Combination therapy of glucocorticoid with ginsenoside

Abstract

The invention relates to a combination therapy of glucocorticoid and ginsenoside. The present invention relates to a ginsenoside formulation comprising a PPT-type ginsenoside at least partially integrated in a micelle, preferably at least partially integrated in the outer layer of the micelle, said micelle having a molecular weight of at least 10,000 Da, and to ginsenoside for use in a method for preventing or treating side effects of a glucocorticoid (GC) treatment, the methods comprise administering ginsenoside to a subject suffering from or at risk of suffering from one or more GC treatment side effects. The present invention also relates to a combination of glucocorticoid (GC) and ginsenoside for use in a method of preventing or treating inflammatory diseases, wherein the method comprises administering GC and PPT ginsenoside in specific molar ratios.

Description

[0001] This invention relates to ginsenosides for treating the side effects of glucocorticoid (GC) therapy, combinations of GC and ginsenosides for treating inflammatory diseases, ginsenoside preparations, and pharmaceutical compositions comprising ginsenosides and GC. The invention also relates to methods for preparing such ginsenoside preparations and pharmaceutical compositions, and products obtainable by methods for preparing ginsenoside preparations.

[0002] GCs are widely used to treat overactive inflammatory responses and chronic inflammatory diseases [1-3]. For example, GCs such as dexamethasone, prednisolone, and beclomethasone are synthetic analogs of the steroid hormone cortisol, the main GC hormone secreted by the adrenal glands in our bodies after stress. GCs can diffuse across the cell membrane and bind to and activate intracellular receptors—glucocorticoid receptors (GRs) [1-3]. Upon activation, GR dimers translocate to the cell nucleus, where they act as transcription factors to regulate the transcription of a wide range of target genes. They regulate gene transcription through several mechanisms. First, they bind to DNA sequences called glucocorticoid response elements (GREs), thereby altering the transcription of response genes [1-3]. This mode of action is called transactivation because, in most cases, it results in an increase in gene transcription. Second, GRs can induce alterations in gene transcription by interacting with other transcription factors, such as NF-κB and AP-1, and thereby regulate their activity. Because this type of activity is inhibitory in most cases, this mechanism is generally referred to as transinhibition [4-6].

[0003] Genetic cytokines (GCs) are potent immunosuppressants and anti-inflammatory drugs. They inhibit the production of pro-inflammatory cytokines and other effector molecules, alter monocyte recirculation, and induce lymphocyte death [5,6]. Traditionally, the anti-inflammatory function of GCs has been based on the trans-inhibitory activity of glycogenophores (GRs), but recent years have clearly demonstrated that the transactivation of anti-inflammatory genes also plays an important role. The main problem limiting the clinical use of GCs, especially in patients requiring long-term treatment, is the severity of their side effects, which include diabetes, decreased cortisol levels, slowed growth, impaired wound healing, tissue degeneration, osteoporosis, hypertension, weight gain, and muscle weakness [5-11]. Most of the side effects of GCs are caused by the transactivation activity of GRs [7-9], although the trans-inhibitory activity of GRs has also been shown to cause decreased cortisol levels and osteoporosis.

[0004] Another problem associated with treatment for chronic GC is resistance to the therapy. Over time, the therapeutic effect may diminish due to decreased GR activity, and higher doses and longer treatment durations are often required to elicit anti-inflammatory activity, which may exacerbate side effects

[12] . Although the exact mechanisms underlying acquired GC resistance remain to be fully elucidated, ligand-mediated GR downregulation (homology downregulation) is considered a hallmark of acquired GC resistance [12,13].

[0005] Therefore, there is an urgent need to develop more selective anti-inflammatory GC drugs that maintain anti-inflammatory activity without causing serious side effects and GC therapy resistance.

[0006] The inventors recognized that one or more of the above-mentioned drawbacks could be overcome by administering ginsenosides to subjects who are suffering from or at risk of suffering from the side effects of GC treatment.

[0007] Therefore, the present invention relates to ginsenosides in a method for preventing or treating side effects of GC treatment, the method comprising administering ginsenosides to a subject who is suffering from one or more side effects of GC treatment or who is at risk of suffering from one or more side effects of GC treatment.

[0008] The inventors further realized that when GC and ginsenosides are applied in a specific molar ratio, the side effects of GC treatment can be effectively prevented or treated while maintaining the anti-inflammatory effect.

[0009] Therefore, the present invention also relates to a pharmaceutical composition comprising GC, ginsenosides and a pharmaceutically acceptable carrier, wherein the molar ratio of GC to ginsenosides is from about 1:1 to about 1:2000.

[0010] The inventors further recognized that the solubility of the ginsenosides, and optionally the GC, can be significantly improved when the ginsenosides are at least partially integrated into micelles. This improved solubility is particularly advantageous when relatively high concentrations of ginsenosides are required, for example, to enable the application of relatively high concentrations of ginsenosides to a subject in need, or to facilitate the preparation of pharmaceutical compositions containing ginsenosides.

[0011] Therefore, the present invention also relates to a ginsenoside formulation comprising PPT-type ginsenosides at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, wherein the micelles have a molecular weight of at least 10,000 Da. Attached Figure Description

[0012] Figure 1In a zebrafish caudal fin trauma assay, Rg1 induced an additive anti-inflammatory effect against beclomethasone. (A) Chemical structures of synthesized GC beclomethasone and ginsenoside Rg1, with glucose groups indicated in blue. (B) Schematic diagram of the caudal fin trauma assay design. At 72 hours post-fertilization (hpf), Tg(mpx:GFP) was... i114 / mpeg1:mCherry umsF001 (C) Zebrafish larvae of strain 1 were treated with (different combinations of) compounds for 6 hours (2 hours before and 4 hours after trauma). Caudal fin trauma was performed at the indicated location (red line) 74 hours after fertilization. Larvae were imaged using a fluorescence microscope 78 hours after fertilization, and the area used for quantifying fluorescently labeled neutrophils and macrophages (i.e., the area behind the caudal fin vein) is indicated (red box). (C) Effects of solvent (Veh), different doses of beclomethasone (Bec), Rg1, and 10 μM beclomethasone combined with different doses of Rg1 on the number of neutrophils migrating to the trauma site. Data shown (mean ± SEM) are pooled data from three independent experiments, with 15 larvae per group per experiment. (D) Number of neutrophils and macrophages migrating to the trauma site after treatment with solvent, 10 μM beclomethasone, 50 μM Rg1, or 10 μM beclomethasone combined with 50 μM Rg1. The data shown (mean ± SEM) are pooled data from three independent experiments, with 20 larvae per group per experiment. (E) Representative fluorescence microscopy images of wound-induced (GFP-labeled) neutrophil migration after treatment with solvent, beclomethasone, dexamethasone, Rg1, or Rg1 combined with beclomethasone. Scale bar: 100 μm. (F) Effects of beclomethasone, Rg1, and beclomethasone combined with Rg1 on il1b and il6 expression. qPCR expression analysis was performed using total RNA from unwound and wounded larvae treated with solvent, 10 μM beclomethasone, 50 μM Rg1, or 10 μM beclomethasone combined with 50 μM Rg1. Relative expression levels in (F) are normalized to ppial expression levels in zebrafish. The data shown are mean ± SEM from three independent experiments (each experiment performed in triplicate, with two being technical replicates). The statistical significance of C, D and F is shown below: compared with the trauma solvent group, P<0.05 (*), 0.01 (**) and 0.001 (***); compared with the beclomethasone group, the combined treatment P<0.05 (#), 0.01 (##) and 0.001 (###).

[0013] Figure S1(A) Representative fluorescence microscopy images of wound-induced migration of (GFP-labeled) neutrophils and (mCherry-labeled) macrophages after treatment with solvent, beclomethasone (10 μM), Rg1 (50 μM), or Rg1 (50 μM) combined with beclomethasone (10 μM). Scale bar: 100 μm. White boxes indicate counting areas. (B) Representative fluorescence microscopy images of wound-induced neutrophil migration after treatment with dexamethasone (10 μM) or Rg1 (50 μM) combined with dexamethasone (10 μM). Scale bar: 100 μm. (C) Number of neutrophils migrating to the wound site after treatment with solvent, 10 μM dexamethasone, or 10 μM dexamethasone combined with 50 μM Rg1. Data shown (mean ± SEM) are pooled data from three independent experiments, with 15 larvae used per group per experiment. The statistical significance in (C) is as follows: compared with the solvent group, P<0.001 (***); compared with the dexamethasone group, the combined treatment P<0.001 (###).

[0014] Figure 2Rg1 binds to the GR receptor and triggers an anti-inflammatory effect superimposed on beclomethasone in the HeLa cell line. (A) Relative GR binding affinity was determined in vitro using the PolarScreen glucocorticoid receptor (GR) competition assay. The fluorescence polarization level is plotted in the figure, reflecting the binding of the fluorescent ligand, which was competitively displaced from the receptor by increasing concentrations of the compounds beclomethasone (Bec), dexamethasone (Dex), and Rg1. The IC50 value for each compound is also indicated in the figure. The data shown are mean ± SEM from two independent experiments (each performed in triplicate). (B) Nuclear translocation level of GR in HeLa cells after treatment with beclomethasone and Rg1 (determined using immunocytochemistry and confocal microscopy) and relative translocation level (mean ± SEM from three independent experiments, each performed in triplicate). (C) Representative confocal microscopy images of GR nuclear translocation levels in HeLa cells after treatment with beclomethasone and Rg1, showing GR (green) and DAPI staining (blue). (D) Effects of beclomethasone, Rg1, and beclomethasone combined with Rg1 on IL1B and IL8 expression. Expression analysis was performed using total RNA from HeLa cells by qPCR, with untreated HeLa cells, cells treated with TNF-α, and cells co-treated with solvent, 0.01 μM beclomethasone, 20 μM Rg1, or 0.01 μM beclomethasone combined with 20 μM Rg1. Relative expression levels were normalized to the expression levels of 18S rRNA in HeLa cells and are displayed on a log2 scale. Data in A are mean ± SEM from two independent experiments (each performed in duplicate), and data in B and D are mean ± SEM from three independent experiments (each performed in triplicate, with two technical replicates). The statistical significance of A and D is shown below: compared with the trauma solvent group, P<0.05 (*), 0.01 (**) and 0.001 (***); compared with the beclomethasone group, the combined treatment P<0.05 (#), 0.01 (##) and 0.001 (###).

[0015] Figure S2Rg1 alleviates the side effects induced by beclomethasone and dexamethasone. (A) Representative image of the caudal fin of zebrafish larvae at 5 days post-fertilization (dpf), who were traumatized 2 days post-fertilization and treated with Rg1 (50 μM) alone or in combination with beclomethasone (10 μM) or dexamethasone (10 μM). Scale bar: 100 μm. (B) Caudal fin length regenerated 5 days post-fertilization after truncation 2 days post-fertilization and treatment with solvent (Veh), 10 μM dexamethasone (Dex), or 10 μM dexamethasone in combination with 50 μM Rg1 from 0 to 5 days post-fertilization. (C) Tg(9xGCRE-HSV.UI23:EGFP) 24 hours after exposure to the treatment shown 2 days post-fertilization. ia20 The relative EGFP intensity of zebrafish larvae in the reporting strain. The data (mean ± SEM) shown in B and C are pooled data from three independent experiments, with 15 larvae per group per experiment. Statistical significance in C is as follows: P < 0.001 (***) compared with the solvent group; P < 0.001 (###) compared with the dexamethasone group.

[0016] Figure 3Rg1 combined treatment significantly reduced beclomethasone-induced side effects. (A) Caudal fin length 5 days post-fertilization after caudal fin trauma 2 days post-fertilization, and treatment with solvent (Veh), 10 μM beclomethasone (Bec), 50 μM Rg1, or 10 μM beclomethasone combined with 50 μM Rg1 from 0 to 5 days post-fertilization. Data shown (mean ± SEM) are pooled data from three independent experiments, with 15 larvae per group per experiment. (B) Larval length 5 days post-fertilization after 5 days of treatment with solvent, beclomethasone, Rg1, or beclomethasone combined with Rg1. Data shown (mean ± SEM) are pooled data from three independent experiments, with 15 larvae per group per experiment. (C, D) Systemic glucose levels were determined colorimetrically from samples taken from larvae 5 days post-fertilization. Larvae were treated with solvent, 10 μM beclomethasone, 50 μM Rg1, or 10 μM beclomethasone combined with 50 μM Rg1 from day 0 to day 4 post-fertilization (in C), and with different doses of beclomethasone (0.1 μM, 1 μM, 5 μM, 10 μM, and 15 μM) alone or in combination with 50 μM Rg1 (in D). Data shown are mean ± SEM values ​​from three independent experiments (each experiment performed in triplicate, with two being technical replicates). (E, F) Systemic cortisol levels, measured by ELISA from samples of larvae 5 days post-fertilization, treated from day 0 to 4 post-fertilization with solvent, 10 μM beclomethasone, 50 μM Rg1, or 10 μM beclomethasone combined with 50 μM Rg1 (in E), and with different doses of beclomethasone (0.1 μM, 1 μM, 5 μM, 10 μM, and 15 μM) alone or in combination with 50 μM Rg1 (in F). (G) Tg(9xGCRE-HSV.UI23:EGFP) 24 hours after exposure to the treatments shown (starting 2 days post-fertilization). ia20 The relative EGFP intensity of zebrafish larvae (3 days post-fertilization) in the reporting strain. Data shown (mean ± SEM) are pooled from three independent experiments, each using 15 larvae. (H) Tg(9xGCRE-HSV.UI23:EGFP) 24 hours after exposure to the treatment shown (starting 2 days post-fertilization). ia20Representative fluorescence microscopy images of zebrafish larvae (3 days post-fertilization) from the report strain. Scale bar: 100 μm. (I) Total RNA from 3-day post-fertilization larvae (uninjured or treated with solvent, beclomethasone, Rg1, or beclomethasone combined with Rg1) was used to investigate fkbp5 and pck1 mRNA levels by qPCR. Treatment lasted for 6 hours (2 hours before and 4 hours after injury). Relative expression values ​​were normalized to ppial expression values ​​and displayed on a log2 scale. Data shown are mean ± SEM of three independent experiments (each experiment performed in triplicate, with two technical replicates). In Figures A, B, C, E, G, and I, the statistical significance is as follows: compared with the solvent group and the trauma solvent group in (I), P < 0.05 (*), 0.01 (**), and 0.001 (***); compared with the beclomethasone group, the combined treatment P < 0.05 (#), 0.01 (##), and 0.001 (###). In Figures D and F, the statistical significance is as follows: compared with the Rg1 group, P < 0.05 (*), 0.01 (**), and 0.001 (***).

[0017] Figure 4 Long-term treatment with a combination of beclomethasone and Rg1 did not decrease GC sensitivity. The mRNA levels of fkbp5 (A), pck1 (B), nfkbiaa (C), and gr (D) were investigated by qPCR using total RNA from 5-day post-fertilization larvae (untraumatized, after short-term or long-term treatment with solvent, beclomethasone (10 μM), Rg1 (50 μM), or beclomethasone (10 μM) and Rg1 (50 μM). Short-term treatment lasted 6 hours. In long-term treatment, larvae were treated for 5 days (from day 0 to day 5 post-fertilization). Relative expression values ​​were normalized to ppial expression values ​​and displayed on a log2 scale. Data presented are mean ± SEM of three independent experiments (each experiment performed in triplicate, with two technical replicates). Statistical significance is shown below: compared with the solvent group, P<0.001 (***); compared with beclomethasone alone, the combined treatment P<0.001 (###); compared with the corresponding short-term treatment, the long-term treatment P<0.05 (+), 0.01 (++) and 0.001 (+++).

[0018] Figure S4Rg1 restores the GC sensitivity of HeLa cells reduced by prolonged high-dose GC treatment. (A, B) Effects of Rg1 and beclomethasone, as single or combined treatments, on relative IL1B mRNA levels (measured by qPCR) at different time points after TNF-α treatment during short-term treatment (A) or long-term treatment (B). The control group reflects the solvent treatment group in the absence of TNF-α. (C, D) Effects of Rg1 combined with dexamethasone treatment on IL1B (C) and NFKBIA (D) expression after short-term or long-term treatment. In A through D, short-term treatment included 6 hours of combined treatment with the compound and TNF-α; in long-term treatment, cells were treated with the compound for 30 hours, including 24 hours of compound treatment plus 6 hours of combined treatment with the compound and TNF-α. Relative expression values ​​are normalized to 18S rRNA expression values ​​and displayed on a log2 scale. Data shown are the mean ± SEM of three independent experiments (each experiment performed in triplicate, with two technical replicates). Statistical significance was as follows: Compared with the corresponding solvent group, P < 0.05 (*), 0.01 (**), and 0.001 (***); compared with beclomethasone or dexamethasone alone, the combined treatment P < 0.05 (#), 0.01 (##), and 0.001 (###). Compared with the corresponding short-term treatment, the long-term treatment P < 0.05 (+), 0.01 (++), and 0.001 (+++).

[0019] Figure 5Rg1 restored the GC sensitivity of HeLa cells reduced by long-term high-dose GC treatment. (A to G) The relative mRNA levels of IL1B (A), MMP9 (B), IL8 (C), FKBP5 (D), NFKBIA (E), GILZ (F), and SGK1 (G) in HeLa cells were determined by qPCR. In A, HeLa cells were treated (short-term or long-term) with escalating doses of beclomethasone (0.01 μM, 0.1 μM, and 1 μM), Rg1 (20 μM, 100 μM, and 500 μM), or Rg1 (20 μM, 100 μM, and 500 μM) in combination with beclomethasone (0.01 μM). In B to G, HeLa cells were treated with solvent, beclomethasone (1 μM), Rg1 (20 μM), or beclomethasone in combination with Rg1 in the presence and absence of TNF-α. Short-term treatment consisted of 6 hours of combined treatment with the compound and TNF-α; long-term treatment involved 30 hours of compound treatment, comprising 24 hours of compound treatment followed by 6 hours of combined treatment with the compound and TNF-α. Relative expression values ​​were normalized to 18S rRNA expression values ​​and presented on a log2 scale. Data shown in (A to G) are the mean ± SEM of three independent experiments (each performed in triplicate, with two technical replicates). Statistical significance was as follows: compared with the corresponding solvent group, P < 0.05 (*), 0.01 (**), and 0.001 (***); compared with beclomethasone, the combined treatment P < 0.05 (#), 0.01 (##), and 0.001 (###); compared with the corresponding short-term treatment, the long-term treatment P < 0.05 (+), 0.01 (++), and 0.001 (+++).

[0020] Figure S5 Representative gel images of Western blots show that Rg1 inhibits beclomethasone-induced downregulation of GR in HeLa cells. (A) Western blot images of GR (and β-actin) obtained from (repeated) protein samples of HeLa cells after short-term (6 h) treatment with the specified compound. (B) Western blot images of GR (and β-actin) obtained from (repeated) protein samples of HeLa cells after long-term (24 h) treatment with the specified compound. (C) Western blot images of GR (and β-actin) obtained from protein samples of HeLa cells after long-term (24 h) treatment with the specified compound, with and without cycloheximide (5 μg / ml). The integrated intensities of the GR and β-actin bands were determined using ImageJ software.

[0021] Figure 6Rg1 inhibits beclomethasone-induced downregulation of GR in HeLa cells. (A) The effect of beclomethasone (1 μM) and / or Rg1 (20 μM) treatment on GR mRNA levels after short-term and long-term treatments in the presence and absence of actinomycin D (1 ng / ml) was determined by qPCR. Relative expression values ​​were normalized to 18S rRNA expression levels. Data shown are mean ± SEM of three independent experiments (each performed in triplicate, with two technical replicates) and plotted on a log2 scale. (B) The effect of beclomethasone and / or Rg1 treatment on GR protein levels after short-term and long-term treatments (6 h and 24 h, respectively) was determined by Western blotting. (C) The effect of beclomethasone and / or Rg1 treatment on GR protein levels after long-term treatment in the presence and absence of cycloheximide (5 μg / ml). The data shown are normalized to β-actin expression levels and are the mean ± SEM of four independent experiments (in A, each performed in duplicate) or the mean ± SEM of six independent experiments (in B, each measured once). Statistical significance is as follows: compared with the corresponding solvent group, P < 0.05 (*), 0.01 (**), and 0.001 (***); compared with beclomethasone alone, the combined treatment P < 0.05 (#), 0.01 (##), and 0.001 (###); compared with the corresponding short-term treatment, the long-term treatment P < 0.05 (+), 0.01 (++), and 0.001 (+++).

[0022] Figure 7 The anti-inflammatory effects of monosaccharidated ginsenosides require Gr function but not glucosinolate β2 (Gba2). A. Structures of the compounds used for treatment: beclomethasone (Bec), protopanaxadiol (PPT), F1, Rh1, and Rg1. Glucose (Glc) groups conjugated to the steroid backbone are indicated in blue. B. Schematic diagram of the experimental method. From Tg(mpx:GFP) i114 / mpeg1:mCherry-F umsF001Zebrafish larvae of the strain were subjected to caudal fin trauma 74 hours post-fertilization. Chemical compound treatment began 2 hours before trauma and continued for 4 hours post-traumatically. At this time point, the number of neutrophils migrating to the trauma area (indicated by a red box) was measured. C. Number of neutrophils and macrophages migrating after compound treatment 4 hours post-traumatically. D. Number of neutrophils migrating after compound treatment 4 hours post-traumatically, with and without the Gba2 inhibitor MZ31. E. Relative mRNA levels of il1b, il6, il8, mmp9, and mmp13, measured by qPCR before and after trauma, and after compound treatment. Data shown are mean ± SEM, and C and D represent the mean of pooled data from three independent experiments (n=60), or E represents the mean from three independent experiments (each experiment performed in triplicate). Statistical significance was determined using one-way or two-way ANOVA and Tukey's post-hoc test. Significant differences from the trauma solvent group (in C and E) or the corresponding solvent group (Veh / Veh or Veh / MZ31; in D) are indicated by *** (P < 0.001). Differences from solvent groups treated with the same compound (shaded bars vs. unshaded bars) are indicated by ### (P < 0.001). Significant differences from the PPT group (in E) are indicated by $$$ (P < 0.001), and differences from the F1 group (in E) are indicated by &&& (P < 0.001).

[0023] Figure S7 Acute embryo toxicity tests (FETs) of ginsenosides and beclomethasone in fish embryos were studied (A, B). Embryos were exposed to different concentrations of the compounds to assess their toxicity. The experiments included a negative control (nC), a solvent control (sC), and a positive control (pC). A. Hatching rate (%) at 48 and 72 hours post-fertilization. B. Survival rate (%) at 96 and 120 hours post-fertilization. C. Wild-type (gr) + / + ) and Gr defect type (gr - / - The number of neutrophils migrating in individuals after treatment with Bec, PPT, F1, Rh1, or Rg1. Data shown are mean ± SEM, where A and B represent the mean of data from two independent experiments (n=100) and C represents the mean of aggregate data from three independent experiments (n=60). Statistical significance was determined using one-way or two-way ANOVA and Tukey post-hoc tests. (The data is compared with the solvent (NC) group (at the same time point) or the corresponding solvent group (gr) in (A, B). + / + or gr - / - Significant differences in (C) are indicated by *** (P<0.001), and are consistent with those treated with the same compound. + / +Significant differences between groups (shaded bars and unshaded bars; C) are indicated by ### (P<0.001).

[0024] Figure 8 Compared with beclomethasone, ginsenosides showed a significant reduction in side effects. A. Systemic glucose levels in zebrafish larvae 5 days post-fertilization (from 2 hours post-fertilization to 5 days post-fertilization) after treatment with beclomethasone or ginsenosides PPT, F1, Rh1, or Rg1, as determined by colorimetric assay. B. Systemic glucose levels in larvae 5 days post-fertilization after trauma 2 days post-fertilization, and after compound treatment with or without MZ31 2 hours post-fertilization to 5 days post-fertilization. C. Systemic cortisol levels in zebrafish larvae 5 days post-fertilization (from 2 hours post-fertilization to 4 days post-fertilization) after treatment with beclomethasone or ginsenosides PPD, F2, or Rb1, as determined by ELISA. D. Systemic cortisol levels in larvae 5 days post-fertilization after trauma 2 days post-fertilization, and after compound treatment with or without MZ31 2 hours post-fertilization to 4 days post-fertilization. E. Body length of zebrafish larvae at 5 days post-fertilization after compound treatment, from 2 hours to 5 days post-fertilization. F. Regenerated caudal fin length of larvae at 5 days post-fertilization after trauma at 2 days post-fertilization, and after compound treatment with or without MZ31 (from 2 hours to 5 days post-fertilization). G. Representative fluorescence microscopy images of embryos at 3 days post-fertilization from the experimental group shown in H. Scale bar: 200 μm. H. Relative GFP fluorescence level 24 hours after compound treatment in embryos of the Tg (9x GCRE-HSV.UI23:EGFP) strain (a reporter strain with Gr trans-activation activity) at 3 days post-fertilization. I. Relative mRNA levels of fkbp5 determined by qPCR before and after trauma and after compound treatment. Data shown in A, B, C, D, and I are mean ± SEM values ​​and are the mean of three independent experiments (three replicates per experiment). Data shown in E, F, and H are mean ± SEM, representing the mean of data from three independent experiments (n=60). Statistical significance was determined using one-way or two-way ANOVA and Tukey post-hoc tests. Significant differences from the solvent group (in A, C, E, H) or the corresponding solvent group (Veh / Veh or Veh / MZ31; in B, D, F) are indicated by * (P<0.05), ** (P<0.01), *** (P<0.001); differences from groups treated with the same compound (shaded and unshaded bars; in B, D, F) are indicated by # (P<0.05), ## (P<0.01), ### (P<0.001).

[0025] Figure S8 A. Figure 2F shows a representative bright-field microscopic image of the caudal fin of larvae in the experimental group 5 days after fertilization. Arrows indicate truncation sites; any tissue observed to the right of this point represents regenerated tissue. Scale bar: 100 μm. B. Relative mRNA levels of pck1 and nfkbiaa, determined by qPCR before and after trauma and after compound treatment. Data shown are mean ± SEM values ​​and are the mean of three independent experiments (each performed in triplicate). Statistical significance was determined using two-way ANOVA and Tukey's post-hoc test. Significant differences from the solvent group are indicated by *** (P < 0.001).

[0026] Figure 9 Anti-inflammatory effects and hypothetical side effects of ginsenoside Re on post-traumatic injury induced by caudal fin truncation in zebrafish.

[0027] A: Effects of Re and GC (prednisone) on the number of neutrophils at the truncated site; B: Effects of Re and GC (prednisone) on the number of macrophages at the truncated site; C: Effects of Re and GC (prednisone) on systemic glucose levels; D: Effects of Re and GC (prednisone) on systemic cortisol levels; and E: Effects of Re and GC (prednisone) on regenerating tissues. Veh: solvent.

[0028] Figure 10 A. Appearance of ginsenoside aqueous solution after micelle formation. From left to right: 10 mg / mL pure Re dissolved in ethanol before micelle formation; 10 mg / mL pure Re dissolved in H2O after micelle formation; ethanol solution of ginseng extract enriched with 150 mg / mL Re / Rg1 obtained by purification and decolorization with D101 and D941 resins, respectively; ginseng extract enriched with 150 mg / mL Re / Rg1 after micelle formation. B: Microscopic view (400x) of Re crystals formed when the Re film is dissolved in H2O during the micelle formation process.

[0029] Figure 11 Size exclusion chromatography was used to determine the molecular weight and composition of micelles. One mL of an aqueous solution containing Re / Rg1-enriched ginseng extract and dexamethasone was loaded onto a 17 mL Sephadex G50 size exclusion column after micelle formation and eluted with H2O at a flow rate of 0.4 mL / min. Two mL of the fraction was collected. The column was calibrated using blue dextran (MW 2,000,000 Da) and erythrosine (MW 880 Da) as molecular weight markers (see arrows for marker elution peaks). The presence and concentrations of ginsenosides Re, Rg1, and dexamethasone were determined and calculated using high-performance liquid chromatography (HPLC).

[0030] Figure 12HPLC chromatograms of fractions related to or obtained during size exclusion chromatography. A: HPLC chromatogram and peak positions of pure dexamethasone at 203 nm (top) and 241 nm (bottom); B: HPLC chromatogram of the 15th fraction eluted at 30 mL at 203 nm, showing Rg1 and Re as major peaks; C: HPLC chromatogram of the 15th fraction eluted at 30 mL at 241 nm, showing the presence of dexamethasone.

[0031] Figure 13 The percentage of inhibition of neutrophil migration to the wound site by 10 μM beclomethasone, 80 μM Rg1, and the combination of 10 μM beclomethasone and 80 μM Rg1. Summary of the Invention

[0032] This invention relates to a ginsenoside preparation comprising PPT-type ginsenosides at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, wherein the molecular weight of the micelles is at least 10,000 Da, preferably at most 100,000 Da.

[0033] In a preferred embodiment, the ginsenoside is a PPT-type ginsenoside, preferably selected from Rg1, Re, and combinations thereof.

[0034] This invention also relates to a method for preparing a ginsenoside preparation according to the invention, the method comprising:

[0035] (a) Provide ginsenoside extracts from plants or plant parts of the Panax genus, preferably Panax ginseng;

[0036] (b) The ginsenoside extract is contacted with an aqueous solvent mixture, preferably wherein the aqueous solvent mixture comprises a mixture of water and a non-aqueous solvent, the non-aqueous solvent being preferably an alcohol solvent, more preferably selected from methanol, ethanol, propanol and isopropanol, to obtain a ginsenoside mixture;

[0037] (c) At least partially removing the aqueous solvent mixture from the ginsenoside mixture; and optionally...

[0038] (d) The ginsenoside mixture obtained in step c) is reconstituted in water to obtain a ginsenoside preparation.

[0039] In a preferred method, the method includes a column chromatography step of using a non-polar resin, preferably a D101 macroporous resin, as the stationary phase to perform column chromatography on the ginsenoside extract; and / or a decolorization step of the ginsenoside extract, preferably using a D941 macroporous resin.

[0040] The present invention also relates to ginsenoside preparations obtainable by the method of the present invention, which preferably contain PPT-type ginsenosides at least partially integrated in micelles, preferably at least partially integrated in the outer layer of micelles, wherein the micelles have a molecular weight of at least 10,000 Da and / or a molecular weight of at most 100,000 Da.

[0041] In the preferred ginsenoside preparations that can be obtained by the method of the present invention, the ginsenosides are PPT-type ginsenosides, preferably selected from Rg1, Re and combinations thereof.

[0042] Furthermore, the present invention relates to ginsenosides in a method for preventing or treating side effects of GC treatment, the method comprising administering ginsenosides to a subject who is suffering from one or more side effects of GC treatment or who is at risk of suffering from one or more side effects of GC treatment.

[0043] Preferably, the side effects are selected from GC-induced diabetes, GC-induced decreased cortisol levels, GC-induced decreased growth rate, GC-induced impaired wound healing, GC-induced tissue degeneration, GC-induced osteoporosis, GC-induced hypertension, GC-induced weight gain, and GC-induced muscle weakness.

[0044] The present invention also relates to ginsenoside preparations according to the invention for use according to the invention.

[0045] The present invention also relates to a combination of GC (preferably selected from beclomethasone and dexamethasone) and ginsenosides in a method for the prevention or treatment of inflammatory diseases, wherein the method comprises administering GC and ginsenosides to a subject in need at a molar ratio of about 1:1 to about 1:2000.

[0046] The molar ratio is between about 1:4 and about 1:1000, more preferably between about 1:8 and about 1:20.

[0047] In a preferred embodiment, the present invention relates to a combination for use according to the invention, wherein the GC is administered at a therapeutically ineffective dose, preferably at a dose of up to 0.07 mg / kg / day, more preferably at a dose of up to 0.007 mg / kg / day; and / or wherein the ginsenoside is administered at a therapeutically ineffective dose, preferably at a dose of up to 5.5 mg / kg / day, more preferably at a dose of up to 0.05 mg / kg / day.

[0048] In a preferred embodiment, the present invention relates to a combination for use according to the invention, wherein the inflammatory disease is selected from asthma, allergic rhinitis, hay fever, urticaria, atopic eczema, chronic obstructive pulmonary disease, inflammation of joints, muscles and tendons, lupus, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), giant cell arteritis and polymyalgia rheumatica, and multiple sclerosis.

[0049] Preferably, the present invention relates to a combination for use according to any one of claims 8 to 13, wherein the ginsenoside is at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, the micelles having a molecular weight of at least 10,000 Da and / or a molecular weight of at most 100,000 Da. Preferably, the ginsenoside is a PPT-type ginsenoside, preferably selected from Rg1, Re, and combinations thereof.

[0050] The present invention also relates to a pharmaceutical composition comprising GC, ginsenosides (preferably wherein the ginsenosides are protopanaxadiol (PPT) type ginsenosides, more preferably selected from Rg1 and Re and combinations thereof), and a pharmaceutically acceptable carrier, wherein the molar ratio of the GC to the ginsenosides is between about 1:1 and about 1:2000, preferably between about 1:4 and about 1:1000, more preferably between 1:8 and 1:20.

[0051] Preferably, in the pharmaceutical composition according to the invention, the ginsenoside is at least partially integrated in micelles, more preferably at least partially integrated in the outer layer of the micelles, the micelles having a molecular weight of at least 10,000 Da and / or at most 100,000 Da.

[0052] This invention relates to a pharmaceutical composition according to the invention, wherein the pharmaceutical composition is formulated as a cream, lotion, balm, hydrogel, ointment, foam, gel, spray, tablet, capsule, lozenge, external solution, external suspension, aerosol, injection, and syrup. More preferably, the pharmaceutical composition is formulated as a hydrogel.

[0053] The present invention also relates to pharmaceutical compositions for methods of preventing or treating inflammatory diseases, preferably wherein the inflammatory diseases are selected from asthma, allergic rhinitis, hay fever, urticaria, atopic eczema, chronic obstructive pulmonary disease, inflammation of joints, muscles and tendons, lupus, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), giant cell arteritis and polymyalgia rheumatica, and multiple sclerosis. Detailed Implementation

[0054] Unless otherwise specified, the term "or" as used herein is defined as "and / or".

[0055] Unless otherwise specified, the term “a / an” as used herein is defined as “at least one”.

[0056] In this text, the terms "substantially" or "basically" are generally used to indicate that something possesses the general characteristics or functions of a specified thing. When referring to quantifiable characteristics, these terms are especially used to indicate that the characteristic accounts for at least 75%, more particularly at least 90%, and even more particularly at least 95%.

[0057] In the context of this application, the term “about” generally means a deviation from a given value of 15% or less, particularly 10% or less, and even more particularly 5% or less.

[0058] When referring to a singular noun, the plural form is included unless the context indicates that only the singular should be referred to.

[0059] Ginsenosides are a class of steroidal glycosides and triterpenoid saponins found in plants of the genus *Panax*. Ginsenosides are usually designated as "Rx," where "x" indicates chromatographic polarity in alphabetical order, although some ginsenosides use other names, such as ginsenoside F1. Therefore, Ra is one of the least polar ginsenosides known to date, while Rb is more polar than Ra.

[0060] Ginsenosides are characterized by the presence of a dammarane skeleton—also known by the systematic name (1R,3aR,3bR,5aS,9aS,9bR,11aR)-1-[(2R)-6-methylheptane-2-yl]-3a,3b,6,6,9a-pentamethylhexadecyl-1H-cyclopenta[a]phenanthrene]—typically with a sugar moiety attached to it, although ginsenosides without a sugar moiety have also been described, such as ginsenoside "glycoside-PPT". Ginsenosides are generally divided into two major subclasses: 20(S)-protopanaxadiol (PPD) type ginsenosides (Formula I) and 20(S)-protopanaxtriol (PPT) type ginsenosides (Formula II). The difference between PPD and PPT type ginsenosides lies in the position of the sugar moiety attached to the dammarane skeleton. In PPT-type ginsenosides, the sugar moiety is linked to the 6-position of the backbone, while in PPD-type ginsenosides, the sugar moiety is linked to the 3-position of the backbone.

[0061]

[0062] Formula (I)

[0063]

[0064] Equation (II)

[0065] Examples of protopanaxadiol-type ginsenosides include Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, and Rs1. Examples of protopanaxadiol-type ginsenosides include Re, Rf, Rg1, Rg2, and Rh1. In addition, some rare ginsenosides are described, such as octylene saponin F11 (24-R-pseudoginsenoside) and pentacyclic oleanane saponin Ro (3,28-O-disaccharide chain saponin).

[0066] "Glucocorticoids" (GCs) in this article refer to a class of steroid hormones that bind to glucocorticoid receptors. GCs are commonly used to treat (chronic) inflammatory diseases, including eczema, asthma, and rheumatoid arthritis. Cortisol, also known as hydrocortisone, is an endogenous GC. Synthetic analogues of cortisol are also described, which typically have one or more different properties compared to cortisol. Examples of synthetic GCs include dexamethasone, betamethasone, prednisolone, methylprednisolone, triamcinolone, dexamethasone acetate, fludrocortisone acetate, aldosterone, beclomethasone, hydrocortisone, or their prodrugs.

[0067] As used in this article, "prodrug" refers to a glucocorticoid analogue that is converted into biologically active GC in the body. Prodrugs of GC include cortisone and prednisone.

[0068] The term "micelle" refers to a substantially spherical particle comprising an outer layer and an inner cavity, the outer layer containing one or more amphiphilic molecules. The outer layer typically has a hydrophilic outer surface and a hydrophobic inner center. In aqueous solutions, the hydrophilic portion of the micelle is typically in contact with the aqueous medium, while the hydrophobic portion is isolated from it. The inner cavity is usually formed by the outer layer and may enclose one or more small molecules, such as therapeutic molecules.

[0069] In the context of this application, the hydrophilic portion of micelles is typically formed by the hydrophilic portion of ginsenosides (typically the sugar portion), while the hydrophobic portion of micelles is typically formed by the hydrophobic portion of ginsenosides (typically the dammarane portion of ginsenosides).

[0070] As used herein, the term "therapeutic effective amount" refers to the amount of an active ingredient (e.g., GC or ginsenosides) administered for the necessary time that is effective in providing an anti-inflammatory effect to the recipient. The term "therapeutic ineffective amount" refers to any amount of an active ingredient (e.g., GC or ginsenosides) below the therapeutic effective amount, which, when administered for a sufficiently long period and in the absence of another anti-inflammatory compound, would not exhibit a substantially significant anti-inflammatory effect on the recipient.

[0071] Ginsenosides used to treat side effects of GC treatment

[0072] Although GC is an effective anti-inflammatory drug, long-term use can have some drawbacks.

[0073] For example, long-term use of glucocorticoids (GC) can reduce glucocorticoid receptor (GR) activity, thereby increasing GC resistance. Therefore, to maintain the effectiveness of GC, it is typically necessary to increase the dosage.

[0074] However, long-term use of GC is particularly associated with the occurrence of serious side effects, including diabetes, decreased cortisol levels, slowed growth, impaired wound healing, tissue degeneration, osteoporosis, high blood pressure, weight gain, and muscle weakness.

[0075] As the dosage increases, the occurrence and severity of these side effects usually worsen, and increasing the dosage is often necessary to overcome the decrease in glucocorticoid receptor activity and maintain therapeutic efficacy.

[0076] The inventors unexpectedly discovered that ginsenosides can effectively prevent or reduce the side effects of GC treatment and can also prevent GC resistance. This will significantly increase treatment options for anti-inflammatory diseases.

[0077] Unwilling to be bound by any theory, the inventors believe that ginsenosides, especially Rg1, act as competitive antagonists of glucocorticoid receptors (see, for example, examples and...). Figure 3 D、 Figure 3 F). More specifically, the inventors hypothesize that the selective binding of ginsenosides to glucocorticoid receptors induces trans-inhibition rather than trans-activation, the latter of which is associated with the occurrence of side effects.

[0078] Therefore, one aspect of the present invention relates to ginsenosides in a method for preventing or treating side effects of GC treatment, the method comprising administering ginsenosides to a subject who is suffering from one or more side effects of GC treatment or who is at risk of suffering from one or more side effects of GC treatment.

[0079] Ginsenosides

[0080] The ginsenosides used according to the present invention can, in principle, be any ginsenosides that exert anti-inflammatory effects and can alleviate the side effects of GC treatment.

[0081] The ginsenosides are preferably 20(S)-protopanaxadiol (PPD) type ginsenosides or 20(S)-protopanatriol (PPT) type ginsenosides, and more preferably PPT type ginsenosides.

[0082] Preferably, the ginsenosides are selected from Rg1, Re, Rf, Rg2, F1, aglycone-PPT and Rh1 or combinations thereof; more preferably, the at least one ginsenoside is selected from Rg1 and Re or combinations thereof.

[0083] Rg1 is a PPT-type ginsenoside with the molecular formula Cp. 42 H 72 O 14It has a molecular weight of 801 g / mol. It also has other chemical names, such as (3β,6α,12β)-3,12-dihydroxydammar-24-ene-6,20-diylbis-β-D-glucopyranoside, ginsenoside A2, ginsenoside g1, ginsenoside A, ginsenoside Rg1, notoginsenoside C1, and notoginsenoside Rg1. The molecular structure of Rg1 is as follows... Figure 1 As shown in Figure A.

[0084] In addition, Re is another type of PPT ginsenoside with the molecular formula C. 48 H 82 O 18 It has a molecular weight of 947.15 g / mol. It also has the following chemical names: (3β,6α,12β)-20-(β-D-glucopyranoside)-3,12-dihydroxydammar-24-en-6-yl 2-O-(6-deoxy-α-L-mannopyranoside)-β-D-glucopyranoside, bamboo saponin IVc, ginsenoside B2, NSC308877, ginsenoside Re, and notoginsenoside Re.

[0085] Preferably, the ginsenoside is selected from Re, Rf, Rg1, Rg2, Rh1 or a combination thereof; more preferably, the ginsenoside contains Rg1, Re or a combination of Rg1 and Re; even more preferably, if the ginsenoside contains a combination of Re and Rg1, the weight ratio of Rg1 to Re is between about 1:2 and about 2:1; more preferably, it is between about 1:1.5 and about 1.5:1.

[0086] The ginsenosides can be obtained from natural sources or prepared through synthesis.

[0087] For example, the ginsenosides can be obtained from plants of the genus Panax. Preferably, the ginsenosides can be obtained from one or more of the following species: Panax bipinnatifidus, Panax, elegantior, P. ginseng, P. japonicus, P. major, P. notoginseng, P. omeiensis, P. pseudoginseng, P. quinquefolius, P. sikkimensis, P. sinensis, P. stipuleanatus, P. trifolius, P. vietnamensis, P. wangianus, and P. zingiberensis.

[0088] Preferably, the ginsenosides can be obtained from P. quinquefolius (also known as American ginseng), P. japonicus (also known as Japanese ginseng), P. ginseng (also known as Korean ginseng) or P. notoginseng (also known as South China ginseng).

[0089] Preferably, the ginsenosides can be obtained from one or more of the roots, stems, fruits, rhizomes, flowers, or leaves of plants in the genus *Panax*, and more preferably from one or more of the roots, stems, fruits, rhizomes, flowers, or leaves of *P. ginseng*, *P. japonicus*, *P. notoginseng*, or *P. quinquefolius*.

[0090] The content of ginsenosides in ginseng plants is typically in the range of 1 wt.% to 10 wt.% based on plant dry weight, for example, between about 2 wt.% and 8 wt.% based on plant dry weight, between about 3 wt.% and about 6 wt.%, and especially about 4 wt.%.

[0091] The ginsenosides can be isolated from ginseng plants using any suitable method known in the art. For example, the ginsenosides can be obtained by drying a part of a ginseng plant (e.g., the root) and then extracting the dried plant or plant part with a suitable solvent. Methods for preparing ginseng extracts are described in the Chinese Pharmacopoeia 2015 (English Edition), Part I, pp. 524-526.

[0092] Optionally, the plant part (e.g., the root of the ginseng plant) is steamed at 100°C for at least two hours before drying. The extract obtained using this method is typically referred to as "red ginseng".

[0093] Ginseng extracts are also commercially available, for example, G115 from P. ginseng (Pharmaton SA, Switzerland) and NAGE from P. quinquefolius (Canadian Phytopharmaceuticals Corporation, Canada).

[0094] Preferably, the ginsenoside content in the ginseng extract is at least 1 wt.%, more preferably at least 2 wt.%, at least 3 wt.%, and at least 4 wt.%. Typically, the ginsenoside content in the ginseng extract is at most 99 wt.%, more preferably at most 98 wt.%, and even more preferably at most 95 wt.%.

[0095] The preferred range of ginsenosides in ginseng extract is between about 4 wt.% and about 50 wt.% based on the dry weight of ginseng extract, more preferably between about 8 wt.% and about 20 wt.%.

[0096] In one embodiment, the ginsenosides are provided in the form of ginseng extract.

[0097] The ginseng extract may selectively contain one or more components other than ginsenosides, such as ginseng protein and ginseng carbohydrates.

[0098] Alternatively, the ginsenosides are provided in isolated form, i.e., essentially isolated from naturally occurring components of the Panax genus. Isolated ginsenosides are commercially available, for example, ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rh1 (CAS 63223-86-9), ginsenoside Re (CAS 52286-59-6), and ginsenoside Rg1 (CAS 22427-39-0) are all commercially available from Sigma-Aldrich.

[0099] If a chemical synthesis route is known, isolated ginsenosides can optionally be provided in synthetic form. For example, Anufriev et al. (Carbohydr Res. 1997; 304(2):179-182) described the chemical synthesis of ginsenoside Rg3.

[0100] Preferably, the purity of the ginsenosides is at least 90 wt.%. More preferably, the purity of the ginsenosides is at least 92 wt.%, at least 94 wt.%, at least 96 wt.%, at least 98 wt.%, at least 99 wt.%, for example, 100 wt.%.

[0101] micelles

[0102] The inventors further recognized that the solubility of ginsenosides in water is a limiting factor in their medical use for treating inflammatory diseases, preventing side effects caused by GC treatment, or preparing pharmaceutical combinations according to the invention. Furthermore, it is well known that ginsenosides are readily degraded in the gastrointestinal tract, leading to their inactivation and hindering their systemic application.

[0103] Therefore, the inventors developed a novel ginsenoside preparation that overcomes one or more of the aforementioned drawbacks.

[0104] Therefore, the ginsenosides used according to the present invention are preferably at least partially integrated into the micelles, and more preferably integrated into the outer layer of the micelles.

[0105] The inventors realized that when ginsenosides are formulated into micelles, their solubility in water can be significantly improved and their stability in the intestine can also be improved compared to ordinary ginsenoside preparations (in which ginsenosides are not integrated into the micelles (the outer layer of the micelles)).

[0106] This increased water solubility is beneficial because it allows for sufficiently high concentrations of ginsenosides in an aqueous medium, which is often necessary to counteract one or more side effects of GC treatment or to achieve anti-inflammatory effects.

[0107] Furthermore, improved intestinal stability is beneficial in enhancing the systemic efficacy of the ginsenosides.

[0108] In principle, micelles containing at least one ginsenoside can be prepared using any suitable method known in the art, depending on the type of ginsenoside to be integrated into the micelles, preferably at least partially integrated into the outer layer of the micelles. For example, micelles containing one or more PPD-type ginsenosides integrated therein can be prepared by slowly evaporating a solution of PPD-type ginsenosides in an organic or aqueous solvent (e.g., ethanol or a mixture of ethanol and water) under reduced pressure, and then slowly dissolving the resulting ginsenoside film in water to obtain micelles containing one or more PPD-type ginsenosides integrated therein.

[0109] However, the inventors unexpectedly realized that when this method was applied using PPT-type ginsenoside Re, crystallization of Re was observed, but micelle formation did not occur. Example 4 demonstrates this.

[0110] The inventors unexpectedly discovered that micelles containing at least one PPT-type ginsenoside can be prepared using (crude) ginsenoside extracts containing at least one PPT-type ginsenoside, instead of isolated PPT-type ginsenosides, as substrates.

[0111] Therefore, the present invention also relates to a ginsenoside formulation comprising PPT-type ginsenosides at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, wherein the micelles have a molecular weight of at least 10,000 Da.

[0112] The ginsenosides mentioned are as defined in the "Ginsenosides" section above, provided that they are PPT-type ginsenosides.

[0113] The molecular weight of the micelles is at least 10,000 Da, preferably at least 20,000 Da, at least 40,000 Da, at least 50,000 Da, at least 60,000 Da, at least 70,000 Da, at least 80,000 Da, at least 90,000 Da, and most preferably at least 100,000 Da.

[0114] The molecular weight of micelles is typically up to 100,000,000 Da, preferably up to 10,000,000 Da, and most preferably up to 1,000,000 Da.

[0115] The molecular weight of the micelles is preferably in the range of about 10,000 Da to about 100,000,000 Da, more preferably in the range of about 50,000 Da to about 10,000,000 Da, and even more preferably in the range of about 80,000 Da to about 1,000,000 Da.

[0116] The molecular weight can be determined using any suitable method known in the art, such as size exclusion chromatography or high performance liquid chromatography, particularly the method described in Example 4.

[0117] Typically, micelles contain about 10 to about 100,000 ginsenoside molecules, more preferably about 20 to about 10,000 ginsenoside molecules, more preferably about 50 to about 10,000 ginsenoside molecules, and especially about 100 to about 1,000 ginsenoside molecules.

[0118] The solubility of the ginsenoside formulation according to the present invention, or ginsenosides at least partially integrated in micelles (outer layer), in an aqueous medium (preferably water) is preferably at least 0.03 mmol / L, more preferably at least 0.05 mmol / L, at least 0.1 mmol / L, at least 1 mmol / L, at least 5 mmol / L, at least 10 mmol / L, at least 50 mmol / L, at least 0.1 mol / L, at least 0.15 mol / L, at least 0.2 mol / L, at least 0.25 mol / L, and most preferably at least 0.3 mol / L.

[0119] Typically, the solubility of the ginsenoside formulation according to the present invention, or ginsenosides at least partially integrated in micelles (outer layer), in an aqueous medium (preferably water) is about 0.03 mmol / L to about 10 mol / L, preferably about 0.05 mmol / L to about 5 mol / L, more preferably about 0.1 mmol / L to about 2 mol / L, about 1 mmol / L to about 1 mol / L, even more preferably about 5 mmol / L to about 0.5 mol / L, and especially about 10 mmol / L to about 0.3 mol / L.

[0120] The solubility of the ginsenoside preparations according to the present invention, or ginsenosides at least partially integrated in micelles (outer layer), in an aqueous medium (preferably water) is preferably at least 30 mg / L, more preferably at least 50 mg / L, at least 100 mg / L, at least 1 g / L, at least 5 g / L, at least 10 g / L, at least 50 g / L, at least 100 g / L, at least 150 g / L, at least 200 g / L, at least 250 g / L, and more preferably at least 300 g / L.

[0121] The solubility of the ginsenoside formulation according to the present invention, or the ginsenoside at least partially integrated in micelles (outer layer), in an aqueous medium (preferably water) is preferably about 30 mg / L to about 1000 g / L, more preferably about 50 mg / L to about 5000 g / L, more preferably about 100 mg / L to about 2000 g / L, more preferably about 1 g / L to about 1000 g / L, about 5 g / L to about 500 g / L, and especially about 10 g / L to about 300 g / L.

[0122] Particularly good results have been obtained with ginsenoside formulations according to the invention, or with ginsenosides at least partially integrated in micelles (outer layer) comprising at least partially integrated Rg1 and / or Re, preferably wherein Rg1 and Re are present in a weight ratio of about 1:2 to about 2:1, more preferably about 1:1 to about 1:1.5. Preferably, the micelles have a molecular weight of about 50,000 Da to about 100,000 Da.

[0123] The present invention also relates to a method for preparing ginsenoside formulations according to the present invention, or ginsenosides at least partially integrated in micelles (outer layer), the method comprising:

[0124] (a) Provide ginsenoside extracts from plants or plant parts of the Panax genus, preferably Panax ginseng;

[0125] (b) Contact the ginsenoside extract with an aqueous solvent mixture to obtain a ginsenoside mixture;

[0126] (c) At least partially removing the aqueous solvent mixture from the ginsenoside mixture; and optionally...

[0127] (d) The ginsenoside mixture obtained in step c) is reconstituted in water to obtain the ginsenoside preparation according to the present invention.

[0128] The ginsenoside extract can be obtained by any suitable method known in the art, such as boiling dried ginseng plants or plant parts in water and then filtering the aqueous phase to obtain crude ginsenoside extract.

[0129] The amount of water used in this article depends on the amount of dried plant or dried plant parts to be extracted. Those skilled in the art can select an appropriate amount of water based on their general knowledge and the information provided herein.

[0130] Preferably, before or during contacting the ginsenoside extract with the aqueous solvent mixture in step b, one or more purification steps are performed on the ginsenoside extract. Preferably, a non-polar resin is used as the stationary phase for column chromatography of the ginsenoside extract.

[0131] The non-polar resin is preferably D101, D201, D113, D285, D296, D941, D945, DM130, DM131, Dt, HPD100, HPD300, NKA, LK37, LK1300S, LK20, 388, Amberlite IRA900, AB-8, Amberlite XAD16, SA-2, or LX-TS4.

[0132] Preferably, a suitable aqueous solvent mixture is used to elute ginsenosides from a nonpolar resin to obtain a purified ginsenoside extract. The aqueous solvent mixture is preferably a mixture of water and a non-aqueous solvent. Preferably, the non-aqueous solvent has a boiling point lower than that of water. Preferably, the aqueous solvent mixture is a mixture of alcohols in water, for example, a mixture of about 40% to about 80% alcohols in water. More preferably, the aqueous solvent is a mixture of methanol, ethanol, propanol, or isopropanol in water, and most preferably a mixture of about 40% to about 80% ethanol in water.

[0133] Preferably, the nonpolar resin is first washed with a polar solvent (preferably water), and then ginsenosides are eluted from the nonpolar resin using the aqueous solvent mixture defined above.

[0134] Preferably, the ginsenoside extract undergoes a decolorization step. Preferably, the purified ginsenoside extract is contacted with a decolorizing agent (preferably D941 macroporous resin). Preferably, the ginsenoside extract undergoes a decolorization step after contact with a non-polar resin. Preferably, the decolorization step is carried out in step b), for example, the ginsenoside extract is contacted with an aqueous solvent mixture, and the aqueous solvent mixture containing the ginsenoside extract is contacted with the decolorizing agent.

[0135] The amounts of solvent, nonpolar resin, and decolorizing agent used in this article depend on the amount of ginsenoside extract to be treated by this method. Those skilled in the art can select appropriate amounts of nonaqueous solvent, polar solvent, nonpolar resin, and decolorizing agent based on their general knowledge and the information provided herein.

[0136] In step c, the aqueous solvent mixture is preferably removed from the ginsenoside mixture. This can be achieved by any suitable method known in the art, preferably by vacuum evaporation. Alternatively, the ginsenoside mixture can be heated, typically to a temperature of 30°C to 90°C, for example, 40°C to about 80°C, to promote the evaporation of the (partial) aqueous solvent mixture.

[0137] Preferably, the ginsenoside mixture obtained in step c) is dissolved in water to obtain the ginsenoside preparation according to the present invention.

[0138] Alternatively, the non-aqueous solvent present in the aqueous solvent mixture may be at least partially evaporated to obtain an aqueous fraction containing the ginsenoside preparation according to the invention or an aqueous solvent mixture rich in water.

[0139] The ginsenosides in the ginsenoside formulations according to the present invention are at least partially integrated into micelles, preferably at least partially integrated into the outer layer of the micelles. This can be determined by any suitable analytical method known in the art, such as size exclusion chromatography, optionally coupled with high performance liquid chromatography, as described in Example 4, for example.

[0140] Alternatively or additionally, the micelles preferably further comprise GC at least partially integrated therein, more preferably the GC selected from dexamethasone and beclomethasone. In this embodiment, ginsenosides are preferably integrated in the outer layer of the micelles, and the GC is preferably present in the inner lumen of the micelles. The micelles can be prepared by the method described above, provided that in step b), the GC is preferably contacted with the ginsenoside extract.

[0141] This invention also relates to a ginsenoside preparation that can be obtained by the method of this invention.

[0142] The ginsenoside preparation preferably comprises PPT-type ginsenosides at least partially integrated in micelles, preferably at least partially integrated in the outer layer of micelles, wherein the molecular weight of the micelles is at least 10,000 Da and / or at most 100,000 Da.

[0143] Furthermore, the ginsenosides are preferably PPT-type ginsenosides, and more preferably selected from Rg1, Re, and combinations thereof.

[0144] Glucocorticoids

[0145] As is known to those skilled in the art, there are various types of glucocorticoids (GCs). Examples include short-acting glucocorticoids (such as cortisol or cortisone), intermediate-acting glucocorticoids (such as prednisone), long-acting glucocorticoids (such as dexamethasone and betamethasone), and mineralocorticoids (such as fludrocortisone).

[0146] The GC used in the combination for use according to the invention can be any natural or synthetic GC or its prodrug. Preferably, the GC is selected from dexamethasone, betamethasone, prednisolone, methylprednisolone, triamcinolone, dexamethasone acetate, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, hydrocortisone, and beclomethasone, or their prodrugs; preferably, the prodrug is selected from cortisone or prednisone.

[0147] In one implementation, the GC is preferably a long-acting GC. Alternatively or additionally, the GC is beclomethasone or dexamethasone.

[0148] Advantageously, the combination used according to the invention allows for the use of GCs that are typically accompanied by very serious side effects and are therefore not usually administered to the subject in treatment. However, since the presence of ginsenosides can alleviate the side effects, such GCs can be used in the combination used according to the invention. This advantageously broadens the options for treating inflammatory diseases in subjects in need.

[0149] Medical use

[0150] Side effects of GC treatment can be any side effects caused by (long-term) use of one or more GCs. Examples of possible side effects include diabetes, decreased cortisol levels, slowed growth, impaired wound healing, tissue degeneration, osteoporosis, high blood pressure, weight gain, and muscle weakness.

[0151] Preferably, the side effects of GC treatment are selected from decreased cortisol levels, slowed growth rate, and impaired wound healing.

[0152] Therefore, another aspect of the present invention relates to ginsenosides in methods for preventing or treating (GC-induced) diabetes, (GC-induced) decreased cortisol levels, (GC-induced) slowed growth, (GC-induced) impaired wound healing, GC-induced tissue degeneration, (GC-induced) osteoporosis, (GC-induced) hypertension, (GC-induced) weight gain, and (GC-induced) muscle weakness, the method comprising administering ginsenosides to a subject suffering from or at risk of suffering from one or more GC treatment side effects. Preferably, the present invention relates to ginsenosides in methods for preventing or treating (GC-induced) decreased cortisol levels, (GC-induced) slowed growth, and (GC-induced) impaired wound healing.

[0153] The object can be any object suffering from GC-induced side effects, preferably a mammal, and more preferably a human.

[0154] Specifically, the object is an object that has received or previously received GC treatment. Preferably, the object receives a certain amount of GC per day.

[0155] Typically, the subject receives long-term treatment with one or more GCs for a duration of, for example, at least 2 days, preferably at least 7 days, preferably at least 14 days, more preferably at least 21 days, more preferably at least 28 days, more preferably at least 35 days, more preferably at least 6 weeks, preferably at least 8 weeks, more preferably at least 12 weeks, more preferably at least 16 weeks, at least 20 weeks, at least 25 weeks, at least 30 weeks, at least 35 weeks, at least 40 weeks, at least 45 weeks, at least 50 weeks, more preferably at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years, at least 3.5 years, at least 5 years, and most preferably at least 10 years.

[0156] Typically, the subject has received one or more GC treatments for approximately 6 weeks to 10 years, approximately 8 weeks to 5 years, approximately 12 weeks to 3 years, and approximately 16 weeks to 1 year.

[0157] Preferably, the subject is receiving treatment for GC treatment side effects while receiving one or more GC treatments. Alternatively, the subject has received GC treatment before receiving the side effect treatment. Preferably, the subject received one or more GC treatments less than 5 years prior to receiving the GC treatment side effect treatment; more preferably less than 4 years, 3 years, 2 years, 1 year, 9 months, 6 months, 4 months, 2 months, and most preferably less than 1 month prior to receiving the GC treatment side effect treatment.

[0158] Typically, treatment for one or more of the GCs is performed approximately 1 week to 5 years, preferably approximately 2 weeks to 4 years, approximately 4 weeks to 3 years, approximately 2 months to 2 years, or for example, approximately 4 months to 1 year prior to treatment for the side effects of the GC treatment.

[0159] Typical dosages for GC treatment vary between about 0.5 mg and about 150 mg, for example, about 1 mg to about 100 mg of GC, especially about 4 mg to about 50 mg, preferably about 5 mg to about 20 mg. Generally, the dosage of GC is about 0.007 mg / kg to about 2 mg / kg, for example, 0.01 mg / kg to about 1.5 mg / kg, especially about 0.05 mg / kg to about 0.7 mg / kg, preferably about 0.07 mg / kg to about 0.3 mg / kg. Typically, the dosage of GC is about 0.2 mg / kg. 2 Body surface area approximately 85 mg / m² 2 Body surface area, preferably approximately 0.5 mg / m² 2 Body surface area approximately 60 mg / m² 2 Body surface area, more preferably about 2 mg / m² 2 Body surface area approximately 30 mg / m² 2 Body surface area, for example, about 3 mg / m² 2 Body surface area approximately 15 mg / m² 2Body surface area.

[0160] Those skilled in the art should understand that the dosage of ginsenosides depends on the specific circumstances, such as the patient, the side effects to be treated, and the dosage of GC that the subject is currently receiving or has previously received.

[0161] Typical doses of ginsenosides range from about 4 mg to about 400 mg, for example, from about 30 mg to about 70 mg. Generally, the dose of GC is from about 0.05 mg / kg to about 5.5 mg / kg, for example, from about 0.4 mg / kg to about 1.0 mg / kg.

[0162] Ginsenosides can be administered to the subject in a single dose or in multiple doses per day, such as 2 to 4 doses per day, provided that the total amount administered each day is the same.

[0163] Preferably, especially when the subject is receiving GC treatment, the molar amount of ginsenosides applied to the subject is at least equal to the molar amount of GC. More preferably, the application of ginsenosides in molar excess relative to GC is preferably at least 4 times the molar excess, more preferably at least 8 times, and even more preferably at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times, or at least 2000 times.

[0164] Preferably, the molar ratio of ginsenosides to GC applied to the object is about 1 (GC) to 2000 (ginsenosides), more preferably about 4 to about 1000, about 6 to about 100, or about 8 to about 20.

[0165] Ginsenosides can be administered in any suitable manner. Ginsenosides can be applied topically, intestinally, or parenterally. Furthermore, ginsenosides can be administered systemically, such as orally, sublingually, buccally, or rectally. Alternatively or additionally, the combination can be administered topically, such as transdermally, or by inhalation or nasal administration (orally). The combination can also be administered by injection, such as subcutaneously, intravenously, or intramuscularly. Preferably, ginsenosides are administered orally or transdermally.

[0166] Ginsenosides are preferably administered in a formulation containing a suitable carrier, preferably an aqueous carrier, and more preferably a hydrogel. In one specific embodiment, the ginsenosides are at least partially integrated into micelles, preferably into the outer layer of the micelles, the micelles having a molecular weight of at least 10,000 Da, and the ginsenosides are administered in a formulation containing an aqueous carrier. Such ginsenosides are particularly effective in treating or preventing GC.

[0167] The present invention also relates to a method for treating GC treatment side effects, wherein the GC treatment side effects are preferably (GC-induced) diabetes, (GC-induced) decreased cortisol levels, (GC-induced) slowed growth, (GC-induced) impaired wound healing, (GC-induced) tissue degeneration, (GC-induced) osteoporosis, (GC-induced) hypertension, (GC-induced) weight gain, and (GC-induced) muscle weakness, the method comprising administering ginsenosides to a subject who is suffering from or at risk of suffering from one or more GC treatment side effects.

[0168] The present invention also relates to the use of ginsenosides in the preparation of a medicament for treating the side effects of GC treatment, wherein the GC treatment side effects are preferably (GC-induced) diabetes, (GC-induced) decreased cortisol levels, (GC-induced) slowed growth rate, (GC-induced) impaired wound healing, (GC-induced) tissue degeneration, (GC-induced) osteoporosis, (GC-induced) hypertension, (GC-induced) weight gain, and (GC-induced) muscle weakness.

[0169] Combination of ginsenosides and GC in methods for the prevention or treatment of inflammatory diseases

[0170] The inventors further recognized that the combined treatment of ginsenosides and GC has several advantages over using GC or ginsenosides alone as anti-inflammatory drugs.

[0171] For example, ginsenosides advantageously reduce resistance to GC treatment. Without being bound by any theoretical framework, it is believed that ginsenosides can advantageously stabilize receptor mRNA and receptor proteins, thereby preventing the downregulation of glucocorticoid receptors (GR) and thus reducing sensitivity to GC treatment (Example 1).

[0172] Furthermore, the inventors discovered that ginsenosides and GC exhibit a favorable synergistic anti-inflammatory effect. This is advantageous because only lower doses of GC and ginsenosides are needed to achieve an effective anti-inflammatory response. Figure 1 c). Using lower doses of GC also advantageously reduces the risk of one or more side effects commonly associated with GC treatment.

[0173] Therefore, the present invention also relates to a combination of GC and ginsenosides in a method for preventing or treating inflammatory diseases. The inventors further recognize that when GC and ginsenosides are applied in a specific molar ratio, the side effects of GC treatment can be effectively prevented or treated while maintaining the anti-inflammatory effect.

[0174] Unwilling to be bound by any theory, the inventors believe that ginsenosides act as competitive antagonists to glucocorticoid receptors by inhibiting their dimerization (see Example 1).

[0175] Therefore, the present invention relates to a combination of GC and ginsenosides in a method for preventing or treating inflammatory diseases, wherein the method comprises administering GC and ginsenosides to a subject in need in a molar ratio of about 1:1 to about 1:2000, preferably about 1:4 to about 1:1000, more preferably 1:8 to 1:20.

[0176] The ginsenosides and the GC are as described above.

[0177] Preferably, the molar ratio of the GC to the ginsenoside is about 1:2 to about 1:1000, more preferably 1:4 to about 1:500, even more preferably about 1:6 to about 1:250, and even more preferably about 1:7 to about 1:100, especially about 1:8 to about 1:20. It is not intended to be bound by any theory that the optimal competitive antagonistic effect of the ginsenoside is obtained within these molar ratio ranges. Furthermore, within these molar ratio ranges, the solubility of a therapeutically effective amount of the ginsenoside is feasible, particularly when the ginsenoside is administered in a micellar formulation as described above.

[0178] The GC and the ginsenosides can be administered together or separately. Therefore, in one embodiment, the GC and the ginsenosides are administered in the form of a pharmaceutical composition according to the invention.

[0179] Alternatively, the GC and the ginsenosides can be applied alone. The GC and the ginsenosides are preferably applied within a time window of 1 hour or less, more preferably within a time window of 45 minutes or less, for example, within a time window of 30 minutes or less, and particularly preferably within a time window of 15 minutes or less. Most preferably, the GC and the ginsenosides are applied substantially simultaneously.

[0180] Alternatively, the ginsenosides may be applied shortly before or after GC application, for example, about 60 minutes before or after GC application, preferably about 45 minutes before or after GC application, more preferably about 30 minutes before or after GC application, for example, 15 minutes before or after GC application.

[0181] As previously mentioned, GC and ginsenosides have an advantageous synergistic effect, together achieving an anti-inflammatory effect. This synergistic effect allows for the application of GC and ginsenosides at lower doses than would be required to induce an anti-inflammatory effect if GC and ginsenosides were used alone.

[0182] Therefore, the present invention preferably relates to a combination of GC and ginsenosides for use according to the invention, wherein the GC and / or the ginsenosides are administered at therapeutically ineffective amounts. In other words, the amount of GC and / or the ginsenosides administered is preferably lower than the amount administered when GC or ginsenosides are used alone to treat the same object with the same anti-inflammatory disease.

[0183] Preferably, the application amount of the GC and / or the ginsenosides is at most 80% of the therapeutically effective amount, more preferably at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, or at most 5%. Preferably, the application amount of the GC and / or the ginsenosides is from about 1% to about 80% of the therapeutically effective amount, more preferably from about 2% to about 75%, from about 3% to about 70%, from about 4% to about 65%, from about 5% to about 60%, for example, from about 10% to about 50%.

[0184] As those skilled in the art will understand, the dosage of GC or ginsenosides prescribed to an individual typically depends on the patient, the patient's age, the disease, the type and dosage form of the GC or ginsenoside, and the specific circumstances. For example, the severity of the disease may require a higher dose to be effective. Similarly, as described herein, patients who have been taking GC long-term may develop at least partial resistance to GC treatment, which usually also requires a higher dose to achieve therapeutic effect. Furthermore, the dosage of systemically applied GC is typically different from that of topically applied GC.

[0185] Skilled technicians can select the appropriate dosage (preferably corresponding to the ineffective dosage) based on the information provided in this article, general knowledge, and a reasonable amount of trial and error.

[0186] Typical dosages of GC for regular administration can be found in medical registers, such as the Dutch "geneesmiddeleninformatiebank" (https: / / www.geneesmiddeleninformatiebank.nl / ords / f?p=111:1:0::NO:RP,1:P0_DOMAIN,P0_LANG:H,EN; accessed May 7, 2024).

[0187] As a rule of thumb, a dose of 5 mg prednisone is equivalent to approximately 0.75 mg dexamethasone, 0.6 mg betamethasone, 4 mg methylprednisolone, 4 mg triamcinolone, 5 mg prednisolone, 20 mg hydrocortisone, or 25 mg cortisone and 2 mg fludrocortisone (Nicolaides NC, Pavlaki AN, Maria Alexandra MA, et al. Glucocorticoid Therapy and Adrenal Suppression. [Updated 19 October 2018]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Table 1: [Glucocorticoid Equivalencies] (). Available at: https: / / www.ncbi.nlm.nih.gov / books / NBK279156 / table / adrenal_glucocorticoid-therapy-and-adrenal-suppression; accessed on May 7, 2024.

[0188] The table below also lists the commonly used GC dosages for subjects requiring treatment. In the table, the dosage range expressed in mg / kg corresponds to the total body weight (kg) of the subject requiring treatment. In the table, the dosage range expressed in mg / m² corresponds to the total body weight (kg) of the subject requiring treatment. 2 The dosage range expressed as body surface area covers the total surface area (m²) of the object requiring treatment. 2 As those skilled in the art will understand, when GC is applied topically (e.g., externally to the skin), the local concentration of GC applied to the infected area of ​​the skin may exceed the concentrations shown in the table below.

[0189]

[0190] Therefore, the typical amount of GC in the combination of GC and ginsenosides according to the present invention (used in the method according to the present invention) varies between about 0.01 mg and about 150 mg, preferably about 0.1 mg and about 100 mg, more preferably about 0.5 mg and about 50 mg, and most preferably about 1 mg and about 20 mg of GC.

[0191] Preferably, the GC dosage is from about 0.001 mg / kg to about 2.5 mg / kg, more preferably from about 0.007 mg / kg to about 1.5 mg / kg, more preferably from about 0.014 mg / kg to about 0.7 mg / kg, and most preferably from 0.03 mg / kg to about 0.3 mg / kg. Preferably, the dosage is at most 2.5 mg / kg, more preferably at most 1.4 mg / kg, at most 0.7 mg / kg, at most 0.3 mg / kg, at most 0.07 mg / kg, and most preferably at most 0.007 mg / kg, particularly at most 0.0001 mg / kg. Preferably, the GC dosage is about 0.01 mg / kg. 2 Body surface area approximately 100 mg / m² 2 Body surface area, preferably about 0.05 mg / m² 2 Body surface area approximately 60 mg / m² 2 Body surface area, more preferably about 0.1 mg / m² 2 Body surface area approximately 30 mg / m² 2 The optimal body surface area is approximately 0.2 mg / m². 2 Body surface area approximately 15 mg / m² 2 Body surface area. Preferably, the dose of GC is at most about 60 mg / m². 2 Body surface area, more preferably up to about 30 mg / m² 2 Body surface area, more preferably up to about 15 mg / m² 2 Body surface area, or even more preferably up to about 5 mg / m² 2 Body surface area, for example, at most 2 mg / m² 2 Body surface area, more preferably up to about 1 mg / m² 2 The optimal body surface area is approximately 0.2 mg / m². 2 Body surface area.

[0192] The dosage can be provided to the subject as a single dose or as multiple doses per day, such as 2 to 4 doses per day, provided that the total amount applied each day is approximately the same.

[0193] Therefore, the present invention preferably relates to a combination of GC and ginsenosides for use according to the invention, wherein the dosage of GC is at most about 150 mg / day, preferably at most 100 mg / day, more preferably at most 50 mg / day, more preferably at most 25 mg / day, more preferably at most 10 mg / day, more preferably at most 5 mg / day, even more preferably at most 4 mg / day, at most 3 mg / day, at most 2 mg / day, at most 1 mg / day, even more preferably at most 0.5 mg / day, particularly at most 0.1 mg / day, and even more particularly at most 0.01 mg / day. Preferably, the dosage of GC is from about 0.001 mg / day to about 150 mg / day, preferably from about 0.01 mg / day to about 100 mg / day, more preferably from about 0.1 mg / day to about 50 mg / day, and most preferably from about 0.5 mg / day to about 20 mg / day.

[0194] In a preferred embodiment, the present invention relates to a combination of GC and ginsenosides for use according to the present invention, wherein the GC and the amount thereof are shown in the table below.

[0195]

[0196] Therefore, in a particularly preferred embodiment, the present invention relates to a combination of GC and ginsenosides for use in the method according to the invention, wherein the GC is dexamethasone, and wherein the GC is administered at a therapeutically ineffective dose, preferably from about 0.007 mg / kg to about 0.3 mg / kg, more preferably from about 0.009 mg / kg to about 0.14 mg / kg, more preferably from about 0.01 mg / kg to about 0.11 mg / kg, more preferably from about 0.014 mg / kg to about 0.07 mg / kg; or wherein the GC is beclomethasone, and wherein the GC is administered at a therapeutically ineffective dose, preferably from about 0.001 mg / dose to about 0.3 mg / dose, more preferably from about 0.05 mg / dose to about 0.2 mg / dose, even more preferably from about 0.1 mg / dose to about 0.15 mg / dose; and / or the dosage is from about 0.001 mg / kg to about 0.005 mg / kg, more preferably from about 0.01 mg / kg to about 0.03 mg / kg.

[0197] The dosage of ginsenosides (preferably Rg1 or Re or a combination thereof) typically varies between about 4 mg and about 400 mg, preferably about 10 mg to about 300 mg, more preferably about 20 mg to about 200 mg, more preferably about 30 mg to about 150 mg, and most preferably about 40 mg to about 70 mg. Preferably, the dosage of ginsenosides (preferably Rg1, Re or a combination thereof) is about 0.05 mg / kg to about 5.5 mg / kg, more preferably about 0.1 mg / kg to about 4 mg / kg, more preferably about 0.2 mg / kg to about 3 mg / kg, more preferably about 0.3 mg / kg to about 2 mg / kg, and most preferably about 0.4 mg / kg to about 1.0 mg / kg. Preferably, the dosage of ginsenosides (preferably Rg1, Re or a combination thereof) is at most 5.5 mg / kg, more preferably at most 4 mg / kg, at most 3 mg / kg, at most 2 mg / g, at most 1 mg / kg, more preferably at most 0.5 mg / kg, and especially at most 0.05 mg / kg.

[0198] Preferably, the dosage of ginsenosides (preferably Rg1, Re, or a combination thereof) is about 1.5 mg / m². 2 Body surface area approximately 250 mg / m² 2 Body surface area, more preferably about 5 mg / m² 2 Body surface area approximately 200 mg / m² 2 Body surface area, preferably about 10 mg / m² 2 Body surface area approximately 150 mg / m² 2 Body surface area, approximately 15 mg / m² 2 Body surface area approximately 100 mg / m² 2 Body surface area, most preferably about 20 mg / m² 2 Body surface area approximately 50 mg / m² 2 Body surface area.

[0199] The dosage can be provided to the subject as a single dose or as multiple doses per day, such as 2 to 4 doses per day, provided that the total amount applied each day is the same.

[0200] Therefore, the present invention preferably relates to a combination of GC and ginsenosides for use according to the invention, wherein the ginsenosides (preferably Rg1 or Re or a combination thereof) are administered at a dose of up to about 400 mg / day, preferably up to 200 mg / day, up to 120 mg / day, more preferably up to 70 mg / day, up to 20 mg / day, and even more preferably up to 4 mg / day.

[0201] The combination can be administered in any suitable manner. For example, the combination can be administered systemically, such as by oral administration, sublingual administration, buccal administration, or rectal administration. Alternatively or additionally, the combination can be administered locally, such as transdermally, by (oral) inhalation, by nasal administration, or by intraocular administration. The combination can also be administered by injection, such as subcutaneous injection, intravenous injection, or intramuscular injection.

[0202] Optionally, the GC may be administered via a different route of administration than the ginsenosides. Preferably, the GC and the ginsenosides are administered via the same method of administration, preferably transdermal (topical), inhalation, or oral administration.

[0203] The combination is preferably administered in a formulation containing a suitable carrier (preferably an aqueous carrier). In one specific embodiment, the ginsenosides present in the formulation are at least partially integrated into micelles, preferably into the outer layer of the micelles, the micelles having a molecular weight of at least 10,000 Da, and are preferably administered in a formulation containing an aqueous medium (e.g., a hydrogel). This composition is particularly effective in achieving anti-inflammatory effects while preventing the occurrence of side effects.

[0204] The object can be any object that suffers from or is susceptible to an inflammatory disease (preferably chronic inflammation). Preferably, the object is a mammal, more preferably a human.

[0205] In principle, the inflammatory disease can be any inflammatory disease that can be alleviated by activating glucocorticoid receptors.

[0206] Examples include, but are not limited to, asthma, allergic rhinitis, hay fever, urticaria (hives), atopic dermatitis, chronic obstructive pulmonary disease (COPD), inflammation of joints, muscles and tendons, lupus, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), giant cell arteritis and polymyalgia rheumatica, and multiple sclerosis (MS).

[0207] Therefore, the present invention also relates to combinations of GC and ginsenosides for the prevention or treatment of asthma, allergic rhinitis, hay fever, urticaria (hives), atopic eczema, chronic obstructive pulmonary disease (COPD), inflammation of joints, muscles and tendons, lupus, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), giant cell arteritis and polymyalgia rheumatica, or multiple sclerosis (MS).

[0208] The present invention also relates to a method for treating inflammatory diseases, preferably selected from asthma, allergic rhinitis, hay fever, urticaria (hives), atopic eczema, chronic obstructive pulmonary disease (COPD), inflammation of joints, muscles and tendons, lupus, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), giant cell arteritis and polymyalgia rheumatica, or multiple sclerosis (MS), the method comprising administering a combination of GC and ginsenosides to the subject in need.

[0209] The present invention also relates to the use of the combination of GC and ginsenosides in the preparation of a medicament for treating inflammatory diseases, preferably selected from asthma, allergic rhinitis, hay fever, urticaria (hives), atopic dermatitis, chronic obstructive pulmonary disease (COPD), inflammation of joints, muscles and tendons, lupus, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), giant cell arteritis and polymyalgia rheumatica, or multiple sclerosis (MS), including administering the combination of GC and ginsenosides to a subject in need.

[0210] In a specific embodiment, the present invention preferably relates to a combination of GC (preferably selected from dexamethasone and beclomethasone) and ginsenosides (preferably PPT-type ginsenosides, more preferably selected from Rg1, Re and combinations thereof) in a method for preventing or treating inflammatory diseases, wherein the method comprises administering GC and ginsenosides to a subject in need in a molar ratio of about 1:1 to about 1:2000.

[0211] The target group is typically human beings who are in need, preferably adults, such as humans who are at least 18 years of age or older.

[0212] Pharmaceutical Composition

[0213] The present invention also relates to a pharmaceutical composition comprising ginsenosides, a pharmaceutically acceptable carrier, and preferably GC, more preferably wherein the molar ratio of GC to ginsenosides is preferably from about 1:1 to about 1:2000. The ginsenosides, GC, and preferred molar ratio are as described above.

[0214] Drug carrier

[0215] The pharmaceutical compositions according to the invention comprise a pharmaceutically acceptable carrier. The carrier may, in principle, be any pharmaceutically acceptable carrier known in the art, such as a liquid carrier (preferably an aqueous carrier) or a solid carrier (e.g., lactose).

[0216] Preferably, the pharmaceutically acceptable carrier is a water-based carrier, more preferably a hydrogel. Using this carrier, the pharmaceutical composition can be formulated into a topical preparation, such as a cream. Such pharmaceutical compositions are particularly suitable for dermatological applications, such as for eczema.

[0217] Alternatively, the drug carrier may also be a lipid carrier, or a combination of a liquid carrier and an aqueous carrier.

[0218] Preferably, the pharmaceutical compositions according to the invention are formulated into creams, lotions, balms, hydrogels, ointments, foams, gels, sprays, tablets, capsules, lozenges, external solutions, external suspensions, aerosols, injections, and syrups, most preferably hydrogels.

[0219] The pharmaceutical compositions according to the present invention may also contain one or more additives, such as humectants, stabilizers, dispersants, UV stabilizers, plasticizers, emulsifiers, softeners, preservatives, or pH adjusters.

[0220] In one specific embodiment, the present invention relates to a pharmaceutical composition comprising GC (preferably selected from dexamethasone and betamethasone), ginsenosides (preferably PPT-type ginsenosides, preferably selected from Rg1, Re, and combinations thereof), and a pharmaceutically acceptable carrier, preferably wherein the molar ratio of GC to ginsenosides is from about 1:1 to about 1:2000, more preferably from about 1:8 to about 1:20. Preferably, the ginsenosides are at least substantially integrated into micelles, preferably into the outer layer of the micelles, and the micelles have a molecular weight of at least 10,000 Da. This pharmaceutical composition has been found to be particularly effective in achieving anti-inflammatory effects while reducing the occurrence of side effects, especially those associated with GC treatment.

[0221] The present invention also relates to a method for preparing a pharmaceutical composition according to the invention. The method comprises mixing ginsenosides, a drug carrier, and optionally GC to obtain a pharmaceutical composition according to the invention.

[0222] Preferably, the method comprises mixing the ginsenoside preparation according to the invention with GC (preferably dexamethasone or beclomethasone) and a drug carrier (preferably hydrogel) to obtain the pharmaceutical composition according to the invention.

[0223] Alternatively or additionally, the method includes mixing ginsenosides, at least partially integrated in the outer layer of micelles with a molecular weight of at least 10,000 Da, with a drug carrier and preferably GC. Optionally, the GC may be at least partially integrated in the micelles (the inner lumen of the micelles), and the micelles containing the GC are mixed with the drug carrier to obtain a pharmaceutical composition according to the invention.

[0224] This invention also relates to the following provisions.

[0225] 1. A combination of glucocorticoids (GC) and ginsenosides in a method for the prevention or treatment of inflammatory diseases, wherein the method comprises administering GC and ginsenosides to a subject in need in a molar ratio of about 1:1 to about 1:2000.

[0226] 2. The combination of uses according to Clause 1, wherein the molar ratio is between about 1:4 and about 1:1000, more preferably between about 1:8 and about 1:20.

[0227] 3. A combination of uses according to any of the foregoing clauses, wherein the GC is administered at a therapeutically ineffective dose, preferably at a dose of up to 0.07 mg / kg / day, more preferably at a dose of up to 0.007 mg / kg / day.

[0228] 4. A combination of uses according to any of the preceding clauses, wherein the ginsenoside is administered at a therapeutically ineffective dose, preferably at a dose of up to 5.5 mg / kg / day, more preferably at a dose of up to 0.05 mg / kg / day.

[0229] 5. A combination of uses according to any of the foregoing clauses, wherein the GC is selected from beclomethasone and dexamethasone.

[0230] 6. A combination of uses according to any of the preceding clauses, wherein the inflammatory disease is selected from asthma, allergic rhinitis, hay fever, urticaria, atopic eczema, chronic obstructive pulmonary disease, inflammation of joints, muscles and tendons, lupus, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, giant cell arteritis and polymyalgia rheumatica, and multiple sclerosis.

[0231] 7. A combination of uses according to any of the preceding clauses, wherein the ginsenoside is at least partially integrated in the micelles, preferably at least partially integrated in the outer layer of the micelles, the micelles having a molecular weight of at least 10,000 Da.

[0232] 8. A combination of uses according to any of the foregoing clauses, wherein the ginsenoside is a PPT-type ginsenoside, preferably selected from Rg1, Re and combinations thereof.

[0233] 9. The combination of uses according to clauses 7 or 8 above, wherein the molecular weight of said micelles is at most 100,000 Da.

[0234] 10. A ginsenoside preparation comprising a PPT-type ginsenoside at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, wherein the micelles have a molecular weight of at least 10,000 Da.

[0235] 11. A ginsenoside preparation according to Clause 10, wherein the ginsenoside is a PPT-type ginsenoside, preferably selected from Rg1, Re and combinations thereof.

[0236] 12. A ginsenoside preparation according to clause 10 or 11, wherein the molecular weight of said micelles is at most 100,000 Da.

[0237] 13. The ginsenoside preparation according to any one of clauses 10 to 12 further comprises GC, preferably wherein the GC is encapsulated in the micelles.

[0238] 14. Ginsenosides in a method for preventing or treating side effects of glucocorticoid therapy, the method comprising administering ginsenosides to a subject who is suffering from or at risk of suffering from side effects of one or more glucocorticoid therapy.

[0239] 15. Ginsenosides used according to Clause 14, wherein the side effects are selected from GC-induced diabetes, GC-induced decreased cortisol levels, GC-induced decreased growth rate, GC-induced impaired wound healing, GC-induced tissue degeneration, GC-induced osteoporosis, GC-induced hypertension, GC-induced weight gain, and GC-induced muscle weakness.

[0240] 16. Ginsenosides for use according to Clause 14 or 15, wherein the subject is administered the ginsenosides at a dose of about 0.7 mg / kg / day to about 1.5 mg / kg / day.

[0241] 17. A ginsenoside preparation according to any one of claims 14 to 16 for use in any one of claims 10 to 13.

[0242] 18. A pharmaceutical composition comprising GC, ginsenosides and a pharmaceutically acceptable carrier, wherein the molar ratio of GC to ginsenosides is from about 1:1 to about 1:2000, preferably from about 1:4 to about 1:1000, more preferably from 1:8 to 1:20.

[0243] 19. The pharmaceutical composition according to Clause 18, wherein the ginsenoside is a protopanaxadiol (PPT) type ginsenoside, preferably selected from Rg1 and Re and combinations thereof.

[0244] 20. A pharmaceutical composition according to clause 18 or 19, wherein the ginsenoside is at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, the micelles having a molecular weight of at least 10,000 Da.

[0245] 21. A pharmaceutical composition according to any one of clauses 18 to 20, wherein the pharmaceutical composition is formulated as a cream, lotion, balm, hydrogel, ointment, foam, gel, spray, tablet, capsule, lozenge, topical solution, topical suspension, aerosol, and syrup.

[0246] 22. A pharmaceutical composition according to any one of clauses 18 to 21 used in a method for the prevention or treatment of inflammatory diseases.

[0247] 23. A pharmaceutical composition for use according to Clause 22, wherein the inflammatory disease is selected from asthma, allergic rhinitis, hay fever, urticaria, atopic eczema, chronic obstructive pulmonary disease, inflammation of joints, muscles and tendons, lupus, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), giant cell arteritis and polymyalgia rheumatica, and multiple sclerosis.

[0248] 24. A method for preparing a ginsenoside preparation according to any one of clauses 10 to 13, comprising:

[0249] (a) Provide ginsenoside extracts from plants or plant parts of the Panax genus, preferably Panax ginseng;

[0250] (b) Contact the ginsenoside extract with an aqueous solvent mixture to obtain a ginsenoside mixture;

[0251] (c) At least partially removing the aqueous solvent mixture from the ginsenoside mixture; and optionally...

[0252] (d) The ginsenoside mixture obtained in step c) is reconstituted in water to obtain a ginsenoside preparation.

[0253] 25. The method according to Clause 24, wherein the aqueous solvent mixture comprises a mixture of water and a non-aqueous solvent, wherein the non-aqueous solvent is preferably an alcohol solvent, more preferably selected from methanol, ethanol, propanol and isopropanol.

[0254] 26. The method according to clause 24 or 25, including a column chromatography step of using a nonpolar resin, preferably D101 macroporous resin, as the stationary phase, on the ginsenoside extract.

[0255] 27. The method according to any one of clauses 24 to 26, including a decolorization step of the ginsenoside extract, preferably using D941 macroporous resin.

[0256] 28. A ginsenoside preparation, which can be obtained by any one of the methods in clauses 24 to 27.

[0257] 29. A ginsenoside preparation according to Clause 28, comprising a PPT-type ginsenoside at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, wherein the molecular weight of the micelles is at least 10,000 Da.

[0258] 30. A ginsenoside preparation according to clause 28 or 29, wherein the ginsenoside is a PPT-type ginsenoside, preferably selected from Rg1, Re and combinations thereof.

[0259] 31. A ginsenoside preparation according to Clause 30, wherein the molecular weight of said micelles is at most 100,000 Da.

[0260] For clarity and brevity, the features described herein are part of the same or different implementations; however, it should be understood that the scope of the invention may include implementations having all or some of the described features.

[0261] The present invention is demonstrated through the following embodiments.

[0262] Example

[0263] Example 1: Combined processing of Rg1 and GC

[0264] Materials and Methods

[0265] Zebrafish strains and maintenance

[0266] Zebrafish (Danio rerio) were fed and handled in accordance with the guidelines of the Zebrafish Model Organism Database (http: / / zfin.org) and the instructions of the Leiden University Local Animal Welfare Committee. Zebrafish were exposed to a 14-hour light / 10-hour dark cycle to maintain their diurnal rhythm. Fertilization occurred naturally at the start of the light period. Eggs were collected and incubated in egg water (containing 60 μg / mL Instant Ocean sea salt and 0.0025% methylene blue) at 28°C. The following zebrafish strain was used in this study: the double transgenic strain Tg(mpx:GFP). i114 / mpeg1:mCherry umsF001 ), in which neutrophils and macrophages were fluorescently labeled (green and red), respectively [14,15]; and Tg(9xGCRE-HSV.UI23:EGFP) ia20 The reporter strains, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by a promoter containing GRE

[16] .

[0267] chemical substances

[0268] The chemical compounds beclomethasone, dexamethasone, Rg1, TNF-α, actinomycin D and cyclohexylimide were all purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0269] Zebrafish larvae tail fin trauma test

[0270] In the caudal fin trauma experiment, 20 larvae were used in each experimental group at 2 or 3 days post-fertilization (dpf). For trauma, the larvae were anesthetized in egg water containing 0.02% buffered ethyl aminobenzoate (tricaine; Sigma-Aldrich). The larvae were placed in culture dishes coated with 2% agarose and observed under a Leica M165C stereomicroscope (Leica Microsystems, Wetzlar, Germany), and the caudal fin was partially amputated using a 1-mm sapphire blade (World Precision Instruments, Sarasota, Florida, USA). For quantification of leukocyte migration, the larvae were fixed overnight in 4% paraformaldehyde (PFA) at 4°C. The following day, the fixed larvae were washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20 and stored at 4°C until imaging.

[0271] In the caudal fin trauma experiment, larvae were treated with a solvent (0.01% DMSO), beclomethasone (or dexamethasone), and / or Rg1 diluted in egg water at a specified concentration for 6 hours (2 hours before trauma and 4 hours after trauma).

[0272] Using Leica MZ16FA fluorescence stereomicroscopy (Leica Microsystems) to study Tg(mpx:GFP) i114 / mpeg1:mCherry umsF001 Imaging of juvenile strains was performed. Macrophages were detected based on their red (mCherry) fluorescence, and neutrophils were detected based on their green (EGFP) fluorescence. To quantify the number of macrophages and neutrophils recruited to the wound area, cells in specific regions of the caudal fin were identified by blinded manual counting.

[0273] Determination of Gr transactivation activity in zebrafish larvae

[0274] To investigate Gr transactivation activity in zebrafish larvae, Tg(9xGCRE-HSV.UI23:EGFP) was used. ia20The reporter strain expresses enhanced green fluorescent protein (EGFP) under the control of a promoter containing a series of nine GREs

[16] . To investigate the effects of solvent, 10 μM beclomethasone (or dexamethasone), 50 μM Rg1, and 10 μM beclomethasone (or dexamethasone) in combination with 50 μM Rg1, 15 embryos per group (2 days post-fertilization) were treated with the indicated compounds for 24 hours and fixed for visualization to measure whole-body fluorescence intensity using a Leica MZ16FA fluorescence stereomicroscope (Leica Microsystems) supported by LAS 3.7 software. The integrated intensity of the EGFP signal in the larvae was determined using ImageJ software.

[0275] Measurement of larval body length and caudal fin regeneration

[0276] To determine the length of regenerated caudal fin tissue or larval body length after trauma, embryos (15 per group) were chemically treated (beclomethasone / dexamethasone (10 μM), or Rg1 (50 μM), or beclomethasone / dexamethasone (10 μM) combined with Rg1 (50 μM)) starting 2 hours post-fertilization and continuing until 5 days post-fertilization. The solution was changed daily during this period. For regeneration experiments, the caudal fin was traumatized 2 days post-fertilization. To determine larval length and caudal fin regeneration, larvae were fixed overnight at 4°C with 4% PFA 5 days post-fertilization. The following day, imaging was performed using a Leica MZ16FA fluorescence stereomicroscope (Leica Microsystems) supported by LAS 3.7 software, and the length of the newly generated caudal fin tissue or the entire larva was measured using ImageJ software. The newly generated tissue could be visually distinguished from the old tissue, allowing for precise measurement of the size of the regenerated tissue.

[0277] Measurement of whole-body glucose in zebrafish larvae

[0278] Two hours post-fertilization, zebrafish embryos were chemically treated (solvent, 10 μM beclomethasone, 50 μM Rg1, or 10 μM beclomethasone combined with 50 μM Rg1), with the solution changed daily until 5 days post-fertilization. For dose-response curves, 0.1 μM, 1 μM, 5 μM, 10 μM, or 15 μM beclomethasone alone, or in combination with 50 μM Rg1, were introduced. Five days post-fertilization, larvae (15 embryos per sample) were collected in Eppendorf tubes, washed with oocyte fluid (3 times, 10 minutes each time), and incubated for 1 hour. Subsequently, 100 μL of ice-cold glucose buffer was added to each sample, and homogenized using BulletBlender® at 8,000 rpm for 3 minutes. The homogenate was then centrifuged at 11,000 rpm for 8 minutes at 4°C, and the supernatant was stored at -20°C. Whole-body glucose concentrations were determined using a glucose colorimetric assay kit (Cayman Chemical, Ann Arbor, Michigan, USA) following the manufacturer's instructions. For each experiment, three biological replicates were used for each treatment group, and two technical replicates were performed for the colorimetric assay.

[0279] Measurement of whole-body cortisol in zebrafish larvae

[0280] Two hours post-fertilization, zebrafish embryos were chemically treated (solvent, 10 μM beclomethasone, 50 μM Rg1, or 10 μM beclomethasone plus 50 μM Rg1), with the solution changed daily until 96 hours post-fertilization. For dose-response curves, 0.1 μM, 1 μM, 5 μM, 10 μM, or 15 μM beclomethasone alone, or in combination with 50 μM Rg1, were introduced. Treatment was then stopped, and the larvae were incubated in egg water until 5 days post-fertilization to avoid cross-reactivity with the cortisol antibody used in the ELISA. Treatment was then stopped, and the egg water was changed. Five days post-fertilization, the larvae were collected in Eppendorf tubes (30 embryos per sample), and 100 μl of ice-cold egg water was added. After removing excess water, the samples were frozen in an ethanol (EtOH) / dry ice bath. Subsequently, the homogenate was homogenized using BulletBlender® at 8,000 rpm for 3 minutes. Ethyl acetate was added to the homogenate. The homogenate was then centrifuged at 11,000 rpm for 8 minutes at 4°C, and the supernatant was collected and evaporated. 150 μl of 0.2% bovine serum albumin (Sigma-Aldrich) dissolved in PBS was added to the sample, and the sample was frozen. Whole-body cortisol levels in zebrafish larvae were determined by ELISA (Demeditec Diagnostics GmbH, Kiel-Welse, Germany) according to the manufacturer's instructions. For each experiment, three biological replicates were used for each treatment group, and two ELISA replicates were performed using the technique.

[0281] Competitive glucocorticoid receptor binding assay

[0282] To determine the relative in vitro binding affinity of different compounds for human GR, PolarScreen was used. TM The glucocorticoid receptor competition assay kit (Thermo Fisher Scientific, Waltham, MAS, USA) was used and the procedure was followed according to the manufacturer's instructions. In short, the test compound was first dissolved in DMSO to prepare a 10 mM stock concentrate, which was then further diluted with GR buffer (100 mM potassium phosphate (pH 7.4), 200 mM Na₂MoO₄, 1 mM EDTA, and 20% DMSO). The serially diluted compounds were then transferred to Corning® black 384-well plates, and Fluormone GSred was added. TM The full-length GR protein (partially purified receptor, dissolved in storage buffer) was then added. The plates were incubated at room temperature in the dark for 4 hours, and fluorescence polarization was measured using a CLARIOStar microplate reader (BMGLabtech, Ortenberg, Germany). The obtained values ​​were normalized to the maximum (ligand-free) and minimum control values ​​(10 μM dexamethasone), and the relative percentage of polarization was determined. The IC50 value for each compound was calculated by fitting a dose-response curve.

[0283] Cell culture

[0284] HeLa (human cervical cancer) cells were cultured in Duchenne modified Eagle (DMEM) high-glucose (HG) medium supplemented with 10% fetal bovine serum (FCS) and 10% glutamine (Sigma-Aldrich) without phenol red. Cells were maintained at 37°C and 5% CO2. Twenty-four hours before the addition treatment, the medium was replaced with DMEM HG containing 10% activated charcoal-inactivated serum and 10% Glutamax (Sigma-Aldrich). Cells were exposed to short-term and long-term compound treatments. In short-term treatment, cells were treated for 6 hours with TNF-α (10 ng / ml, Sigma-Aldrich) and solvent (0.01% DMSO), beclomethasone (0.01 μM, 0.1 μM or 1 μM) or dexamethasone (1 μM), Rg1 (20 μM, 100 μM or 500 μM), or beclomethasone (0.01 μM or 1 μM, or dexamethasone (1 μM)) in combination with Rg1 (20 μM, 100 μM, 500 μM). In long-term treatment, cells were treated for 24 hours with one of the following: solvent (0.01% DMSO), beclomethasone (0.01 μM, 0.1 μM, or 1 μM) or dexamethasone (1 μM), Rg1 (20 μM, 100 μM, or 500 μM), or beclomethasone (0.01 μM or 1 μM, or dexamethasone (1 μM)) combined with Rg1 (20, 100, or 200 μM), with or without the addition of actinomycin D (1 ng / ml) or cycloheximide (5 μg / ml). Subsequently, TNF-α (10 ng / ml) was added concurrently with the compound treatment for 6 hours (this final step was not added in the actinomycin D and cycloheximide experiments).

[0285] Immunocytochemistry

[0286] To determine the nuclear translocation of GR, immunocytochemical staining was performed on GR cells in HeLa cells. Cells were seeded in Nunc TM Lab-Tek TM II Chamber Slide TMCells were cultured for 48 hours in DMEM HG medium (phenol red-free) supplemented with 10% activated charcoal-inactivated serum and 100 mM Glutamax (Sigma-Aldrich) in a Thermo Fisher Scientific system. Subsequently, cells were treated with escalating doses of beclomethasone or Rg1 for 6 hours and fixed overnight at 4°C with 4% PFA. Cells were washed three times with PBS for 5 minutes each time; then washed for 15 minutes with PBS containing 0.1% Tween 20 (PBST); and finally washed for 30 minutes with PBST containing 3% BSA. Then, GR primary antibody (glucocorticoid receptor (D6H2L) XP® rabbit mAb (Cell Signaling Technology, Danvers University, Massachusetts, USA) diluted 1:1000 with PBST containing 3% BSA was added, and incubated overnight at 4°C. The following day, the samples were washed four times with PBS for 5 minutes each time. Then, add Alexa Fluor 488® goat anti-rabbit antibody (Sigma-Aldrich) diluted (1:500) with PBST containing 3% BSA and incubate at room temperature for 2 hours. Then wash the sample four times with PBS for 5 minutes each time. Use ProLong containing DAPI... TM Cell slides were mounted with Diamond anti-fading mounting medium (Thermo Fisher) and imaged using a Leica TCS SP8 confocal microscope (Leica Microsystems) with a 40x (1.25 NA) objective. Each treatment group was run three times in triplicate. In each experiment, 40 randomly selected cells from each treatment group were analyzed. Relative nuclear translocations of GR were quantified by measuring the integrated fluorescence intensity of the nucleus and whole cells using Fiji ImageJ v1.53c software, and these values ​​were corrected against the mean background fluorescence intensity. The percentage of nuclear translocation was assessed by determining the percentage (in%) of the corrected density in the nucleus relative to the corrected density in the whole cell.

[0287] Quantitative PCR (qPCR) analysis

[0288] To determine gene expression levels in zebrafish larvae, larvae aged 3 days post-fertilization were used under traumatic and untraumatized conditions, as well as short-term and long-term treatments. Fifteen larvae were collected from each group in TRIzol reagent (Thermo Fisher), and total RNA was isolated using the miRNeasy mini kit (Qiagen, Germany). For HeLa cell experiments, cells were seeded in 6-well plates, treated, and then removed from the wells using TRIzol reagent. mRNA was isolated using the miRNeasy mini kit (Qiagen) according to the manufacturer's instructions. DNA-free... TM RNA samples were DNase-treated using a DNA removal kit (Thermo Fisher Scientific). cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), with 1 μg of RNA per sample. For qPCR, 10 μM forward and 10 μM reverse primers, 12.5 μl of IQ SYBR Green Supermix (Bio-Rad Laboratories), and 2 μl of cDNA were added to the qPCR reaction mixture. The total volume of each mixture was 25 μl, which was divided into two 12.5 μl aliquots. qPCR reactions were performed on a MyiQ Monochrome Real-Time PCR Detection System (Bio-Rad Laboratories), with initial denaturation for 3 minutes at 95°C, followed by 40 cycles, each consisting of 15 seconds at 95.5°C, 15 seconds at 60°C, and 30 seconds at 72°C. The cycle threshold (Ct value, i.e., the number of cycles required to reach the fluorescence intensity threshold) was determined for each sample. Gene expression levels for each sample were normalized to the expression levels of pipial (peptidyl prolyl isomerase Ab (cyclosporine A)) in zebrafish samples and 18S rRNA expression levels in human cells. Fold changes (compared to the corresponding control group) for each sample were calculated using the ΔΔCt method. Three biological replicates were used for each treatment group in each experiment, and all reactions were performed in duplicate. The qPCR primer sequences used for zebrafish and HeLa cell experiments are listed in Table S1.

[0289] Table S1: qPCR primers for zebrafish and humans (Zebrafish and human primers)

[0290]

[0291] Protein blot

[0292] Western blotting was performed to determine GR protein levels in HeLa cells. Cells were seeded in 6-well plates as previously described. Cells were exposed to short-term and long-term compound treatments. In the short-term treatment, cells were treated for 6 hours with solvent (0.01% DMSO), beclomethasone (1 μM), Rg1 (20 μM), or beclomethasone combined with Rg1 (1 μM and 20 μM, respectively). In the long-term treatment, cells were treated for 30 hours with one of the following: solvent, beclomethasone (1 μM), Rg1 (20 μM), or beclomethasone combined with Rg1 (20 μM and 1 μM, respectively), with or without cycloheximide (5 μg / ml). Cells were harvested using trypsin (0.25% (v / v) trypsin dissolved in PBS, EDTA-free), washed twice with PBS, and centrifuged. After centrifugation at 3000 rpm for 3 minutes, the cell pellet was stored at -80°C until use. Vibra-Cell was used in potassium phosphate (KPi lysis buffer; 25 mM K2HPO4-KH2PO4 (pH 6.5) + 0.1% (v / v) Triton X-100 (Merck, Darmstadt, Germany)) TMCell homogenization was performed using a VCX 130 sonicator (Sonics, Newtown, Connecticut, USA) on ice (20% amplitude, 3 seconds on, 3 seconds off, 4 cycles). Total protein concentration in the homogenate was determined using a Quickstart Bradford Protein Assay Kit (Bio-Rad Laboratories) and measured using an EMax®plus microplate reader (Molecular Devices, Sunnyvale, California, USA). HeLa cell homogenate (30 μg protein dissolved in 10 μl of solution) was added to 30 μl of Laemmli sample buffer, vortexed, and boiled at 98°C for 5 minutes. 15 μl samples were loaded in duplicate onto 12% gradient pre-prepared SDS-PAGE gels (Bio-Rad Laboratories) and run at 90 V for approximately 2 hours (repeat was not used in the cycloheximide assay). All experiments were performed four times (six times for the cycloheximide assay). After gel electrophoresis, proteins were transferred to a nitrocellulose membrane (Thermo Fisher Scientific) using a Bio-Rad Power Pac Basic Mini electrophoresis system at 100V for 1 hour. The membrane was washed three times for 15 minutes each time with blocking buffer (Tris-buffered saline (TBST) containing 0.1% Tween-20 and 3% BSA) at room temperature. A 1:500 dilution of GR primary antibody (recombinant rabbit monoclonal antibody against glucocorticoid receptor (2D8, Thermo Fisher Scientific)) dissolved in TBST containing 3% BSA was added to the blot membrane, and the membrane was incubated overnight at 4°C. The next day, the blot membrane was washed three times for 5 minutes each time with TBST. Then, a 1:2000 dilution of HRP-conjugated anti-rabbit secondary antibody (Sigma-Aldrich) dissolved in TBST containing 3% BSA was added, and the membrane was incubated at room temperature for 2 hours. The blot membrane was washed three times for 5 minutes each time with TBST containing 3% BSA, followed by two more washes with TBST for 5 minutes each time. Finally, mix the blot membrane with 10 ml of Pierce. TMEnhanced chemiluminescence (ECL) protein blot substrate (Thermo Fisher Scientific) was incubated for 1 to 2 minutes. Chemiluminescence was detected using a ChemiDocMP imaging system (Bio-Rad Laboratories) with an exposure time of 1 minute. The blot membrane was washed three times with TBST for 5 minutes each time, followed by washing for 1 hour in TBST containing 3% BSA. β-actin antibody (MA1-91399, Thermo Fisher Scientific) was diluted 1:2000 in TBST containing 3% BSA, added to the blot membrane, and incubated overnight at 4°C. The next day, the blot membrane was washed three times with TBST containing 3% BSA for 5 minutes each time. Then, HRP-conjugated anti-mouse secondary antibody (Sigma-Aldrich) diluted 1:2000 in TBST containing 3% BSA was added, and the membrane was incubated for 2 hours at room temperature. Finally, the blot membrane was washed three times with TBST for 5 minutes each time. The blot membrane was incubated with 10 ml of ECL substrate for 1 to 2 minutes, and then the chemiluminescent signal was visualized. The intensity of the GR and β-actin bands was quantified using ImageJ software, and the GR level was normalized to the β-actin level.

[0293] Statistical analysis

[0294] Statistical analysis of the experiments was performed using GraphPad Prism software. One-way or two-way ANOVA was used, and Tukey's post-hoc test was performed. Statistical analysis of the qPCR data was performed on log2-transformed data. The significance level was set at P < 0.05, and the different significance levels in the figure are shown below: *P < 0.05; **P < 0.01; ***P < 0.001; Combined treatment compared to beclomethasone or dexamethasone alone: ​​#P < 0.05; ##P < 0.01; ###P < 0.001; Long-term treatment compared to corresponding short-term treatment: +P < 0.05; ++P < 0.01; +++P < 0.001.

[0295] result

[0296] When administered in combination with GC, Rg1 exhibits synergistic anti-inflammatory effects.

[0297] To investigate the combined effects of Rg1 and beclomethasone, we first measured the anti-inflammatory effects of beclomethasone, Rg1, and combinations of these compounds based on neutrophil migration 4 hours post-traumatic stress in zebrafish tail fin trauma assays. The chemical structures of the compounds are as follows: Figure 1 As shown in Figure A, and the experimental procedure diagram is as follows: Figure 1As shown in Figure B, beclomethasone was administered at concentrations of 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM, and Rg1 was administered at concentrations of 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM. The results indicated that beclomethasone concentrations below 10 μM had no effect on neutrophil migration to the wound site, while concentrations of 20 μM and 40 μM significantly reduced neutrophil migration. Figure 1 C). For Rg1, concentrations of 40 μM and below do not alter neutrophil migration, while concentrations of 80 μM and 160 μM do. Figure 1 C). For combined treatment, 10 μM beclomethasone was combined with different concentrations of Rg1 (10 μM, 20 μM, 40 μM, and 80 μM). Compared with the solvent-treated control group, all beclomethasone combined with Rg1 treatment groups showed reduced neutrophil migration to the wound site, and the higher the Rg1 dose, the stronger the inhibitory effect. Figure 1 C). Clearly, the combined treatment with Rg1 and beclomethasone had a synergistic effect on the anti-inflammatory effect of beclomethasone. Furthermore, the percentage of inhibition of neutrophil migration to the wound site by 10 μM beclomethasone, 80 μM Rg1, and the combined use of 10 μM beclomethasone and 80 μM Rg1 was calculated. Results are as follows... Figure 13 As shown, this clearly supports the conclusion that beclomethasone and Rg1 have a synergistic effect.

[0298] Based on these observations, we further investigated the anti-inflammatory effect of the combination of 10 μM beclomethasone and 50 μM Rg1. This treatment significantly reduced neutrophil migration but had no effect on macrophage migration. Figure 1 D, E, S1A), as previously shown in this experiment with each GC treatment

[17]

[18] . Furthermore, we investigated the effect of Rg1 in combination with another commonly used synthetic GC, dexamethasone. Similar to what was observed with beclomethasone, the data indicated that 10 μM dexamethasone did not affect neutrophil migration, but treatment with 50 μM Rg1 significantly inhibited neutrophil migration to the site of injury (D, E, S1A), as previously shown with treatment with each GC in this experiment

[17]

[18] . Figure S1 (B, C). These results indicate that the additive anti-inflammatory effect of Rg1 was observed not only when treated in combination with beclomethasone, but also when treated in combination with other GR agonists.

[0299] To explain the observed effects and generalize the results obtained in the zebrafish model to humans, we first investigated the binding of beclomethasone and Rg1 to human GR in vitro using competitive ligand binding assays. The results showed that beclomethasone has a high relative affinity for GR, as indicated by an IC50 value of 4.9 nM (…). Figure 2A). The relative binding affinity of Rg1 was significantly lower than that of beclomethasone (approximately 1 / 4,000), reflected in an IC50 of 22 μM ( Figure 2 A). Immunocytochemistry of HeLa cell cultures was used to determine the translocation of GR to the nucleus 6 hours after treatment with different concentrations of beclomethasone (0.01 μM, 0.1 μM, and 1 μM) and Rg1 (5 μM, 10 μM, and 20 μM). Compared with the solvent, the 0.01 μM beclomethasone concentration significantly increased the translocation level, and the largest nuclear translocations were observed after treatment with 0.1 μM and 1 μM beclomethasone. Figure 2 B, C). Compared with solvent treatment, Rg1 only increased the translocation level at 20 μM, and only induced partial translocation (B, C). Figure 2 B, C).

[0300] To investigate the anti-inflammatory effects of the combination of Rg1 and beclomethasone in human cells, we determined the anti-inflammatory effects of beclomethasone, Rg1, and combinations of these compounds after short-term treatment (6 hours) based on the pro-inflammatory genes IL1B and IL8. For this purpose, beclomethasone was administered at a concentration of 0.01 μM and Rg1 at a concentration of 20 μM, neither of which triggered the maximum translocation of GR. HeLa cells treated with TNF-α induced the expression of IL1B and IL8. This induction was slightly inhibited by treatment with 0.01 μM beclomethasone and 20 μM Rg1 alone. Figure 2 D). Interestingly, Rg1 (20 μM) in combination with beclomethasone (0.01 μM) strongly and significantly inhibited the induction of IL1B and IL8 after (6 h) treatment. Figure 2 D).

[0301] To confirm the anti-inflammatory effect of the combined application of Rg1 and GC, we measured the mRNA levels of two inflammation-related genes, il1b and il6, by qPCR at 4 hours post-traumatic time. Figure 1 F). Previous studies have shown that the cytokine-encoding genes il1b and il6 are trans-inhibited in this assay after treatment with beclomethasone or Rg1

[17] . The results showed that the cytokine-encoding genes il1b and il6 were upregulated after injury, and that 10 μM beclomethasone and 50 μM Rg1, as individual treatments, slightly reduced the expression of these genes (F). Figure 1 F). Interestingly, the combined treatment further significantly reduced the expression of il1b and il6 (F). Figure 1 F).

[0302] Ginsenoside Rg1 antagonizes GC-induced side effects in zebrafish

[0303] In zebrafish larvae, various biomarkers can be studied as indicators of simulated GC side effects: tissue regeneration

[17] , larval length, glucose and cortisol levels

[19] , and the expression of several Gr target genes. In this study, we used these biomarkers to examine whether co-treatment with Rg1 could alleviate GC-induced side effects.

[0304] First, caudal fin regeneration assays were performed, in which the caudal fin was amputated 2 days post-fertilization (similar to a trauma test), and the length of the regenerated fin tissue was measured 5 days post-fertilization. Zebrafish were incubated with compound treatments from 0 to 5 days post-fertilization. Our data showed that solvent-treated larvae fully regenerated the amputated caudal fin; beclomethasone (10 μM) inhibited regeneration; while Rg1 (50 μM) had no effect on the regeneration process, as previously shown

[17] . Interestingly, the combined treatment of Rg1 (50 μM) and beclomethasone (10 μM) inhibited regeneration, but this inhibition was significantly reduced compared to beclomethasone treatment ( Figure 3 A, S2A). Therefore, our data indicate that Rg1 reduces the inhibitory effect of beclomethasone-induced tissue regeneration. To confirm this result using another GC assay, we investigated the effect of Rg1 in combination with dexamethasone (10 μM) in the same assay ( Figure S2 A, B). The results showed that Rg1 also alleviated the dexamethasone-induced inhibition of the regeneration process, suggesting that Rg1 generally antagonizes the inhibition of regeneration by GC.

[0305] Secondly, we investigated the effects of the Rg1 / beclomethasone combination on zebrafish growth. To this end, we measured larval length at 5 days post-fertilization after exposing zebrafish to the compound treatment for the entire five-day period (from day 0 to day 5 post-fertilization). Our data showed that larval length was significantly reduced with beclomethasone treatment compared to solvent treatment. However, Rg1 treatment did not affect larval length, nor did the combined treatment affect (…). Figure 3 B), which suggests that Rg1 antagonized the effect of beclomethasone on juvenile growth.

[0306] Next, we investigated the effects of beclomethasone treatment on systemic glucose and cortisol levels five days post-fertilization, indicators of GC-induced metabolic and endocrine disorders, respectively. For glucose measurements (using a colorimetric method), larvae were treated for five days (days 0 to 5 post-fertilization); to determine the effect on cortisol levels (by ELISA), larvae were treated for four days (days 0 to 4 post-fertilization). Our results showed that, compared to solvent-treated larvae, beclomethasone-treated larvae exhibited elevated systemic glucose levels and decreased systemic cortisol levels. Figure 3C, E). In contrast, Rg1 had no effect on systemic glucose and cortisol concentrations. In larvae treated with a combination of beclomethasone and Rg1, systemic glucose levels were unaffected, while systemic cortisol concentrations were only slightly lower compared to solvent-treated larvae. Figure 3 (C, E). These data indicate that Rg1 antagonizes the effects of beclomethasone on the metabolic and endocrine systems.

[0307] To determine whether the antagonistic effect of Rg1 on beclomethasone's effects on glucose and cortisol levels is competitive or non-competitive, dose-response curves for beclomethasone (0.1 μM, 1 μM, 5 μM, 10 μM, and 15 μM) were generated in the presence and absence of Rg1 (50 μM). Our data indicate a dose-dependent effect of beclomethasone on systemic glucose and cortisol levels. Interestingly, there were significant differences between the beclomethasone-treated group and the group treated with the same concentration of beclomethasone in combination with 50 μM Rg1, and the curve for the combination treatment shifted to the right compared to the beclomethasone dose-response curve, suggesting that Rg1 acts as a competitive antagonist of beclomethasone. Figure 3 D, F).

[0308] It is generally believed that many side effects of GC treatment are a result of GR transactivation activity. To investigate whether Rg1 inhibits the transactivation activity of beclomethasone-activated Gr in zebrafish, Tg(9xGCRE-HSV.UI23:EGFP) was used. ia20 Report on a zebrafish strain. In this strain, EGFP gene expression is driven by a promoter containing a GRE element, thus Gr transactivation activity can be determined by measuring the intensity of EGFP in larvae.

[0309] Two days after fertilization, the larvae were treated for 24 hours, then fixed and observed under a stereomicroscope to determine the EGFP signal intensity within them. The relative fluorescence intensity was determined by normalizing the EGFP signal to the solvent group. Our results showed that both beclomethasone and dexamethasone significantly increased the relative intensity of EGFP. Figure 3 G, H, S2C). Importantly, Rg1 treatment did not affect EGFP signaling, but in the combined treatment group, Rg1 eliminated beclomethasone (and dexamethasone)-induced EGFP signal enhancement. Figure 3 (G, H, S2C). These data indicate that Rg1 antagonizes GC-induced Gr transactivation activity in zebrafish.

[0310] To further investigate the inhibitory effect of Rg1 on the transactivation activity of beclomethasone-activated Gr, the expression levels of two endogenous Gr target genes, pck1 and fkbp5, were measured by qPCR in traumatized zebrafish larvae treated for 6 hours 3 days post-fertilization. Our data indicate that although beclomethasone upregulated the expression of pck1 and fkbp5, Rg1 had no effect on the expression of these genes. Figure 3 I). Interestingly, the combined treatment with beclomethasone / Rg1 had no effect on the expression of either of these genes. Figure 3 I). These data confirm the antagonistic effect of Rg1 on the transactivation activity of Gr in zebrafish.

[0311] Long-term combined treatment with beclomethasone and Rg1 did not reduce GC sensitivity.

[0312] We investigated whether long-term beclomethasone treatment induced GC resistance in a zebrafish model. To this end, we investigated the effects of beclomethasone on endogenous Gr target genes fkbp5, pck1, and nfkbiaa at day 5 post-fertilization after short-term (6 hours) and long-term (5 days, from day 0 to day 5 post-fertilization) treatment. Our data showed that beclomethasone (10 μM) upregulated fkbp5 (…) after short-term treatment. Figure 4 A), pck1 ( Figure 4 B) and nfkbiaa ( Figure 4 The expression of C was increased, but this upregulation of pck1 and nfkbiaa was significantly reduced after long-term treatment. Figure 4 A, B), while the upward adjustment of fkbp5 did not decrease ( Figure 4 A). Rg1 (50 μM) had no effect on the expression of these genes after either short-term or long-term treatment. Importantly, Rg1 (50 μM) in combination with beclomethasone (10 μM) similarly upregulated the expression of these genes after both short-term and long-term treatment, indicating that larvae did not lose GC sensitivity during long-term combined treatment. Figure 4 A to Figure 4 C).

[0313] To investigate the effects of beclomethasone and Rg1 treatments on gr expression levels in zebrafish larvae, qPCR analysis was performed 5 days post-fertilization after short-term and long-term treatments. Figure 4 D). Experimental results showed that beclomethasone only inhibited gr mRNA levels after long-term treatment. Rg1 did not affect gr mRNA levels, but long-term combined treatment alleviated the inhibitory effect of beclomethasone on gr expression. In fact, the combined treatment group did not show any inhibition of gr mRNA concentration. Figure 4D). These data indicate that Rg1 can alleviate beclomethasone-induced gr expression inhibition, which could explain why GC sensitivity was not reduced after Rg1 / beclomethasone combined treatment.

[0314] To investigate the effect of Rg1 on beclomethasone-induced decrease in GR sensitivity in human cells, we first determined the effects of beclomethasone, Rg1, and combinations thereof on the pro-inflammatory gene IL1B in HeLa cells after short-term (6 h) and long-term (30 h) treatment. For this purpose, beclomethasone was administered at concentrations of 0.01 μM, 0.1 μM, and 1 μM, and Rg1 was administered at concentrations of 20 μM, 100 μM, and 500 μM. Cells were then treated with TNF-α for the last 6 h. This TNF-α treatment increased IL1B expression, and after short-term treatment, 0.1 μM and 1 μM beclomethasone strongly inhibited this increase. However, after long-term treatment, we observed a significant decrease in the inhibitory effect of these high doses of beclomethasone. Figure 5 A). All three doses of Rg1 resulted in decreased IL1B expression after short-term treatment, with no significant difference after long-term treatment. Similarly, Rg1 combined with beclomethasone inhibited the induction of IL1B expression after both short-term and long-term treatment, indicating no loss of GC sensitivity during long-term combined treatment. Similar data were obtained regarding the effect of Rg1 (20 μM) combined with dexamethasone (1 μM) on IL1B expression. Figure S4 C).

[0315] To investigate the effect of Rg1 on the reduced GC sensitivity after long-term treatment in more detail, the effects of two different concentrations of beclomethasone (0.01 μM and 1 μM) combined with Rg1 (20 μM) on IL1B expression at several time points during short-term and long-term treatments in combination with TNF-α treatment were studied. The results showed that high-dose beclomethasone (1 μM) exhibited a strong inhibitory effect on IL1B expression during short-term treatment, while low concentration (0.01 μM) showed almost no effect. The inhibitory effect of high-dose beclomethasone was relatively stable over 6 hours, and Rg1 had a slight additional effect when co-administered. Figure S4 A). However, after long-term treatment, high-dose beclomethasone (1 μM) did not significantly inhibit IL1B expression, while low-dose (0.01 μM) did have a significant inhibitory effect. Figure S4 (B) Interestingly, Rg1 combined with high-dose beclomethasone treatment strongly inhibited IL1B. These data suggest that the reduced GC sensitivity of HeLa cells after long-term high-dose beclomethasone treatment can be prevented by combined treatment with Rg1.

[0316] Subsequently, we investigated the effects of Rg1 (20 μM), beclomethasone (1 μM), and Rg1 combined with beclomethasone on the expression of pro-inflammatory genes MMP9 and IL8 after short-term and long-term treatment. The results showed that the inhibitory effect of beclomethasone was significantly reduced after long-term treatment. In contrast, Rg1 slightly inhibited the expression of MMP9 and IL8 after short-term treatment, and the inhibition was stronger after long-term treatment. Figure 5 B, C). Interestingly, Rg1 combined with beclomethasone significantly reduced the expression of both genes after both short-term and long-term treatment. Figure 5 (B, C). These data confirm that the combined Rg1 treatment prevents the decrease in GC sensitivity observed after long-term GC processing.

[0317] In addition to its effect on GR trans-inhibitory activity in HeLa cells, we also investigated the effects of beclomethasone (1 μM) and Rg1 (50 μM) on GR trans-activation activity. To this end, we measured the expression of GR target genes FKBP5, NFKBIA, GILZ, and SGK1 after short-term and long-term treatments. Beclomethasone strongly increased the expression of these genes after short-term treatment (…). Figure 5 (D to G). Beclomethasone-induced FKBP5 expression levels increased over time (i.e., long-term treatment resulted in higher expression levels than short-term treatment), while beclomethasone-induced NFKBIA, GILZ, and SGK1 expression levels decreased over time. Rg1 alone did not affect the expression of these genes after either short-term or long-term treatment. In short-term combined treatment, Rg1 antagonized transactivation activity, reducing beclomethasone-induced expression of the studied genes; while in long-term combined treatment, Rg1 eliminated the beclomethasone-induced increase in FKBP5 expression, slightly increased the transactivation activity of NFKBIA and GILZ, and did not significantly alter SGK1 expression. Figure 5 D to G). Similar data were also observed for NFKBIA expression with the dexamethasone (1 μM) / Rg1 (20 μM) combination. Figure S4 D). These data suggest that decreased sensitivity after long-term GC treatment is not a common feature of all genes transactivated by GR, but for genes exhibiting this decreased sensitivity, Rg1 co-treatment may prevent this decrease.

[0318] Rg1 inhibits beclomethasone-induced homologous downregulation of GR.

[0319] To explain the observed changes in GC sensitivity and to confirm the observed effect on zebrafish GR expression in human cells, we assessed GR mRNA and protein levels in HeLa cells after short-term (6 h) and long-term (24 h) treatment with beclomethasone (1 μM) and Rg1 (20 μM) by qPCR and Western blot analysis. Our data show that beclomethasone inhibited GR expression at both the mRNA and protein levels, and this inhibitory effect increased over time, while Rg1 did not affect GR mRNA or GR protein levels. Figure 6 A, B, S5A, B). Interestingly, the Rg1 / beclomethasone combined treatment, compared with solvent treatment, did not affect GR mRNA concentration after either short-term or long-term treatment. Figure 6 A) only caused a slight decrease in GR protein levels in short-term treatment and had no effect on protein levels in long-term treatment. Figure 6 (B, S5A, B). These data indicate that beclomethasone reduces GR mRNA and GR protein levels, while Rg1 does not affect these levels; however, combined treatment with Rg1 can inhibit beclomethasone-induced GR expression reduction.

[0320] To differentiate the effects on transcription and translation from those on mRNA and protein stability, we investigated the effects of long-term beclomethasone / Rg1 co-treatment in the presence of the transcription inhibitor actinomycin D (1 ng / ml) or the protein synthesis inhibitor cycloheximide (5 μg / ml). This allowed us to determine the effects of different treatments on mRNA and protein stability. Our results showed that, as expected, actinomycin D and cycloheximide significantly reduced GR mRNA and protein concentrations in HeLa cells treated with the solvent, beclomethasone, Rg1, or Rg1 combined with beclomethasone, although the effect on mRNA levels in the beclomethasone-treated group was not statistically significant. Figure 6 A, C, S5C). When we compared the mRNA levels in the actinomycin D treatment groups, we found that beclomethasone reduced mRNA levels when transcription was blocked, while Rg1 and beclomethasone / Rg1 did not change GR mRNA levels ( Figure 6 A). Clearly, beclomethasone, rather than Rg1, reduces the stability of GR mRNA. Similar data were observed at the protein level in the presence of cyclohexylimide. Figure 6 (C, S5C) indicates that beclomethasone affects protein stability in a similar way to its effect on mRNA levels, while Rg1 has no effect. In summary, these data suggest that the effect of beclomethasone on GR expression levels is largely due to reduced mRNA and protein stability, and that this stability reduction can be prevented by co-treatment with Rg1.

[0321] Example 2: Anti-inflammatory effects of PPT, F1, Rg1 and Rh1 and their influence on glucose and cortisol levels, larval length and post-traumatic tissue regeneration

[0322] Materials and Methods

[0323] Zebrafish strains and maintenance

[0324] Zebrafish (Danio rerio) were fed and handled in accordance with the guidelines of the Zebrafish Model Organism Database (http: / / zfin.org) and the instructions of the Leiden University Local Animal Welfare Committee. Zebrafish were exposed to a 14-hour light / 10-hour dark cycle to maintain their diurnal rhythm. Fertilization occurred naturally at the start of the light period. Eggs were collected and incubated in egg water (containing 60 μg / ml Instant Ocean sea salt and 0.0025% methylene blue) at 28°C. The following zebrafish strains were used in this study: AB / TL wild-type and the transgenic strain Tg(mpx:GFP). i114 / mpeg1:mCherry umsF001 [14,15] and Tg(9xGCRE-HSV.UI23:EGFPia 20

[16] and the mutant strain grs357

[20] .

[0325] chemical substances

[0326] The chemical compounds beclomethasone, prednisolone, dexamethasone, dexamethasone glucuronide (GDex), PPT, Rh1, F1, Rg1, actinomycin D, and cyclohexylimide were all purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Glucose-prednisolone (GPdn) was synthesized in our laboratory as described above

[21] . MZ31 was kindly provided by Prof. Dr. Hans Aerts (Leiden University, Netherlands).

[0327] Fish embryo acute toxicity test (FET)

[0328] Fish embryo acute toxicity (FET) tests were performed to determine the tolerable dose of the compounds under investigation. Due to the relatively high cost of the ginsenosides under investigation, the OECD FET guideline (No. 236 http: / / dx.doi.org / 10.1787 / 4.9789264203709-en)

[22] was adapted from a previously published protocol that had been successfully used to test nanoparticles

[23] , [17,21]. Three test concentrations (100 μM, 120 μM, and 150 μM) of Rg1, Rh1, and F1, and three test concentrations (25 μM, 50 μM, and 75 μM) of PPT were used. Four test concentrations (5 μM, 10 μM, 20 μM, and 25 μM) of beclomethasone were used. The stock solution was prepared with DMSO and finally diluted with egg water (60 μg / mL Instant Ocean sea salt and 0.0025% methylene blue) to achieve a final DMSO concentration of 0.01%. The following controls were used: negative control (nC, egg water), solvent control (sC, egg water containing 0.01% DMSO), and positive control (pC, egg water containing 4 mg / L 3,4-dichloroaniline (Sigma-Aldrich, St. Louis, Missouri, USA).

[0329] Embryos were collected approximately 1.5 hours post-fertilization and allocated to standard 24-well plates (10 embryos per well), with 2 mL of test solution added to each well. The transgenic line Tg (mpx: GFPi114 / mpeg1:mCherry-FumsF001) was used for appropriate comparisons with other experiments in this study. Five 24-well plates were prepared, each with three wells containing Rg1, F1, or Rh1 solution (100 μM, 120 μM, 150 μM), three wells containing PPT solution (25 μM, 50 μM, and 75 μM), two wells for nC and sC, and one well for pC (leaving seven empty wells in each plate). All solutions were changed daily.

[0330] The plates were maintained at 26–27°C and exposed to a 12-hour light-dark cycle. The mean survival rate (%) of all experimental groups was determined at 96 and 120 hours post-fertilization. Hatching was also recorded at 48 and 72 hours post-fertilization. The survival rates of the studied compounds and pC-treated groups in the five independent plates were averaged. At 96 hours post-fertilization, both hatching rates (nC and sC ≥ 80%) and survival rates (nC and sC ≥ 90%, pC ≤ 70%) met the test validity criteria.

[0331] Zebrafish larvae tail fin trauma test

[0332] In the caudal fin trauma experiment, larvae 2 or 3 days after fertilization were anesthetized in egg water containing 0.02% buffered ethyl aminobenzoate (tricaine; Sigma-Aldrich). The larvae were placed in petri dishes coated with 2% agarose and observed under a Leica M165C stereomicroscope (Leica Microsystems, Wetzlar, Germany), and the caudal fin was partially severed using a 1-mm sapphire blade (World Precision Instruments, Sarasota, Florida, USA).

[0333] Quantitative analysis of leukocyte migration

[0334] In experiments using leukocyte migration as an indicator, if the larvae were to be treated with chemicals, treatment began 2 hours before truncation (pretreatment) and continued for 4 hours after tail fin truncation (20 larvae per group unless otherwise specified). In some experiments, treatment with MZ31 (10 μM) was initiated 2 days post-fertilization and continued for 24 hours during pretreatment and treatment with other chemicals. Four hours post-truncation, the larvae were fixed overnight in 4% paraformaldehyde (PFA) at 4°C. The following day, the fixed larvae were washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20 and stored at 4°C until imaging. The images from Tg (mpx:GFP) were analyzed using a Leica MZ16FA fluorescence stereomicroscope (Leica Microsystems) supported by LAS 3.7 software. i114 / mpeg1:mCherry umsF001 Imaging was performed on juveniles of the strain. Macrophages were detected based on their red (mCherry) fluorescence, and neutrophils were detected based on their green (EGFP) fluorescence. To quantify the number of macrophages and neutrophils recruited to the wound area, cells in specific regions of the caudal fin were identified by blinded manual counting. When using grus... 357 During the juvenile stage of the mutant strain, neutrophils were labeled by specifically staining myeloperoxidase (MPX) positive cells using the TSA fluorescein assay kit (PerkinElmer) according to the manufacturer's instructions.

[0335] Measurement of larval body length and caudal fin regeneration

[0336] In experiments using the length of regenerated caudal fin tissue or larval body length as indicators after trauma, chemical treatment was initiated 2 hours post-fertilization and continued until 5 days post-fertilization, as instructed (15 larvae per group). The solution was changed daily during this period. For regeneration experiments, the caudal fin was severed 2 days post-fertilization. Five days post-fertilization, larvae were fixed overnight at 4°C with 4% PFA. To determine larval length and caudal fin regeneration, larvae were imaged using a Leica MZ16FA fluorescence stereomicroscope supported by LAS 3.7 software. The length of the entire larva or newly formed tissue was measured using ImageJ software. Newly formed tissue can be visually distinguished from old tissue, allowing for accurate measurement of its length.

[0337] Measurement of whole-body glucose in zebrafish larvae

[0338] Two hours post-fertilization, zebrafish embryos were chemically treated with or without MZ31, with the solution changed daily until five days post-fertilization. Caudal fin trauma was performed two days post-fertilization. Five days post-fertilization, larvae (15 per sample) were placed in ovipositor for 1 hour, then collected in Eppendorf tubes and washed with ovipositor (3 times, 10 minutes each). Subsequently, 100 μL of ice-cold glucose buffer was added to each sample, and homogenized using BulletBlender® at 8,000 rpm for 3 minutes. The homogenate was then centrifuged at 11,000 rpm for 8 minutes at 4°C, and the supernatant was stored at -20°C. Whole-body glucose concentrations were determined using a glucose colorimetric assay kit (Cayman Chemical, Ann Arbor, Michigan, USA) according to the manufacturer's instructions. Three biological replicates were used for each treatment group in each experiment, and two technical replicates were used for colorimetric determination.

[0339] Measurement of whole-body cortisol in zebrafish larvae

[0340] Zebrafish embryos 2 hours post-fertilization were chemically treated with or without MZ31, with the solution changed daily until 96 hours post-fertilization. Caudal fin trauma was performed 2 days post-fertilization. At 96 hours post-fertilization, treatment was stopped, and larvae were incubated in egg water until 5 days post-fertilization to avoid cross-reactivity of the compounds with the cortisol antibody used in the ELISA. 5 days post-fertilization, larvae were collected into Eppendorf tubes (30 larvae per sample) and 100 μl of ice-cold egg water was added. After removing excess water, the samples were frozen in an ethanol (EtOH) / dry ice bath. Subsequently, homogenization was performed using BulletBlender® at 8,000 rpm for 3 minutes. Ethyl acetate was added to the homogenate. The homogenate was centrifuged at 11,000 rpm for 8 minutes at 4°C, and the supernatant was collected and evaporated. 150 μl of 0.2% bovine serum albumin (Sigma-Aldrich) dissolved in PBS was added to the sample, and the sample was frozen. Whole-body cortisol levels in zebrafish larvae were determined using the Cortisol-free in Saliva ELISA kit (Demeditec Diagnostics GmbH, Kiel-Werssel, Germany) according to the manufacturer's instructions. For each experiment, three biological replicates were used for each treatment group, and two technical replicates were performed for the ELISA.

[0341] Determination of Gr transactivation activity in zebrafish larvae

[0342] To investigate Gr transactivation activity in zebrafish larvae, Tg(9xGCRE-HSV.UI23:EGFP) was used. ia20 The reporter strain expressed enhanced green fluorescent protein (EGFP) under the control of a promoter containing a series of nine GREs

[16] . To investigate the effects of the chemical treatment, zebrafish embryos (15 per group) 2 days post-fertilization were treated with the indicated compound for 24 hours and fixed for visualization to measure whole-body fluorescence intensity using a Leica MZ16FA fluorescence stereomicroscope (Leica Microsystems) supported by LAS 3.7 software. The integrated intensity of the EGFP signal in the larvae was determined using ImageJ software.

[0343] overexpression of gba2 in zebrafish

[0344] To overexpress gba2 in zebrafish, a plasmid (pDEST-zeo-zGBA2) containing cDNA encoding the zebrafish gba2 gene fused to the CMV promoter was used [21,23,24]. The plasmid (1 nl / egg, final concentration 80 pg / egg) diluted in nuclease-free water was injected into single-celled zebrafish embryos using an Automated Microinjection System Version 3 AMS-03 (Life Science Methods BV, Leiden, Netherlands). Following injection, chemical treatment was performed as instructed.

[0345] Quantitative PCR (qPCR) analysis

[0346] To determine the mRNA levels of specific genes in zebrafish, 3-day-old post-fertilization larvae (wounded and unwounded) were used, and total RNA was isolated using the miRNeasy mini kit 6 hours after chemical treatment (2 hours before and 4 hours after trauma), as instructed. For each sample, 15 larvae per group were collected in TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and total RNA was isolated using the miRNeasy mini kit (Qiagen, Hilden, Germany). For a similar experiment in HeLa cells, cells were seeded in 6-well plates, chemically treated for 6 hours, and then removed from the wells using TRIzol reagent. Total RNA was isolated using the miRNeasy mini kit according to the manufacturer's instructions. DNA-free... TMRNA samples were DNase-treated using a DNA removal kit (ThermoFisher Scientific). cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), with 1 μg of RNA per sample. For PCR, 10 μM forward and 10 μM reverse primers, 12.5 μl of iQ SYBR Green Supermix (Bio-Rad), and 2 μl of cDNA were added to the qPCR reaction mixture. The total volume of each mixture was 25 μL, which was divided into two 12.5 μL aliquots. The reaction was performed on a MyiQ Monochrome Real-Time PCR Detection System (Bio-Rad Laboratories) with an initial denaturation temperature of 95 °C for 3 minutes, followed by 40 cycles, each consisting of 15 seconds at 95.5 °C, 15 seconds at 60 °C, and 30 seconds at 72 °C. The cycle threshold (Ct value, i.e., the number of cycles required to reach the fluorescence intensity threshold) was determined for each sample. Gene expression levels for each sample were normalized to the expression levels of pipial (peptidyl prolyl isomerase Ab (cyclosporine A)) in zebrafish samples and 18S rRNA expression levels in human cells. Fold changes (compared to the corresponding control group) for each sample were calculated using the ΔΔCt method. Three biological replicates were used for each treatment group in each experiment, and all reactions were performed in duplicate. The qPCR primer sequences for zebrafish and HeLa cell experiments are listed in Tables 1 and 2, respectively.

[0347] Table 1. qPCR primers for zebrafish

[0348]

[0349] Table 2. Human qPCR primers

[0350]

[0351] Cell culture and transfection

[0352] HeLa cells (human cervical cancer cells) were purchased from ATCC and cultured in Duchenne Modified Eagle (DMEM) high-glucose (HG) medium supplemented with 10% fetal bovine serum (FCS) and 10% Glutamax (Sigma-Aldrich) without phenol red. Cells were maintained at 37°C and 5% CO2. Twenty-four hours before treatment, cells were seeded in 6-well plates, allowing adhesion with or without MZ31 (1 μM). MZ31 was added overnight. After adhesion and reaching 80% confluence, cells were treated with the indicated chemicals for 6 hours with or without TNF-α (10 ng / ml) (Sigma-Aldrich) and / or MZ31 (1 μM).

[0353] To overexpress GBA2 in HeLa cells, a plasmid (pDEST-zeo-hGBA2) [21,23,24] containing cDNA encoding the human GBA2 gene fused to the CMV promoter was transfected into HeLa cells with approximately 70% confluence. The plasmid was mixed with FuGENEHD transfection reagent (Promega, Madison, Wisconsin, USA) and 500 μl of serum-free DMEM. The mixture was incubated at room temperature for 20 minutes and then added to HeLa cells cultured in supplemented DMEM. The medium was changed after two days, and the cells were chemically treated for 6 hours as instructed.

[0354] Statistical analysis

[0355] Statistical analysis of the experiments was performed using GraphPad Prism software, employing one-way or two-way ANOVA, and Tukey post-hoc tests were conducted.

[0356] The anti-inflammatory effects of monosaccharidated ginsenosides depend on glucocorticoid receptor function, rather than the deglycosylation effect of glucocorticoid β2.

[0357] To investigate whether monosacchariylated ginsenosides (such as F1 and Rh1) have similar effects and dependence on Gr and / or Gba2, we studied their anti-inflammatory effects in zebrafish larvae. To explore the structure-activity relationships of these compounds, we used ginsenoside protopanaxadiol (PPT), F1, Rh1, and Rg1, as well as the glucocorticoid beclomethasone (Bec) as positive controls (compound structures are shown in Figure 1). Figure 7As shown in A). PPT did not undergo glycosylation. F1 and Rh1 consist of the aglycone PPT and a glucose group attached to the C-20 or C-6 position of the PPT backbone, respectively. Rg1 consists of the aglycone PPT and a glucose group attached to the C-6 position and a glucose group attached to the C-20 position. First, to determine the appropriate dosage of ginsenosides in this study, we performed an acute embryo toxicity test (FET), exposing zebrafish embryos to a range of concentrations of these compounds (from 0 to 120 hours post-fertilization) and determining the effects on hatching and survival. Figure S7 (A, B). For PPT, no effect was observed up to 50 μM, while a significant effect was observed at 75 μM. For F1, Rh1, and Rg1, no effect was observed up to 100 μM, while a slight effect was observed at 120 μM. Based on these results, we selected a dose of 50 μM for PPT and 100 μM for F1, Rh1, and Rg1.

[0358] We used these dosages to determine the anti-inflammatory effects of these compounds in zebrafish larvae. We induced localized inflammation in zebrafish larvae by trauma to the tail fin 3 days post-fertilization (dpf) (see schematic diagram). Figure 7 B). The results showed that, similar to Bec treatment, treatment of larvae with ginsenosides PPT, F1, Rh1, and Rg1 inhibited neutrophil migration without affecting macrophage migration. Figure 7 C). For all compounds, the inhibitory effect on neutrophil migration was eliminated in zebrafish larvae of Gr-deficient mutant strains. Figure S7 C) indicates that the anti-inflammatory effect of the ginsenosides studied is mediated by Gr.

[0359] Furthermore, we investigated whether the anti-inflammatory effects of monosaccharide-based ginsenosides also require the participation of Gba2 using the specific GBA2 inhibitor MZ31. Treatment with MZ31 did not affect the reduction in neutrophil migration caused by monosaccharide-based ginsenosides F1 and Rh1 or aglycone ginsenoside PPT. In contrast, this inhibitor eliminated the effect of polysaccharide-based ginsenoside Rg1. Figure 7 D). In summary, these data indicate that the anti-inflammatory effects of monosaccharide ginsenosides F1 and Rh1 are independent of Gba2. This suggests that adding a single glucose group at the C-6 or C-20 position of PPT does not interfere with its activation of Gr, while the presence of glucose groups at both sites requires deglycosylation by Gba2 to activate Gr.

[0360] To further investigate the anti-inflammatory effects of ginsenosides, we studied their influence on the expression of genes encoding pro-inflammatory proteins. Our results indicate that ginsenosides selectively inhibit inflammation-induced gene expression. All ginsenosides studied effectively inhibited the expression levels of il1b, il6, and mmp9, similar to Bec (…). Figure 7 E). However, ginsenosides were less effective than Bec in inhibiting the expression of il8 and mmp13. PPT and F1 showed only slight effects on the expression of these two genes, while no effect was observed on Rh1 and Rg1. Figure 7 (E). The difference between Rh1 and Rg1 and PPT and F1 lies in the glucose group at the C-6 position. Based on these data, we hypothesize that the glucose group at the C-6 position in Rh1 and Rg1 cannot be cleaved by Gba2, while this enzyme can effectively remove the glucose group at the C-20 position in F1. Therefore, PPT and F1 have similar effects on il8 and mmp13 expression, and Rh1 and Rg1 are also similar.

[0361] Glycosylation of ginsenosides leads to a significant reduction in side effects.

[0362] To evaluate the side effects of monosaccharide-based ginsenosides, zebrafish embryos were treated with Bec, PPT, F1, Rh1, and Rg1, and their effects on glucose and cortisol levels, larval length, and post-traumatic tissue regeneration were monitored. Bec treatment, as a positive control, resulted in elevated glucose levels (…). Figure 8 A) Decreased cortisol levels ( Figure 8 C) Shortened larval length ( Figure 8 E), and inhibits tissue regeneration ( Figure 8 F, S8A). Ginsenoside PPT does not affect glucose levels (F, S8A). Figure 8 A), but it reduced cortisol concentration ( Figure 8 C) indicates a small effect on larval length. Figure 8 E), and significantly inhibited the regeneration of the caudal fin after trauma ( Figure 8 F, S8A). Interestingly, glycosylated ginsenosides F1, Rh1, and Rg1 did not show any side effects, either on glucose levels (F, S8A). Figure 8 A) Cortisol concentration ( Figure 8 C) Juvenile body length ( Figure 8 E) or tissue regeneration ( Figure 8 Neither F nor S8A showed any side effects. Therefore, we conclude that glycosylation of PPT eliminates all its side effects.

[0363] Interestingly, F1 did not affect glucose levels after trauma. Figure 8 B), but it has a slight effect on cortisol levels and tissue regeneration. Figure 8D, F, S8A). The latter's effect depends on Gba2, as confirmed by the Gba2 inhibitor MZ31. Figure 8 (D, F), suggesting that F1 undergoes deglycosylation post-traumatically due to enhanced Gba2 activity. In contrast, Rh1 and Rg1 did not show any effect on glucose or cortisol levels or tissue regeneration post-traumatically. Figure 8 (B, D, F, S8A). These results indicate that Gba2 can cleave the glucose group at the C20 position in ginsenoside F1 (thus converting it to PPT), but cannot cleave the glucose group at the C6 position in Rh1 and Rg1, thus the latter two ginsenosides remain glycosylated at that position. As a result, C-6 glycosylation (such as Rh1) also eliminated the side effects of PPT after trauma, but C-20 glycosylation (such as F1) failed to eliminate the side effects of PPT, possibly because the increased local Gba2 activity after trauma caused glucose to be cleaved.

[0364] To investigate whether ginsenosides PPT, F1, and Rh1 can trigger GR transactivation activity, we used the fish reporter strain Tg (9xGCRE-HSV.UI23:EGFP), which fused the GFP gene with a promoter containing GRE. ia20 Treatment of the larvae of this strain with beclomethasone significantly enhanced the GFP signal throughout the larvae, while ginsenosides PPT, F1, Rh1, and Rg1 did not alter the fluorescence intensity. Figure 8 G, H). Furthermore, these ginsenosides did not increase the expression of fkbp5, pck1, and nfkbiaa, which are known target genes for GR transactivation activity. Figure 8 (I, S8B). These data indicate that the non-induction of GR transactivation activity by ginsenosides is a general characteristic.

[0365] Example 3: Effects of ginsenoside Re on zebrafish

[0366] In addition to Rg1, the effects of ginsenoside Re on anti-inflammatory activity and probable side effects were also tested in zebrafish as described in Example 1. The results showed that Re inhibited the migration of neutrophils to the wounded area of ​​the caudal fin, but did not inhibit the migration of macrophages. Figure 9 A, B). Unlike glucocorticoids, no side effects on systemic glucose and cortisol levels were observed after the addition of Re. Figure 9 C, D). Furthermore, Re has no effect on regenerated tissue (C, D). Figure 9 E).

[0367] Example 4: Preparation of micelles from ginseng leaves

[0368] Eight grams of dried ginseng leaves were boiled in 350 mL of water, filtered, and loaded onto a 20 mL bed volume (BV) column packed with macroporous resin (D101 type). After thorough washing with water (15 BV), ginsenosides were eluted with 60% EtOH. The ginsenoside solution was decolorized using a 20 mL BV column packed with D941 type macroporous resin. Subsequently, the solvent was slowly evaporated at 80 °C under reduced pressure (375 hPa) using a glass vial and an evaporator (P12-Multivapor, Buchi). After the ethanol and water evaporated, a transparent film formed on the wall of the glass vial. The film was dissolved in H2O to obtain a clear solution (see [link to product description]). Figure 12 A), which contains approximately 15 mg / mL of ginsenosides.

[0369] Meanwhile, 100 mg of pure Re was dissolved in 10 mL of ethanol. This yielded a clear solution (see [link to solution]). Figure 12 A). Ethanol was slowly removed at 80°C using a glass vial and an evaporator (P12-Multivapor) under low pressure (375 hPa). A transparent film re-formed on the glass tube wall. This film was dissolved in 10 mL of H2O to obtain a turbid solution (see [link to solution]). Figure 12 A). Microscopic examination revealed Re crystals ( Figure 12 B), micelles were not obtained.

[0370] Micelle formation of the ginsenoside extract obtained from dried leaves was replicated in the presence of 0.6 mg dexamethasone. First, dexamethasone was dissolved in ethanol at a concentration of 10 mg / mL, and 0.6 mg of dexamethasone was added to the ethanol ginsenoside fraction obtained after purification and decolorization using D101 and D941 resins, respectively, as described above. The Re and Rg1 content in this ethanol ginsenoside fraction was determined by HPLC to be 4 mg / 10 mL (see below). The molecular ratio of dexamethasone to Re / Re1 in this solution was approximately 5:70. The ethanol fraction was slowly removed using a glass vial and evaporator at 80 °C and under reduced pressure (375 hPa) to obtain a thin film. This film was dissolved in water to obtain a clear aqueous solution. The content and presence of micelles were evaluated by size exclusion chromatography as described below.

[0371] The size and content of micelles were determined by size exclusion chromatography. 1 mL of ginsenoside / dexamethasone aqueous solution was loaded onto a Sephadex G50 size exclusion column (25 cm × 1 cm) and eluted with water by gravity. Blue dextran (MW = 2,000,000 Da) and erythrosine (880 Da) were used as molecular weight markers. 2.0 mL of the fraction was collected. The fraction was dried and dissolved in 70% methanol, and the ginsenoside content was analyzed by HPLC. The HPLC conditions were as follows:

[0372] - HPLC equipment: HPLC / DAD Aligent 1200 series, with a quaternary pump connected to the DAD and autosampler.

[0373] - Stationary phase: Kinetex 2.6μm, C18 100Å, 100×4.6mm — Phenomenex, USA

[0374] - Mobile phase:

[0375] ○ A: 0.03% ultrapure H2O solution of o-phosphoric acid (H3PO4)

[0376] ○ B: 0.03% o-phosphoric acid (H3PO4) acetonitrile solution (LiChrosolv, Millipore-Merck, Darmstadt, Germany; high-quality far-UV)

[0377] - Gradient:

[0378]

[0379] - condition

[0380] ○ Flow rate: 0.8 ml / min

[0381] ○ Injection volume: 20μL

[0382] ○ Column temperature: 35℃

[0383] ○ Detection wavelengths: 203nm and 241nm

[0384] Figure 13 Size exclusion chromatography is shown, which displays the molecular weight and content of micelles.

[0385] After eluting 30 mL, micelles with the highest concentration and high content of ginsenosides Re and Rg1 were eluted. Figure 13 (Figure 14B). Based on the elution curves of molecular weight labeled blue dextran (MW 2,000,000 Da; peak elution at 17 mL) and erythrosine (MW 880 Da; peak elution at 55 mL), the size of the micelles mainly containing Re and Rg1 was estimated to be approximately 80,000 Da.

[0386] These results indicate that the micelles contain approximately 50 to 100 molecules primarily composed of Re, Rg1, or both. Interestingly, dexamethasone was also found to co-elute with micelles containing Re and Rg1. Furthermore, dexamethasone (monomer with a molecular weight of 392.5 Da) was expected to elute after erythrosine (e.g., in fractions after 55 mL), but it was not detected in these fractions. This suggests that dexamethasone is encapsulated in micelles composed of Re, Rg1, or both. Additionally, the results indicate that high concentrations of PPT-type ginsenosides can be obtained in water using this method, optionally in combination with dexamethasone.

[0387] References

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Claims

1. A ginsenoside preparation comprising protopanaxadiol (PPT) type ginsenosides, wherein the protopanaxadiol (PPT) type ginsenosides are at least partially integrated in micelles, preferably at least partially integrated in the outer layer of the micelles, wherein the molecular weight of the micelles is at least 10,000 Da.

2. The ginsenoside preparation according to claim 1, wherein the molecular weight of the micelles is at least 100,000 Da.

3. The ginsenoside preparation according to any one of the preceding claims, wherein the ginsenoside is selected from Re, Rf, Rg1, Rg2, Rh1 or a combination thereof, preferably wherein the ginsenoside is Rg1, Re or a combination thereof.

4. A pharmaceutical composition comprising a ginsenoside preparation according to any one of the preceding claims and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, further comprising GC, preferably wherein the GC is dexamethasone, beclomethasone, or a combination thereof.

6. The pharmaceutical composition of claim 5, wherein the GC is at least partially encapsulated in the micelles.

7. The pharmaceutical composition according to claims 4 to 6, wherein the pharmaceutical composition is formulated into a cream, lotion, balm, hydrogel, ointment, foam, gel, spray, tablet, capsule, lozenge, external solution, external suspension, aerosol, injection or syrup.

8. The pharmaceutical composition according to claims 5 to 7, wherein the molar ratio of GC to ginsenoside is about 1:1 to about 1:2000, preferably about 1:4 to about 1:1000, more preferably 1:8 to 1:

20.

9. A ginsenoside preparation according to claims 1 to 3 or a drug according to claims 4 to 8, used in a method for the prevention or treatment of inflammatory diseases.

10. The ginsenoside preparation or pharmaceutical composition for use according to claim 9, wherein the inflammatory disease is selected from asthma, allergic rhinitis, hay fever, urticaria, atopic eczema, chronic obstructive pulmonary disease, inflammation of joints, muscles and tendons, lupus, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, giant cell arteritis and polymyalgia rheumatica, and multiple sclerosis.

11. The ginsenoside preparation or pharmaceutical composition for use according to claim 9 or 10, wherein the GC is administered at a therapeutically ineffective dose, preferably wherein the dose of the GC is at most 2.5 mg / kg, more preferably at most 1.5 mg / kg, more preferably at most 0.07 mg / kg, and most preferably at most 0.007 mg / kg.

12. The ginsenoside preparation or pharmaceutical composition for use according to claims 8 to 11, wherein the ginsenoside is administered at a therapeutically ineffective dose, preferably at a dose of up to 5.5 mg / kg, more preferably at a dose of up to 4 mg / kg, up to 3 mg / kg, up to 2 mg / g, up to 1 mg / kg, more preferably at a dose of up to 0.5 mg / kg, and particularly at a dose of up to 0.05 mg / kg.

13. A method for preparing ginsenoside preparations according to claims 1 to 3, comprising: (a) Provide ginsenoside extracts from plants or plant parts of the Panax genus, preferably Panax ginseng; (b) Contact the ginsenoside extract with an aqueous solvent mixture to obtain a ginsenoside mixture; (c) At least partially removing the aqueous solvent mixture from the ginsenoside mixture; and optionally... (d) The ginsenoside mixture obtained in step c) is reconstituted in water to obtain a ginsenoside preparation.

14. The method of claim 13, wherein the aqueous solvent mixture comprises a mixture of water and a non-aqueous solvent, wherein the non-aqueous solvent is preferably an alcohol solvent, more preferably selected from methanol, ethanol, propanol and isopropanol.

15. The method according to claim 13 or 14, comprising using a nonpolar resin, preferably D101 macroporous resin, as the stationary phase to perform column chromatography on the ginsenoside extract.

16. The method according to any one of claims 13 to 15, comprising a decolorization step of the ginsenoside extract, preferably using D941 macroporous resin.

17. A ginsenoside preparation, which can be obtained by the method according to any one of claims 14 to 16.

18. A combination of glucocorticoids (GC) and ginsenosides in a method for the prevention or treatment of inflammatory diseases, wherein the method comprises administering GC and PPT type ginsenosides to a subject in need in a molar ratio of about 1:1 to about 1:2000.

19. The combination according to claim 18, wherein the GC is administered at a therapeutically ineffective dose, preferably at a dose of up to 2.5 mg / kg, more preferably at a dose of up to 1.5 mg / kg, more preferably at a dose of up to 0.07 mg / kg / day, and most preferably at a dose of up to 0.007 mg / kg / day.

20. The combination according to claim 18 or 19, wherein the ginsenoside is selected from Re, Rf, Rg1, Rg2, Rh1 or a combination thereof, preferably wherein the ginsenoside is Rg1, Re or a combination thereof, and / or wherein the GC is dexamethasone, beclomethasone or a combination thereof.

21. The combination according to claim 18, wherein the ginsenoside is at least partially integrated in the micelles, preferably at least partially integrated in the outer layer of the micelles, and the micelles have a molecular weight of at least 10,000 Da, preferably at least 100,000 Da.

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