Preparation method of paeoniflorin

Abstract

The invention discloses a preparation method of paeoniflorin, and belongs to the technical field of natural pharmaceutical chemistry. The method comprises the following specific steps: firstly, extracting crude extracts from white peony root bark and doghead tablets, and then carrying out extraction, alcohol precipitation and silica gel column chromatography to obtain high-content paeoniflorin. The optimal extraction method is obtained by changing the proportion of the solvent for extraction, and it is found that the yield of paeoniflorin is up to 1.72% and the content of paeoniflorin is up to 95.62% when the white peony root bark and the doghead tablets are extracted with the solvent with the alcohol-water volume ratio of 7: 3. The method is simple in preparation step, mild in condition, high in extraction and separation efficiency and small in loss, the content of the obtained paeoniflorin is high, the radix paeoniae alba resource is fully utilized, and comprehensive utilization of the resource is achieved.

Description

Technical field:

[0001] The invention belongs to the technical field of natural medicinal chemistry, and particularly relates to a method for preparing paeoniflorin from white peony bark and dog head slices. Background technology:

[0002] Radix Paeoniae Alba (Radix Paeoniae Alba) from Bozhou is one of Anhui's "Four Greatest" precious and authentic medicinal herbs, with high medicinal value. White peony root, the dried root of the Ranunculaceae plant, is slightly cold in nature and bitter and sour in flavor. It has the properties of calming the liver and relieving pain, nourishing blood and regulating menstruation, and astringing yin and stopping sweating. It is used for headaches, dizziness, rib pain, abdominal pain, and menstrual irregularities. It is one of the main ingredients in the classic Song Dynasty formula Siwu Decoction (prepared rehmannia root, angelica root, white peony root, and Chuanxiong).

[0003] The active ingredient in white peony is total glucosides of white peony, which includes paeoniflorin, paeoniflorin, hydroxypeoniflorin, peony phenol, benzoylpeoniflorin and other active ingredients. Currently, the main chemical indicator for evaluating the quality of white peony is the content of paeoniflorin, and its molecular formula is C 23 H 28 O 11 , with a molecular weight of 480. Paeoniflorin is an off-white powder with a melting point of 196°C. It has good water solubility in methanol, ethanol, and other solvents. It is stable at pH 2-6 and unstable under alkaline conditions. Paeoniflorin is a pinane monoterpene glycoside compound, and its chemical structure is shown below.

[0004]

[0005] Peony is a perennial herbaceous plant of the genus Paeonia in the family Paeoniaceae, consisting of four parts: roots, stems, leaves, and flowers. The roots are thick and dark brown in color; the stems are hairless, and the leaflets are narrowly ovate, elliptical, or lanceolate; the flowers are borne at the stem top and in the leaf axils. The roots of peony are used medicinally and are the source of the important Chinese medicinal herb "white peony." According to the 2020 edition of the Chinese Pharmacopoeia, the dried roots of peony are dug up in summer and autumn, washed, and the head, tail, and fine roots are removed. They are then boiled in boiling water and the outer skin removed or peeled and boiled again. After drying, the peeled white peony is used as a raw material. Chinese patent CN115677796A reports a method for extracting paeoniflorin from white peony root. This patent involves immersing white peony root powder in an alcohol-water solvent for ultrasonic extraction, followed by extraction with ether at a temperature of 10-20°C. The paeoniflorin extract is adsorbed and eluted using a macroporous resin with a mobile phase of 20%-40% ethanol. The eluate is concentrated to obtain paeoniflorin, effectively removing the benzoic acid content and improving the purity of paeoniflorin to 93%. Chinese patent CN109021043A reports a method for extracting paeoniflorin. This patent involves dissolving white peony root material in 8-10 times its volume of 60-80°C hot water, dynamically stirring at a speed of 60-70 r / min for 100-120 minutes, and extracting 2-3 times. The extract is then applied to a macroporous adsorption resin column, eluted first with water and then with 70-80% ethanol, and the ethanol eluate is collected. The eluate is decolorized with activated carbon and filtered, and the filtrate is then concentrated to an alcohol-free state, followed by addition of ethyl acetate for crystallization. The purity of the paeoniflorin is improved after crystallization. Chinese patent CN108707172A reports a method for preparing high-purity paeoniflorin. This patent involves extracting sliced ​​white peony root using 6-8 times the volume of water as a solvent in a flash extractor. The supernatant is passed through a macroporous resin column. After dynamic adsorption saturation, it is gradiently eluted with an ethanol-water mixed solvent at 0-20% and 50%-60% ethanol. The eluate from the 50%-60% ethanol elution is separated and purified using semi-preparative high-performance liquid chromatography. The above three patents can all produce paeoniflorin with high purity, but in the process of preparing paeoniflorin, other parts of the white peony root, such as white peony bark and dog head slices, are wasted. The content of paeoniflorin in white peony bark and dog head slices is relatively high, and paeoniflorin can be extracted from these parts as the active ingredient of a single prescription medicine, realizing the comprehensive utilization of resources and improving the economic value of crops.

[0006] The 2020 edition of the Chinese Pharmacopoeia specifies that the specific medicinal part of white peony root is the root, "removing the head, tail, and rootlets, and boiling in boiling water before removing the outer bark or peeling and then boiling." These removed parts, such as the head, tail, rootlets, and outer bark, actually contain the active ingredient paeoniflorin, and at a considerable concentration. However, local farmers often discard these parts as waste when processing white peony, which is a pity, especially the bark, which accounts for approximately 5% of the total root mass. Although the paeoniflorin content in white peony bark is lower than that in the peeled root, it is still considered waste, and making full use of this waste can reduce resource waste. White peony root strips vary in thickness and shape. Thicker root strips or root tips are more likely to curl at the edges when sliced, forming a "dog's head" shape. These strips account for approximately 10% of the root mass. Goutou slices, a type of white peony slice, are named for their irregular shape, wrinkled and curled edges, and overall "dog's head" resemblance. The ingredients in goutou slices are essentially the same as those in white peony. Regarding the distribution of white peony root, white peony root bark, dog head slices, etc., see the attached Figure 1 . As a single-ingredient medicine, paeoniflorin can be used in the fields of medicine such as nervous system protection, osteoarthritis, urinary system, and cardiovascular protection. For these medicines, it is equivalent whether the active ingredient paeoniflorin is extracted from the peeled root or extracted from white peony bark or dog head slices, and there is no difference. The present application utilizes white peony bark and dog head slices to prepare paeoniflorin, which can greatly reduce the loss of peony resources and improve the utilization rate of white peony. Moreover, the separation method is simple and easy to operate, the conditions are mild, the cost is low, and a high content of paeoniflorin can be obtained, which is of great significance to the extraction and application of paeoniflorin, and improves the development of paeoniflorin in the field of pharmaceutical applications. At present, no related technology has been published or announced. Summary of the invention:

[0007] The present invention aims to provide a method for preparing paeoniflorin from white peony bark and dog head slices. White peony bark and dog head slices are used as raw materials for research. The active ingredients in the white peony bark and dog head slices are first extracted, and the crude extracts are separated and purified to obtain a high-content paeoniflorin. The specific technical scheme of the present invention is as follows:

[0008] A method for preparing paeoniflorin, comprising the following specific steps:

[0009] (1) Wash fresh white peony root, remove the dirt on the surface, peel off the skin, dry the skin and the "dog head" part, crush them, and sieve them to obtain raw material powder; weigh the raw material powder, put it into a flask, add mixed solvent A, maintain an appropriate mass volume ratio of the raw material powder to the mass volume of the solvent, extract at a certain temperature, for a certain time, and extract 2-3 times; combine the extracts, then rotary evaporate the extracts, recover the solvent, and the obtained extract is the crude extract.

[0010] (2) Solvent B is added to the above crude extract to fully dissolve and disperse it, and solvent C is added to fully extract 2-3 times until the aqueous phase is clear; the extracted aqueous phase is rotary evaporated to an extract, and precipitated with a certain concentration of ethanol, and then the upper liquid is concentrated to obtain a concentrate, which is weighed.

[0011] (3) The concentrate in step (2) is dispersed in ethanol, and a certain amount of 200-300 mesh silica gel powder is added, and the mixture is rotary evaporated again to obtain a mixed powder of the concentrate and silica gel powder, which is then dry-packed into a column.

[0012] (4) Using mixed solvent D as eluent, after the target substance passes through the column, eluent D is obtained, and eluent D is rotary evaporated to obtain white powder paeoniflorin.

[0013] Furthermore, the mixed solvent A in the above step (1) is a mixed solvent having a volume ratio of ethanol to deionized water of 7:3;

[0014] The raw material powder in the above step (1) is white peony root bark and dog head slices;

[0015] The ratio of the mass of the raw material powder to the mass volume of the solvent in the above step (1) is 1:(12-16) (g:mL);

[0016] In the above step (1), the extraction temperature of the raw material is 70-80° C., and the extraction time is 120-180 min;

[0017] In the above step (2), solvent B is deionized water and solvent C is ethyl acetate;

[0018] The concentration of ethanol used in the alcohol precipitation in the above step (2) is 85-95wt%;

[0019] The amount of silica gel powder added in the above step (3) is 1 to 2 times the mass of the concentrate, and the amount of silica gel used for column loading is 2 to 4 times the mass of the mixed powder;

[0020] The mixed solvent D in the above step (4) is a mixed solvent having a volume ratio of ethyl acetate to methanol of (14-18):1;

[0021] The invention provides a method for preparing paeoniflorin from white peony bark and dog head slices, and adopts a relatively simple extraction and purification method to obtain high-content paeoniflorin. Description of the drawings:

[0022] Figure 1 is a physical picture of the various parts of the white peony root Attachment Figure 2 This is a photo of the prepared paeoniflorin sample.

[0023] Attachment Figure 3 This is the ESI-MS (positive) mass spectrum of the prepared paeoniflorin.

[0024] Attachment Figure 4 For the preparation of paeoniflorin 1 H-NMR spectrum.

[0025] Attachment Figure 5 This is the UV absorption spectrum of the prepared paeoniflorin.

[0026] Among them, the attached Figure 2 It can be seen that the prepared paeoniflorin is a white powder. Figure 3 It can be obtained that ESI-MS (positive) m / z: 481 [M+H] + , the molecular weight of this substance is 480, combined with Figure 4 of 1 The molecular formula was confirmed to be C by H-NMR. 23 H 28 O 11 , which is completely consistent with the molecular weight of paeoniflorin. Figure 5 Paeoniflorin powder is dissolved in ethanol. The UV absorption spectrum of the solution shows an absorption peak at 230 nm. Because the aglycone in the structure of paeoniflorin is linked to a glycosyl group, there is an absorption peak for the glycosyl group at 200 nm. In summary, this method proves that high-purity paeoniflorin can be obtained from white peony bark and dog head slices. Specific implementation method:

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0028] Example 1: Preparation of paeoniflorin using white peony bark as raw material in an alcohol-water volume ratio of 7:3 (preferred solution)

[0029] (1) 50 g of white peony root bark raw material was placed in a 1 L round-bottom flask, followed by addition of 245 mL of anhydrous ethanol and 105 mL of deionized water; heating extraction was performed for 2 h, and the extract was separated; 245 mL of anhydrous ethanol and 105 mL of deionized water were added to the round-bottom flask again, and heating extraction was performed for 1 h, and the extract was separated for the second time; the two extracts were combined, and the solvent was recovered by rotary evaporation at 65°C to obtain 8.00 g of crude extract;

[0030] (2) Solvent B was added to the crude extract to fully dissolve and disperse it, and solvent C was added for extraction. The extraction was repeated twice until the aqueous phase was clear. The aqueous phase after extraction was rotary evaporated to an extract, and precipitated with 90 wt% ethanol. The supernatant was then concentrated to obtain a concentrate, which was weighed to obtain 4.10 g of concentrate.

[0031] (3) The concentrate in step (2) was dispersed in ethanol, and 8.00 g of 200-300 mesh silica gel powder was added, and the mixture was rotary evaporated again to obtain a mixed powder of the concentrate and silica gel powder, which was then dry-packed into a column;

[0032] (4) Using mixed solvent D as the eluent, after the target product passes through the column, eluent D is obtained. Eluent D is subjected to rotary evaporation to obtain 0.73 g of white powdered paeoniflorin with a yield of 1.46% and a purity of 95.43%.

[0033] Example 2: Preparation of paeoniflorin using white peony bark as raw material and alcohol-water volume ratio of 6:4

[0034] (1) 50 g of white peony root bark raw material was placed in a 1 L round-bottom flask, followed by addition of 210 mL of anhydrous ethanol and 120 mL of deionized water; heating extraction was performed for 2 h, and the extract was separated; 210 mL of anhydrous ethanol and 120 mL of deionized water were added to the round-bottom flask again, and heating extraction was performed for 1 h, and the extract was separated for the second time; the above two extracts were combined, and the solvent was recovered by rotary evaporation at 65°C to obtain 6.70 g of crude extract;

[0035] (2) Solvent B was added to the crude extract to fully dissolve and disperse it, and solvent C was added for extraction. The extraction was repeated twice until the aqueous phase was clear. The aqueous phase after extraction was rotary evaporated to an extract, and precipitated with 90 wt% ethanol. The supernatant was then concentrated to obtain a concentrate, which was weighed to obtain 3.60 g of concentrate.

[0036] (3) The concentrate in step (2) was dispersed in ethanol, and 7.00 g of 200-300 mesh silica gel powder was added, and the mixture was rotary evaporated again to obtain a mixed powder of the concentrate and silica gel powder, which was then dry-packed into a column;

[0037] (4) Using mixed solvent D as the eluent, after the target product passes through the column, eluent D is obtained. Eluent D is subjected to rotary evaporation to obtain 0.61 g of white powdered paeoniflorin with a yield of 1.22% and a purity of 94.26%.

[0038] Example 3: Preparation of paeoniflorin using white peony bark as raw material and alcohol-water volume ratio of 8:2

[0039] (1) 50 g of white peony root bark raw material was placed in a 1 L round-bottom flask, followed by addition of 280 mL of anhydrous ethanol and 70 mL of deionized water; heating extraction was performed for 2 h, and the extract was separated; 280 mL of anhydrous ethanol and 70 mL of deionized water were added to the round-bottom flask again, and heating extraction was performed for 1 h, and the extract was separated for the second time; the above two extracts were combined, and the solvent was recovered by rotary evaporation at 65°C to obtain 7.80 g of crude extract;

[0040] (2) Solvent B was added to the crude extract to fully dissolve and disperse it, and solvent C was added for extraction. The extraction was repeated twice until the aqueous phase was clear. The aqueous phase after extraction was rotary evaporated to an extract, and precipitated with 90 wt% ethanol. The supernatant was then concentrated to obtain a concentrate, which was weighed to obtain 3.50 g of concentrate.

[0041] (3) The concentrate in step (2) was dispersed in ethanol, and 7.00 g of 200-300 mesh silica gel powder was added, and the mixture was rotary evaporated again to obtain a mixed powder of the concentrate and silica gel powder, which was then dry-packed into a column;

[0042] (4) Using mixed solvent D as the eluent, after the target product passes through the column, eluent D is obtained. Eluent D is subjected to rotary evaporation to obtain 0.68 g of white powdered paeoniflorin with a yield of 1.36% and a purity of 93.71%.

[0043] Example 4: Preparation of paeoniflorin by doubling the raw materials and solvents of Example 1

[0044] (1) 100 g of white peony root bark raw material was placed in a 2 L round-bottom flask, followed by the addition of 490 mL of anhydrous ethanol and 210 mL of deionized water; the extraction was heated for 2 h, and the extract was separated; 490 mL of anhydrous ethanol and 210 mL of deionized water were added to the round-bottom flask again, and the extraction was heated for 1 h, and the extract was separated for the second time; the two extracts were combined, and the solvent was recovered by rotary evaporation at 65 ° C to obtain 17.10 g of crude extract;

[0045] (2) Solvent B was added to the crude extract to fully dissolve and disperse it, and solvent C was added for extraction. The extraction was repeated twice until the aqueous phase was clear. The aqueous phase after extraction was rotary evaporated to an extract, and precipitated with 90 wt% ethanol. The supernatant was then concentrated to obtain a concentrate, which was weighed to obtain 8.52 g of concentrate.

[0046] (3) The concentrate in step (2) was dispersed in ethanol, and 17.00 g of 200-300 mesh silica gel powder was added, and the mixture was rotary evaporated again to obtain a mixed powder of the concentrate and silica gel powder, which was then dry-packed into a column;

[0047] (4) Using mixed solvent D as the eluent, after the target product passes through the column, eluent D is obtained. Eluent D is subjected to rotary evaporation to obtain 1.48 g of white powdered paeoniflorin with a yield of 1.48% and a purity of 95.62%.

[0048] Example 5: Preparation of paeoniflorin using dog head slices as raw material and alcohol-water volume ratio of 7:3

[0049] (1) 50 g of dog head slices raw material was placed in a 1 L round-bottom flask, followed by addition of 245 mL of anhydrous ethanol and 105 mL of deionized water; heating extraction for 2 h, and separation of the extract; 245 mL of anhydrous ethanol and 105 mL of deionized water were added to the round-bottom flask again, heating extraction for 1 h, and separation of the extract for the second time; the above two extracts were combined, and the solvent was recovered by rotary evaporation at 65 ° C to obtain 8.30 g of crude extract;

[0050] (2) Solvent B was added to the crude extract to fully dissolve and disperse it, and solvent C was added for extraction. The extraction was repeated twice until the aqueous phase was clear. The aqueous phase after extraction was rotary evaporated to an extract, and precipitated with 90 wt% ethanol. The supernatant was then concentrated to obtain a concentrate, which was weighed to obtain 4.43 g of concentrate.

[0051] (3) The concentrate in step (2) was dispersed in ethanol, and 9.00 g of 200-300 mesh silica gel powder was added, and the mixture was rotary evaporated again to obtain a mixed powder of the concentrate and silica gel powder, which was then dry-packed into a column;

[0052] (4) Using mixed solvent D as the eluent, after the target product passes through the column, eluent D is obtained. Eluent D is subjected to rotary evaporation to obtain 0.86 g of white powdered paeoniflorin with a yield of 1.72% and a purity of 92.47%.

[0053] Example 6: Preparation of paeoniflorin using peeled white peony root as raw material and alcohol-water volume ratio of 7:3

[0054] (1) 50 g of peeled white peony root was placed in a 1 L round-bottom flask, followed by the addition of 245 mL of anhydrous ethanol and 105 mL of deionized water; the mixture was heated and extracted for 2 h, and the extract was separated; 245 mL of anhydrous ethanol and 105 mL of deionized water were added to the round-bottom flask again, and the mixture was heated and extracted for 1 h, and the extract was separated for the second time; the two extracts were combined, and the solvent was recovered by rotary evaporation at 65 ° C to obtain 10.7 g of crude extract;

[0055] (2) Solvent B was added to the crude extract to fully dissolve and disperse it, and solvent C was added for extraction. The extraction was repeated twice until the aqueous phase was clear. The aqueous phase after extraction was rotary evaporated to an extract, and precipitated with 90 wt% ethanol. The supernatant was then concentrated to obtain a concentrate, which was weighed to obtain 6.10 g of concentrate.

[0056] (3) The concentrate in step (2) was dispersed in ethanol, and 12.00 g of 200-300 mesh silica gel powder was added, and the mixture was rotary evaporated again to obtain a mixed powder of the concentrate and silica gel powder, which was then dry-packed into a column;

[0057] (4) Using mixed solvent D as the eluent, after the target product passes through the column, eluent D is obtained. Eluent D is subjected to rotary evaporation to obtain 1.24 g of white powdered paeoniflorin with a yield of 2.48% and a purity of 94.20%.

[0058] Table 1 lists the yields and contents of paeoniflorin extracted from different raw materials in the above examples.

[0059] Table 1 Paeoniflorin yield and content in Examples 1 to 6

[0060]

[0061] In summary, the present invention obtains paeoniflorin by utilizing white peony bark and dog head slices, extracts crude extracts from white peony bark and dog head slices, and then obtains high-purity paeoniflorin through extraction, alcohol precipitation, and silica gel column chromatography. And using mass spectrometry, nuclear magnetic resonance, ultraviolet, these testing technologies, it is proved that the prepared product is high-purity paeoniflorin. As can be seen from the data in Table 1, the solvent extraction effect with a volume ratio of ethanol to deionized water of 7:3 is better. Among them, Example 4 is an experiment magnified twice by Example 1, and its paeoniflorin yield and content are slightly greater than Example 1. The yield of paeoniflorin in white peony bark reaches 1.48%, and the content reaches 95.62%, indicating that Example 1 has good repeatability. According to the conclusions of Example 5 and Example 6, the yield of paeoniflorin obtained from the same 50g of peeled white peony root is 2.48%, and the yield of paeoniflorin obtained from dog head slices is 1.72%. Although the content of paeoniflorin in white peony root bark and dog head slices is slightly lower than that in peeled white peony root, white peony root bark is a waste, and dog head slices have low medicinal value. The present invention makes full use of these raw materials. Therefore, the present invention uses white peony root bark and dog head slices to prepare paeoniflorin, which greatly improves the utilization rate of white peony root medicinal materials, and the yield is higher than the current literature reported value.

[0062] Therefore, the present invention provides a method for preparing paeoniflorin from white peony bark and dog head slices. The present invention has fewer preparation steps, simple extraction and separation methods, mild conditions, low loss, and high purity of the obtained paeoniflorin, making full use of white peony resources.

[0063] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing paeoniflorin, characterized in that: The following steps are involved: (1) Wash fresh white peony root, remove the dirt on the surface, peel off the skin, dry the skin and the "dog head" part, crush them, and sieve them to obtain a raw material powder; weigh the raw material powder, put it into a flask, add mixed solvent A, maintain an appropriate mass volume ratio of the raw material powder to the solvent, extract at a certain temperature, for a certain time, and extract 2-3 times; combine the extracts, and then rotary evaporate the extracts to recover the solvent. The obtained extract is the crude extract; (2) Solvent B is added to the crude extract to fully dissolve and disperse it, and solvent C is added to fully extract 2-3 times until the aqueous phase is clear; the extracted aqueous phase is rotary evaporated to an extract, and precipitated with a certain concentration of ethanol, and then the supernatant is concentrated to obtain a concentrate, which is weighed; (3) adding ethanol to disperse the concentrate in step (2), adding a certain amount of 200-300 mesh silica gel powder, and re-evaporating to obtain a mixed powder of the concentrate and silica gel powder, and performing dry column loading; (4) Using mixed solvent D as the eluent, after the target product passes through the column, obtain eluent D, which is then rotary evaporated to obtain white powder paeoniflorin; The mixed solvent A in the above step (1) is a mixed solvent with a volume ratio of ethanol to deionized water of 7:3; The raw material powder in the above step (1) is white peony root bark and dog head slices; The ratio of the mass of the white peony root bark powder in the above step (1) to the mass volume of the solvent is 1: (12-16) (g: mL); The extraction temperature of the white peony root bark in the above step (1) is 70-80° C., and the extraction time is 120-180 min; In the above step (2), solvent B is deionized water and solvent C is ethyl acetate; The concentration of ethanol used in the alcohol precipitation in the above step (2) is 85-95 wt / %; The amount of silica gel powder added in the above step (3) is 1 to 2 times the mass of the concentrate, and the amount of silica gel used for column loading is 2 to 4 times the mass of the mixed powder; The mixed solvent D described in the above step (4) is a mixed solvent with a volume ratio of ethyl acetate to methanol (14-18):

1.

2. A method for preparing paeoniflorin according to claim 1, wherein the effect of preparing paeoniflorin according to the method is characterized in that: The yield of paeoniflorin in white peony bark is 1.46%, and its content is 95.43%; the yield of paeoniflorin in dog head slices is 1.72%, and its content is 92.47%.

Images