Application of lupin or lupin extract in preparation of drugs for relieving reproductive toxicity of chemotherapeutic drugs

Abstract

The invention relates to the technical field of medicines, and discloses an application of lupin or an extract thereof in preparation of a drug for relieving reproductive toxicity of chemotherapeutic drugs, the lupin is selected from lupin plants, preferably lupin variety Jenabillup, and the chemotherapeutic drugs comprise busulfan, cyclophosphamide or cis-platinum. The administration dosage of the lupin or the extract thereof is 0.1-100 mg / kg body weight, the administration period is continuous administration for at least 5 weeks, experiments show that the lupin can significantly improve the sperm concentration and sperm motility of chemotherapy model animals and improve pathological damage of testicular tissues, the mechanism relates to antioxidant, anti-inflammatory and anti-apoptosis effects, and the lupin or the extract thereof can be used for preparing a chemotherapy model animal. The invention provides a safe and effective new strategy for fertility protection of chemotherapy patients.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of lupin or its extracts in the preparation of drugs that reduce the reproductive toxicity of chemotherapy drugs. Background Technology

[0002] Malignant tumors pose a serious threat to human health, and chemotherapy is currently one of the most important clinical treatments for malignant tumors. Busulfan is a bifunctional alkylating agent widely used clinically for the treatment of chronic myeloid leukemia, bone marrow transplant pretreatment, and plays a particularly important role in the treatment of childhood cancer. However, while exerting its anti-tumor effects, busulfan can produce serious toxic side effects on normal tissues and cells. Studies have shown that busulfan has significant toxic effects on the male reproductive system, manifested as decreased sperm concentration, reduced sperm motility, and testicular tissue damage, which in severe cases can lead to permanent infertility.

[0003] The reproductive toxicity mechanism induced by busulfan involves multiple aspects. First, busulfan can induce oxidative stress damage in testicular tissue by generating large amounts of reactive oxygen species (ROS), leading to increased levels of malondialdehyde (MDA), a lipid peroxidation product, while reducing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Second, busulfan can directly damage testicular Sertoli cells and spermatogenic cells, disrupting the integrity of the seminiferous epithelium and inhibiting spermatogenesis. Furthermore, busulfan can exacerbate reproductive damage through inducing apoptosis and interfering with endocrine regulation.

[0004] Other commonly used chemotherapy drugs, such as cyclophosphamide and cisplatin, also exhibit significant reproductive toxicity. Cyclophosphamide, as a nitrogen mustard alkylating agent, can cause testicular atrophy and increased sperm abnormality rates; cisplatin, as a platinum compound, can lead to DNA damage and apoptosis in spermatogenic cells. The reproductive toxicity of these chemotherapy drugs poses a significant challenge to young cancer patients, especially children and adolescents who wish to have children.

[0005] Currently, protective measures against the reproductive toxicity of chemotherapy drugs are very limited. Clinically, fertility preservation is mainly achieved through methods such as sperm cryopreservation and testicular tissue cryopreservation; however, these methods are complex, expensive, and have limited applicability to pediatric patients. Regarding drug intervention, although some studies have reported that certain antioxidants, such as vitamin E and N-acetylcysteine ​​(NAC), have a certain protective effect against the reproductive toxicity of chemotherapy drugs, their clinical efficacy and safety still need further verification. Therefore, developing safe and effective adjuvant drugs to reduce the reproductive toxicity of chemotherapy drugs has significant clinical value and social implications.

[0006] Natural products have received extensive attention in the field of protecting against the toxic and side effects of chemotherapy due to their characteristics of multi-target and low toxicity. In recent years, it has been found that various plant extracts have a protective effect on chemotherapy-induced reproductive damage. For example, the extract of Trogopterus dung can reverse busulfan-induced testicular damage by activating the arginine biosynthesis pathway, reducing oxidative stress and inflammatory responses. However, most of these studies have focused on traditional Chinese medicine compounds or single compounds, and relatively few studies have been conducted on edible plant raw materials with rich nutrition and high safety.

[0007] Lupinus is a general term for plants of the genus Lupinus in the legume family. There are about 400 species globally, mainly distributed in the Mediterranean region, the Americas, and Africa. As an important edible bean, lupinus has nutritional characteristics such as high protein content, low fat content, and rich dietary fiber. Lupinus angustifolius, also known as blue lupin, is one of the lupin cultivars with the largest global planting area. Sweet lupin varieties obtained through breeding improvement, such as Jenabillup cultivated in Australia, have the advantages of low alkaloid content (less than 500 mg / kg), good palatability, and high safety, and have been widely used in the food and feed industries.

[0008] Modern research shows that lupinus is rich in various bioactive components. Its protein content can reach 35 - 45%, containing all the essential amino acids for the human body, especially rich in leucine and lysine. Lupinus also contains abundant polyphenolic compounds, including flavonoids (luteolin, apigenin, isoflavones), phenolic acids (protocatechuic acid, p-hydroxybenzoic acid), etc., which have significant antioxidant activity. In addition, the dietary fiber in lupinus can regulate the intestinal microecology, and functional components such as γ-aminobutyric acid (GABA) have a protective effect on the nervous system. Clinical studies have shown that the intake of lupinus can reduce blood sugar, improve blood lipids, and reduce body weight, and has a protective effect on type 2 diabetes and cardiovascular diseases.

[0009] Although the nutritional value and various health effects of lupinus have been widely recognized, its application research in the field of male reproductive health is still blank. Searching domestic and foreign literature databases, no relevant reports on the use of lupinus to reduce the reproductive toxicity of chemotherapy drugs have been found. Given the high nutritional value, various bioactive components, and good safety of lupinus, exploring its application in protecting against chemotherapy-induced reproductive toxicity has important research value and practical significance. Summary of the Invention

[0010] Aiming at the problem of the lack of safe and effective adjuvant drugs for reducing the reproductive toxicity of chemotherapy drugs in the prior art, the present invention first proposes to apply lupinus or its extract to reduce the reproductive toxicity of chemotherapy drugs, in order to provide a new option for the protection of fertility in chemotherapy patients.

[0011] This invention provides the use of lupin or its extract in the preparation of a drug to reduce the reproductive toxicity of chemotherapy drugs, wherein the lupin is selected from plants of the genus Lupinus.

[0012] In some embodiments, the lupins are selected from one or more of sweet lupins, narrow-leaved lupins, white lupins, or yellow lupins, preferably the sweet lupin variety Jenabillup.

[0013] In some implementations, the chemotherapy drug is one or more of busulfan, cyclophosphamide, or cisplatin.

[0014] In some embodiments, the extract is an aqueous extract, an alcoholic extract, an enzymatic extract, or a supercritical extract. Extract.

[0015] In some embodiments, the reproductive toxicity includes one or more of the following: decreased sperm concentration, decreased sperm motility, testicular tissue damage, or apoptosis of spermatogenic epithelial cells.

[0016] In some embodiments, the dosage of the lupin or its extract is 0.1-100 mg / kg body weight, preferably 50-100 mg / kg body weight.

[0017] In some implementations, the dosing cycle is at least 5 weeks of continuous dosing, and the dosing frequency is once daily.

[0018] In some implementations, the dosage form of the drug is an oral liquid, capsule, tablet, granule, powder, or functional food.

[0019] In some implementations, the drug may also include a pharmaceutically acceptable carrier or excipient.

[0020] In some implementations, the drug is used concurrently with chemotherapy drugs or is started before chemotherapy begins.

[0021] The beneficial effects of this invention include the following aspects:

[0022] This invention is the first to discover that lupin has a significant protective effect against chemotherapy-induced male reproductive toxicity. Experimental results show that lupin administration significantly increases sperm concentration and sperm motility in busulfan-based animal models and improves testicular tissue pathological damage, suggesting that lupin has good reproductive protective efficacy.

[0023] Lupins are a traditional edible plant with a long history of consumption and a good safety record. Sweet lupin varieties have low alkaloid content, making them suitable as a health food or auxiliary drug ingredient for long-term use.

[0024] This invention fills the gap in the application of lupins in the field of chemotherapy-induced reproductive toxicity protection, and provides a new fertility protection strategy for chemotherapy patients, especially young patients with fertility needs. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from its spirit and scope, and all such modifications and variations fall within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] The term lupin refers to the general term for plants in the genus *Lupinus* of the legume family, including but not limited to narrow-leaved lupin (*Lupinus angustifolius*), white lupin (*Lupinus albus*), yellow lupin (*Lupinus luteus*), and multi-leaved lupin (*Lupinus polyphyllus*). The term sweet lupin refers to lupin varieties with low alkaloid content obtained through breeding improvement; their total alkaloid content is typically below 500 mg / kg.

[0028] The term Jenabillup refers to a sweet lupin variety developed in Australia. It belongs to the narrow-leaved lupin species and is characterized by low alkaloid content, high protein content, and strong adaptability.

[0029] The term "reproductive toxicity of chemotherapy drugs" refers to the toxic side effects of chemotherapy drugs on the reproductive system, mainly manifested as impaired sperm production, sperm function damage, gonadal endocrine disorders, and decreased fertility.

[0030] The term busulfan refers to a bifunctional alkylating agent, chemically named 1,4-butanediol dimethyl sulfonate, with the molecular formula [missing information]. It is used clinically for the treatment of chronic myeloid leukemia and pretreatment for bone marrow transplantation.

[0031] The effective amount mentioned in this invention refers to the amount of lupin or its extract sufficient to produce the desired biological effect, and this amount can be adjusted according to factors such as route of administration, patient condition, and combination therapy.

[0032] The sources of the raw materials used in this invention are not specifically limited and can be obtained commercially or prepared according to conventional methods in the field.

[0033] Example 1: Preparation of Lupin Flour

[0034] Mature sweet lupin (Jenabillup variety) seeds were selected, washed, dried, and then ground using a grinder. The powder was passed through a 100-mesh standard sieve to obtain whole lupin flour. Its main nutritional components were determined: protein content 42.3%, fat content 6.8%, dietary fiber content 28.5%, ash content 3.2%, and total alkaloid content 185 mg / kg. The lupin flour was sealed and stored in a cool, dry place for later use.

[0035] Example 2 Preparation of lupin aqueous extract

[0036] 100 g of lupin powder prepared in Example 1 was added to 10 times (1000 mL) of purified water and extracted by stirring in an 80°C water bath for 2 hours. The filtrate was collected by filtration. The residue was then added to 8 times the amount of purified water, and the extraction was repeated once. The two filtrates were combined. The combined filtrate was concentrated under reduced pressure to a relative density of 1.10-1.15 (measured at 50°C), and freeze-dried to obtain lupin aqueous extract powder. The extract yield was determined to be 35.2%, the polyphenol content was 4.85 g GAE / 100g (calculated as gallic acid equivalent), and the flavonoid content was 2.12 g RE / 100g (calculated as rutin equivalent).

[0037] Example 3 Preparation of Lupin Alcohol Extract

[0038] 100 g of lupin powder prepared in Example 1 was added to 800 mL of 70% ethanol solution and extracted by stirring in a 60°C water bath for 1.5 hours. The filtrate was collected by filtration. The residue was then extracted again with 6 times the amount of 70% ethanol, and the extraction was repeated once. The two filtrates were combined. The combined filtrate was subjected to reduced pressure to recover ethanol, concentrated to a relative density of 1.12-1.18 (measured at 50°C), diluted with an appropriate amount of purified water, and freeze-dried to obtain lupin alcohol extract powder. The extract yield was determined to be 28.6%, with a polyphenol content of 7.23 g GAE / 100g, a flavonoid content of 3.85 g RE / 100g, and an isoflavone content of 1.56 g / 100g.

[0039] Example 4 Preparation of enzymatic hydrolysis extract from lupins

[0040] 100 g of lupin powder prepared in Example 1 was added to 15 times the amount of purified water, the pH was adjusted to 5.0, and 0.5% (by weight) of cellulase and 0.3% (by weight) of pectinase were added. The mixture was enzymatically hydrolyzed in a 50°C water bath for 4 hours. After hydrolysis, the enzyme was inactivated at 95°C for 10 minutes, cooled, and filtered. The filtrate was concentrated under reduced pressure and freeze-dried to obtain the lupin enzymatic extract. The extract yield was 42.5%, the soluble protein content was 28.3%, the polyphenol content was 5.68 g GAE / 100g, and the DPPH free radical scavenging rate (IC50) was 125.6 μg / mL.

[0041] Example 5: Supercritical Lupin Preparation of extracts

[0042] Take 200 g of the lupin powder prepared in Example 1 and place it in a supercritical fluid. In the extraction apparatus. Extraction conditions set: extraction pressure 25 MPa, extraction temperature 45℃. The extraction was performed at a flow rate of 20 L / h for 2 hours, using anhydrous ethanol as an entrainer (5% of the raw material mass). After extraction, the extract was collected in a separator, and the ethanol was evaporated under reduced pressure to obtain lupin supercritical fluid. Extract. The extract yield was determined to be 8.5%, the total flavonoid content was 12.35 g RE / 100g, and the β-sitosterol content was 2.8%.

[0043] Example 6: Preparation of Lupin Oral Solution

[0044] Prescription composition: 50 g of lupin water extract (Example 2), 150 g of sucrose, 0.25 g of potassium sorbate, and purified water to 1000 mL.

[0045] Preparation method: Dissolve lupin aqueous extract in an appropriate amount of purified water; separately dissolve sucrose in water by heating to make a syrup; dissolve potassium sorbate in a small amount of water. Mix the above solutions evenly, add purified water to a final volume of 1000 mL, stir well, allow to stand and filter, fill into 10 mL oral liquid bottles, seal, and sterilize with flowing steam at 100℃ for 30 minutes. Each oral liquid contains 0.5 g of lupin aqueous extract, equivalent to 1.42 g of raw drug.

[0046] Example 7 Preparation of Lupin Capsules

[0047] Prescription composition: 200 g of lupin alcohol extract (Example 3), 80 g of microcrystalline cellulose, 4 g of magnesium stearate, and 16 g of hydroxypropyl methylcellulose.

[0048] Preparation method: Lupin alcohol extract was mixed evenly with microcrystalline cellulose and hydroxypropyl methylcellulose to form a soft mass, which was then granulated through a 20-mesh sieve and dried at 60℃ for 2 hours. After granulation, magnesium stearate was added and mixed evenly, and the mixture was filled into No. 0 capsules, with each capsule containing 0.3 g. Each capsule contains 0.2 g of lupin alcohol extract, equivalent to 0.70 g of the raw drug.

[0049] Example 8: Preparation of Lupin Tablets

[0050] Prescription composition: 300 g of lupin enzymatic hydrolysis extract (Example 4), 120 g of lactose, 60 g of starch, 15 g of sodium carboxymethyl starch, and 5 g of magnesium stearate.

[0051] Preparation method: The enzymatic hydrolysis extract of lupin is mixed evenly with lactose and starch. A soft mass is prepared using 10% starch slurry as a binder, granulated through a 16-mesh sieve, and dried at 60℃ until the moisture content is less than 3%. After granulation, sodium carboxymethyl starch and magnesium stearate are added and mixed evenly, and then compressed into tablets. Each tablet weighs 0.5 g and contains 0.3 g of enzymatic hydrolysis extract of lupin, equivalent to 0.71 g of the raw drug.

[0052] Example 9: Preparation of Lupin Granules

[0053] Prescription composition: 100 g of lupin water extract (Example 2), 150 g of dextrin, and 250 g of sucrose.

[0054] Preparation method: Mix lupin aqueous extract, dextrin, and sucrose evenly, add an appropriate amount of purified water to form a soft mass, granulate through a 14-mesh sieve, dry at 60℃ until the moisture content is less than 5%, granulate, and package. Each bag contains 10 g of lupin aqueous extract, which is equivalent to 5.68 g of raw drug.

[0055] Example 10 Preparation of functional foods made from lupins

[0056] Formula composition: 500 g lupin whole bean flour (Example 1), 300 g oat flour, 150 g brown rice flour, 50 g fructooligosaccharides.

[0057] Preparation method: Mix the above raw materials evenly according to the specified ratio, add an appropriate amount of purified water, and extrude and expand the mixture using a twin-screw extruder. Set the temperature to 120-160℃ and the screw speed to 150 rpm. Cut the extrudate into appropriate lengths, dry at 80℃ until the moisture content is below 8%, and package after cooling. Each serving (50 g) contains 25 g of lupin whole bean flour.

[0058] Example 11: Effects of lupins on sperm quality in normal mice

[0059] Objective: To investigate the effects of different doses of lupin on sperm concentration and sperm motility in normal mice.

[0060] Experimental materials: 3-week-old male ICR mice, weighing 18-22 g, purchased from the experimental animal center. Lupin whole bean powder (Example 1).

[0061] Experimental Methods: Forty mice were randomly divided into four groups of ten each. The control group received 10 mL / kg of physiological saline via gavage daily. The low-dose group received 0.1 mg / kg body weight of lupin powder suspension via gavage daily. The medium-dose group received 10 mg / kg body weight of lupin powder suspension via gavage daily. The high-dose group received 100 mg / kg body weight of lupin powder suspension via gavage daily. All groups received the medication for five consecutive weeks.

[0062] Twenty-four hours after the last administration, mice were sacrificed, and the epididymis was removed, chopped, and placed in physiological saline at 37°C for 15 minutes to allow sperm to swim out. Sperm concentration and motility parameters were measured using a computer-aided sperm analysis system (CASA).

[0063] The experimental results are shown in Table 1.

[0064] Table 1. Effects of lupins on sperm parameters in normal mice (n=10, ± s)

[0065] Group Sperm concentration ( / mL) Sperm motility (%) Forward motility rate (%) Blank control group 28.5 ± 4.2 72.3 ± 6.8 45.2 ± 5.5 Low-dose group (0.1 mg / kg) 29.8 ± 3.9 74.5 ± 5.9 47.8 ± 4.8 Medium dose group (10 mg / kg) 32.6 ± 4.5 78.2 ± 5.2* 52.3 ± 5.1* High-dose group (100 mg / kg) 38.2 ± 5.1** 85.6 ± 4.6** 61.5 ± 4.9**

[0066] Note: Compared with the blank control group, *P<0.05, **P<0.01.

[0067] Experimental conclusion: High dose (100 mg / kg) of lupin powder can significantly increase sperm concentration and sperm motility in normal mice, while medium dose (10 mg / kg) can also improve sperm motility and progressive sperm rate, suggesting that lupin has a promoting effect on male reproductive function.

[0068] Example 12: Protective effect of lupins against busulfan-induced reproductive toxicity in mice.

[0069] Objective: To investigate the protective effect of lupins against busulfan-induced reproductive toxicity in mice.

[0070] Experimental materials: 3-week-old male ICR mice, weighing 18-22 g. Lupin whole bean powder (Example 1). Busulfan injection.

[0071] Experimental Methods: Sixty mice were randomly divided into 6 groups of 10 mice each. Normal control group: single intraperitoneal injection of saline, followed by daily oral administration of saline; Model control group: single intraperitoneal injection of busulfan 20 mg / kg, followed by daily oral administration of saline; Positive control group: single intraperitoneal injection of busulfan 20 mg / kg, followed by daily oral administration of vitamin E 100 mg / kg; Low-dose lupin group: single intraperitoneal injection of busulfan 20 mg / kg, followed by daily oral administration of lupin powder 0.1 mg / kg; Medium-dose lupin group: single intraperitoneal injection of busulfan 20 mg / kg, followed by daily oral administration of lupin powder 10 mg / kg; High-dose lupin group: single intraperitoneal injection of busulfan 20 mg / kg, followed by daily oral administration of lupin powder 100 mg / kg.

[0072] Lupin powder was prepared into a suspension of appropriate concentration using physiological saline and administered one day before busulfan injection, with continuous administration for 5 weeks. Mice were sacrificed 24 hours after the last administration, and sperm parameters, testicular histopathological changes, and serum biochemical indicators were examined.

[0073] Sperm parameter detection: The epididymis was cut into small pieces and placed in physiological saline at 37°C. The sperm concentration, sperm motility, and progressive motility rate were measured using the CASA system.

[0074] Testicular histopathological examination: One testis was taken and fixed in 4% paraformaldehyde, routinely dehydrated, embedded in paraffin, sectioned (5 μm thick), stained with hematoxylin and eosin (HE), and the morphological changes of the testicular tissue were observed under an optical microscope.

[0075] Serum biochemical markers were detected: blood was collected from the eyeballs, serum was separated, and serum testosterone (T), follicle-stimulating hormone (FSH), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured using an ELISA kit.

[0076] Experimental results:

[0077] The sperm parameter results are shown in Table 2.

[0078] Table 2. Effects of lupins on sperm parameters in bukhart-induced mice (n=10, ...). ± s)

[0079] Group Sperm concentration ( / mL) Sperm motility (%) Forward motility rate (%) normal control group 32.4 ± 4.8 78.5 ± 5.2 52.8 ± 4.9 Model control group 8.6 ± 2.3 25.3 ± 6.8 12.5 ± 3.8 Positive drug group 18.5 ± 3.6 52.6 ± 5.9 32.4 ± 5.2 low-dose lupin group 15.2 ± 3.2 45.8 ± 6.5 28.6 ± 4.8 Lupin medium dose group 22.8 ± 4.1 58.5 ± 5.6 38.2 ± 5.5 High-dose lupin group 28.5 ± 4.5 72.3 ± 5.8 48.6 ± 5.1

[0080] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0081] The results of serum biochemical indicators are shown in Table 3.

[0082] Table 3. Effects of lupin on serum biochemical parameters in bucetan-induced model mice (n=10, ± s)

[0083] Group T (nmol / L) FSH (mIU / mL) SOD (U / mL) MDA (nmol / L) normal control group 18.6 ± 2.5 15.8 ± 2.2 165.2 ± 12.8 4.85 ± 0.92 Model control group 8.2 ± 1.8 8.5 ± 1.6 98.5 ± 10.2 12.56 ± 1.85 Positive drug group 12.8 ± 2.1 11.2 ± 1.9 135.6 ± 11.5 7.25 ± 1.12 low-dose lupin group 10.5 ± 1.9 10.2 ± 1.8 125.8 ± 10.8 8.65 ± 1.25 Lupin medium dose group 14.2 ± 2.2 12.5 ± 2.0 148.2 ± 11.2 6.52 ± 1.05 High-dose lupin group 16.8 ± 2.4 14.5 ± 2.1 158.6 ± 12.2 5.28 ± 0.98

[0084] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0085] Histopathological observation of testicular tissue: In the normal control group, the testicular tissue structure was intact, the seminiferous tubules were regularly arranged, the levels of spermatogenic cells were clearly defined, and a large number of mature sperm were visible in the lumen. In the model control group, the testicular tissue showed obvious pathological changes, with atrophy and deformation of the seminiferous tubules, shedding and reduction of seminiferous epithelial cells, sparse or absent sperm in the lumen, and significant interstitial edema. The degree of pathological damage to the testicular tissue was reduced to varying degrees in the positive control group and the lumen dose groups. Among them, the testicular tissue morphology of the high-dose lumen group was close to that of the normal control group, the seminiferous tubule structure was relatively intact, spermatogenic cells at all levels were visible, and a large number of sperm were visible in the lumen.

[0086] Experimental conclusions: Busulfan significantly reduced sperm concentration and motility in mice, decreased serum T, FSH, and SOD levels, increased MDA content, and caused significant pathological damage to testicular tissue. Lupin administration dose-dependently improved these indicators, and high-dose (100 mg / kg) lupin showed better protective effects than the positive control drug vitamin E, suggesting that lupin has a good protective effect against busulfan-induced reproductive toxicity.

[0087] Example 13: Protective effect of lupin aqueous extract against busulfan-induced reproductive toxicity in mice.

[0088] Objective: To compare the protective effects of lupin aqueous extract and whole bean powder against busulfan-induced reproductive toxicity in mice.

[0089] Experimental Methods: Forty male ICR mice were randomly divided into four groups of 10 mice each. The normal control group received a single intraperitoneal injection of saline solution followed by daily gavage. The model control group received a single intraperitoneal injection of busulfan 20 mg / kg followed by daily gavage. The lupin powder group received a single intraperitoneal injection of busulfan 20 mg / kg followed by daily gavage of lupin powder 100 mg / kg. The lupin water extract group received a single intraperitoneal injection of busulfan 20 mg / kg followed by daily gavage of lupin water extract 35 mg / kg (equivalent to 100 mg / kg lupin powder). Sperm parameters were measured after 5 weeks of continuous administration.

[0090] The experimental results are shown in Table 4.

[0091] Table 4. Effects of different forms of lupin on sperm parameters in bukhart model mice (n=10, ± s)

[0092] Group Sperm concentration ( / mL) Sperm motility (%) normal control group 31.8 ± 4.5 76.8 ± 5.5 Model control group 9.2 ± 2.5 26.5 ± 6.2 Lupin whole bean powder group 27.5 ± 4.2 70.2 ± 5.8 Lupin water extract group 29.2 ± 4.8 73.5 ± 5.2

[0093] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0094] Experimental conclusion: Both lupin water extract and whole lupin powder have good protective effects against bucetamine-induced reproductive toxicity, and their effects are comparable, suggesting that the effective components of lupin can be obtained through water extraction.

[0095] Example 14: Protective effect of lupin alcohol extract against busulfan-induced reproductive toxicity in mice.

[0096] The experimental method was the same as in Example 13, except that the lupin water extract group was replaced with the lupin alcohol extract group (dose 28.6 mg / kg, equivalent to 100 mg / kg whole bean flour).

[0097] Experimental results: The sperm concentration in the lupin extract group was (30.5 ± 4.6). / mL, sperm motility rate was (75.2 ± 5.5)%, which was not significantly different from the normal control group (P>0.05) and significantly different from the model control group (P<0.05).

[0098] Experimental conclusion: Lupin alcohol extract also has a good protective effect against busulfan-induced reproductive toxicity, and the effect is slightly better than that of water extract, which may be related to the higher content of polyphenols and flavonoids in alcohol extract.

[0099] Example 15: Protective effect of lupins against cyclophosphamide-induced reproductive toxicity in mice.

[0100] Objective: To investigate the protective effect of lupins against reproductive toxicity induced by cyclophosphamide, another commonly used chemotherapy drug.

[0101] Experimental Methods: Thirty male ICR mice were randomly divided into three groups of 10 mice each. The normal control group received intraperitoneal injections of saline solution daily, followed by oral administration of saline solution daily. The model control group received intraperitoneal injections of cyclophosphamide 50 mg / kg daily for 5 consecutive days, followed by oral administration of saline solution daily. The lupin group received intraperitoneal injections of cyclophosphamide 50 mg / kg daily for 5 consecutive days, followed by oral administration of lupin powder 100 mg / kg daily. Lupin administration began 3 days before cyclophosphamide injection and continued for 4 weeks after the cyclophosphamide injection ended. Sperm parameters were measured after the last administration.

[0102] The experimental results are shown in Table 5.

[0103] Table 5. Effects of lupins on sperm parameters in cyclophosphamide-induced model mice (n=10, ± s)

[0104] Group Sperm concentration ( / mL) Sperm motility (%) Sperm deformity rate (%) normal control group 30.2 ± 4.2 75.5 ± 5.8 8.2 ± 1.5 Model control group 12.5 ± 3.5 35.2 ± 6.5 28.5 ± 4.2 Lupin Group 25.8 ± 4.5 65.8 ± 5.2 12.5 ± 2.8

[0105] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0106] Experimental conclusion: Lupin also has a protective effect against cyclophosphamide-induced reproductive toxicity, significantly increasing sperm concentration and motility and reducing sperm abnormality rate, suggesting that the reproductive protective effect of lupin is broad-spectrum and can be used for the protection against reproductive toxicity induced by various chemotherapy drugs.

[0107] Example 16: Preliminary Study on the Mechanism of Action of Lupinus

[0108] Experimental objective: To preliminarily explore the mechanism by which lupin protects against the reproductive toxicity of chemotherapy drugs.

[0109] Experimental Methods: Based on Example 12, the following detection indicators were added: Antioxidant enzyme activity in testicular tissue: The activities of SOD, CAT, GSH-Px, and the content of MDA in testicular tissue homogenate were measured. Inflammatory factors in testicular tissue: The contents of TNF-α, IL-6, and IL-1β in testicular tissue homogenate were measured using an ELISA kit. Apoptosis-related proteins in testicular tissue: The expression of Bax, Bcl-2, and Caspase-3 proteins was detected using Western blot.

[0110] Experimental results:

[0111] The antioxidant indices of testicular tissue are shown in Table 6.

[0112] Table 6. Effects of lupin on antioxidant indices in the testes of bucephalan model mice (n=10, ± s)

[0113] Group SOD (U / mg prot) CAT (U / mg prot) GSH-Px (U / mg prot) MDA (nmol / mg prot) normal control group 85.6 ± 8.5 42.5 ± 5.2 125.8 ± 12.5 2.85 ± 0.45 Model control group 45.2 ± 6.8 22.8 ± 4.5 68.5 ± 8.2 8.52 ± 1.25 High-dose lupin group 78.5 ± 7.5 38.6 ± 4.8 112.5 ± 10.8 3.56 ± 0.52

[0114] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0115] Inflammatory factors in testicular tissue are shown in Table 7.

[0116] Table 7. Effects of lupin on testicular inflammatory factors in bukurine-induced model mice (n=10, ± s)

[0117] Group TNF-α (pg / mg prot) IL-6 (pg / mg prot) IL-1β (pg / mg prot) normal control group 25.6 ± 4.2 18.5 ± 3.5 12.8 ± 2.5 Model control group 85.2 ± 10.5 65.8 ± 8.5 48.5 ± 6.2 High-dose lupin group 35.8 ± 5.5 28.5 ± 4.8 18.5 ± 3.2

[0118] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0119] Apoptosis-related protein expression: In the model control group, Bax expression was significantly upregulated, Bcl-2 expression was significantly downregulated, the Bax / Bcl-2 ratio was increased, and Caspase-3 expression was increased in testicular tissue. The high-dose lupin group reversed the above changes, reduced Bax expression, increased Bcl-2 expression, decreased the Bax / Bcl-2 ratio, and inhibited Caspase-3 expression.

[0120] Experimental conclusions: The mechanism by which lupin protects against the reproductive toxicity of chemotherapy drugs may involve the following aspects: enhancing the antioxidant capacity of testicular tissue, increasing the activity of SOD, CAT, and GSH-Px, and reducing MDA levels; inhibiting the inflammatory response of testicular tissue, and reducing the expression of inflammatory factors such as TNF-α, IL-6, and IL-1β; inhibiting spermatogenic cell apoptosis, regulating the Bax / Bcl-2 balance, and inhibiting Caspase-3 activation.

[0121] Example 17: The effect of lupin administration timing

[0122] Experimental objective: To compare the effects of different administration times (prophylactic and therapeutic administration) on the protective effect of lupins.

[0123] Experimental Methods: Forty male ICR mice were randomly divided into four groups of 10 mice each. The normal control group received a single intraperitoneal injection of saline solution followed by daily gavage. The model control group received a single intraperitoneal injection of busulfan 20 mg / kg followed by daily gavage. The prevention group received 100 mg / kg lupin powder via gavage starting one week before busulfan injection and continuing for 6 weeks. The treatment group received 100 mg / kg lupin powder via gavage starting on the day of busulfan injection and continuing for 5 weeks. Sperm parameters were measured after the last administration.

[0124] The experimental results are shown in Table 8.

[0125] Table 8. Effects of different administration times on the protective effect of lupin (n=10, ± s)

[0126] Group Sperm concentration ( / mL) Sperm motility (%) normal control group 30.5 ± 4.5 76.2 ± 5.5 Model control group 8.8 ± 2.2 24.8 ± 6.5 Prevention Group 29.2 ± 4.2 73.5 ± 5.2 Treatment group 25.5 ± 4.8 68.2 ± 5.8

[0127] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0128] Experimental conclusion: Prophylactic administration (starting before chemotherapy) has a better protective effect than therapeutic administration (starting at the same time as chemotherapy). It is recommended to start taking lupin preparations before chemotherapy to obtain the best reproductive protection effect.

[0129] Example 18 Safety evaluation of long-term administration of lupins

[0130] Experimental objective: To evaluate the safety of long-term administration of lupins.

[0131] Experimental Methods: Thirty male ICR mice were randomly divided into three groups of 10 each. The control group received daily oral saline; the low-dose group received daily oral lupin powder (100 mg / kg, recommended dose); and the high-dose group received daily oral oral lupin powder (500 mg / kg, 5 times the recommended dose). Treatment continued for 12 weeks. Body weight was recorded weekly, and complete blood count and blood biochemical indicators were measured every 4 weeks. Pathological examinations of major organs were performed at the end of the experiment.

[0132] Experimental Results: Mice in all groups maintained good mental condition, normal diet and activity levels, and no abnormal deaths occurred during the experiment. There was no significant difference in body weight gain among the groups (P>0.05). Complete blood count (WBC, RBC, HGB, PLT) and blood biochemical indicators (ALT, AST, BUN, Cr) were all within the normal range, with no significant differences among the groups (P>0.05). Pathological examination showed that the structures of major organs such as the heart, liver, spleen, lungs, and kidneys were normal in all groups, with no obvious pathological changes.

[0133] Experimental conclusion: Lupinus has good safety profile after long-term administration (12 weeks), with no obvious toxic reactions observed at 5 times the recommended dose. This suggests that lupinus has high safety as an edible plant material and is suitable as a raw material for long-term health food or adjuvant drugs.

[0134] Example 19: Interaction between lupins and chemotherapy drugs

[0135] Experimental objective: To investigate whether lupin affects the antitumor activity of busulfan.

[0136] Experimental Methods: A mouse model of L1210 leukemia was established. Forty mice were randomly divided into four groups of ten mice each. The control group received intraperitoneal injection of saline; the tumor control group received L1210 leukemia cells but no treatment; the busulfan group received L1210 leukemia cells and intraperitoneal injection of busulfan 10 mg / kg once daily for five consecutive days; the combined group received L1210 leukemia cells, intraperitoneal injection of busulfan 10 mg / kg, and gavage administration of lupin powder 100 mg / kg. The survival time of mice in each group was observed, and the life extension rate was calculated.

[0137] Results: The mean survival time in the tumor control group was (12.5 ± 1.8) days; the mean survival time in the busulfan group was (22.8 ± 2.5) days, with a life extension rate of 82.4%; and the mean survival time in the combined group was (23.2 ± 2.8) days, with a life extension rate of 85.6%. There was no significant difference in survival time between the busulfan group and the combined group (P>0.05).

[0138] Experimental conclusion: The combined use of lupins did not affect the antitumor activity of busulfan, suggesting that lupins can be safely used in combination with chemotherapy drugs, protecting reproductive function without affecting the efficacy of chemotherapy.

[0139] Example 20 Comparison of different lupin varieties

[0140] Objective: To compare the protective effects of different lupin varieties against the reproductive toxicity of chemotherapy drugs.

[0141] Experimental Methods: Whole lupin powders from sweet lupin (Jenabillup variety), narrow-leaved lupin, white lupin, and yellow lupin were used in the experiment. Fifty male ICR mice were randomly divided into 5 groups (n=10 per group). The model control group received a single intraperitoneal injection of busulfan 20 mg / kg, followed by daily gavage with saline. The sweet lupin group received a single intraperitoneal injection of busulfan 20 mg / kg, followed by daily gavage with 100 mg / kg sweet lupin powder. The narrow-leaved lupin group received a single intraperitoneal injection of busulfan 20 mg / kg, followed by daily gavage with 100 mg / kg narrow-leaved lupin powder. The white lupin group received a single intraperitoneal injection of busulfan 20 mg / kg, followed by daily gavage with 100 mg / kg white lupin powder. The yellow lupin group received a single intraperitoneal injection of busulfan 20 mg / kg, followed by daily gavage with 100 mg / kg yellow lupin powder. Sperm parameters were measured after 5 weeks of continuous administration.

[0142] The experimental results are shown in Table 9.

[0143] Table 9. Effects of different lupin varieties on sperm parameters in bukhart model mice (n=10, ± s)

[0144] Group Sperm concentration ( / mL) Sperm motility (%) Model control group 9.5 ± 2.5 26.8 ± 6.5 Sweet Lupin Group 28.8 ± 4.5 72.5 ± 5.5 Section Narrow-leaved Lupinus 26.5 ± 4.2 68.8 ± 5.8 White Lupin Group 24.2 ± 4.5 65.2 ± 6.2 Yellow Lupin Group 22.8 ± 4.8 62.5 ± 6.5

[0145] Note: Different superscript letters in the table indicate significant differences between groups (P<0.05).

[0146] Experimental conclusion: Different lupin varieties all exhibited protective effects against busulfan-induced reproductive toxicity, with sweet lupin (Jenabillup variety) showing the best effect, possibly due to its low alkaloid content and high polyphenol content. It is recommended to prioritize sweet lupin varieties for the preparation of drugs that alleviate chemotherapy-induced reproductive toxicity.

[0147] The lupin or its extract of the present invention can effectively reduce the reproductive toxicity induced by chemotherapy drugs, and its mechanism of action involves synergistic effects at multiple levels.

[0148] From an antioxidant perspective, chemotherapy drugs such as busulfan can induce oxidative stress damage in testicular tissue by generating large amounts of reactive oxygen species (ROS). Lupins are rich in polyphenolic compounds (such as luteolin, apigenin, and isoflavones) and flavonoids, which have significant free radical scavenging capabilities and metal ion chelating effects, effectively capturing reactive oxygen species such as superoxide anions, hydroxyl radicals, and hydrogen peroxide generated during the metabolism of chemotherapy drugs. Experimental results show that lupin administration can significantly increase the activity of antioxidant enzymes such as SOD, CAT, and GSH-Px in testicular tissue and reduce the level of lipid peroxidation product MDA, indicating that lupins can counteract oxidative damage caused by chemotherapy drugs by enhancing the function of the endogenous antioxidant system.

[0149] From an anti-inflammatory perspective, chemotherapy drugs can activate inflammatory signaling pathways in testicular tissue, inducing the massive release of inflammatory factors such as TNF-α, IL-6, and IL-1β, further exacerbating tissue damage. Isoflavones in lupins possess phytoestrogenic activity and can regulate the expression of inflammation-related genes; polyphenols can inhibit NF-κB signaling pathway activation and reduce the production of pro-inflammatory factors. The experimental results of this invention confirm that lupin administration can significantly reduce the level of inflammatory factors in testicular tissue, suggesting that its anti-inflammatory effect is one of the important mechanisms for protecting reproductive function.

[0150] From a cell protection perspective, chemotherapy drugs can directly damage testicular Sertoli cells and spermatogenic cells, inducing apoptosis. Lupins can protect spermatogenic cells from chemotherapy drug damage by regulating the Bax / Bcl-2 balance, inhibiting the expression of pro-apoptotic proteins, upregulating the expression of anti-apoptotic proteins, blocking the mitochondrial-mediated apoptosis pathway, and reducing Caspase-3 activation. Furthermore, the high-quality protein and essential amino acids in lupins can provide sufficient nutrients for the repair of damaged cells, promoting the structural and functional recovery of testicular tissue.

[0151] From an endocrine regulation perspective, chemotherapy drugs can interfere with the hypothalamic-pituitary-gonadal axis, leading to abnormal levels of hormones such as testosterone and FSH. Lupin administration can improve serum testosterone and FSH levels, suggesting that it may support spermatogenesis by regulating endocrine function.

[0152] In summary, lupin exerts a protective effect against chemotherapy-induced reproductive toxicity through a synergistic effect of multiple mechanisms, including antioxidant, anti-inflammatory, anti-apoptotic, and endocrine-regulating mechanisms. This multi-target, multi-pathway mode of action makes lupin a promising protective agent against chemotherapy-induced reproductive toxicity.

[0153] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. The application of lupin or its extract in the preparation of drugs to reduce the reproductive toxicity of chemotherapy drugs, wherein, The lupins are selected from plants of the genus Lupinus.

2. The application according to claim 1, characterized in that, The lupins are selected from one or more of sweet lupins, narrow-leaved lupins, white lupins, or yellow lupins, with the sweet lupin variety Jenabillup being the preferred choice.

3. The application according to claim 1, characterized in that, The chemotherapy drug is one or more of busulfan, cyclophosphamide, or cisplatin.

4. The application according to claim 1, characterized in that, The extract is a water extract, an alcohol extract, an enzymatic extract, or a supercritical CO2 extract.

5. The application according to claim 1, characterized in that, The reproductive toxicity includes one or more of the following: decreased sperm concentration, decreased sperm motility, testicular tissue damage, or apoptosis of spermatogenic epithelial cells.

6. The application according to any one of claims 1 to 5, characterized in that, The dosage of the lupin or its extract is 0.1-100 mg / kg body weight, preferably 50-100 mg / kg body weight.

7. The application according to claim 6, characterized in that, The dosing cycle is at least 5 weeks of continuous administration, with a dosing frequency of once daily.

8. The application according to any one of claims 1 to 5, characterized in that, The dosage form of the drug is oral liquid, capsule, tablet, granule, powder, or functional food.

9. The application according to any one of claims 1 to 5, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

10. The application according to any one of claims 1 to 5, characterized in that, The drug is used concurrently with chemotherapy drugs or is started before chemotherapy begins.