Method for simultaneously extracting and separating flavonoid and alkaloid components in mulberry leaves

Abstract

The invention discloses a method for simultaneously extracting and separating flavonoid and alkaloid components in mulberry leaves, which comprises the following steps: firstly, adjusting the pH value of an aqueous solution of a deep eutectic solvent to form a uniform single-phase solution, adding mulberry leaf powder into the single-phase solution, carrying out ultrasonic extraction, centrifuging after the extraction is finished, and taking a supernatant; adjusting the pH value of the extracting solution again to form a two-phase system, and respectively obtaining an upper-layer eutectic solvent phase and a lower-layer water phase. The conditions of the extraction step are as follows: the concentration of the eutectic solvent is 30%-70%, the solid-to-liquid ratio is 1: 5 g / mL-1: 40 g / mL, the extraction time is 20 min-100 min, and the extraction pH is 5.5-9.5; in the separation step, the conditions of the separation step are as follows: the separation pH is 1-5. Through HPLC (High Performance Liquid Chromatography) analysis, a flavonoid component (isoquercitrin) is mainly distributed in an upper-layer deep-eutectic solvent phase, an alkaloid component (deoxynojirimycin) is mainly distributed in a lower-layer water phase, and the reusability is good.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction and separation, and specifically relates to a method for extracting and separating flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) components from mulberry leaves by using a low eutectic solvent. Background Art

[0002] Mulberry leaves are the dried leaves of white mulberry and Chinese mulberry of the Moraceae family, and are commonly used in traditional Chinese medicine. Mulberry leaves mainly include chemical components such as flavonoids, alkaloids, and polysaccharides. Among them, flavonoids mainly include isoquercetin, rutin, quercetin, etc., and alkaloids are mainly polyhydroxy alkaloids, including deoxynojirimycin and buckwheat alkaloids, which are considered to be the main active ingredients of mulberry leaves and have multiple pharmacological effects such as hypoglycemic and anti-tumor. At present, the extraction of mulberry leaf chemical components mainly uses traditional solvents such as water, methanol, and ethanol. The obtained extract contains both flavonoids and alkaloids. In order to further separate these two types of components, column chromatography, liquid-liquid extraction and other methods are needed. These complex extraction and separation processes are time-consuming, inefficient, and costly, and a large amount of toxic organic solvents are also used. Therefore, it is necessary to develop a green and efficient extraction and separation method to promote the development and utilization of flavonoids and alkaloids in mulberry leaves.

[0003] Deep eutectic solvent (DES) is a green solvent formed by hydrogen bond donors and hydrogen bond acceptors connected by hydrogen bonds. It has the advantages of good solubility, non-volatility, and simple preparation. It is expected to replace traditional organic solvents and be used in the field of extraction and separation. At the same time, due to the structural designability of DES, different hydrogen bond donors and hydrogen bond acceptors can be selected as needed to synthesize specific DES to achieve efficient or targeted separation of target components. In recent years, more and more studies have used DES to extract natural products from traditional Chinese medicine, and achieved satisfactory results.

[0004] The DES / H2O two-phase system is often composed of DES and an inorganic salt aqueous solution (phosphate, sulfate, etc.). Compared with other conventional two-phase liquid systems, it has the advantages of mild operating conditions and easy phase separation. In the past decade, the DES / H2O two-phase system has developed rapidly and has become an effective method for the enrichment and separation of various chemical components. In recent years, stimulus-responsive DES / H2O systems have gradually developed, and their single-phase / two-phase states can be reversibly switched with changes in experimental conditions (temperature, pH, CO2, etc.). Compared with conventional DES / H2O two-phase systems, this type of system is easy to recycle and reuse DES, which is more in line with the concept of green chemistry and sustainable development. Among them, the pH-responsive DES / H2O system has low energy consumption, simple equipment and easy to expand production in actual operation, and has gradually attracted widespread attention.

[0005] However, there is no study on the application of pH-responsive DES / H2O system for the extraction and further separation of different types of natural products from traditional Chinese medicine. Summary of the invention

[0006] In order to overcome the problems of the prior art, the purpose of the present invention is to provide a method for simultaneously extracting and separating flavonoids and alkaloids in mulberry leaves, which is a method for extracting and separating flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) from mulberry leaves using a low eutectic solvent. The method is green and environmentally friendly, has good specificity and extraction and separation effects, and is simple to operate.

[0007] In order to achieve the above-mentioned purpose of the invention, the purpose of the present invention is achieved by the following technical solutions:

[0008] A method for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves is to use a deep eutectic solvent (DES) to extract and separate flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) from mulberry leaves. Specifically, at room temperature, the deep eutectic solvent DES and water are mixed in a certain molar ratio, and a NaOH solution is added thereto to adjust the pH to obtain a uniform single-phase solution. Then a certain amount of mulberry leaf powder is added, ultrasonic extraction is performed for a certain period of time, and after the extraction is completed, centrifugation is performed to remove the mulberry leaf residue to obtain the upper extract.

[0009] In order to further separate the flavonoids and alkaloids, hydrochloric acid solution was added to the extract to adjust the pH to promote the separation of the solution, and the upper DES phase and the lower aqueous phase were obtained respectively. The flavonoids (isoquercetin) component was mainly distributed in the upper DES phase, while the alkaloids (deoxynojirimycin) component was mainly distributed in the lower aqueous phase.

[0010] The chemical structures of isoquercetin and deoxynojirimycin are shown below:

[0011]

[0012] Specifically, in the above extraction method, the deep eutectic solvent (DES) is shown in Table 1.

[0013] Table 1. Deep eutectic solvents (DES) used in the present invention

[0014]

[0015] In order to optimize the extraction conditions, the concentration of the deep eutectic solvent (DES) is selected to be 30% to 70% (preferably 50%); the solid-liquid ratio is 1:5 g / mL to 1:40 g / mL (preferably 1:10 g / mL); the extraction time is 20 min to 100 min (preferably 80 min); and the pH is 5.5 to 9.5 (preferably 6.5) for extraction. In addition, the pH is selected to be 1 to 5 (preferably 3) for separation.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0017] The present invention adopts a deep eutectic solvent (DES) ultrasound-assisted extraction and separation-HPLC detection method, applies Origin statistical analysis software, and uses an HPLC method to simultaneously determine the content of isoquercetin and deoxynojirimycin in the extract (diluted with an equal volume of methanol) to obtain the optimal extraction and separation process. The specific method of the present invention using DES to extract and separate flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) from mulberry leaves is as follows:

[0018] 1. Preparation and characterization of deep eutectic solvents (DES). The DES used in this study was mixed with hydrogen bond donors and hydrogen bond acceptors in a molar ratio of 1:1. Each DES was obtained by continuous stirring at 60°C for 2 hours. They are all uniform and transparent liquids. Then, the infrared spectra of each DES were measured using a Fourier transform infrared spectrometer for structural characterization.

[0019] 2. pH-responsive DES / H2O system: The phase diagram of each DES / H2O system under different pH conditions was determined by turbidity point titration to obtain the phase separation of each DES and water.

[0020] 3. Determine the best DES as the extraction agent, use 2-methyl-2,4-pentanediol (MPD) as the hydrogen bond donor, compare the extraction efficiency and separation effect of different hydrogen bond acceptors (pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid and nonanoic acid) on the target compound, and finally determine [hexanoic acid][2-methyl-2,4-pentanediol] ([HexA][MPD]) as the best extraction agent.

[0021] 4. Using [HexA][MPD] as the extraction agent, single factor analysis experiments were used to investigate the effects of different solid-liquid ratios, extraction time, pH, and DES concentration on the extraction efficiency of isoquercetin and deoxynojirimycin. The final solid-liquid ratio was 1:10 g / mL; extraction time was 80 min; pH was 6.5; and DES concentration was 50%. According to the separation effect of isoquercetin and deoxynojirimycin, the separation pH was determined to be 3.

[0022] 5. Use the optimal factor level in step 4 to extract and separate the flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) components in mulberry leaves, evaluate the extraction and separation effects, and recover DES by column chromatography to evaluate the recyclability of DES.

[0023] The present invention provides a new idea and method for the extraction and separation of natural products. The optimal extraction and separation conditions established by the present invention are highly efficient and can efficiently extract and separate flavonoids and alkaloid compounds from mulberry leaves. At the same time, the extraction medium DES in the method can be recycled and reused, is environmentally friendly and pollution-free, and is suitable for industrial production, providing a scientific basis for the further development and utilization of mulberry leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This paper describes the process of extracting and separating flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) from mulberry leaves using a pH-responsive deep eutectic solvent (DES) / H2O system.

[0025] Figure 2 Infrared spectra of hexanoic acid (HexA), 2-methyl-2,4-pentanediol (MPD) and [HexA][MPD].

[0026] Figure 3 Phase diagrams of different DES / H2O systems under different pH conditions.

[0027] Figure 4 The phase separation of [HexA][MPD] (50%, v / v) and water under different pH conditions.

[0028] Figure 5 The extraction and separation efficiency of isoquercetin (A) and deoxynojirimycin (B) in mulberry leaves using different DES / H2O systems.

[0029] Figure 6 Effects of [HexA][MPD] concentration (A), extraction time (B), solid-liquid ratio (C) and pH value (D) on the extraction efficiency of isoquercetin and deoxynojirimycin from mulberry leaves.

[0030] Figure 7 The distribution ratios of isoquercetin (A) and deoxynojirimycin (B) in the [HexA][MPD] / H2O two-phase system under different pH conditions.

[0031] Figure 8 The reusability of [HexA][MPD] aqueous solution in extracting isoquercetin and deoxynojirimycin from mulberry leaves (the extraction rate is expressed as the percentage of the measured value relative to the maximum measured value). DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0033] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0034] Example 1

[0035] A method for extracting and separating flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) from mulberry leaves using a deep eutectic solvent, specifically comprising the following steps:

[0036] (1) Extraction step: At room temperature, a low eutectic solvent (DES) and water were mixed in the same volume ratio, and NaOH solution was added thereto to adjust the pH to obtain a uniform single-phase solution. Then, 0.6 g of mulberry leaf powder was accurately weighed, and 6 mL of the above 50% DES aqueous solution was added. Ultrasonic extraction was performed for 80 min, and centrifugation was performed to obtain the upper extract. (2) Separation step: In order to further separate the flavonoids and alkaloids, hydrochloric acid solution was added to the extract to adjust the pH to promote the separation of the solution, and the upper DES phase and the lower aqueous phase were obtained, respectively. Figure 1 In the experiment, the extraction and separation conditions such as DES type, DES concentration, solid-liquid ratio, extraction time, pH, etc. were changed in turn to investigate their effects on the extraction and separation efficiency. Each experiment was performed three times in parallel.

[0037] 1. Structural Characterization of Deep Eutectic Solvents (DES)

[0038] In the present invention, the inventors used organic acids of different chain lengths (pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid and nonanoic acid) as hydrogen bond donors and 2-methyl-2,4-pentanediol (MPD) as hydrogen bond acceptors to synthesize a series of DES with polarity gradient changes. The synthesis process of DES mainly involves the formation of hydrogen bonds between hydrogen bond donors and hydrogen bond acceptors. Therefore, the inventors characterized the structure of the synthesized DES through infrared spectra to confirm the existence of hydrogen bonds therein. Taking [HexA][MPD] as an example, its infrared spectrum is as follows Figure 2 [HexA][MPD] contains all the characteristic absorption peaks of the hydrogen bond donor hexanoic acid and the hydrogen bond acceptor MPD, and no new absorption peaks are generated, indicating that no chemical reaction (breaking or forming of chemical bonds) occurs during the synthesis of [HexA][MPD]. In addition, the infrared spectra of hexanoic acid or MPD have a peak at 3000-3600cm -1 There is a characteristic absorption peak of -OH between them, and after the formation of [HexA][MPD], the absorption peak at this position becomes broader and stronger, proving the existence of hydrogen bonds and confirming the synthesis of [HexA][MPD].

[0039] 2. Phase diagram of pH-responsive DES / H2O system

[0040] At room temperature and pressure, the prepared deep eutectic solvent (DES) was mixed with water in different volume ratios to obtain a two-phase solution, with the upper layer being the DES phase and the lower layer being the water phase. Then, NaOH solution was added dropwise to the solution and vortexed to adjust the pH of the solution until a uniform single-phase solution was formed, and the pH value of the solution and the DES concentration at this time were recorded. This process was repeated to generate a series of data to establish the phase diagram of the DES / H2O system. Figure 3 As shown in the figure, [PenA][MPD], [HexA][MPD], [HepA][MPD], [OctA][MPD] and [NonA][MPD] can all form a pH-responsive two-phase system with water. When the position corresponding to the DES concentration and pH value is below the curve, the system forms two phases with large polarity differences, namely the upper DES phase and the lower aqueous phase, while when the position is above the curve, the system forms a uniform single phase. In addition, for the DES / H2O system in two phases, changes in pH value can significantly affect the volume of the upper DES phase and the lower aqueous phase. Taking [HexA][MPD] as an example, as the pH value of the system increases, the volume of the upper DES phase gradually increases, while the volume of the lower aqueous phase gradually decreases ( Figure 4 ), indicating that pH value can affect the phase separation of DES and water, which may further affect the separation of flavonoids and alkaloids in mulberry leaves.

[0041] 3. Selection of Deep Eutectic Solvent (DES)

[0042] Different DES were selected to extract and separate flavonoids (isoquercetin) and alkaloids (deoxynojirimycin) from mulberry leaves, and the extraction and separation efficiencies of the two target compounds were compared. Figure 5 As shown in the figure, flavonoids are mainly distributed in the DES phase, while alkaloids with relatively large polarity are mainly distributed in the water phase. Figure 5 A), as the alkyl chain of the organic acid is extended, the content in the DES phase first increases and then decreases, and correspondingly, the content in the water phase first decreases and then increases. Among them, the content of isoquercetin in the [HexA][MPD] phase is the highest, and at the same time, the content in the corresponding water phase is the lowest. For deoxynojirimycin ( Figure 5 B), as the alkyl chain of the organic acid was extended, the content in the DES phase did not change significantly, while the content in the aqueous phase tended to decrease gradually. Among them, the content of [PenA][MPD] in the aqueous phase was slightly higher than that in the aqueous phase of [HexA][MPD]. Based on the extraction and separation efficiency of isoquercetin and deoxynojirimycin, [hexanoic acid][2-methyl-2,4-pentanediol] ([HexA][MPD]) was finally selected as the optimal DES for subsequent experimental condition optimization.

[0043] IV. Selection of [HexA][MPD] concentration

[0044] According to the above extraction conditions, other experiments were the same, and the effects of different [HexA][MPD] concentrations (v / v) of 30%, 40%, 50%, 60%, and 70% on the extraction efficiency of the target compound were investigated. Figure 6 As shown in A, when the [HexA][MPD] concentration increased from 30% to 50%, the extraction efficiency of isoquercetin increased, while when the [HexA][MPD] concentration increased further, the extraction efficiency of isoquercetin decreased slightly. On the other hand, the extraction efficiency of deoxynojirimycin decreased with the increase of [HexA][MPD] concentration. Based on the above results, 50% (v / v) was selected as the optimal [HexA][MPD] concentration.

[0045] 5. Selection of extraction time

[0046] According to the above extraction conditions, other experiments were the same, and the effects of different extraction times of 20, 40, 60, 80, and 100 min on the extraction efficiency of the target compound were investigated. Figure 6 As shown in Figure B, when the extraction temperature increases from 20 min to 80 min, the extraction efficiency of the target compound gradually increases. After further extending the extraction time, the extraction efficiency of the target compound no longer increases, indicating that the extraction process has reached equilibrium. Therefore, 80 min was selected as the optimal extraction time.

[0047] 6. Selection of solid-liquid ratio

[0048] According to the above extraction conditions, other experiments were the same, and the effects of different solid-liquid ratios of 1:5, 1:10, 1:20, and 1:40 g / mL on the extraction efficiency of the target compound were investigated. Figure 6 As shown in Figure C, when the solid-liquid ratio reaches 1:10 g / mL, the extraction efficiency reaches the maximum value. If the solid-liquid ratio is further increased, the extraction efficiency will decrease. Therefore, 1:10 g / mL is selected as the optimal solid-liquid ratio.

[0049] 7. Choice of pH value

[0050] According to the above extraction conditions, other experiments were the same, and the effects of different pH values ​​of 6.5, 7.5, 8.5, and 9.5 on the extraction efficiency of the target compound were investigated. Figure 6 As shown in Figure 3, the solvent pH has a great influence on the extraction efficiency of deoxynojirimycin. In the pH range where [HexA][MPD] and water form a single phase, the extraction efficiency of deoxynojirimycin decreases with the increase of pH, and the extraction efficiency is the highest at pH 6.5. On the other hand, the solvent pH has no significant effect on the extraction efficiency of isoquercetin. Therefore, 6.5 was selected as the optimal solvent pH.

[0051] 8. Optimization of separation conditions of [HexA][MPD] / H2O two-phase system

[0052] According to the above conditions, other experiments were the same, and the effects of different pH values ​​of 5, 4, 3, 2, and 1 on the separation of target compounds were investigated. Figure 7 As shown in the figure, when the pH decreases from 5 to 3, the distribution ratio of isoquercetin in the two phases does not change significantly, while the proportion of deoxynojirimycin in the aqueous phase gradually increases. When the pH further decreases from 3 to 1, the proportion of isoquercetin in the [HexA][MPD] phase gradually decreases, while the distribution ratio of deoxynojirimycin in the two phases does not change significantly. In view of this, the optimal pH for the separation of the [HexA][MPD] / H2O two-phase system is selected as 3.

[0053] IX. Evaluation of the reuse of [HexA][MPD]

[0054] Using methanol as the eluent, 50 mL of Sephadex LH-20 dextran gel was filled into an open glass column, and the [HexA][MPD] phase after the mulberry leaf extraction and separation was separated by column chromatography. First, [HexA][MPD] was eluted with 20 mL of methanol, and then the chemical components of the mulberry leaf were eluted with 30 mL of methanol. Then, a rotary evaporator was used to remove the methanol in the eluent to obtain [HexA][MPD] and the chemical components of the mulberry leaf, respectively. Finally, the obtained [HexA][MPD] was reused for extraction. Figure 8 As shown, after [HexA][MPD] was reused three times, the extraction efficiency of isoquercetin and deoxynojirimycin did not decrease significantly, indicating that [HexA][MPD] has good reusability.

[0055] Although the present application has been described through embodiments, those skilled in the art will appreciate that there are many modifications and variations to the present application without departing from the spirit of the present application, and it is intended that the appended claims include these modifications and variations without departing from the spirit of the present application.

Claims

1. A method for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves, characterized in that: The invention uses a low eutectic solvent to simultaneously extract and separate flavonoids and alkaloids from mulberry leaves; the flavonoid chemical component is isoquercetin, and the alkaloid chemical component is deoxynojirimycin; the structural formulas of the two compounds are as follows: 。 2. The method for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves according to claim 1, characterized in that: The following steps are involved: (1) Extraction step: adjust the pH value of the low eutectic solvent DES aqueous solution to form a uniform single-phase solution, add mulberry leaf powder into it and perform ultrasonic extraction, centrifuge after the extraction, and collect the supernatant; (2) Separation step: The pH value of the extract is adjusted again to form a two-phase system, and an upper low eutectic solvent DES phase and a lower aqueous phase are obtained respectively. The flavonoid components are mainly distributed in the upper DES phase, and the alkaloid components are mainly distributed in the lower aqueous phase.

3. A method for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves according to claim 1 or 2, characterized in that: The low eutectic solvent is any one of [PenA][MPD], [HexA][MPD], [HepA][MPD], [OctA][MPD] and [NonA][MPD].

4. A method for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves according to claim 1 or 2, characterized in that: In the extraction step, the extraction conditions are as follows: low eutectic solvent concentration 30% ~ 70%, solid-liquid ratio 1:5g / mL ~ 1:40 g / mL, extraction time 20 min ~ 100 min, extraction pH 5.5 ~ 9.5; in the separation step, the separation conditions are as follows: separation pH 1 ~ 5.

5. A method for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves according to claim 1 or 2, characterized in that: In the extraction step, the extraction conditions are as follows: low eutectic solvent concentration of 50%, solid-liquid ratio of 1:10 g / mL, extraction time of 80 min, and extraction at pH 6.5; in the separation step, the separation conditions are as follows: pH 3.

6. Use of low eutectic solvents for simultaneous extraction and separation of flavonoids and alkaloids from mulberry leaves.

7. The use according to claim 6, characterized in that The low eutectic solvent is any one of [PenA][MPD], [HexA][MPD], [HepA][MPD], [OctA][MPD] and [NonA][MPD]; the flavonoid chemical component is isoquercetin, and the alkaloid chemical component is deoxynojirimycin.

8. The use according to claim 6, characterized in that The following steps are involved: (1) Extraction step: The pH of the DES aqueous solution is adjusted to form a uniform single-phase solution, mulberry leaf powder is added thereto, and ultrasonic extraction is performed. After the extraction is completed, centrifugation is performed and the supernatant is collected; (2) Separation step: The pH of the extract is adjusted again to form a two-phase system, and an upper DES phase and a lower aqueous phase are obtained respectively. The flavonoid components are mainly distributed in the upper DES phase, and the alkaloid components are mainly distributed in the lower aqueous phase.

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