A rhododendron extract having immunosuppressive function, and a preparation method and application thereof

Abstract

The application discloses a Rhododendron molle flower extract with immunosuppressive activity and a preparation method and application thereof. The Rhododendron molle flower extract is prepared by taking dried flower powder of Rhododendron molle as raw material and 95% ethanol as an extracting agent under the following conditions: condensation reflux extraction for 2 hours at 60 DEG C and a material-liquid ratio of 1:10. The extract is filtered and collected, and the above extract is combined and concentrated under vacuum and reduced pressure to obtain the Rhododendron molle flower extract. The extract has an inhibitory rate of 61.41% on T lymphocyte proliferation induced by abrin and an inhibitory rate of 51.54% on B lymphocyte proliferation induced by lipopolysaccharide at a concentration of 100 mg / mL, and has no cytotoxicity on normal mouse spleen lymphocytes. The application provides a candidate research material for researching and developing a new immunosuppressant, and provides a scientific basis for developing and utilizing the Rhododendron molle resources.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to an extract of Rhododendron molle with immunosuppressive function, its preparation method, and its application. Background Technology

[0002] Autoimmune diseases are chronic conditions, including rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, affecting approximately 9.4% of the global population. Patients typically exhibit abnormal immune responses and inflammation, characterized by immune cell dysbiosis, immune cell activation, upregulation of inflammatory mediators, and tissue damage. Because the underlying causes are not fully understood, most patients cannot be completely cured. Currently, clinically used immunosuppressants mainly fall into five categories: cytokine inhibitors, such as cyclosporin A (CsA), tacrolimus (FK506), and rapamycin; DNA synthesis inhibitors, such as mycophenolate mofetil; corticosteroids, such as prednisone; anti-lymphocyte antibodies, such as anti-lymphocyte globulins; and other immunosuppressants, such as sotrastaurin (AEB071), fingolimod (FTY720), and cyclophosphamide. Most of these immunosuppressants function based on T-cell-related immune signaling pathways. For example, after CsA and FK506 enter the human body, they form complexes with cyclin and FK506-binding protein 12 (FKBP12), respectively. Both of these complexes can inhibit intracellular calcineurin and interfere with the T cell nuclear factor pathway, thereby inhibiting T cell activation. Rapamycin, on the other hand, forms a complex with FKBP12 in cells by inhibiting mTOR phosphorylation, thus blocking the transmission of proliferation signals. Tofacitinib exerts its immunosuppressive effect by inhibiting JAK3 phosphorylation.

[0003] *Rhododendron radendum* Fang is an evergreen shrub belonging to the genus *Rhododendron* in the family Ericaceae. It typically grows in mountain thickets or under forests of *Pinus armandii* and *Quercus acutissima* at altitudes of 3000-4100 meters. It is mainly distributed in western and southwestern Sichuan, China. This species has high ornamental value due to its beautiful flowers. The roots, leaves, flowers, and fruits of *Rhododendron radendum* Fang are all used medicinally, possessing astringent, hemostatic, anti-inflammatory, analgesic, blood-activating, stasis-removing, wind-dispelling, dampness-removing, and skin-whitening effects. It is mainly used to treat traumatic bleeding and swelling caused by falls and injuries, bone pain, joint pain, and limb numbness caused by rheumatic arthritis, as well as dull complexion and lack of radiance due to insufficient qi and blood. However, there are few reports on the active ingredients and pharmacological activities of extracts from *Rhododendron radendum* Fang; only one article provides a preliminary identification and content determination of the anthocyanin extract. Summary of the Invention

[0004] The purpose of this invention is to address the lack of research on the active components of Rhododendron molle and to provide an extract of Rhododendron molle with immunosuppressive function, its preparation method, and its application.

[0005] The first objective of this invention is to provide a method for preparing an extract of Rhododendron molle with immunosuppressive function, comprising the following steps:

[0006] Fresh Rhododendron molle is extracted with ethanol or an aqueous ethanol solution. The extract is then concentrated to obtain Rhododendron molle extract, which is the Rhododendron molle extract.

[0007] Further optimization involves sequentially extracting the extract of Rhododendron molle with petroleum ether, ethyl acetate, and n-butanol, followed by concentrating the extract under reduced pressure to obtain four different polar fractions of Rhododendron molle: petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. The ethyl acetate fraction is the Rhododendron molle extract.

[0008] Further optimization involved using petroleum ether-ethyl acetate as the eluent in a gradient elution at volume ratios of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, and 0:1. The fractions were collected and subjected to TLC thin-layer chromatography with petroleum ether:ethyl acetate = 30:1-1:1, v / v as the developing solvent. The fraction exhibiting a dark red spot when petroleum ether:ethyl acetate = 2:1, Rf = 0.5-0.6, and sulfuric acid ethanol was collected; this fraction was identified as the extract of Rhododendron molle.

[0009] Preferably, the extraction of fresh Rhododendron molle with ethanol or an aqueous ethanol solution, followed by concentration of the extract to obtain Rhododendron molle extract, involves freeze-drying and pulverizing the fresh Rhododendron molle, then performing reflux extraction with 95% ethanol at 60°C and a material-to-liquid ratio of 1:10 g / ml. The extract is then collected by filtration and vacuum-depressurized concentration to obtain Rhododendron molle extract.

[0010] A second objective of this invention is to provide the application of the above-mentioned Rhododendron simsii extract in the preparation of immunosuppressants.

[0011] A third objective of this invention is to provide an immunosuppressant comprising the above-mentioned Rhododendron molle extract as an active ingredient.

[0012] This invention, through experiments, revealed that the crude extract of *Rhododendron simsii* and all its components had no significant cytotoxic effect on normal mouse spleen cells, but exhibited a certain degree of inhibitory effect on the proliferation of ConA-induced mouse T lymphocytes and LPS-induced mouse B lymphocytes. Using an immunosuppressive activity-guided separation strategy, the highly active fraction of the crude extract, the EADJ-H segment, was successfully identified. At a final concentration of 100 μg / mL, this fraction inhibited the proliferation of ConA-induced mouse T lymphocytes and LPS-induced mouse B lymphocytes by 73.87% and 56.97%, respectively. Simultaneously, the positive control agents cyclosporine A (CsA) and mycophenolate mofetil (MMF) at a final concentration of 20 μM inhibited the proliferation of T cells and B cells by 91.48% and 70.56%, respectively. These results indicate that the EADJ-H segment of *Rhododendron simsii* possesses significant immunosuppressive activity.

[0013] This invention utilizes an immunosuppressive activity-guided separation strategy to successfully isolate the fraction EADJ-H, which exhibits significant immunosuppressive activity, from the 95% ethanol extract of dried flower powder of Rhododendron radendum Fang. The combined components of this fraction demonstrate significant immunosuppressive activity and hold promise for research and development of combined immunosuppressant applications. Furthermore, it provides candidate active components for the creation of novel immunosuppressive drugs and offers a scientific basis for the development and utilization of natural active substances from the medicinal plant Rhododendron radendum. Attached Figure Description

[0014] Figure 1 This is a flowchart of the extraction and separation process of active ingredients from Rhododendron molle.

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

[0016] Figure 3 It is the EADJ-H flow. 13 C-NMR spectrum (CDCl3). Detailed Implementation

[0017] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0018] Example 1:

[0019] The components of Rhododendron molle 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, packing materials, and instruments used in the experiment: Anhydrous ethanol, petroleum ether, ethyl acetate, and n-butanol, among other organic reagents, were purchased from Guangzhou Chemical Reagent Factory; normal-phase silica gel column chromatography silica gel powder (60-100 mesh, 100-200 mesh, and 200-300 mesh) was purchased from Qingdao Ocean Chemical Co., Ltd.; thin-layer chromatography silica gel plates GF254 were purchased from Qingdao Spectro-Separation Materials Co., Ltd. The following instruments were used: 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), cryogenic cooling circulating pump (DLSB-5 / 20, Zhengzhou Great Wall Science & Industry Trade Co., Ltd., China), and 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 molle

[0023] like Figure 1 As shown, firstly, fresh Rhododendron molle was freeze-dried and pulverized into a fine powder. Then, it was extracted with 95% ethanol at 60°C and a material-to-liquid ratio of 1:10 under reflux for 2 hours. This process was repeated three times. The extracts from the three extractions were filtered and collected, combined, and then concentrated under vacuum to obtain Rhododendron molle extract (DJ). Subsequently, the extract was dispersed into an aqueous suspension with a certain amount of distilled water. Different polar fractions were then repeatedly extracted with petroleum ether, ethyl acetate, and n-butanol (v:v = 1:2, volume ratio of water to organic solvent) until the extract was completely clear. The extract was then concentrated under reduced pressure to obtain four different polar fractions of Rhododendron molle: petroleum ether fraction (PEDJ), ethyl acetate fraction (EADJ), n-butanol fraction (nButDJ), and aqueous fraction (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 a volume ratio of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, 0:1. The fractions were collected, and then combined according to the Rf value 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, EADJ-J; Finally, the above fractions were tested for cytotoxicity and immunosuppressive activity on normal mouse splenocytes in vitro, so as to accurately lock the strongly active fraction EADJ-H segment (petroleum ether: ethyl acetate = 2:1, Rf = 0.5 - 0.6, sulfuric acid ethanol shows dark red spots), and preliminary nuclear magnetic hydrogen spectrum and carbon spectrum analysis were carried out 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, different extraction fractions and different elution fractions of Rhododendron moulmainense.

[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 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 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, batch number: SCXK(Guangdong)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 approximately 20g, were euthanized by cervical dislocation in a laminar flow hood. Their bodies were disinfected with 75% medical alcohol for 3-5 minutes. The spleens were removed using sterilized surgical forceps and ground in a mesh. The cells were continuously washed with PBS phosphate and filtered to obtain a spleen cell suspension. The cell suspension was centrifuged for 5 minutes (1000 rpm), and the supernatant was discarded. An appropriate amount of erythrocyte lysis buffer was added, and lysis was carried out in the dark for 1 minute. Immediately afterward, 8 mL of RPMI-1640 medium was added to terminate the lysis reaction, and the cells were centrifuged for 5 minutes (1000 rpm), and the supernatant was discarded. The cells were then washed twice with RPMI-1640 medium and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to completely remove the erythrocyte lysis buffer. The cells were resuspended and diluted in RPMI-1640 medium containing 10% fetal bovine serum, and then counted.

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

[0033] The above spleen cell suspension was prepared at a ratio of 1×10⁴ cells per well. 6 Cells were seeded at a concentration of 180 μL / well in a 96-well plate. For the control group, 20 μL of PBS buffer containing 5% DMSO was added to each well. For the experimental group, 20 μL of the test sample with a final concentration of 100 μg / mL was added to each well. For the positive control group, 20 μL of CsA or MMF with a final concentration of 20 μM was added to each well. After 36 h of incubation, 25 μL of MTT solution with a concentration of 5 mg / mL was added to each well. After incubation at 37°C for 4 h, the 96-well plate was centrifuged at 3000 rpm for 10 minutes, the supernatant was discarded, and 100 μL of DMSO solvent was added to each well. The plate was then shaken slowly on a shaker in the dark for 15 min until the formazan crystals were completely dissolved. The OD value at 490 nm was measured using a microplate reader.

[0034] The cell survival rate is calculated as follows: Survival rate (%) = Mean OD value of the drug treatment group / Mean OD value of the blank control group × 100%.

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

[0036] This experiment used in vitro models of T and B lymphocyte proliferation induced by ConA and LPS, respectively, to screen and measure the immunosuppressive activity of samples. A suspension of lysed erythrocytes was prepared according to the above protocol, and then a concentration of 5 × 10⁻⁶ was determined. 5180 μL of cells were seeded into each well of a 96-well plate. The non-model group received no ConA or LPS inducers; the model group received either 5 μg / mL ConA or 10 μg / mL LPS stimulants to simulate T and B cell proliferation and differentiation, respectively. 20 μL of PBS buffer containing 5% DMSO was added to each well in the non-model and negative control groups. 20 μL of different concentrations of selected samples and positive control agents (CsA or MMF) were added to each well in the stimulant groups. After culturing for 36 h at 37°C with 5% CO2, 25 μL of 5 mg / mL MTT solution was added to each well. After 4 h of incubation, the 96-well plate was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and 100 μL of DMSO was added to each well. The plate was then slowly shaken on a shaker in the dark for 15 min until the formazan crystals were completely dissolved. The OD value at 490 nm was measured using a microplate reader. 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: The results are shown in Table 2. The crude extract of *Rhododendron pubescens* and all its components showed no significant cytotoxic effect on normal mouse spleen cells. Subsequently, based on the immunosuppressive activity-guided separation strategy, the highly active fraction of the crude extract, the EADJ-H fraction, was successfully identified. As shown in Table 2, the EADJ-H fraction with a final concentration of 100 μg / mL inhibited the proliferation of ConA-induced mouse T lymphocytes and LPS-induced mouse B lymphocytes by 73.87% and 56.97%, respectively. Simultaneously, the positive control agents cyclosporine A (CsA) and mycophenolate mofetil (MMF) with a final concentration of 20 μM inhibited the proliferation of T cells and B cells by 91.48% and 70.56%, respectively. These results indicate that the EADJ-H fraction of *Rhododendron pubescens* possesses significant immunosuppressive activity. Therefore, this invention provides candidate research materials for the study and development of novel 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: All data are expressed as mean ± SD (n = 3). CsA is the positive control drug for the T cell proliferation model, MMF is the positive control drug for the B cell proliferation model, and NT indicates not tested.

Claims

1. A method for preparing an extract of Rhododendron molle with immunosuppressive function, characterized in that, Includes the following steps: Fresh Rhododendron molle was freeze-dried, pulverized, and extracted by reflux with 95% ethanol at 60°C and a material-to-liquid ratio of 1:10 g / ml. The extract was collected by filtration and then concentrated under vacuum and reduced pressure to obtain Rhododendron molle extract. The extract of Rhododendron molle was extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The extract was then concentrated under reduced pressure to obtain four different polar fractions of Rhododendron molle: petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction. The ethyl acetate fraction was eluted using petroleum ether-ethyl acetate gradients at volume ratios of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, and 0:

1. The fractions were collected and subjected to TLC with petroleum ether:ethyl acetate ratios of 30:1–1:1 (v / v). The fraction exhibiting a dark red spot when petroleum ether:ethyl acetate ratios of 2:1 (Rf = 0.5–0.6) and sulfuric acid ethanol was collected; this fraction was the extract of Rhododendron molle.

2. An extract of Rhododendron molle obtained according to the preparation method of claim 1.

3. An immunosuppressant, characterized in that, The active ingredient includes the rhododendron extract as described in claim 2.

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