A total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect, and its preparation method and application

Abstract

The invention discloses a total alkaloid extract of Dendrobium officinale with anti-gastrointestinal tumor effect, and a preparation method and application thereof. The preparation method comprises the following steps: (1) taking Dendrobium officinale stem powder, adding acid-alcohol extracting solution to carry out leaching treatment first, and obtaining a crude extract of total alkaloid of Dendrobium officinale; (2) extracting the crude extract of total alkaloid of Dendrobium officinale, standing for phase separation, and then rotary evaporation treatment to obtain a total alkaloid extract; (3) dissolving the total alkaloid extract with anhydrous ethanol to obtain a total alkaloid loading solution; (4) mixing the total alkaloid loading solution with a macroporous resin, and after static adsorption, first washing impurities with an ethanol aqueous solution, and then statically desorbing with ethanol to obtain an ethanol eluate; (5) blowing the ethanol eluate with nitrogen at room temperature to a constant weight. The components of the total alkaloid extract of Dendrobium officinale obtained by the method of the invention play a significant synergistic regulatory role among each other, and have a significant inhibitory effect on the vitality of digestive tract tumor cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect, and a preparation method and application thereof. Background Art

[0002] Dendrobium officinale Kimura et Migo has been used in my country as a traditional Chinese herbal medicine and health food for thousands of years. According to the 2020 edition of the Chinese Pharmacopoeia (Volume 1), Dendrobium officinale is sweet, slightly cold, and enters the stomach and kidney meridians. It mainly nourishes the stomach and produces fluid, nourishes yin and clears heat, and is rich in a variety of secondary metabolites, such as polysaccharides, flavonoids, and alkaloids.

[0003] Alkaloids are one of the important active ingredients of Dendrobium plants. Compared with other Dendrobium plants, Dendrobium officinale is rich in indole alkaloids and isoquinoline alkaloids. Although the alkaloid content is low, it has high activity and has high development value. The total alkaloid extract of Dendrobium officinale has complex components, and the functions and polarities of alkaloids with different structures vary greatly. However, there are no reports on the extraction of alkaloids with anti-gastrointestinal tumor effects from Dendrobium officinale. Summary of the invention

[0004] The main purpose of the present invention is to provide a total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect and a preparation method and application thereof.

[0005] To achieve the above object, the present invention provides a method for preparing a total alkaloid extract of Dendrobium officinale having an anti-digestive tract tumor effect, comprising the following steps:

[0006] (1) taking the powder of Dendrobium officinale stem, adding the acid-alcohol extract to perform leaching treatment, then performing ultrasonic extraction treatment, centrifuging and separating the supernatant, repeating the above extraction process on the precipitate, centrifuging and separating the supernatant again, combining the two supernatants, and obtaining a crude extract of total alkaloids of Dendrobium officinale;

[0007] (2) adding ammonia water to the crude extract of total alkaloids of Dendrobium officinale to adjust the pH to alkaline, and then mixing with an equal volume of dichloromethane solution, standing for phase separation after extraction, collecting the lower dichloromethane phase, and then rotary evaporating to obtain a total alkaloid extract;

[0008] (3) dissolving the total alkaloid extract with anhydrous ethanol, and then diluting it with distilled water, and using anhydrous ethanol and distilled water to form an ethanol aqueous solution with a concentration of 20% as a solvent to obtain a total alkaloid loading solution;

[0009] (4) mixing the total alkaloid loading solution with the macroporous resin, and after static adsorption, first washing away impurities with an ethanol aqueous solution, and then statically desorbing with ethanol to obtain an ethanol eluate;

[0010] (5) The ethanol eluate is blown with nitrogen at room temperature to a constant weight, thereby obtaining a total alkaloid extract of Dendrobium officinale with anti-gastrointestinal tumor effect.

[0011] Furthermore, in step (1), the acid-alcohol extract contains 0.05% to 0.2% by volume of hydrochloric acid (V 盐酸 :V 水 ) and 70% to 90% ethanol (V 乙醇 :V 水 ); the solid-liquid ratio of Dendrobium officinale powder and acid-alcohol extract is 1g:10-30mL.

[0012] Furthermore, in step (1), the leaching treatment temperature is 20 to 30° C., and the leaching treatment time is 24 to 36 hours.

[0013] Furthermore, in step (1), the ultrasonic extraction treatment time is 30 to 60 minutes, the ultrasonic extraction treatment temperature is 30 to 60° C., and the ultrasonic extraction treatment frequency is 120 to 180 Hz.

[0014] Furthermore, in step (1), the centrifugal separation speed is 5000-7000 rpm, and the centrifugal separation time is 20-30 min.

[0015] Furthermore, in step (2), the volume concentration of ammonia water is 28%, and the pH value of the total alkaloid crude extract is adjusted to 9.0-11.0 by ammonia water; the volume concentration of the dichloromethane solution is 99.5%, the rotary evaporation temperature is 40-60°C, and the rotary evaporation speed is 350-400rpm.

[0016] Furthermore, in step (3), the concentration of the total alkaloid loading solution is 300 to 500 μg / mL.

[0017] Furthermore, in step (4), the macroporous resin is any one of D101, HPD100, and X-5.

[0018] Furthermore, in step (4), the solid-liquid ratio of the macroporous resin to the total alkaloid loading solution is 0.5 g: 5-15 mL; the static adsorption and desorption temperatures are 25-45° C., the static adsorption and desorption times are 12-24 h, respectively; the static adsorption and desorption are carried out on a shaker at a shaking speed of 100-150 rpm; the ethanol volume fraction of the ethanol aqueous solution is 60% (V 乙醇 :V 水 ).

[0019] The present invention also provides a total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect, which is prepared according to the above preparation method.

[0020] The present invention also provides a use of the total alkaloid extract of Dendrobium officinale in preparing an anti-digestive tract tumor drug.

[0021] The beneficial effects of the present invention are embodied in:

[0022] The present invention adopts acid-alcohol assisted ultrasonic extraction of total alkaloids from Dendrobium officinale, utilizes the characteristics of macroporous resin with both adsorption and sieving, purifies alkaloids through macroporous resin, and efficiently obtains high-purity total alkaloids from Dendrobium officinale with anti-gastrointestinal tumor effect. Experiments show that the components of the total alkaloid extract of Dendrobium officinale obtained by the method of the present invention play a significant synergistic regulatory role, and have a significant inhibitory effect on the vitality of digestive tract tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a flow chart of the preparation method of the total alkaloid extract of Dendrobium officinale.

[0024] Figure 2 This is a graph showing the experimental results of Example 6 of the present invention showing the effect of the total alkaloid extract of Dendrobium officinale on the vitality of digestive tract tumor cells.

[0025] Figure 3 This is a graph showing the experimental results of Example 6 of the present invention showing the effect of the total alkaloid extract of Dendrobium officinale on the vitality of digestive tract tumor cells. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with embodiments:

[0027] The various raw materials and equipment used in the following examples, unless otherwise specified, are all commercially available products known in the art. The stem powder of Dendrobium officinale is a two-year-old superfine powder of Dendrobium officinale purchased from Anhui Ningguo Wanhutang Co., Ltd. Macroporous resins D101, HPD100, and X-5 were all purchased from Nankai Technology Co., Ltd.

[0028] Example 1

[0029] Preparation of total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect

[0030] See also Figure 1 , the steps include:

[0031] (1) Weigh 40 g of Dendrobium officinale stem powder and add an acid-alcohol extract (the solvent of the acid-alcohol extract is water containing 0.05% hydrochloric acid (V)) at a solid-liquid ratio of 1 g:12.5 mL. 盐酸 :V 水 ) and 70% ethanol (V 乙醇 :V 水)), extract uniformly at 25 ° C for 24 hours, then ultrasonically extract at 60 ° C and 120 Hz for 50 minutes, then centrifuge at 6000 rpm for 25 minutes, collect the supernatant, repeat the above extraction steps on the precipitate obtained by centrifugation, centrifuge again, collect the supernatant, combine the two supernatants, and obtain a crude extract of total alkaloids.

[0032] (2) The pH of the crude total alkaloid extract was adjusted to 10.0 with 28% ammonia water, and an equal volume of 99.5% dichloromethane solution was added and mixed. After extraction, the mixture was allowed to stand for phase separation, and the lower dichloromethane phase was collected and rotary evaporated at 50° C. and 370 rpm until no obvious liquid flow occurred, thereby obtaining a total alkaloid extract of Dendrobium officinale.

[0033] (3) The total alkaloid extract of Dendrobium candidum was completely dissolved in anhydrous ethanol, and then distilled water was added to dilute it, and the anhydrous ethanol and distilled water formed an ethanol aqueous solution with a concentration of 20% (V 乙醇 :V 水 ) as the solvent and the total alkaloid extract of Dendrobium officinale as the solute to obtain a total alkaloid loading solution with a concentration of 350 μg / mL.

[0034] (4) The macroporous resin D101 and the alkaloid loading solution were mixed evenly at a solid-liquid ratio of 0.5 g:10 mL, and static adsorption was carried out at 25 °C and 120 rpm on a shaking table for 24 h. The ethanol volume fraction was 60% (V 乙醇 :V 水 ) was rinsed with ethanol-water solution for 10 min to remove impurities, and then static desorption was carried out with ethanol at 25°C and 120 rpm shaking for 24 h to obtain ethanol eluate.

[0035] (5) The ethanol eluate is blown with nitrogen at room temperature to a constant weight, thereby obtaining a total alkaloid extract of Dendrobium officinale that is rich in alkaloid compounds and has anti-gastrointestinal tumor effects.

[0036] Example 2

[0037] Preparation of total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect

[0038] See also Figure 1 , the steps include:

[0039] (1) Weigh 40 g of Dendrobium officinale stem powder and add an acid-alcohol extract (the solvent of the acid-alcohol extract is water containing 0.1% hydrochloric acid (V)) at a solid-liquid ratio of 1 g:10 mL. 盐酸 :V 水 ) and 80% ethanol (V 乙醇 :V 水)), extract uniformly at 20°C for 36h, then extract ultrasonically at 60°C and 140Hz for 60min, then centrifuge at 7000rpm for 20min, collect the supernatant, repeat the above extraction steps on the precipitate obtained by centrifugation, centrifuge again, collect the supernatant, combine the two supernatants to obtain a crude extract of total alkaloids.

[0040] (2) The pH value of the crude total alkaloid extract was adjusted to 9.0 with 28% ammonia water, and an equal volume of 99.5% dichloromethane solution was added and mixed. After extraction, the mixture was allowed to stand for phase separation, and the lower dichloromethane phase was collected and rotary evaporated at 40° C. and 400 rpm until no obvious liquid flow occurred, thereby obtaining a total alkaloid extract of Dendrobium officinale.

[0041] (3) The total alkaloid extract of Dendrobium candidum was completely dissolved in anhydrous ethanol, and then distilled water was added to dilute it, and the anhydrous ethanol and distilled water formed an ethanol aqueous solution with a concentration of 20% (V 乙醇 :V 水 ) was used as the solvent and the total alkaloid extract of Dendrobium officinale was used as the solute to obtain a total alkaloid loading solution with a concentration of 300 μg / mL.

[0042] (4) The macroporous resin HPD100 and the alkaloid loading solution were mixed evenly at a solid-liquid ratio of 0.5 g:5 mL, and static adsorption was carried out at 35 °C and 100 rpm for 18 h. The ethanol volume fraction was 60% (V 乙醇 :V 水 ) was rinsed with ethanol-water solution for 10 min to remove impurities, and then static desorption was carried out with ethanol at 35°C and 100 rpm shaking for 18 h to obtain ethanol eluate.

[0043] (5) The ethanol eluate is blown with nitrogen at room temperature to a constant weight, thereby obtaining a total alkaloid extract of Dendrobium officinale that is rich in alkaloid compounds and has anti-gastrointestinal tumor effects.

[0044] Example 3

[0045] Preparation of total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect

[0046] See also Figure 1 , the steps include:

[0047] (1) Weigh 40 g of Dendrobium officinale stem powder and add an acid-alcohol extract (the solvent of the acid-alcohol extract is water containing 0.2% hydrochloric acid (V)) at a solid-liquid ratio of 1 g:30 mL. 盐酸 :V 水 ) and 90% ethanol (V 乙醇 :V 水)), extract uniformly at 30°C for 30h, then extract ultrasonically at 30°C and 180Hz for 30min, then centrifuge at 5000rpm for 30min, collect the supernatant, repeat the above extraction steps on the precipitate obtained by centrifugation, centrifuge again, collect the supernatant, combine the two supernatants to obtain a crude extract of total alkaloids.

[0048] (2) The pH value of the crude extract of total alkaloids was adjusted to 11.0 with 28% ammonia water, and an equal volume of 99.5% dichloromethane solution was added and mixed. After extraction, the mixture was allowed to stand for phase separation, and the lower dichloromethane phase was collected and rotary evaporated at 60° C. and 350 rpm until no obvious liquid flow occurred, thereby obtaining a total alkaloid extract of Dendrobium officinale.

[0049] (3) The total alkaloid extract of Dendrobium candidum was completely dissolved in anhydrous ethanol, and then distilled water was added to dilute it, and the anhydrous ethanol and distilled water formed an ethanol aqueous solution with a concentration of 20% (V 乙醇 :V 水 ) was used as the solvent and the total alkaloid extract of Dendrobium officinale was used as the solute to obtain a total alkaloid loading solution with a concentration of 400 μg / mL.

[0050] (4) The macroporous resin X-5 and the alkaloid loading solution were mixed evenly at a solid-liquid ratio of 0.5 g:15 mL, and static adsorption was performed at 45 °C and 150 rpm on a shaking table for 12 h. The ethanol volume fraction was 60% (V 乙醇 :V 水 ) was rinsed with ethanol-water solution for 10 min to remove impurities, and then static desorption was carried out with ethanol at 45°C and 150 rpm shaking for 12 h to obtain ethanol eluate.

[0051] (5) The ethanol eluate is blown with nitrogen at room temperature to a constant weight, thereby obtaining a total alkaloid extract of Dendrobium officinale that is rich in alkaloid compounds and has anti-gastrointestinal tumor effects.

[0052] Example 4

[0053] Preparation of total alkaloid extract of Dendrobium officinale with anti-digestive tract tumor effect

[0054] See also Figure 1 , the steps include:

[0055] (1) Weigh 40 g of Dendrobium officinale stem powder and add an acid-alcohol extract (the solvent of the acid-alcohol extract is water containing 0.15% hydrochloric acid (V)) at a solid-liquid ratio of 1 g:25 mL. 盐酸 :V 水 ) and 75% ethanol (V 乙醇 :V 水)), extract uniformly at 25 ° C for 32 hours, then ultrasonically extract at 50 ° C and 150 Hz for 40 minutes, then centrifuge at 5000 rpm for 30 minutes, collect the supernatant, repeat the above extraction steps on the precipitate obtained by centrifugation, centrifuge again, collect the supernatant, combine the two supernatants to obtain a crude extract of total alkaloids.

[0056] (2) The pH of the crude total alkaloid extract was adjusted to 11.0 with 28% ammonia water, and an equal volume of 99.5% dichloromethane solution was added and mixed. After extraction, the mixture was allowed to stand for phase separation, and the lower dichloromethane phase was collected and rotary evaporated at 55° C. and 350 rpm until no obvious liquid flow occurred, thereby obtaining a total alkaloid extract of Dendrobium officinale.

[0057] (3) The total alkaloid extract of Dendrobium candidum was completely dissolved in anhydrous ethanol, and then distilled water was added to dilute it, and the anhydrous ethanol and distilled water formed an ethanol aqueous solution with a concentration of 20% (V 乙醇 :V 水 ) as the solvent and the total alkaloid extract of Dendrobium officinale as the solute to obtain a total alkaloid loading solution with a concentration of 350 μg / mL.

[0058] (4) The macroporous resin X-5 and the alkaloid loading solution were mixed evenly at a solid-liquid ratio of 0.5 g:12 mL, and static adsorption was performed at 30°C and 150 rpm on a shaking table for 20 h. The ethanol volume fraction was 60% (V 乙醇 :V 水 ) was rinsed with ethanol aqueous solution for 10 min to remove impurities, and then static desorption was carried out with ethanol at 30°C and 150 rpm shaking for 20 h to obtain ethanol eluate.

[0059] (5) The ethanol eluate is blown with nitrogen at room temperature to a constant weight, thereby obtaining a total alkaloid extract of Dendrobium officinale that is rich in alkaloid compounds and has anti-gastrointestinal tumor effects.

[0060] Example 5

[0061] Analysis of the components of total alkaloid extracts from Dendrobium officinale

[0062] The components of the total alkaloid extract of Dendrobium candidum obtained in Example 1 were analyzed by UPLC / MS / MS technology, and a total of 655 compounds were detected, of which 60 were alkaloid compounds, accounting for 9.16%, as shown in Table 1, including 22 isoquinoline alkaloids, 12 indole alkaloids, 5 pyrrole alkaloids, 7 pyridine alkaloids, 5 quinoline alkaloids, 2 terpenoid alkaloids, 3 organic amine alkaloids, and 4 others. According to the peak area size, indole alkaloids Harmane and Norharman accounted for 41% and 36% respectively, and isoquinoline alkaloids Palmatine and Norisoboldin accounted for 7% and 4% respectively.

[0063] Table 1 Alkaloid compounds in the total alkaloid extract of Dendrobium candidum

[0064]

[0065]

[0066] Example 6

[0067] Effects of total alkaloid extracts from Dendrobium officinale on the vitality of digestive tract tumor cells

[0068] Cell lines and cell culture: Human gastric cancer cell lines HGC-27, MKN-45, SGC-7901, and colorectal cancer cell lines SW620, HCT 116, and HT 29 were selected. The cells were placed in RPMI-1640 medium containing 10% FBS and cultured in a carbon dioxide incubator with a CO2 concentration of 5% and a culture temperature of 37°C.

[0069] The cell viability was determined by the MTT method: the logarithmic phase cells were inoculated in a 96-well plate, and a 200-fold diluted DMSO solution and different concentrations of the total alkaloid extract of Dendrobium officinale prepared in Example 1 (1.25, 2.5, 5, 10, 20 and 40 nmol / mL) were used, and the cells were cultured in a carbon dioxide incubator for 24 h, the supernatant was carefully aspirated, 200 μL of a 10% MTT solution with a concentration of 0.5 mg / mL was added to each well, and the cells were further cultured in a carbon dioxide incubator for 4 h, the MTT solution was carefully aspirated, 150 μL of a DMSO solution was added to each well, the absorbance was measured at 490 nm using an enzyme reader, and the half inhibitory concentration (IC) was calculated using the 4-Parameter method. 50 value).

[0070] Cell survival rate (%) = [(OD 给药 -OD 空白 ) / OD 空白 ]×100%;

[0071] The results are as follows Figure 2 and Figure 3 The total alkaloid extract of Dendrobium officinale had a significant inhibitory effect on the cell viability of three colorectal cancer cells SW620, HCT116, HT29, and three gastric cancer cells HGC-27, MKN-45, SGC-7901, and the inhibitory effect showed a dose-dependent change. The IC 50 The values ​​were 18.221nmol / mL, 21.077nmol / mL, and 27.711nmol / mL, respectively, and the IC 50 The values ​​were 18.072 nmol / mL, 22.008 nmol / mL, and 28.853 nmol / mL, respectively.

[0072] The experiment showed that after 24 hours of incubation, the IC50 value of the total alkaloid extract for colorectal cancer cells SW620 was 18.221nmol / mL, the IC50 value for colorectal cancer cells HCT 116 was 21.077nmol / mL, the IC50 value for colorectal cancer cells HT29 was 27.711nmol / mL, the IC50 value for gastric cancer cells HGC-27 was 18.072nmol / mL, the IC50 value for gastric cancer cells MKN-45 was 22.008nmol / mL, and the IC50 value for gastric cancer cells SGC-7901 was 28.853nmol / mL.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a total alkaloid extract of Dendrobium officinale having anti-digestive tract tumor effect, characterized in that: The following steps are involved: (1) taking the stem powder of Dendrobium officinale, adding the acid-alcohol extract to perform leaching treatment, then performing ultrasonic extraction treatment, centrifuging and separating the supernatant, repeating the above extraction process on the precipitate, centrifuging and separating the supernatant again, combining the two supernatants to obtain a crude extract of total alkaloids of Dendrobium officinale; The acid-alcohol extract contains 0.05% to 0.2% hydrochloric acid and 70% to 90% ethanol; the solid-liquid ratio of the Dendrobium officinale powder to the acid-alcohol extract is 1 g: 10 to 30 mL; (2) adding ammonia water to the crude extract of total alkaloids of Dendrobium officinale to adjust the pH to alkaline, and then mixing with an equal volume of dichloromethane solution, allowing the phases to stand after extraction, collecting the lower dichloromethane phase, and then rotary evaporating to obtain a total alkaloid extract; The volume concentration of ammonia water is 28%, and the pH value of the total alkaloid crude extract is adjusted to 9.0-11.0 by ammonia water; the volume concentration of the dichloromethane solution is 99.5%, the rotary evaporation temperature is 40-60°C, and the rotary evaporation speed is 350-400 rpm; (3) dissolving the total alkaloid extract with anhydrous ethanol, and then diluting it with distilled water, and using anhydrous ethanol and distilled water to form an ethanol aqueous solution with a concentration of 20% as a solvent to obtain a total alkaloid loading solution; (4) The total alkaloid loading solution is mixed with the macroporous resin, and after static adsorption, the impurities are first washed away with an ethanol aqueous solution, and then static desorption is performed with ethanol to obtain an ethanol eluate; The macroporous resin is any one of D101, HPD100, and X-5; the material-liquid ratio of the macroporous resin to the total alkaloid loading solution is 0.5 g: 5-15 mL; the static adsorption and desorption temperatures are 25-45° C., the static adsorption and desorption times are 12-24 h, respectively, and the static adsorption and desorption are carried out on a shaker at a shaker speed of 100-150 rpm; the ethanol volume fraction of the ethanol aqueous solution is 60%; (5) The ethanol eluate was nitrogen blown to a constant weight at room temperature to obtain a total alkaloid extract of Dendrobium officinale with anti-gastrointestinal tumor effect.

2. The method for preparing the total alkaloid extract of Dendrobium officinale having anti-digestive tract tumor effect according to claim 1, characterized in that: In step (1), the leaching temperature is 20 to 30° C., and the leaching time is 24 to 36 hours.

3. The method for preparing the total alkaloid extract of Dendrobium officinale having anti-digestive tract tumor effect according to claim 1, characterized in that: In step (1), the ultrasonic extraction treatment time is 30 to 60 minutes, the ultrasonic extraction treatment temperature is 30 to 60° C., and the ultrasonic extraction treatment frequency is 120 to 180 Hz.

4. The method for preparing the total alkaloid extract of Dendrobium officinale having anti-digestive tract tumor effect according to claim 1, characterized in that: In step (3), the concentration of the total alkaloid loading solution is 300-500 μg / mL.

5. A total alkaloid extract of Dendrobium officinale having anti-digestive tract tumor effect, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the total alkaloid extract of Dendrobium officinale as claimed in claim 5 in the preparation of an anti-digestive tract tumor drug, wherein the digestive tract tumor is gastric cancer or rectal cancer.

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