A traditional Chinese medicine compound composition for protecting sperm DNA integrity, and a preparation method and application thereof

Abstract

This invention discloses a traditional Chinese medicine compound composition for protecting sperm DNA integrity, its preparation method, and its application. The composition comprises the following components by mass: 10-30 parts Codonopsis pilosula, 10-20 parts Citrus reticulata, 10-30 parts Taraxacum mongolicum, 10-30 parts Agrimonia pilosa, 10-30 parts Astragalus membranaceus, 10-30 parts Rhodiola rosea, 6-10 parts Astragalus complanatus, 6-10 parts Morus alba, 6-10 parts Schisandra chinensis, 6-10 parts Curcuma zedoaria, 6-10 parts Carthamus tinctorius, and 6-10 parts Citrus reticulata seed. This traditional Chinese medicine compound composition can tonify the spleen and kidneys, clear heat and detoxify, invigorate blood circulation and regulate qi, and balance the yin and yang of kidney essence. It effectively protects sperm DNA integrity and reduces the increase in sperm DNA fibrillation (DFI) caused by sperm DNA damage, demonstrating definite clinical efficacy. The traditional Chinese medicine compound composition of this invention is convenient to take in clinical use, has good patient tolerance, and no adverse reactions have been found with long-term oral administration. After being made into a standardized traditional Chinese medicine preparation, it can further promote the standardized treatment and use of traditional Chinese medicine.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine compound formulas, specifically relating to a traditional Chinese medicine compound formula composition for protecting sperm DNA integrity and its preparation method. Background Technology

[0002] The World Health Organization (WHO) lowered its minimum standards for male semen quality in 1990 and 2010, respectively. Influenced by environmental pollution (PM2.5, formaldehyde, benzene, and other organic chemicals), food safety (food additives), unhealthy lifestyle habits (smoking, drinking, prolonged sitting, sauna use, etc.), high temperatures, and ionizing radiation, the incidence of male infertility is rising annually. Traditional assessments of male fertility include semen analysis and sperm abnormality rate. Semen analysis parameters reflect sperm concentration, quantity, motility, and liquefaction time, while sperm abnormality rate parameters reflect sperm morphological integrity. However, neither can reveal the integrity of the genetic material (DNA) carried by sperm. Clinical studies show that approximately 15% of men of reproductive age have normal semen analysis and sperm abnormality rate results but are still unable to conceive, which is closely related to low DNA integrity in sperm. Sperm DNA is the sole carrier of paternal genetic material. The integrity of sperm nuclear DNA and mid-sperm mitochondrial DNA not only reflects the macroscopic function and morphology of sperm but also reveals sperm quality at the microscopic level, playing a crucial role in offspring health. The applicant has conducted extensive clinical and basic research in the early stages. When male sperm is affected by endogenous and exogenous factors, resulting in DNA single-strand and double-strand breaks, a significantly elevated sperm DNA fragmentation index (DFI) and a decreased sperm mitochondrial membrane potential (MMP) can be rapidly and accurately detected clinically using flow cytometry. Existing research has demonstrated that sperm DNA fragmentation, i.e., reduced integrity, not only severely affects fertilization success rates leading to pregnancy failure but also significantly increases the probability of recurrent miscarriages and embryonic arrest in early pregnancy, and even directly impacts the pregnancy outcome of assisted reproductive technologies.

[0003] Modern medical research indicates that, in addition to the effects of varicocele, reproductive tract pathogen infection, environmental pollution, and unhealthy lifestyle habits, mechanisms that may lead to sperm DNA damage include spermatocyte apoptosis during testicular spermatogenesis, abnormal changes in sperm chromosomes during epididymal maturation, and the effects of oxidative stress during vas deferens transport and seminal vesicle storage. Currently, there are no targeted drugs in clinical practice that can treat sperm DNA damage and protect sperm DNA integrity; limited treatment options are confined to the single pathway of anti-oxidative stress. The few available drugs include L-carnitine oral solution, thioctic acid capsules, vitamin C tablets, natural vitamin E soft capsules, and coenzyme Q10. Their common drawbacks include: (1) lack of evidence-based medicine, with no treatment guidelines or expert consensus recommending drugs with definite efficacy, and the clinical efficacy is uncertain, with only small sample sizes of clinical literature reports; (2) long-term oral administration may cause varying degrees of gastrointestinal reactions (L-carnitine oral solution, coenzyme Q10), oral treatment may affect blood sugar and cause obvious esophageal irritation symptoms (thioctic acid capsules), long-term oral administration may cause urinary tract stones (vitamin C tablets), and although natural vitamin E capsules have fewer side effects, long-term high-dose oral administration may cause platelet aggregation and thrombosis; (3) the clinical use of the above drugs is all off-label use, which involves medical ethics controversies and medical safety risks. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a traditional Chinese medicine compound composition that protects the integrity of sperm DNA. When applied to the treatment of male infertility, it can not only protect the integrity of sperm DNA and reduce sperm DFI, but also make it more acceptable to patients and avoid the side effects of oral drug treatment. It can also improve the fertilization success rate of male infertility patients and reduce the incidence of recurrent miscarriage and embryonic arrest in early pregnancy, so as to achieve the goal of eugenics.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A traditional Chinese medicine compound composition for protecting sperm DNA integrity, comprising the following components in parts by weight: Codonopsis pilosula 10-30 parts, Citrus reticulata 10-20 parts, Taraxacum mongolicum 10-30 parts, Agrimonia pilosa 10-30 parts, Astragalus membranaceus 10-30 parts, Rhodiola rosea 10-30 parts, Astragalus complanatus 6-10 parts, Morus alba 6-10 parts, Schisandra chinensis 6-10 parts, Curcuma zedoaria 6-10 parts, Carthamus tinctorius 6-10 parts, Citrus reticulata seed 6-10 parts.

[0007] Preferably, the traditional Chinese medicine compound composition for protecting sperm DNA integrity of the present invention comprises the following components in parts by weight: 15 parts Codonopsis pilosula, 15 parts Citrus reticulata, 15 parts Taraxacum mongolicum, 15 parts Agrimonia pilosa, 10 parts Astragalus membranaceus, 10 parts Rhodiola rosea, 6 parts Astragalus complanatus, 6 parts Morus alba, 6 parts Schisandra chinensis, 6 parts Curcuma zedoaria, 6 parts Carthamus tinctorius, and 6 parts Citrus reticulata seed.

[0008] Traditional Chinese medicine theory holds that the kidneys store essence and govern reproduction. The kidneys store the innate essence inherited from one's parents, and the kidneys' role in reproduction relies on this stored essence to produce sperm; kidney essence is the material source for sperm production. In his published academic paper, the applicant proposed that dysfunction of the "yang transforming qi and yin forming" function is the fundamental pathogenesis of male infertility. Specifically, when the "yang transforming qi" function is impaired, the yang qi in the kidneys cannot warm, promote, and disperse the already produced sperm, resulting in weak sperm motility. When the "yin forming" function is impaired, the yin qi in the kidneys cannot condense into tangible sperm, resulting in low sperm count and a high rate of sperm abnormalities. When both "yang transforming qi" and "yin forming" functions are impaired, kidney essence is deficient, and yin and yang are imbalanced, resulting in sperm DNA damage and increased fragmentation rate. Furthermore, modern environmental pollution, food safety issues, pathogen infections, and ionizing radiation can lead to damp-heat toxins descending to the kidneys (testes) and seminal vesicles (prostate and seminal vesicles), stagnating in the meridians and obstructing qi and blood, thus causing disease with a complex nature of deficiency and excess. Based on the above-mentioned TCM pathogenesis theory, the inventor believes that protecting sperm DNA integrity and reducing sperm DFI should not be limited to simply tonifying the kidneys or simply detoxifying. Instead, it should combine tonification and elimination, regulate the balance of Yin and Yang, and supplement with blood-activating and qi-blood meridian unblocking methods to adapt to the complex pathogenesis of sperm DNA damage.

[0009] This invention innovates in the four properties, five flavors, meridian tropism, main therapeutic effects, and the compatibility of principal, assistant, adjuvant, and guide herbs in traditional Chinese medicine:

[0010] Goji berries: pungent and warm in nature, they enter the liver and stomach meridians, and can soothe the liver and stomach, regulate qi and relieve pain, and eliminate stagnation. Goji berry fruit contains various flavonoids such as hesperidin and citric acid, while the volatile oil in the peel contains various alkenes such as pinene, myrcene, and limonene, as well as various chemical components such as hesperidin and limonene. Modern pharmacological studies have shown that the various active ingredients in goji berries can regulate the body's immune function and have anti-aging, anti-tumor, and blood sugar and lipid-regulating effects.

[0011] Codonopsis pilosula: Sweet and neutral in nature, it enters the spleen and lung meridians, and can strengthen the spleen and lungs, nourish blood and promote body fluid production. Codonopsis pilosula contains various chemical components such as codonopsis polysaccharides, codonopsis glycosides, amino acids, trace elements, phytosterols, flavonoids, and phenolic acids. Modern pharmacological studies have shown that Codonopsis pilosula decoction not only regulates gastrointestinal motility and protects the gastric mucosa, but also has anti-hypoxia, anti-ischemia, anti-radiation, anti-aging, and memory and cognitive improvement effects.

[0012] Dandelion: Bitter, sweet, and cold in nature, it enters the liver and stomach meridians. It can clear heat and detoxify, reduce swelling and dissipate nodules, clear the liver and improve eyesight, and promote diuresis and relieve strangury. Dandelion is an essential medicine for treating various carbuncles and boils caused by internal and external heat toxins. It mainly contains organic acids such as caffeic acid, chlorogenic acid, and pseudotaraxasterol palmitic acid, as well as flavonoids such as quercetin, luteolin, and geraniol. Its volatile oil contains ethanol and trans-caryophyllene. Dandelion decoction has a strong inhibitory effect on various bacteria, can activate macrophages, stimulate the body's immunity, and has anti-endotoxin, anti-tumor, hepatoprotective, choleretic, and diuretic effects.

[0013] Agrimony: Bitter, astringent, and neutral in nature, it enters the heart and liver meridians. It can detoxify and reduce swelling, astringe and stop bleeding, and tonify and strengthen the body. Modern pharmacological studies have shown that Agrimony mainly contains luteolin-7-glucoside, quercetin, and rutin, as well as tannins and vitamin K, and has anti-inflammatory, anti-tumor, immune-enhancing, hypoglycemic, and hypotensive effects.

[0014] Raw Astragalus: Sweet and warm in nature, it enters the spleen and lung meridians. It can tonify the middle energizer and replenish qi, generate fluids and nourish blood, strengthen the exterior and stop sweating, and promote diuresis and reduce swelling. In addition to glycosides, polysaccharides, and flavonoids, raw Astragalus also contains amino acids, proteins, riboflavin, folic acid, vitamin D, and various trace elements. Raw Astragalus can promote RNA and protein synthesis, resulting in vigorous cell growth and extended lifespan. It has clear therapeutic effects in anti-fatigue, anti-aging, anti-tumor, anti-radiation, anti-inflammatory, and immune-enhancing aspects.

[0015] Rhodiola rosea: sweet, bitter, and neutral in nature; it enters the spleen, lung, and heart meridians, and can invigorate qi and promote blood circulation. Rhodiola rosea mainly contains rhodioloside, rhodioloside aglycone, flavonoids, organic acids, polysaccharides, and inorganic elements. It has clear anti-aging, anti-stress, antiviral, anti-radiation, immune-regulating, and memory-improving effects.

[0016] Astragalus complanatus seeds: sweet and warm in nature, entering the liver and kidney meridians, they can tonify the kidneys and strengthen yang, consolidate essence and reduce urination, nourish the liver and improve eyesight. Modern pharmacological studies have shown that Astragalus complanatus seeds contain various amino acids, polypeptides, proteins, phenols, tannins, alkaloids, and flavonoids. Modern pharmacological studies have also shown that Astragalus complanatus seeds can enhance the body's non-specific and specific immune functions, and have anti-fatigue, anti-radiation, anti-tumor, and anti-aging effects.

[0017] Mulberry: Sweet, sour, and cold in nature, it enters the heart, liver, and kidney meridians, and can nourish yin and blood, promote body fluid production and moisten dryness. Mulberries contain cyanidin-glucoside, linoleic acid, and other fatty oils, while the volatile oil contains eucalyptol, geraniol, and other organic acids, carotene, and various vitamins. Mulberries can delay aging, increase the activity of glutathione peroxidase and catalase, enhance superoxide dismutase activity, promote the recovery of aging T-cell function, and enhance the body's immunity.

[0018] Schisandra chinensis: Sour, sweet, and warm in nature, it enters the lung, heart, and kidney meridians. It has astringent and consolidating properties, replenishes qi and generates fluids, and tonifies the kidneys and calms the mind. Modern pharmacological studies show that Schisandra chinensis mainly contains organic acids, tannins, vitamins, and resins, and its volatile oil contains schisandrin, among other things. Schisandra chinensis has adaptogenic effects similar to ginseng, enhancing the body's defense against non-specific stimuli, strengthening cellular immunity, and increasing superoxide dismutase activity, thus improving immunity, anti-oxidation, and anti-aging effects.

[0019] Curcuma zedoaria: pungent, bitter, and warm in nature, it enters the liver and spleen meridians. It can break up blood stasis, promote qi circulation, eliminate stagnation, and relieve pain. The volatile oil of Curcuma zedoaria mainly contains pinene, curcumol, eugenol, and curcumone, which can inhibit the growth of various pathogenic bacteria, have anti-inflammatory effects, and promote the recovery of microarterial blood flow and improve local microcirculation.

[0020] Safflower: pungent and warm in nature, it enters the heart and liver meridians, and can promote blood circulation, remove blood stasis, and relieve pain. Safflower mainly contains safflower yellow pigment, safflower quinone glycoside, neosafflower glycoside, safflower glycoside, and safflower oil, which can inhibit platelet aggregation, reduce whole blood viscosity, and its extract can significantly improve the body's tolerance to hypoxia. It also has anti-inflammatory, sedative, and analgesic effects.

[0021] Orange seeds: bitter and neutral in nature, they enter the liver and kidney meridians and can regulate qi, disperse stagnation, and relieve pain.

[0022] The herbal compound composition of this invention contains ingredients such as Citrus reticulatae, Codonopsis pilosula, Astragalus membranaceus, Rhodiola rosea, Astragalus complanatus, and Schisandra chinensis, which have anti-aging effects; Taraxacum mongolicum and Agrimonia pilosa not only have anti-inflammatory effects but also enhance and stimulate the body's immunity; Mulberry not only has anti-aging effects but also increases immunity and resists oxidative stress; Curcuma zedoaria and Carthamus tinctorius can improve blood circulation and enhance the body's tolerance to hypoxia. Modern medical research shows that protecting DNA integrity exerts its therapeutic effects through multiple pathways and targets, including anti-aging, enhancing immunity, anti-inflammatory and anti-oxidative stress effects, and improving blood circulation. Therefore, the herbal compound composition of this invention has the effect of protecting sperm DNA integrity through multiple pathways and targets.

[0023] In this invention, the herbal compound formula uses *Citrus reticulata*, *Codonopsis pilosula*, *Taraxacum mongolicum*, and *Agrimonia pilosa* as the principal herbs. Traditional Chinese medicine considers the spleen to be the foundation of acquired constitution and the kidneys to be the foundation of innate constitution. The innate essence in the kidneys relies on the spleen and stomach to transform and transport the essence of food and water for continuous nourishment. Of the four principal herbs, *Codonopsis pilosula* tonifies qi and strengthens the spleen, while *Agrimonia pilosa* tonifies deficiency; the synergy of these two herbs is called "tonification." *Taraxacum mongolicum* has been an essential medicine for treating various internal and external heat toxins, carbuncles, and boils since ancient times, clearing heat and detoxifying without harming essence. *Agrimonia pilosa* also has the effect of clearing heat and detoxifying; the synergy of these two herbs is called "elimination." *Citrus reticulata* promotes qi circulation and resolves stagnation, ensuring that tonification is not cloying and also aiding in the effects of clearing heat and detoxifying. These four principal herbs combine "elimination and tonification." *Astragalus membranaceus*, *Rhodiola rosea*, *Astragalus complanatus*, *Morus alba*, and *Schisandra chinensis* are the assistant herbs. *Astragalus membranaceus*, compared to *Astragalus membranaceus* prepared with roasted, not only has a stronger qi-tonifying effect but also generates fluids and nourishes blood, assisting the principal herb *Codonopsis pilosula* in tonifying the middle jiao and replenishing qi. Astragalus complanatus, mulberry fruit, and schisandra fruit all enter the kidney meridian. The combination of these three herbs warms the kidneys and invigorates yang, nourishes yin and tonifies the kidneys, and strengthens essence. This synergistic effect of yin and yang complements the principal herb, replenishing the innate essence. Safflower and turmeric are adjuvant herbs. Both are pungent and warm, promoting blood circulation and removing blood stasis. Turmeric also has the effect of breaking up qi stagnation. As the saying goes, qi flows smoothly when warmed, and blood flows smoothly when qi is present. The combination of these two herbs warms yang, promotes qi circulation, and removes blood stasis, assisting the principal and adjuvant herbs to ensure that tonification is not cloying and that elimination is not stagnant. Orange seed is the guiding herb, with a mild nature, entering the liver and kidney meridians. It acts as a guiding herb, directing the other herbs downwards to the kidneys (testes) to reach the affected area. The entire formula, with its combination of principal, adjuvant, and guiding herbs, achieves a synergistic effect of tonification and elimination, promoting blood circulation and qi flow, and regulating the balance of yin and yang in the kidney essence. Clinical use has shown significant efficacy in protecting sperm DNA integrity and reducing sperm DFI.

[0024] Furthermore, the traditional Chinese medicine compound composition for protecting sperm DNA integrity of the present invention can be formulated into any one of the following: ointment, tablet, capsule or granule.

[0025] Furthermore, the present invention also provides a method for preparing the above-mentioned traditional Chinese medicine compound composition for protecting sperm DNA integrity, comprising the following steps:

[0026] (1) Mix the following ingredients in the formula: Codonopsis pilosula, Citrus reticulata, Taraxacum mongolicum, Agrimonia pilosa, Astragalus membranaceus, Rhodiola rosea, Astragalus complanatus, Morus alba, Schisandra chinensis, Curcuma zedoaria, Carthamus tinctorius, and Citrus reticulata seeds, and soak them in water.

[0027] (2) Heat and decoct to obtain an aqueous extract;

[0028] (3) Concentrate the aqueous extract to obtain the extract.

[0029] Preferably, in step (1), each time the herbs are soaked in water at 8 to 10 times their total weight; the soaking time is 30 to 60 minutes each time.

[0030] Preferably, in step (2), the water is boiled 2 to 3 times, each time for 30 to 60 minutes, and the water extracts are combined.

[0031] Preferably, in step (3), the aqueous extract is concentrated under reduced pressure to obtain the extract, and the temperature of the reduced pressure concentration is controlled at 50 to 70 degrees Celsius.

[0032] Specifically, when tablets are required, the concentrated extract is mixed with carrier lactose or corn starch, granulated, dried, and lubricant magnesium stearate is added if necessary. The mixture is then compressed into tablets.

[0033] Specifically, when it is necessary to make capsules, the concentrated extract is mixed evenly with the carrier lactose or corn starch, granulated, and then encapsulated to make capsules.

[0034] Specifically, when it is necessary to make granules, the concentrated extract is mixed evenly with the carrier lactose or corn starch, granulated, dried, and made into granules.

[0035] Finally, the application of the above-mentioned traditional Chinese medicine compound composition in the preparation of drugs that reduce sperm DNA fragmentation index (DFI) is also within the scope of protection of this invention.

[0036] The reasons that can lead to an elevated sperm DNA fragmentation index (DFI) include: air and environmental pollution (PM2.5, formaldehyde, benzene and other organic chemicals), food safety (some food additives), unhealthy lifestyle habits (smoking, drinking, prolonged sitting, sauna, etc.), high-temperature working environment, and the effects of ionizing radiation (radar, electromagnetic waves). Beneficial effects

[0037] (1) The traditional Chinese medicine compound composition for protecting sperm DNA integrity of the present invention is scientifically formulated according to the four natures and five flavors of traditional Chinese medicine, and the principles of monarch, minister, assistant and guide. The components can coordinate and promote each other, and play a synergistic role. Traditional Chinese medicine does not have a specific disease name for sperm DNA damage, and it is mostly classified into the category of seeking offspring or infertility. Traditional Chinese medicine theory believes that the kidney stores essence and governs reproduction. Male infertility is mostly attributed to the kidney, that is, insufficient kidney essence or deficiency of kidney essence. The present invention proposes that when the functions of "yang transforming qi and yin forming shape" are both dysfunctional, it will lead to deficiency of kidney essence and imbalance of yin and yang, which manifests as sperm DNA damage and increased fragmentation rate. In addition, modern unhealthy lifestyle habits, environmental pollution, food safety, ionizing radiation and other effects lead to damp-heat evil toxins flowing down to the kidney and seminal chamber, stagnating the qi and blood in the meridians and causing disease. The nature of the disease is a mixture of deficiency and excess. Therefore, traditional Chinese medicine treatment should adhere to the principle of combining tonification and elimination, supplemented by blood-activating and purging. The herbal compound composition of this invention can tonify the spleen and kidneys, clear heat and detoxify, and simultaneously activate blood circulation and regulate the balance of yin and yang in the kidney essence. This effectively protects the integrity of sperm DNA and reduces the increase in sperm DNA fibrillation (DFI) caused by sperm DNA damage, demonstrating definite clinical efficacy. The herbal compound composition of this invention is convenient to take in clinical use, well-tolerated by patients, and no adverse reactions have been observed with long-term oral administration. Furthermore, its formulation into a standardized traditional Chinese medicine preparation can further promote the standardized treatment and use of traditional Chinese medicine.

[0038] (2) The clinical indications of this invention are clear, there are no medical ethics and medical safety risks associated with off-label use, and it is safe with no obvious side effects in clinical use. Currently, all drugs used in clinical practice, including traditional Chinese medicines, do not have indications in their instructions to protect sperm DNA integrity, reduce sperm DNA damage, or lower sperm DFI.

[0039] (3) The clinical efficacy of the present invention is definite. Compared with sperm energy metabolism agents (levocarnitine oral solution) and antioxidant stress drugs (vitamin C, natural vitamin E soft capsules, thioctic acid capsules, coenzyme Q10, etc.) and existing traditional Chinese medicine for tonifying the kidney and promoting sperm production, the traditional Chinese medicine compound composition of the present invention has a definite clinical efficacy in protecting the integrity of sperm DNA and can effectively reduce sperm DFI.

[0040] (4) Based on innovative TCM theory, this invention proposes a treatment principle of combining tonification and elimination with blood-activating to regulate the balance of Yin and Yang in the kidney essence, according to the TCM pathogenesis of sperm DNA damage and the combination of principal, assistant, and adjuvant herbs. Traditional Chinese medicine preparations for treating male infertility, such as Wuzi Yanzong Wan, Qilin Wan, Huangjing Zanyu Capsules, and Jujing Wan, all take traditional kidney essence deficiency as the main pathogenesis and use simple tonification—tonifying the kidney and generating sperm—as the treatment principle. They have not re-understood the pathogenesis of sperm DNA damage from the microscopic level of modern medicine. The concept of simple tonification based on the treatment principle is vague and does not clearly define whether it is tonifying kidney Yin, warming and tonifying kidney Yang, or both. Therefore, it is difficult to grasp the important pathogenesis and exert the therapeutic effect. The compound TCM composition of this invention, supported by the inventor's innovative TCM theory, clearly clarifies that while tonifying, it also clears heat and detoxifies, eliminates damp-heat evil toxins, combines tonification and elimination with blood-activating and stasis-removing. The pathogenesis is clear, the treatment method is appropriate, and the combination of herbs is reasonable. It is an innovation in prescription and medication based on TCM theory and clinical practice. Detailed Implementation

[0041] The present invention can be better understood from the following embodiments. Example 1

[0042] The traditional Chinese medicine compound composition contains the following components in parts by weight: Codonopsis pilosula 15 parts, Citrus reticulata 15 parts, Taraxacum mongolicum 15 parts, Agrimonia pilosa 15 parts, Astragalus membranaceus 10 parts, Rhodiola rosea 10 parts, Astragalus complanatus 6 parts, Morus alba 6 parts, Schisandra chinensis 6 parts, Curcuma zedoaria 6 parts, Carthamus tinctorius 6 parts, Citrus reticulata seed 6 parts.

[0043] Preparation method:

[0044] The following ingredients were mixed according to the formula: Codonopsis pilosula, Citrus reticulata, Taraxacum mongolicum, Agrimonia pilosa, Astragalus membranaceus, Rhodiola rosea, Astragalus complanatus, Morus alba, Schisandra chinensis, Curcuma zedoaria, Carthamus tinctorius, and Citrus reticulata seed. After soaking in water for half an hour with 10 times the total weight of the herbs, the mixture was decocted three times, 45 minutes each time. The aqueous extracts were combined and concentrated under reduced pressure at 60 degrees Celsius to obtain the extract, which is the Chinese herbal compound extract. Example 2

[0045] The traditional Chinese medicine compound composition contains the following components in parts by weight: Codonopsis pilosula 10 parts, Citrus reticulata 10 parts, Taraxacum mongolicum 10 parts, Agrimonia pilosa 10 parts, Astragalus membranaceus 10 parts, Rhodiola rosea 10 parts, Astragalus complanatus 8 parts, Morus alba 8 parts, Schisandra chinensis 8 parts, Curcuma zedoaria 8 parts, Carthamus tinctorius 8 parts, Citrus reticulata seed 6 parts.

[0046] Preparation method:

[0047] The following herbs were mixed according to the formula: Codonopsis pilosula, Citrus reticulata, Taraxacum mongolicum, Agrimonia pilosa, Astragalus membranaceus, Rhodiola rosea, Astragalus complanatus, Morus alba, Schisandra chinensis, Curcuma zedoaria, Carthamus tinctorius, and Citrus reticulata seed. After soaking in water at 8 times the total weight of the herbs for half an hour, the mixture was decocted twice, 30 minutes each time. The aqueous extracts were combined and concentrated under reduced pressure at 60 degrees Celsius to obtain the extract, which is the Chinese herbal compound extract. Example 3

[0048] The traditional Chinese medicine compound composition contains the following components in parts by weight: Codonopsis pilosula 30 parts, Citrus reticulata 20 parts, Taraxacum mongolicum 30 parts, Agrimonia pilosa 30 parts, Astragalus membranaceus 30 parts, Rhodiola rosea 30 parts, Astragalus complanatus 10 parts, Morus alba 10 parts, Schisandra chinensis 10 parts, Curcuma zedoaria 10 parts, Carthamus tinctorius 10 parts, Citrus reticulata seed 10 parts.

[0049] Preparation method:

[0050] Mix the following ingredients in the prescribed amounts: Codonopsis pilosula, Citrus reticulata, Taraxacum mongolicum, Agrimonia pilosa, Astragalus membranaceus, Rhodiola rosea, Astragalus complanatus, Morus alba, Schisandra chinensis, Curcuma zedoaria, Carthamus tinctorius, and Citrus reticulata seed. Soak the mixture in water for half an hour with 8 to 10 times the total weight of the herbs. Then decoct the mixture 2 to 3 times, each time for 30 to 60 minutes. Combine the aqueous extracts and concentrate them under reduced pressure at 60 degrees Celsius to obtain the extract, which is the compound extract of traditional Chinese medicine.

[0051] 1. Clinical Data

[0052] 1.1 Source of Cases

[0053] All cases observed were male infertility patients (sperm DNA damage) who visited the andrology clinic of Jiangsu Provincial Hospital of Traditional Chinese Medicine. The cases that met the inclusion criteria were randomly divided into a positive control group of 30 cases and a treatment group of 30 cases.

[0054] 1.2 Inclusion Criteria

[0055] (1) Meets the Western medical diagnostic criteria for male infertility (sperm DNA damage);

[0056] (2) Age 18-50 years old;

[0057] (3) Disease duration ≥ 1 year;

[0058] (4) Sperm concentration ≥15×10 6 / ml;

[0059] (5) Percentage of progressively motile sperm (PR) ≥ 32%;

[0060] (6) The rate of normal sperm morphology is ≥4% or the rate of abnormal sperm morphology is ≤96%;

[0061] (7) Sperm DNA Fragmentation Index (DFI) > 30%.

[0062] 1.3 Exclusion criteria:

[0063] (1) Spouse has reproductive dysfunction;

[0064] (2) Male immunological infertility;

[0065] (3) Male endocrine dysfunction;

[0066] (4) Infertility caused by congenital malformation of male reproductive organs or reproductive tract, or by sperm expulsion obstruction;

[0067] (5) Taking anti-epileptic, anti-tumor or other drugs that have clear toxic side effects on sperm quality within the past month; taking Western medicine or traditional Chinese medicine for the treatment and improvement of sperm quality within the past month;

[0068] (6) Comorbid severe cardiovascular and cerebrovascular diseases and hematopoietic system diseases;

[0069] (7) Those with liver or kidney dysfunction.

[0070] 1.4 Rejection, detachment, and exit criteria:

[0071] (1) Exclusion criteria: those who do not meet the diagnostic criteria, meet the exclusion criteria, have poor compliance and cannot follow the treatment plan and have regular follow-up visits;

[0072] (2) Dropout criteria: Cases included in the observation, randomly assigned to groups, who failed to complete the treatment cycle or were lost to follow-up after the treatment cycle ended;

[0073] (3) Withdrawal criteria: Patients with poor compliance who fail to take medication regularly; or whose condition continues to worsen during treatment and who are deemed to have stopped taking medication by the attending physician; or who experience allergic reactions or serious adverse events.

[0074] 1.5 Grouping Method

[0075] Male infertility (sperm DNA damage) patients who visited the Department of Andrology at Jiangsu Provincial Hospital of Traditional Chinese Medicine between March 2022 and April 2023 were selected. Those who met the inclusion criteria and did not meet the exclusion criteria were randomly divided into a treatment group and a positive control group using a random number table.

[0076] 1.5 General Information

[0077] Positive control group: 30 cases, aged 25 - 41 (33.1 ± 7.9) years old, with a disease course of 1 - 3 (2.2 ± 0.8) years; treatment group: 30 cases, aged 26 - 39 (32.7 ± 6.3) years old, with a disease course of 1 - 3 years (2.4 ± 0.6) years. After statistical analysis, there was no significant difference in age and disease course between the two groups ( p > 0.05).

[0078] 2 Methods

[0079] 2.1 Treatment methods

[0080] (1) The positive control group was given fertility guidance to the patients [advising the patients to live regularly, quit bad living habits (smoking, drinking, staying up late), guiding diet (eating more fresh vegetables and fruits, having a light diet, supplementing high-quality protein, avoiding soy products, etc.), avoiding the influence of environmental factors (wearing protective masks to reduce the impact of air pollution, avoiding working in high-temperature and radiation environments), exercising moderately 3 times a week, and maintaining a good emotional state]. At the same time, natural vitamin E soft capsules were orally administered, 1 capsule each time, 3 times a day, after meals; levocarnitine was orally administered, 1 vial each time, 2 times a day, after meals; the treatment course was 12 weeks.

[0081] (2) The treatment group was given the oral administration of the traditional Chinese medicine compound preparation prepared in Example 1 of the present invention while giving fertility guidance to the patients. The Chinese medicinal materials were uniformly purchased by the Traditional Chinese Medicine Preparation Department of Jiangsu Provincial Hospital of Traditional Chinese Medicine and were decocted by the Traditional Chinese Medicine Preparation Department on behalf of the patients according to the operation process of the automatic decocting machine. Administration method: 2 times a day, 1 bag each time, 200 ml per bag, taken warm half an hour after meals.

[0082] 2.2 Observation time points and observation indexes

[0083] The follow-up observation period was 12 weeks, which were carried out before treatment and 12 weeks after treatment respectively. The changes of indexes such as sperm DFI (sperm DNA fragmentation index), seminal plasma ROS (reactive oxygen species), seminal plasma SOD (superoxide dismutase), and seminal plasma MDA (malondialdehyde) of the patients were observed. If there were any changes in the patient's condition, examinations were carried out at any time, and the examination time and results were recorded in a timely manner.

[0084] Curative effect judgment criteria

[0085] Recovery: The spouse is pregnant (and there is no early miscarriage or embryo arrest in the early stage); Marked effect: The spouse is not pregnant, but DFI ≤ 15% after treatment; Effective: The spouse is not pregnant, but 15% < DFI < 30% after treatment; Ineffective: The spouse is not pregnant, DFI > 30%.

[0086] 2.3 Statistical methods

[0087] SPSS 23.0 software was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation. Paired samples were used before and after treatment to ensure normal distribution and homogeneity of variance. t For tests that do not conform to a normal distribution and have unequal variances, a multi-sample rank-sum test is used, and data are expressed as median interquartile range; categorical data are expressed using the χ² test. 2 For ordinal data, the rank-sum test is used. p <0.05 indicates a statistically significant difference.

[0088] 3 Results

[0089] 3.1 Clinical efficacy rates of the two treatment groups

[0090] After 12 weeks of treatment, in the treatment group, 4 cases were cured (due to the spouse's pregnancy), 10 cases showed significant improvement, 13 cases showed improvement, and 3 cases showed no improvement, with a total effective rate of 90.0%; in the control group, 0 cases were cured, 4 cases showed significant improvement, 9 cases showed improvement, and 17 cases showed no improvement, with a total effective rate of 43.3%. The difference in effective rates between the two groups was statistically significant according to the chi-square test. p <0.05), see Table 1 for details.

[0091] Table 1: Clinical efficacy rates of the two treatment groups

[0092]

[0093] Note: *Compared with the treatment group, p<0.05 was considered statistically significant according to the chi-square test.

[0094] 3.2 Changes in sperm DFI and oxidative stress indicators before and after treatment in both groups

[0095] Before treatment: In the treatment group, the sperm DFI was 40.04±4.39%, and the seminal plasma ROS levels were 9.62±0.81μM, MDA levels were 16.69±1.01μM, and SOD levels were 58.79±12.00μM; in the control group, the sperm DFI was 41.04±6.27%, and the seminal plasma ROS levels were 9.68±0.80μM, MDA levels were 16.56±0.87μM, and SOD levels were 58.23±11.93μM. Statistical analysis showed no significant differences in sperm DFI, seminal plasma ROS, MDA, and SOD levels between the two groups before treatment, indicating that the data were comparable between the two groups before treatment. p >0.05).

[0096] After treatment: In the treatment group, the sperm DFI decreased to 16.61±4.20%, the ROS level in seminal plasma decreased to 5.06±0.52μM, the MDA level decreased to 11.95±1.94μM, and the SOD level increased to 139.05±14.71μM; in the control group, the sperm DFI decreased to 32.66±5.23%, the ROS level in seminal plasma decreased to 7.08±0.51μM, the MDA level decreased to 15.27±1.23μM, and the SOD level increased to 100.65±10.59μM (see Table 2 for details).

[0097] Table 2: Changes in sperm DFI, seminal plasma ROS, MDA, and SOD levels before and after treatment in both groups

[0098]

[0099] Note: ◆ Compared with before treatment p >0.05, ■ Compared with before treatment p <0.05, ▲ Compared with the control group after treatment p <0.05

[0100] As shown in Table 2, after 12 weeks of treatment, the sperm DFI in the treatment group using the traditional Chinese medicine compound preparation decreased significantly, with a much greater reduction than that in the control group. Considering the overall clinical efficacy of the two groups, the treatment group showed significantly better clinical efficacy than the control group. Furthermore, after treatment, both groups showed a decrease in seminal plasma ROS and MDA levels and an increase in SOD levels. This indicates that both the traditional Chinese medicine compound preparation and vitamin E combined with L-carnitine have the effect of reducing oxidative stress damage. Although there was no statistically significant difference in the reduction of seminal plasma ROS and MDA levels between the treatment and control groups after treatment compared to pre-treatment levels (p>0.05), the reduction in the treatment group was greater than that in the control group. The increase in SOD levels in the treatment group was significantly greater than that in the control group, and the difference between the two groups was statistically significant. This further confirms that the traditional Chinese medicine compound preparation is more effective than vitamin E combined with L-carnitine in improving anti-oxidative stress damage, thereby reducing sperm DNA damage and lowering the DFI value.

[0101] This invention provides a traditional Chinese medicine compound composition for protecting sperm DNA integrity, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A traditional Chinese medicine compound composition for protecting the integrity of sperm DNA, characterized in that, It is made from the following components by weight: Codonopsis pilosula 10-30 parts, Citrus reticulata 10-20 parts, Taraxacum mongolicum 10-30 parts, Agrimonia pilosa 10-30 parts, Astragalus membranaceus 10-30 parts, Rhodiola rosea 10-30 parts, Astragalus complanatus 6-10 parts, Morus alba 6-10 parts, Schisandra chinensis 6-10 parts, Curcuma zedoaria 6-10 parts, Carthamus tinctorius 6-10 parts, Citrus reticulata seed 6-10 parts.

2. The traditional Chinese medicine compound composition for protecting sperm DNA integrity according to claim 1, characterized in that, It is made from the following components by weight: 15 parts Codonopsis pilosula, 15 parts Citrus reticulata, 15 parts Taraxacum mongolicum, 15 parts Agrimonia pilosa, 10 parts Astragalus membranaceus, 10 parts Rhodiola rosea, 6 parts Astragalus complanatus, 6 parts Morus alba, 6 parts Schisandra chinensis, 6 parts Curcuma zedoaria, 6 parts Carthamus tinctorius, and 6 parts Citrus reticulata seed.

3. The traditional Chinese medicine compound composition for protecting sperm DNA integrity according to claim 1, characterized in that, It can be any one of the following: ointment, tablet, capsule, or granule.

4. A method for preparing the traditional Chinese medicine compound composition for protecting sperm DNA integrity as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Mix the following ingredients in the formula: Codonopsis pilosula, Citrus reticulata, Taraxacum mongolicum, Agrimonia pilosa, Astragalus membranaceus, Rhodiola rosea, Astragalus complanatus, Morus alba, Schisandra chinensis, Curcuma zedoaria, Carthamus tinctorius, and Citrus reticulata seeds, and soak them in water. (2) Heat and decoct to obtain an aqueous extract; (3) Concentrate the aqueous extract to obtain the extract.

5. The method for preparing the traditional Chinese medicine compound composition for protecting sperm DNA integrity according to claim 4, characterized in that, In step (1), soak the herbs in water at a rate of 8 to 10 times their total weight each time; the soaking time is 30 to 60 minutes each time.

6. The method for preparing the traditional Chinese medicine compound composition for protecting sperm DNA integrity according to claim 4, characterized in that, In step (2), decoct with water 2-3 times, each time for 30-60 minutes, and combine the water extracts.

7. The method for preparing the traditional Chinese medicine compound composition for protecting sperm DNA integrity according to claim 4, characterized in that, In step (3), the aqueous extract is concentrated under reduced pressure to obtain the extract, and the temperature of the reduced pressure concentration is controlled at 50-70 degrees Celsius.

8. The method for preparing the traditional Chinese medicine compound composition for protecting sperm DNA integrity according to claim 4, characterized in that, It also includes step (4), which involves mixing the concentrated extract with carrier lactose or corn starch, granulating, drying, adding magnesium stearate as a lubricant if necessary, mixing evenly, and then compressing into tablets.

9. The method for preparing the traditional Chinese medicine compound composition for protecting sperm DNA integrity according to claim 4, characterized in that, It also includes step (4), which involves mixing the concentrated extract with carrier lactose or corn starch, granulating it, and then filling it into capsules.

10. The use of the traditional Chinese medicine compound composition according to claim 1 in the preparation of a drug for reducing sperm DNA fragmentation index.

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