added via a constant flow pump. After standing overnight, the precipitate was removed by centrifugation. The supernatant was then lyophilized again after removing the ethanol by rotary evaporation to obtain a crude extract of phenolic substances from young barley leaves.

[0061] Step 2, Liquid-Liquid Extraction

[0062] Using a separatory funnel, mix 50 mL of crude polyphenols (0.03 g / mL) with 50 mL of petroleum ether solution for 3-5 min, repeating twice. The petroleum ether should be in the upper layer and the aqueous layer in the lower layer; collect the petroleum ether extract. Repeat the extraction twice. Alternatively, pour 50 mL of chloroform into a separatory funnel; the chloroform should be in the lower layer and the water in the upper layer; collect the chloroform extract. Or, pour 50 mL of ethyl acetate into a separatory funnel; the ethyl acetate should be in the upper layer and the water in the lower layer; collect the ethyl acetate extract. Similarly, for n-butanol, the n-butanol should be in the upper layer and the water in the lower layer; collect the n-butanol extract. Rotary evaporate and then freeze-dry the petroleum ether extract, chloroform extract, ethyl acetate extract, and n-butanol extract, respectively.

[0063] Implementation results: Figure 1 It can be seen that the total phenolic contents of the petroleum ether extract, chloroform extract, ethyl acetate extract, and n-butanol extract were 5650, 4721, 8202, and 15410 mg GAE / 100g DW, respectively, significantly lower than those in Example 1. From Figure 2-4 It was found that the DPPH radical scavenging rate, ABTS radical scavenging rate, and FRAP radical scavenging rate were significantly lower than those in Example 1. This indicates that liquid-liquid extraction alone cannot effectively purify polyphenols and obtain barley leaf polyphenols with high antioxidant activity. The results also show that the n-butanol extract has higher antioxidant activity among the four organic reagents, indicating that polyphenols with higher antioxidant activity have similar polarity to n-propanol.

[0064] Example 7

[0065] This embodiment relates to a method for extracting, separating, and purifying polyphenols from young barley leaves with high antioxidant activity. The steps are as follows:

[0066] Step 1: Preparation of crude polyphenols from young barley leaves

[0067] After defatting with petroleum ether, the whole powder of young barley leaves was mixed with 80% methanol solution at a ratio of 1g:20mL. The mixture was ultrasonically sonicated at 40℃ for 30min using 480W, followed by centrifugation at 3000×g for 5min. The supernatant was collected. The precipitate was extracted twice more under the same ultrasonic conditions. The supernatants from the three extractions were combined and lyophilized under reduced pressure at 55℃ using a rotary evaporator. The lyophilized product was prepared into a 2mg / mL solution. Under magnetic stirring, three volumes of 100% ethanol were slowly added via a constant flow pump. After standing overnight, the precipitate was removed by centrifugation. The supernatant was then lyophilized again after removing the ethanol by rotary evaporation to obtain a crude extract of phenolic substances from young barley leaves.

[0068] Step 2, Liquid-Liquid Extraction

[0069] Using a separatory funnel, mix 50 mL of crude polyphenols (0.03 g / mL) with 50 mL of n-butanol solution for 3-5 min, repeating twice. The n-butanol layer should be on top, and the aqueous layer on the bottom. Collect the n-butanol extract. Repeat the extraction twice, then rotary evaporate 100 mL of n-butanol and freeze-dry.

[0070] Step 3: Activation of macroporous resin

[0071] The macroporous resin was soaked in 95% ethanol for 24 hours and then washed with deionized water until no white floating matter and no alcohol odor remained. The resin was then soaked in twice its volume of 5% hydrochloric acid solution and 2% sodium hydroxide solution for 4 hours and then washed with deionized water until neutral.

[0072] Step 4: Purification of polyphenols from young barley leaves

[0073] The pretreated AB-8 saturated wet resin was wet-packed into a chromatography column. The polyphenols obtained in step 2 were loaded at a concentration of 2 mg / mL and a flow rate of 2 BV / min for 4 BV. After removing most impurities with 4 BV of deionized water, the target analyte was eluted with 70% ethanol at a flow rate of 2.5 BV / min. The collected 6 BV eluent was used to remove the ethanol using a rotary evaporator, and then freeze-dried under vacuum to obtain the purified barley young leaf polyphenol extract.

[0074] Implementation results: Figure 1 It can be seen that the total phenol content of the polyphenol extract in Example 7 is 34789 mg GAE / 100 g DW, which is derived from... Figure 2-4It was found that the DPPH radical scavenging rate was 159568 μmol TE / g, the ABTS radical scavenging rate was 163802 μmol TE / g, and the FRAP radical scavenging rate was 193708 μmol TE / g. These figures are higher than in Example 1. This is because liquid-liquid extraction with n-butanol first yields polyphenols with higher antioxidant activity, achieving a preliminary purification effect, reducing the loading capacity of the macroporous resin, and thus improving the purification effect of the macroporous resin on polyphenols, resulting in polyphenols with higher antioxidant activity. The polyphenols from the young barley leaves of Example 7 were identified by UPLC-MS. The identified substances included baicalin-7-diglucoside and chrysin 6-C-glucoside. The following substances were isolated from young barley leaves for the first time: 8-C-arabinoside, quercetin-3-O-neohesperidoside, quercetin-3-O-robinobioside, quercetin-3-O-rutinose, quercetin-7-rutinoside, rutin, apigenin-7-rutinoside, daidzein 7,4'-di-O-glucoside, and isorhamnetin-3-rutinoside. The combined effect of these substances resulted in the highest antioxidant activity in Example 7.

[0075] Comparative Example 1

[0076] This comparative example relates to a method for extracting, separating, and purifying polyphenols from young barley leaves with high antioxidant activity. The steps are as follows:

[0077] Step 1: Preparation of crude polyphenols from young barley leaves

[0078] After defatting with petroleum ether, the whole powder of young barley leaves was mixed with 80% methanol solution at a ratio of 1g:20mL. The mixture was ultrasonically sonicated at 40℃ for 30min using 480W, followed by centrifugation at 3000×g for 5min. The supernatant was collected. The precipitate was extracted twice more under the same ultrasonic conditions. The supernatants from the three extractions were combined and lyophilized under reduced pressure at 55℃ using a rotary evaporator. The lyophilized product was prepared into a 2mg / mL solution. Under magnetic stirring, three volumes of 100% ethanol were slowly added via a constant flow pump. After standing overnight, the precipitate was removed by centrifugation. The supernatant was then lyophilized again after removing the ethanol by rotary evaporation to obtain a crude extract of phenolic substances from young barley leaves.

[0079] Step 2: Ultrafiltration membrane separation

[0080] Pretreatment of ultrafiltration membranes: The filter membrane in the ultrafiltration tube was soaked in 10% glycerol overnight, washed with water, and then centrifuged with deionized water for 20 min. 8 mL (0.1 g / 10 mL) of crude polyphenol solution was mixed with 10 mL of water and poured into 3 kDa and 1 kDa ultrafiltration tubes respectively. The ultrafiltration tubes were centrifuged in a low-temperature centrifuge for 1 h (in two separate centrifugations) at a speed of 5000 x g and a temperature of 10 °C. The filtrates with values ​​greater than 3 kDa, 1–3 kDa, and less than 1 kDa were collected and freeze-dried.

[0081] Results: In Comparative Example 1, the total phenolic content of the purified products with greater than 3 kDa, 1–3 kDa, and less than 1 kDa was 8362, 7504, and 1482 mg GAE / 100g DW, respectively, significantly lower than that in Example 1. The DPPH radical scavenging rates were 46597, 38904, and 8237 μmol TE / g, respectively; the ABTS radical scavenging rates were 15394, 83203, and 23492 μmol TE / g, respectively; and the FRAP radical scavenging rates were 46302, 23404, and 9732 μmol TE / g, respectively, significantly lower than those in Example 1. This indicates that ultrafiltration membrane separation is not an effective method for purifying polyphenols and obtaining high antioxidant activity barley leaf polyphenols.

[0082] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A method for extracting, separating and purifying high-antioxidant-activity young-leaf highland barley polyphenols, characterized in that, The method comprises the following steps: S1, after the whole powder of the young leaf of highland barley is defatted with petroleum ether, the defatted whole powder is mixed with a 70-90% methanol solution, and ultrasonic treatment is performed at an ultrasonic temperature of 40-50 DEG C, centrifugation is performed, the precipitate is mixed with 70-90% methanol, ultrasonic treatment is performed, centrifugation is performed, the supernatant is combined, rotary evaporation is performed, and then freeze-drying is performed, ethanol precipitation is performed, centrifugation is performed, the supernatant is rotary evaporated, and then freeze-drying is performed, so as to obtain a crude extract of phenolic substances of the young leaf of highland barley; S2, liquid-liquid extraction is performed, the crude extract of phenolic substances of the young leaf of highland barley is dissolved in water, a separatory funnel is used to mix the crude polyphenol solution with an organic solvent n-butanol, the mixing is repeated twice, n-butanol is in the upper layer, and water is in the lower layer, the extraction liquid is collected, the extraction is repeated twice, and then rotary evaporation and freeze-drying are performed on the extraction liquid; S3, activation of a macroporous resin, the macroporous resin is soaked in 90-95% ethanol for 12-24 h, deionized water is used for washing until there is no white floating substance and no alcohol smell, the resin is soaked in 3-5% hydrochloric acid solution and 2-4% sodium hydroxide solution with a volume twice that of the resin for 4-6 h, and then deionized water is used for washing until the resin is neutral; S4, after the activated saturated wet resin is wetly filled in a chromatographic column, the polyphenol is dissolved in a 70-90% methanol solution to prepare a solution, the solution is loaded, deionized water is used for elution to remove impurities, and then 70-75% ethanol is used for elution to obtain the target product, the eluent is collected, ethanol is removed through rotary evaporation, and then vacuum freeze-drying is performed, so as to obtain the polyphenol extract of the young leaf of highland barley.

2. The method of extraction, isolation and purification as claimed in claim 1, wherein, In step S1, the ratio of the whole powder to the 70-90% methanol solution is 1 g:20 mL-1 g:25 mL.

3. The method of extraction, isolation and purification as claimed in claim 1, wherein, In step S1, the ultrasonic power is 480-560 W, and the ultrasonic time is 30-60 min.

4. The method of claim 1, wherein the method is a method of extraction, separation and purification. In step S1, after centrifugation, the residue precipitate is mixed with 70-90% methanol for ultrasonic extraction 2-3 times.

5. The method of claim 1, wherein the method is a method of extraction, separation and purification. In step S1, three volumes of ethanol are used for precipitation to remove polysaccharide substances.

6. The method of claim 1, wherein the method is a method of extraction, separation and purification. In step S2, the mass / volume fraction of the crude extract of phenolic substances of the young leaf of highland barley to water is 0.03-0.05 g / mL, and the volume ratio of the crude polyphenol solution to the organic reagent n-butanol is 1:1-1:1.

5.

7. The method of extraction, separation and purification as claimed in claim 1, wherein, In step S3, AB-8 macroporous resin is selected.

8. The method of extraction, isolation and purification as claimed in claim 1, wherein, In step S4, the concentration of the loading solution is 2-3 mg / mL, the loading speed is 2-3 BV / min, 4 BV of deionized water is used for removing impurities, and then 70-75% ethanol is used for elution to obtain the target product at a flow rate of 2.5-3 BV / min.

9. A high-antioxidant-activity polyphenol of the young leaf of highland barley, which is prepared by the method according to any one of claims 1-8.

10. Use of a high-antioxidant-activity polyphenol of the young leaf of highland barley, which is prepared by the method according to any one of claims 1-8, in the preparation of health-care food.

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