Application of rhododendron dauricum and hippophae rhamnoides leaf composition in preparation of psoriasis treatment medicine

Abstract

The invention discloses application of a folium rhododendri daurici and hippophae rhamnoides leaf composition in preparation of a medicine for treating psoriasis. Belongs to the technical field of traditional Chinese medicine compositions. The invention provides application of a combination of rhododendron dauricum and hippophae rhamnoides leaves in preparation of a medicine for treating psoriasis, it is found for the first time that the combination of rhododendron dauricum serving as a main medicine and hippophae rhamnoides leaves serving as an adjuvant according to the mass ratio of (1-5): 1 is better in psoriasis treatment effect compared with single rhododendron dauricum or hippophae rhamnoides leaves, the skin lesion condition and PASI score of the back of a mouse with psoriasis can be remarkably reduced, and the application prospect is broad. According to the present invention, the psoriasis treatment effect is substantially improved, the psoriasis mouse epidermal thickness is reduced, the mouse spleen swelling condition is relieved, the spleen index is reduced, the psoriasis related inflammatory factor IL-17 and TNF-alpha content in the mouse serum is reduced, the psoriasis treatment effect is substantially improved, and the research results show that the dahurian rhododendron leaf and the hippophae rhamnoides l leaf have the synergistic effect on psoriasis treatment, the treatment effect of the compound is superior to that of a positive drug methotrexate, and a new direction is provided for psoriasis treatment.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine composition technology, and more specifically to the application of the combination of Rhododendron molle and Hippophae rhamnoides leaves in the preparation of drugs for treating psoriasis. Background Technology

[0002] Psoriasis (PSO) is a chronic, relapsing, inflammatory, systemic disease mediated by the immune system and induced by both genetic and environmental factors. Its pathogenesis involves the Th17 / IL-23 immune axis, the TNF-α-mediated inflammatory pathway, and excessive proliferation of keratinocytes. Clinically, it manifests as dark red plaques or infiltrated erythema covered with white or silvery-white scales. Currently, the main treatments for PSO include corticosteroids, immunosuppressants, and biologics. These treatments have various limitations, including significant side effects with long-term use and rebound effects upon discontinuation. While biologics are relatively safe, they are expensive. Therefore, finding low-cost, safer, and more effective anti-psoriasis drugs remains a pressing need. In recent years, Traditional Chinese Medicine (TCM) has gained considerable attention. The advantages of TCM in treating psoriasis lie in its holistic approach and syndrome differentiation, individualized treatment that addresses both local symptoms and systemic imbalances, relatively fewer side effects, and a tendency to reduce recurrence.

[0003] Sea buckthorn leaves are the leaves of Hippophae rhamnoides L., a plant belonging to the Elaeagnaceae family. In the prior art, the main technical solutions for the treatment of psoriasis with sea buckthorn leaves are as follows, such as application number 202111401896.0, invention title: A sea buckthorn leaf composition for improving psoriasis and its application, which uses sea buckthorn leaves as the main component for the treatment of psoriasis, but the therapeutic effect of sea buckthorn leaves alone is not good.

[0004] Rhododendron dauricum folium, derived from the dried leaves of *Rhododendron dauricum* L. (family Ericaceae), is listed in all editions of the *Pharmacopoeia of the People's Republic of China*. Other rhododendrons used in folk and ethnic medicine, including *Rhododendron dauricum* (primarily from the high-altitude Qinghai-Tibet Plateau, a Tibetan medicine, often used for its leaves and flowers) and *Rhododendron simsii* (primarily from the Yunnan-Guizhou region, commonly used in Miao medicine), are not yet included in the pharmacopoeia. The content of components in medicinal materials from different origins varies significantly in traditional Chinese medicine; these materials belong to the same genus but different subgenuses or groups, indicating a distant phylogenetic relationship. Furthermore, there is no research on the treatment of psoriasis with *Rhododendron dauricum folium*. Currently available traditional Chinese medicine compositions for treating psoriasis are complex and have unsatisfactory efficacy.

[0005] Therefore, how to develop simple formulations with good therapeutic effects for treating psoriasis is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of the combination of rhododendron and sea buckthorn leaves in the preparation of drugs for treating psoriasis. It is the first time that the combination of rhododendron as the main drug and sea buckthorn leaves as the auxiliary drug in a mass ratio of 1-5:1 has been found to have a better therapeutic effect on psoriasis than that of the positive drug methotrexate.

[0007] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0008] The primary objective of this application is to provide the use of a combination of rhododendron and sea buckthorn leaves in the preparation of a drug for treating psoriasis.

[0009] As a preferred technical solution, the application combines rhododendron and sea buckthorn leaves in a mass ratio of 1-5:1.

[0010] Another object of this application is to provide: a combination extract of rhododendron for treating psoriasis, comprising a mixed aqueous extract of rhododendron and sea buckthorn leaves.

[0011] As a preferred technical solution, the rhododendron and sea buckthorn leaves are mixed in a mass ratio of 1-5:1 and then subjected to water extraction.

[0012] As a preferred technical solution, the water extraction process is as follows:

[0013] (1) Weigh out the rhododendron and sea buckthorn leaves according to a mass ratio of 1-5:1, crush them, and soak them in water.

[0014] (2) After soaking, the extract was subjected to multiple water extractions at 90-100℃, the filtrates were combined, centrifuged, concentrated and dried to prepare the combined extract of *Rhododendron molle*.

[0015] As a preferred technical solution, the water extraction is performed 2-3 times, with each extraction lasting 30-60 minutes; the centrifugation speed is 8000-10000 rpm; and the concentration is performed using vacuum concentration.

[0016] Another object of this application is to provide: a medicine for treating psoriasis, comprising the combination of the aforementioned rhododendron and sea buckthorn leaves and / or the extract of the aforementioned rhododendron combination.

[0017] As a preferred technical solution, the drug for treating psoriasis also includes other active ingredients for treating psoriasis and / or pharmaceutical excipients.

[0018] Another object of this application is to provide the use of the aforementioned Rhododendron simsii extract and / or the aforementioned medicine in the treatment of psoriasis.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides the application of the combination of rhododendron and sea buckthorn leaves in the preparation of drugs for treating psoriasis. It is the first time that the combination of rhododendron as the main drug and sea buckthorn leaves as the excipient, in a mass ratio of 1-5:1, has a better effect on treating psoriasis than rhododendron and sea buckthorn leaves alone. It can significantly reduce the skin lesions on the back of mice and the PASI score, alleviate splenomegaly in mice, reduce the spleen index, and reduce the content of inflammatory factors in the serum of mice, which greatly improves the treatment effect of psoriasis. Furthermore, the study found that rhododendron and sea buckthorn leaves have a synergistic effect in the treatment of psoriasis, and their therapeutic effect is better than that of the positive control drug methotrexate.

[0021] This invention fully utilizes the natural medicinal value of sea buckthorn leaves and rhododendron, providing a new treatment option for psoriasis patients through multi-dimensional synergistic effects of anti-inflammation and immunosuppression. It effectively reduces skin inflammation caused by psoriasis, improves dryness, relieves itching, reduces inflammatory cells and inflammatory factors, promotes the restoration of balance, is safe and has no toxic side effects, and reduces recurrence. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the psoriasis mouse modeling and drug administration process.

[0024] Figure 2 See: Skin lesion appearance of psoriatic mice on day 6 after treatment in each treatment group.

[0025] Figure 3 The following describes the changes in skin lesions in psoriatic mice from day 0 to day 6 after treatment in each treatment group. Rows 1-8 from top to bottom correspond to the following groups: Control, IMQ, MTX, IMQ+Shā:Xing = 0:1, IMQ+Shā:Xing = 1:0, IMQ+Shā:Xing = 1:1, IMQ+Shā:Xing = 1:5, and IMQ+Shā:Xing = 1:10, where Shā represents sea buckthorn leaves and Xing represents rhododendron. Columns 1-6 from left to right represent the changes in skin lesions in each group from day 1 to day 6.

[0026] Figure 4 The change in PASI score after treatment of psoriasis mice in each treatment group is shown.

[0027] Figure 5 Image: HE staining micrographs of psoriasis mice after treatment with each treatment group (×20).

[0028] Figure 6 To: the effect of each treatment group on spleen size in psoriasis mice.

[0029] Figure 7 The effect of each treatment group on the spleen index in psoriasis mice.

[0030] Figure 8 The effect of each treatment group on serum TNF-α in psoriasis mice.

[0031] Figure 9 To: The effect of each treatment group on serum IL-17 in psoriasis mice. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Experimental animals: The experimental animals used in the embodiments of this application were male SPF-grade BALB / C mice aged 7-8 weeks, purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0034] Husbandry conditions: Mice were housed together in opaque plastic cages (32cm×22cm×16cm, L×W×H) with 2.5cm of sawdust bedding, maintaining a 12h diurnal rhythm. They were provided with free access to food and water. The ambient temperature was 18-24℃, and the relative humidity was 50%-70%.

[0035] All experimental procedures were conducted in accordance with the ethical requirements for laboratory animals at Jiamusi University, and humane care was provided in accordance with the 3R principle of laboratory animal use. All experimental procedures were carried out at the SPF-grade animal experimental facility of the Laboratory Animal Center of Jiamusi University.

[0036] Example 1

[0037] A combination extract of Rhododendron molle was prepared using the following method:

[0038] (1) Weigh out 1 part each of clean sea buckthorn leaves and rhododendron according to the mass ratio, crush them, and soak them in triple-distilled water at a material-to-liquid ratio of 1:10 (g / ml) for 12 hours;

[0039] (2) Water decoction method: extract at 100℃ for 1 hour, filter; add an equal amount of water, extract again at 100℃ for 1 hour, filter, and combine the two filtrates;

[0040] (3) Centrifuge the combined filtrate at 8000 r / min for 10 min; concentrate it using a rotary evaporator. Set the rotary evaporator to 70℃, 0.1 mPa, and 30 r / min. After concentration, dry it in a 60℃ oven and grind it in a mortar to obtain the dry powder of the Manshanhong combination extract.

[0041] Example 2

[0042] A combination extract of Rhododendron molle was prepared using the following method:

[0043] (1) Weigh out 1 part of clean sea buckthorn leaves and 5 parts of rhododendron according to the mass ratio, crush them, and add triple-distilled water at a material-to-liquid ratio of 1:10 (g / ml) to soak for 12 hours;

[0044] (2) Water decoction method: extract at 100℃ for 1 hour, filter; add an equal amount of water, extract again at 90℃ for 1 hour, filter, and combine the two filtrates.

[0045] (3) Centrifuge the combined filtrate at 10000r / min for 10min; concentrate it using a rotary evaporator. Set the rotary evaporator to 70℃, 0.1mPa, and 30r / min. After concentration, dry it in a 60℃ oven and grind it in a mortar to obtain the dry powder of the Manshanhong combination extract.

[0046] Comparative Example 1

[0047] A combination extract of Rhododendron molle was prepared using the following method:

[0048] (1) Weigh out 1 part of clean sea buckthorn leaves and 10 parts of rhododendron according to the mass ratio, crush them, and add them to triple-distilled water at a material-liquid ratio of 1:10 (g / ml) and soak for 12 hours.

[0049] (2) Water decoction method: extract at 100℃ for 1 hour, filter; add an equal amount of water, extract again at 100℃ for 1 hour, filter, and combine the two filtrates;

[0050] (3) Centrifuge the combined filtrate at 8000 r / min for 10 min; concentrate it using a rotary evaporator. Set the rotary evaporator to 70℃, 0.1 mPa, and 30 r / min. After concentration, dry it in a 60℃ oven and grind it in a mortar to obtain the dry powder of the Manshanhong combination extract.

[0051] Comparative Example 2

[0052] Extract 1 is prepared by the following method:

[0053] (1) Weigh out 1 part of clean rhododendron, crush it, and soak it in triple-distilled water at a ratio of 1:10 (g / ml) for 12 hours;

[0054] (2) Water decoction method: extract at 100℃ for 1 hour, filter; add an equal amount of water, extract again at 100℃ for 1 hour, filter, and combine the two filtrates;

[0055] (3) Centrifuge the combined filtrate at 8000 r / min for 10 min; concentrate it using a rotary evaporator. Set the rotary evaporator to 70℃, 0.1 MPa, and 30 r / min. After concentration, dry it in a 60℃ oven and grind it in a mortar to obtain the dry powder of extract 1.

[0056] Comparative Example 3

[0057] Extract 2 is prepared by the following method:

[0058] (1) Weigh out 1 part of clean sea buckthorn leaves, crush them, and soak them in triple-distilled water at a ratio of 1:10 (g / ml) for 12 hours.

[0059] (2) Water decoction method: extract at 100℃ for 1 hour, filter; add an equal amount of water, extract again at 100℃ for 1 hour, filter, and combine the two filtrates;

[0060] (3) Centrifuge the combined filtrate at 8000 r / min for 10 min; concentrate it using a rotary evaporator. Set the rotary evaporator to 70℃, 0.1 mPa, and 30 r / min. After concentration, dry it in a 60℃ oven and grind it with a mortar to obtain the dry powder of extract 2.

[0061] Experimental grouping, construction of a mouse model of psoriasis, and validation of its effects (details as follows) Figure 1 )

[0062] (1) Experimental groups (6 mice per group):

[0063] Blank control group: Normal mice as controls;

[0064] IMQ model group: Model group mouse control;

[0065] Positive control group (methotrexate MTX);

[0066] Extract groups (0:1, 1:0, 1:1, 1:5, 1:10);

[0067] (2) Administration method:

[0068] Except for the blank control group, mice in the other groups were treated with 62.5 mg / d IMQ (5% imiquimod cream, Sichuan Mingxin Pharmaceutical Co., Ltd.) on their backs for 5 days. Animals in the positive control group and extract group were administered the drug by gavage from Day 1 of modeling until Day 7. The blank control group and IMQ model group were administered 0.9% saline by gavage from Day 1 of modeling until Day 7. Specific administration methods are as follows:

[0069] Blank control group: Normal mice were administered 200 μL of 0.9% saline by gavage;

[0070] IMQ model group: Model group mice were administered 200 μL of 0.9% saline by gavage;

[0071] Positive control group: Model group mice were administered 0.1 mg / kg / d methotrexate (MTX) by gavage (dissolved in 200 μL of gavage solution);

[0072] Extract group: Model mice were administered the extract (6 mg / kg / d dissolved in 200 μL gavage solution) by gavage.

[0073] (3) Evaluation of Results

[0074] The psoriasis lesion area and severity index (PASI) of the skin lesions on the backs of mice in each group were recorded, and the mice were weighed daily. On day 7, all mice were euthanized using the CO2 asphyxiation method, and the skin lesions were observed. Figures 2-4 ), and HE staining was performed on mice in each treatment group ( Figure 5 ) and PASI score (scoring table as shown in Table 1), spleen and serum were collected to measure spleen size and spleen index ( Figures 6-7 ) and the levels of inflammatory factors in serum ( Figures 8-9 The specific process and results are as follows:

[0075] Table 1 PASI Severity Score

[0076]

[0077] Effects of different groups on skin lesions in mice

[0078] from Figure 2 and Figure 3 The skin lesion appearance images show that the skin on the back of the blank group was smooth and without erythema; the skin of the IMQ group showed significant erythema, scaling and thickening; and the skin lesions of the MTX group were significantly improved. All extract groups showed varying degrees of relief, with the 1:1 and 1:5 ratio groups showing a significant reduction in erythema and scaling, and the skin texture returning to near normal.

[0079] PASI is a comprehensive skin lesion severity index used to assess erythema, scaling, thickness, and total score. The score is positively correlated with severity. Figure 4 It can be seen that the PASI total score in the IMQ group peaked on day 5 and was significantly higher than that in the control group. All scores in the MTX group decreased significantly. Among the extract groups, the 1:1 and 1:5 ratio groups showed the most significant improvement on days 4-6, with a significant decrease in total score (P<0.01), similar to the MTX group. The 0:1, 1:0, and 1:10 groups showed improvement, but the magnitude was smaller.

[0080] HE staining was used to observe pathological changes in the dorsal skin of mice in different treatment groups (magnification: ×20). Figure 5 It can be seen that the skin structure of the blank control group was intact and no lesions were observed; the IMQ model group showed typical psoriasis-like manifestations, including epidermal hyperkeratosis, acanthosis, and inflammatory cell infiltration; the positive drug MTX group showed significant pathological improvement. After treatment with different ratios of Rhododendron dauricum and Hippophae rhamnoides, the IMQ&Hippophae rhamnoides:Rhododendron dauricum = 1:1-5 group showed the best skin repair effect.

[0081] Effects of different groups on spleen size and spleen index in mice

[0082] The spleen index is one of the indicators reflecting the body's immune status. Mice were weighed daily from Day 0 to Day 7, and the average weight was recorded. On Day 7, mice were sacrificed, and their spleens were harvested and weighed. The spleen size was compared to a graduated pathological specimen, and the spleen index was calculated. The experimental results are as follows: Figure 6-7 As shown.

[0083] Spleen index (%) = [Spleen weight (g) / Mouse body weight (g)] * 100%

[0084] Results analysis: From Figure 6 and Figure 7 The spleen size and spleen index of mice showed that the spleen of mice in the IMQ group was significantly enlarged, and the spleen index was about 3 times higher than that of the control group. The spleen index of the 1:1 and 1:5 ratio groups was significantly reduced (P<0.0001), which was significantly better than that of the positive drug MTX group; other ratio groups showed a decrease, but it was not significant.

[0085] Sea buckthorn leaves can significantly reduce spleen index, which aligns with the treatment strategy for PSO; furthermore, combining raspberry and sea buckthorn leaves in a 1:1-5:1 ratio consistently improves spleen index compared to using either alone, and through… Figure 7 It can be seen that the two have a synergistic effect.

[0086] Effects of different groups on serum TNF-α levels in mice

[0087] Elevated serum TNF-α levels are a characteristic change in the inflammatory response of PSO. To determine the effect of different groups on TNF-α levels, a double-antibody sandwich ELISA method was used to measure TNF-α levels. The specific steps are as follows:

[0088] (1) Prepare solid-phase antibody by coating microplates with purified human TNF-α antibody;

[0089] (2) The sample (containing human TNF-α) and HRP-labeled human TNF-α antibody were added sequentially to the microwells coated with monoclonal antibody to form an "antibody-antigen-enzyme-labeled antibody" complex.

[0090] (3) After thoroughly washing to remove unbound components, the substrate TMB is added for color development; TMB is first converted to blue under HRP catalysis, and then treated with acid to generate a yellow final product. The color intensity is positively correlated with the TNF-α content in the sample.

[0091] (4) The absorbance (OD value) was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The concentration of TNF-α in the sample was calculated using a standard curve. The experimental results are as follows: Figure 8 As shown.

[0092] Results Analysis: Figure 8 It was found that serum TNF-α levels were significantly elevated in the IMQ group (approximately 850 pg / mL). Different proportions of the extract significantly reduced serum TNF-α levels in psoriasis model mice. Furthermore, the combination of *Rosa laevigata* and *Hippophae rhamnoides* leaves in a ratio of 1:1 to 5:1 resulted in the greatest reduction in TNF-α, superior to either ingredient alone. Figure 8 It can be seen that the two have a synergistic effect.

[0093] Effects of different groups on serum IL-17 levels in mice

[0094] Elevated serum IL-17 levels are a characteristic change in the inflammatory response of PSO. To determine the effect of different groups on IL-17 levels, a double-antibody sandwich ELISA method was used to measure IL-17 levels. The specific steps are as follows:

[0095] (1) Prepare solid-phase antibody by coating microplates with purified human IL-17 antibody;

[0096] (2) The sample (containing human IL-17) and HRP-labeled human IL-17 antibody were added sequentially to the microwells coated with monoclonal antibody to form an "antibody-antigen-enzyme-labeled antibody" complex.

[0097] (3) After thorough washing to remove unbound components, the substrate TMB was added for color development. TMB was first converted to blue under HRP catalysis, and then acid treatment was performed to generate a yellow final product. The color intensity was positively correlated with the IL-17 content in the sample.

[0098] (4) The absorbance (OD value) was measured at 450 nm using an ELISA reader. The concentration of IL-17 in the sample was calculated using a standard curve. The experimental results are as follows: Figure 9 As shown.

[0099] Results analysis: IL-17 levels were significantly elevated in the IMQ group, significantly decreased in the 1:1 and 1:5 ratio groups (P<0.05), and showed no significant changes in the other ratio groups.

[0100] In summary, based on the above indicators, extracts of sea buckthorn leaf and rhododendron significantly improved IMQ-induced psoriatic lesions, reduced PASI scores, inhibited splenomegaly, and significantly downregulated serum TNF-α and IL-17 levels. The combined therapeutic effects were best at ratios of 1:1 and 1:5, suggesting a synergistic effect between the two extracts.

[0101] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0102] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of the combination of rhododendron and sea buckthorn leaves in the preparation of drugs for treating psoriasis.

2. The application according to claim 1, characterized in that, The rhododendron and sea buckthorn leaves are combined in a mass ratio of 1-5:

1.

3. A combination extract of Rhododendron molle for treating psoriasis, characterized in that, It includes a mixed water extract of rhododendron and sea buckthorn leaves.

4. The rhododendron extract according to claim 3, characterized in that, The rhododendron and sea buckthorn leaves were mixed at a mass ratio of 1-5:1 and then water-extracted.

5. The rhododendron extract according to claim 4, characterized in that, The water extraction process is as follows: (1) Weigh out the rhododendron and sea buckthorn leaves according to a mass ratio of 1-5:1, crush them, and soak them in water. (2) After soaking, the extract was subjected to multiple water extractions at 90-100℃, the filtrates were combined, centrifuged, concentrated and dried to prepare the combined extract of *Rhododendron molle*.

6. The rhododendron extract according to claim 5, characterized in that, The water extraction is performed 2-3 times, with each extraction lasting 30-60 minutes; the centrifugation speed is 8000-10000 rpm; and the concentration is performed under reduced pressure.

7. A medicine for treating psoriasis, characterized in that, It includes the combination of Rhododendron molle and sea buckthorn leaves as described in any one of claims 1-2 and / or the Rhododendron molle combination extract as described in any one of claims 3-6.

8. The medicament for treating psoriasis according to claim 7, characterized in that, It also includes other active ingredients and / or pharmaceutical excipients for treating psoriasis.

9. The use of the rhododendron extract according to any one of claims 3-6 and / or the drug according to any one of claims 7-8 in the treatment of psoriasis.

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