Litchi seed extract, preparation method and application thereof

Abstract

The application discloses a litchi kernel extract, a preparation method thereof and a medicine for preventing and / or treating prostatitis, and is prepared from litchi kernels through the following steps of ethanol refluxing, concentrating, n-butanol extraction, macroporous resin adsorption and ethanol gradient elution. The litchi kernel extract can reduce the release level of inflammatory factors, treat prostatitis and provide a new idea and method for treating prostatitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a litchi seed extract, its preparation method, and a drug for the prevention and / or treatment of prostatitis. Background Technology

[0002] Prostatitis is a common disease in adult men, with a lifetime prevalence of 1.8% to 8.2%. Its etiology is complex, causing a wide range of signs and symptoms, such as urogenital pain, lower urinary tract symptoms (urinary or urinary retention symptoms), psychological problems, and sexual dysfunction, significantly impacting men's quality of life. Prostatitis is mainly classified into four types: Type I acute bacterial prostatitis; Type II chronic bacterial prostatitis; Type III prostatodynia, also known as chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS); and Type IV non-bacterial prostatitis. CP / CPPS accounts for 90% of all prostatitis cases, and its etiology is complex and not fully understood. Etiological factors include inflammation-mediated abnormal pelvic floor neuromuscular activity, lower urinary tract epithelial cell dysfunction, and immune abnormalities.

[0003] Currently, common treatments for prostatitis include: quinolone antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), and physical therapy (using microwave, radiofrequency, and other technologies to enhance antibacterial efficacy and relieve pain). Antibiotic therapy is less effective because the prostate-blood barrier between the prostate acini and microcirculation hinders the passage of water-soluble antibiotics, significantly reducing treatment efficacy. Furthermore, long-term antibiotic use can easily induce serious side effects such as pseudomembranous colitis, diarrhea, and the growth of drug-resistant intestinal bacteria. Antibiotic therapy is also ineffective for patients with nonbacterial prostatitis. Anti-inflammatory and analgesic drugs, along with physical therapy, can only serve as adjunctive treatments to reduce inflammation and relieve pain; they do not provide a cure.

[0004] The seed of the litchi (Litchi chinensis Sonn.), a plant in the Sapindaceae family, is a dried, mature seed. It is associated with the liver and kidney meridians and is believed to have the effects of promoting qi circulation, dispersing stagnation, dispelling cold, and relieving pain. It can be used to treat abdominal pain due to cold hernia and testicular swelling. Records of its cultivation in China date back to the Han Dynasty over 2000 years ago, and it is known as the "King of Fruits." Globally, the litchi industry has significant economic value, accounting for a large proportion of the total agricultural output. However, the seeds, leaves, and peel of the litchi tree are usually discarded. Studies have shown that they are rich in saponins, flavonoids, and polyphenols, possessing significant medicinal value. Litchi seed extract exhibits a wide range of pharmacological activities, such as anti-inflammatory, antiviral, antioxidant, and antitumor effects. However, research on the effects of litchi seed extract on prostatitis is scarce. Therefore, studying the therapeutic effects of litchi seed extract on prostatitis is of great significance for exploring new methods for treating prostatitis and expanding the application scope of litchi seeds. Summary of the Invention

[0005] To address the aforementioned issues with prostatitis medications and to expand new ideas and methods for prostatitis treatment, this invention provides the following technical solution.

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

[0007] (1) The litchi seeds were crushed, extracted by ethanol reflux, and concentrated to obtain an extract;

[0008] (2) The extract was extracted with n-butanol to obtain n-butanol extract;

[0009] (3) Dissolve the n-butanol extract in water, adsorb it through a macroporous resin and elute it with an ethanol gradient, collect the elution product with 30%-70% ethanol, and remove the ethanol to obtain the extract.

[0010] Preferably, the ethanol concentration in step (1) is ≥70%, for example: 70%, 75%, 80%, 85%, 90%, 95%, 100%.

[0011] Furthermore, the ethanol is refluxed 1-3 times, for example: once, twice, or three times.

[0012] Furthermore, the ethanol reflux time is 1-2 hours per cycle, for example: 1 hour, 1.5 hours, or 2 hours.

[0013] Preferably, the gradient elution in step (3) includes elution with water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol.

[0014] Preferably, the macroporous resin is D101 macroporous resin.

[0015] Preferably, the extract is a product eluted with 30% ethanol, 50% ethanol, or 70% ethanol, and more preferably a product eluted with 30% ethanol.

[0016] In one specific embodiment of the present invention, the preparation method of the litchi seed extract is as follows: weigh litchi seed medicinal material, crush it, and extract it three times by reflux with 70% ethanol, each time for 1.5 hours. Combine the filtrates, concentrate under reduced pressure, and recover the ethanol until it is tasteless to obtain litchi seed extract. Disperse the extract with water, extract it three times with n-butanol, recover the organic solvent, dissolve it with a small amount of water, and use D101 macroporous adsorption resin to perform gradient elution with water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol. Recover the solvent under reduced pressure to obtain the product eluted with 30% ethanol.

[0017] In a second aspect, the present invention provides a litchi seed extract, which is prepared according to the preparation method described in the first aspect.

[0018] Thirdly, the present invention provides a medicament for the prevention and / or treatment of prostatitis, comprising the litchi seed extract described in the second aspect.

[0019] Preferably, the dosage of the prostatitis drug is 25-100 mg / kg / day, for example: 25 mg / kg / day, 30 mg / kg / day, 40 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day, 70 mg / kg / day, 80 mg / kg / day, 90 mg / kg / day, 100 mg / kg / day.

[0020] Preferably, the drug can be in any dosage form or administration method, especially oral dosage forms, which can be selected by those skilled in the art as appropriate, including, but not limited to, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, oral tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, solutions, emulsions, suspensions, controlled-release formulations (e.g., instantaneous-release formulations, sustained-release formulations, sustained-release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa-adhesive films), injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous infusions, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories (e.g., rectal suppositories, vaginal suppositories), nasal preparations, pulmonary preparations (inhalers), drops, etc.

[0021] Preferably, the drug further includes pharmaceutically permissible excipients.

[0022] Preferably, the pharmaceutically permissible excipients include sweeteners (specifically, sucrose, xylitol, fructooligosaccharides, cyclamate, stevia, aspartame, etc.), flavoring agents (such as fragrances, flavorings, etc.), gelling agents (specifically, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, etc.), clarifying agents (specifically, chitosan, gelatin, etc.), preservatives (specifically, benzoic acid and its salts, sorbic acid and its salts, parabens, etc.), disintegrants (specifically, low-substituted hydroxypropyl cellulose, crospovidone, sodium carboxyacetic acid starch, croscarmellose sodium, starch, etc.), and binders (specifically, hydroxypropyl cellulose, hydroxypropyl methylcellulose). The ingredients include: povidone, copovidone, pregelatinized starch, etc.; lubricants (such as stearic acid, magnesium stearate, sodium fumarate stearate, etc.); wetting agents (such as polyoxyethylene sorbitan fatty acid ester, poloxamer, polyoxyethylene castor oil derivatives, etc.); suspending agents (such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, copovidone, sodium carboxymethyl cellulose, methylcellulose, etc.); stabilizers (such as citric acid, fumaric acid, succinic acid, etc.); fillers (such as starch, sucrose, lactose, microcrystalline cellulose, etc.); and binders (such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone, etc.).

[0023] Fourthly, the present invention provides the use of the litchi seed extract in the preparation of a drug for the prevention and / or treatment of prostatitis.

[0024] Preferably, the prostatitis drug reduces the expression of TNF-α, IL-6, PGE2 and p-p65 proteins in prostatitis tissue.

[0025] Preferably, the prostatitis drug regulates the NF-κB signaling pathway and / or the MAPK signaling pathway.

[0026] Furthermore, the prostatitis drug inhibits the expression of p-IKK-α / β and p-IκBα proteins and upregulates the expression of IκBα protein.

[0027] Furthermore, the prostatitis drug inhibits the expression of p-ERK, p-p38, and p-JNK proteins.

[0028] The beneficial effects of this invention are:

[0029] The litchi seed extract of this invention can treat prostatitis by regulating the NF-κB and MAPK signaling pathways, reducing the phosphorylation level of related target proteins in the signaling pathways, and decreasing the release level of inflammatory factors, thus providing a new approach and method for the treatment of prostatitis. At the same time, this invention expands the application scope of litchi seeds and increases the economic value of litchi. Attached Figure Description

[0030] Figure 1 The diagram shows the preparation process of drugs for prostatitis.

[0031] Figure 2 The figure shows the effects of LCS2, LCS3, and LCS4 on LPS-induced NO secretion in RAW264.7 cells, where A represents LCS2, B represents LCS3, and C represents LCS4.

[0032] Figure 3 The diagram shows the rat model of prostatitis and the method of drug administration.

[0033] Figure 4 The image shows the effect of LCS2 on the complete blood count of rats, where A represents the white blood cell count, B represents the neutrophil count, and C represents the percentage of neutrophils.

[0034] Figure 5 The figure shows the effect of LCS2 on inflammatory factors in rats, where A represents the level of TNF-α in rat serum, B represents the level of IL-6 in rat serum, C represents the level of PGE2 in rat serum, D represents the level of PSA in rat serum, E represents the level of TNF-α in rat prostate tissue, F represents the level of IL-6 in rat prostate tissue, G represents the level of PGE2 in rat prostate tissue, and H represents the level of PSA in rat prostate tissue.

[0035] Figure 6 The image shows the HE staining results of rat prostate tissue, where A represents the prostate HE staining image (20X) and B represents the inflammation score;

[0036] Figure 7 The image shows the IHC staining results (20X) of rat prostate tissue, where A represents TNF-α expression in prostate tissue, B represents IL-6 expression in prostate tissue, and C represents PGE2 expression in prostate tissue.

[0037] Figure 8 The image shows the HE staining (10X) results of rat heart, liver, spleen, lung, and kidney tissues;

[0038] Figure 9 The figure shows the effect of LCS2 on the expression of p-p65 protein in rat prostate tissue. A represents p-p65 protein expression, B represents statistical analysis of p-p65 protein, and C represents immunohistochemical detection of p-p65 expression in prostate tissue (20X).

[0039] Figure 10 The figure shows the effect of LCS2 on the expression of COX-2 and iNOS proteins in rat prostate tissue, where A represents the expression of COX-2 and iNOS proteins, and B represents the statistical analysis results of COX-2 and iNOS proteins.

[0040] Figure 11 The figure shows the effect of LCS2 on the expression of NF-κB signaling pathway proteins in rat prostate tissue, where A represents the expression of NF-κB signaling pathway proteins and B represents the statistical analysis results of NF-κB signaling pathway proteins.

[0041] Figure 12 The figure shows the effect of LCS2 on the expression of MAPK signaling pathway proteins in rat prostate tissue, where A represents the expression of MAPK signaling pathway proteins and B represents the statistical analysis results of MAPK signaling pathway proteins.

[0042] In the above figures # P<0.05, ## P<0.01, ### P<0.001 vs. blank control group; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group (n = 7). Detailed Implementation

[0043] 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.

[0044] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0045] Example 1: Extraction of litchi seeds and separation and identification of the extract

[0046] according to Figure 1 The technical route shown is used to extract litchi seeds, and the specific process is as follows:

[0047] 1. Weigh 15 kg of litchi seed medicinal material, crush it, and extract it three times with 70% ethanol under reflux for 1.5 hours each time. Combine the filtrates, concentrate under reduced pressure, and recover the ethanol until it is tasteless to obtain litchi seed extract. Disperse the extract with water, and extract it three times with n-butanol. After recovering the organic solvent, obtain the n-butanol fraction (326 g).

[0048] 2. Take 200g of n-butanol fraction sample and dissolve it in a small amount of pure water. Use D101 macroporous adsorption resin and perform gradient elution with pure water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol. Recover the solvent under reduced pressure to obtain 5 fractions: LCS1 (pure water), LCS2 (30% ethanol), LCS3 (50% ethanol), LCS4 (70% ethanol) and LCS5 (95% ethanol).

[0049] Example 2 Screening of anti-inflammatory activity of litchi seed extract

[0050] The anti-inflammatory activities of LCS2, LCS3, and LCS4 were evaluated using the Griess reagent kit. RAW264.7 cells were pre-treated with different concentrations of LCS2, LCS3, and LCS4 for 2 hours, followed by the addition of LPS to induce an inflammatory response in the cells for 18 hours. Results are shown below. Figure 2 .

[0051] Depend on Figure 2 It can be seen that LCS2 can significantly reduce the content of Nitrite and has an anti-inflammatory effect, while the anti-inflammatory effects of LCS3 and LCS4 are not obvious. Therefore, LCS2 was selected for subsequent cell and animal experiments.

[0052] Example 3: The effect of LCS2 in treating prostatitis in rats

[0053] according to Figure 3 The process for constructing a rat prostatitis model is as follows:

[0054] After 7 days of acclimatization, 60 rats were randomly divided into 6 groups (n=10 per group) according to their body weight: normal control group, LPS model control group (3 mg / kg), low-dose LCS2 group (25 mg / kg), medium-dose LCS2 group (50 mg / kg), high-dose LCS2 group (100 mg / kg), and positive control group for tamsulosin hydrochloride sustained-release capsules (THSRC) (0.023 mg / kg). Body weight was recorded daily before administration, and rat diet was controlled, but water intake was not restricted. LCS2 and THSRC were dissolved in physiological saline and administered to SD rats by gavage once daily for 7 days. The normal control group and model group were administered an equal volume of physiological saline by gavage at corresponding time points. After 7 days, the blank control group received no treatment, while the model group, the three LCS2 dose groups, and the positive control group were all intraperitoneally injected with LPS (3 mg / kg) to stimulate an inflammatory response.

[0055] Eight hours after LPS induction, rats were generally anesthetized by injection of sodium pentobarbital, and blood samples were collected from the abdominal aorta for blood routine tests and inflammatory factor determination. The rats were then sacrificed, and prostate tissue samples were collected. One portion was fixed with 4% paraformaldehyde and stored at room temperature for histological section staining and observation. The other portion was cryopreserved at -80°C for Western blotting and ELISA analysis. Simultaneously, heart, liver, spleen, lung, and kidney tissues from the rats were collected, fixed with 4% paraformaldehyde, stored at room temperature, and histological section staining and observation were performed to assess the safety of LCS2.

[0056] Blood routine test results as follows Figure 4As shown, compared with the blank control group, the LPS model group rats had significantly increased white blood cell count, neutrophil count, and neutrophil percentage. Compared with the LPS model group, the treatment groups (LCS2 25, 50, 100 mg / kg) and the THSRC positive control group had significantly reduced white blood cell count, neutrophil count, and neutrophil percentage in their blood. This indicates that LCS2 can significantly reduce the release of white blood cells and neutrophils related to the inflammatory response and the percentage of neutrophils in whole blood of rats stimulated by LPS.

[0057] Results of inflammatory factor assays in rat blood and prostate tissue: Figure 5 As shown, compared with the normal group, the levels of inflammatory factors TNF-α, IL-6, PGE2, and PSA in the blood and prostate tissue of rats in the LPS model group were significantly increased, indicating that the inflammation model was successfully established. Compared with the LPS group, after administration of different doses of LCS2 and THSRC positive control drug, the levels of TNF-α, IL-6, PGE2, and PSA in the blood and prostate tissue of rats were significantly reduced in a dose-dependent manner. This indicates that LCS2 has a significant inhibitory effect on inflammatory factors in the serum and prostate tissue of inflamed rats.

[0058] Results of rat prostate tissue sections Figure 6 As shown, in the normal group, the prostate gland ducts of rats contained a large amount of dark red secretions, with no obvious inflammatory cell infiltration in the interstitium, and no pathological changes of inflammation were observed in the tissue; the tissue morphology and structure were normal. In the LPS model group, the dark red secretions in the prostate gland ducts of rats disappeared, the duct structure was destroyed, and a large number of inflammatory cells infiltrated the interstitium, indicating significant damage to the prostate tissue structure. This indicates that the LPS-induced rat inflammation model has been successfully established. Compared with the model group, the number of inflammatory cells and neutrophils in the prostate interstitium of the LCS2 (25, 50, 100 mg / kg) group and the THSRC positive control group was significantly reduced, and dark red secretions could be seen in the ducts; the prostate tissue morphology and structure were normal. This indicates that LCS2 can alleviate the damage to the prostate tissue of inflamed rats, and its effect is comparable to that of tamsulosin hydrochloride sustained-release capsules as a positive control.

[0059] Immunohistochemical results of rat prostate tissue as follows Figure 7 As shown, the release levels of inflammatory factors TNF-α, IL-6, and PGE2 proteins in the prostate tissue of rats in the LPS model group were significantly increased compared with those in the control group. After treatment with LCS2 (25, 50, 100 mg / kg) and THSRC positive control drug, the expression of TNF-α, IL-6, and PGE2 proteins in the tissue was significantly reduced compared with the model group. This indicates that LCS2 has a significant inhibitory effect on inflammatory factors in the prostate tissue of inflamed rats.

[0060] HE staining results of rat organ tissue sections are as follows: Figure 8 As shown, the heart, liver, spleen, lungs, and kidneys of the rats in the LCS2 group were intact and not damaged, indicating the safety of LCS2.

[0061] The expression results of p-p65 protein in rat prostate tissue are as follows: Figure 9 As shown, compared with the normal group, the expression of p-p65 protein in the prostate tissue of rats in the LPS model group was significantly increased. Compared with the model group, the expression of p-p65 protein in the THSRC positive control group and the LCS2 group was significantly decreased after treatment. This indicates that LCS2 can inhibit the expression of p-p65 protein in rat prostate tissue.

[0062] The expression results of COX-2 and iNOS proteins in rat prostate tissue are as follows: Figure 10 As shown, compared with the normal group, the expression levels of COX-2 and iNOS proteins in the prostate tissue of the LPS group were significantly increased, indicating that the inflammation model was successfully established. Compared with the model group, both the LCS2 treatment group and the THSRC positive control group significantly inhibited the expression levels of COX-2 and iNOS proteins in the prostate tissue. This indicates that LCS2 can inhibit the expression of COX-2 and iNOS proteins in LPS-stimulated rat prostate tissue.

[0063] The results of NF-κB signaling pathway protein expression in rat prostate tissue are as follows: Figure 11 As shown, compared with the normal group, the expression levels of p-IKK-α / β and p-IκBα proteins in the prostate tissue of rats in the LPS group were significantly increased, while the expression of IκBα protein was significantly decreased. Compared with the model group, the expression of p-IKK-α / β and p-IκBα proteins in prostate tissue was significantly inhibited under THSRC positive drug and LCS2 treatment, but the expression of total IKKα and IKKβ proteins was not affected, while the expression of IκBα protein was significantly upregulated. This indicates that LCS2 can regulate the phosphorylation level of related proteins by modulating the NF-κB signaling pathway, thereby improving the LPS-induced inflammatory response in rats.

[0064] The results of MAPK signaling pathway protein expression in rat prostate tissue are as follows: Figure 12 As shown, compared with the control group, the expression of p-ERK, p-p38, and p-JNK proteins in the prostate tissue of rats in the LPS model group was significantly increased in a dose-dependent manner. Compared with the model group, both the THSRC positive control group and the LCS2 group significantly reduced the expression of p-ERK, p-p38, and p-JNK proteins in the prostate tissue, but the expression of total p38, JNK, and ERK proteins was unaffected. This indicates that LCS2 can also exert an anti-inflammatory effect in the LPS-constructed inflammation model by regulating the phosphorylation level of key proteins through the MAPK signaling pathway, which is closely related to inflammation.

[0065] 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 a litchi seed extract in the preparation of a medicament for the prevention and / or treatment of prostatitis, characterized in that: The preparation method of the litchi seed extract includes the following steps: (1) The litchi seeds were crushed, extracted by reflux with ethanol of ≥70% concentration, and concentrated to obtain an extract; (2) The extract was extracted with n-butanol to obtain n-butanol extract; (3) Dissolve the n-butanol extract in water, adsorb it through a macroporous resin, elute it with an ethanol gradient, collect the elution product with 30% ethanol, and remove the ethanol to obtain the extract.

2. The application according to claim 1, characterized in that: The ethanol in step (1) is refluxed 1-3 times, with a reflux time of 1-2 hours per reflux.

3. The application according to claim 1, characterized in that: The gradient elution in step (3) includes gradient elution with water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol.

4. The application according to claim 1, characterized in that: The macroporous resin is D101 macroporous resin.

5. The application according to claim 3, characterized in that: Weigh the litchi seed medicinal material, crush it, and extract it three times with 70% ethanol under reflux for 1.5 hours each time. Combine the filtrates, concentrate under reduced pressure, and recover the ethanol until it is tasteless to obtain litchi seed extract. Disperse the extract with water, extract it three times with n-butanol, recover the organic solvent, dissolve it with a small amount of water, and use D101 macroporous adsorption resin to perform gradient elution with water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol. Recover the solvent under reduced pressure to obtain the product eluted with 30% ethanol.

6. A drug for the prevention and / or treatment of prostatitis, characterized in that: The drug is made from litchi seed extract and pharmaceutically permissible excipients; The preparation method of the litchi seed extract includes the following steps: (1) The litchi seeds were crushed, extracted by reflux with ethanol of ≥70% concentration, and concentrated to obtain an extract; (2) The extract was extracted with n-butanol to obtain n-butanol extract; (3) Dissolve the n-butanol extract in water, adsorb it through a macroporous resin, elute it with an ethanol gradient, collect the elution product with 30% ethanol, and remove the ethanol to obtain the extract.

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