Rhododendron mucronatum extract with immunosuppression function as well as preparation method and application of rhododendron mucronatum extract

Abstract

The invention discloses a rhododendron simsii extract with immunosuppressive activity as well as a preparation method and application of the rhododendron simsii extract. The method comprises the following steps: by taking dry flower micro powder of rhododendron simsii as a raw material and 95% ethanol as an extracting agent, carrying out condensation reflux extraction for 2 hours under the conditions that the temperature is 60 DEG C and the material-liquid ratio is 1: 10, filtering and collecting an extracting solution, mixing the extracting solutions, and carrying out vacuum and vacuum concentration to obtain a rhododendron simsii extract; when the concentration of the extract is 100 mg / mL, the inhibition rates of the extract on T lymphocyte proliferation induced by concanavalin and B lymphocyte proliferation induced by lipopolysaccharide are 61.41% and 51.54% respectively, and the extract has no cytotoxicity on splenic lymphocytes of normal mice. The invention provides a candidate research material for research and development of a novel immunosuppressor, and also provides a scientific basis for development and utilization of rhododendron molle resources.

Description

Technical Field

[0001] The invention belongs to the field of medical biotechnology, and specifically relates to a Rhododendron pubescens extract with immunosuppressive function, a preparation method and application thereof. Background Art

[0002] Autoimmune diseases are chronic diseases, including rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus, and the global prevalence of patients accounts for about 9.4% of the total population. Patients usually have abnormal immune responses and inflammation, manifested as disordered immune cell populations, immune cell activation, upregulation of inflammatory mediators and tissue damage. Since the root cause of the disease is still unclear, most patients cannot be completely cured. Currently, the immunosuppressants used in clinical practice mainly include five categories: cytokine inhibitors, such as cyclosporin A (CsA), tacrolimus (FK506) and rapamycin; DNA synthesis inhibitors, such as mycophenolate mofetil; adrenocortical hormones, such as prednisone; anti-lymphocyte antibodies, such as anti-lymphocyte globulin; and other immunosuppressants, such as Sotrastaurin (AEB071), Fingolimod (FTY720), cyclophosphamide, etc. Among them, most immunosuppressants work based on T cell-related immune response signaling pathways. For example, after entering the human body, CsA and FK506 form complexes with cyclophilin and FK506 binding protein 12 (FKBP12), respectively. Both complexes can inhibit intracellular calcineurin, interfere with the T cell nuclear factor pathway, and thus inhibit T cell activation. The complex formed by rapamycin and FKBP12 in cells blocks the transmission of proliferation signals by inhibiting the phosphorylation of mTOR. Tofacitinib can inhibit the phosphorylation of JAK3, thereby exerting an immunosuppressive effect.

[0003] Rhododendron radendum Fang is a plant of the genus Rhododendron in the family Ericaceae. It is an evergreen shrub that grows mostly in mountain thickets at an altitude of 3000-4100 meters or under Pinus armandii and Quercus alpina forests. It is mainly distributed in western and southwestern Sichuan, China. This species has a high horticultural ornamental value due to its beautiful flowers. The roots, leaves, flowers and fruits of Rhododendron radendum Fang can be used as medicine, which has the effects of astringent hemostasis, swelling and pain relief, blood circulation and blood stasis, wind and dampness removal, spot lightening and whitening. It is mainly used to treat traumatic bleeding, blood stasis and swelling caused by traumatic injuries, bone pain, joint pain, limb numbness caused by rheumatic arthritis, and dull complexion caused by insufficient qi and blood. However, there are few reports on the pharmacological activities of the active ingredients and extracts of Rhododendron radendum Fang. Only one literature has conducted a preliminary identification and content determination of the flower pigment extract of Rhododendron radendum Fang. Summary of the invention

[0004] The purpose of the present invention is to provide a Rhododendron pubescens extract with immunosuppressive function and a preparation method and application thereof in view of the insufficient research on the active ingredients of Rhododendron pubescens.

[0005] The first object of the present invention is to provide a method for preparing a Rhododendron pubescens extract having an immunosuppressive function, comprising the following steps:

[0006] The fresh Rhododendron pubescens flowers are extracted with ethanol or ethanol water solution, and the extract is concentrated to obtain the Rhododendron pubescens flower extract, which is the Rhododendron pubescens extract.

[0007] Further preferred method is to extract the Rhododendron pubescens extract with petroleum ether, ethyl acetate and n-butanol in sequence, and then concentrate the extract under reduced pressure to obtain four different polarity parts of Rhododendron pubescens, namely: petroleum ether part, ethyl acetate part, n-butanol part, water part, and the ethyl acetate part is the Rhododendron pubescens extract.

[0008] Further preferably, the ethyl acetate portion is eluented with petroleum ether-ethyl acetate, and gradient elution is performed with a volume ratio of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, and 0:1. The fractions are collected and chromatographed by TLC thin layer chromatography with petroleum ether:ethyl acetate = 30:1-1:1, v / v as the developing solvent. The components with petroleum ether:ethyl acetate = 2:1, Rf = 0.5-0.6 and dark red spots shown by ethanolic sulfuric acid are collected, which is the Rhododendron pubescens extract.

[0009] Preferably, the extracting of fresh Rhododendron pubescens with ethanol or ethanol aqueous solution and concentrating the extract to obtain the Rhododendron pubescens extract is performed by freeze-drying the fresh Rhododendron pubescens, crushing it, performing condensation reflux extraction with 95% ethanol at a temperature of 60°C and a solid-liquid ratio of 1:10 g / ml, filtering and collecting the extract, and concentrating the extract under vacuum and reduced pressure to obtain the Rhododendron pubescens extract.

[0010] The second object of the present invention is to provide the use of the above Rhododendron pubescens extract in the preparation of immunosuppressants.

[0011] The third object of the present invention is to provide an immunosuppressant comprising the above Rhododendron pubescens extract as an active ingredient.

[0012] The present invention has found through experiments that the crude extract of Rhododendron pubescens and all its components have no obvious cytotoxic effect on normal mouse spleen cells, but show a certain degree of inhibitory effect on the proliferation of mouse T lymphocytes induced by ConA and mouse B lymphocytes induced by LPS. According to the immunosuppressive activity-oriented separation strategy, the highly active fraction of the crude extract of Rhododendron pubescens, the EADJ-H segment, was successfully tracked. Its inhibition rates on the proliferation of mouse T lymphocytes induced by ConA and mouse B lymphocytes induced by LPS were 73.87% and 56.97% respectively at a final concentration of 100 μg / mL; at the same time, the positive drugs cyclosporine A (CsA) and mycophenolate mofetil (MMF) at a final concentration of 20 μM inhibited the proliferation rates of T cells and B cells by 91.48% and 70.56% respectively. This result shows that the EADJ-H segment of Rhododendron pubescens has a significant immunosuppressive activity.

[0013] The present invention successfully tracked and obtained the fraction EADJ-H with significant immunosuppressive activity from the 95% ethanol extract of the dried flower powder of Rhododendronradendum Fang through an immunosuppressive activity-guided tracking and separation strategy. The combined components of this part showed significant immunosuppressive activity and are expected to be used in the research and development of combined application of immunosuppressants. It also provides candidate active components for the creation of new immunosuppressive drugs and provides a scientific basis for the development and utilization of natural active substances in the medicinal plant Rhododendronradendum Fang resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a flow chart of extraction and separation of active ingredients from Rhododendron pubescens;

[0015] Figure 2 It is EADJ-H 1 H-NMR spectrum (CDCl3);

[0016] Figure 3 It is EADJ-H 13 C-NMR spectrum (CDCl3). DETAILED DESCRIPTION

[0017] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0018] Embodiment 1:

[0019] The components of Rhododendron pubescens were extracted and separated by freeze-drying pretreatment, 95% ethanol reflux extraction, organic solvent extraction and normal silica gel column chromatography.

[0020] 1.1. Reagents, fillers and instruments used in the experiment: organic reagents such as anhydrous ethanol, petroleum ether, ethyl acetate and n-butanol were purchased from Guangzhou Chemical Reagent Factory; silica gel powder for normal phase silica gel column chromatography (60-100 mesh, 100-200 mesh and 200-300 mesh) was purchased from Qingdao Ocean Chemical Co., Ltd.; thin layer chromatography silica gel plate GF254 was purchased from Qingdao Spectrum Separation Materials Co., Ltd. Nuclear magnetic resonance spectrometer (Bruker, with TMS as internal standard, Germany), rotary evaporator (Eyela N-1100, Shanghai Ailang Instrument Co., Ltd., China), water bath (Eyela SB-1100, Shanghai Ailang Instrument Co., Ltd., China), circulating water vacuum pump (Eyela A-1000S, Japan), low temperature cooling circulating pump (DLSB-5 / 20, Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd., China), analytical balance (Precisa, Shanghai Tianmei Balance Instrument Co., Ltd., China).

[0021] 1.2 Experimental Methods

[0022] 1.2.1 Extraction and separation of active ingredients from Rhododendron pubescens

[0023] like Figure 1 As shown, first, the fresh sample of Rhododendron tomentosa was freeze-dried and crushed into a micro powder state, and 95% ethanol was used for condensation reflux extraction at a temperature of 60°C and a solid-liquid ratio of 1:10 for 2 hours, and the above operation was repeated three times. The extracts were filtered and collected three times, and then vacuum and reduced pressure concentration were performed after merging to obtain Rhododendron tomentosa extract (DJ); then, the extract was dispersed into an aqueous suspension with a certain amount of distilled water, and different polar parts were repeatedly extracted with petroleum ether, ethyl acetate and n-butanol (v:v=1:2, the volume ratio of water to organic solvent) in turn until the extract was completely clarified, and then the extract was concentrated under reduced pressure to obtain four different polar parts of Rhododendron tomentosa, namely: petroleum ether part (PEDJ), ethyl acetate (EADJ), n-butanol part (nButDJ), and water part (WDJ).

[0024] Subsequently, according to the screening results of immunosuppressive activity, the ethyl acetate (EADJ) fraction with the most significant activity was subjected to normal-phase silica gel column chromatography. Petroleum ether - ethyl acetate was used as the eluent, and the ethyl acetate fraction (EADJ) of the Rhododendron moulmainense extract was gradient eluted at volume ratios of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, and 0:1. The fractions were collected, and then they were combined according to the Rf values of TLC thin-layer chromatography (petroleum ether: ethyl acetate = 30:1 - 1:1, v / v) and the color development of different components. After vacuum concentration, the following 10 components were obtained: EADJ-A, EADJ-B, EADJ-C, EADJ-D, EADJ-E, EADJ-F, EADJ-G, EADJ-H, EADJ-I, and EADJ-J. Finally, the above fractions were tested for cytotoxicity and immunosuppressive activity on normal mouse splenocytes in vitro to accurately lock in the strongly active EADJ-H segment (petroleum ether: ethyl acetate = 2:1, Rf = 0.5 - 0.6, dark red spots appeared with sulfuric acid - ethanol), and preliminary 1H NMR and 13C NMR analyses were performed on this segment ( Figure 2 and 3 ).

[0025] Example 2:

[0026] The MTT method was used to screen the immunosuppressive activity of the total extract of Rhododendron moulmainense, different extraction fractions, and different elution fractions.

[0027] 2.1 Test reagents: RPMI - 1640 medium. All fractions of the Rhododendron moulmainense extract were dissolved in dimethyl sulfoxide (DMSO) and diluted to the required final concentration: 100 μg / mL. The stimulators were selected as the T lymphocyte stimulator concanavalin A (ConA) and the B lymphocyte stimulator lipopolysaccharide (LPS) solution with a final concentration of 5 μg / mL. The positive control drugs were selected as the T lymphocyte inhibitor cyclosporine A (CsA) and the B lymphocyte inhibitor mycophenolate mofetil (MMF) solution with a final concentration of 20 μg / mL.

[0028] The BALB / c mice used in this experiment were purchased from the Guangdong Provincial Laboratory Animal Center, with the batch number: SCXK (Yue) 2022 - 0002.

[0029] 2.2 Experimental method

[0030] 2.2.1 Preparation of mouse splenocytes

[0031] Male BALB / c mice aged 6-8 weeks and weighing about 20g were killed by cervical dislocation in a clean bench, and the body surface was disinfected with 75% medical alcohol for 3-5 minutes. The mouse spleen was removed with high-temperature sterilized surgical forceps and placed in a Mesh net for grinding. It was continuously rinsed with PBS phosphate and filtered to obtain a spleen cell suspension. The cell suspension was centrifuged for 5 minutes (speed 1000rpm) and the supernatant was discarded. After adding an appropriate amount of red blood cell lysis solution and lysing in the dark for 1 minute, 8mL of RPMI-1640 medium was immediately added to terminate the lysis reaction, and centrifuged for 5 minutes (speed 1000rpm), and the supernatant was discarded. Then it was washed twice with RPMI-1640 medium, centrifuged at 1000rpm for 5min, and the supernatant was discarded to completely remove the red blood cell lysis solution. RPMI-1640 medium containing 10% fetal bovine serum was added to resuspend the cells for dilution and count.

[0032] 2.2.2 Cytotoxic effects of the test samples on normal mouse spleen cells

[0033] The spleen cell suspension was added at 1×10 6 The concentration of cells was inoculated on a 96-well plate (180 μL / well). 20 μL of PBS buffer containing 5% DMSO was added to each well of the blank control group, 20 μL of the test sample with a final concentration of 100 μg / mL was added to each well of the experimental group, and 20 μL of CsA or MMF with a final concentration of 20 μM was added to the positive control group. After 36 hours of action, 25 μL of MTT solution with a concentration of 5 mg / mL was added to each well. After incubation in a 37°C incubator for 4 hours, the 96-well plate was centrifuged at 3000 r / min for 10 minutes, the supernatant was discarded, 100 μL of DMSO solvent was added to each well, and it was shaken slowly on a shaker in the dark for 15 minutes. After the formazan crystals were completely dissolved, the OD value at 490 nm was measured using an enzyme marker.

[0034] The cell survival rate was calculated as follows: survival rate (%) = mean OD value of the drug administration group / mean OD value of the blank control group × 100%.

[0035] 2.2.3 Inhibitory effect of the test samples on ConA and LPS-induced T and B lymphocyte proliferation

[0036] In this experiment, ConA and LPS were used to induce T and B lymphocyte proliferation in vitro to screen the immunosuppressive activity of the test samples. The spleen lymphocyte suspension was prepared after lysing red blood cells according to the above scheme, and then the concentration of 5×10 5Cells were inoculated on 96-well plates, 180 μL per well, of which no ConA or LPS inducer was added as the non-model group; ConA with a final concentration of 5 μg / mL or LPS stimulator was added as the model group, which was used to simulate T and B cell proliferation and differentiation, respectively. 20 μL of PBS buffer containing 5% DMSO was added to each well of the non-model group and the negative control group of the model group, and 20 μL of different concentrations of selected samples and positive drugs (CsA or MMF) were added to each well of the stimulator group. After 36 hours of incubation in an incubator at 37°C with 5% CO2, 25 μL of 5 mg / mL MTT solution was added to each well. After 4 hours of action, the 96-well plate was centrifuged at 3000 r / min for 10 minutes and the supernatant was discarded. 100 μL of DMSO was added to each well, and it was shaken slowly on a shaker in the dark for 15 minutes. After the formazan crystals were completely dissolved, the OD value at 490 nm was measured with an enzyme marker. The proliferation inhibition rate was calculated as follows: proliferation inhibition rate (%) = (mean OD of negative control in model group - mean OD of test group) / (mean OD of negative control in model group - mean OD of negative control in non-model group) × 100%, with three replicates for each sample.

[0037] 2.2.4, Experimental results: As shown in Table 2, the crude extract of Rhododendron pubescens and all its components had no obvious cytotoxic effect on normal mouse spleen cells. Subsequently, according to the immunosuppressive activity-oriented separation strategy, the highly active fraction of the crude extract of Rhododendron pubescens, the EADJ-H segment, was successfully tracked. As shown in Table 2, the EADJ-H segment fraction with a final concentration of 100 μg / mL had an inhibition rate of 73.87% and 56.97% on the proliferation of mouse T lymphocytes induced by ConA and mouse B lymphocytes induced by LPS, respectively; at the same time, the positive drugs cyclosporine A (CsA) and mycophenolate mofetil (MMF) with a final concentration of 20 μM had an inhibition rate of 91.48% and 70.56% on the proliferation rate of T cells and B cells, respectively. This result shows that the EADJ-H segment of Rhododendron pubescens has a significant immunosuppressive activity. Therefore, the present invention provides candidate research materials for the research and development of new immunosuppressants, and also provides a scientific basis for the development and utilization of Rhododendron pubescens resources.

[0038] Table 2 Cytotoxic effects on mouse lymphocytes and immunosuppressive effects on ConA (5 μg / mL) and LPS (10 μg / mL)-induced mouse lymphocyte proliferation

[0039]

[0040]

[0041] Note: Data are expressed as mean ± SD (n = 3), CsA is the positive control drug for T cell proliferation model, MMF is the positive control drug for B cell proliferation model, NT means not tested.

Claims

1. A method for preparing a Rhododendron pubescens extract having an immunosuppressive function, characterized in that: The following steps are involved: The fresh Rhododendron pubescens flowers are extracted with ethanol or ethanol-water solution, and the extract is concentrated to obtain the Rhododendron pubescens flower extract, which is the Rhododendron pubescens extract.

2. The preparation method according to claim 1, characterized in that: The method comprises extracting the Rhododendron pubescens with petroleum ether, ethyl acetate and n-butanol in sequence, and then concentrating the extract under reduced pressure to obtain four different polarity parts of Rhododendron pubescens, which are: petroleum ether part, ethyl acetate part, n-butanol part and water part. The ethyl acetate part is the Rhododendron pubescens extract.

3. The preparation method according to claim 2, characterized in that: The ethyl acetate fraction was eluted with petroleum ether-ethyl acetate at a volume ratio of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, and 0:1 for gradient elution. The fractions were collected and chromatographed by TLC with petroleum ether:ethyl acetate = 30:1-1:1, v / v as the developing solvent. The components with petroleum ether:ethyl acetate = 2:1, Rf = 0.5-0.6 and dark red spots shown by ethanolic sulfuric acid were collected, which was the Rhododendron pubescens extract.

4. The preparation method according to claim 1, characterized in that: The method of extracting fresh Rhododendron pubescens with ethanol or ethanol aqueous solution and concentrating the extract to obtain Rhododendron pubescens extract is as follows: freeze-drying the fresh Rhododendron pubescens, crushing it, using 95% ethanol at a temperature of 60 DEG C and a solid-liquid ratio of 1:10 g / ml to perform condensation reflux extraction, filtering and collecting the extract, and concentrating the extract under vacuum and reduced pressure to obtain the Rhododendron pubescens extract.

5. A Rhododendron pubescens extract obtained according to the preparation method of claim 1, 2, 3 or 4.

6. Use of the Rhododendron pubescens extract according to claim 5 in the preparation of immunosuppressants.

7. An immunosuppressant, characterized in that The invention comprises the Rhododendron pubescens extract according to claim 5 as an active ingredient.

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