A method for simultaneously extracting and separating flavonoids and alkaloids in mulberry leaves

Abstract

The application discloses a method for simultaneously extracting and separating flavonoids and alkaloids in mulberry leaves, which comprises the following steps: firstly, adjusting the pH of a eutectic solvent aqueous solution to form a homogeneous single-phase solution; then adding mulberry leaf powder into the solution and performing ultrasonic extraction; after the extraction is completed, centrifuging and taking the supernatant; and secondly, adjusting the pH of the extraction solution to form a two-phase system, and obtaining an upper eutectic solvent phase and a lower water phase respectively. The extraction step conditions are as follows: the eutectic solvent concentration is 30%-70%, the solid-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 separation step conditions are as follows: the separation pH is 1-5. Through HPLC analysis, flavonoids (isoquercitrin) are mainly distributed in the upper eutectic solvent phase, and alkaloids (deoxyacteoside) are mainly distributed in the lower water phase, and the reusability is good.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant extraction and separation, and particularly relates to a method for extracting and separating flavonoids (isoquercitrin) and alkaloids (deoxyacteoside) from mulberry leaves by using a eutectic solvent. BACKGROUND

[0002] Mulberry leaves are the dried leaves of Morus alba L. and Morus nigra Linn, which are commonly used in traditional Chinese medicine. Mulberry leaves mainly contain flavonoids, alkaloids, polysaccharides and other chemical components. Flavonoids mainly include isoquercitrin, rutin, quercitrin, etc., and alkaloids mainly include polyhydroxy alkaloids, including deoxyacteoside, fagomine, etc. They are considered to be the main active components of mulberry leaves, and have multiple pharmacological effects such as hypoglycemic and anti-tumor effects. At present, traditional solvents such as water, methanol and ethanol are mainly used for the extraction of chemical components in mulberry leaves. 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 solvents (DES) are green solvents formed by hydrogen bond donors and hydrogen bond acceptors through hydrogen bonding. They have good solubility, non-volatility, simple preparation and other advantages, and are expected to replace traditional organic solvents and be applied to the field of extraction and separation. At the same time, due to the designability of the structure of DES, different hydrogen bond donors and hydrogen bond acceptors can be selected 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 satisfactory results have been achieved.

[0004] DES / H2O two-phase systems are usually composed of DES and inorganic salt aqueous solution (phosphate, sulfate, etc.). Compared with other conventional two-phase liquid systems, they have the advantages of mild operating conditions and easy phase separation. In the past decade, DES / H2O two-phase systems have developed rapidly and have become an effective method for the enrichment and separation of various chemical components. In recent years, stimulus-responsive DES / H2O systems have gradually developed. Their single-phase / two-phase state 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 realize the recycling of DES, and is more in line with the concept of green chemistry and sustainable development. Among them, pH-responsive DES / H2O systems have low energy consumption, simple equipment and are easy to scale up in actual operation, and have gradually attracted widespread attention.

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

[0006] To overcome the problems of the prior art, the purpose of the present application 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 (isoquercitrin) and alkaloids (deoxyacteoside) from mulberry leaves using deep eutectic solvents (DES), and is green, environmentally friendly, specific, and easy to operate.

[0007] To achieve the above-mentioned purpose of the application, the purpose of the present application is achieved by the following technical solutions:

[0008] A method for simultaneously extracting and separating flavonoids and alkaloids in mulberry leaves, which is a method for extracting and separating flavonoids (isoquercitrin) and alkaloids (deoxyacteoside) from mulberry leaves using deep eutectic solvents (DES), and specifically, at room temperature, deep eutectic solvents (DES) and water are mixed in a certain molar ratio, and NaOH solution is added to adjust the pH to obtain a homogeneous single-phase solution. Then a certain amount of mulberry leaf powder is added, and ultrasonic extraction is performed for a certain time. After extraction is completed, centrifugation is performed, the mulberry leaf residue is removed, and the upper extraction liquid is obtained.

[0009] To further separate the flavonoids and alkaloids, hydrochloric acid solution is added to the extraction liquid to adjust the pH to promote the separation of the solution into layers, and the upper DES phase and the lower water phase are obtained. The flavonoids (isoquercitrin) are mainly distributed in the upper DES phase, and the alkaloids (deoxyacteoside) are mainly distributed in the lower water phase.

[0010] The chemical structures of isoquercitrin and deoxyacteoside are as follows:

[0011]

[0012] Specifically, in the above-mentioned extraction method, the deep eutectic solvents (DES) are shown in Table 1.

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

[0014]

[0015] To optimize the extraction conditions, the concentration of deep eutectic solvents (DES) is selected to be 30% to 70% (preferably 50%); the solid-liquid ratio is selected to be 1:5 g / mL to 1:40 g / mL (preferably 1:10 g / mL); the extraction time is selected to be 20 min to 100 min (preferably 80 min); and the pH is selected to be 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] Advantages: Compared with the prior art, the present application has the following advantages:

[0017] The present application adopts a low eutectic solvent (DES) ultrasonic-assisted extraction separation-HPLC detection method, applies Origin statistical analysis software, simultaneously determines the contents of isoquercitrin and deoxyacteoside in the extract (diluted by an equal volume of methanol) by using the HPLC method, and obtains the optimal extraction and separation process. The specific method for extracting and separating flavones (isoquercitrin) and alkaloids (deoxyacteoside) components from mulberry leaves by using the DES is as follows:

[0018] 1. Preparation and characterization of the low eutectic solvent (DES): The DES used in the present research is mixed by a hydrogen bond donor and a hydrogen bond acceptor at a molar ratio of 1:1, continuously stirred at 60°C for 2 hours, and then each DES is obtained, which is a uniform transparent liquid. Then, the infrared spectrum of each DES is measured by using a Fourier infrared spectrometer for structure characterization.

[0019] 2. pH-responsive DES / H2O system: The phase diagram of each DES / H2O system under different pH conditions is determined by using the cloud point titration method, so as to obtain the phase separation of each DES and water.

[0020] 3. Determination of the optimal DES as an extractant: 2-methyl-2,4-pentanediol (MPD) is used as the hydrogen bond donor, and different hydrogen bond acceptors (pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid and nonanoic acid) are compared in terms of the extraction efficiency and separation effect of the target compounds, and finally [hexanoic acid][2-methyl-2,4-pentanediol] ([HexA][MPD]) is determined as the optimal extractant.

[0021] 4. Using [HexA][MPD] as the extractant, the effects of different solid-liquid ratios, extraction times, pH values and DES concentrations on the extraction efficiency of isoquercitrin and deoxyacteoside are investigated by using single-factor analysis experiment, and finally the solid-liquid ratio is determined as 1:10 g / mL, the extraction time is 80 min, the pH value is 6.5, and the DES concentration is 50%. According to the separation effect of isoquercitrin and deoxyacteoside, the pH value for separation is determined as 3.

[0022] 5. The flavones (isoquercitrin) and alkaloids (deoxyacteoside) components in mulberry leaves are extracted and separated by using the optimal factor levels in step 4, the extraction and separation effects are evaluated, and the DES is recovered by column chromatography, and the recycling property of the DES is evaluated.

[0023] The application provides a new idea and method for extraction and separation of natural products, and the optimal extraction and separation conditions established in the application have high efficiency, and can efficiently extract and separate flavonoids and alkaloids from mulberry leaves. Meanwhile, the extraction medium DES in the method can be recycled and utilized, is environment-friendly and non-polluting, and is suitable for industrial production, thereby providing a scientific basis for further development and utilization of mulberry leaves. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The process for extracting and separating flavonoids (isoquercitrin) and alkaloids (deoxy-ajmalicine) in mulberry leaves in a pH-responsive deep eutectic solvent (DES) / H2O system.

[0025] Figure 2 The infrared spectrograms of hexanoic acid (HexA), 2-methyl-2,4-pentanediol (MPD) and [HexA][MPD].

[0026] Figure 3 The 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 different DES / H2O systems on isoquercitrin (A) and deoxy-ajmalicine (B) in mulberry leaves.

[0029] Figure 6 The influence of [HexA][MPD] concentration (A), extraction time (B), solid-liquid ratio (C) and pH value (D) on the extraction efficiency of isoquercitrin and deoxy-ajmalicine in mulberry leaves.

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

[0031] Figure 8 The reusability of [HexA][MPD] aqueous solution for extracting isoquercitrin and deoxy-ajmalicine in mulberry leaves (the extraction rate is represented as the percentage of the measured value relative to the maximum measured value). DETAILED DESCRIPTION

[0032] The application will be further described below in combination with specific embodiments, but the protection scope of the application is not limited to this:

[0033] The materials, reagents and instruments used in the following examples can be obtained from commercial channels unless otherwise specified.

[0034] Example 1

[0035] A method for extracting and separating flavonoids (isoquercitrin) and alkaloids (deoxyacteoside) components from mulberry leaves using deep eutectic solvents, specifically comprising the following steps:

[0036] (1) Extraction step: At room temperature, mix deep eutectic solvents (DES) and water in the same volume ratio, and add NaOH solution to adjust the pH to obtain a homogeneous single-phase solution. Then accurately weigh 0.6 g of mulberry leaf powder, add 6 mL of the above 50% DES aqueous solution, ultrasonic extraction for 80 min, centrifuge, and obtain the upper extraction liquid. (2) Separation step: In order to further separate the flavonoids and alkaloids components, add hydrochloric acid solution to the extraction liquid to adjust the pH to promote the solution to separate into layers, and obtain the upper DES phase and the lower water phase, respectively, as shown in Figure 1 In the experiment, the DES type, DES concentration, solid-liquid ratio, extraction time, pH, and other extraction and separation conditions were changed in turn to investigate their effects on the extraction and separation efficiency. Each experiment was repeated three times.

[0037] I. Structural characterization of deep eutectic solvents (DES)

[0038] In the present application, the inventors synthesized a series of DES with varying polarity gradients using different chain lengths of organic acids (pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, and nonanoic acid) as hydrogen bond donors and 2-methyl-2,4-pentanediol (MPD) as a hydrogen bond acceptor. 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 by infrared spectrogram to confirm the presence of hydrogen bonds. For example, the infrared spectrogram of [HexA][MPD] is shown in 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 (breakage or formation of chemical bonds) occurred during the synthesis of [HexA][MPD]. In addition, the infrared spectrogram of hexanoic acid or MPD has a characteristic absorption peak of -OH between 3000-3600 cm -1 After the formation of [HexA][MPD], the absorption peak at this position becomes wider and stronger, proving the existence of hydrogen bonds and confirming the synthesis of [HexA][MPD].

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

[0040] The prepared deep eutectic solvent (DES) was mixed with water at different volume ratios at room temperature and normal pressure 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 homogeneous single-phase solution was formed, and the solution pH value and 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. As shown in Figure 3 , [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 a large difference in polarity, i.e., the upper DES phase and the lower water phase, while when the position is above the curve, the system forms a homogeneous single phase. In addition, for the DES / H2O system in two phases, the change in pH value can significantly affect the volume of the upper DES phase and the lower water 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 water phase gradually decreases Figure 4 , indicating that the pH value can affect the phase separation of DES and water, and thus may affect the separation effect of flavonoids and alkaloids in mulberry leaves.

[0041] III. Selection of deep eutectic solvent (DES)

[0042] Different DESs were selected for the extraction and separation of flavonoids (isoquercitrin) and alkaloids (deoxyjirinin) in mulberry leaves, and the extraction and separation efficiency of the two target compounds was compared. As shown in Figure 5 , flavonoids are mainly distributed in the DES phase, while alkaloids with relatively high polarity are mainly distributed in the water phase. For isoquercitrin 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 isoquercitrin in [HexA][MPD] is the highest, and the content in the corresponding water phase is the lowest. For deoxyjirinin Figure 5 B), as the alkyl chain of the organic acid is extended, the content in the DES phase does not change significantly, while the content in the water phase has a tendency to decrease. Among them, the content of [PenA][MPD] in the water phase is slightly higher than that of [HexA][MPD]. Considering the extraction and separation efficiency of isoquercitrin and deoxyjirinin, [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 are the same, respectively, to investigate the different [HexA] [MPD] concentration (v / v) 30%, 40%, 50%, 60%, 70% on the extraction efficiency of target compounds. As shown in Figure 6 A, when the [HexA] [MPD] concentration from 30% to 50% increase, the extraction efficiency of isoquercitrin, and when the [HexA] [MPD] concentration further increases, the extraction efficiency of isoquercitrin slightly decreased. On the other hand, the extraction efficiency of deoxyactinomycin decreased with the increase of [HexA] [MPD] concentration. In summary, the above results, select 50% (v / v) as the best [HexA] [MPD] concentration.

[0045] Five, the choice of extraction time

[0046] According to the above extraction conditions, other experiments are the same, respectively, to investigate the different extraction time 20, 40, 60, 80, 100 min on the extraction efficiency of target compounds. As shown in Figure 6 B, when the extraction temperature from 20 min to 80 min, the extraction efficiency of target compounds gradually increased, further extension of extraction time, the extraction efficiency of target compounds no longer increase, prompt the extraction process has reached equilibrium. Therefore, select 80 min as the best extraction time.

[0047] Six, the choice of solid-liquid ratio

[0048] According to the above extraction conditions, other experiments are the same, respectively, to investigate the different solid-liquid ratio 1:5, 1:10, 1:20, 1:40 g / mL on the extraction efficiency of target compounds. As shown in Figure 6 C, when the solid-liquid ratio reaches 1:10 g / mL, the extraction efficiency to the maximum, continue to increase the solid-liquid ratio, the extraction efficiency decreased. Therefore, select 1:10 g / mL as the best solid-liquid ratio.

[0049] Seven, the choice of pH value

[0050] According to the above extraction conditions, other experiments are the same, respectively, to investigate the different pH value for 6.5, 7.5, 8.5, 9.5 on the extraction efficiency of target compounds. As shown in Figure 6 D, the solvent pH has a greater impact on the extraction efficiency of deoxyactinomycin, in the [HexA] [MPD] and water to form a single phase of the pH range, the extraction efficiency of deoxyactinomycin decreased with the increase of pH, the extraction efficiency of pH 6.5 is the highest. On the other hand, the extraction efficiency of isoquercitrin has no obvious effect of solvent pH. Therefore, select 6.5 as the best solvent pH value.

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

[0052] According to the above conditions, other experiments are the same, respectively, to investigate the influence of different pH 5, 4, 3, 2, 1 on the separation effect of target compounds. As shown in Figure 7 When the pH is reduced from 5 to 3, the distribution ratio of isoquercitrin in the two phases has no obvious change, and the ratio of deoxynojirimycin in the aqueous phase gradually increases. When the pH is further reduced from 3 to 1, the ratio of isoquercitrin in the [HexA][MPD] phase gradually decreases, and the distribution ratio of deoxynojirimycin in the two phases has no obvious change. In view of this, the optimal pH for separation of [HexA][MPD] / H2O two-phase system is 3.

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

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

[0055] Although the present application is described by way of embodiments, those skilled in the art know that there are many modifications and changes 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 changes without departing from the spirit of the present application.

Claims

1. A method for simultaneous extraction and separation of flavonoids and alkaloids in mulberry leaves, characterized in that, The application discloses a method for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves by using a deep eutectic solvent; the flavonoid chemical component is isoquercitrin, and the alkaloid chemical component is deoxyacteoside; and the structural formulae of the two compounds are shown as follows: ; comprising the steps of: (1) an extraction step: adjusting the pH value of a deep eutectic solvent DES aqueous solution to form a uniform single-phase solution, adding mulberry leaf powder into the solution, and then performing ultrasonic extraction; after the extraction is completed, centrifugation is performed, and the supernatant is taken; The deep eutectic solvent is any one of [PenA][MPD], [HexA][MPD], [HepA][MPD], [OctA][MPD] and [NonA][MPD]; The extraction conditions are as follows: the concentration of the deep eutectic solvent is 30% to 70%, the solid-liquid ratio is 1:5 g / mL to 1:40 g / mL, the extraction time is 20 min to 100 min, and the extraction pH value is 5.5 to 9.5; (2) a separation step: adjusting the pH value of the extraction liquid again to form a two-phase system, obtaining an upper deep eutectic solvent DES phase and a lower water phase, respectively, and the flavonoid chemical component is mainly distributed in the upper DES phase, and the alkaloid chemical component is mainly distributed in the lower water phase; the separation pH value is 1 to 5.

2. The method according to claim 1, characterized in that, In the extraction step, the extraction conditions are as follows: the concentration of the deep eutectic solvent is 50%, the solid-liquid ratio is 1:10 g / mL, the extraction time is 80 min, and the pH value is 6.5; and in the separation step, the separation conditions are as follows: the pH value is 3.

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