Method for extracting lonicera caerulea polyphenol from lonicera caerulea

Abstract

The invention relates to the technical field of polyphenol extraction, and provides a method for extracting lonicera caerulea polyphenol from lonicera caerulea. The method for extracting the lonicera caerulea polyphenol from the lonicera caerulea comprises the following steps: mixing the lonicera caerulea with a deep eutectic solvent, and performing ultrasonic treatment to obtain the lonicera caerulea polyphenol extract. The green and efficient deep eutectic solvent is adopted to replace a traditional organic solvent, so that the risk of environmental pollution is remarkably reduced, and the selectivity of bioactive substances in polyphenol is also improved. In addition, ultrasonic treatment is adopted, solute diffusion can be remarkably accelerated, lonicera caerulea polyphenol is promoted to be released from cell tissues, and therefore the extraction time is shortened, and the extraction efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of polyphenol extraction, and in particular to a method for extracting blue loquat polyphenols from blue loquat. Background Art

[0002] Blue indigo fruit is a deciduous shrub of the genus Lonicera in the family Caprifoliaceae. It is extremely cold-resistant and can overwinter in the open. The fruit and leaves of blue indigo fruit contain blue indigo polyphenols, which include various bioactive substances such as anthocyanins, phenolic acids, and flavonoids. These polyphenols have antioxidant, anti-inflammatory, and antibacterial properties, and are closely linked to human health. They are widely used in functional foods, medicines, and cosmetics.

[0003] Currently, polyphenol extraction methods primarily involve solvent extraction. Traditional solvent extraction methods use organic solvents, which have poor selectivity for extracting bioactive components from polyphenols. Furthermore, organic solvents may remain in the final product, impacting both humans and the environment. Summary of the Invention

[0004] The present invention aims to provide a method for extracting polyphenols from Lonicera edulis. The method provided by the present invention can improve the selectivity of bioactive substances in polyphenols and reduce the risk of environmental pollution.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for extracting polyphenols from Lonicera edulis fruit comprises the following steps:

[0007] The blue loquat fruit is mixed with a deep eutectic solvent and subjected to ultrasonic treatment to obtain a blue loquat fruit polyphenol extract.

[0008] Preferably, the deep eutectic solvent comprises a hydrogen bond acceptor, a hydrogen bond donor and water, the hydrogen bond acceptor comprises choline chloride, and the hydrogen bond donor comprises one of fructose, glucose, malic acid, citric acid, lactic acid, urea, ethylene glycol, glycerol or 1,4-butanediol.

[0009] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the deep eutectic solvent is 1:(1-6), and the volume fraction of water in the deep eutectic solvent is 20-40%.

[0010] Preferably, the mass ratio of the blue loquat fruit to the deep eutectic solvent is 1:30-60.

[0011] Preferably, the temperature of the ultrasonic treatment is 0-35° C., and the time is 10-20 min; the ultrasonic power of the ultrasonic treatment is 150-250 W, and the ultrasonic frequency is 20-25 kHz.

[0012] Preferably, after the ultrasonic treatment, the method further comprises: centrifuging the obtained mixed extract to obtain a supernatant; and filtering the supernatant to obtain a blue loquat polyphenol extract.

[0013] Preferably, the rotation speed of the centrifugal treatment is 6000-10000 rpm, the temperature is 4-25° C., and the time is 10-20 min; and the pore size of the filter membrane used in the filtration is 0.22 μm.

[0014] Preferably, the Lonicera edulis fruit includes Lonicera edulis fruit or Lonicera edulis leaves.

[0015] Preferably, before the blue loquat fruit is mixed with the deep eutectic solvent, the method further comprises: freezing, crushing and sieving the blue loquat fruit in sequence.

[0016] Preferably, the freezing temperature is -80 to -20°C, and the freezing time is 12 to 48 hours; and the sieving mesh size is 100 meshes.

[0017] The present invention provides a method for extracting polyphenols from blueberries, comprising the following steps: mixing blueberries with a deep eutectic solvent and subjecting them to ultrasonic treatment to obtain a blueberries polyphenol extract. The present invention uses a green and efficient deep eutectic solvent to replace traditional organic solvents, significantly reducing the risk of environmental pollution and improving the selectivity of bioactive substances in the polyphenols. Furthermore, ultrasonic treatment can significantly accelerate solute diffusion and promote the release of blueberries polyphenols from cell tissues, thereby shortening the extraction time and improving the extraction efficiency.

[0018] Furthermore, the present invention effectively avoids high-temperature destruction of polyphenols by precisely controlling the temperature of ultrasonic treatment, maintains the natural antioxidant activity of polyphenols, maximizes the protection of heat-sensitive components (such as anthocyanins), ensures the high biological activity of the blue loquat polyphenol extract, and further enhances its antioxidant, anti-inflammatory, antibacterial and other functional properties, thereby improving the quality and application value of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flow chart of extracting polyphenols from blueberries in the present invention;

[0020] Figure 2 Fourier transform infrared spectra of different types of deep eutectic solvents;

[0021] Figure 3 Bar graph showing the total content of blue loquat polyphenols extracted from fruits and leaves of different species of blue loquat using 80% methanol solvent and different deep eutectic solvents. DETAILED DESCRIPTION

[0022] The present invention provides a method for extracting polyphenols from Lonicera edulis, comprising the following steps:

[0023] The blue loquat fruit is mixed with a deep eutectic solvent and subjected to ultrasonic treatment to obtain a blue loquat fruit polyphenol extract.

[0024] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0025] As an embodiment of the present invention, the blue indigo carp includes blue indigo carp fruits or blue indigo carp leaves, specifically blue fairy fruits, bud fruits, blue fairy leaves or bud leaves.

[0026] As an embodiment of the present invention, the blue honeysuckle is further pretreated before being mixed with the deep eutectic solvent; the pretreatment may include freezing, crushing and sieving the blue honeysuckle in sequence. As an embodiment of the present invention, the freezing temperature may be -80 to -20°C, and the time may be 12 to 48 hours, specifically 20 hours. Freezing the blue honeysuckle can stabilize its cell structure, which is beneficial for subsequent extraction. After freezing, the frozen blue honeysuckle is transferred to a freeze dryer and freeze-dried to a constant weight state; the crushing is carried out in a crusher; the mesh size of the sieving may be 100 meshes.

[0027] As an embodiment of the present invention, the deep eutectic solvent includes a hydrogen bond acceptor, a hydrogen bond donor and water, the hydrogen bond acceptor includes choline chloride, and the hydrogen bond donor includes one of fructose, glucose, malic acid, citric acid, lactic acid, urea, ethylene glycol, glycerol or 1,4-butanediol.

[0028] As an embodiment of the present invention, the preparation method of the deep eutectic solvent comprises the following steps:

[0029] The hydrogen bond acceptor, the hydrogen bond donor and water are mixed, heated and stirred to obtain a deep eutectic solvent.

[0030] As an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor and the hydrogen bond donor of the deep eutectic solvent is 1: (1 to 6), specifically 1: 2; the volume fraction of water in the deep eutectic solvent is 20 to 40%, specifically 30%, and adding water to the mixed solution of the hydrogen bond acceptor and the hydrogen bond donor can reduce the viscosity of the deep eutectic solvent. The temperature of the heating and stirring can be 60 to 80°C; the present invention has no special limitation on the time of the heating and stirring, and the heating and stirring is carried out until a uniform and stable liquid is formed. As an embodiment of the present invention, after the heating and stirring are completed, the obtained deep eutectic solvent is cooled to below 40°C, specifically 15 to 30°C, and poured into a glass bottle and sealed for storage.

[0031] As an embodiment of the present invention, the mass ratio of the blue loquat fruit to the deep eutectic solvent can be 1:30 to 60.

[0032] As one embodiment of the present invention, the ultrasonic treatment temperature can be 0-35°C, the time can be 10-20 minutes, specifically 15 minutes; the ultrasonic power of the ultrasonic treatment can be 150-250W, specifically 210W, and the ultrasonic frequency can be 20-25kHz. The equipment used for the ultrasonic treatment is preferably a probe-type ultrasonic system. In the present invention, the mixture of blue loquat fruit and deep eutectic solvent is preferably placed in a water bath for the ultrasonic treatment.

[0033] As an embodiment of the present invention, after the ultrasonic treatment, the method further comprises: centrifuging the obtained mixed extract to obtain a supernatant; and filtering the supernatant to obtain a blueberry polyphenol extract.

[0034] In a specific embodiment of the present invention, the blue honeysuckle polyphenols extracted from the blue elf fruit mainly include cyanidin-3-glucoside anthocyanin. The content of cyanidin-3-glucoside extracted using a deep eutectic solvent can be 540-1510 mg / 100 g DW, specifically 554.53±6.33-1487.20±13.76 mg / 100 g DW. The content of cyanidin-3-glucoside extracted using a choline chloride-lactic acid deep eutectic solvent is the highest. The blue honeysuckle polyphenols extracted from the bud fruit mainly include cyanidin-3-glucoside anthocyanin. The content of cyanidin-3-glucoside extracted using a deep eutectic solvent can be 700-1610 mg / 100g DW, specifically 739.70±34.97-1551.13±58.88 mg / 100g DW. The content of cyanidin-3-glucoside extracted using a choline chloride-lactic acid deep eutectic solvent is the highest. The polyphenols extracted from the leaves of the blueberry fruit mainly consisted of chlorogenic acid. The chlorogenic acid content was higher when extracted using choline chloride-malic acid, choline chloride-citric acid, choline chloride-lactic acid, and choline chloride-1,4-butanediol deep eutectic solvents, ranging from 395 to 565 mg / 100 g DW (440.12±40.47, 491.87±48.72, 598.10±50.29, and 514.81±46.32 mg / 100 g DW, respectively). The polyphenols extracted from the buds of the blueberry fruit mainly consisted of kaempferol-glucoside. The chlorogenic acid content was higher when extracted using choline chloride-glycerol deep eutectic solvents, reaching 470.59±42.95 mg / 100 g DW.

[0035] As an embodiment of the present invention, the rotation speed of the centrifugal treatment can be 6000-10000 rpm, the temperature can be 4-25° C., and the time can be 10-20 min; the pore size of the filter membrane used in the filtration is 0.22 μm.

[0036] As an embodiment of the present invention, the polyphenol extract of Lonicera caerulea is stored at 4°C.

[0037] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] S1: Freeze the Smurf fruit at -20°C for 20 hours, transfer the frozen Smurf fruit to a freeze dryer, and freeze-dry to a constant weight; grind the freeze-dried blue indigo fruit with a grinder, and sieve through a 100-mesh sieve to obtain Smurf fruit powder.

[0040] S2: Choline chloride is used as a hydrogen bond acceptor, and fructose, glucose, malic acid, citric acid, lactic acid, urea, ethylene glycol, propylene glycol and 1,4-butanediol are used as hydrogen bond donors, respectively. The hydrogen bond acceptor and the hydrogen bond donor are mixed in a molar ratio of 1:2, heated at 80°C with constant stirring, and water with a volume fraction of 30% of a deep eutectic solvent is added to obtain deep eutectic solvent groups (1) to (9); the deep eutectic solvent groups (1) to (9) are cooled to 20°C and sealed for storage.

[0041] S3: Take the Smurf fruit powder obtained in S1, add the deep eutectic solvents (1) to (9) prepared in S2 respectively and mix thoroughly, the mass ratio of blue indigo fruit to deep eutectic solvent is 1:30; place the mixture of blue indigo fruit and deep eutectic solvent in a water bath, maintain the temperature at 30°C, and use a probe-type ultrasonic system to ultrasonically treat the mixture for 15 minutes at an ultrasonic power of 210W and an ultrasonic frequency of 20kHz to obtain mixed extracts (1) to (9).

[0042] S4: The mixed extracts (1) to (9) obtained in S3 were taken separately, and centrifuged at 8000 rpm for 15 min at 20°C to obtain supernatants (1) to (9). The supernatants (1) to (9) were filtered using a 0.22 μm filter membrane to obtain blue indigo polyphenol extracts (1) to (9) of the blue smurf fruit, and stored at 4°C.

[0043] Example 2

[0044] The method is basically the same as Example 1, except that the blue elf fruit is replaced with the bud fruit, and the blue elongated indigo fruit polyphenol extracts (1) to (9) of the bud fruit are obtained.

[0045] Example 3

[0046] The method is basically the same as Example 1, except that the Smurf fruit is replaced with Smurf leaf, and the Smurf leaf polyphenol extracts (1) to (9) of the blueberry fruit are obtained.

[0047] Example 4

[0048] The method is basically the same as Example 1, except that the blueberry fruit is replaced with bud leaves to obtain groups (1) to (9) of blueberry polyphenol extracts of bud leaves.

[0049] Comparative Example 1

[0050] The method is basically the same as Example 1, except that the deep eutectic solvent is replaced with a methanol solvent with a volume fraction of 80%, thereby obtaining an 80% methanol-extracted blue loquat fruit polyphenol extract.

[0051] Comparative Example 2

[0052] The method is basically the same as Example 2, except that the deep eutectic solvent is replaced with a methanol solvent with a volume fraction of 80%, thereby obtaining an 80% methanol-extracted blueberry polyphenol extract of the bud fruit.

[0053] Comparative Example 3

[0054] The method is basically the same as Example 3, except that the deep eutectic solvent is replaced with a methanol solvent with a volume fraction of 80%, thereby obtaining an 80% methanol-extracted polyphenol extract of the blueberry leaves.

[0055] Comparative Example 4

[0056] The method is basically the same as Example 4, except that the deep eutectic solvent is replaced with a methanol solvent with a volume fraction of 80%, thereby obtaining an 80% methanol-extracted blueberry polyphenol extract of buds and leaves.

[0057] Test Example 1

[0058] Fourier transform infrared spectrometer at 4000 cm -1 and 400cm -1 The deep eutectic solvent was analyzed as follows. Figure 2 shown.

[0059] Test Example 2

[0060] 20 μL of 1-3 mg / mL of the blue loquat polyphenol extracts extracted from Examples 1-4 and Comparative Examples 1-4 and 90 μL of deionized water were added to a 96-well plate, and then 10 μL of 1 mol / L Folin phenol reagent was added. The mixture was incubated for 5 minutes in a dark environment at room temperature. Subsequently, 80 μL of 75 g / L Na2CO3 solution was added to the 96-well plate, and the mixture was incubated for 2 hours in a dark environment at room temperature. The absorbance of the sample was then measured at 765 nm using a microplate reader. The results are expressed in milligrams of gallic acid equivalents per gram of sample dry weight (mg GAE / g DW). The results are shown in Table 1. Figure 3 shown.

[0061] Test Example 3

[0062] Utilizing LC-HR-TOF / MS 2 The identification and quantitative analysis of polyphenol components in blue loquat fruit extract were carried out by high performance liquid chromatography-mass spectrometry (LC-HR-TOF / MS 2 ) Qualitative and quantitative analysis was performed on the polyphenol extracts of Indigofera edulis extracted from the fruits and leaves of Indigofera edulis in Examples 1-4 and Comparative Examples 1-4. The polyphenol extracts were separated using a Phenomenex Luna C18 column (5 μm, 250 mm x 46 mm). Flow rate: 1 mL / min; column temperature: 25°C; injection volume: 5 μL. The polyphenol elution program is shown in Table 1; the mass spectrometry positive ion conditions are shown in Table 2; and the mass spectrometry negative ion conditions are shown in Table 3. The contents of various bioactive substances in the polyphenol extracts of Indigofera edulis extracted from different fruits and leaves of Indigofera edulis in Examples 1-4 and Comparative Examples 1-4 using different solvents are shown in Table 4.

[0063] Table 1 Polyphenol elution procedure

[0064]

[0065] Table 2 Mass spectrometry positive ion conditions

[0066] Parameter Primary mass spectrometry ESI-MS <![CDATA[Secondary mass spectrometry ESI-MS 2 > Scan TypeScanType TOF TOF IonSprayVoltageFloating (ISVF) 5500V 5500V Spray gas SourceGas1(GS1) 55psi 55psi Auxiliary heating gas SourceGas2 (GS2) 55psi 55psi CurtainGas (CUR) 35psi 35psi Temperature (TEM) 550℃ 550℃ Declustering Potential (DP) 100V 100V Collision Energy (CE) 10V 40V Collision Energy Spread (CES) 20V

[0067] Table 3 Mass spectrometry negative ion conditions

[0068]

[0069]

[0070] Table 4 Contents of various bioactive substances in polyphenol extracts from different blueberry fruits and leaves extracted using different solvents (mg / 100g DW)

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] Table 4 shows that the present invention detected three types of anthocyanins in the blue Lonicera edulis fruit and a total of 21 polyphenolic compounds in the blue Lonicera leaves. Among them, cyanidin-3-glucoside is the main anthocyanin in the blue Lonicera fruit; chlorogenic acid is the phenolic acid with the highest content in the blue Lonicera leaves; and kaempferol-glucoside is the main flavonoid compound in the buds. Compared with the traditional 80% methanol extraction method, the blue Lonicera polyphenol extract obtained by extraction using a choline chloride-lactic acid deep eutectic solvent showed an increase of cyanidin-3-glucoside by 1.96% in the blue Lonicera buds and 2.28% in the blue Lonicera fruit. Compared to 80% methanol, the kaempferol-glucoside content of the blue indigo fruit polyphenol extract from the bud leaves, extracted using a choline chloride-glycerol deep eutectic solvent, increased by 11.57%. The chlorogenic acid content of the blue indigo fruit polyphenol extract from the blue elf leaves, extracted using a choline chloride-lactic acid deep eutectic solvent, increased significantly by 47.60%. These results demonstrate that green deep eutectic solvents not only improve polyphenol extraction efficiency but also exhibit strong component selectivity, providing an effective technical means for the targeted extraction of functional active substances from different tissues of the blue indigo fruit.

[0077] Figure 1 This is a flow chart of extracting polyphenols from blueberries from blueberries; Figure 1 It can be seen that in the present invention, the blue honeysuckle is pretreated and a deep eutectic solvent is prepared, and the pretreated blue honeysuckle and the deep eutectic solvent are extracted by ultrasonic treatment to finally obtain the blue honeysuckle polyphenol extract.

[0078] Figure 2 The following are Fourier transform infrared spectra of different types of deep eutectic solvents. Figure 2 It can be seen that after the synthesis of the deep eutectic solvent, the main characteristic absorption peaks of choline chloride with alcoholamines and carboxylic acids are still retained in the deep eutectic solvent, indicating that their functional groups have not undergone any chemical reaction and are in a certain stable state, indicating that strong hydrogen bonding is formed between choline chloride and hydrogen bond donors such as sugars (choline chloride-fructose, choline chloride-glucose), carboxylic acids (choline chloride-malic acid, choline chloride-citric acid and choline chloride-lactic acid) and alcoholamines (choline chloride-urea, choline chloride-ethylene glycol, choline chloride-glycerol and choline chloride-1,4-butanediol).

[0079] Figure 3 The total content of polyphenols extracted from the fruits and leaves of different species of blue loquat fruit by 80% methanol solvent and different kinds of deep eutectic solvents is shown in the bar graph. Figure 3As shown in the data, for the bud fruit, the total polyphenol content of the blue loquat polyphenol extracts extracted using deep eutectic solvents ranged from 59.30 to 111.13 mg GAE / g DW, while the total polyphenol content of 80% methanol solvent was 80.49 mg GAE / g DW. The extraction yields of phenolic compounds using different deep eutectic solvents also varied significantly. The choline chloride-fructose deep eutectic solvent significantly exceeded the extraction yields of other solvents, with a total polyphenol yield of 111.13 mg GAE / g DW, a 1.38-fold increase compared to 80% methanol. The total polyphenol content extracted using the choline chloride-glucose deep eutectic solvent was second only to the choline chloride-fructose deep eutectic solvent, reaching 94.28 mg GAE / g DW, a 1.17-fold increase compared to 80% methanol. For Smurf fruit, the choline chloride-fructose and choline chloride-1,4-butanediol deep eutectic solvents achieved high extraction yields, reaching total polyphenol contents of 126.3 mg GAE / g DW and 74.87 mg GAE / g DW, respectively. These yields were significantly increased by 2-fold and 1.2-fold compared to the conventional 80% methanol solvent (62.36 mg GAE / g DW). For bud leaves, the total polyphenol content extracted using deep eutectic solvents ranged from 5.75 to 12.40 mg GAE / g DW. The choline chloride-glycerol deep eutectic solvent achieved the highest total polyphenol extraction yield, 1.3 times that of the 80% methanol solvent. For Smurf leaves, the total polyphenol content extracted by deep eutectic solvents was 9.15 to 15.48 mg GAE / g DW. The extraction rate of choline chloride-urea deep eutectic solvents was significantly higher than that of 80% methanol, which was 1.25 times that of 80% methanol.

[0080] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for extracting polyphenols from Lonicera edulis, comprising the following steps: The blue loquat fruit is mixed with a deep eutectic solvent and subjected to ultrasonic treatment to obtain a blue loquat fruit polyphenol extract.

2. The method according to claim 1, characterized in that The deep eutectic solvent includes a hydrogen bond acceptor, a hydrogen bond donor and water, the hydrogen bond acceptor includes choline chloride, and the hydrogen bond donor includes one of fructose, glucose, malic acid, citric acid, lactic acid, urea, ethylene glycol, glycerol or 1,4-butanediol.

3. The method according to claim 2, characterized in that The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the deep eutectic solvent is 1:(1-6), and the volume fraction of water in the deep eutectic solvent is 20-40%.

4. The method according to claim 1, wherein The mass ratio of the blue loquat fruit to the deep eutectic solvent is 1:30-60.

5. The method according to claim 1, wherein The temperature of the ultrasonic treatment is 0-35° C., and the time is 10-20 min. The ultrasonic power of the ultrasonic treatment is 150-250 W, and the ultrasonic frequency is 20-25 kHz.

6. The method according to claim 1, characterized in that After the ultrasonic treatment, the method further comprises: centrifuging the obtained mixed extract to obtain a supernatant; and filtering the supernatant to obtain a blue loquat polyphenol extract.

7. The method according to claim 6, characterized in that The rotation speed of the centrifugal treatment is 6000-10000 rpm, the temperature is 4-25° C., and the time is 10-20 min; the pore size of the filter membrane used in the filtration is 0.22 μm.

8. The method according to claim 1, characterized in that The blue honeysuckle includes blue honeysuckle fruits or blue honeysuckle leaves.

9. The method according to claim 8, characterized in that Before the blue loquat fruit is mixed with the deep eutectic solvent, the method further comprises: freezing, crushing and sieving the blue loquat fruit in sequence.

10. The method according to claim 9, characterized in that The freezing temperature is -80 to -20°C, and the freezing time is 12 to 48 hours; and the sieving mesh number is 100 meshes.

Images