Anti-melanin tumor compound containing pan jade extract and preparation method of anti-melanin tumor compound

Abstract

The invention belongs to the field of mineral extraction and compound preparation thereof, and particularly relates to an anti-melanin tumor compound containing a pan jade extract and a preparation method of the anti-melanin tumor compound. The compound is prepared from the following components in parts by weight: 20 to 25 parts of pan jade extract, 40 to 50 parts of fiveleaf gynostemma herb extract, 15 to 20 parts of praeruptorin B, 10 to 12 parts of baicalein and 5 to 8 parts of betulin. The preparation method of the compound comprises the following steps: mixing the pan jade extract, the fiveleaf gynostemma herb extract, the praeruptorin B, the baicalein and the betulin in proportion, adding the mixture into absolute ethyl alcohol, stirring for 30 minutes at room temperature, and finally drying at 40-50 DEG C to obtain the melanin tumor resisting compound. According to the anti-melanoma compound provided by the invention, the pan jade extract is modified by adopting the rapeseed polypeptide, and the fiveleaf gynostemma herb extract, the praeruptorin B and the like are scientifically matched, so that a multi-component, multi-target and multi-level synergistic anti-tumor effect is realized, and the defect of serious side effects of traditional chemotherapy is overcome while the melanoma inhibition effect is improved.

Description

Technical Field

[0001] This application belongs to the field of mineral extraction and preparation of its complexes, specifically relating to an anti-melanoma complex containing Panlong jade extract and its preparation method. Background Technology

[0002] In modern medicine and health science, natural extracts, rich in bioactive components and exhibiting low adverse reaction rates, have garnered significant attention. Mineral medicines, with their long history and diverse varieties, embody the profound depth and breadth of traditional Chinese medicine. Statistics show that over 370 kinds of mineral-based medicinal materials are recorded in Chinese classics. Among them, the *Classic of Mountains and Seas* (Shan Hai Jing), as an early exploration of mineral medicines, records information on over 60 kinds of mineral-based medicinal materials. Furthermore, the latest 2020 edition of the *Chinese Pharmacopoeia* has carefully selected and included 23 kinds of mineral medicines, further confirming their important position in the modern medical system.

[0003] Jade, since ancient times, has been deeply loved for its warm touch and elegant color. However, its medicinal and health benefits are equally undeniable. As early as two thousand years ago, jade was used in the medical field. The saying "Jade is the most beautiful of stones, sweet in taste, neutral in nature, and non-toxic" not only summarizes the characteristics of jade but also reveals its medicinal value. In 2015, Jilin Sanhe Mining Development Co., Ltd. discovered Panlong jade near a lead-zinc mine in the Longgang Mountains of Panshi City, Jilin Province. Panlong jade, with its black, blackish-green, and purplish-black colors, is rich in various trace mineral elements such as Li, As, B, Ba, Cr, Se, and Cu. Subsequently, many scholars have conducted in-depth research on the medicinal value of Panlong jade.

[0004] For example, Chinese patent CN112022872 A discloses an extract of Panlong black jade, its preparation method, and its uses. This application mentions obtaining Panlong black jade extract through extraction with methanol, ethanol, or water. It was found that the methanol or ethanol extracts of Panlong jade can effectively inhibit the proliferation of melanocytes, with the ethanol extract showing the best effect in inhibiting B16F10, with an IC50 of 60.74±5.72 μg / μL. Wang Li et al. conducted mineral element analysis of Panlong jade and Panlong black jade, and a preliminary study on the tumor cell cytotoxic activity of Panlong black jade extract. The results showed that Panlong jade and Panlong black jade contain abundant mineral elements, including Se, Sr, and other trace elements related to human physiological functions. The extract of Panlong black jade has certain cytotoxic activity against tumor cell lines. Zhu Zhiheng et al. conducted experimental research on the anti-inflammatory and analgesic effects of Panlong jade thermotherapy on rheumatic knee osteoarthritis, clarifying that Panlong jade thermotherapy has the effects of promoting blood circulation, removing blood stasis, anti-inflammatory, and analgesic.

[0005] However, while existing treatments for melanoma have improved survival rates for advanced melanoma, immunotherapy (such as PD-1 / CTLA-4 inhibitors) extends survival for some patients by activating the immune system to attack tumors, it can also cause severe autoimmune damage, such as colitis and hepatitis. Targeted therapy is fast-acting but prone to resistance and may induce squamous cell carcinoma of the skin when used alone. Traditional chemotherapy has limited efficacy and often leads to bone marrow suppression and neurotoxicity.

[0006] Therefore, based on the characteristics of Panlong jade, this application attempts to provide a complex and preparation method for inhibiting melanoma that can effectively inhibit melanoma with low toxicity and good biocompatibility, overcoming the shortcomings of traditional methods. Summary of the Invention

[0007] In view of the limited efficacy of traditional chemotherapy and the fact that it often leads to bone marrow suppression and neurotoxicity, this application, based on the properties of Panlongyu, attempts to provide a complex for inhibiting melanoma with low toxicity and good biocompatibility, as well as a preparation method, to overcome the shortcomings of traditional methods.

[0008] The technical solution of this application is as follows:

[0009] On one hand, this application provides an anti-melanoma complex containing Panlongyu extract, comprising the following components in parts by weight: 20-25 parts of Panlongyu extract, 40-50 parts of Gynostemma pentaphyllum extract, 15-20 parts of angelica dahurica extract, 10-12 parts of baicalin, and 5-8 parts of betulin; wherein the Panlongyu extract is obtained by modifying rapeseed peptides.

[0010] Preferably, the components include the following parts by weight: 22 parts of Panlongyu extract, 43 parts of Gynostemma pentaphyllum extract, 17 parts of angelica dahurica extract, 11 parts of baicalin, and 7 parts of betulin.

[0011] Preferably, the specific preparation process of the Panlongyu extract modified with rapeseed peptides is as follows:

[0012] Panlong jade extraction: Panlong jade is crushed and sieved to obtain Panlong jade powder. Then, solvent is added and ultrasonic treatment is performed. The supernatant is centrifuged, filtered, and the filtrate is evaporated dry, then re-dissolved, and finally vacuum dried to obtain Panlong jade extract.

[0013] Rapeseed peptide modification: Panlong jade extract, rapeseed peptide powder and deionized water were mixed, heated and the pH value was adjusted, and the mixture was stirred to obtain a mixed solution. Finally, ethanol was added to the mixed solution, and the mixture was centrifuged and freeze-dried to obtain the rapeseed peptide modified Panlong jade extract.

[0014] Preferably, the Panlong jade powder is passed through a 400-mesh sieve, the solvent is either methanol or ethanol, the ratio of Panlong jade powder to solvent is 1g:(50-100mL), the ultrasonic treatment time is 1-2h, and the filtration is a 0.22μm filter membrane.

[0015] Preferably, the mass ratio of Panlong jade extract, polypeptide powder and deionized water is 1:2:(5-10), the pH value is 7-8, the heating temperature is 40-60℃, the stirring time is 30-60 min, and the volume of ethanol added is 8-10 times that of the mixed solution.

[0016] Preferably, the extraction process of the Gynostemma pentaphyllum extract is as follows:

[0017] First, crush the stems and leaves of Gynostemma pentaphyllum to 40-60 mesh, then soak them in a citric acid buffer solution with a pH of 5.6-6.4 for 30 minutes. The mass of the citric acid buffer solution should be 10 times that of the stems and leaves of Gynostemma pentaphyllum.

[0018] Add 0.2% to 1.0% of cellulase by weight of Gynostemma pentaphyllum stems and leaves, and sonicate at 45 to 50°C for 40 to 50 minutes. Finally, inactivate the enzyme to obtain the enzymatic hydrolysate.

[0019] Finally, anhydrous ethanol was added for precipitation for 12 hours, filtered, and dried at 60°C to obtain Gynostemma pentaphyllum extract. The amount of anhydrous ethanol added should be no less than twice the volume of the enzymatic hydrolysate.

[0020] On the other hand, this application provides a method for preparing an anti-melanoma complex containing Panlongyu extract, specifically as follows: 20-25 parts of Panlongyu extract, 40-50 parts of Gynostemma pentaphyllum extract, 15-20 parts of angelica dahurica extract, 10-12 parts of baicalein, and 5-8 parts of betulin are mixed in an anhydrous ethanol in a certain proportion, stirred at room temperature for 30 minutes, and finally dried at 40-50°C to obtain the anti-melanoma complex.

[0021] The beneficial effects of this application are:

[0022] The Panlong jade extract selected in this application is rich in various trace elements, such as selenium, zinc, and iron. These trace elements have important biological functions. Through the preparation method of this application, these beneficial components in Panlong jade are effectively extracted and maintained in the composition. The trace elements in the Panlong jade extract can inhibit the growth and spread of tumor cells directly or indirectly through regulating the body's immune system. Rapeseed polypeptides, as hydrolyzed products of plant protein, not only retain the nutritional value of protein but also have high bioavailability. Its rich amino acids, peptides, and other active ingredients have positive effects on enhancing the body's immunity and promoting cell repair. The unique structure and properties of rapeseed polypeptides can also promote the absorption of minerals and trace elements. At the same time, rapeseed polypeptides can form a protective layer in the gastrointestinal tract, reducing the inflammatory response of the gastrointestinal mucosa and protecting the gastrointestinal tract from the stimulation of harmful substances. Therefore, when rapeseed polypeptides are combined with Panlong jade extract, they can promote the absorption rate of trace elements in Panlong jade and further enhance its effect in inhibiting the proliferation of melanoma. The combination of Panlong jade extract and rapeseed peptides enhances their effect in inhibiting melanoma through synergistic action.

[0023] The complex in this application, containing extracts of *Panax japonicus*, *Gynostemma pentaphyllum*, and baicalein, works synergistically to enhance the immune system, strengthen anti-tumor immune responses, inhibit immunosuppressive cells and factors, and improve the tumor immune microenvironment. The antioxidant and anti-inflammatory effects of components such as baicalein and betulin effectively scavenge free radicals that may be generated when the active ingredients, such as those from *Panax japonicus* extract, are released, reducing inflammatory responses, protecting normal cells, and inhibiting the chronic inflammatory environment upon which tumor growth depends, thus achieving an anti-melanoma effect.

[0024] Gynostemma pentaphyllum extract is rich in saponins, which can inhibit tumor cell proliferation, induce apoptosis, inhibit tumor angiogenesis, and regulate immune function. It complements and synergizes with Panlongyu extract in terms of immunomodulation.

[0025] As a coumarin compound, white angelica dahurica extract can inhibit tumor cell DNA topoisomerase, interfere with cell cycle progression, and induce tumor cell differentiation and apoptosis.

[0026] Baicalein, as a flavonoid compound, possesses potent antioxidant and anti-inflammatory effects. It can scavenge free radicals, reduce oxidative stress damage, inhibit the production of pro-inflammatory factors, and suppress tumor cell proliferation, invasion, and metastasis by inhibiting multiple kinase signaling pathways, while also inducing apoptosis. Its antioxidant activity provides protection for other components and synergistically inhibits inflammatory responses in the tumor microenvironment.

[0027] Betulin belongs to the lupinane-type triterpenoids, which mainly induce tumor cell apoptosis through the mitochondrial pathway and may inhibit tumor cell migration and invasion.

[0028] All components of this complex are derived from natural plants or minerals and contain no chemically synthesized additives. Compared to the side effects of traditional chemotherapy drugs, this natural complex, based on its component sources and synergistic mechanism, is expected to have lower systemic toxicity and better biocompatibility.

[0029] In summary, the anti-melanoma complex provided in this application enhances the bioavailability and efficacy of the active ingredients by modifying Panlongyu extract with rapeseed peptides. Based on Gynostemma pentaphyllum extract, angelica dahurica extract, baicalin, and betulin, it achieves a multi-component, multi-target, and multi-level synergistic anti-tumor effect. While improving the inhibitory effect on melanoma, it overcomes the severe side effects of traditional chemotherapy drugs, demonstrating comprehensive advantages of high efficiency, low toxicity, and good biocompatibility. Detailed Implementation

[0030] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with preferred embodiments, is provided below.

[0031] Preparation Examples 1-3 provide a method for preparing Panlong jade extract:

[0032] Preparation Example 1

[0033] Panlong jade collected from the Longgang Mountains in Panshi City, Jilin Province, was mechanically pulverized through a 400-mesh sieve to obtain Panlong jade powder. Then, 10g of Panlong jade powder was added to 350mL of methanol, ultrasonically treated for 1h, centrifuged at 4000rpm for 30min, and the supernatant was filtered through a 0.22μm filter membrane. The filtrate was then evaporated at 55℃, redissolved in 1mL of methanol, and finally vacuum dried at 80℃ to obtain the Panlong jade extract.

[0034] Take 10g of rapeseed meal, wash it twice, filter to remove the filtrate, and dry it at 50℃ for 3 hours to obtain dried rapeseed meal. Then, take 8g of the dried rapeseed meal and mix it with 25mL of acetone, stir at room temperature for 1 hour, centrifuge to remove the acetone, and obtain defatted rapeseed meal. Then, add 6g of defatted rapeseed meal to 25g of water and 0.12g of Bacillus subtilis protease to adjust the pH to 8 for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 45℃, and the mixture is sonicated for 15 minutes, followed by stirring for 40 minutes. The sonication and stirring are repeated once. Finally, the mixture is placed in a 70℃ water bath for 1 hour. Bacillus subtilis protease was inactivated for 2 minutes, and the mixture was centrifuged at 4000 rpm for 30 minutes to obtain a rapeseed polypeptide solution. The solution was then freeze-dried at -70℃ to remove moisture and obtain rapeseed polypeptide powder. 0.5 g of Panlong jade extract, 1 g of rapeseed polypeptide powder and 8 g of deionized water were mixed, and the pH was adjusted to 7. The temperature was raised to 50℃ and stirred for 45 minutes to obtain a mixed solution. Finally, 81 mL of ethanol was added to the mixed solution, and the mixture was centrifuged at 4000 rpm for 30 minutes. The solution was then freeze-dried at -70℃ to obtain the rapeseed polypeptide-modified Panlong jade extract.

[0035] Preparation Example 2

[0036] Panlong jade collected from the Longgang Mountains in Panshi City, Jilin Province, was mechanically pulverized through a 400-mesh sieve to obtain Panlong jade powder. Then, 10g of Panlong jade powder was added to 250mL of ethanol, ultrasonically treated for 1.5h, centrifuged at 4000rpm for 30min, and the supernatant was filtered through a 0.22μm filter membrane. The filtrate was then evaporated at 55℃, redissolved in 1mL of ethanol, and finally vacuum dried at 80℃ to obtain the Panlong jade extract.

[0037] Take 10g of sunflower seed meal, wash it twice, filter to remove the filtrate, and dry it at 50℃ for 3 hours to obtain dried sunflower seed meal. Then, take 8g of the dried sunflower seed meal and mix it with 20mL of ethyl acetate, stir at room temperature for 1 hour, centrifuge to remove the ethyl acetate, and obtain defatted sunflower seed meal. Then, add 6g of defatted sunflower seed meal to 20g of water and 0.06g of Novo protease to adjust the pH to 8 for enzymatic hydrolysis. The hydrolysis temperature is 50℃, and the mixture is sonicated for 15 minutes, followed by stirring for 40 minutes. The sonication and stirring are repeated once. Finally, the mixture is subjected to a 70℃ water bath. Bacillus subtilis protease was inactivated for 10 min, and the mixture was centrifuged at 4000 rpm for 30 min to obtain a rapeseed polypeptide solution. The solution was then dried at -70℃ to remove moisture and obtain rapeseed polypeptide powder. 0.5 g of Panlong jade extract, 1 g of sunflower polypeptide powder and 5 g of deionized water were mixed, and the pH was adjusted to 8. The temperature was raised to 60℃ and stirred for 30 min to obtain a mixed solution. Finally, 60 mL of ethanol was added to the mixed solution, and the mixture was centrifuged at 4000 rpm for 30 min. The solution was then freeze-dried at -70℃ to obtain the rapeseed polypeptide-modified Panlong jade extract.

[0038] Preparation Example 3

[0039] Panlong jade collected from the Longgang Mountains in Panshi City, Jilin Province, was mechanically pulverized through a 400-mesh sieve to obtain Panlong jade powder. Then, 10g of Panlong jade powder was added to 500mL of methanol, ultrasonically treated for 2h, centrifuged at 4000rpm for 30min, and the supernatant was filtered through a 0.22μm filter membrane. The filtrate was then evaporated at 55℃, redissolved in 1mL of methanol, and finally vacuum dried at 80℃ to obtain the Panlong jade extract.

[0040] Take 10g of rapeseed meal, wash it twice, filter to remove the filtrate, and dry it at 50℃ for 3 hours to obtain dried rapeseed meal. Then, take 8g of the dried rapeseed meal and mix it with 30mL of acetone, stir at room temperature for 1 hour, centrifuge to remove the acetone, and obtain defatted rapeseed meal. Then, add 6g of defatted rapeseed meal to 30g of water and 0.24g of Carsberg protease to adjust the pH to 7 for enzymatic hydrolysis at 40℃, sonicate for 15min, stir for 60min, and repeat the sonication and stirring once. Finally, bathe in a 70℃ water bath. Bacillus subtilis protease was inactivated for 15 min, and the mixture was centrifuged at 4000 rpm for 30 min to obtain a rapeseed polypeptide solution. The solution was then freeze-dried at -70℃ to remove moisture and obtain rapeseed polypeptide powder. 0.5 g of Panlong jade extract, 1 g of rapeseed polypeptide powder and 10 g of deionized water were mixed, and the pH was adjusted to 7. The temperature was raised to 40℃ and stirred for 60 min to obtain a mixed solution. Finally, 88 mL of ethanol was added to the mixed solution, and the mixture was centrifuged at 4000 rpm for 30 min and freeze-dried at -70℃ to obtain the rapeseed polypeptide-modified Panlong jade extract.

[0041] Preparation Examples 4-6 provide a method for preparing Gynostemma pentaphyllum extract, as detailed below:

[0042] Preparation Example 4

[0043] Soak 10g of Gynostemma pentaphyllum stems and leaves in 100g of pH 6 citric acid buffer solution for 30 minutes;

[0044] Accurately weigh 0.05g of cellulase (cellulase activity of 10000U / g), add it to the soaked mixture, and perform ultrasonic enzymatic hydrolysis at 45℃ for 45 minutes. After the enzymatic hydrolysis is completed, raise the temperature to 80℃ and maintain it for 10 minutes to inactivate the enzyme, and obtain the enzymatic hydrolysate.

[0045] Add anhydrous ethanol four times the volume of the enzymatic hydrolysate to the enzymatic hydrolysate, allow it to precipitate for 12 hours, filter the mixture, collect the residue, and dry it in an oven at 60°C until constant weight to obtain Gynostemma pentaphyllum extract.

[0046] Preparation Example 5

[0047] Soak 10g of Gynostemma pentaphyllum stems and leaves in 100g of citric acid buffer solution (pH 5.6) for 30 minutes;

[0048] Accurately weigh 0.05g of cellulase (cellulase activity of 10000U / g), add it to the soaked mixture, and perform ultrasonic enzymatic hydrolysis at 50℃ for 40 minutes. After the enzymatic hydrolysis is completed, raise the temperature to 80℃ and maintain it for 10 minutes to inactivate the enzyme, and obtain the enzymatic hydrolysate.

[0049] Add anhydrous ethanol twice the volume of the enzyme hydrolysate to the enzyme hydrolysate, precipitate for 12 hours, filter, collect the filter residue, and dry it in an oven at 60°C to constant weight to obtain Gynostemma pentaphyllum extract.

[0050] Preparation Example 6

[0051] Soak 10g of Gynostemma pentaphyllum stems and leaves in 100g of citric acid buffer solution (pH 6.4) for 30 minutes;

[0052] Accurately weigh 0.05g of cellulase (cellulase activity of 10000U / g), add it to the soaked mixture, and perform ultrasonic enzymatic hydrolysis at 46℃ for 50 minutes. After the enzymatic hydrolysis is completed, raise the temperature to 80℃ and maintain it for 10 minutes to inactivate the enzyme, and obtain the enzymatic hydrolysate.

[0053] Add anhydrous ethanol three times the volume of the enzymatic hydrolysate to the enzymatic hydrolysate, allow it to precipitate for 12 hours, filter the mixture, collect the residue, and dry it in an oven at 60°C until constant weight to obtain Gynostemma pentaphyllum extract.

[0054] Examples 1 to 4 provide a method for preparing an anti-melanoma complex containing Panlong jade extract, as detailed below:

[0055] Example 1

[0056] This embodiment provides a method for preparing an anti-melanoma complex containing Panlong jade extract:

[0057] 22 parts of Panlongyu extract prepared in Preparation Example 1, 43 parts of Gynostemma pentaphyllum extract prepared in Preparation Example 4, 17 parts of angelica dahurica extract, 11 parts of baicalein extract and 7 parts of betulinol were mixed and added to anhydrous ethanol. The mixture was stirred at room temperature for 30 minutes and then dried at 45°C to obtain the anti-melanoma complex.

[0058] Example 2

[0059] This embodiment provides a method for preparing an anti-melanoma complex containing Panlong jade extract:

[0060] 25 parts of the Panlongyu extract prepared in Preparation Example 2, 40 parts of the Gynostemma pentaphyllum extract prepared in Preparation Example 5, 15 parts of angelica dahurica extract, 12 parts of baicalin extract, and 8 parts of betulinol were mixed in anhydrous ethanol in a certain proportion, stirred at room temperature for 30 minutes, and finally dried at 40°C to obtain the anti-melanoma complex.

[0061] Example 3

[0062] This embodiment provides a method for preparing an anti-melanoma complex containing Panlong jade extract:

[0063] 20 parts of the Panlongyu extract prepared in Preparation Example 3, 50 parts of the Gynostemma pentaphyllum extract prepared in Preparation Example 5, 15 parts of angelica dahurica extract, 10 parts of baicalin extract and 5 parts of betulinol were mixed in anhydrous ethanol in a certain proportion, stirred at room temperature for 30 minutes, and finally dried at 45°C to obtain the anti-melanoma complex.

[0064] Example 4

[0065] This embodiment provides a method for preparing an anti-melanoma complex containing Panlong jade extract:

[0066] 21 parts of Panlongyu extract prepared in Preparation Example 1, 43 parts of Gynostemma pentaphyllum extract prepared in Preparation Example 6, 20 parts of angelica dahurica extract, 10 parts of baicalin extract and 6 parts of betulinol were mixed in anhydrous ethanol in proportion, stirred at room temperature for 30 minutes, and finally dried at 50°C to obtain the anti-melanoma complex.

[0067] Comparative Example 1

[0068] The difference between this comparative example and Example 1 is that no Gynostemma pentaphyllum extract was added to this comparative example.

[0069] Comparative Example 2

[0070] The difference between this comparative example and Example 1 is that no Panlongyu extract was added to this comparative example.

[0071] Comparative Example 3

[0072] The difference between this comparative example and Example 1 is that no angelica dahurica extract was added to this comparative example.

[0073] Comparative Example 4

[0074] The difference between this comparative example and Example 1 is that the Panlongyu extract in this comparative example has not been modified with peptides.

[0075] Comparative Example 5

[0076] The difference between this comparative example and Example 1 is that this comparative example did not contain Gynostemma pentaphyllum extract and angelica dahurica extract.

[0077] Comparative Example 6

[0078] The difference between this comparative example and Example 1 is that this comparative example did not contain Panlongyu extract and Baihuaqianhu ethyl.

[0079] Comparative Example 7

[0080] The difference between this comparative example and Example 1 is that no Panlongyu extract or Gynostemma pentaphyllum extract was added to this comparative example.

[0081] Anti-tumor performance test:

[0082] Experimental materials

[0083] RPMI-1640 and DMEM culture media (Gibco, USA); fetal bovine serum (Biological Industries, Israel); methylthiazolyl ltetrazolium (MTT, Sigma, USA); DMSO (Sigma, USA). Mouse melanoma cells B16F10 and human melanoma cells A375 were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0084] Experimental methods

[0085] Take cells in the logarithmic growth phase and adjust the density to 2 × 10⁻⁶. 5 Cells were seeded at a concentration of 10% fetal bovine serum (FBS) in RPMI-1640 or DMEM medium in 96-well plates and cultured at 37°C, 5% CO2, and saturated humidity for 24 h. After cell attachment, 100 μL of drug-containing medium was added according to the grouping. A total of 12 groups were formed (blank control group, Examples 1-4, and Comparative Examples 1-7), with a drug concentration of 100 μg / μL, and incubated for 48 h.

[0086] 10 μL of MTT (5 g / L) was added to each well of cells, and after incubation for 4 h, the supernatant was discarded. 100 μL of LDMSO was then added to each well, and the mixture was shaken to mix. The OD value of each well was measured using a microplate reader (wavelength 570 nm). The results were recorded, and the cell proliferation rate was calculated. The experiment was repeated three times. Cell proliferation rate = (OD value of each experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. The half-maximal inhibitory concentration (IC50) for tumor cell growth was also calculated, and the results are shown in Table 1.

[0087] Table 1

[0088]

[0089]

[0090] As can be seen from Table 1, the best inhibitory effect was observed in Example 1: B16F10 cells: IC50 = 15.2 μg / mL; A375 cells: IC50 = 18.5 μg / mL. This is because the formulation in Example 1 has a better inhibitory effect on melanoma. The extract of Panlongyu (after peptide modification) synergistically enhances the anti-tumor activity with Gynostemma pentaphyllum saponins and Imperatorin. At the same time, it can be seen that the inhibitory effects of Examples 1 to 4 are all better than those of Comparative Examples 1 to 7. It is evident that the absence of the core component has a greater impact on inhibiting the proliferation of melanoma (see Comparative Examples 1 to 4). Furthermore, observation of Comparative Examples 5 to 7 shows that the synergistic attenuation of the inhibitory effect due to the absence of two components is observed.

[0091] It is evident that the anti-melanoma complex provided in this application enhances the bioavailability and efficacy of its active ingredients by modifying Panlongyu extract with rapeseed peptides, and scientifically combines Gynostemma pentaphyllum extract, angelica dahurica extract, baicalein, and betulin to achieve a synergistic anti-tumor effect with multiple components, multiple targets, and multiple levels. While improving the inhibitory effect on melanoma, it overcomes the core defects of traditional chemotherapy drugs, such as high toxicity and severe side effects, and demonstrates the comprehensive advantages of high efficiency, low toxicity, and good biocompatibility.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A melanoma-fighting complex containing Panlong jade extract, characterized in that, The product comprises the following components in parts by weight: 20-25 parts of Panlongyu extract, 40-50 parts of Gynostemma pentaphyllum extract, 15-20 parts of angelica dahurica extract, 10-12 parts of baicalin, and 5-8 parts of betulin; wherein the Panlongyu extract is obtained by modifying rapeseed peptides.

2. The anti-melanoma complex containing Panlong jade extract according to claim 1, characterized in that, The components include the following parts by weight: 22 parts of Panlongyu extract, 43 parts of Gynostemma pentaphyllum extract, 17 parts of angelica dahurica extract, 11 parts of baicalin, and 7 parts of betulin.

3. The anti-melanoma complex containing Panlong jade extract according to claim 1, characterized in that, The specific preparation process of the Panlongyu extract modified with rapeseed polypeptides is as follows: Panlong jade extraction: Panlong jade is crushed and sieved to obtain Panlong jade powder. Then, solvent is added and ultrasonic treatment is performed. The supernatant is centrifuged, filtered, and the filtrate is evaporated dry, then re-dissolved, and finally vacuum dried to obtain Panlong jade extract. Rapeseed peptide modification: Panlong jade extract, rapeseed peptide powder and deionized water were mixed, heated and the pH value was adjusted, and the mixture was stirred to obtain a mixed solution. Finally, ethanol was added to the mixed solution, and the mixture was centrifuged and freeze-dried to obtain the rapeseed peptide modified Panlong jade extract.

4. The anti-melanoma complex containing Panlong jade extract according to claim 3, characterized in that, The Panlong jade powder is passed through a 400-mesh sieve, the solvent is either methanol or ethanol, the ratio of Panlong jade powder to solvent is 1g:(50-100mL), the ultrasonic treatment time is 1-2h, and the filtration is a 0.22μm filter membrane.

5. The anti-melanoma complex containing Panlong jade extract according to claim 3, characterized in that, The mass ratio of Panlong jade extract, polypeptide powder and deionized water is 1:2:(5-10), the pH value is 7-8, the heating temperature is 40-60℃, the stirring time is 30-60 min, and the volume of ethanol added is 8-10 times that of the mixed solution.

6. The anti-melanoma complex containing Panlong jade extract according to claim 1, characterized in that, The extraction process of the Gynostemma pentaphyllum extract is as follows: First, crush the stems and leaves of Gynostemma pentaphyllum to 40-60 mesh, then soak them in a citric acid buffer solution with a pH of 5.6-6.4 for 30 minutes. The mass of the citric acid buffer solution should be 10 times that of the stems and leaves of Gynostemma pentaphyllum. Add 0.2% to 1.0% of cellulase by weight of Gynostemma pentaphyllum stems and leaves, and sonicate at 45 to 50°C for 40 to 50 minutes. Finally, inactivate the enzyme to obtain the enzymatic hydrolysate. Finally, anhydrous ethanol was added for precipitation for 12 hours, filtered, and dried at 60°C to obtain Gynostemma pentaphyllum extract. The amount of anhydrous ethanol added should be no less than twice the volume of the enzymatic hydrolysate.

7. A method for preparing an anti-melanoma complex containing Panlong jade extract as described in any one of claims 1 to 6, characterized in that, Specifically, 20-25 parts of Panlongyu extract, 40-50 parts of Gynostemma pentaphyllum extract, 15-20 parts of angelica dahurica extract, 10-12 parts of baicalin, and 5-8 parts of betulin were mixed in anhydrous ethanol and stirred at room temperature for 30 minutes. Finally, the mixture was dried at 40-50°C to obtain the anti-melanoma complex.

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