CN106420871B - Preparation method of total notoginsenoside rich in Fe and Fd and monomers Fe and Fd thereof - Google Patents

Abstract

The invention provides a method for preparing total notoginsenoside and monomer Fe and Fd thereof which are rich in Fe and Fd. The preparation method comprises the following steps: (1) adding a pseudo-ginseng medicinal material into warm water, soaking for 10-200 min, and then adding an ethanol water solution or water for extraction to obtain an extracting solution, wherein the pseudo-ginseng medicinal material comprises pseudo-ginseng leaves and/or pseudo-ginseng flowers; (2) loading the extractive solution onto macroporous adsorbent resin column, and eluting with ethanol water solution to obtain Notoginseng radix total saponin rich in rare notoginsenoside Fe and Fd. The preparation method can greatly improve the content of the rare notoginsenoside Fe and Fd in the total saponin, and can obtain notoginsenoside Fe and Fd monomers by a simple separation and purification method.

Description

Fe, Fd-rich Panax notoginseng saponins and preparation method of their monomers Fe and Fd

technical field

The invention belongs to the technical field of natural medicine preparation, in particular to a preparation method of Fe, Fd-rich Panax notoginseng saponins and its monomers Fe and Fd.

Background technique

Panax notoginseng (Panax notoginseng (Burk.) FHChen) is the dry root of Panax notoginseng (Burk.) FHChen. Studies have found that Panax notoginseng leaves have the effects of calming the nerves, lowering blood pressure, analgesia, and anti-inflammatory. However, its utilization rate is only 5%, and most of the available resources are discarded. The leaves and flowers of Panax notoginseng contain a large amount of dammarane-type protopanaxadiol-type ginsenosides, with ginsenosides Rb ₃ , Rb ₂ and Rc as the main components, while the content of notoginsenosides Fe and Fd is extremely low, so its There are few reports of pharmacological effects. In order to further develop Panax notoginseng leaves/flowers and rare ginsenosides, how to obtain Panax notoginseng saponins rich in Fe and Fd of Panax notoginseng saponins, and how to quickly prepare a large number of chemical monomers of Panax notoginseng saponins Fe and Fd is an urgent problem to be solved.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a preparation method of Fe, Fd-rich Panax notoginseng saponins and monomers Fe and Fd. The preparation method of the invention can greatly increase the content of rare Panax notoginseng saponins Fe and Fd in the total saponins, and simultaneously can obtain high content of Panax notoginseng saponins Fe and Fd monomers through a simple separation and purification method.

In order to achieve the above object, the present invention provides a kind of preparation method of Fe, Fd-rich Panax notoginseng saponins and its monomers Fe and Fd, comprising the steps:

(1) soaking the medicinal materials of Panax notoginseng in warm water for 10-200 min, and then adding an aqueous ethanol solution or water for extraction to obtain an extract, wherein the medicinal materials of Panax notoginseng include Panax notoginseng leaves and/or Panax notoginseng flowers;

(2) Putting the extract on a macroporous adsorption resin column and eluting it with an aqueous ethanol solution to obtain total notoginseng saponins rich in rare notoginseng saponins Fe and Fd.

Wherein, in step (1), the Panax notoginseng medicinal material is preferably a powdered Panax notoginseng medicinal material, that is, a Panax notoginseng medicinal material powder, and more preferably a Panax notoginseng medicinal material powder with a specification of 20-60 meshes.

The temperature of the warm water is preferably 10 to 80°C, more preferably 40 to 70°C.

The soaking time is preferably 30-90 min.

When soaking, the mass-volume ratio of the notoginseng medicinal materials and warm water is preferably 1:1 to 1:10. Here, the mass-volume ratio is the conventional meaning in the art, and its unit can be kg/L or g/L. For example, the mass-to-volume ratio of 1kg of Panax notoginseng medicinal materials soaked in 1L of warm water is 1:1.

In step (1), the concentration of the aqueous ethanol solution is conventional in the art, and its concentration can be any value from 0 to 100% (v/v), preferably 70 to 90%. The mass-volume ratio is preferably 1:1～1:100, more preferably 1:20～1:25, here, the mass-volume ratio is the conventional meaning in the art, and its unit can be kg/L or g/ mL, for example, the mass-volume ratio of 1kg of Panax notoginseng medicinal materials extracted with 1L of ethanol aqueous solution is 1:1.

In step (1), the extraction is a conventional operation in the field, preferably any one of percolation extraction, thermal reflux extraction and ultrasonic extraction; the extraction time is preferably 1-6 hours, more preferably 90-150 minutes.

In step (2), before applying the extract to the macroporous adsorption resin column, it is also preferable to concentrate the extract under reduced pressure, and then apply the concentrated extract to the macroporous adsorption resin column; if in step (1) If an ethanol aqueous solution is added to the extract, the extract is preferably concentrated under reduced pressure until there is no alcohol smell, and then the concentrated extract is placed on a macroporous adsorption resin column.

In step (2), the macroporous adsorption resin is preferably any one of X-5, AB-8, DM-301, NK-2, NKA-2, NK-9, D-101 and WLD.

In step (2), the eluting with ethanol aqueous solution can be conventional in the art, preferably, firstly, washing with 0-20% (v/v) ethanol aqueous solution, discarding the washing solution, and then washing with 40-100% (v/v) ethanol solution. v/v) Elution with an aqueous ethanol solution, and the eluate was collected.

In step (2), preferably, before washing with ethanol aqueous solution, wash with water until the effluent is colorless.

Preferably, in order to obtain high-content rare Panax notoginseng saponins Fe and Fd monomers, the preparation method of the Fe, Fd-rich Panax notoginseng saponins and monomers Fe and Fd thereof also comprises the following steps:

(3) adsorbing the total notoginseng saponins rich in rare notoginseng saponins Fe and Fd obtained in step (2) on a silica gel column, eluting with chloroform-methanol and ethyl acetate-methanol respectively, collecting and combining three The fractions of heptasaponins Fe and Fd are recovered under reduced pressure to dry the solvent to obtain the monomers of notoginsenosides Fe and Fd.

Wherein, in the chloroform-methanol, the volume ratio of chloroform to methanol is preferably 20:1 to 1:1; in the ethyl acetate-methanol, the volume ratio of ethyl acetate to methanol is preferably 10:1 to 1:1: 1.

In the present invention, the above-mentioned preferred conditions can be arbitrarily combined on the basis of common knowledge in the art, to obtain each preferred embodiment of the present invention.

The raw materials or reagents used in the present invention are commercially available unless otherwise specified.

The present invention has achieved the following beneficial effects:

The product obtained by the preparation method of the present invention has a high yield of total saponins, the content of total saponins can reach 84% (vanillin-perchloric acid UV method), mainly contains components such as notoginsenoside Fe and Fd, and notoginsenoside Fe The enrichment degree and content of Panax notoginseng saponins and Fd are relatively high, in which the Fe content of Panax notoginseng saponins can reach 5.1% (HPLC method), and the content of Panax notoginseng saponins Fd can reach 8.8% (HPLC method). The purity of the monomers is all above 90%, so it can be seen that the process of the present invention has high process efficiency, less time-consuming, low cost and high efficiency, and is conducive to industrialized high-quality production.

Description of drawings

Fig. 1 is the HPLC chromatogram of the methanol extract of Panax notoginseng leaves (A) and water decoction (B), wherein 1-3 are ginsenosides Rc, Rb ₂ and Rb ₃ , and 4-5 are notoginsenosides Fe and Fd;

Fig. 2A and Fig. 2B are the result graphs of the effect of water immersion temperature on conversion efficiency;

Figure 3A and Figure 3B are graphs of the effect of water immersion time on conversion efficiency;

Fig. 4 is the concentrated solution (A) without macroporous adsorption resin treatment in the embodiment of the present invention 1, and the HPLC of the prepared Panax notoginseng saponins composition product (B) rich in rare Panax notoginseng saponins Fe and Fd In the figure, 1-3 are ginsenosides Rc, Rb ₂ and Rb ₃ , and 4-5 are notoginsenosides Fe and Fd;

Fig. 5 is the HPLC chromatogram of the monomers of notoginsenoside Fe(A) and notoginsenoside Fd(B) separated in Example 1 of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

In view of the fact that the leaves and flowers of Panax notoginseng contain a large amount of dammarane-type protopanaxadiol-type ginsenosides, with ginsenosides Rc, Rb ₂ and Rb ₃ as the main components, while the content of Panax notoginseng saponins Fe and Fd is extremely low, so There are few reports on its pharmacological effects. In order to strengthen the research on rare Panax notoginseng saponins Fe and Fd, it is necessary to first increase the content of rare Panax notoginseng saponins Fe and Fd, and separate and enrich them. In the course of a large number of experiments and explorations, the applicant was pleasantly surprised to find that if the medicinal materials of Panax notoginseng were soaked in warm water for a certain period of time before alcohol extraction (or without alcohol extraction), it would help. The formation and transformation of Fe and Fd of Panax notoginseng saponins increase the content of Fe and Fd of rare Panax notoginseng saponins. Further, if the medicinal materials of Panax notoginseng are pulverized to a certain mesh size, and the temperature and soaking time of warm water are optimized, the conversion efficiency of Fe and Fd can be further improved. The applicant has finally obtained the experimental conditions of the present invention through a large number of exploration experiments and repeated experimental verifications. The results show that ideal effects can be achieved within the scope of the experimental parameters of the present invention.

The applicant found in the previous research that in the methanol or ethanol extract of Panax notoginseng leaves, the content of notoginsenoside Fe and Fd was extremely low, the content of notoginsenoside Fe and Fd in the water decoction was increased, and the ginsenoside Rc and Rb ₃ content decreased accordingly, and the results are shown in Figure 1. Further research found that in the presence of pure water, ginsenosides Rc and Rb ₃ can be hydrolyzed into notoginsenosides Fe and Fd under the action of a certain enzyme. After further research, the applicant found that the efficiency of this conversion has a greater correlation with the temperature. After repeated experimental verification, it was found that the conversion efficiency at 50°C was the highest, and as the temperature continued to increase, the conversion rate began to slow. Slow down, see Figure 2A and Figure 2B for results. At the same time, the applicant also investigated the soaking time and optimized the soaking time. The results are shown in Figure 3A and Figure 3B.

Example 1

Pulverize 20g of Panax notoginseng leaves into 40 mesh powder, add 80mL of water to soak at 50°C for 0.5 hours, then add 400mL of 70% ethanol for reflux extraction for 60 minutes, collect the extract, filter, and recycle the filter residue, and continue to add 400mL of 70% ethanol. Reflux extraction with ethanol for 60 minutes, combine the extracts, and concentrate under reduced pressure to a relative density of 1.11; put the concentrate on a D-101 type macroporous adsorption resin column, wherein the volume ratio of macroporous adsorption resin to concentrate is 2:1, and the column The ratio of diameter to column height is 1:15, first wash with water until the effluent is colorless, then wash with 15 times the column volume of 20% ethanol, discard the washing liquid, and finally use 60% ethanol to elute all, The eluate was collected, and the solvent was recovered to dryness to obtain 2.5 g of the total saponin composition of Panax notoginseng leaves rich in rare Panax notoginseng saponins Fe and Fd. After the vanillin-perchloric acid UV method, the total saponin purity was 84%. The HPLC method showed that the Fe content of Panax notoginseng saponins was 5.1%, and the content of Panax notoginseng saponins Fd was 8.8%. In the present embodiment, the concentrated solution (A) without macroporous adsorption resin treatment, and the total saponin composition product (B) of Panax notoginseng leaves rich in rare Panax notoginseng saponins Fe and Fd obtained by macroporous adsorption resin treatment The HPLC chromatogram is shown in Figure 4, wherein 1-3 are ginsenosides Rc, Rb ₂ and Rb ₃ , and 4-5 are notoginsenosides Fe and Fd.

The prepared total saponins were adsorbed on 2 times the amount of silica gel, volatilized to dryness, and used for later use; loaded into a column, 10 times the amount of total saponins was used for column chromatography on silica gel, packed with chloroform wet method, and eluted with eluent repeatedly. , to make the silica gel fully sink and close; for sample loading and elution, slowly add the sample to the chromatography column, use chloroform-methanol (volume ratio 5:1) for isocratic elution, and collect in sections. 4 times the column volume, the fractions containing Notoginsenoside Fe and Fd were combined after detection by thin layer chromatography, the solvent was recovered under reduced pressure to dryness, and then silica gel column chromatography was used, and ethyl acetate-methanol (volume ratio 5:1) was used. ) isocratic elution, segmented collection, and the collection amount of each fraction is 3 times of column volumes, after TLC detection, merge notoginsenoside Fe and Fd fractions respectively, and the solvent is recovered under reduced pressure to dryness to obtain notoginsenoside Fe54 .6mg and Fd 56.7mg. The purity detected by HPLC was 90.6% and 90.8% respectively, and the HPLC chromatograms of the separated notoginsenoside Fe(A) and notoginsenoside Fd(B) monomers were shown in FIG. 5 .

Example 2

Pulverize 10 g of Panax notoginseng flowers into 50 mesh powder, add 100 mL of water to soak at 40°C for 1.5 hours, then add 150 mL of 90% ethanol for ultrasonic extraction for 60 minutes, collect the extract, filter, and concentrate under reduced pressure to a relative density of 1.15; On the X-5 type macroporous adsorption resin column, the volume ratio of macroporous adsorption resin and concentrated solution is 1.5:1, and the ratio of column diameter to column height is 1:10. First wash with water until the effluent is colorless, and then use Rinse with 10 times the column volume of 20% ethanol, discard the rinse solution, and finally elute with 70% ethanol by volume. Seven flower total saponins 2.02g, the total saponin purity was 64.7% by vanillin-perchloric acid UV method. The HPLC method showed that the Fe content of Panax notoginseng saponins was 4.23%, and the content of Panax notoginseng saponins Fd was 7.16%.

The prepared total saponins were adsorbed on 3 times the amount of silica gel, volatilized to dryness, and used for later use; loaded into a column, 5 times the amount of total saponins on column chromatography silica gel was packed with chloroform wet method, and eluted with eluent repeatedly. , to make the silica gel fully sink and close; for sample loading and elution, slowly add the sample to the chromatography column, elute with chloroform-methanol (volume ratio 20:1) isocratic, and collect in sections. 4 times the column volume, merge the fractions containing notoginsenoside Fe and Fd after detection by thin layer chromatography, recover the solvent under reduced pressure to dryness, then use silica gel column chromatography, use ethyl acetate-methanol (volume ratio 3:1) ) isocratic elution, sectional collection, the collection amount of every stream is 3 times of column volumes, after TLC detection, merges respectively notoginsenoside Fe and Fd stream, and the solvent is recovered under reduced pressure to dry notoginsenoside Fe 21. 0 mg and Fd 23.4 mg. The HPLC detected purities were 82.1% and 73.5%, respectively.

Example 3

Pulverize 15g of Panax notoginseng leaves into 60 mesh powder, add 90mL of water to infiltrate at 70°C for 0.5 hours, then add 150mL of 80% ethanol for ultrasonic extraction for 30 minutes, collect the extract, filter, recover the filter residue, and concentrate under reduced pressure to a relative density of 1.08; Put the concentrated solution on a DM-301 macroporous adsorption resin column, wherein the volume ratio of the macroporous adsorption resin to the concentrated solution is 1:2, and the ratio of the column diameter to the column height is 1:12, first wash with water until the effluent is colorless , then rinsed with 10 times the column volume of 10% ethanol, discarded the rinse solution, and finally eluted with 80% ethanol by volume, collected the eluent, and recovered the solvent to dryness to obtain Fe rich in rare Panax notoginseng saponins. The total saponins of Panax notoginseng and Fd were 3.10 g, and the purity of total saponins was 41.0% by vanillin-perchloric acid UV method. The HPLC method showed that the Fe content of Panax notoginseng saponins was 4.24%, and the content of Panax notoginseng saponins Fd was 8.24%.

The obtained total saponins were adsorbed on 5 times the amount of silica gel, volatilized to dryness, and used for later use; the column was loaded, and 15 times the amount of total saponins was used for column chromatography on silica gel. , to make the silica gel fully sink and close; for sample loading and elution, slowly add the sample to the chromatography column, elute with chloroform-methanol (volume ratio 15:1) isocratic, and collect in sections. 4 times the column volume, the fractions containing Notoginsenoside Fe and Fd were combined after detection by thin layer chromatography, the solvent was recovered under reduced pressure to dryness, then silica gel column chromatography was used, and ethyl acetate-methanol (volume ratio 10:1) was used. ) isocratic elution, sectional collection, the collection amount of every fraction is 3 times of column volumes, after TLC detection, merge notoginsenoside Fd and Fe fractions respectively, and the solvent is recovered under reduced pressure to dryness to obtain notoginsenoside Fe18 .3mg and Fd 20.1mg. The purity detected by HPLC was 81.3% and 70.6%, respectively.

The above embodiment only enumerates some of the conditions applicable to the technical solution of the present invention, such as the temperature of warm water, soaking time, etc. In fact, within the scope of protection of the appended claims of the present invention, ideal effects can be achieved, that is, compared to The prior art greatly increases the content of Fe and Fd in the total saponins of Panax notoginseng, which will not be listed here.

The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above-mentioned specific embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principle of the present invention, any modifications, equivalent replacements, improvements, etc. made should be included within the protection scope of the present invention.

Claims (14)

1. a preparation method of the total saponins of Panax notoginseng rich in Fe, Fd, is characterized in that, comprises the steps: (1) adding the medicinal materials of Panax notoginseng into warm water for 10 to 200 minutes, and then adding an aqueous ethanol solution or water for extraction to obtain an extract, wherein the medicinal materials of Panax notoginseng are selected from Panax notoginseng leaves and/or Panax notoginseng flowers; the warm water The temperature is 40~50℃; (2) Put the extract on a macroporous adsorption resin column and elute with an aqueous ethanol solution to obtain the total notoginseng saponins rich in rare notoginseng saponins Fe and Fd. 2 . The preparation method according to claim 1 , wherein, in step (1), the medicinal material of Panax notoginseng is powder of medicinal material of Panax notoginseng . 3 . The preparation method according to claim 2 , wherein in step (1), the Panax notoginseng medicinal material is 20-60 mesh Panax notoginseng medicinal material powder. 4 . 4. The preparation method according to claim 1, wherein in step (1), the soaking time is 30-90 min; During soaking, the mass-volume ratio of the Panax notoginseng medicinal materials to warm water is 1:1 to 1:10. 5 . The preparation method according to claim 1 , wherein in step (1), the concentration of the ethanol aqueous solution is 70-90%, and the mass volume ratio of the Panax notoginseng medicinal material to the ethanol aqueous solution is 1. 6 . :1~1:100. 6 . The preparation method according to claim 5 , wherein in step (1), the mass-volume ratio of the medicinal material of Panax notoginseng to the aqueous ethanol solution is 1:20-1:25. 7 . 7 . The preparation method according to claim 1 , wherein in step (1), the extraction is any one of percolation extraction, thermal reflux extraction and ultrasonic extraction; 7 . The extraction time is 1-6h. 8 . The preparation method according to claim 7 , wherein, in step (1), the extraction time is 90-150 min. 9 . 9 . The preparation method according to claim 1 , wherein in step (2), before applying the extract to the macroporous adsorption resin column, the extract is further concentrated under reduced pressure, and then the concentrated Macroporous adsorption resin column on the extract; If an ethanol aqueous solution is added for extraction in step (1), the extract is concentrated under reduced pressure until there is no alcohol smell, and then the concentrated extract is placed on a macroporous adsorption resin column. The preparation method according to claim 1, wherein in step (2), the macroporous adsorption resin is X-5, AB-8, DM-301, NK-2, NKA-2, NK -9, any of D-101 and WLD. 11. The preparation method according to claim 1, characterized in that, in step (2), the washing with an aqueous ethanol solution is to first wash with a 0-20% (v/v) aqueous ethanol solution, and discard the washing solution, Then eluted with 40~100% (v/v) ethanol aqueous solution, and collected the eluate. 12 . The preparation method according to claim 11 , wherein, in step (2), before elution with the ethanol aqueous solution, the effluent is washed with water until the effluent is colorless. 13 . 13. a preparation method of Notoginsenoside Fe and Fd monomer, is characterized in that, also comprises the steps: (1) adding the medicinal materials of Panax notoginseng into warm water for 10 to 200 minutes, and then adding an aqueous ethanol solution or water for extraction to obtain an extract, wherein the medicinal materials of Panax notoginseng are selected from Panax notoginseng leaves and/or Panax notoginseng flowers; the warm water The temperature is 40~50℃; (2) Putting the extract on a macroporous adsorption resin column and eluting it with an aqueous ethanol solution to obtain Panax notoginseng saponins rich in rare Panax notoginseng saponins Fe and Fd; (3) Adsorbing the total notoginseng saponins rich in rare notoginseng saponins Fe and Fd obtained in step (2) on a silica gel column, eluting with chloroform-methanol and ethyl acetate-methanol respectively, collecting and combining three The fractions of heptasaponins Fe and Fd are recovered under reduced pressure to dry the solvent to obtain the monomers of notoginsenosides Fe and Fd. 14. The preparation method according to claim 13, wherein in step (3), in the chloroform-methanol, the volume ratio of chloroform to methanol is 20:1 to 1:1; In the ethyl acetate-methanol, the volume ratio of ethyl acetate to methanol is 10:1 to 1:1.

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