A tetrastigma hemsleyanum extract, a preparation method thereof, and a medicine for preventing and / or treating thymus atrophy

Abstract

The application discloses a tetrastigma hemsleyanum extract, a preparation method thereof and a medicine for preventing and treating thymic atrophy, and the tetrastigma hemsleyanum extract is prepared by the following steps: crushing dried tetrastigma hemsleyanum plants, extracting by ethanol reflux, and concentrating under reduced pressure to obtain a tetrastigma hemsleyanum crude extract; dissolving the tetrastigma hemsleyanum crude extract in water, extracting by n-butanol, and recovering the solvent to obtain the tetrastigma hemsleyanum extract. The tetrastigma hemsleyanum extract can obviously improve thymic atrophy and has a good anti-thymic atrophy effect, and therefore has great clinical significance and practical value for exploring a new method for preventing and / or treating thymic atrophy and expanding the application range of the tetrastigma hemsleyanum.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a *Trifolium repens* extract, its preparation method, and a drug for preventing and / or treating thymic atrophy. Background Technology

[0002] *Trifolium repens*, also known as golden thread gourd or snake aconite, belongs to the genus *Trifolium* of the Vitaceae family. The entire plant can be used medicinally, possessing properties such as clearing heat and detoxifying, promoting blood circulation and resolving stagnation, dispelling wind and resolving phlegm, and reducing inflammation and relieving pain. It is commonly used for febrile convulsions, inflammation, and anti-tumor effects, and is a unique and precious medicinal herb in my country. The main components of *Trifolium repens* include flavonoids, phenols, triterpenoids, and fatty acids.

[0003] The emergence and spread of novel viruses often necessitate the absence of effective drugs in the short term. After viral infection, in addition to the direct suppression of the immune system, immune organs (such as the thymus) simultaneously become targets of viral attack, leading to atrophy and functional decline. This affects the differentiation, maturation, migration, and formation of immune memory in T cells, thereby suppressing cellular immunity and further reducing the body's ability to fight viruses and recover. Therefore, restoring immunity is of great significance for patients with viral infections. At this time, targeting the improvement of immune organ function and enhancing the body's immunity will greatly help patients rebuild their own immunity to resist viral attacks and promote post-recovery.

[0004] Currently, there are no drugs specifically for thymic atrophy in clinical practice. Hormones or immunomodulators are often used as adjunctive therapies. Furthermore, there are few reports on the effects of Tripterygium wilfordii extract on thymic atrophy. Therefore, studying the anti-thymic effect of Tripterygium wilfordii extract is of great clinical significance and practical value for exploring new methods for the prevention and / or treatment of thymic atrophy and expanding the application scope of Tripterygium wilfordii. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems and expand the efficacy and application scope of Tripterygium wilfordii, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a method for preparing a *Trifolium repens* extract, comprising the following steps:

[0007] S1, the dried Trifoliate Orchid plant is crushed, and the crude extract of Trifoliate Orchid is obtained by reflux extraction with ethanol and concentration.

[0008] S2, the crude extract of *Trifolium repens* is extracted with n-butanol, back-extracted with water, and the solvent is recovered to obtain the *Trifolium repens* extract.

[0009] Preferably, the *Trifolium repens* plant comprises *Trifolium repens* stems and leaves, for example: *Trifolium repens* stems and leaves, or a mixture of *Trifolium repens* stems and leaves and roots.

[0010] Furthermore, fresh *Trifolium repens* plants can be washed and dried.

[0011] Preferably, the drying process is at least one of air drying, sun drying, hot air drying, microwave drying, or freeze drying.

[0012] Preferably, the powder obtained after pulverization in S1 is 80 mesh or finer.

[0013] Preferably, the ethanol concentration in S1 is ≥50%, and the ethanol reflux time is 3-9 hours. Preferably, the ethanol concentration is 50%, 75%, or 90%.

[0014] Preferably, the ethanol reflux in S1 is ethanol reflux at different concentrations, and the reflux time for each concentration is 1-3h, for example: 1h, 1.5h, 2h, 2.5h, 3h.

[0015] Preferably, step S1 further includes a step of refluxing ethanol and then filtering, and collecting the filtrate after filtration.

[0016] Preferably, step S1 further includes the step of concentrating the extract to obtain an extract, and then drying the extract.

[0017] Furthermore, the extract is dried by at least one of the following methods: air drying, sun drying, hot air drying, microwave drying, or freeze drying.

[0018] In one embodiment of the present invention, the preparation method of the *Trifolium repens* extract is as follows: Dried *Trifolium repens* plants are pulverized into coarse powder, passed through an 80-mesh sieve, and 75% ethanol is added. The mixture is then refluxed twice, each time for 2 hours. The filtrates are filtered and combined, and the solvent is recovered by vacuum concentration to obtain a crude *Trifolium repens* extract, which is then stored at low temperature. The extract is dissolved in water and extracted three times with twice the amount of n-butanol solution, followed by back-extraction with an equal amount of water. The n-butanol layer is then collected, and the solvent is recovered under reduced pressure until no alcohol odor remains, thus obtaining the *Trifolium repens* extract.

[0019] Preferably, the *Trifolium repens* plant comprises *Trifolium repens* stems and leaves, for example: *Trifolium repens* stems and leaves, or a mixture of *Trifolium repens* stems and leaves and roots.

[0020] In a second aspect, the present invention provides a *Trifolium repens* extract, which is prepared according to the preparation method described in the first aspect.

[0021] Preferably, the *Trifolium repens* extract comprises *Trifolium repens* stem and leaf extract.

[0022] Thirdly, the present invention provides a medicament for preventing and / or treating thymic atrophy, comprising the *Trifolium repens* extract described in the second aspect.

[0023] Preferably, the dosage of the drug is 0.01-10g of raw medicinal material / kg, for example: 0.01g of raw medicinal material / kg, 0.05g of raw medicinal material / kg, 0.1g of raw medicinal material / kg, 0.2g of raw medicinal material / kg, 0.5g of raw medicinal material / kg, 0.7g of raw medicinal material / kg, 1.0g of raw medicinal material / kg, 1.5g of raw medicinal material / kg, 2g of raw medicinal material / kg, 2.5g of raw medicinal material / kg, 3g of raw medicinal material / kg. Raw drug weight / kg, 3.5g raw drug weight / kg, 4g raw drug weight / kg, 4.5g raw drug weight / kg, 5g raw drug weight / kg, 5.5g raw drug weight / kg, 6g raw drug weight / kg, 6.5g raw drug weight / kg, 7g raw drug weight / kg, 7.5g raw drug weight / kg, 8g raw drug weight / kg, 8.5g raw drug weight / kg, 9g raw drug weight / kg, 9.5g raw drug weight / kg, 10g raw drug weight / kg.

[0024] Preferably, the drug is a tablet, capsule, solution, cream, gel, or emulsion.

[0025] Preferably, the drug further includes pharmaceutically permissible excipients.

[0026] Furthermore, the excipients are selected from one or more combinations of diluents, absorbents, wetting agents, adhesives, disintegrants, lubricants, solvents, pH adjusters, buffers, antioxidants, metal ion chelators, antibacterial agents, or isotonic adjusters.

[0027] The beneficial effects of this invention are:

[0028] The *Tripterygium wilfordii* extract of this invention can resist thymic atrophy and increase the white blood cell count in the body's blood, providing a new approach and method for the prevention and / or treatment of thymic atrophy. At the same time, this invention expands the application scope of *Tripterygium wilfordii* and increases its economic value. Attached Figure Description

[0029] Figure 1 The image shows the HE staining results of the thymus of rats after administration of crude extract of Trifolium repens;

[0030] Figure 2 The image shows the HE staining results of the thymus of rats after administration of extracts from *Tripterygium wilfordii* treated with different solvents. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0032] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0033] Experimental materials in the examples:

[0034] SPF-grade male ICR (4 weeks old): provided by the Experimental Animal Center of Hangzhou Medical College, experimental animal production license number: SCXK(Zhe)2019-0002.

[0035] Water bottles, cages, bedding: experimental animal use license number: SYXK(Zhe)2018-0011

[0036] Example 1 Preparation of crude extract of Tetrastigma hemsleyanum

[0037] 1.1 Preparation method of crude extract of Tetrastigma hemsleyanum stems and leaves: Take 15 g of dry powder of Tetrastigma hemsleyanum stems and leaves, divide it into three equal parts and place them in ethanol with concentrations of 50%, 75%, and 90% respectively for extraction for 3 h. After filtration, combine the filtrates, evaporate and concentrate to obtain an extract, and dry to obtain 0.8175 g of crude extract of Tetrastigma hemsleyanum stems and leaves, which is diluted for use as needed.

[0038] 1.2 Preparation method of crude extract of Tetrastigma hemsleyanum tubers: Take 15 g of dry powder of Tetrastigma hemsleyanum tubers, divide it into three equal parts and place them in ethanol with concentrations of 50%, 75%, and 90% respectively for extraction for 3 h. After filtration, combine the filtrates, evaporate and concentrate to obtain an extract, and dry to obtain 0.5626 g of crude extract of Tetrastigma hemsleyanum roots, which is diluted for use as needed.

[0039] Example 2 Effect of crude extract of Tetrastigma hemsleyanum on anti-thymus atrophy

[0040] Select 60 SPF-grade male ICR (4 weeks old), 18 - 20 g, and raise them in a clean-grade animal room at a temperature of 18 - 26 °C, relative humidity of 40% - 70%, with 12 h of alternating light and dark, and ventilation 22 times per hour. Water bottles, cages, bedding, etc. are sterilized by high-pressure steam, and the water bottles and bedding are replaced 3 times a week.

[0041] After 3 days of adaptive feeding of the mice, randomly divide the 60 male ICR mice into 6 groups according to body weight, namely blank control group, model group, high-dose group of crude extract of Tetrastigma hemsleyanum stems and leaves (2 g of crude drug amount / kg), low-dose group of crude extract of Tetrastigma hemsleyanum stems and leaves (1 g of crude drug amount / kg), high-dose group of crude extract of Tetrastigma hemsleyanum tubers (2 g of crude drug amount / kg), low-dose group of crude extract of Tetrastigma hemsleyanum tubers (1 g of crude drug amount / kg), with 10 mice in each group. Each treatment group is continuously administered orally for 14 d. On the 13th and 14th days of administration, the model group and each treatment group are intraperitoneally injected with a sterile Poly(I∶C) saline solution (16 mg / kg) to establish a model. 24 h after the last administration, weigh the animals and take blood from the inner canthus of the eye for peripheral blood cell analysis. After decapitation, take the thymus for weighing, observe its morphology and calculate the organ index. Take part of the thymus and place it in formalin solution for pathological detection. The results are shown in Table 1, Table 2 and Figure 1 .

[0042] The formula for calculating the thymus index is: Thymus index (mg / g) = Thymus mass (mg) / Body weight (g)

[0043] Peripheral blood cell analysis: 150 μL of anticoagulated peripheral blood was collected and blood cell differential count was performed using a fully automated blood cell analyzer.

[0044] HE staining histopathological examination: The thymus was fixed in 4% formaldehyde solution for one week and then paraffin sections (5 μm thick) were prepared. Hematoxylin-eosin staining was performed and histopathological changes in each group were observed.

[0045] Table 1. Thymus mass and thymus index

[0046]

[0047]

[0048] Note: Compared with the blank control group, ＊＊ P < 0.01; compared with the model group, # P < 0.05 ## P < 0.01.

[0049] Table 1 shows that the thymus of the model group mice was significantly atrophied, with a highly significant reduction in thymus weight and thymus index compared to the blank control group (P < 0.01), and the thymus atrophied by 41.09%. In the treatment groups, both the high- and low-dose groups of the crude extract of *Tripterygium wilfordii* stems and leaves showed good efficacy, with a significantly improved thymus index compared to the model group (P < 0.01). This indicates that the efficacy of the crude extract of *Tripterygium wilfordii* stems and leaves in preventing thymus atrophy is significantly better than that of the crude extract of *Tripterygium wilfordii* tuberous roots at the same dose.

[0050] Table 2. Detection of peripheral blood cell levels

[0051]

[0052] Note: Compared with the blank control group, ＊＊ P < 0.01; compared with the model group, # P < 0.05.

[0053] As shown in Table 2, after modeling, the white blood cell count in the peripheral blood of the model group was significantly lower than that of the blank control group (P < 0.01). Among the treatment groups, the high-dose group of crude extract of *Tripterygium wilfordii* stems and leaves showed a significant increase compared to the model group (P < 0.05), while the low-dose groups of crude extracts of *Tripterygium wilfordii* stems and leaves, low-dose groups of crude extracts of *Tripterygium wilfordii* roots, and high-dose groups of crude extracts of *Tripterygium wilfordii* stems and roots all showed increases compared to the model group. This indicates that the crude extract of *Tripterygium wilfordii* stems and leaves can reverse the decrease in white blood cell levels in model mice, improve the body's immune level, and alleviate thymic atrophy.

[0054] Depend on Figure 1 It can be seen that in the blank control group, the boundary between the thymic cortex and medulla was clear, the thymic cells in the cortex were tightly arranged, and a large number of lymphocytes were visible. The thymic epithelial cells and thymic corpuscle epithelial cells in the medulla were clearly visible, and no abnormalities such as apoptosis were observed. In the model group, a large number of thymic cortical cells died, the cortex was lightly stained, the number of epithelioid cells and lymphocytes decreased sharply, a large number of medullary thymic epithelial cells underwent apoptosis, lymphocytes were visible in patches, the boundary between the cortex and medulla disappeared, macrophages increased, and the damage was more severe. In the drug-treated groups, the high- and low-dose groups of *Trifolium repens* stem and leaf extract showed a clearer boundary between the thymic cortex and medulla, reduced apoptosis compared to the model group, visible thymic corpuscles, and no significant inflammatory cell infiltration, demonstrating better efficacy than the same dose of crude extract from the tuberous root. This indicates that the crude extract of *Trifolium repens* stem and leaf extract can significantly improve thymic atrophy.

[0055] Example 3: Preparation of n-butanol extract from stems and leaves of *Trifolium repens*

[0056] The specific process is as follows:

[0057] S1. Grinding: Take dried stems and leaves of *Clerodendrum trifolium*, grind them into coarse powder, and then pass them through an 80-mesh sieve.

[0058] S2. Extraction: Take 200g of crude powder and add 2L of 75% ethanol. Reflux and extract twice, 2 hours each time. Filter and combine the filtrates.

[0059] S3. Concentration: After reducing the pressure and recovering the solvent, a crude extract of Tripterygium wilfordii is obtained and stored at low temperature.

[0060] S4. Extraction: Dissolve the extract in an appropriate amount of water, extract three times with twice the amount of n-butanol solution, then back-extract once with an equal amount of water. Take the n-butanol layer, add an appropriate amount of water, and reduce the pressure to recover the solvent until there is no alcohol odor, to obtain the n-butanol extract of the stems and leaves of *Trifolium repens*.

[0061] Comparative Example 1: Preparation of Ethyl Trifoliate Oryza sativa Extract

[0062] The extraction process is the same as in Example 3, except that the extraction solvent is ethyl acetate.

[0063] Comparative Example 2: Preparation of Petroleum Ether Extract of Trifoliate Orange

[0064] The extraction process is the same as in Example 3, except that the extraction solvent is petroleum ether.

[0065] Comparative Example 3: Preparation of Trifoliate Orchid Water Extract

[0066] The aqueous extract of *Trifolium repens* was obtained by drying the extract in the remaining water after organic solvent extraction in Examples 3, 1, and 2.

[0067] Example 4: Effect of *Trifolium repens* stem and leaf extract on thymic atrophy

[0068] One hundred SPF-grade male ICRs (4 weeks old, 18-20g) were selected and housed in a clean-grade animal room at a temperature of 18-26℃ and a relative humidity of 40%-70%. The lighting and dark conditions were alternated for 12 hours each, and ventilation was provided 22 times per hour. Water bottles, cages, and bedding were sterilized by autoclaving, and water bottles and bedding were changed 3 times a week.

[0069] After 3 days of acclimatization, 100 male ICR mice were randomly divided into 10 groups based on body weight: blank control group, model group, high-dose water extract group, low-dose water extract group, high-dose n-butanol group, low-dose n-butanol group, high-dose ethyl acetate group, low-dose ethyl acetate group, high-dose petroleum ether group, and low-dose petroleum ether group, with 10 mice in each group. Based on the yields of the *Trifolium repens* stem and leaf extracts in Examples 3 and Comparative Examples 1-3, the dosages were adjusted to the same crude drug level through different dilution ratios (see Table 3).

[0070] Each treatment group received oral administration for 14 consecutive days. On days 13 and 14 of administration, the model group and each treatment group were intraperitoneally injected with sterile Poly(I:C) saline solution (16 mg / kg) to induce the model. Twenty-four hours after the last administration, animal weight was measured, and blood was drawn from the inner canthus of the eye for peripheral blood cell analysis. Animals were euthanized by cervical dislocation, and their thymus was harvested and weighed. Its morphology was observed, and organ indices were calculated. A portion of the thymus was placed in formalin solution for pathological examination. Results are shown in Tables 4 and 5. Figure 2 .

[0071] Table 3 Dosage

[0072]

[0073] Table 4. Body Weight and Organ Index

[0074]

[0075] Note: Compared with the blank control group, ＊＊ P < 0.01; compared with the model group, # P < 0.05 ## P < 0.01.

[0076] As shown in Table 4, the body weight, thymus, and thymus index of the model group mice were significantly lower than those of the blank control group (P < 0.01). Among the drug administration groups, only the high and low doses of n-butanol extract significantly improved the thymus weight (P < 0.05) and thymus index (P < 0.01) of the model mice. The results indicate that the part of the stem and leaves of *Trifolium repens* that has the therapeutic effect on thymus atrophy is the n-butanol extract.

[0077] Table 5. Detection of peripheral blood cell levels

[0078]

[0079]

[0080] Note: Compared with the blank control group, ＊＊ P < 0.01; compared with the model group, # P < 0.05 ## P < 0.01.

[0081] As shown in Table 5, compared with the blank control group, the level of white blood cells in the peripheral blood of mice in the model group was significantly decreased after modeling (P < 0.01), while the percentages of neutrophils, lymphocytes, monocytes, and eosinophils were not significantly different from those in the blank control group (P > 0.05). In the drug-treated groups, the high dose of n-butanol extract had a highly significant effect on increasing the level of white blood cells in the peripheral blood of model mice (P < 0.01), and the number of white blood cells in the low dose n-butanol group was also significantly improved compared with the model group (P < 0.05). This result is consistent with the thymus index results, indicating that the n-butanol extract of *Trifolium repens* stems and leaves can significantly reverse the decrease in white blood cell levels in model mice, improve the body's immune level, and improve thymus atrophy.

[0082] Depend on Figure 2 It can be seen that in the blank control group, the boundary between the cortex and medulla of the thymus tissue was clear, the thymocytes in the cortex were tightly arranged, and a large number of lymphocytes were visible. The thymic epithelial cells and thymic corpuscle epithelial cells in the medulla were clearly visible, with no apoptosis or inflammation. In the model group, a large number of cortical cells died, resulting in pale staining of the cortex. Epithelial-like cells and lymphocytes were exhausted, the medulla was deeply stained, and lymphocytes proliferated within it, showing patchy lymphocytes. The boundary between the cortex and medulla disappeared, macrophages increased, phagocytosis was obvious, and a small number of "starry sky" patterns appeared, indicating more severe damage. The high- and low-dose n-butanol groups showed significant improvement in the deep staining of the medulla and the pale staining of the cortex, and the boundary between the cortex and medulla was clearer, with an increased number of thymocytes compared to the model group. This indicates that the n-butanol extract from the stems and leaves of *Trifolium repens* can significantly improve thymic atrophy.

[0083] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. The use of *Trifolium repens* extract in the preparation of drugs for the prevention and / or treatment of thymic atrophy, characterized in that: The preparation method of the *Trifolium repens* extract includes the following steps: S1, the dried Trifoliate orange plant is crushed, and the crude extract of Trifoliate orange is obtained by reflux extraction and concentration with ethanol, wherein the ethanol concentration is ≥50% and the ethanol reflux time is 3-9 h. S2, the crude extract of *Trifolium repens* is extracted with n-butanol, back-extracted with water, and the solvent is recovered to obtain the *Trifolium repens* extract.

2. The application according to claim 1, characterized in that: The ethanol concentration is 50%, 75%, or 90%.

3. The application according to claim 1 or 2, characterized in that: S1 also includes the steps of refluxing ethanol, filtering, and collecting the filtrate.

4. The application according to any one of claims 1-3, characterized in that: S1 also includes a step of concentration followed by drying.

5. The application according to any one of claims 1-4, characterized in that: The aforementioned *Trifolium repens* plant includes the stems and leaves of *Trifolium repens*.

6. The application according to claim 5, characterized in that: Take dried *Trifolium repens* plants, crush them into coarse powder, pass them through an 80-mesh sieve, add 75% ethanol, and reflux extract twice, 2 hours each time. Filter and combine the filtrates, concentrate under reduced pressure to recover the solvent, and obtain a crude extract of *Trifolium repens*. Store it at low temperature. Take the extract, dissolve it in water, extract it three times with twice the amount of n-butanol solution, and then back-extract it once with an equal amount of water. Take the n-butanol layer, add water, and recover the solvent under reduced pressure until there is no alcohol odor, to obtain the *Trifolium repens* extract.

7. The application according to any one of claims 1-6, characterized in that: The drug also includes pharmaceutically permissible excipients.

8. The application according to claim 7, characterized in that: The drug is in the form of tablets, capsules, solutions, creams, or emulsions.

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