Preparation method of a semen smilacis glabrae extract and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine extracts, and particularly relates to a preparation method of a radix croci extract and application thereof. The preparation method comprises the following steps: placing radix croci in ethanol, ultrasonic treatment, and extraction to obtain the radix croci extract. The preparation method of the radix croci extract is simple in operation and low in cost. By respectively regulating ultrasonic conditions and the amount of an extraction solvent, a haematin content of the prepared radix croci extract is high, and the purity is high. Through a DPPH free radical scavenging rate experiment, it is shown that the selection of an extraction agent also has a certain influence on the antioxidant effect of haematin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine extract, and particularly relates to a preparation method of Daemonorops draco extract and application thereof. BACKGROUND

[0002] Blood is a traditional precious Chinese medicine, known as "the blood of the holy medicine", which has the effects of activating blood, relieving pain, removing blood stasis, and stopping bleeding. The 2010 edition of Chinese Pharmacopoeia records blood as the resin exuded from the fruit of Daemonorops draco Bl. of the Palmaceae family, which is processed. Blood has a wide range of pharmacological effects and is widely used in clinical practice, such as treating cervical erosion, uterine fibroids, and cervical bleeding in gynecology, treating coronary heart disease in cardiovascular department, treating hemorrhoids, bedsores, and surface wounds in surgery, and treating epistaxis, oral ulcers, and retinal branch vein obstruction in ophthalmology. Daemonorops draco contains about 57.5% red resin, from which bloodletting and bloodletting are separated, which are both derivatives of flavonoids, and bloodletting is one of the important active substances.

[0003] Chinese invention patent CN106279090A discloses a method for separating and purifying bloodletting from Daemonorops draco husk, which specifically comprises the following steps: A: adding an ethanol solution with a volume fraction of 95% to Daemonorops draco husk, the amount of which is 6.075-7.290 times the mass of the husk; B: extracting the mixture obtained in step A by ultrasonic extraction and concentrating it to a extract. This method can obtain red resin containing bloodletting remaining in the discarded husk of Daemonorops draco. Chinese another invention patent CN106946836A discloses a process for extracting bloodletting from Daemonorops draco, which is prepared by the steps of crushing, primary decolorization, crude crystallization, secondary decolorization, macroporous adsorption resin separation and purification, concentration and crystallization, and drying. Chinese another invention patent CN106588849A discloses a method for separating and purifying bloodletting, which comprises the following steps: (1) obtaining medicinal blood as raw material; (2) dissolving the blood in step (1) with an organic solution, and taking the supernatant; (3) adding an equal volume of water to the supernatant in step (2) for back extraction, and taking the water phase layer; (4) vacuum concentrating and vacuum drying the water phase layer in step (3) to obtain bloodletting powder. However, the above-mentioned patents have the problems of low yield, complex steps, and high production cost in extracting bloodletting.

[0004] Therefore, there is an urgent need in the art for a preparation method of Daemonorops draco extract with high yield, simple process, and low cost. SUMMARY

[0005] The present application provides a preparation method of Daemonorops draco extract and application thereof to solve the problems in the prior art.

[0006] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0007] A preparation method of a radix luffae extract, comprising placing radix luffae in ethanol, ultrasonic extraction, and obtaining the radix luffae extract, wherein the mass-volume ratio of the radix luffae to the ethanol is 1g:80-200mL.

[0008] Preferably, the mass concentration of the ethanol is 85-95%.

[0009] Preferably, the temperature of the ultrasonic extraction is 40-80℃, the time is 50-70min, and the power is 60-100W.

[0010] More preferably, the temperature of the ultrasonic extraction is 60℃, the time is 60min, and the power is 80W.

[0011] Preferably, the ultrasonic extraction is followed by suction filtration, rotary evaporation, and dilution with water.

[0012] Preferably, the temperature of the rotary evaporation is 55-65℃.

[0013] Preferably, the temperature of the water is 70-85℃.

[0014] Preferably, the volume ratio of the water to the concentrated solution after the rotary evaporation is 8-10:1-2.

[0015] More preferably, the volume ratio of the water to the concentrated solution after the rotary evaporation is 9:1.

[0016] Preferably, the solvent for the extraction is selected from any one of petroleum ether, ethyl acetate, and n-butanol.

[0017] Preferably, the volume ratio of the solvent for the extraction to the solution after dilution with water is 0.7-1.3:1.

[0018] The present application also provides the use of the above-mentioned radix luffae extract in the preparation of an antioxidant drug.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The preparation method of the radix luffae extract provided by the present application is simple to operate and low in cost. By respectively regulating the ultrasonic conditions and the amount of the extraction solvent, the content and purity of the hemin in the radix luffae extract are high, which has obvious advantages over the method for preparing hemin in the prior art. The DPPH free radical scavenging rate experiment shows that the selection of the extraction agent also has a certain influence on the antioxidant effect of the hemin. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 HPLC chromatogram of hemin in QLJ① layer in Example 1.

[0022] Figure 2 HPLC chromatogram of dracorhodin for QLJ ② layer in Example 1.

[0023] Figure 3 HPLC chromatogram of dracorhodin for QLJ ③ layer in Example 1.

[0024] Figure 4 HPLC chromatogram of dracorhodin for QLJ ④ layer in Example 1.

[0025] Figure 5 HPLC chromatogram of dracorhodin perchlorate control. DETAILED DESCRIPTION

[0026] It is worth mentioning that the raw materials used in the present application are all ordinary commercially available products.

[0027] Example 1

[0028] A preparation method of a extract of dracaenae cornea, the specific steps are:

[0029] 1g of dracaenae cornea is added with 100mL of 95% ethanol, and the dracaenae cornea is extracted by ultrasonic heating, the ultrasonic temperature is set to 60℃, the ultrasonic time is set to 60min, and the ultrasonic power is set to 80W, then the extraction is filtered, and 5mL of dracaenae cornea extraction concentrate is obtained by rotary evaporation at 65℃, then 45mL of 85℃ distilled water is added to dilute the dracaenae cornea dilution liquid to 50mL, finally, 50mL of petroleum ether, 50mL of ethyl acetate and 50mL of n-butanol are used to directly extract the dracaenae cornea dilution liquid respectively, and three extraction parts are separated, the 95% ethanol extraction part, the petroleum ether, the ethyl acetate and the n-butanol extraction part are respectively named as QLJ ①, QLJ ②, QLJ ③ and QLJ ④, and different extraction layer paste of dracaenae cornea is obtained by evaporating dry in a 90℃ water bath.

[0030] Example 2

[0031] A preparation method of a extract of dracaenae cornea, the specific steps are:

[0032] 1g of dracaenae cornea is added with 80mL of 85% ethanol, and the dracaenae cornea is extracted by ultrasonic heating, the ultrasonic temperature is set to 40℃, the ultrasonic time is set to 70min, and the ultrasonic power is set to 60W, then the extraction is filtered, and 5mL of dracaenae cornea extraction concentrate is obtained by rotary evaporation at 55℃, then 50mL of 70℃ distilled water is added to dilute the dracaenae cornea dilution liquid to 55mL, finally, 40mL of petroleum ether, 40mL of ethyl acetate and 40mL of n-butanol are used to directly extract the dracaenae cornea dilution liquid respectively, and three extraction parts are separated, the 85% ethanol extraction part, the petroleum ether, the ethyl acetate and the n-butanol extraction part are respectively named as QLJ ①, QLJ ②, QLJ ③ and QLJ ④, and different extraction layer paste of dracaenae cornea is obtained by evaporating dry in a 90℃ water bath.

[0033] Example 3

[0034] A preparation method of a Herba Epimedii extract, the specific steps being:

[0035] 1 g of Herba Epimedii is added with 200 mL of 90% ethanol, and the Herba Epimedii is extracted by ultrasonic heating, the ultrasonic temperature being set to 80°C, the ultrasonic time being set to 50 min, and the ultrasonic power being set to 100 W, after which the Herba Epimedii is extracted by filtration, 5 mL of Herba Epimedii extraction concentrate is rotary evaporated at 65°C, 40 mL of 85°C distilled water is added to dilute the Herba Epimedii dilution liquid to 45 mL, and finally 60 mL of petroleum ether, 60 mL of ethyl acetate and 60 mL of n-butanol are used to directly extract the Herba Epimedii dilution liquid, and three extraction parts are separated, the 90% ethanol extraction part, the petroleum ether extraction part, the ethyl acetate extraction part and the n-butanol extraction part are respectively named as QLJ①, QLJ②, QLJ③ and QLJ④, and the different extraction layer paste of Herba Epimedii is obtained by evaporating to dryness in a 90°C water bath.

[0036] Comparative Example 1

[0037] A preparation method of a Herba Epimedii extract, the specific steps being:

[0038] 1 g of Herba Epimedii is added with 50 mL of 95% ethanol, and the Herba Epimedii is extracted by ultrasonic heating, the ultrasonic temperature being set to 60°C, the ultrasonic time being set to 60 min, and the ultrasonic power being set to 80 W, after which the Herba Epimedii is extracted by filtration, 5 mL of Herba Epimedii extraction concentrate is rotary evaporated at 65°C, 45 mL of 85°C distilled water is added to dilute the Herba Epimedii dilution liquid to 50 mL, and finally 50 mL of petroleum ether, 50 mL of ethyl acetate and 50 mL of n-butanol are used to directly extract the Herba Epimedii dilution liquid, and three extraction parts are separated, the 95% ethanol extraction part, the petroleum ether extraction part, the ethyl acetate extraction part and the n-butanol extraction part are respectively named as QLJ①, QLJ②, QLJ③ and QLJ④, and the different extraction layer paste of Herba Epimedii is obtained by evaporating to dryness in a 90°C water bath.

[0039] Comparative Example 2

[0040] A preparation method of a Herba Epimedii extract, the specific steps being:

[0041] 1g of the Herba Epimedii was added with 300 mL of 95% ethanol, and the Herba Epimedii was extracted by ultrasonic heating. The ultrasonic temperature was set to 60°C, the ultrasonic time was set to 60 min, and the ultrasonic power was set to 80 W. Then, the Herba Epimedii was filtered, and 5 mL of concentrated extract of the Herba Epimedii was obtained by rotary evaporation at 65°C. Then, 45 mL of 85°C distilled water was added to dilute the concentrated extract to 50 mL of diluted extract of the Herba Epimedii. Finally, 50 mL of petroleum ether, 50 mL of ethyl acetate, and 50 mL of n-butanol were directly used to extract the diluted extract of the Herba Epimedii, respectively, and 3 extracted parts were separated. The 95% ethanol extracted part, the petroleum ether extracted part, the ethyl acetate extracted part, and the n-butanol extracted part were named as QLJ①, QLJ②, QLJ③, and QLJ④, respectively. The different extracted layer paste of the Herba Epimedii was obtained by evaporating the above-mentioned four parts in a 90°C water bath.

[0042] Comparative Example 3

[0043] A preparation method of a Herba Epimedii extract includes the following specific steps.

[0044] 1g of the Herba Epimedii was added with 300 mL of 95% ethanol, and the Herba Epimedii was extracted by ultrasonic heating. The ultrasonic temperature was set to 60°C, the ultrasonic time was set to 60 min, and the ultrasonic power was set to 80 W. Then, the Herba Epimedii was filtered, and 5 mL of concentrated extract of the Herba Epimedii was obtained by rotary evaporation at 65°C. Then, 45 mL of 85°C distilled water was added to dilute the concentrated extract to 50 mL of diluted extract of the Herba Epimedii. Finally, 50 mL of petroleum ether, 50 mL of ethyl acetate, and 50 mL of n-butanol were directly used to extract the diluted extract of the Herba Epimedii, respectively, and 3 extracted parts were separated. The 95% ethanol extracted part, the petroleum ether extracted part, the ethyl acetate extracted part, and the n-butanol extracted part were named as QLJ①, QLJ②, QLJ③, and QLJ④, respectively. The different extracted layer paste of the Herba Epimedii was obtained by evaporating the above-mentioned four parts in a 90°C water bath.

[0045] 1.77 mg of hemin perchlorate control was precisely weighed and determined, 3% phosphoric acid methanol solution was precisely added and diluted to 10 mL in a volumetric flask, and then shaken to obtain a hemin perchlorate control solution (128.54 μg of hemin was contained in every 1 mL of hemin perchlorate) (the weight of hemin = the weight of hemin perchlorate / 1.377). The hemin perchlorate control solution was configured to have concentrations of 11.0625, 22.125, 44.25, 88.5, and 177 μg / mL, respectively.

[0046] 100 mg of the different extracted layer samples of the Herba Epimedii in Examples 1-3 and Comparative Examples 1-3 were precisely weighed, respectively, 3% phosphoric acid methanol solution was added and diluted to 10 mL in a volumetric flask, and then shaken to obtain a sample solution with a concentration of 10 mg / mL.

[0047] The content of dracorhodin in different extraction layers in Examples 1-3 and Comparative Examples 1-3 was detected by HPLC, wherein the HPLC detection column was ZORBAX SB-C18; the mobile phase A was 0.2% acetic acid aqueous solution, the mobile phase B was acetonitrile, the gradient elution program was shown in Table 1; the detection wavelength was 440 nm; the injection volume was 5 μL; and the flow rate was 1.0 mL / min.

[0048] Table 1 Gradient elution program

[0049]

[0050] The standard curve of the reference dracorhodin perchlorate was y = 9.1405x - 5.5804, R = 0.9995, and the concentration linear range was 11.0625-177 μg / mL. 2

[0051] The HPLC chromatogram of dracorhodin in QLJ① layer is shown in Figure 1; the HPLC chromatogram of dracorhodin in QLJ② layer is shown in Figure 2; the HPLC chromatogram of dracorhodin in QLJ③ layer is shown in Figure 3; the HPLC chromatogram of dracorhodin in QLJ④ layer is shown in Figure 4; and the HPLC chromatogram of the reference dracorhodin perchlorate is shown in Figure 5. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

[0052] The content of dracorhodin in different extraction layers in Examples 1-3 and Comparative Examples 1-3 is shown in Table 2.

[0053] From Table 2, it can be seen that the content of dracorhodin was the highest in the ethyl acetate extraction layer (QLJ③), followed by the ethanol extraction part (QLJ①), and therefore, the ethyl acetate extraction might have an enrichment effect on dracorhodin and compounds with similar polarity.

[0054] Table 2 Content and purity of dracorhodin in different extraction layers of Eupholia lindleyana Baill.

[0055]

[0056] Effect Example: DPPH free radical scavenging rate experiment

[0057] Preparation of DPPH ethanol solution: 20 mg of DPPH was weighed, dissolved in anhydrous ethanol, and diluted to a 250 mL volumetric flask, which was stored at 0-4 °C in the dark and used immediately, and was effective within 4 h.

[0058] Preparation of the test solution: the Eupholia lindleyana Baill. extracts in QLJ①, QLJ②, QLJ③ and QLJ④ layers in Example 1 were prepared into 1, 0.5, 0.25 and 0.125 mg / mL test solutions. ​​​​​​

[0059] The positive control group selects 1 mg / mL vitamin C, which is stored at 0-4°C in the dark. It is prepared and used immediately.

[0060] The negative control group selects distilled water.

[0061] Specific experimental steps: add each reagent according to the ratio of Table 3, mix well, react at room temperature in the dark for 30 min, and then measure the absorbance values of A, B, and C tubes at 517 nm using an enzyme marker.

[0062] Table 3 Liquid-solid ratio

[0063]

[0064] The DPPH free radical scavenging rate calculation formula is:

[0065] Scavenging rate (%) = [(B+C)-A] ÷ B × 100%.

[0066] In the formula: A is the absorbance value of tube A at 517 nm; B is the absorbance value of tube B at 517 nm; and C is the absorbance value of tube C at 517 nm.

[0067] Result analysis: the DPPH scavenging rate of the positive control group is 98.05%, the DPPH scavenging rate of the negative control group is 2.79%, and the DPPH scavenging rate of the test solution is shown in Table 4. It can be seen that the antioxidant effect is QLJ ③ > QLJ ④ > QLJ ② > QLJ ①, and the above HPLC detection of dracorhodin content is QLJ ③ > QLJ ① > QLJ ④ > QLJ ②, which shows that the extractant also has certain influence on the antioxidant effect.

[0068] Table 4 DPPH scavenging rate of different extract layers of Kirin extract

[0069]

[0070] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by ordinary skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing a dragon's blood extract, characterized in that, The extraction process involves placing draconis in ethanol, sonicating, and then extracting. The mass-to-volume ratio of draconis to ethanol is 1 g:80 mL, 1 g:100 mL, or 1 g:200 mL. The sonication temperature is 40℃, 60℃, or 80℃, the time is 50 min, 60 min, or 70 min, and the power is 60 W, 80 W, or 100 W. The mass concentration of the ethanol is 85%, 90%, or 95%. The extraction solvent is selected from any one of petroleum ether, ethyl acetate, and n-butanol.

2. The preparation method according to claim 1, characterized in that, The process involves ultrasonication followed by filtration, rotary evaporation, and dilution with water. The volume ratio of the extraction solvent to the diluted solution is 0.7-1.3:

1.

3. The preparation method according to claim 2, characterized in that, The temperature of the rotary evaporation is 55-65℃.

4. The preparation method according to claim 2, characterized in that, The temperature of the water is 70-85℃.

5. The preparation method according to claim 2, characterized in that, The volume ratio of water to the concentrated liquid after rotary evaporation is 8-10:

1.

6. A *Dracaena cochinchinensis* extract prepared by the preparation method according to any one of claims 1-5.

7. The use of the *Dracaena cochinchinensis* extract as described in claim 6 in the preparation of antioxidant drugs.

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