Separation and purification method of mangiferin monomer in aquilaria sinensis leaves

Abstract

The invention provides a separation and purification method of mangiferin monomers in aquilaria sinensis leaves, and relates to the technical field of processing and extraction of traditional Chinese medicinal materials. The method for separating and purifying the mangiferin monomer in the aquilaria sinensis leaves comprises the following steps: step 1, pretreating bay leaves; step 2, extracting and concentrating; step 3, extracting and concentrating; step 4, storing at low temperature; and step 5, washing and suction filtration. The method comprises the steps of aquilaria sinensis leaf raw material pretreatment, extraction, concentration, extraction, concentration, low-temperature storage, pure water washing and suction filtration, and finally high-purity mangiferin is obtained, the separation and purification method is simpler, the process is green and environmentally friendly, the cost is low, the product quality is good, the method is suitable for industrial and large-scale production, aquilaria sinensis leaves are adopted as the raw material, and rich mangiferin is contained; according to the mangiferin preparation method, the mangiferin is high in purity, the variety and number of other impurity components are small, the raw material does not contain allergic components, pesticides are not used in the raw material production process, and the prepared product is high-quality and high-purity mangiferin without allergens and organic pesticide residues.

Description

Technical Field

[0001] The invention relates to the technical field of processing and extracting traditional Chinese medicinal materials, and in particular to a method for separating and purifying mangiferin monomers in Aquilaria sinensis leaves. Background Art

[0002] Aquilaria sinensis, also known as agarwood, agarwood tree, Wanxiang, tooth incense, daughter incense, etc., is a plant of the genus Aquilaria of the family Thymelaeaceae, an evergreen tree, a unique national second-level protected plant in my country, planted in Guangdong, Hainan, Guangxi, Fujian and other places, and is a precious medicinal material commonly used in traditional Chinese medicine. The heartwood of Aquilaria sinensis is pungent, bitter, and slightly warm. It has the effects of lowering qi and regulating the middle, warming the kidney and relieving pain. It is often used for chest and abdominal pain, chest tightness, vomiting and hiccups, abdominal rumbling and diarrhea, and shortness of breath. The heartwood of Aquilaria sinensis contains resin, which can be used as a spice raw material and a special medicine for treating stomach diseases. The bark fiber is flexible, white and delicate, and can be used as a raw material for high-grade paper and artificial cotton. The wood can be used to extract aromatic oils, and the flowers can be used to make extracts. It is a plant with high economic benefits.

[0003] Mangiferin, also known as mangoside and Guanzhimuning, is a tetrahydroxypyrrolidone carbon glycoside, belonging to the class of bispyrrolidone compounds. Mangiferin has the effects of relieving asthma, relieving cough and reducing phlegm, relieving fever, calming, anti-inflammatory, analgesic, anti-oxidation, anti-hyperlipidemia, protecting liver and promoting bile secretion, immunomodulatory, anti-bacterial, anti-viral, anti-tumor, anti-tuberculosis, anti-diabetes, anti-obesity, anti-radiation, anti-depression, anti-neurasthenia, preventing and treating hyperuricemia and gout, inhibiting bone absorption, etc., and is widely used in the pharmaceutical, cosmetics and health care products industries.

[0004] Since mangiferin has a wide range of uses, there are many patents for extracting, separating and refining this compound from natural products. A Chinese invention patent application entitled "Method for preparing high-purity mangiferin" (application number 200610079234.5) discloses a method for extracting mangiferin from plants containing mangiferin as raw materials, preferably mango leaves, almond leaves and rhizoma anemarrhenae, through methyl (ethyl) alcohol aqueous solution extraction, resin decolorization, concentration and drying, recrystallization, etc. The purity of mangiferin obtained by this invention is ≥90%. A Chinese invention patent application entitled "A method for extracting mangiferin" (application number 200710066354.6) discloses a method for extracting mangiferin from mango leaves or almond leaves as raw materials, through high-temperature and high-pressure solvent extraction, membrane concentration, macroporous resin separation and purification, etc. The purity of mangiferin obtained by this invention is more than 60%, and the solvent used is water or methanol, ethanol, propanol, butanol (water) solution. The Chinese invention patent application entitled "Method for preparing mangoside" (application number 200910175781.7) discloses a method for obtaining mangoside from mango leaves as raw materials through crushing, enzyme treatment, petroleum ether degreasing, ethanol water extraction, spray drying, dissolution, centrifugal filtration, concentration and drying, and recrystallization twice; the purity of mangoside obtained by this invention can reach 98%. The Chinese invention patent application entitled "A method for preparing mangoside" (application number 200910234065.1) discloses a method for extracting mangoside from mango leaves or almond leaves as raw materials through immersion extraction with saturated lime water solution, macroporous resin adsorption, water and different concentrations of ethanol solution segmented elution, reduced pressure concentration crystallization, methanol acidification recrystallization and other processes; the purity of mangoside obtained by this process is more than 95%.

[0005] The above disclosed existing mangiferin production process has the following problems:

[0006] The raw materials commonly used are mango leaves, almond leaves, and Anemarrhena asphodeloides, which have low mangoside content and many impurities, and have low yield per unit weight, high cost, and low profit;

[0007] Mango leaves are commonly used as raw materials because the tree is prone to insects and pesticide use often exceeds the standard, resulting in mangoside containing organic pesticide residues;

[0008] Mango leaves are commonly used as raw materials. Since this raw material contains a large amount of organic acid allergens, the application of mangoside products extracted from it in the pharmaceutical, health care and cosmetic industries is greatly limited;

[0009] The extraction process generally uses toxic organic solvents such as butanol and petroleum ether, which pollute the environment. The mangoside products produced contain residual organic solvents, which affects safety.

[0010] The commonly available mangiferin products have low purity.

[0011] In summary, the existing separation and purification processes of mangiferin are relatively complicated and difficult to operate. The use of multiple organic solvents and macroporous resins significantly increases production costs and affects safety.

[0012] Therefore, those skilled in the art provide a method for separating and purifying mangiferin monomers in Aquilaria sinensis leaves to solve the problems raised in the above background technology. Summary of the invention

[0013] 1. Technical issues to be resolved

[0014] In view of the shortcomings of the prior art, the present invention provides a method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves, which solves the problems that the existing separation and purification process of mangiferin is relatively complicated, the operation is difficult, the use of multiple organic solvents and macroporous resins significantly increases the production cost and affects the safety.

[0015] (II) Technical solution

[0016] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0017] A method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves comprises the following steps:

[0018] Step 1. Pretreatment of bay leaves

[0019] Put the white atractylodes leaves into a drying oven for drying, and then crush or pulverize them for later use;

[0020] Step 2. Extraction and concentration

[0021] The pretreated Aquilaria sinensis leaf fragments are decocted with a solvent for multiple times, filtered to obtain an Aquilaria sinensis alcohol solution, combined with the filtrate, concentrated to recover the solvent, and obtained an Aquilaria sinensis leaf concentrate;

[0022] Step 3. Extraction and concentration

[0023] The above concentrate was dissolved in pure water to obtain a concentrated solution of Aquilaria sinensis leaves, which was then diluted with water to obtain a solution of Aquilaria sinensis leaves, which was repeatedly extracted with petroleum ether, the solvent was recovered, the aqueous phase was collected, and the aqueous phase was repeatedly extracted with n-butanol, the aqueous phase was discarded, and the solvent was recovered from the organic layer to obtain a crude product of mangiferin;

[0024] Step 4. Low temperature storage

[0025] The crude mangiferin product is dissolved in pure water and stored at low temperature, and mangiferin crystals are precipitated;

[0026] Step 5. Wash and filter

[0027] The mangiferin crystals obtained above are repeatedly washed with pure water, filtered, dried and crushed to obtain the mangiferin monomer product.

[0028] Preferably, the Aquilaria sinensis leaves in step 1 are dried by oven drying or sun drying at a drying temperature of 30 to 80°C.

[0029] Preferably, in step 1, the Aquilaria sinensis leaves can be crushed to 10 mesh to 100 mesh after drying.

[0030] Preferably, the extraction method in step 2 is reflux extraction, the extraction solvent used is 0% to 80% methanol-water solution, the extraction material-liquid ratio is 1:5 to 1:15, the extraction is performed 2 to 4 times, each time for 10 to 120 minutes, and the extraction temperature is 40 to 80°C.

[0031] Preferably, the petroleum ether used for extraction in step 3 is petroleum ether with a boiling point of 60-90° C. and a volume fraction of 100%, and the amount used is 0.5-5 times the volume of the concentrated solution, and the n-butanol used is n-butanol with a volume fraction of 100%, and the amount used is 0.5-5 times the volume of the aqueous phase after petroleum ether extraction.

[0032] Preferably, the extraction times of petroleum ether and n-butanol in step 3 is 2 to 5 times.

[0033] Preferably, the water in step 4 is purified water, and the amount used is 5 to 20 times that of the crude mangiferin.

[0034] Preferably, the low temperature condition in step 4 is 2-8°C.

[0035] Preferably, the storage time in step 4 is 4 to 48 hours.

[0036] Preferably, the washing and filtration in step 5 are performed 2 to 6 times, the drying temperature is 40-80° C., and the drying time is 2-48 hours.

[0037] (III) Beneficial effects

[0038] The present invention provides a method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves. It has the following beneficial effects:

[0039] 1. The present invention provides a method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves, which comprises pretreatment of Aquilaria sinensis leaves raw materials, extraction, concentration, extraction, concentration, low-temperature storage, pure water washing, and suction filtration to finally obtain high-purity mangiferin. The separation and purification method is simpler, the process is green and environmentally friendly, the cost is low, the product quality is good, and it is suitable for industrial and large-scale production.

[0040] 2. The present invention provides a method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves, which uses Aquilaria sinensis leaves as raw materials, is rich in mangiferin, and the types and quantities of other impurity components are relatively small. The raw materials do not contain allergic components, and no pesticides are used in the production process of the raw materials. The prepared product is high-quality and high-purity mangiferin without allergens and organic pesticide residues, and can be widely used in the medicine, health care products, and cosmetics industries.

[0041] 3. The present invention provides a method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves. The entire process uses pure water as solvent, which is pollution-free to the environment. The prepared product has no harmful organic residues and is safer. The solvent separation technology is used, and the process steps are simple, stable, and feasible, with a short production cycle, less manpower consumption, and lower cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the detection spectrum of the present invention;

[0043] Figure 2 This is the nuclear magnetic spectrum 1 of the present invention;

[0044] Figure 3 This is the second nuclear magnetic spectrum of the present invention. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The specific embodiment of the present invention provides a method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves, comprising the following process:

[0048] Step 1. Pretreatment of bay leaves

[0049] Put the white agarwood leaves into a drying oven for drying, and then crush or pulverize them for standby use, the drying method is drying or sun drying, the drying temperature is 30-80°C, and the leaves are crushed into 10-100 meshes;

[0050] Step 2. Extraction and concentration

[0051] The pretreated Aquilaria sinensis leaf fragments are decocted with a solvent for multiple times, filtered to obtain an Aquilaria sinensis alcohol solution, the filtrate is combined, and the solvent is concentrated to recover the Aquilaria sinensis leaf concentrate, the extraction method is reflux extraction, the extraction solvent used is 0% to 80% methanol-water solution, the extraction material-liquid ratio is 1:5 to 1:15, the extraction is performed 2 to 4 times, each time for 10 to 120 minutes, and the extraction temperature is 40 to 80°C;

[0052] Step 3. Extraction and concentration

[0053] The above concentrate is dissolved in pure water to obtain a concentrated solution of Aquilaria sinensis leaves, which is then diluted with water to obtain a solution of Aquilaria sinensis leaves, which is repeatedly extracted with petroleum ether, the solvent is recovered, the aqueous phase is collected, and the aqueous phase is repeatedly extracted with n-butanol, the aqueous phase is discarded, and the solvent is recovered from the organic layer to obtain a crude product of mangiferin, wherein the petroleum ether used for extraction is a petroleum ether with a boiling point of 60 to 90° C. and a volume fraction of 100%, and the amount used is 0.5 to 5 times that of the concentrated solution, and the n-butanol used for extraction is a n-butanol with a volume fraction of 100%, and the amount used is 0.5 to 5 times the volume of the aqueous phase after the petroleum ether extraction, and the number of extractions of petroleum ether and n-butanol is 2 to 5 times;

[0054] Step 4. Low temperature storage

[0055] Dissolve the crude mangiferin in pure water and store it at low temperature until mangiferin crystals precipitate. The pure water is purified water, the amount used is 5 to 20 times that of the crude mangiferin, the low temperature condition is 2 to 8°C, and the storage time is 4 to 48 hours;

[0056] Step 5. Wash and filter

[0057] The mangiferin crystals obtained above are repeatedly washed with pure water, filtered, dried and crushed to obtain mangiferin monomer product. The washing and filtering times are 2 to 6 times, the drying temperature is 40-80° C., and the drying time is 2 to 48 hours.

[0058] Embodiment 1:

[0059] The method for separating and purifying mangiferin monomers from white atractylodes leaves comprises the following steps:

[0060] 1) Weigh 100 g of fresh Aquilaria sinensis leaves, dry them at 50°C, and crush them into 50 mesh;

[0061] 2) adding 1000 mL of 20% methanol aqueous solution at a material-liquid ratio of 1:10, heating to 50° C. and extracting for 30 min, filtering, extracting the residue once more by the same method, combining the two filtrates, concentrating and recovering the solvent, and obtaining a concentrate of Aquilaria sinensis leaves;

[0062] 3) The filtrate was concentrated under reduced pressure at 60° C. to 1000 mL, and after cooling to 25° C., it was extracted with petroleum ether (60-90) for 3 times, 500 mL each time, and the petroleum ether portion was discarded. The aqueous phase portion was extracted with n-butanol for 3 times, 500 mL each time, and the n-butanol portions were combined and concentrated under reduced pressure at 60° C. until no solvent flowed out, and the organic layer recovered the solvent to obtain a crude product of mangiferin;

[0063] 4) Add 100 mL of pure water to the residue, heat to 60° C. and stir for 30 min, cool to 4° C. for crystallization for 12 hours, filter, wash the solid with 4° C. cold water 5 times, 20 mL each time, filter to dryness to obtain mangiferin, dry in vacuo at 50° C., and grind to obtain 1.8 g of mangiferin product. The purity detected by HPLC was 96.3%.

[0064] Embodiment 2:

[0065] 1) Weigh 1 kg of fresh Aquilaria sinensis leaves, dry them at 70°C, and crush them into 100 mesh;

[0066] 2) adding 8 L of 50% methanol aqueous solution at a material-liquid ratio of 1:8, heating to 70° C. and extracting for 40 min, filtering, extracting the residue twice again by the same method, combining the filtrates from the three times, concentrating and recovering the solvent, and obtaining a concentrate of Aquilaria sinensis leaves;

[0067] 3) The filtrate was concentrated under reduced pressure at 50°C to 5 L, and after cooling to 20°C, it was extracted with petroleum ether (60-90) for 3 times, 5 L each time, and the petroleum ether portion was discarded. The aqueous phase portion was extracted with n-butanol for 5 times, 2 L each time, and the n-butanol portions were combined and concentrated under reduced pressure at 60°C until no solvent flowed out. The organic layer recovered the solvent to obtain a crude product of mangiferin;

[0068] 4) Add 1L of pure water to the residue, heat to 70°C and stir for 60min, cool to 2°C for crystallization for 24h, filter with suction, wash the solid with 2°C cold water 3 times, 200mL each time, filter with suction to dry to obtain mangiferin, dry with air at 70°C, and grind to obtain 18.7g of mangiferin product, with a purity of 94.6% as determined by HPLC.

[0069] The invention discloses a method for separating and purifying mangiferin monomers from aquilaria sinensis leaves, comprising raw material pretreatment, extraction, concentration, extraction, concentration, low-temperature storage, pure water washing and suction filtration of aquilaria sinensis leaves, and finally obtaining high-purity mangiferin. The separation and purification method is simpler, the process is green and environmentally friendly, the cost is low, the product quality is good, and the product is suitable for industrial and large-scale production.

[0070] Isolation and identification of mangiferin:

[0071] The specific steps include:

[0072] S1. Bay leaf extract

[0073] 1) Put 2.5 kg of Aquilaria sinensis leaves into a drying oven and dry them, then crush or grind them;

[0074] 2) Put the crushed medicinal materials into a non-woven bag and put it into a decoction machine, add 70% methanol to soak the medicinal materials, set the temperature to 75°, decoct for 30 minutes, and pour out the filtrate;

[0075] 3) After this operation is performed three times, the three filtrates are combined and concentrated to recover the solvent;

[0076] S2. Extraction

[0077] 1) Take 7 L of Aquilaria sinensis leaf concentrate and extract it at a volume ratio of 1:1, petroleum ether (7 L): concentrate (7 L). This operation is repeated 3 times, and the organic phases are combined to recover the solvent.

[0078] 2) The aqueous phase after petroleum ether extraction was further extracted with n-butanol (7 L): aqueous phase solvent (7 L) at a volume ratio of 1:1. This operation was repeated three times. The organic phases were combined, the solvent was recovered, and the aqueous phase was collected and stored.

[0079] 3) Since the organic reagent is recycled, the water content is relatively high. After the extraction, the aqueous phase is 14L. After the organic layer solvent is concentrated, it is dissolved with an appropriate amount of pure water and taken out for later use;

[0080] S3. Separation and purification

[0081] 1) Prepare the resin

[0082] Weigh 50 g of D101 resin, wash with 3 L of methanol until it turns milky white and yellowish brown, and replace the methanol with 3 L of pure water;

[0083] Use 3L of 3% dilute hydrochloric acid to activate the resin, and then replace it with pure water;

[0084] Then use 3L of 3% sodium hydroxide to activate the resin and replace it with pure water;

[0085] Finally, the residual organic phase was washed with pure methanol and replaced with pure water;

[0086] 2) Sample loading

[0087] Wet loading was used, 1.4 L of sample solvent was drained into the column with a glass rod, and 1.5 L of pure water was used to elute until the fraction was slightly colored;

[0088] 3) Elution with different concentrations of eluent

[0089] ①, 20% methanol elution: elution fractions 1-17, elution solvent 5L, among which fraction 6 becomes darker in color, fractions 8 and 9 are the darkest in color, and fractions 9 and 17 are analyzed and tested;

[0090] ②, 50% methanol elution: elution fractions 18-38, elution solvent 9.5 L, of which fractions 23-28 are brown-red, fractions 19-22 and 29-38 are brown-yellow, and fractions 22 and 28 are analyzed and tested;

[0091] ③, 80% methanol elution: elution fractions 39-54, elution solvent 9L, fraction 44 for analysis and detection;

[0092] ④, pure methanol elution: elution fractions 55-68, elution solvent 9L, other fractions 59 and 60 are darker in color, fraction 60 is analyzed and tested;

[0093] S4. Precipitation sample treatment method

[0094] The solvent storage temperature after extraction was -5° and the storage time was 72h. After taking the sample out of the refrigerator, precipitation was found, which was judged as precipitation;

[0095] Method 1: Heating ultrasound (45°), time 10 min, the precipitate dissolution effect is poor;

[0096] Method 2: Wash the precipitate with pure water and filter, repeat the operation 3 times, and take samples of the filter cake for analysis and the purity is >95%.

[0097] Conclusion: Solvents of different concentrations can elute the mangiferin component.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for separating and purifying mangiferin monomers from Aquilaria sinensis leaves, characterized in that: The process includes: Step 1. Pretreatment of bay leaves Put the white atractylodes leaves into a drying oven for drying, and then crush or grind them for later use; Step 2. Extraction and concentration The pretreated Aquilaria sinensis leaf fragments are decocted with a solvent for multiple times, filtered to obtain an Aquilaria sinensis alcohol solution, combined with the filtrate, concentrated to recover the solvent, and obtained an Aquilaria sinensis leaf concentrate; Step 3. Extraction and concentration The above concentrate was dissolved in pure water to obtain a concentrated solution of Aquilaria sinensis leaves, which was then diluted with water to obtain a solution of Aquilaria sinensis leaves, which was repeatedly extracted with petroleum ether, the solvent was recovered, the aqueous phase was collected, and the aqueous phase was repeatedly extracted with n-butanol, the aqueous phase was discarded, and the solvent was recovered from the organic layer to obtain a crude product of mangiferin; Step 4. Low temperature storage The crude mangiferin product is dissolved in pure water and stored at low temperature, and mangiferin crystals are precipitated; Step 5. Wash and filter The mangiferin crystals obtained above are repeatedly washed with pure water, filtered, dried and crushed to obtain the mangiferin monomer product.

2. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: In the step 1, the Aquilaria sinensis leaves are dried by oven drying or sun drying at a drying temperature of 30 to 80°C.

3. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: In the step 1, the Aquilaria sinensis leaves can be crushed to 10 meshes to 100 meshes after being dried.

4. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: The extraction method in step 2 is reflux extraction, the extraction solvent used is 0% to 80% methanol-water solution, the extraction material-liquid ratio is 1:5 to 1:15, the extraction is performed 2 to 4 times, each time for 10 to 120 minutes, and the extraction temperature is 40 to 80°C.

5. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: The petroleum ether used for extraction in step 3 is 100% petroleum ether with a boiling point of 60-90° C., and the amount used is 0.5-5 times the volume of the concentrated solution. The n-butanol used is 100% n-butanol with a volume fraction of 0.5-5 times the volume of the water phase after petroleum ether extraction.

6. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: In the step 3, the extraction times of petroleum ether and n-butanol are 2 to 5 times.

7. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: The water in step 4 is purified water, and the amount used is 5 to 20 times that of the crude mangiferin.

8. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: The low temperature condition in step 4 is 2-8°C.

9. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: The storage time in step 4 is 4 to 48 hours.

10. The method for separating and purifying mangiferin monomer from Aquilaria sinensis leaves according to claim 1, characterized in that: The washing and filtration times in step 5 are 2 to 6 times, the drying temperature is 40-80° C., and the drying time is 2-48 hours.

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