Separation and purification process of benzophenone compounds in aquilaria sinensis leaves

Abstract

The invention provides a separation and purification process of benzophenone compounds in aquilaria sinensis leaves, and relates to the technical field of processing and extraction of traditional Chinese medicinal materials. The process for separating and purifying the benzophenone compounds in the aquilaria sinensis leaves comprises the following steps: step 1, step 2 and step 3: extracting the aquilaria sinensis leaves; step 4, step 5 and step 6: extraction of aquilaria sinensis leaves; step 7, step 8 and step 9: separating compounds in a water phase part; performing HPLC detection on the target fraction; and identifying the structure of a water-phase part compound. According to the present invention, the separation and purification process comprises aquilaria sinensis leaf raw material pretreatment, extraction, concentration, extraction, water phase part compound separation and structure identification, the benzophenone compound is finally obtained, the separation and purification method is simple, the process is environmentally friendly, the cost is low, the product quality is good, and the method is suitable for industrial and large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine processing and extraction, and specifically to a separation and purification process for benzophenone compounds in Aquilaria sinensis leaves. Background Art

[0002] Aquilaria sinensis, also known as native agarwood, is the resin-containing wood of Aquilaria agallocha and Aquilaria sinensis of the Thymelaeaceae family. It is pungent and bitter in taste, slightly warm in nature, and has the effects of promoting qi circulation to relieve pain, warming the middle-jiao to arrest vomiting, and assisting qi transformation to relieve asthma. It is commonly used for treating chest and abdominal distending pain, stomach cold and vomiting, kidney deficiency and qi reversal, etc. The resin-containing heartwood of the Aquilaria sinensis tree can be used as a raw material for spices and is a specific 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 xylem can be used to extract aromatic oil, and the flowers can be used to prepare extract. It is a plant with relatively high economic benefits.

[0003] With the in-depth research on the active ingredients in Aquilaria sinensis leaves, it is found that the extracts of Aquilaria sinensis leaves have various pharmacological activities such as hypoglycemic, anti-asthmatic, analgesic, anti-inflammatory, antioxidant, anti-tumor and anti-cancer effects. However, since only the resin-containing wood of the Aquilaria sinensis tree can be used medicinally, other parts such as Aquilaria sinensis leaves are all treated as waste, resulting in a large amount of resource waste.

[0004] Although the prior art has reported that Aquilaria sinensis has anti-tumor effects, on the one hand, the chemical components in Aquilaria sinensis leaves are rich, and it is still necessary to further study which effective parts have anti-tumor effects and which effective parts have better anti-tumor effects in the prior art; on the other hand, there are also various types of tumors, and it is still necessary to further study which effective parts are effective against which tumors and which tumors have the best effects.

[0005] It is found through experiments that different extraction solvents, such as methanol and ethanol, and different extraction solvent concentrations, such as 20% methanol, 50% methanol, 80% methanol, and pure methanol, all have different effects on the extraction effect of the components in Aquilaria sinensis leaves.

[0006] Therefore, the technical personnel in this field have provided a separation and purification process for benzophenone compounds in Aquilaria sinensis leaves to solve the problems raised in the above background art. Summary of the Invention

[0007] (1) Technical Problems to be Solved

[0008] In view of the deficiencies of the prior art, the present invention provides a separation and purification process for benzophenone compounds in Aquilaria sinensis leaves, which solves the problems that the existing separation and purification processes of benzophenone compounds in Aquilaria sinensis leaves are all relatively complex, with large operation difficulty and high production cost.

[0009] (2) Technical Solutions

[0010] To achieve the above purposes, the present invention is realized through the following technical solutions:

[0011] A process for the separation and purification of benzophenone compounds from Aquilaria sinensis leaves, comprising the following steps:

[0012] Step 1. Put the Aquilaria sinensis leaves into a drying oven for drying. After drying, crush or pulverize them for later use;

[0013] Step 2. Put the pulverized medicinal materials into a non-woven bag and place it in a decocting machine. Add 15 L of methanol with a concentration of 70% for extraction, and squeeze out the filtrate;

[0014] Step 3. Repeat the operation in Step 2 twice, and combine the two filtrates. Recover the solvent under reduced pressure to obtain 7 L of an extraction concentrate;

[0015] Step 4. Measure 7 L of the Aquilaria sinensis leaf concentrate, extract it twice with petroleum ether at a volume ratio of 1:1, and combine the petroleum ether phases. Recover the solvent under reduced pressure to obtain the petroleum ether extraction component of Aquilaria sinensis leaves;

[0016] Step 5. For the aqueous phase after petroleum ether extraction, extract it twice with n-butanol at a volume ratio of 1:1, combine the n-butanol phases, and recover the solvent under reduced pressure to obtain the n-butanol extraction component of Aquilaria sinensis leaves;

[0017] Step 6. The aqueous phase after extraction is about 8 L, take it out for later use;

[0018] Step 7. Weigh 2 kg of D101 resin, soak it in 6 L of industrial methanol overnight. Take a glass chromatography column, clean it, and replace the methanol with pure water;

[0019] Step 8. Use a glass rod to drain the 8 L sample solution of the aqueous layer after extraction in Step 6 into the column, and the sample loading flow rate is 2 bv / h;

[0020] Step 9. Elute with 20% methanol-aqueous solution, 50% methanol-aqueous solution, 80% methanol-aqueous solution, and pure methanol solution at a flow rate of 2 bv / h respectively with a volume of 4 bv each. Collect the eluates in segments, conduct liquid phase detection, and search for the target fractions.

[0021] Furthermore, the drying method of the Aquilaria sinensis leaves in Step 1 is drying by baking or sunning, and the drying temperature is 30 - 80 °C.

[0022] Furthermore, after drying, the Aquilaria sinensis leaves in Step 1 can be pulverized to 20 - 80 mesh.

[0023] Furthermore, the ratio of extraction material to liquid in Step 2 is 1:5 to 1:15, the extraction temperature is 75 °C, and the extraction time is 30 min.

[0024] Furthermore, the petroleum ether used for extraction in Step 4 is petroleum ether with a boiling point of 60 - 90 °C and a volume fraction of 100%.

[0025] Further, the n-butanol used in Step 5 is n-butanol with a volume fraction of 100%.

[0026] Further, the glass chromatography column in Step 7 is packed with a column size of 8 cm × 50 cm and a volume of approximately 2.5 L.

[0027] Further, the pure water in Step 7 is purified water, and the dosage is 5 to 20 times that of industrial methanol.

[0028] (III) Beneficial Effects

[0029] The present invention provides a separation and purification process for benzophenone compounds in Aquilaria sinensis leaves. It has the following beneficial effects:

[0030] 1. The present invention provides a separation and purification process for benzophenone compounds in Aquilaria sinensis leaves, including pretreatment of Aquilaria sinensis leaf raw materials, extraction, concentration, extraction, separation and structure identification of compounds in the aqueous phase part, and finally obtaining benzophenone compounds. 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 industrialized and large-scale production.

[0031] 2. The present invention provides a separation and purification process for benzophenone compounds in Aquilaria sinensis leaves. The entire purification process uses pure water as a solvent, which has no pollution to the environment. The separated and purified benzophenone compounds have no harmful organic residues, and the safety is higher. Moreover, the solvent extraction separation technology is adopted, and the process steps are simple, stable, and feasible. The production cycle is short, the labor consumption is small, and the cost is lower. Description of the Drawings

[0032] Figure 1 It is the HPLC detection result diagram of the 20% methanol elution section of the present invention;

[0033] Figure 2 It is the HPLC detection result diagram of the 50% methanol elution section of the present invention;

[0034] Figure 3 It is the HPLC detection result diagram of the 80% methanol elution section of the present invention;

[0035] Figure 4 It is the preparation spectrum diagram of the 50% methanol elution sample of the present invention;

[0036] Figure 5 It is the liquid phase detection spectrum diagram of peak1 of the present invention;

[0037] Figure 6 It is the nuclear magnetic spectrum diagram of peak1 of the present invention;

[0038] Figure 7 It is the liquid phase detection spectrum diagram of peak2 of the present invention;

[0039] Figure 8 The peak2 NMR spectrum of the present invention;

[0040] Figure 9 The peak3 liquid phase detection spectrum of the present invention;

[0041] Figure 10 The peak3 NMR spectrum of the present invention;

[0042] Figure 11 The peak4 liquid phase detection spectrum of the present invention;

[0043] Figure 12 The peak4 NMR spectrum of the present invention. Detailed implementation manners

[0044] Next, the technical solutions in the specific implementation manners of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the specific implementation manners of the present invention. Obviously, the described specific implementation manners are only a part of the specific implementation manners of the present invention, rather than all of the specific implementation manners. Based on the specific implementation manners of the present invention, all other specific implementation manners obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0045] Example:

[0046] The embodiment of the present invention provides a separation and purification process for benzophenone compounds in Aquilaria sinensis leaves, including the following steps:

[0047] Step 1. Put the Aquilaria sinensis leaves into a drying oven for drying. The drying method is drying by baking or sunning. The drying temperature is 60 °C. After drying, it is crushed or pulverized for standby, and pulverized to 40 meshes;

[0048] Step 2. Put the pulverized medicinal materials into a non-woven bag and place it in a decocting machine, add 15 L of methanol with a concentration of 70% for extraction, squeeze out the filtrate. The extraction material-liquid ratio is 1:5 to 1:15, the extraction temperature is 75 °C, and the extraction time is 30 min;

[0049] Step 3. Repeat the operation in Step 2 twice, and combine the two filtrates, and recover the solvent under reduced pressure to obtain 7 L of extraction concentrate;

[0050] Step 4. Measure 7 L of the Aquilaria sinensis leaf concentrate, extract it twice with petroleum ether at a volume ratio of 1:1. The petroleum ether used for extraction is petroleum ether with a boiling point of 80 °C and a volume fraction of 100%, and combine the petroleum ether phases, and recover the solvent under reduced pressure to obtain the petroleum ether extraction component of Aquilaria sinensis leaves;

[0051] Step 5. For the aqueous phase after petroleum ether extraction, extract it twice with n-butanol with a volume fraction of 100% at a volume ratio of 1:1, combine the n-butanol phases, and recover the solvent under reduced pressure to obtain the n-butanol extraction component of Aquilaria sinensis leaves;

[0052] Step 6. The aqueous phase after extraction is about 8 L and is taken out for standby;

[0053] Step 7. Weigh 2 kg of D101 resin, soak it in 6 L of industrial methanol overnight, clean a glass chromatography column. The glass chromatography column is 8 cm × 50 cm with a volume of 2.5 L, and replace the methanol with pure water. The pure water is purified water and the dosage is 10 times that of industrial methanol;

[0054] Step 8. Drain the 8 L sample solution of the aqueous layer after extraction in Step 6 into the column with a glass rod, and the sample loading flow rate is 2 bv / h;

[0055] Step 9. Elute with 4 bv of 20% methanol - aqueous solution, 50% methanol - aqueous solution, 80% methanol - aqueous solution, and pure methanol solution at a flow rate of 2 bv / h respectively, collect the eluate in fractions, conduct liquid phase detection, and search for the target fraction.

[0056] HPLC detection of the target fraction:

[0057] Take samples of the liquid phases after elution with 20% methanol - aqueous solution, 50% methanol - aqueous solution, 80% methanol - aqueous solution, and pure methanol solution respectively, and conduct HPLC detection;

[0058] The chromatographic conditions for HPLC detection are shown in Table 1 below:

[0059] Table 1 Chromatographic conditions for HPLC detection

[0060]

[0061]

[0062] The detection results are as Figures 1 - 3 shown. HPLC detection finds that the target compound is mainly concentrated in the 50% methanol elution section of macroporous resin column chromatography.

[0063] The compounds in the 50% methanol elution section of macroporous resin column chromatography are prepared and separated by the following method:

[0064] 1) Concentrate the 50% methanol elution section of macroporous resin column chromatography under reduced pressure to obtain 68.8 g of brown solid;

[0065] 2) Weigh 60 g of the brown solid in 1), add 300 mL of 30% acetonitrile - aqueous solution, dissolve it by ultrasonic treatment, and filter through a 0.45 - um filter membrane to obtain an agarwood leaf sample solution with a concentration of 200 mg / mL;

[0066] 3) Preparation was carried out using a DAC-50 dynamic axial compression preparative chromatography system. Single chromatographic peak fractions were collected according to the elution profile, and the preparation was repeated 10 times. Each preparation fraction was detected and confirmed by analytical HPLC, and then the same fractions were combined, concentrated under reduced pressure, and dried in vacuo to obtain 4 monomeric products of compounds, denoted as peak1, peak2, peak3, and peak4;

[0067] 4) 15 mg of each product was taken separately for high performance liquid chromatography analysis and nuclear magnetic resonance detection to determine the compound structure.

[0068] The conditions for preparative liquid phase purification are shown in Table 2 below:

[0069] Table 2 Conditions for preparative liquid phase purification

[0070]

[0071] The preparative chromatogram is as Figure 4 shown.

[0072] Identification of the compound structure in the aqueous phase part:

[0073] Samples of peak1, peak2, peak3, and peak4 were taken separately for HPLC and NMR analysis to determine their compound structures;

[0074] For the structure identification of peak1, by comparison with the literature, it was determined that peak1 is 2,3-C-glucoside-2,4,6,4'-tetrahydroxybenzophenone. The HPLC detection chromatogram of peak1 is as Figure 5 shown, and the NMR chromatogram is as Figure 6 shown.

[0075] For the structure identification of peak2, by comparison with the literature, it was determined that peak2 is 2-O-α-rhamnoside-4,6,4'-trihydroxybenzophenone. The HPLC detection chromatogram of peak2 is as Figure 7 shown, and the NMR chromatogram is as Figure 8 shown.

[0076] For the structure identification of peak3, by comparison with the literature, it was determined that peak3 is mangiferin. The HPLC detection chromatogram of peak3 is as Figure 9 shown, and the NMR chromatogram is as Figure 10 shown.

[0077] For the structure identification of peak4, by comparison with the literature, it was determined that peak4 is 3,5-di-C-glucoside-2,4,6,4'-tetrahydroxybenzophenone. The HPLC detection chromatogram of peak4 is as Figure 11 shown, and the NMR chromatogram is as Figure 12 shown.

[0078] The separation and purification process of the present invention includes the pretreatment of Aquilaria sinensis leaf raw materials, extraction, concentration, extraction, and the separation and structure identification of compounds in the aqueous phase part, and finally obtains benzophenone compounds. 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 industrialized and large-scale production.

[0079] In the separation and purification process of the present invention, pure water is used as the solvent in all purification process flows, which has no pollution to the environment. The separated and purified benzophenone compounds have no harmful organic residues, and the safety is higher. Moreover, the solvent extraction and separation technology is adopted, the process steps are simple, stable and feasible, the production cycle is short, the labor consumption is less, and the cost is lower.

[0080] Although specific embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these specific embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A separation and purification process for benzophenone compounds in Aquilaria sinensis leaves, characterized in that: The following steps are involved: Step 1. Put the white agarwood leaves into a drying oven for drying, and then crush or grind them for later use; Step 2. Put the crushed medicinal materials into a non-woven bag and put it into a decoction machine, add 15L of 70% methanol for extraction, and squeeze out the filtrate; Step 3. Repeat the operation of step 2 twice, combine the two filtrates, recover the solvent under reduced pressure, and obtain 7L of extract concentrate; Step 4. Take 7 L of the Aquilaria sinensis leaf concentrate, extract it twice with petroleum ether at a volume ratio of 1:1, combine the petroleum ether phases, and recover the solvent under reduced pressure to obtain the Aquilaria sinensis leaf petroleum ether extract component; Step 5. The aqueous phase after the petroleum ether extraction is extracted twice with n-butanol at a volume ratio of 1:1, the n-butanol phases are combined, and the solvent is recovered under reduced pressure to obtain the n-butanol extract component of Aquilaria sinensis leaves; Step 6. After extraction, the aqueous phase is about 8 L, which is taken out for later use; Step 7. Weigh 2 kg of D101 resin, soak it in 6 L of industrial methanol overnight, clean the glass chromatography column, and replace the methanol with pure water; Step 8. Drain 8L of sample solution from the water layer after extraction in step 6 into the column using a glass rod at a flow rate of 2 bv / h; Step 9. Elute with 4 bv of 20% methanol-water solution, 50% methanol-water solution, 80% methanol-water solution and pure methanol solution at a flow rate of 2 bv / h, collect the eluate in sections, perform liquid phase detection, and search for the target fraction.

2. The separation and purification process of benzophenone compounds in 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 separation and purification process of benzophenone compounds in Aquilaria sinensis leaves according to claim 1, characterized in that: In the step 1, the Aquilaria sinensis leaves can be crushed into 20 to 80 meshes after being dried.

4. The separation and purification process of benzophenone compounds in Aquilaria sinensis leaves according to claim 1, characterized in that: In step 2, the solid-liquid ratio is 1:5 to 1:15, the extraction temperature is 75° C., and the extraction time is 30 min.

5. The separation and purification process of benzophenone compounds in Aquilaria sinensis leaves according to claim 1, characterized in that: The petroleum ether used for extraction in step 4 is petroleum ether with a boiling point of 60-90° C. and a volume fraction of 100%.

6. The separation and purification process of benzophenone compounds in Aquilaria sinensis leaves according to claim 1, characterized in that: The n-butanol used in step 5 is n-butanol with a volume fraction of 100%.

7. The separation and purification process of benzophenone compounds in Aquilaria sinensis leaves according to claim 1, characterized in that: The glass chromatography column in step 7 is packed with 8 cm×50 cm and has a volume of about 2.5 L.

8. The separation and purification process of benzophenone compounds in Aquilaria sinensis leaves according to claim 1, characterized in that: The pure water in step 7 is pure water, and the amount used is 5 to 20 times that of industrial methanol.

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