Process for enriching and purifying maqui berry anthocyanin by connecting macroporous resin and cation exchange resin in series

Abstract

The invention discloses a process for enriching and purifying maqui berry anthocyanin by connecting macroporous resin and cation exchange resin in series, and belongs to the technical field of separation and purification of plant extracts. The core of the process is that efficient and high-selectivity purification of the maqui berry anthocyanin crude extract is realized through ordered combination of two chromatography media with different separation mechanisms. The preparation method comprises the following steps: firstly, dissolving a maqui berry crude extract containing about 35% of anthocyanin into an acidic buffer solution with a specific pH value to form a raw material solution; secondly, the raw material liquid passes through a pretreated non-polar macroporous adsorption resin column, and the physical adsorption effect of the raw material liquid is utilized; through the synergistic effect of two-step series chromatography, the purity of the product is remarkably improved, the performance and the service life of the cation exchange resin are effectively protected, the technological process is mild, efficient and easy to amplify, the obtained product is stable in quality, and the method is suitable for the fields of food, health care products and medicine.

Description

A process for enriching and purifying maquill anthocyanins using a tandem combination of macroporous resin and cation exchange resin Technical Field

[0001] This invention relates to the field of natural product separation and purification technology, and more specifically, to a method for purifying plant extracts using chromatographic chromatography, particularly a method for efficiently enriching and purifying anthocyanin active ingredients in maquill fruits using a series process of macroporous adsorption resin and strongly acidic cation exchange resin. Background Technology

[0002] As a fruit rich in polyphenolic compounds, the anthocyanin content in the extract of *Mallotus arvense* is particularly prominent, with the main components being glycoside derivatives of delphinidin and cyanidin. Modern pharmacological studies have shown that *Mallotus arvense* anthocyanins possess powerful antioxidant, anti-inflammatory, vision-improving, and blood sugar-regulating bioactivities, demonstrating significant application potential in functional foods, health products, and pharmaceuticals. However, the crude extract of *Mallotus arvense* obtained through conventional solvent extraction is extremely complex in composition. Besides the target product anthocyanins, it also contains a large amount of sugars (such as fructose and glucose), organic acids (such as citric acid and malic acid), phenolic compounds (such as gallic acid and caffeic acid), flavonols, and small amounts of proteins and inorganic salts. The presence of these impurities not only significantly reduces the relative content of anthocyanins, affecting product efficacy and standardization, but may also adversely affect product stability, color, flavor, and bioavailability. Therefore, efficient separation and purification of the crude extract of *Mallotus arvense* to remove ineffective or interfering components is a key technical step in achieving its high-value application.

[0003] Currently, various industrial techniques are used to purify anthocyanins, but each has its own insurmountable technical bottlenecks and pain points, limiting the large-scale preparation of high-quality maquilli anthocyanin products: Single adsorption resin purification processes suffer from poor selectivity and limited purity improvement. Macroporous adsorption resins are currently the most widely used plant polyphenol purification technology. This method mainly relies on intermolecular van der Waals forces and hydrophobic interactions to separate the target analyte from impurities. When processing crude maquilli extract, operators can utilize macroporous resins to effectively adsorb anthocyanins and other phenolic compounds, while allowing highly polar impurities such as sugars and organic acids to permeate and flow out with the water flow. However, its core challenge lies in the non-specificity of adsorption. Anthocyanins share certain similarities in molecular structure and polarity with other polyphenolic compounds such as phenolic acids and flavonols, resulting in similar adsorption behaviors on macroporous resins. During the elution stage, it is difficult to effectively separate anthocyanins from these structurally similar polyphenol impurities by simply adjusting the concentration of the eluent (such as ethanol). They are often eluted together, making it difficult for the purity of the final product to consistently exceed a certain threshold (usually below 45%), thus failing to meet the quality requirements of high-specification products.

[0004] The purification process using a single cation exchange resin is susceptible to contamination and suffers from low efficiency. Cation exchange chromatography is a highly selective separation method developed based on the characteristic that anthocyanins exist as positively charged flavylium ions under acidic conditions. Theoretically, it can precisely capture anthocyanin cations while allowing neutral (such as most sugars) or acidic (such as phenolic acids) impurities to escape. However, in practice, directly loading the complex crude extract of maquillia onto a cation exchange resin column presents serious challenges. The extremely high content (sometimes exceeding 50% of dry matter) of sugars in the crude extract results in high viscosity, easily clogging the pores between resin particles and the micropores on the surface, forming a "barrier membrane" that severely hinders the contact between anthocyanins and the exchange groups inside the resin. This not only leads to a sharp decline in the dynamic adsorption capacity of the resin and low processing efficiency but also makes the resin cleaning and regeneration process extremely difficult and time-consuming, significantly shortening the lifespan of the expensive ion exchange resin and substantially increasing production costs.

[0005] Traditional solvent extraction or precipitation methods are environmentally unfriendly and the products are easily degraded: Some traditional chemical separation methods, such as liquid-liquid extraction using different organic solvents or stepwise precipitation by adjusting pH, are feasible on a laboratory scale, but they present many problems in industrial production. First, these methods typically consume large amounts of organic solvents, such as ethyl acetate and n-butanol, which not only increases production costs and operational risks, but also poses a serious threat to the environment due to their volatilization and wastewater treatment, which is inconsistent with the development trend of green chemistry. Second, the glycosidic bonds and conjugated systems in the anthocyanin molecular structure are sensitive to strong acids, strong bases, high temperatures, and certain organic solvents. Repeated solvent handling and drastic pH changes can easily trigger degradation or isomerization, leading to loss of product activity and reduced yield. In addition, these methods usually have low selectivity, cumbersome separation steps, and are difficult to implement in continuous and automated production.

[0006] In summary, existing single purification techniques fail to simultaneously achieve high selectivity, high processing efficiency, mildness, and environmental friendliness when processing complex masquilli anthocyanin crude extracts. Therefore, developing an innovative purification method that integrates the advantages of different separation mechanisms, has a rational process flow, and can stably produce high-purity masquilli anthocyanin products has become a pressing technical challenge in this field. Summary of the Invention

[0007] To address the aforementioned problems, this invention proposes a process for enriching and purifying maquill anthocyanins using a tandem combination of macroporous resin and cation exchange resin, which more accurately solves the problems mentioned in the background section.

[0008] This invention is achieved through the following technical solution: This invention proposes a process for enriching and purifying mariberry anthocyanins using a tandem macroporous resin and cation exchange resin, comprising the following steps: S1: Preparing a raw material solution by dissolving crude mariberry anthocyanin extract in an acidic buffer solution with a pH less than 3.0; S2: Performing macroporous resin chromatography by loading the raw material solution obtained in S1 onto a macroporous adsorption resin column, sequentially performing adsorption, deionized water rinsing, and aqueous ethanol elution, collecting the first eluent containing anthocyanins; S3: Preparing an intermediate sample by processing the first eluent obtained in S2 at a set temperature... S3: Reduced pressure concentration to remove ethanol solvent, obtaining an anthocyanin intermediate concentrate; S4: Cation exchange resin chromatography, the anthocyanin intermediate concentrate obtained in S3 is diluted with an acidic buffer solution with a pH value less than 3.0 and loaded onto a pre-converted H+ type strongly acidic cation exchange resin column, and sequentially subjected to sample adsorption, acidic water rinsing and acidic aqueous ethanol solution elution, collecting the second eluent containing anthocyanins; S5: Preparation of the finished product, the second eluent obtained in S4 is concentrated under reduced pressure at a set temperature, the solvent is recovered and then dried to obtain the purified anthocyanin product of Maquiberry.

[0009] The macroporous adsorption resin in S2 is a non-polar or weakly polar polystyrene-based macroporous adsorption resin, specifically selected from one or more of AB-8 type resin or HPD-100 type resin.

[0010] The macroporous resin chromatography operating parameters in S2 are as follows: the anthocyanin concentration in the feed solution is controlled at 1-2 mg / mL, and the loading flow rate is controlled at 1 times the bed volume / hour; the volume of deionized water rinsing is 2-3 times the bed volume; the ethanol volume fraction of the aqueous ethanol solution is 50%, the elution volume is 5 times the bed volume, and the elution flow rate is controlled at 1-2 times the bed volume / hour.

[0011] The temperature for vacuum concentration in S3 is controlled to be no higher than 50°C.

[0012] The strongly acidic cation exchange resin in S4 is a styrene-based sulfonic acid cation exchange resin, specifically selected from one or more of the following: 001×7 type resin, 732 type resin, Amberlite IR120 type resin, or Lewatit S100 type resin.

[0013] The pretreatment of the strongly acidic cation exchange resin in S4 includes: sequentially treating it with an acid solution to convert it to the H+ form, and then washing it with deionized water until the pH of the effluent is close to neutral.

[0014] The cation exchange resin chromatography operating parameters in S4 are as follows: the loading concentration of the anthocyanin intermediate concentrate after dilution is controlled at 1-2 mg / mL, the loading flow rate is controlled at 1 times the bed volume / hour, and the ratio of the wet-filled resin bed volume to the loading liquid volume is 1:5 to 1:10.

[0015] The acidic water in S4 is an acidic aqueous solution with a pH of 2.0, and the rinsing volume is 1-2 times the bed volume; the acidic aqueous ethanol solution is an aqueous solution containing 1% hydrochloric acid and 50-60% ethanol by volume, and the elution flow rate is controlled at 1 times the bed volume / hour.

[0016] The temperature for vacuum concentration in S5 is controlled to be no higher than 45°C.

[0017] The drying method in S5 is either spray drying or freeze drying.

[0018] Compared with existing technologies, this invention provides a process for enriching and purifying malkiberry anthocyanins using a tandem macroporous resin and cation exchange resin, which has the following beneficial effects: This process, by first pretreating with a macroporous resin, utilizes its strong adsorption capacity for moderately polar substances and its weak retention characteristics for highly polar substances to effectively remove a large amount of highly polar water-soluble impurities such as sugars and organic acids from the raw materials. This effectively protects the subsequent cation exchange resin, thereby avoiding physical blockage of the resin pores and contamination of the exchange sites, significantly extending the service life of the cation exchange resin, and improving its dynamic adsorption capacity and overall purification efficiency.

[0019] This process, which uses a macroporous resin and a cation exchange resin in series to enrich and purify anthocyanins from maquilli, fully utilizes the specific electrostatic adsorption of anthocyanins in the form of flavonoid cations by a strongly acidic cation exchange resin under specific acidic conditions (pH < 3.0) after initial purification with the macroporous resin. This achieves precise separation of the target product, anthocyanins, from structurally similar but electrically neutral or weakly acidic phenolic acid impurities. As a result, it breaks through the purity bottleneck of a single adsorption resin, significantly improves the final purity of the product (from the initial 35% to over 50%), and enhances the flavor and color of the product.

[0020] This process for enriching and purifying anthocyanins from maquillib is achieved by combining macroporous resin and cation exchange resin in a tandem manner. This scientifically and systematically combines resin chromatography techniques with different separation mechanisms, namely physical adsorption and ion exchange, and systematically optimizes key process parameters such as sample loading, rinsing, and elution at each stage. This allows the entire purification process to be carried out gently at room temperature and pressure, thereby maximizing the preservation of the natural structure and bioactivity of anthocyanins, effectively increasing the total product yield, and significantly reducing the amount of organic solvent used and waste liquid discharged. Attached Figure Description

[0021] Figure 1 is a schematic diagram of the process for enriching and purifying maquill anthocyanins using a macroporous resin and a cation exchange resin in series, as proposed in this invention. Detailed Implementation

[0022] To more clearly and completely illustrate the technical solution of the present invention, the present invention will be further described below with reference to the accompanying drawings. Embodiments

[0023] This embodiment describes a process for enriching and purifying maquill anthocyanins using a tandem macroporous resin and a cation exchange resin.

[0024] S1: Preparation of Raw Material Solution A batch of crude anthocyanin extract powder from *Mallotus arvensis* was taken, and its anthocyanin content was determined to be 35.2% by high-performance liquid chromatography (HPLC) using a UV detector and external standard method. 100 g of the crude extract was accurately weighed and added to 2 L of 0.1 M citric acid-sodium citrate buffer solution, which had been pre-adjusted to pH 2.8 with concentrated citric acid. The solution was magnetically stirred at room temperature for 30 minutes until the extract powder was completely dissolved, forming a clear, transparent purple-red solution. A sample was taken and the anthocyanin concentration in the solution was determined to be 1.75 mg / mL, which was used as the raw material solution to be processed.

[0025] S2: Pretreatment of macroporous resin for chromatography: Take AB-8 type macroporous adsorption resin and soak it in excess ethanol for 24 hours to allow it to swell fully. Wet-pack the resin into a chromatography column (column dimensions: inner diameter 5cm, column height 50cm), with a bed volume (BV) of approximately 1 liter. Wash the column sequentially with 5 BV of ethanol and then 5 BV of deionized water at a flow rate of 2 BV / h to complete the pretreatment.

[0026] Sample loading and adsorption: The feed solution prepared by S1 was pumped into the pretreated macroporous resin column at a flow rate of 1 BV / h (1 L / h) using a peristaltic pump. The color change of the effluent was monitored in real time at the outlet. When the effluent began to show a distinct purplish-red color, indicating that anthocyanins had broken through, the sample loading was stopped immediately.

[0027] Rinsing to remove impurities: After the sample loading is completed, immediately pump 2.5 BV (2.5 liters) of deionized water at a flow rate of 2 BV / h to quickly rinse the resin bed, so as to thoroughly remove strong polar impurities such as sugars, inorganic salts and organic acids that are physically retained between resin particles and on the surface.

[0028] Elution: After rinsing, replace the eluent with a 50% (v / v) aqueous ethanol solution. Elute at a flow rate of 1.5 BV / h (1.5 L / h), for a total eluent volume of 5 BV (5 L). Monitor the absorbance at 520 nm using a UV-Vis spectrophotometer online and collect the eluent in segments based on color intensity. Combine the intermediate eluent segments with the darkest color (approximately 3 BV) as the first eluent.

[0029] S3: Preparation of intermediate sample. The collected first eluent was transferred to a rotary evaporator and concentrated under reduced pressure at a vacuum of -0.08 MPa and a water bath temperature of 48°C until no obvious ethanol odor was emitted. Approximately 500 mL of alcohol-free anthocyanin intermediate concentrate was obtained.

[0030] S4: Pretreatment of cation exchange resin for chromatography: Take 001×7 type strong acid cation exchange resin and wet-pack it into another chromatography column (column dimensions: inner diameter 3cm, column height 40cm), with a bed volume of approximately 250 mL. Treat sequentially with 5 BV of 5% HCl solution at a flow rate of 2 BV / h to completely convert the resin to the H+ form. Then continuously rinse with deionized water at the same flow rate until the pH of the effluent reaches 4.5, and set aside for use.

[0031] Sample loading and adsorption: The intermediate concentrate obtained from S3 was diluted with 0.1M citrate-sodium citrate buffer (pH 2.8) to an anthocyanin concentration of approximately 1.5 mg / mL, with a total volume of approximately 1.5 L. This diluted solution was then loaded into a pretreated cation exchange resin column at a flow rate of 1 BV / h (250 mL / h).

[0032] Rinsing to remove impurities: After sample loading, pump 1.5 BV (375 mL) of pH 2.0 hydrochloric acid aqueous solution at a flow rate of 1 BV / h to further rinse and remove impurities such as phenolic acids that may be physically adsorbed.

[0033] Elution: Replace the eluent with a 55% ethanol aqueous solution containing 1% HCl. Perform gradient elution at a flow rate of 1 BV / h, and collect the darkest purple-red main band to obtain the second eluent.

[0034] S5: Immediately after preparing the finished product, the collected second eluent was transferred to a rotary evaporator and concentrated under reduced pressure at a vacuum of -0.09 MPa and a water bath temperature of 42°C to recover ethanol. The resulting concentrate was freeze-dried to obtain a deep purple-red powder. The powder was weighed and its anthocyanin content was determined by high-performance liquid chromatography (HPLC) using a UV detector and external standard method.

[0035] Results: 35.8 grams of purified product powder were finally obtained, and the anthocyanin content was found to be 51.5%. Based on the anthocyanins in the crude extract, the total recovery rate was 52.4%. Example

[0036] This embodiment describes a process for purifying maquill anthocyanins using HPD-100 macroporous resin and Amberlite IR120 cation exchange resin in series.

[0037] Except for the following parameters, the remaining steps are basically the same as in Example 1: S2: The macroporous resin used is HPD-100. The eluent is a 50% (v / v) aqueous solution of ethanol, and the elution flow rate is 1 BV / h.

[0038] S4: The cation exchange resin used is Amberlite IR120. The eluent is a 60% aqueous solution of ethanol containing 1% HCl.

[0039] S5: The drying method is spray drying, with an inlet air temperature of 160℃ and an outlet air temperature of 80℃.

[0040] Results: 36.5 grams of purified product powder were finally obtained, with an anthocyanin content of 50.8% and a total recovery rate of 52.8%.

[0041] Comparative Example 1: Purification using only macroporous resin To verify the superiority of the tandem process of the present invention, a comparative example was set up using only macroporous resin for purification.

[0042] Take an equal amount of crude extract as in Example 1, and operate exactly according to S1 and S2 of Example 1. The first eluent collected in S2 is concentrated and freeze-dried directly according to the conditions of S5 in Example 1 to obtain the final product.

[0043] Results: 45.2 grams of product powder were obtained, and the anthocyanin content was found to be 41.3%.

[0044] Results Analysis and Comparison: As shown in the table above, the tandem process employed in this invention (Example 1) has a slightly lower overall recovery rate compared to the single process using only macroporous resin (Comparative Example 1). However, the anthocyanin purity of the final product significantly increased from 41.3% to 51.5%, an improvement of over 24%. This fully demonstrates that the added cation exchange resin step plays a crucial and irreplaceable role in removing phenolic acid impurities that exhibit similar adsorption behavior to anthocyanins on macroporous resins, thus overcoming the purity bottleneck of single adsorption resins and obtaining high-specification refined products. This is of decisive significance for meeting the application needs of the high-end market.

[0045] Finally, it should be noted that the basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification, and therefore remain within the spirit and scope of the exemplary embodiments of this specification. Furthermore, this specification uses specific terms to describe embodiments of this specification. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that "an embodiment," "one embodiment," or "an alternative embodiment" mentioned twice or more in different locations in this specification do not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined. Moreover, unless expressly stated in the claims, the order of processing elements and sequences, the use of numbers and letters, or other names described in this specification are not intended to limit the order of the processes and methods of this specification.

[0046] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 process for enriching and purifying malkiberry anthocyanins by tandem use of macroporous resin and cation exchange resin, characterized in that, Includes the following steps: S1: Prepare the raw material solution by dissolving the crude anthocyanin extract of *Mallotus arvensis* in an acidic buffer solution with a pH value less than 3.0; S2: Perform macroporous resin chromatography by loading the raw material solution obtained in S1 onto a macroporous adsorption resin column, and sequentially performing adsorption, rinsing with deionized water, and elution with aqueous ethanol solution, collecting the first eluent containing anthocyanins; S3: Prepare the intermediate sample by concentrating the first eluent obtained in S2 under reduced pressure at a set temperature to remove the ethanol solvent, obtaining an anthocyanin intermediate concentrated sample; S4: Perform cation exchange resin chromatography. Dilute the anthocyanin intermediate concentrate obtained in S3 with an acidic buffer solution with a pH value less than 3.0, and load it onto a pre-converted H+ type strongly acidic cation exchange resin column. Perform sample adsorption, acidic water rinsing, and acidic aqueous ethanol elution in sequence, and collect the second eluent containing anthocyanins. S5: Prepare the finished product. Concentrate the second eluent obtained in S4 under reduced pressure at a set temperature, recover the solvent, and dry it to obtain the purified anthocyanin product of Maquiberry.

2. The process according to claim 1, characterized in that, The macroporous adsorption resin in S2 is a non-polar or weakly polar polystyrene-based macroporous adsorption resin, specifically selected from one or more of AB-8 type resin or HPD-100 type resin.

3. The process according to claim 1 or 2, characterized in that, The macroporous resin chromatography operating parameters in S2 are as follows: the anthocyanin concentration in the feed solution is controlled at 1-2 mg / mL, and the loading flow rate is controlled at 1 times the bed volume / hour; the volume of deionized water rinsing is 2-3 times the bed volume; the ethanol volume fraction of the aqueous ethanol solution is 50%, the elution volume is 5 times the bed volume, and the elution flow rate is controlled at 1-2 times the bed volume / hour.

4. The process according to claim 1, characterized in that, The temperature for vacuum concentration in S3 is controlled to be no higher than 50°C.

5. The process according to claim 1, characterized in that, The strongly acidic cation exchange resin in S4 is a styrene-based sulfonic acid cation exchange resin, specifically selected from one or more of the following: 001×7 type resin, 732 type resin, Amberlite IR120 type resin, or Lewatit S100 type resin.

6. The process according to claim 1 or 5, characterized in that, The pretreatment of the strongly acidic cation exchange resin in S4 includes: sequentially treating it with an acid solution to convert it to the H+ form, and then washing it with deionized water until the pH of the effluent is close to neutral.

7. The process according to claim 1, characterized in that, The cation exchange resin chromatography operating parameters in S4 are as follows: the loading concentration of the anthocyanin intermediate concentrate after dilution is controlled at 1-2 mg / mL, the loading flow rate is controlled at 1 times the bed volume / hour, and the ratio of the wet-filled resin bed volume to the loading liquid volume is 1:5 to 1:

10.

8. The process according to claim 1 or 7, characterized in that, The acidic water in S4 is an acidic aqueous solution with a pH of 2.0, and the rinsing volume is 1-2 times the bed volume; the acidic aqueous ethanol solution is an aqueous solution containing 1% hydrochloric acid and 50-60% ethanol by volume, and the elution flow rate is controlled at 1 times the bed volume / hour.

9. The process according to claim 1, characterized in that, The temperature for vacuum concentration in S5 is controlled to be no higher than 45°C.

10. The process according to claim 1 or 9, characterized in that, The drying method in S5 is either spray drying or freeze drying.

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