Method for extracting triterpenoids from mangrove forest plant hedyotis diffusa

Abstract

The invention discloses a method for extracting triterpenoids from mangrove forest plant hedyotis diffusa, and relates to the technical field of chemical engineering, the method comprises the following steps: S1, drying and grinding hedyotis diffusa leaves into powder; s2, adding ethanol with the concentration of 80% into the leaf powder, and filtering after ultrasonic-assisted extraction to obtain an extracting solution; and S3, filling a column by using D301 macroporous adsorption resin, adsorbing triterpenoid components in the extracting solution by using a D301 macroporous adsorption resin column, and eluting by using ethanol as an eluent after full adsorption to obtain an eluent, namely the high-concentration triterpenoid purified extracting solution. According to the method disclosed by the invention, the triterpenoid components in the hedyotis diffusa are extracted by ultrasonic assistance, and the ethanol which is low in price and low in toxicity is used as an extraction solvent of the triterpenoid and an eluent of macroporous resin, so that the method is economical and environment-friendly; and an extraction solvent and an eluent are ethanol with the volume fraction of 80%, so that the extraction rate of the triterpenoids in the leaves of the hemsleya amabilis is optimal, and the storage cost of raw materials during industrial batch preparation can be reduced.

Description

Technical Field

[0001] The invention relates to the technical field of chemical engineering, and in particular to a method for extracting triterpenoid components from a mangrove plant, Cibotium obesum. Background Art

[0002] Mangrove plants contain a variety of chemical components due to their special growth environment. Plant triterpenoids are widely present in nature and have multiple physiological activities, and are an important class of chemical components of mangrove plants. Silver leaf tree (Heritiera Littoralis Dryand) belongs to a kind of mangrove plant. At present, the research on the chemical components of silver leaf tree is mostly focused on total flavonoids, tannins, lignin, etc. The extraction, separation and purification process of triterpenoid components of silver leaf tree in the prior art CN1580071A adopts petroleum ether, ethyl acetate and n-butanol for purification and elution, but these eluents are all toxic and easy to pollute the environment. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for extracting triterpenoid components from the mangrove plant Lycopodiella cernua.

[0004] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0005] Provided is a method for extracting triterpenoid components from a mangrove plant, the method comprising the following steps:

[0006] S1: Dry and grind the leaves of the silverleaf tree into powder;

[0007] S2: adding 80% ethanol to the leaf powder, filtering to obtain an extract after ultrasonic-assisted extraction;

[0008] S3: Use D301 macroporous adsorption resin to fill the column, use D301 macroporous adsorption resin column to adsorb the triterpenoid components in the extract, and use ethanol as the eluent to elute and filter after sufficient adsorption. The filtrate obtained is a high-concentration triterpenoid purified extract.

[0009] Furthermore, in step S2, the usage ratio of leaf powder to 80% concentration ethanol is 1 g: 60-70 mL.

[0010] Furthermore, in step S2, the usage ratio of leaf powder to 80% concentration ethanol is 1 g:70 mL.

[0011] Furthermore, the ultrasonic-assisted extraction in step S2 specifically adopts a power of 210W to 250W, and the ultrasonic extraction time is 23 to 27 minutes.

[0012] Furthermore, the ultrasonic-assisted extraction in step S2 specifically uses a power of 240 W and an ultrasonic extraction time of 25 min.

[0013] Furthermore, the specific steps of using ethanol for elution and filtration after sufficient adsorption in step S3 are as follows:

[0014] A1: After sufficient adsorption, use distilled water to elute the resin column at a flow rate of 1 BV / h to remove water-soluble impurities until the effluent is colorless;

[0015] A2: The resin column washed with water in A1 is eluted with 75% to 90% ethanol by volume and 3 to 5 BV of eluent to obtain a purified extract of triterpenes.

[0016] Furthermore, the resin column after washing with A1 was eluted with 80% by volume ethanol and 4 BV of eluent to obtain a purified extract of triterpenes.

[0017] Furthermore, before adding D301 macroporous adsorption resin between the containers, the D301 macroporous adsorption resin needs to be pretreated. The specific steps are as follows:

[0018] B1: Soak the purchased D301 macroporous adsorption resin in anhydrous ethanol until it is fully swollen, and pour out the floating matter and anhydrous ethanol;

[0019] B2: Use deionized water to wash the fully swollen D301 macroporous adsorption resin until no white turbidity appears;

[0020] B3: Wet-fill the column with washed D301 macroporous adsorption resin, and then wash with deionized water until the effluent has no alcohol taste;

[0021] B4: Soak D301 macroporous adsorption resin in 4% hydrochloric acid solution for 4 hours, then wash with water until the effluent is neutral;

[0022] B5: Soak the D301 macroporous adsorption resin in a 4% sodium hydroxide solution for 4 hours, and then wash with water until the effluent is neutral to complete the pretreatment.

[0023] The beneficial effects of the present invention are:

[0024] The invention uses ultrasound to assist in extracting triterpenoid components from silverleaf trees, and uses ethanol, which is inexpensive and less toxic, as a triterpenoid extraction solvent and an eluent for macroporous resins, which is economical and environmentally friendly. The extraction solvent and the eluent are ethanol with a volume fraction of 80%, at which time the extraction rate of triterpenoid components in silverleaf tree leaves is optimal, and the reserve cost of raw materials during industrial batch preparation can also be reduced.

[0025] The entire extraction and purification process is time-saving, has high extraction efficiency, and can be effectively purified. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the standard curve of oleanolic acid;

[0027] Figure 2 It is the fitting curve diagram of the effect of solid-liquid ratio on triterpene extraction rate;

[0028] Figure 3 It is the fitting curve diagram of the effect of ultrasonic power on triterpene extraction rate;

[0029] Figure 4 It is the fitting curve diagram of the effect of ultrasound time on triterpene extraction rate;

[0030] Figure 5 It is the fitting curve diagram of the effect of eluent concentration on triterpene content;

[0031] Figure 6 It is a line graph showing the effect of different macroporous resins on triterpene resolution;

[0032] Figure 7 This is a fitting curve diagram showing the effect of the concentration of the extract on the triterpene extraction rate. DETAILED DESCRIPTION

[0033] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0034] Example 1 Pretreatment of D301 macroporous adsorption resin

[0035] B1: Soak the D301 macroporous adsorption resin purchased from Anhui Sanxing Resin in anhydrous ethanol until it is fully swollen, and pour out the floating matter and anhydrous ethanol;

[0036] B2: Use deionized water to wash the fully swollen D301 macroporous adsorption resin until no white turbidity appears;

[0037] B3: Wet-fill the column with washed D301 macroporous adsorption resin, and then wash with deionized water until the effluent has no alcohol taste;

[0038] B4: Soak D301 macroporous adsorption resin in 4% hydrochloric acid solution for 4 hours, then wash with water until the effluent is neutral;

[0039] B5: Soak the D301 macroporous adsorption resin in 4% sodium hydroxide solution for 4 hours, wash with water until the effluent is neutral, and then complete the pretreatment for use.

[0040] Example 2

[0041] Extracting triterpenoid components from silverleaf tree comprises the following steps:

[0042] S1: Grind the leaves of the silverleaf tree into powder, sieve them through a 40-mesh sieve, and weigh 5 g of the sieved leaf powder;

[0043] S2: Add 80% ethanol to the sieved leaf powder, and use an ultrasonic generator of model KQ-300DE at room temperature with a power set to 210W to 250W for 23min to 27min to complete the initial extraction of triterpenoid components, and filter to obtain an extract and a solid filtrate; the ratio of leaf powder to 80% ethanol is 1g:60~70mL.

[0044] S3: Fill a column with 15 g of D301 macroporous adsorption resin using the method of Example 1, and use the D301 macroporous adsorption resin column to adsorb the triterpenoid components in the extract. After sufficient adsorption, use ethanol as an eluent to elute and filter. The specific steps are as follows:

[0045] A1: After sufficient adsorption, use distilled water to elute the resin column to remove water-soluble impurities until the effluent is colorless;

[0046] A2: The resin column washed with water in A1 is eluted with 75% to 90% ethanol by volume and 4 BV of eluent to obtain a purified triterpenoid extract (hereinafter referred to as purified extract). In specific implementation, the amount of eluent can also be 3 BV or 5 BV.

[0047] The above method was used to set up multiple extraction schemes, and the parameters of each scheme are shown in Table 1;

[0048] Table 1

[0049]

[0050] The triterpene content was determined for the extracts extracted from each group of solutions and the purified solution obtained by purification. The specific steps of the content determination were as follows:

[0051] Standard curve drawing: accurately weigh 5.0 mg of oleanolic acid standard, dissolve it in anhydrous ethanol, then transfer it to a 50 mL volumetric flask and make up the volume, mix it evenly, and prepare a 0.10 mg / mL oleanolic acid standard solution. Accurately prepare 0.1, 0.2, 0.4, 0.8, and 1.6 mL oleanolic acid standard solutions and place them in 10 mL volumetric flasks respectively. Volatilize the solvent in a 90 ° C water bath, quickly shake and add 0.4 mL of freshly prepared 5% vanillin-glacial acetic acid and 1 mL of perchloric acid, put them in a 65 ° C water bath and heat them for 15 minutes. After taking them out, immediately cool them to room temperature, add glacial acetic acid to make up the volume, mix them evenly, and place them in the dark for color development for 15 minutes. Set the wavelength to 546 nm and measure the absorbance. Set the absorbance and the concentration of the standard solution as the ordinate and abscissa of the standard curve for drawing, establish a regression equation, and obtain the standard curve equation: y = 3.8048x + 0.0051, and its regression coefficient R 2 =0.9991.

[0052] Accurately pipette 0.4 mL of the extract and purified solution obtained from each group of schemes into a 10 mL volumetric flask, add 0.4 mL of 5% vanillin-glacial acetic acid and 1 mL of perchloric acid and shake well quickly, heat in a 65°C water bath for 15 min, cool to room temperature, add glacial acetic acid to make up to volume, and shake well. Set the wavelength to 546 nm, measure the absorbance three times in parallel and take the average value. Substitute into the standard curve equation to obtain the triterpene content (mg / mL), and calculate the mass of triterpene in the extract or purified solution; thereby calculate the extraction rate of triterpene in the extract;

[0053] Determine and calculate the purity of triterpenes in the extract before purification and the purity of triterpenes in the purified solution after purification;

[0054] The calculation formula of extraction rate is:

[0055] The formula for calculating purity is:

[0056] When calculating and measuring the purity of the triterpenes in the extract, after the mass of the triterpenes in the extract is measured and calculated, the extract is freeze-dried, and the mass of the solid obtained by freeze-drying is the solid content of the extract. Before the purification process, the freeze-dried solid is re-concentrated to 50 mL and used as the extract to perform step S3 again to obtain a purified solution;

[0057] When calculating and measuring the purity of triterpenes in the purified solution, after the mass of triterpenes in the purified solution is measured and calculated, the purified solution is freeze-dried, and the mass of the solid obtained by freeze-drying is the solid content of the purified solution.

[0058] The purity difference before and after purification by the method of the present invention was calculated based on the purity of the triterpene in the extract and the purity of the triterpene in the purified solution, and the results are shown in Table 2;

[0059] Table 2

[0060]

[0061] According to the data in Table 2, the extraction rates of groups 1, 2, 3, 4 and 5 were obtained as follows: Figure 2 The fitting curve diagram of the effect of the usage ratio of leaf powder to 80% ethanol on the triterpene extraction rate is shown in the figure, wherein the ordinate is the extraction rate and the abscissa is the usage ratio of leaf powder to 80% ethanol (hereinafter referred to as the solid-liquid ratio); Figure 2 It can be seen that when the solid-liquid ratio is 1:60, the extraction rate changes slowly, that is, when the solid-liquid ratio is greater than or equal to 1:60, the effect of 80% ethanol on the extraction rate is small. The extraction rate when the solid-liquid ratio is 1:80 is slightly different from that when it is 1:70. Considering cost saving, the best solution is when the solid-liquid ratio is 1:70, that is, the usage ratio of leaf powder to 80% ethanol is 1g:70mL.

[0062] The extraction rates of groups 4, 6, 7, 8 and 9 were prepared as follows Figure 3 The fitting curve of the effect of ultrasonic power on triterpene extraction rate is shown in FIG. Figure 3 It can be seen that when the ultrasonic power is 240W, the extraction rate is optimal. According to the fitting curve, when the ultrasonic power is less than 210W and greater than 250W, the extraction rate decreases significantly. Therefore, when extracting triterpenes from silver leaves, the ultrasonic power is best between 210W and 250W.

[0063] The extraction rates of groups 4, 10, 11, 12 and 13 were prepared as follows Figure 4 The fitting curve of the effect of ultrasonic time on triterpene extraction rate is shown in FIG. Figure 4 It can be seen that the extraction rate is highest when the ultrasonic time is 25 minutes; according to the fitting curve, the extraction rate decreases significantly when the ultrasonic time is less than 23 minutes and greater than 27 minutes. Therefore, when extracting triterpenes from silver leaves, the best ultrasonic extraction time is 23 minutes to 27 minutes.

[0064] The extraction rates of groups 4, 14, 15, 16 and 17 were prepared as follows Figure 5 The fitting curve of the effect of eluent volume fraction on triterpene purity is shown by Figure 5 It can be seen that when the eluent volume fraction is 80%, the purity of triterpenes in the purified liquid is the highest and the purity difference is the largest; according to the fitting curve, when the eluent volume fraction is less than 75% and greater than 90% min, the purity of triterpenes decreases significantly. Therefore, when extracting triterpenes from silver leaves, the optimal eluent volume fraction is 75% to 90%.

[0065] In summary, when the ultrasonic generator is set to 240W and lasts for 25min; the ratio of leaf powder to 80% ethanol is 1g:70mL, the extraction rate is optimal; after elution with 80% ethanol by volume and 4BV of eluent, the content of triterpenoid components is the highest.

[0066] Example 3

[0067] Other macroporous resins used in the prior art include S-8, D101, AB-8 and ADS-7 for the extraction and purification of triterpenes. Except for the different types of macroporous resins, the other experimental parameters remain the same, namely: ultrasonic power of 240 W, ultrasonic time of 25 min; the ratio of leaf powder to 80% ethanol concentration of 1 g: 70 mL; 80% ethanol by volume and 4 BV of eluent are used for elution.

[0068] The purified solution was tested and the results were as follows: Figure 6 As shown by Figure 6 It can be seen that the D301 macroporous resin has the best resolution rate for the triterpenoid components in the leaves of the silver leaf tree using the extraction and purification method provided by the present invention, and its adsorption rate is second only to the S-8 macroporous resin; therefore, after using ethanol to extract the triterpenoids from the leaves of the silver leaf tree, when using ethanol as an eluent to purify the triterpenoid components, the D301 macroporous resin is significantly superior to other existing commonly used macroporous resins.

[0069] Example 4

[0070] The ethanol with a volume fraction of 70%, 75%, 80%, 85% and 90% was used as the extracting solution, and the other experimental parameters were kept the same: at room temperature, the ultrasonic power was 240W, the ultrasonic time was 25min; the ratio of leaf powder to extracting solution was 1g:70mL, and the triterpenoid components in the silver leaf tree leaf powder after sieving 5g of 40 mesh sieve were extracted. The extraction rate was determined and calculated, and the results are as follows Figure 7 As shown by Figure 7 It can be seen that when the triterpenoid components in the leaves of the silver leaf tree are extracted under ultrasound assistance, the volume fraction of the extract has a very significant effect. The extraction rate of the extract with a volume fraction of 80% is significantly greater than that of other volume fractions. Whether the volume fraction increases or decreases, the extraction rate will decrease.

Claims

1. A method for extracting triterpenoid components from the mangrove plant Cibotium obesum, characterized in that: The steps include: S1: Dry and grind the leaves of the silverleaf tree into powder; S2: adding 80% ethanol to the leaf powder, filtering to obtain an extract after ultrasonic-assisted extraction; S3: Use D301 macroporous adsorption resin to fill the column, use D301 macroporous adsorption resin column to adsorb the triterpenoid components in the extract, and use ethanol as the eluent to elute and filter after sufficient adsorption. The filtrate obtained is a high-concentration triterpenoid purified extract.

2. The method for extracting triterpenoid components from the mangrove plant Cibotium obesum according to claim 1, characterized in that: In the step S2, the usage ratio of leaf powder to 80% concentration ethanol is 1 g: 60-70 mL.

3. The method for extracting triterpenoid components from the mangrove plant Cibotium obesum according to claim 2, characterized in that: In the step S2, the usage ratio of leaf powder to 80% concentration ethanol is 1 g: 70 mL.

4. The method for extracting triterpenoid components from the mangrove plant Cibotium obesum according to claim 1, characterized in that: The ultrasonic assisted extraction in step S2 specifically uses a power of 210W to 250W, and the ultrasonic extraction time is 23 to 27 minutes.

5. The method for extracting triterpenoid components from the mangrove plant Cibotium obesum according to claim 4, characterized in that: The ultrasonic assisted extraction in step S2 specifically uses a power of 240 W and an ultrasonic extraction time of 25 min.

6. The method for extracting triterpenoid components from the mangrove plant Cibotium obesum according to claim 1, characterized in that: The specific steps of using ethanol for elution and filtration after sufficient adsorption in step S3 are: A1: After sufficient adsorption, use distilled water to elute the resin column at a flow rate of 1 BV / h to remove water-soluble impurities until the effluent is colorless; A2: The resin column washed with water in A1 is eluted with 75% to 90% ethanol by volume and 3 to 5 BV of eluent to obtain a purified extract of triterpenes.

7. The method for extracting triterpenoid components from the mangrove plant Cibotium obesum according to claim 6, characterized in that: The resin column after A1 washing was eluted using 80% by volume ethanol and 4 BV of eluent.

8. The method for extracting triterpenoid components from the mangrove plant Cibotium obesum according to claim 1, characterized in that: Before adding D301 macroporous adsorption resin between containers, it is also necessary to pre-treat D301 macroporous adsorption resin. The specific steps are as follows: B1: Soak the purchased D301 macroporous adsorption resin in anhydrous ethanol until it is fully swollen, and pour out the floating matter and anhydrous ethanol; B2: Use deionized water to wash the fully swollen D301 macroporous adsorption resin until no white turbidity appears; B3: Wet-fill the column with washed D301 macroporous adsorption resin, and then wash with deionized water until the effluent has no alcohol taste; B4: Soak D301 macroporous adsorption resin in 4% hydrochloric acid solution for 4 hours, then wash with water until the effluent is neutral; B5: Soak the D301 macroporous adsorption resin in a 4% sodium hydroxide solution for 4 hours, and then wash with water until the effluent is neutral to complete the pretreatment.

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