Method for extracting didymoside from physiological dropped citrus fruits

Abstract

The invention relates to the technical field of natural product extraction, in particular to a method for extracting didymoside from physiological dropped citrus fruits. The method comprises the steps of raw material crushing, pretreatment, alkalescent solvent extraction, resin adsorption purification and refining: crushing physiological dropped citrus fruits to 0.4-0.7 cm, soaking in 3-4 times of clear water for 60-120 minutes, extracting at the temperature of 40-70 DEG C by adopting a 3-5% alkalescent carbonate or bicarbonate solution, adsorbing an extracting solution through macroporous resin such as HPD-600, eluting with 75-80% ethanol, and concentrating to obtain a crude extract; and concentrating, crystallizing and drying to obtain the didymoside. The method provided by the invention realizes efficient extraction and purification of didymoside, avoids structural degradation caused by strong base, improves the resource utilization rate, reduces the environmental load, and has good economic benefits and environmental protection values.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, and in particular to a method for extracting lemon balm glycosides from physiologically fallen citrus fruits. Background Technology

[0002] Lemon glycoside is a flavonoid glycoside compound with significant antioxidant and anticancer activities. It is widely found in the leaves, flowers, immature fruits, and mature peels of plants in the Rutaceae family, Citrus genus. It has a stable chemical structure and unique physiological activities. Since its initial discovery in the leaves of peppermint (Mentha) in 1967, researchers have subsequently isolated and identified lemon glycoside from various other plants. Lemon glycoside was found in Citrus aurantium (Zhishi Xiebai Guizhi Tang) in the *Synopsis of Prescriptions of the Golden Chamber* (Jinkui Yaolue).

[0003] With the advancement of modern medical research, the value of lemon balm glycosides in the pharmaceutical field is gradually becoming more prominent.

[0004] However, despite the significant medicinal value of menthol, there is a lack of efficient and low-loss industrial extraction technology for menthol in current technology. Existing citrus processing mainly focuses on extracting active ingredients such as hesperidin, while menthol is usually considered an impurity in this process and is removed through methods such as alkalization precipitation or resin adsorption.

[0005] Furthermore, existing citrus processing technologies suffer from the following drawbacks: First, there is a lack of specific extraction methods for lemon glycosides, hindering efficient industrial-scale extraction. Second, in traditional citrus processing for hesperidin extraction, lemon glycosides are typically removed as impurities, resulting in extremely low recovery rates. Additionally, existing separation processes often require the use of strong acids or alkalis, which not only increases operational risks but also easily leads to the degradation of the lemon glycoside structure, affecting its application value. With the continuous improvement of citrus varieties and the gradual expansion of the planting area of ​​hybrid citrus varieties (with lemon glycoside content greater than 2.5%), traditional citrus processing technologies face a dilemma: retaining lemon glycosides significantly reduces the purity of hesperidin, making it difficult to meet the market requirement of over 95% purity; removing lemon glycosides wastes this high-value component, failing to fully utilize the potential value of citrus resources.

[0006] Therefore, developing an industrial method for the efficient extraction of lemon balm glycosides is of great significance for making full use of citrus resources, improving the recovery rate of lemon balm glycosides, and meeting market demand. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for the efficient separation and purification of lemon balm glycosides from physiologically fallen citrus fruits. The method involves selectively extracting lemon balm glycosides using a weakly alkaline solvent system, and achieving high-purity separation by combining resin adsorption with ethanol elution. This method avoids the adverse effects of traditional strong acid or strong alkali processes on the environment and the target product.

[0008] The technical solution of the present invention is as follows:

[0009] A method for extracting lemon balm glycoside from physiologically fallen citrus fruits is provided. The method includes the following steps: S1, obtaining and crushing physiologically fallen citrus fruits as raw material; S2, pretreating the raw material to obtain pretreated material; S3, extracting the pretreated material using a weakly alkaline extraction solvent, wherein the weakly alkaline extraction solvent is an aqueous solution of a weakly alkaline carbonate or a weakly alkaline bicarbonate, and the extraction temperature is controlled within the range of 40°C to 70°C. After extraction, solid-liquid separation is performed to obtain a crude extract containing lemon balm glycoside; S4, purifying the crude extract using resin adsorption, wherein the crude extract is passed through a macroporous adsorption resin to adsorb lemon balm glycoside, and then eluting the macroporous adsorption resin containing lemon balm glycoside using an aqueous ethanol solution as an eluent, and collecting the eluent; S5, refining the eluent, including concentrating, crystallizing, and drying the eluent to finally obtain lemon balm glycoside.

[0010] Preferably, in step S1, the citrus physiological fruit drop material is pulverized to obtain pulverized material with a particle size of 0.4cm to 0.7cm.

[0011] Preferably, step S2, the step of pretreating the raw materials, specifically includes: soaking the pulverized material in clean water for 60 to 120 minutes, wherein the amount of clean water is 3 to 4 times the weight of the pulverized material; after soaking, draining the soaking water to obtain the pretreated material.

[0012] Preferably, in step S3, the mass fraction concentration of the weakly basic carbonate or weakly basic bicarbonate in the weakly basic extraction solvent is 3% to 5%.

[0013] Preferably, the weakly basic carbonate is selected from at least one of sodium carbonate or potassium carbonate; the weakly basic bicarbonate is selected from at least one of sodium bicarbonate or potassium bicarbonate.

[0014] Preferably, the weakly alkaline extraction solvent is a 4% sodium carbonate aqueous solution, and the extraction temperature is controlled at 50°C.

[0015] Preferably, in step S4, the macroporous adsorption resin used in the resin adsorption purification process is selected from one or more of HPD-600, LX-38, or LX-60 macroporous adsorption resins; and the volume fraction of ethanol in the ethanol aqueous solution is 75% to 80%.

[0016] Preferably, step S5, the refining process specifically includes: S5.1, concentrating the eluent under reduced pressure to obtain an extract; S5.2, adding ethanol to the extract for dissolution, wherein the volume fraction of the ethanol is 70% to 90%; S5.3, adding activated carbon to the dissolved solution for decolorization, and then filtering while hot to remove the activated carbon; S5.4, cooling and allowing the filtered filtrate to crystallize, filtering and collecting the crystals and drying them to obtain the lemon balm glycoside.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. This invention achieves selective extraction of lemon balm glycosides using a weakly alkaline solvent system (pH 8-9.5), avoiding the risk of glycosidic bond hydrolysis under traditional strong alkaline conditions. The extraction rate of lemon balm glycosides is increased by more than 400% compared to traditional processes, and the purity is significantly improved. Furthermore, the combination of resin adsorption and ethanol elution further optimizes the separation process, reduces impurity interference, and improves the recovery rate of the target product.

[0019] 2. In terms of economic benefits, this invention improves traditional citrus processing techniques, transforming lemon balm glycosides, which were originally removed as impurities, into a high-value-added product, thus achieving in-depth development and comprehensive utilization of citrus resources. Without requiring significant new equipment investment, lemon balm glycosides can be produced simultaneously on existing production lines simply by optimizing the extraction and purification processes, effectively reducing the production cost per unit. Simultaneously, this process improves raw material utilization, reduces resource waste, opens up new profit growth points for enterprises, enhances product market competitiveness, and has good industrialization prospects and promotional value. Furthermore, the residue after lemon balm glycoside extraction can be used for hesperidin production, significantly improving resource utilization and achieving dual-efficiency utilization of raw materials.

[0020] 3. Regarding environmental performance, the process of this invention employs a weakly alkaline carbonate extraction system, avoiding the corrosion of equipment and the pressure of wastewater treatment caused by traditional strong alkaline extraction methods, thus reducing the environmental burden during production. Simultaneously, through optimization of processes such as resin adsorption and ethanol elution, the solvent recovery rate is improved, reducing organic solvent emissions and meeting the environmental requirements of green production and sustainable development.

[0021] In summary, this invention provides an efficient, environmentally friendly, and economically feasible method for extracting lemon balm glycosides, which solves the problems of low recovery rate, insufficient purity, and resource waste of lemon balm glycosides in the prior art, and provides technical support for the high-value utilization of physiological fruit drop in citrus. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the process for extracting lemon balm glycosides from physiologically fallen citrus fruits according to the present invention. Detailed Implementation

[0023] This invention provides a method for extracting lemon balm glycosides from physiologically fallen citrus fruits, the process flow of which is as follows: Figure 1 As shown. Figure 1 The document illustrates the entire process sequence and key process conditions. The following section, in conjunction with the appendix... Figure 1 The specific embodiments of the present invention will be described in detail below.

[0024] Example 1

[0025] A method for extracting lemon balm glycosides from physiologically fallen citrus fruits specifically includes the following steps:

[0026] S1. Raw material crushing: 500g of physiologically fallen citrus fruit raw material is put into a crusher for crushing to obtain crushed material with a particle size of 0.6cm. This particle size range is the optimal size verified by experiments, which can improve the subsequent extraction efficiency and facilitate operation and transportation.

[0027] S2. Raw Material Pretreatment: The pulverized material is pretreated by adding 2000ml (by weight-volume) of water for 100 minutes. After soaking, the soaking solution is drained to reduce interference from impurities. This operation removes surface-soluble impurities, providing a purer raw material basis for subsequent extraction.

[0028] S3. Lemon balm extract: Prepare 6000 ml of a 4% sodium carbonate solution as the extraction solvent. Mix the pretreated raw material with the solvent and place it in a heating device for percolation extraction. Control the extraction temperature at 50℃ and the extraction time at 10 hours. During this period, percolation is used to ensure sufficient contact between the solvent and the raw material, ensuring the full release of the active ingredients. After extraction, collect 5000 ml of the percolate through a filtration device, cool to room temperature, and filter to obtain a clear filtrate.

[0029] S4. Resin Adsorption Purification: Selective separation of lemon balm glycosides is achieved using macroporous resin. 2000 ml of regenerated, qualified HPD-600 macroporous resin was selected as the adsorption material. HPD-600 resin is preferred due to its high adsorption and elution rates for lemon balm glycosides. The clarified filtrate was passed through the resin column at a flow rate of 1-3 column volumes / hour. After adsorption, the resin column was washed with deionized water to remove residual impurities. Subsequently, elution was performed using 3000 ml of 75% ethanol solution. The ethanol eluent was recovered to obtain 200 ml of extract. Through this process, the crude extract of lemon balm glycosides was obtained.

[0030] S5. Refining: Further purification of lemon balm glycosides. The eluent is concentrated under reduced pressure to 1 / 5-1 / 10 of its original volume to obtain an extract. 2500 ml of 85% ethanol is added to the extract, followed by 10% (by weight) of activated charcoal. The mixture is stirred thoroughly and heated under reflux for 30-60 minutes. The activated charcoal is removed by filtration while hot, and the ethanol is washed away. After the filtrate cools to room temperature, 90% ethanol solution is added to adjust the crystal precipitation conditions. Crystallization is allowed to occur for 3-5 hours. Finally, the crystals are collected through a filtration device and dried to obtain the lemon balm glycoside product. Experiments show that under the conditions of 85% ethanol concentration and a volume ratio of extract to ethanol of 1:10, the purity of the obtained product can reach over 40%, meeting pharmaceutical standards.

[0031] Example 2

[0032] Unlike Example 1, the extraction temperature in step S3 of this example is 40°C, while the other conditions remain unchanged.

[0033] Example 3

[0034] Unlike Example 1, in this example, the sodium carbonate mass fraction concentration in step S3 is 3%, while the other conditions remain unchanged.

[0035] Example 4

[0036] Unlike Example 1, in this example, the sodium carbonate concentration in step S3 is 5%, while the other conditions remain unchanged.

[0037] Example 5

[0038] Unlike Example 1, in this example, the raw material pretreatment soaking time in step S2 is 120 minutes, the alkaline reagent in step S3 is sodium bicarbonate with a concentration of 5%, the extraction temperature is 60°C, and the other conditions remain unchanged.

[0039] Example 6

[0040] Unlike Example 5, the extraction temperature in step S3 of this example is 50°C, while the other conditions remain unchanged.

[0041] Example 7

[0042] Unlike Example 1, in this example, the alkaline reagent in step S3 is potassium carbonate; the heating temperature is 55°C; the resin type in step S4 is LX-38; the elution ethanol concentration is 80%; and the other conditions remain unchanged.

[0043] Example 8

[0044] Unlike Example 7, the extraction temperature in step S3 of this example is 70°C, while the other conditions remain unchanged.

[0045] Comparative Example 1

[0046] Unlike Example 1, in this example, the alkali reagent in step S3 is sodium hydroxide, while the other conditions remain unchanged.

[0047] Comparative Example 2

[0048] Unlike Example 1, this example uses a traditional process and does not perform the pretreatment for lemon balm glycoside extraction (i.e., steps S2 and S3 are omitted). Hesperidin is extracted directly, and the other conditions remain unchanged.

[0049] Note: All examples and comparative groups are based on the raw material crushing (0.6cm particle size, 500g feed amount), the same batch of fallen citrus fruits (lemon balm glycoside content 3.0±0.2%), and the refining process (85% ethanol + 10% activated carbon) of Example 1. Only the different steps are marked. Parameters not mentioned are completely consistent with those of Example 1.

[0050] Measurements showed that the 4% sodium carbonate solution used in Example 1 of this invention had a pH of approximately 9.2, which is a suitable weakly alkaline environment. The 3%-5% weakly alkaline carbonate or bicarbonate solution specified in this invention typically has a pH range between 8.0 and 9.5. This range ensures effective dissolution of lemon balm glycosides while avoiding glycosidic bond hydrolysis caused by strongly alkaline conditions (such as the sodium hydroxide solution with pH > 12 in Comparative Example 1), thereby guaranteeing the purity and yield of the product.

[0051] Based on the examples listed above, a series of detailed experiments were conducted to verify the extraction effect of lemon balm glycosides under different process conditions. Specifically, the effects of different process parameters, such as temperature, time, solvent type and concentration, on the extraction rate of lemon balm glycosides were compared and analyzed to comprehensively evaluate and verify the extraction effect under each process condition, ensuring the accuracy and reliability of the experimental data and providing a scientific basis for subsequent process optimization.

[0052] I. Experimental Conditions

[0053] Taking the lemon balm extracts from Examples 1-9 and Comparative Example 1 as examples, experiments were conducted to investigate the extraction effects of lemon balm under different processing conditions:

[0054] 1. Yield (%) determination

[0055] Method: Accurately weigh the dried lemon balm glycoside product (m, g), and calculate the yield according to the formula:

[0056] Yield = (m / total amount of lemon balm glycosides in raw material) × 100%

[0057] (Total amount of lemon balm glycosides in raw materials = Amount of fallen citrus fruit added × Initial lemon balm glycoside content, i.e., 500g × 3% = 15g).

[0058] Instrument: Electronic analytical balance (Sartorius CPA225D, accuracy 0.1mg).

[0059] 2. Determination of the purity (%) of lemon balm glycosides

[0060] Methods: High performance liquid chromatography (HPLC) was used. The chromatographic column was an Agilent ZORBAX SB-C18 (4.6×250 mm, 5 μm); the mobile phase was methanol-0.1% phosphoric acid water (55:45, v / v); the flow rate was 1.0 mL / min; the detection wavelength was 330 nm; the column temperature was 30 ℃; and the injection volume was 10 μL.

[0061] Reference solution: Accurately weigh lemon balm glycoside standard (purity ≥98%), dissolve and dilute with methanol to prepare a reference solution of 0.1 mg / mL.

[0062] Test solution: Take 0.1g of the finished product, add methanol and sonicate to dissolve and dilute to 100mL, then filter through a 0.45μm filter membrane.

[0063] Calculation: Using the peak area of ​​the reference standard as a reference, calculate the mass fraction of lemon balm glycoside in the test sample according to the external standard method.

[0064] Instrument: High performance liquid chromatograph (Agilent 1260 model, equipped with diode array detector).

[0065] 3. Determination of total flavonoid content (%)

[0066] Method: The sodium nitrite-aluminum nitrate colorimetric method was used for determination.

[0067] Reference solution: Accurately weigh rutin standard (purity ≥98%), dissolve and dilute with 60% ethanol to prepare a reference solution of 0.2 mg / mL.

[0068] Test solution: Take 0.2g of the finished product, add 60% ethanol and extract by ultrasonication for 30min, make up to 100mL, take 5mL of the supernatant, add 1mL of 5% sodium nitrite, 1mL of 10% aluminum nitrate and 10mL of 4% sodium hydroxide in sequence, mix well and let stand for 15min.

[0069] Detection: The absorbance was measured at a wavelength of 510 nm. A standard curve was plotted using rutin as a standard, and the total flavonoid content was calculated.

[0070] Instrument: UV-Vis spectrophotometer (Shimadzu UV-2600).

[0071] II. Experimental Data and Results

[0072] Table 1 Comparison of Lemon Balm Extraction Effects under Different Processing Conditions

[0073] Group Yield (%) Lemon balm glycoside purity (%) Total flavonoid content (%) Example 1 9.0 40.2 85.3 Example 2 8.4 38.5 81.0 Example 3 7.1 35.8 78.5 Example 4 9.4 37.5 82.6 Example 5 6.4 29.0 45.2 Example 6 5.6 27.5 41.0 Example 7 6.0 35.0 68.0 Example 8 8.0 37.3 81.8 Comparative Example 1 4.2 22.1 30.5 Comparative Example 2 1.8 8.5 15.0

[0074] III. Experimental Conclusions

[0075] 1. The influence of the type of alkaline reagent:

[0076] Sodium carbonate / potassium carbonate showed significantly better extraction efficiency than sodium bicarbonate under conditions of 40–70℃ (P<0.01), with sodium carbonate at a concentration of 4% and 50℃ exhibiting the best overall performance (yield 9.0%, purity 40.2%). Sodium bicarbonate, however, had lower extraction efficiency and was temperature-sensitive; a 10℃ decrease in temperature led to a further 12.5% ​​decrease in yield. While sodium hydroxide could dissolve lemon balm glycosides, the strongly alkaline environment caused hydrolysis of the glycosidic bonds, resulting in a purity of only 22.1%, making it unsuitable for industrial production.

[0077] 2. Optimization of concentration and temperature

[0078] The critical point for sodium carbonate concentration is 4%. Too low a concentration (3%) leads to incomplete extraction (yield 7.1%), while too high a concentration (5%) increases the dissolution of impurities (purity drops to 37.5%).

[0079] Temperature window: 40–70℃ is the effective extraction range. The purity reaches its peak (40.2%) at 50℃. Temperatures below 40℃ or above 70℃ will cause the purity to decrease by ≥3%.

[0080] 3. Technological advantages

[0081] The optimized sodium carbonate extraction process at 4% + 50℃ developed in this study improves the yield by 400% and the purity by 373% compared to the traditional process. It also enables the stepwise extraction of lemon balm glycoside and hesperidin, increasing the raw material utilization rate to over 85%.

[0082] Note: All experimental data are the mean of three parallel experiments. The significance analysis was performed using the t-test (P<0.01 indicates extremely significant difference).

[0083] Throughout the process, the steps are closely interconnected and follow a clear logical sequence. For example, the pulverization process in step S1 provides a suitable particle size for the pretreatment in step S2, while the pretreatment in step S2 lays a clean foundation for the weakly alkaline extraction in step S3. The extract from step S3 achieves preliminary separation of the target components through resin adsorption in step S4, and finally, purification is completed through concentration and crystallization in step S5, demonstrating the integrity and coherence of the process.

[0084] The process flow of this invention first processes the raw materials into a state suitable for extraction through pulverization, and then removes surface impurities through pretreatment to ensure the high efficiency of subsequent extraction. The weakly alkaline extraction stage utilizes a weakly alkaline solvent system to selectively extract lemon balm glycosides, avoiding the adverse effects of traditional strong acid or strong alkali processes on the environment and the target product. The combination of resin adsorption and ethanol elution further optimizes the separation process, reduces impurity interference, and improves the recovery rate of the target product. The concentration and crystallization process, through activated carbon decolorization and ethanol purification, ultimately yields a high-purity lemon balm glycoside product.

[0085] The entire process of this invention is mild and controllable, with low energy consumption, and conforms to the concept of green chemistry. Through the synergistic effect of the above steps, efficient extraction and purification of lemon balm glycosides are achieved, solving the problems of low recovery rate, insufficient purity, and resource waste of lemon balm glycosides in existing technologies, and providing technical support for the high-value utilization of physiological fruit drop in citrus.

[0086] All content not described in detail in this specification belongs to existing technology known to those skilled in the art, and the model parameters of each device are not specifically limited; conventional equipment can be used. Device control components not mentioned in this technical solution are not shown in the figures because they belong to existing technology, and will not be described further here. The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting lemon balm glycosides from physiologically fallen citrus fruits, characterized in that, The method includes the following steps: S1. Obtain and crush citrus physiological fruit drop materials; S2. Pre-treat the raw materials to obtain pre-treated materials; S3. The pretreated material is extracted using a weakly alkaline extraction solvent, wherein the weakly alkaline extraction solvent is an aqueous solution of a weakly alkaline carbonate or a weakly alkaline bicarbonate. The extraction temperature is controlled within the range of 40°C to 70°C. After extraction, solid-liquid separation is performed to obtain a crude extract containing lemon balm glycoside. S4. The crude extract is subjected to resin adsorption purification treatment. This treatment includes passing the crude extract through a macroporous adsorption resin so that lemon balm glycosides are adsorbed by the macroporous adsorption resin. Then, an aqueous ethanol solution is used as an eluent to elute the macroporous adsorption resin adsorbed with lemon balm glycosides, and the eluent is collected. S5. The eluent is purified by: concentrating the eluent under reduced pressure to obtain an extract; adding ethanol to the extract for dissolution and adding activated carbon for decolorization; filtering while hot to remove the activated carbon, cooling the filtrate to allow it to crystallize, and then filtering and drying to finally obtain the lemon balm glycoside.

2. The method for extracting lemon balm glycoside from physiologically fallen citrus fruits according to claim 1, characterized in that, In step S1, the citrus physiological fruit drop material is crushed to obtain crushed material with a particle size of 0.4cm to 0.7cm.

3. The method for extracting lemon balm glycoside from physiologically fallen citrus fruits according to claim 1, characterized in that, In step S2, the pretreatment of the raw materials specifically includes: soaking the pulverized material in clean water for 60 to 120 minutes, wherein the amount of clean water is 3 to 4 times the weight of the pulverized material; after soaking, draining the soaking water to obtain the pretreated material.

4. The method for extracting lemon balm glycoside from physiologically fallen citrus fruits according to claim 1, characterized in that, In step S3, the mass fraction concentration of the weakly basic carbonate or weakly basic bicarbonate in the weakly basic extraction solvent is 3% to 5%.

5. The method for extracting lemon balm glycoside from physiologically fallen citrus fruits according to claim 4, characterized in that, The weakly basic carbonate is selected from at least one of sodium carbonate or potassium carbonate; the weakly basic bicarbonate is selected from at least one of sodium bicarbonate or potassium bicarbonate.

6. The method for extracting lemon balm glycoside from physiologically fallen citrus fruit according to claim 5, characterized in that, The weakly alkaline extraction solvent is a 4% sodium carbonate aqueous solution, and the extraction temperature is controlled at 50°C.

7. The method for extracting lemon balm glycoside from physiologically fallen citrus fruits according to claim 1, characterized in that, In step S4, the macroporous adsorption resin used in the resin adsorption purification process is selected from one or more of HPD-600, LX-38, or LX-60 macroporous adsorption resins; the volume fraction of ethanol in the ethanol aqueous solution is 75% to 80%.

8. The method for extracting lemon balm glycoside from physiologically fallen citrus fruit according to claim 1, characterized in that, In step S5, the volume fraction of the ethanol is 70% to 90%.

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