Method for separating and purifying Rubixanthin from the peel of short-juvenile wild citrus fruit

Abstract

The present invention belongs to the field of natural product extraction technology, specifically relating to a method for isolating and purifying rubixanthin from the peel of the short-lived wild citrus fruit, Citrus aurantium. The method comprises at least the following steps: S1: obtaining a crude total carotenoid extract from the Citrus aurantium peel; S2: isolating and purifying rubixanthin from the crude total carotenoid extract using semi-preparative ultrahigh-performance liquid chromatography (UHPLC) with 100% methanol as mobile phase A and 100% MTBE as mobile phase B. The present invention allows the isolation, identification, and purification of rubixanthin from the Citrus aurantium peel to obtain a high degree of purity.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to a method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit, Citrus aurantium. Background Art

[0002] Carotenoids are an important natural pigment found widely in nature. They are a class of tetraterpenes or triterpenes based on the isoprene skeleton. Due to the varying number of conjugated double bonds in their molecular structure, carotenoids exhibit a wide variety of colors. Furthermore, carotenoids and their derivatives are crucial for human dietary nutrition and health. Among them, carotenoids containing a β-ring structure (such as β-carotene and β-cryptoxanthin) are important precursors of vitamin A, while lutein, a type of oxygenated carotenoid, plays a crucial role in human retinal health. Other carotenoids also play important roles in boosting human immunity, preventing cardiovascular disease, chronic diseases, and cancer. As natural pigments, carotenoids are important natural dyes in the food, cosmetics, and textile industries. Therefore, large-scale industrial production of carotenoids has great potential and economic value.

[0003] Currently, the raw materials for industrial carotenoid production primarily come from plant materials rich in carotenoids. Industrially extracted carotenoids include lycopene, β-carotene, β-cryptoxanthin, lutein, and capsanthin, generating significant economic value in the food and cosmetics sectors.

[0004] As carotenoid research continues to deepen, it's gradually been discovered that brightly colored carotenoids often have greater application potential. This is because their greater number of conjugated double bonds results in a redder color and stronger antioxidant capacity. For example, capsanthin, extracted from chili peppers, is stable and naturally harmless, making it widely used in food processing, cosmetic coloring, and pharmaceutical coatings. Capsanthin also offers significant protection against radiation damage, particularly gamma radiation.

[0005] Other red carotenoids from plant sources include lycopene (primarily from tomatoes), β-limonene (primarily from citrus fruits), and rubixanthin (from rose hips and sweet cherry). Rubixanthin was discovered in rose hips as early as 1934, and the exact structure of rubixanthin and its cis-isomer, gazaniaxanthin, was determined in 1968 (Kuhn and Grundmann. On rubixanthin, a new xanthoyll of the formula C40H56O[J]. Berichte der Deutschen Chemischen Gesellschaft, 1934, 67:339-344; Brown and Weedon. Rubixanthin and gazaniaxanthin[J]. Chemical Communications, 1968, 7:382-384).

[0006] Citrus plants in the Rutaceae family include the genera Citrus, Poncitrus, and Fortunella. Common cultivated citrus fruits (such as tangerines, pomeloes, oranges, mandarins, and lemons) originate from the genus Citrus. Fortunella hindsii is a single wild species within the genus Fortunella. It is an evergreen woody plant with an average fruit weight of 3-5g, a diameter of approximately 2cm, and an orange-yellow to deep red color. Compared to common cultivated citrus fruits, Fortunella hindsii has smaller fruits and grows on shorter trees, earning it the nickname "mini citrus." The juvenile period of Fortunella hindsii is short, with seedlings flowering in the same year and fruiting in the second year. Common citrus seedlings have a juvenile period of 6-8 years. A research team from Huazhong Agricultural University has deciphered the genome information of Fortunella hindsii and established a stable genetic transformation and gene editing system (Zhu C, Zheng X, Huang Y, et al. Genome sequencing and CRISPR / Cas9 gene editing of an early-flowering Mini-Citrus (Fortunella hindsii) [J]. Plant biotechnology journal, 2019, 17(11): 2199-2210.), which is expected to develop this variety into a model plant for citrus functional genomics research. Fortunella hindsii is a nutritious and healthy citrus fruit. Furthermore, its dwarfing characteristics make it suitable for greenhouse cultivation, which is conducive to the development of supporting green production technologies to precisely control the content of active ingredients. It is a potential variety for intelligent and unmanned production. Through large-scale screening of metabolites, the inventors found that the carotenoid composition of the mature peel of the kumquat is quite different from that of common citrus fruits. Only the peel of the kumquat accumulates a large amount of red Rubixanthin, which accounts for about 35.85% of the total carotenoids in the peel. It is the carotenoid with the highest content and is also the main reason for the red trait of the kumquat peel.

[0007] In the prior art, only very trace amounts of rubixanthin carotenoids and their analogs were detected in lemon peel and orange juice from the genus Citrus in the 1960s and 1970s, but they had not been isolated and purified in large quantities (GROSS J, GABAI M, Lifshitz A. Carotenoids in juice of Shamouti orange [J]. Journal of Food Science, 1971, 36(3): 466-473.; CURL AL. The carotenoids of Meyer lemons [J]. Journal of Food Science, 1962, 27(2): 171-176.). Moreover, in recent decades, no studies have confirmed the presence of rubixanthin in lemon and orange fruits. Therefore, it is generally believed that citrus plants are not the source of rubixanthin. The existing technology has only been used to isolate and identify Rubixanthin in trace amounts from other plants (rosehip and sweet cherry), but there are no reports of citrus plants (germplasm resources) that accumulate Rubixanthin in large quantities or technologies for extracting and purifying Rubixanthin in large quantities. In addition, compared to the common β-carotene, β-cryptoxanthin has higher antioxidant activity and therefore has higher anti-inflammatory potential, which is very important for the human body to resist aging and prevent diseases (Katalin Sólyoma et al., Structure-response relationship of carotenoid bioaccessibility and antioxidant activity as affected by the hydroxylation and cyclization of their terminal end group [J], Food Research International, 2014, (66): 107-114.).

[0008] By extensively screening carotenoids from various wild, closely related, and cultivated citrus fruits, the inventors discovered for the first time that Fortunella hindsii, a short-lived wild citrus, can accumulate large quantities of the red carotenoid Rubixanthin, giving it a vibrant red appearance and great ornamental value. It also provides an excellent material for large-scale purification of Rubixanthin. Other reported plant sources of Rubixanthin—such as cherries and rose hips—are only cultivated on a small scale. Citrus, my country's most popular fruit, is expected to provide sufficient plant raw materials for the large-scale production of pure Rubixanthin by modifying its fruit to enrich it with Rubixanthin. This will provide the material foundation and technical support for the innovative use of Rubixanthin natural pigments in health products and food and cosmetics.

[0009] Rubixanthin, also known as 3R-β-carotene-3-ol, rubixanthin, has a molecular formula of C 40 H 56 O, CAS No: 3763-55-1, structural formula is as follows:

[0010] Summary of the Invention

[0011] To address the shortcomings of the existing technology, the present invention provides a method for isolating and purifying Rubixanthin from the peel of the short-lived wild citrus fruit, Fortunella sempervirens (a separate species in the genus Fortunella). This method allows for the isolation and purification of large quantities of Rubixanthin from a crude carotenoid extract from Fortunella sempervirens peel, achieving high purity.

[0012] The technical solutions provided by the present invention are as follows:

[0013] A method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit, Citrus aurantium, comprises the following steps:

[0014] S1: crude total carotenoid extract was obtained from the red peel of ripe fruits of C.

[0015] S2: Rubixanthin was purified from a crude total carotenoid extract using semi-preparative UHPLC with 100% methanol as mobile phase A and 100% MTBE as mobile phase B.

[0016] The inventors have discovered that a large amount of rubixanthin can be accumulated in the peel of the fruit ...

[0017] Specifically, in step S1, the kumquat is a fully mature red kumquat fruit during the winter solstice in December, which turns from green to orange and then completely turns to scarlet. This time point is during the winter solstice in December (National Citrus Breeding Center, Wuhan, Hubei).

[0018] Specifically, step S1 includes the following steps:

[0019] S11: The red peel of the ripe fruit of the kumquat was quickly frozen with liquid nitrogen, then freeze-dried and ground to obtain peel powder;

[0020] S12: Obtaining a crude total carotenoid extract from the peel ground powder using a pigment extractant, wherein the pigment extractant is a mixed solution of n-hexane:acetone:anhydrous ethanol in a volume ratio of 2:(0.9-1.1, preferably 1):(0.9-1.1, preferably 1), and butylated hydroxytoluene (BHT) with a mass fraction of 0.09-0.11‰ is added to the mixed solution.

[0021] Based on this technical solution, the pigment extractant is conducive to fully extracting carotenoids from the peel, and 2,6-di-tert-butyl-4-methylphenol (BHT) has an antioxidant effect, preventing carotenoids from being oxidized during the extraction process.

[0022] Specifically, in step S11, the peel ground powder is obtained by freeze-drying and then grinding, or by quick-freezing with liquid nitrogen and then grinding.

[0023] Specifically, step S12 includes the following steps:

[0024] S121: extracting the peel ground powder with the pigment extractant at 4-25° C. to obtain a pigment extract;

[0025] S122: washing the pigment extract with a saturated sodium chloride solution until it is neutral, discarding the aqueous phase, retaining the upper organic phase, and evaporating the solvent in the organic phase of the pigment extract to obtain a dry carotenoid crude extract;

[0026] S123: re-dissolving the crude carotenoid extract and performing a saponification reaction to convert the esterified carotenoids into a free form (non-esterified carotenoids);

[0027] S124: removing the aqueous phase from the saponification reaction product obtained in step S123, retaining the organic phase, washing the organic phase of the saponification reaction with a saturated sodium chloride solution until neutral, removing the aqueous phase, and evaporating the solvent to obtain the crude extract of free total carotenoids.

[0028] Furthermore, step S1 further includes using HPLC to microseparate and detect Rubixanthin from the carotenoid mixture; in the microseparation and detection, 100% methanol is used as mobile phase A, a methanol:water volume ratio of 4:(0.9 to 1.1, preferably 1) is used as mobile phase B, and 100% MTBE is used as mobile phase C.

[0029] Specifically, the micro-separation and detection of Rubixanthin were performed by the following HPLC method:

[0030] Chromatographic column: YMC C 30 Analytical chromatography column (preferably 4.6 mm × 250 mm, 5 μm);

[0031] Mobile phase A: 100% methanol;

[0032] Mobile phase B: methanol: water = 4: (0.9-1.1, preferably 1);

[0033] Mobile phase C: 100% MTBE;

[0034] The flow rate is 1 ml / min;

[0035] Elution conditions: 95% A from 0 to 6 min; 95-80% A from 6 to 7 min; 80% A for 5 min from 7 to 12 min; 80-30% A from 12 to 32 min; 30% A for 16 min from 32 to 48 min; 30-95% A from 48 to 50 min; equilibrate at 95% A for 10 min, maintaining 5% B during elution; select the full wavelength range scan (200-600 nm) for the visible light-UV absorption wavelength channel; the elution peak at 30-32 min is Rubixanthin.

[0036] The peak eluting at 30-32 min in the chromatogram obtained using this HPLC-I method was identified as Rubixanthin, which was consistent with the standard. This method is referred to as HPLC-I.

[0037] Specifically, in step S2, Rubixanthin is separated and purified in large quantities by the following semi-preparative high performance liquid chromatography (UHPLC) method:

[0038] Chromatographic column: YMC C 30 Semi-preparative chromatography column (preferably 10 mm × 250 mm, 5.0 μm);

[0039] Mobile phase A: 100% methanol;

[0040] Mobile phase B: 100% MTBE;

[0041] Flow rate: 2 ml / min;

[0042] Elution conditions: 0-8 min 60% A; 8-28 min 60-40% A; 28-30 min 40% A hold for 2 min; 30-32 min 40-60% A; 32-37 min 60% A hold for 5 min;

[0043] Visible / ultraviolet absorption channels: UV-vis-1: 450nm or UV-vis-2: 490nm;

[0044] The elution peak at 17.3-17.9 min was collected to obtain purified Rubixanthin.

[0045] The peak eluting at 17.3-17.9 min in the UHPLC chromatogram was identified as rubixanthin, and this peak was collected to obtain purified rubixanthin. This method is referred to as a UHPLC semi-preparative method.

[0046] Preferably, the channels for visible light absorption are UV-vis-1: 450 nm.

[0047] Furthermore, in step S2, the dried product of the crude free carotenoid extract is dissolved in 100% ethyl acetate, centrifuged at room temperature, and the supernatant is filtered through a 0.22 μm filter membrane, and then identified or purified.

[0048] Furthermore, the method further comprises step S3: performing purity analysis and mass spectrometry identification on the purified Rubixanthin;

[0049] In the purity analysis, 100% methanol was used as mobile phase A, methanol:water volume ratio = 4: (0.9 to 1.1, preferably 1) was used as mobile phase B, and 100% MTBE was used as mobile phase C;

[0050] In mass spectrometry identification, a mixture of methanol and water (99:1) containing 0.1% chromatographic grade formic acid was used as mobile phase A, and 100% MTBE was used as mobile phase B.

[0051] Specifically, the following LC-MS method was used for micro-separation and mass spectrometric identification of Rubixanthin:

[0052] The instrument used was an ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS / MS) system (QExactive Plus, Thermo Fisher Scientific, San Jose, CA, USA) equipped with an atmospheric pressure chemical ionization (APCI) source and operated in positive and negative ion modes.

[0053] The mass spectrometry parameters were as follows: Full MS scan parameters: resolution of 70,000 full width at half maximum (FWHM); ion trap dynamic gain control (AGC) of 3e6; maximum injection time (IT) of 100 ms; mass spectrum acquisition range of 100-1400 m / z. ddMS2 scan parameters: 17,500 FWHM; AGC of 1e5; minimum of 8e3; maximum IT of 50 ms;

[0054] Chromatographic column: Thermo C 30 Chromatographic column (2.1mm×150mm, 3.0μm):

[0055] Mobile phase A: methanol: water = 99:1 (containing 0.1% chromatography grade formic acid);

[0056] Mobile phase B: 100% MTBE;

[0057] Elution conditions (variable speed linear gradient elution): 0-6 min, 100% A, flow rate of 0.25 ml / min; 6-6.5 min, flow rate increased to 0.5 ml / min; 6.5-15 min, linear gradient decreased to 80% A; 15-23 min, linear gradient decreased to 10%; 23-25 ​​min, maintained unchanged; 25-26 min, flow rate decreased to 0.25 L / min, linear gradient increased to 100% A.

[0058] The invention also provides a method for separating and purifying Rubixanthin in large quantities, which is extracted from the peel of the short-juvenile wild citrus fruit, Citrus aurantium.

[0059] The inventors have discovered that the peel of the short-juvenile wild citrus fruit, Citrus aurantium, can accumulate a large amount of rubixanthin, and that Rubixanthin with a high purity can be separated and extracted in large quantities from Citrus aurantium.

[0060] The present invention also provides Rubixanthin obtained by the above methods, wherein the purity of the Rubixanthin is greater than or equal to 97.62%.

[0061] Preferably, S1 comprises the following steps:

[0062] S11: Grinding the kumquat peel to obtain peel powder, preferably by vacuum freeze drying;

[0063] S12: Obtaining a crude total carotenoid extract from the peel powder using a pigment extractant, wherein the pigment extractant is a 2:1:1 (v / v) mixed solution of n-hexane:acetone:anhydrous ethanol and contains 0.1‰ BHT.

[0064] Preferably, in the HPLC method for detecting the presence of Rubixanthin in the carotenoid sample of the fruit peel of the Mandarin orange in S2:

[0065] The chromatographic detection system was a Waters 2695 high performance liquid chromatograph, a 2996 secondary array detector, a 717 autosampler, Empower chromatography management software, and a YMC C 30 An analytical column (4.6 mm×250 mm, 5 μm) (Wilmington, NC, USA) was used, and 10 μl of sample was injected each time.

[0066] Mobile phase A: 100% methanol;

[0067] Mobile phase B: methanol: water = 4:1;

[0068] Mobile phase C: 100% MTBE

[0069] The flow rate is 1 ml / min;

[0070] Elution conditions: 0-6 min 95% A; 6-7 min 95-80% A; 7-12 min 80% A for 5 min; 12-32 min 80-30% A; 32-48 min 30% A for 16 min; 48-50 min 30-95% A; 95% A for 10 min, maintaining 5% B during elution.

[0071] The elution peak at 30.5-31.5 min in the HPLC liquid chromatogram is Rubixanthin.

[0072] In this method, the elution peak at 30-32 min is Rubixanthin, which is present in a high content in the peel of the kumquat during the winter solstice ripening period and determines the formation of the red trait of the kumquat fruit.

[0073] Preferably, in the HPLC method for separating, purifying, and extracting Rubixanthin in larger quantities in S2:

[0074] Instrument: Dionex Ultimate 3000 semi-preparative HPLC equipped with Ultimate RS Variable Wavelength Detector.

[0075] Chromatographic column: YMC C 30 Semi-preparative chromatography column (preferably 10 mm × 250 mm, 5.0 μm);

[0076] Mobile phase A: 100% methanol;

[0077] Mobile phase B: 100% MTBE;

[0078] The flow rate is 2 ml / min;

[0079] Elution conditions: 0-8 min 60% A; 8-28 min 60-40% A; 28-30 min 40% A hold for 2 min; 30-32 min 40-60% A; 32-37 min 60% A hold for 5 min. Two visible / UV absorption channels were selected: UV-vis-1: 450 nm, UV-vis-2: 490 nm.

[0080] The elution peak at 17.3-17.9 min was collected to obtain purified Rubixanthin.

[0081] The method is suitable for further separating and purifying Rubixanthin from the total carotenoid extract of the kumquat peel in larger quantities. The semi-preparative liquid phase used in the method has a high flow rate, which greatly accelerates the speed of collecting pure Rubixanthin.

[0082] The beneficial effects of the present invention are as follows:

[0083] The invention identifies for the first time a large amount of accumulated Rubixanthin from the fruit peel of the kumquat at the winter solstice ripening period, separates and purifies the Rubixanthin and extracts it in large quantities, and the obtained Rubixanthin has high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The chromatogram is obtained by detecting the pigment components in the crude extract of total carotenoids using HPLC-I method.

[0085] Figure 2 Semi-preparative UHPLC chromatogram obtained during the purification of Rubixanthin using the UHPLC semi-preparative high-performance liquid chromatography method.

[0086] Figure 3 The chromatograms are obtained by HPLC analysis. Part A is the chromatogram obtained by HPLC analysis of the rubixanthin isolated and purified by the present invention, and Part B is the chromatogram obtained by HPLC analysis of a rubixanthin standard. The two chromatograms are basically consistent, demonstrating the accuracy of the rubixanthin extracted by this method.

[0087] Figure 4 The following are mass spectrometry primary positive ion patterns. Part A shows the mass spectrometry primary positive ion pattern of the rubixanthin isolated and purified by the present invention, while Part B shows the mass spectrometry primary positive ion pattern of a rubixanthin standard. The two patterns are essentially identical, demonstrating the accuracy of the rubixanthin extracted by this method.

[0088] Figure 5 The following are mass spectrometry secondary patterns: Part A shows the mass spectrometry secondary pattern of a purified rubixanthin standard, while Part B shows the mass spectrometry secondary pattern of a rubixanthin standard. The two patterns are essentially identical, further demonstrating the accuracy of rubixanthin extraction using this method.

[0089] Figure 6 This is a comparison of different UHPLC separation effects. Part A in the figure: Chromatogram of carotenoids separated from Aronia sutchuenensis by UHPLC-I method. The black arrow indicates the position of Rubixanthin in the separation chromatogram. The inset corresponds to the black arrow and is the HPLC detection chromatogram after separation of this peak. Part B in the figure: Chromatogram of carotenoids separated from Aronia sutchuenensis by UHPLC-II method. The black arrow indicates the position of Rubixanthin in the separation chromatogram. The inset corresponds to the black arrow and is the HPLC detection chromatogram after separation of this peak. DETAILED DESCRIPTION

[0090] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0091] Unless otherwise specified, the test methods used in the examples of the present invention are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0092] Fortunella hindsii (Fortunella hindsii) originates from the resource garden of the National Citrus Breeding Center at Huazhong Agricultural University. Its color changes completely from green and orange-yellow to red, even vermilion, during the winter solstice in December. For information on Fortunella hindsii, please refer to the following publication: "Deng Xiuxin, Zhang Wencai. Direct Induction of Embryogenic Callus from In Vitro Seedlings of Fortunella hindsii. Fruit Trees in Southern China, 1988."

[0093] Example 1

[0094] 1. Extraction of carotenoids

[0095] 1) Sample Grinding: Take a Kaempferia kumquat fruit (fully ripened on the winter solstice in December, turning from green to orange and ultimately to scarlet). Cut the fruit open with a surgical scalpel and scrape the flesh clean with a steel sampling spoon to obtain the peel. After drying in a vacuum freeze dryer, grind the peel using a sample grinder (or hand mortar and pestle) to obtain a dry peel sample. Alternatively, the fruit can be directly quick-frozen in liquid nitrogen and then ground to obtain a wet peel sample.

[0096] 2) Pigment extraction: Weigh 1 g of dry peel sample (3 g for wet sample) and place it in a 50 ml conical centrifuge tube. Add 35 ml of pigment extraction solution (n-hexane:acetone:anhydrous ethanol = 2:1:1, containing 0.1‰ BHT).

[0097] Add a 1.5 cm diamond-shaped magnetic rotor to each 50 ml conical centrifuge tube containing the sample, shake it manually, and place it on a magnetic stirrer at 1500 rpm in a dark environment at 4 to 25 ° C for 2 hours;

[0098] Centrifuge at 4000 g for 15 min at room temperature, transfer the supernatant to a new 50 ml centrifuge tube, and wash repeatedly three times with saturated NaCl (10%) aqueous solution until neutral, and discard the lower colorless aqueous layer;

[0099] Dispense the upper pigment solution into 10ml centrifuge tubes and evaporate the pigment extract to dryness at 30°C using a vacuum concentrator set to U-AQ mode. Dissolve the resulting solution in 2ml of MTBE and add a 10% KOH-methanol solution (10g KOH, 25ml of first-grade pure water, 75ml of methanol). Mix by oscillation and saponify at room temperature in the dark for 12h.

[0100] After adding 1 ml of MTEB to fully dissolve the pigment, add 2 ml of saturated NaCl solution to facilitate separation. Discard the lower aqueous layer and wash with 4 ml of saturated NaCl solution three times until neutral. The pigment solution was divided into 10 ml centrifuge tubes and concentrated to dryness under vacuum at 30°C using a vacuum concentrator in U-AQ mode to obtain a crude carotenoid extract.

[0101] 2.HPLC detection

[0102] The crude carotenoid extract was dissolved in ethyl acetate (chromatographic grade), centrifuged at 12,000 rpm for 30 min at 4°C, and filtered through a 0.22 μm microporous filter. The filtered liquid was transferred to the inner cannula in the sample loading bottle and prepared for detection. In this example, HPLC-I method was used for detection.

[0103] The chromatographic detection system was a Waters 2695 high performance liquid chromatograph, a 2996 secondary array detector, a 717 autosampler, Empower chromatography management software, and a YMC C 30 Analytical column (4.6 mm × 250 mm, 5 μm), 10 μl of sample was injected each time.

[0104] Mobile phase: Phase A: 100% methanol; Phase B: methanol: water = 4:1; Phase C: 100% MTBE. Elution conditions: 95% A (0-6 min); 95-80% A (6-7 min); 80% A (5 min hold) (7-12 min); 80-30% A (12-32 min); 30% A (16 min hold) (32-48 min); 30-95% A (48-50 min); 95% A (10 min equilibration), maintaining 5% B throughout the elution.

[0105] The results are as follows Figure 1 As shown in the HPLC liquid chromatogram, the elution peak at 30.5-31.5 min indicated by the arrow is Rubixanthin, and the small figure on the right shows its visible light / UV absorption peak.

[0106] 2. Purification of Rubixanthin

[0107] Large-scale separation and purification of the carotenoid rubixanthin: The dried carotenoid was dissolved in 100% ethyl acetate (chromatographic grade) and centrifuged at 12,000 rpm for 30 minutes at room temperature. The supernatant was filtered through a 0.22 μm filter and added to a sample vial. The instrument model and chromatographic conditions used for the semi-preparative separation of rubixanthin are as follows. In this example, semi-preparative UHPLC was used for separation and purification.

[0108] Instrument: Thermo Scientific Dionex Ultimate 3000 semi-preparative HPLC equipped with Ultimate RS Variable Wavelength Detector. Column: YMC C 30 Semi-preparative chromatography column (10 mm × 250 mm, 5.0 μm);

[0109] Mobile phase A: 100% methanol;

[0110] Mobile phase B: 100% MTBE;

[0111] The flow rate is 2 ml / min;

[0112] Elution conditions (multi-step gradient elution): 0-8 min 60% A; 8-28 min 60-40% A; 28-30 min 40% A hold for 2 min; 30-32 min 40-60% A; 32-37 min 60% A hold for 5 min. Select two visible / UV absorption channels: UV-vis-1: 450 nm, UV-vis-2: 490 nm.

[0113] The eluted fractions were collected according to their peak order and peak time, and dried in a vacuum concentrator at 30 degrees. The vacuum concentrator was set to U-AQ mode to obtain purified Rubixanthin. Figure 2 The figure shows the absorbance at 450 nm. The peak time of Rubixanthin is approximately 16.9-19.2 min (indicated by the arrow). Calculations indicate that approximately 91.74 μg of pure Rubixanthin can be extracted and purified from every 1 g of dried peel sample.

[0114] 3. Purity Analysis and Mass Spectrometry Identification

[0115] 1) Purity analysis

[0116] A small amount of the prepared dried product and the Rubixanthin standard were dissolved in an appropriate amount of ethyl acetate and centrifuged at 12,000 rpm for 30 minutes at room temperature. The supernatant was filtered through a 0.22 μm filter and then sampled for analysis. This method was performed using a Waters 2695 HPLC instrument from Waters.

[0117] The chromatographic detection system was a Waters 2695 high performance liquid chromatograph, a 2996 secondary array detector, a 717 autosampler, Empower chromatography management software, and a YMC C 30 Analytical column (4.6 mm × 250 mm, 5 μm), 10 μl of sample was injected each time.

[0118] In this example, HPLC-I method was used for detection

[0119] Mobile phase A: 100% methanol;

[0120] Mobile phase B: methanol: water = 4:1;

[0121] Mobile phase C: 100% MTBE

[0122] The flow rate is 1 ml / min;

[0123] Elution conditions: 0-6 min 95% A; 6-7 min 95-80% A; 7-12 min 80% A for 5 min; 12-32 min 80-30% A; 32-48 min 30% A for 16 min; 48-50 min 30-95% A; 95% A for 10 min, maintaining 5% B during elution.

[0124] The results are as follows Figure 3 As shown, the purity of the Rubixanthin obtained in the present invention is very high, showing a single main peak in HPLC detection, and is comparable to the Rubixanthin standard (C 40 H 56 O, Cas number: 3763-55-1), and the color of pure Rubixanthin is orange-red. The calculated purity is 97.62%.

[0125] 2) Mass spectrometry identification

[0126] Purified rubixanthin and standards were analyzed by mass spectrometry using an ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS / MS) system (Q Exactive Plus, Thermo Fisher Scientific, San Jose, CA, USA) equipped with an atmospheric pressure chemical ionization (APCI) source and operated in positive and negative ion modes.

[0127] Mass spectrometry parameters were as follows: Full MS scan parameters: resolution of 70,000 full width at half maximum (FWHM); ion trap dynamic gain control (AGC) of 3e6; maximum injection time (IT) of 100 ms; and mass spectrum acquisition range of 100–1400 m / z. ddMS2 scan parameters: FWHM of 17,500; AGC of 1e5; minimum value of 8e3; and maximum IT of 50 ms.

[0128] Using Thermo C 30 Chromatographic column (2.1mm×150mm, 3.0μm), 1ul injection each time.

[0129] The mass spectrometry identification LC-MS method used in this example is as follows:

[0130] Mobile phase A: methanol: water = 99:1 (containing 0.1% chromatography grade formic acid);

[0131] Mobile phase B: 100% MTBE;

[0132] Elution conditions (variable speed linear gradient elution): 0-6 min, 100% A, flow rate of 0.25 ml / min; 6-6.5 min, flow rate increased to 0.5 ml / min; 6.5-15 min, linear gradient decreased to 80% A; 15-23 min, linear gradient decreased to 10%; 23-25 ​​min, maintained unchanged; 25-26 min, flow rate decreased to 0.25 L / min, linear gradient increased to 100% A.

[0133] The data were analyzed using the accompanying Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA).

[0134] The results are as follows Figure 4 and Figure 5 As shown, the molecular mass of Rubixanthin obtained in the present invention is 553.426, and the secondary mass spectrum information is also consistent with the standard (C 40 H 56 O, Cas number: 3763-55-1), indicating that the substance isolated from the fortunella is Rubixanthin.

[0135] Example 2

[0136] Rubixanthin purification was performed with reference to Example 1 above, except that the dry peel powder of the short-juvenile wild citrus fruit, Citrus aurantium (a separate wild species distinct from other citrus fruits), was used as the raw material for extraction. The result was that approximately 91.74 μg of pure Rubixanthin with a purity of not less than 97% was obtained per gram of Citrus aurantium peel dry weight.

[0137] Comparative Example 1

[0138] Rubixanthin was purified with reference to Example 1 above, except that the carotenoids in Jaffa orange juice were separated into hydrocarbons, monohydric alcohols, dihydric alcohols, and polyhydric alcohols by column chromatography (MgO-Hyflo Super Cel, packed in a mass ratio of 1:1, eluted with a mixture of ether and acetone). The carotenoids in the different fractions were then separated and identified by thin-layer chromatography (calcium chloride-silica gel G, silica gel G as adsorbent, using a large number of organic reagents such as light petroleum, benzene, ether, acetone, ethyl acetate, dichloromethane, polyethylene, and isopropanol for analysis). After the tedious and laborious separation and identification using a large amount of highly polluting organic reagents, only 0.05% of the total carotenoid content of rubixanthin was detected, and the purity was unknown (GROSS J, GABAI M, Lifshitz A. Carotenoids in juice of Shamouti orange [J]. Journal of Food Science, 1971, 36(3): 466-473.). Previous researchers isolated and identified carotenoids from 500g, 1100g, and 1800g of Meyer lemons. Using n-hexane-99% methanol (v:v = 1.8:1) in a Craig instrument, they performed up to 200 separations to fractionate the different carotenoids. They only identified 0.8% of the total carotenoid content as Rubixanthin-like, but did not provide mass spectrometry or nuclear magnetic resonance analysis information, making it impossible to confirm whether it was Rubixanthin (CURL AL. The carotenoids of Meyer lemons [J]. Journal of Food Science, 1962, 27(2): 171-176.). In short, the rubixanthin / rubixanthin-like content detected by previous researchers in Jaffa orange juice and Meyer lemon peel was very low, making it essentially impossible to isolate, purify, and industrially extract the rubixanthin / rubixanthin-like in large quantities. This method, however, utilizes a chromatographic column as an adsorption carrier and methanol and MTBE as eluents, significantly reducing the amount of organic reagents used. This method, which is the first to identify rubixanthin in Fortunella, reveals that the rubixanthin content in the red peel of mature Fortunella fruit is as high as 35.85%, approximately 700 times that of Jaffa orange juice. More importantly, the purity of the rubixanthin isolated and prepared by this method is as high as 97.62%. Furthermore, the raw materials used in previous studies—Jaffa oranges and Meyer lemons—are hybrids between different species within the genus Citrus. In contrast, the Fortunella used in this invention is a unique wild species within the genus Fortunella. Therefore, this invention achieves the first identification, isolation, and purification of rubixanthin in the Fortunella genus.

[0139] Comparative Example 2

[0140] UHPLC-I method:

[0141] (1) Mobile phase:

[0142] Phase A: methanol: MTBE = 1:1;

[0143] Phase B: methanol: water: MTBE = 6:3:1;

[0144] Flow rate: 2 ml / min;

[0145] (2) Elution conditions (gradient elution)

[0146] 0-20min 92%A; 20-23min 92-82%A; 23-25min 82%-50%A; 25-26min 50%-40%A; 26-36min 40%A maintained for 10min.

[0147] Visible light / ultraviolet absorption channel selection: UV-vis-1: 450nm;

[0148] (3) Separation results

[0149] The elution peak at 21.5-22.1 min in the UHPLC-I chromatogram was collected and identified by HPLC. In addition to the chromatographic peak of Rubixanthin, there was also a chromatographic peak at the same height, indicating that Rubixanthin could not be separated well.

[0150] UHPLC-II method (same as semi-preparative UHPLC used for purification above):

[0151] (1) Mobile phase

[0152] Phase A: 100% methanol;

[0153] Phase B: 100% MTBE;

[0154] Flow rate: 2 ml / min;

[0155] (2) Elution conditions (gradient elution)

[0156] 0-8min 60%A; 8-28min 60-40%A; 28-30min 40%A for 2min; 30-32min

[0157] 40-60% A; 32-37 min 60% A maintained for 5 min.

[0158] Visible light / ultraviolet absorption channel selection: UV-vis-1: 450nm;

[0159] (3) Separation results

[0160] The peak eluting at 17.3-17.9 min in the UHPLC-II chromatogram was collected and identified as Rubixanthin with high purity.

[0161] Comparison of UHPLC-I and UHPLC-II methods:

[0162] like Figure 6 As shown, the UHPLC-II method removes the aqueous phase and replaces it with a pure organic phase, enhancing the separation of carotenoids. Elution conditions are further simplified to a gradient elution of 60-40% A. Because the mobile phase in the UHPLC-II method is a pure organic phase, the collected elution peak solvent does not contain water, making it easier to evaporate to dryness to obtain pure rubixanthin.

[0163] The above are only preferred embodiments of the present invention and do not limit the present invention in any form. Any ordinary technician in this industry can smoothly implement the present invention as shown in the drawings and above. However, any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of protection of the technical solution of the present invention.

Claims

1. A method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit, the method comprising: The following steps are involved: S1: crude extract of total carotenoids was obtained from the peel of the fruit of the genus Citrus aurantium; S2: Rubixanthin was purified from a crude total carotenoid extract using semi-preparative UHPLC with 100% methanol as mobile phase A and 100% MTBE as mobile phase B. In step S1, the kumquat is a fully mature red kumquat fruit during the winter solstice in December; Step S1 includes the following steps: S11: The peel of the kumquat is quickly frozen with liquid nitrogen, then freeze-dried and ground to obtain peel ground powder; S12: obtaining a crude total carotenoid extract from the peel ground powder using a pigment extractant, wherein the pigment extractant is a mixed solution of n-hexane:acetone:anhydrous ethanol in a volume ratio of 2:(0.9-1.1):(0.9-1.1), and butylated hydroxytoluene (BHT) with a mass fraction of 0.09-0.11‰ is added to the mixed solution; In step S11, the peel ground powder is obtained by freeze-drying and then grinding, or by quick-freezing with liquid nitrogen and then grinding; Step S12 specifically includes the following steps: S121: extracting the peel ground powder with the pigment extractant at 4-25° C. to obtain a pigment extract; S122: washing the pigment extract with a saturated sodium chloride solution until it is neutral, discarding the aqueous phase, retaining the upper organic phase, and evaporating the solvent in the organic phase of the pigment extract to obtain a crude carotenoid extract; S123: re-dissolving the crude carotenoid extract and performing a saponification reaction to convert the esterified carotenoid into a free form; S124: removing the aqueous phase from the saponification reaction product obtained in step S123, retaining the organic phase, washing the organic phase of the saponification reaction with a saturated sodium chloride solution until neutral, removing the aqueous phase, and evaporating the solvent to obtain the crude free carotenoid extract; In step S2, Rubixanthin is isolated and purified in large quantities by the following semi-preparative high performance liquid chromatography (UHPLC) method: Chromatographic column: YMC C 30 Semi-preparative chromatography columns; Mobile phase A: 100% methanol; Mobile phase B: 100% MTBE; Flow rate: 2 ml / min; Elution conditions: 0-8 min 60% A; 8-28 min 60-40% A; 28-30 min 40% A hold for 2 min; 30-32 min 40-60% A; 32-37 min 60% A hold for 5 min; Visible / ultraviolet absorption channels: UV-vis-1: 450nm or UV-vis-2: 490nm; The elution peak at 17.3-17.9 min was collected to obtain purified Rubixanthin; In step S2, the dried product of the crude carotenoid extract is dissolved in 100% ethyl acetate, centrifuged at room temperature, and the supernatant is filtered through a 0.22 μm filter membrane, and then separated and purified.

2. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of the species Achyranthes fortunei according to claim 1, characterized in that: Step S1 further includes using HPLC to microseparate and detect Rubixanthin from the carotenoid mixture; in the microseparation and detection, 100% methanol is used as mobile phase A, a methanol:water volume ratio of 4:(0.9-1.1) is used as mobile phase B, and 100% MTBE is used as mobile phase C.

3. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Achyranthes fortunei according to claim 2, characterized in that: In step S1: The micro separation and detection of Rubixanthin were performed by the following HPLC method: Chromatographic column: YMC C 30 Analytical chromatography columns; Mobile phase A: 100% methanol; Mobile phase B: methanol: water = 4: (0.9-1.1); Mobile phase C: 100% MTBE; The flow rate is 1 ml / min; Elution conditions: 0-6 min 95% A; 6-7 min 95-80% A; 7-12 min 80% A for 5 min; 12-32 min 80-30% A; 32-48 min 30% A for 16 min; 48-50 min 30-95% A; 95% A for 10 min, and 5% B was maintained during elution. The elution peak at 30-32 min is Rubixanthin.

4. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Achyranthes fortunei according to claim 1, characterized in that: The method further includes step S3: performing purity analysis and mass spectrometry identification on the isolated and purified Rubixanthin; For purity analysis, 100% methanol was used as mobile phase A, methanol:water volume ratio = 4:(0.9-1.1) was used as mobile phase B, and 100% MTBE was used as mobile phase C; In mass spectrometry identification, a mixture of methanol and water (99:1) containing 0.1% chromatographic grade formic acid was used as mobile phase A, and 100% MTBE was used as mobile phase B.

5. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Achyranthes fortunei according to claim 4, characterized in that: The micro-separation and mass spectrometric identification of Rubixanthin were performed by the following LC-MS method: The instrument is an ultra-high performance liquid chromatography-high resolution tandem mass spectrometry system equipped with an atmospheric pressure chemical ionization ion source and operating in positive and negative ion modes; The mass spectrometry parameters were as follows: Full MS scan parameters: resolution 70,000 half-peak width; ion trap dynamic gain control 3e6; maximum injection time 100 ms; mass spectrum acquisition range 100-1400 m / z; ddMS2 scan parameters: 17,500 FWHM; AGC 1e5; minimum 8e3; maximum IT 50 ms; Chromatographic column: Thermo C 30 Chromatographic column: Mobile phase A: a mixture of methanol and water (99:1) containing 0.1% chromatographic grade formic acid. Mobile phase B: 100% MTBE; Elution conditions: 0-6 min, 100% A, flow rate 0.25 ml / min; 6-6.5 min, flow rate increased to 0.5 ml / min; 6.5-15 min, linear gradient decreased to 80% A; 15-23 min, linear gradient decreased to 10%; 23-25 ​​min, maintained unchanged; 25-26 min, flow rate decreased to 0.25 L / min, linear gradient increased to 100% A.

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