Dendrobium extract as well as preparation method and application thereof

Abstract

The invention relates to a dendrobium extract, and discloses a dendrobium extract as well as a preparation method and application thereof. The preparation method comprises the following steps: removing roots and leaves of dendrobium nobile, and drying the dendrobium nobile at 60-70 DEG C until the weight of the dendrobium nobile is constant, so as to obtain dry dendrobium nobile strips; extracting the obtained dry dendrobium strips for multiple times by adopting a water extraction method to obtain an extracting solution; and carrying out freeze-drying treatment on the extracting solution to obtain dendrobium extract freeze-dried powder. The process is simple, the cost is low, the content of water-soluble flavone and alkaloid is high, and the prepared dendrobium extract can inhibit growth of various tumor cells and induce cell apoptosis and has a remarkable anti-tumor effect.

Description

Technical Field

[0001] The invention relates to a dendrobium extract, in particular to a dendrobium extract and a preparation method and application thereof. Background Art

[0002] Dendrobium not only contains active factors such as polysaccharides and flavonoids, but is also rich in a variety of nutrients such as amino acids, vitamins, proteins, etc. Existing pharmacological studies on Dendrobium have shown that it has anti-tumor effects, but the material basis of its efficacy is still unclear, which makes it difficult for Dendrobium to provide support for anti-tumor drug research. Therefore, it is urgent to provide a Dendrobium extraction method with a clear material basis for its efficacy. Summary of the invention

[0003] In view of the prior art, the present invention provides a dendrobium extract and a preparation method and application thereof.

[0004] In order to solve the above technical problems, the present invention is solved by the following technical solutions:

[0005] A method for preparing a dendrobium extract comprises the following steps:

[0006] Step S1, removing the roots and leaves of Dendrobium officinale, drying at 60-70° C. to a constant weight, and obtaining a dried Dendrobium officinale strip;

[0007] Step S2, extracting the dried Dendrobium officinale strips obtained in step S1 multiple times using a water extraction method to obtain an extract;

[0008] Step S3, freeze-drying the extract to obtain freeze-dried powder of Dendrobium officinale extract.

[0009] Preferably, step S2 specifically includes the following steps:

[0010] Step S21, extracting the dried Dendrobium officinale strips by water extraction, specifically, placing the dried Dendrobium officinale strips in ultrapure water at a temperature of 70-90° C. to dissolve the extract, and the extraction time is 2-4 hours;

[0011] Step S22, after filtering, obtain a primary filtrate and a primary filter residue;

[0012] Step S23, extracting the primary filter residue in step S22 by the same water extraction method as in step S21, and filtering to obtain a secondary filtrate and a secondary filter residue;

[0013] Step S24, repeating step S23 to obtain three filtrates and three filter residues;

[0014] Step S25, combining the filtrates obtained in steps S22-S24, and then concentrating under reduced pressure to obtain an extract.

[0015] Preferably, the material-liquid ratio in steps S22 to S24 is 1:40 (w:v).

[0016] Preferably, the reduced pressure concentration in step S25 is carried out by using a rotary evaporator at a rotation speed of 100 rpm / min and a water bath temperature of 60° C. to concentrate the solution under reduced pressure to 10%-15% of the original volume.

[0017] Preferably, the freeze-drying treatment in step S3 is specifically to place the extract in a freeze dryer to freeze-dry to obtain freeze-dried powder, and then store the freeze-dried powder in a -20°C environment for standby use.

[0018] A dendrobium extract is prepared by adopting a preparation method of the dendrobium extract.

[0019] The invention discloses an application of a dendrobium extract, and the dendrobium extract is applied to a drug for inhibiting the growth of tumor cells.

[0020] Preferably, the tumor cells include mouse melanoma B16 cells, human cervical cancer Hela cells, human liver cancer HepG2 cells, human lung cancer A549 cells, mouse breast cancer 4T1 cells and human melanoma A375 cells.

[0021] Experiments show that Dendrobium extract has a significant inhibitory effect on the above cells, among which the inhibitory effect on B16 cell proliferation is the most obvious, with an IC50 value of 1.38±0.82mg / mL. Dendrobium extract at 2mg / mL can induce B16 cell apoptosis, with an apoptosis rate of 56.53±0.47%, and can block the B16 cell cycle at the G0 / G1 phase.

[0022] The present invention has significant technical effects due to the adoption of the above technical solution:

[0023] (1) On the one hand, the extraction is based on the complete dried strips of Dendrobium, which ensures the integrity of the cell wall of Dendrobium, reduces the leaching of Dendrobium polysaccharides, and at the same time maximizes the content of water-soluble flavonoids and alkaloids in the Dendrobium extract;

[0024] (2) On the other hand, multiple hot water extraction operations can fully dissolve water-soluble flavonoids and alkaloids, thereby increasing their content in the extract.

[0025] (3) The process is simple, the cost is low, the extraction efficiency of water-soluble flavonoids and alkaloids is high, and the prepared Dendrobium extract can inhibit the growth of various tumor cells, induce cell apoptosis, and has a significant anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 UPLC-Q-TOF / MS chromatogram of CDHAE, wherein A is the chromatogram in positive ion mode and B is the chromatogram in negative ion mode.

[0027] Figure 2 UPLC-Q-TOF / MS chromatogram of UDHAE, wherein A is the chromatogram in positive ion mode and B is the chromatogram in negative ion mode.

[0028] Figure 3 Effects of Dendrobium officinale water extract on cell proliferation ( n=3), *P<0.05, **P<0.01, ***P<0.005, ****P<0.001, wherein A is the effect of water extract of Dendrobium officinale on the proliferation of B16 cells, B is the effect of water extract of Dendrobium officinale on the proliferation of Hela cells, C is the effect of water extract of Dendrobium officinale on the proliferation of A375 cells, D is the effect of water extract of Dendrobium officinale on the proliferation of A549 cells, E is the effect of water extract of Dendrobium officinale on the proliferation of 4T1 cells, and F is the effect of water extract of Dendrobium officinale on the proliferation of HepG2 cells.

[0029] Figure 4 The figure shows the effect of UDHAE on cell live and dead staining, where the ordinate represents the live and dead staining dye, and the abscissa represents the administration concentration.

[0030] Figure 5 The figure shows the effect of UDHAE on the cell cycle and apoptosis of B16 cells, where AJ represents the apoptosis and cell cycle inhibition at different concentrations. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0032] Example 1

[0033] This embodiment provides a method for preparing a dendrobium extract. Specifically, the dendrobium selected in this embodiment is Dendrobium huoshanense (D. huoshanense), also known as Dendrobium officinale, which is a perennial herb belonging to the Orchidaceae family and is mainly produced in the Dabie Mountains of Huoshan County, Anhui Province.

[0034] The preparation method of the dendrobium extract specifically comprises the following steps:

[0035] Step S1, removing the roots and leaves of Dendrobium officinale, drying at 60-70° C. to a constant weight, and obtaining a dried Dendrobium officinale strip;

[0036] Step S2, extracting the dried Dendrobium officinale strips obtained in step S1 multiple times using a water extraction method to obtain an extract;

[0037] Step S3, freeze-drying the extract in a freeze dryer to obtain freeze-dried powder, and then storing the freeze-dried powder in a -20°C refrigerator to obtain freeze-dried powder of Dendrobium officinale extract for later use.

[0038] Wherein, step S2 specifically includes the following steps:

[0039] Step S21, extracting the dried dendrobium strips by water extraction, specifically placing the dried dendrobium strips in ultrapure water at a temperature of 70-90° C. to dissolve the extract, the solid-liquid ratio is 1:40 (w:v), that is, the ratio of the mass of the dried dendrobium strips to the volume of the ultrapure water is 1:40, and the extraction time is 2-4 hours;

[0040] Step S22, after filtering, obtain a primary filtrate and a primary filter residue;

[0041] Step S23, extracting the primary filter residue in step S22 by the same water extraction method as in step S21, that is, placing the primary filter residue in ultrapure water at a temperature of 70-90° C. for re-filtration, the material-liquid ratio of the primary filter residue to ultrapure water is 1:40, and the extraction time is 2-4 hours.

[0042] After filtering, a secondary filtrate and a secondary filter residue are obtained;

[0043] Step S24, repeating step S23 to obtain three filtrates and three filter residues;

[0044] In this embodiment, steps S22-S24 are specifically performed in a constant temperature magnetic stirrer at a temperature of 80° C. for 2 hours.

[0045] Step S25, combining the filtrates obtained in steps S22-S24, and then concentrating under reduced pressure to obtain an extract.

[0046] The reduced pressure concentration in step S25 is specifically carried out by using a rotary evaporator at a rotation speed of 100 rpm / min and a water bath temperature of 60° C. to concentrate the solution under reduced pressure to 10%-15% of the original volume.

[0047] Example 2

[0048] This embodiment provides a dendrobium extract, which is prepared by using the preparation method of a dendrobium extract in Example 1.

[0049] Comparative Example 1

[0050] The same as Example 1, except that the preparation method of the Dendrobium extract in this embodiment specifically comprises the following steps:

[0051] Step S1, removing roots and leaves of Dendrobium officinale, drying at 60-70° C. to constant weight to obtain dried Dendrobium officinale strips, and then crushing and sieving the dried Dendrobium officinale strips to obtain crushed Dendrobium officinale;

[0052] Step S2, extracting the crushed dendrobium obtained in step S1 multiple times by water extraction to obtain an extract. The specific extraction method is the same as that in Example 1, and will not be described in detail here;

[0053] Step S3 is the same as step S3 in embodiment 1.

[0054] This comparative example also provides a dendrobium extract prepared based on this comparative example 1.

[0055] Example 3

[0056] In this example, the dendrobium extract obtained in Example 2 was analyzed and compared with the dendrobium extract obtained in Comparative Example 1. For the convenience of description, in this example, the dendrobium extract obtained in Example 2 is referred to as the un-crushed water extract of Dendrobium huoshanense, represented by UDHAE, and the dendrobium extract obtained in Comparative Example 1 is referred to as the crushed water extract of Dendrobium huoshanense, represented by CDHAE.

[0057] 1. Analysis of CDHAE and UDHAE ingredients

[0058] (1) Preliminary treatment: Weigh an appropriate amount of CDHAE and UDHAE (50-100 mg), add 1 mL of water (H2O): acetonitrile (ACN): isopropanol (IPA) (1:1:1, v / v), vortex for 60 s, low-temperature ultrasonic for 30 min, centrifuge at 12000 r / min, 4°C for 10 min, take the supernatant, place at -20°C for 1 h to precipitate protein, centrifuge at 12000 r / min, 4°C for 10 min, take the supernatant, vacuum dry, re-dissolve in 200 μL 50% ACN, vortex, centrifuge at 14000 r / min, 4°C for 15 min, and take the supernatant for detection.

[0059] (2) HPLC parameters were as follows: chromatographic column: Waters HSS T3 (100*2.1mm, 1.8μm); mobile phase: phase A was 0.1% formic acid-water solution, phase B was 0.1% formic acid-acetonitrile; flow rate was 0.3mL / min; column temperature was 40℃; injection volume was 2μL; elution gradient: 0.0-1.0min A / B (100:0V / V), 1.0-9.0min A / B (5:95V / V), 9-13min A / B (5:95V / V), 13.1-17.0min A / B (100:0V / V); during the entire analysis, CDHAE and UDHAE test samples were placed in an automatic sampler at 4℃. In order to avoid the influence of fluctuations in instrument detection signals, samples were analyzed continuously in random order. QC samples were inserted into the PCHE queue to monitor and evaluate the stability of the system and the reliability of experimental data.

[0060] (3) Mass spectrometry conditions: The primary and secondary spectra were collected using the Q Exactive HFX high-resolution mass spectrometry system from Thermo Fisher Scientific, USA.

[0061] The results showed that the water extract of Dendrobium huoshanense adopted different extraction methods, and the final extraction rate and related content determination were obtained through three parallel experiments. The results are shown in Table 1. It can be seen from the table that the extraction rate of CDHAE is higher than that of UDHAE medicinal materials. The polysaccharide content of the two was measured by phenol-sulfuric acid method. The polysaccharide content in CDHAE is higher than that in UDHAE; but for the water-soluble flavonoid content and alkaloid content, the water-soluble flavonoid content and alkaloid content of CDHAE are lower than those of UDHAE.

[0062] Table 1 Information Sheet for Determination of D.huoshanense Aqueous Extract Content

[0063]

[0064] The components of CDHAE and UDHAE were analyzed by LC-MS. The UPLC-Q-TOF / MS results showed that 39 components in CDHAE were identified. Figure 1 As shown in Table 2, these compounds are mainly flavonoids, terpenes, amino acids, etc., of which 13 are flavonoids. In addition, the UPLC-Q-TOF / MS results showed that 70 components in UDHAE were identified. Figure 2 As shown in Table 3, these compounds are mainly flavonoids, terpenes, amino acids, etc., among which 24 flavonoids

[0065] Table 2 Mass spectrometry information of CDHAE chemical components

[0066]

[0067]

[0068] Table 3 Mass spectrometry information of UDHAE chemical components

[0069]

[0070]

[0071]

[0072]

[0073] Example 4

[0074] This embodiment provides an application of a dendrobium extract, in which a dendrobium extract in Example 2 is applied to a drug for inhibiting tumor cell growth.

[0075] In this example, the cytotoxicity was detected by CCK-8 method.

[0076] (1) Cell culture: Take out mouse melanoma (B16) cells, mouse breast cancer (4T1) cells, human cervical cancer (Hela) cells, human liver cancer (HepG2) cells, human melanoma (A375) cells and human lung cancer (A549) cells frozen in liquid nitrogen, thaw in a constant temperature water bath (37°C), and transfer to a culture medium containing 1% (m:v) penicillin and streptomycin mixture and 10% fetal bovine serum (m:v). A375 and A549 cells are placed in DMEM complete culture medium, Hela and HepG2 cells are placed in MEM complete culture medium, and B16 and 4T1 cells are placed in 1640 complete culture medium. Gently blow and mix, transfer to a culture dish, and fill the culture medium to 10mL. The culture conditions are 37°C and 5% CO2. The next day, observe the growth status of the cells. When they grow to 80% of the flat bottom area of ​​the culture dish, they can be passaged (1:2 ratio). They can be used for subsequent experiments only after three generations.

[0077] (2) CDHAE and UDHAE toxicity test: Logarithmic phase cells were collected, and the cell suspension concentration was adjusted to 1×104 cells / mL and plated on a 96-well culture plate, with 100 μL per well and 200 μL PBS (Phosphate-buffered saline) in the outer circle. The culture conditions were 37°C, 5% CO2, and the culture was carried out for 24 hours. The cells were treated with different concentrations of CDHAE and UDHAE (0.5, 1, 2, 4, 8 mg / mL). The experiment set up a drug group and a blank group. Multiple replicate wells were set for each drug. 100 μL of complete medium containing the drug was added to each well and cultured for 24 hours. The next day, the culture medium was discarded, 100 μL of complete medium was added to each well, and then 10 μL of CCK-8 (CellCounting Kit-8) solution was added to each well. The culture was continued for 3 to 4 hours until there was no color change. The absorbance was measured at 450 nm using an ELISA reader, and the data was processed using Graphpad Prism software. The cell proliferation activity (%) was calculated according to the formula: proliferation activity % = (drug group / blank group) × 100%).

[0078] Results Figure 3As shown in the figure, the inhibitory effect on tumor cell proliferation was different after the medicinal materials were treated in different ways. In the dosage range of 0.5-8 mg / mL, UDHAE had an inhibitory effect on B16, 4T1, HepG2, A375, Hela and A549 cells, and there was a significant difference between 4 mg / mL and 8 mg / mL. The inhibitory effect on B16, Hela and HepG2 cells was more obvious, and the effect on B16 cells was the most obvious, IC50=1.38±0.82 mg / mL. The cell survival rate decreased with the increase of the dosage concentration, and it was dose-dependent. The Dendrobium extract at 2 mg / mL could induce B16 cell apoptosis, with an apoptosis rate of 56.53±0.47%, and could block the B16 cell cycle at the G0 / G1 phase.

[0079] There were significant differences between the high concentration (8 mg / mL) of UDHAE and CDHAE groups. CDHAE inhibited the proliferation of A375, 4TI and A549 cells, but had no inhibitory effect on B16, Hela and HepG2 cells. The inhibitory effect on A375 and 4TI cells was more obvious, and the effect on A375 cells was most obvious. Within the dosage range of 0.5-8 mg / mL, IC50=3.95±0.39 mg / mL, and the cell survival rate decreased with the increase of dosage concentration, and was dose-dependent.

[0080] Based on the results of cytotoxicity experiments, we further explored the effects of UDHAE on B16 cells and its anti-tumor effect through cell live-dead staining, flow cytometry and other experiments.

[0081] Results Figure 4 and Figure 5 As shown in the figure, the experimental results of cell live-death staining, cell apoptosis cycle, etc. showed that UDHAE can inhibit the proliferation of tumor cells and induce their apoptosis, which clearly shows that UDHAE is an important component of the anti-tumor effect of Dendrobium huoshanense.

[0082] It is easy to understand that those skilled in the art can combine, split, reorganize, etc. the embodiments of the present application to obtain other embodiments based on one or several embodiments provided in the present application, and these embodiments do not exceed the protection scope of the present application.

[0083] In conclusion, the above is only a preferred embodiment of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the patent of the present invention.

Claims

1. A method for preparing a dendrobium extract, characterized in that: It includes the following steps: Step S1, removing the roots and leaves of Dendrobium officinale, drying at 60-70° C. to a constant weight, and obtaining dried Dendrobium officinale strips; Step S2, extracting the dried Dendrobium officinale strips obtained in step S1 multiple times using a water extraction method to obtain an extract; Step S3, freeze-drying the extract to obtain freeze-dried powder of Dendrobium officinale extract.

2. The method for preparing a dendrobium extract according to claim 1, characterized in that: Step S2 specifically includes the following steps: Step S21, extracting the dried Dendrobium officinale strips by water extraction, specifically, placing the dried Dendrobium officinale strips in ultrapure water at a temperature of 70-90° C. to dissolve the extract, and the extraction time is 2-4 hours; Step S22, after filtering, obtain a primary filtrate and a primary filter residue; Step S23, extracting the primary filter residue in step S22 by the same water extraction method as in step S21, and filtering to obtain a secondary filtrate and a secondary filter residue; Step S24, repeating step S23 to obtain three filtrates and three filter residues; Step S25, combining the filtrates obtained in steps S22-S24, and then concentrating under reduced pressure to obtain an extract.

3. The method for preparing a dendrobium extract according to claim 2, characterized in that: The material-liquid ratio in steps S22 to S24 is 1:

40.

4. The method for preparing a dendrobium extract according to claim 2, characterized in that: The reduced pressure concentration in step S25 is specifically carried out by using a rotary evaporator at a rotation speed of 100 rpm / min and a water bath temperature of 60° C. to concentrate the solution under reduced pressure to 10%-15% of the original volume.

5. The method for preparing a dendrobium extract according to claim 1, characterized in that: The freeze-drying treatment in step S3 specifically includes placing the extract in a freeze dryer to freeze-dry to obtain freeze-dried powder, and then storing the freeze-dried powder in a -20°C environment for standby use.

6. A dendrobium extract, characterized in that: The extract is prepared by the method for preparing the dendrobium extract according to any one of claims 1 to 5.

7. An application of a dendrobium extract, characterized in that: The dendrobium extract according to claim 6 is used in a drug for inhibiting the growth of tumor cells.

8. The use of a dendrobium extract according to claim 7, characterized in that: The tumor cells include mouse melanoma B16 cells, human cervical cancer Hela cells, human liver cancer HepG2 cells, human lung cancer A549 cells, mouse breast cancer 4T1 cells and human melanoma A375 cells.

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