Flavonoids as potential therapeutics in male reproductive disorders

Background Male infertility presents global challenges, as current drug-based treatments demonstrate limited effectiveness due to an incomplete understanding of dysfunctions within the reproductive system. However, there is growing optimism surrounding natural products, particularly flavonoids, which offer promising therapeutic options. Extensive research has unveiled the positive impact of flavonoids on testicular structure, spermatogenesis, and sperm quality. Main body Flavonoids have diverse functions such as immune-stimulating, anti-inflammatory, and antioxidative effects. These properties make them potential inhibitors of male reproductive system problems. This narrative review aims to evaluate the effects of different flavonoids on male reproductive disorders by examining the phytochemical ingredients, traditional applications, potential pharmacological actions, documented effects, and therapeutic applications of flavonoids in functional abnormalities of the male reproductive system. Conclusion This review elaborates on the scientific study findings of flavonoids and recommends their use in male infertility.

pregnancy outcomes [20].Various synthetic medications such as anti-depressants, calcium channel blockers, alpha-adrenergic blockers, and antiepileptic drugs have significant effects on fertility and changes in sperm function and count [21].Long-term use of synthetic drugs may lead to tolerance or dependency potentially leading to withdrawal symptoms [22].Many chemotherapeutic medicines, including neuropsychiatric and antihypertensive medications, can have an impact on sexual function, sperm production, and hormonal levels [23].Flavonoids have been carefully investigated as potential inhibitors for treating problems with the male reproductive system in many nations worldwide [24][25][26][27][28]. Flavonoids are natural antioxidant chemicals with low molecular weight found in plants and fungi.They are polyphenolic and have a fundamental phenyl-benzopyran backbone (C6-C3-C6).Some common sources of flavonoids include citrus fruits, tea, olive oil, red wine, and citrus fruits [29,30].Flavonoids are phytoconstituents that come in a variety of subtypes, including flavanols, catechins, flavones, flavanones, etc. as indicated in Table 1.Flavonoids are said to possess antiviral, anti-inflammatory, anticarcinogenic, anti-apoptotic, and immune-stimulating qualities [31][32][33][34][35]. Several studies have been conducted to demonstrate the benefits of flavonoids and the underlying mechanism in the treatment of disorders associated with the male reproductive system.In the current review the role of flavonoids in the treatment of dysfunction in the male reproductive system, examining their therapeutic benefits and relevant molecular pathways.The article analyzes the role of flavonoids as possible therapeutic agents and provides insights into several illnesses affecting the male reproductive system, including oligospermia, erectile dysfunction, and failure of spermatogenesis.The study includes an extensive literature search from 2003 to 2024, covering research from early developments to current findings about the impact of flavonoids on male reproductive health.Secondary data from reliable online databases, books, and peer-reviewed publications-mainly from Google Scholar, ScienceDirect, and PubMed-were used in this study.Specific keywords such as "flavonoids", "male infertility", "oxidative stress", "spermatogenesis", and "testicular dysfunction" were used to search for relevant literature.

Role of flavonoid in overcoming testis tissue damage
A decrease in weight and anatomical disruption of the testis may prevent the production of sperm [36].This might also have played a role in the declining quantity and quality of sperm.Nna et al. assessed the effect of quercetin, a flavonoid compound, which could effectively help in improving testicular histology and reduce germ cell apoptosis because it reverses the detachment of germ cells from the basal lamina and prevents shedding of immature germ cells against the Wistar rats of four weeks which were induced with testicular toxicity using cadmium [37,38].Guvvala et al. have studied the arsenicinduced mice's testicular spermatogenic degeneration, and damage, including reduced, sperm in seminiferous tubules, epithelial height, and testicular damage was recovered by supplementing with green tea compound, epigallocatechin-3-gallate (EGCG) [26,36].Jahan et al. explored the use of rutin helped to overcome the harmful effects of cisplatin by reducing oxidative stress in the testis and the epididymis which prevents testicular injury [39].Mehfooz et al. concluded that there was a substantial rise in body weight as well as the weight of the testes and epididymis when 200 mg of rutin was given.Rutin's ability to destroy PARP-1 (Poly ADP ribose polymerase-1) in mice testes might explain its protective properties [40].Hassan et al. proposed that rats suffering from lead (Pb) poisoning were able to recover after receiving 80 mg/kg of EGCG.This substance known for its antioxidant properties, demonstrated significant improvements in male sexual organ weight, semen quality, sperm count, and motility [41].

Spermatogenesis and flavonoids
Reduced sexual organ count and poorer sperm quality can interfere with or impede spermatogenesis, the process of producing new eggs [42].Consequently, this further complicates matters about both sperm quality and sexual organs, ultimately decreasing fertility rate reduction in testosterone, or alterations in the amounts of key hormones such as luteinizing hormone (LH) or Folliclestimulating hormone (FSH) that stimulate the production of sperm [43,44].Khorsandi et al. investigated that for 35 days, mice with impairments in spermatogenesis resulting from exposure to titanium dioxide nanoparticles were administered a dosage of 75 mg/kg of quercetin.This treatment demonstrated anti-apoptotic effects via its impact on the hypothalamic-pituitary-testis axis [45].Osawe et al. have investigated that quercetin (20 mg/ kg) and rutin (10 mg/kg) dramatically counteracted the effects of sulfasalazine on steroidogenesis and plasma levels of testosterone, and gonadotropins in experimental rats [46,47].As shown in Table 2, 30 mg/kg of morin was beneficial on spermatogenesis and is ascribed to its unique capacity to safeguard the molecules in the testicles and its notable augmentation of Hypothalamic-Pituitary-Gonadal Axis parameters.Lower luteinizing hormone (LH) levels, however, might prevent spermiation [47,48].A 40 mg/kg increase in LH and an 80-85 mg/kg increase in naringenin may be beneficial for the continued maturation of spermatogonia and the spermiation process [49].Sertoli cells (SCs) and germ cells must interact hormonally and dynamically during spermatogenesis [50,51].The decrease in sex hormone levels is brought on by the number and functionality of Leydig cells and testicular SCs [52][53][54].The physical growth of germ cells is enhanced by hormonal and nutritional substances that are secreted by SCs [55][56][57].Infertility can occur due to dysfunction of the somatic cells (SCs).When exposed to icariin at a concentration of 10 μM, SCs can undergo proliferation.The MEK/ERK (mitogen-activated protein kinase/ERK kinase) pathway is activated to accomplish this, which leads to an increase in the phosphorylated extracellular signal-regulated kinase (pERK) levels.Therefore, the primary mechanism of action by which icariin promotes SC proliferation is mediated through the MEK/ERK pathway [58].
The Leydig cells are located within the connective tissue that surrounds the seminiferous tubules in the testicles and their main function are to produce testosterone which helps in the development of sperm.They play a pivotal role in the male reproductive system [59].It is well-known that naturally occurring flavonoids maintain normal ranges for testosterone levels [60,61].Flavonoids that may raise the gene expressions of 3-β-HSD (3-betahydroxy steroid dehydrogenase), 17-β-HSD (17-betahydroxy steroid dehydrogenase), Cyp11A (Cytochrome P450c 11A), include 10 μM of apigenin, 10 μM of quercetin, 20 mg/kg of luteolin, and 80 mg/kg of EGCG (80 mg/ kg) [43,62].The quantity of testosterone secreted can be increased by promoting steroidogenesis and assisting Leydig cells in functioning [63,64,[149][150][151].The elevation of serum testosterone and the production of the StAR protein may be related to astragalin.Further study is required to determine if it is the primary factor at play and how much of it [65,[152][153][154].According to these results, flavonoids could be crucial for the recovery of spermatogenesis.
Human sperm cryopreservation has advanced significantly in the last several years [66,[155][156][157].This advancement is beneficial for male patients facing challenges such as anejaculation, azoospermia, severe oligospermia, or cancer, as it provides a chance to preserve their fertility.However, the process of freezing and thawing sperm has drawbacks, including a commonly observed decrease in fertility rates.
Cryopreservation also leads to structural and functional changes in sperm cells, resulting in reduced motility and viability, impaired mitochondrial function, and DNA damage [67][68][69].Flavonoids are amphipathic substances that can pass through membranes' lipid bilayers.This characteristic may allow them to protect the membrane of acrosome and spermatozoa from oxidative damage.Therefore, they play a crucial role in ensuring the necessary sperm response for successful conception [29,46].Notably, sperm protection can be provided by a variety of different compounds used as in vitro supplements.Xu et al. used a combination of 1.0 mM of rutin, 0.2 mmol/L of apigenin,10 μg/mL of EGCG, and 5 µmol/L of quercetin can enhance the mitochondrial activity, antioxidant activity, acrosomal activity, membrane integrity of plasma, motility, and reactive oxygen species (ROS) concentration of sperm that are freeze-thawed [70,71].Among these, 1.0 mM rutin was added as a supplement in pig sperm not only improved the post-thaw sperm's kinematic parameters, such as the sperm's physiological traits but also increased the curvilinear line velocity sperm's linear motile sperm and straight-line velocity.In particular, rutin increased the rates of blastocyst formation and cleavage [72].Research has indicated that mitochondria may be quercetin's primary target intercellularly.Rabbit semen supplemented with 5 µmol/L quercetin might be given safeguarding and controlling vital mitochondrial functions, such as oxidative phosphorylation which may have an impact on male gametes' metabolism and behavior [73].
Moreover, these preventive advantages depend on the dose.Studies show that sperm survival is improved at low concentrations; while, they become cytostatic at high concentrations.For instance, the sperm that are frozenthawed either dramatically reduced or remain stable due to apigenin.Similarly, myricetin did not appear to increase sperm adhesion activity, viability, and motility concentrations up to 100 nM [74].
Male reproductive system dysfunction is primarily caused by oxidative stress, apoptosis, related physiological processes, and inflammation.Flavonoids exhibit robust pharmacological properties, such as antioxidant, anti-inflammatory, and anti-apoptotic activities [57,75,158].These effects have shown promise in safeguarding against various degenerative and chronic diseases such as cardiovascular disorders and cancer [76,77].They have

Quercetin To evaluate the effect of quercetin on testicular toxicity and spermatogenesis defects in Wistar rats
The experimental outcomes demonstrated (75 mg/kg) quercetin's role in treating testicular toxicity by enhancing testicular histology and decreasing germ cell apoptosis [37] Epigallocatechin-

3-gallate (EGCG) To evaluate the effect of EGCG (80 mg/kg) on spermatogenic degeneration and testicular damage in mice
The experimental results indicated that EGCG played a role in recovering testicular structure and function, addressing spermatogenic degeneration [52,53] Astragalin To evaluate the effect of astragalin on testicular apoptosis caused by ER stress in varicocele rats The investigation revealed that astragalin (200 mg/kg) reduced oxidative stress and prevented testicular injury, highlighting its role in mitigating ER stressinduced apoptosis [56] Rutin To evaluate the effect of rutin on oxidative stress and cisplatin-induced reproductive toxicity in mice The study's findings showed that rutin (200 mg) reduced oxidative stress and prevented testicular injury, indicating its role in counteracting cisplatin-induced reproductive toxicity [40] Morin To evaluate the effect of morin on spermatogenesis and oxidative stress in rats The findings highlighted that morin (30 mg/kg) improved testicular function and sperm quality, demonstrating its role in enhancing spermatogenesis and reducing oxidative stress [47,133] Naringenin To evaluate the effect of naringenin (40 mg/kg) on spermatogenesis in rats The research outcomes demonstrated that naringenin improved sperm production and quality, underscoring its role in boosting spermatogenesis [49] Icariin To evaluate the effect of icariin (10 μM in vitro) on Sertoli cell proliferation and testicular apoptosis The experimental data confirmed that icariin promoted Sertoli cell proliferation and reduced testicular apoptosis, showing its role in supporting Sertoli cell function [95] Hesperetin To evaluate the effect of hesperetin (50 mg/kg) on testicular apoptosis and oxidative stress in diabetic rats The investigation showed that hesperetin lowered oxidative stress markers and apoptotic cell death in the testes, emphasizing its role in combating diabeticinduced testicular damage [73] Puerarin To evaluate the effect of puerarin (PU) on cadmium (Cd)-induced testicular lactic acid metabolism disruption in Sprague-Dawley rats The study investigated how Cd exposure led to testicular shrinkage, reduced testosterone, lactic acid, and pyruvate levels, weakened antioxidant defenses, increased oxidative stress, and disrupted energy metabolism and signaling pathways.PU treatment counteracted these effects by restoring lactic acid production and mitigating Cd toxicity in the testis [140] Kolaviron To evaluate the effect of kolaviron on busulfan-induced testicular dysfunction and cognitive deficits in male rats The study explored the effect of kolaviron on busulfan-induced reproductive deficits in male rats.Kolaviron mitigated adverse effects on testicular health, spermatogenesis, androgen levels, and sperm integrity, highlighting its role in alleviating male infertility [141] Icariside II To evaluate the effect of icariside II on post-prostatectomy erectile dysfunction in rats The research found that icariside II aids in the recovery of erectile function after cavernous nerve injury by targeting multiple molecular pathways, including NOS/NO, oxidative stress, RhoA/ROCK, TGF-β, Shh, and H2S, and promoting nerve regeneration and functional recovery [142] Dietary carob fruit To evaluate the effect of dietary carob fruit on spermatogenesis and fertility in aging broiler breeder roosters The study found that dietary carob fruit supplementation enhanced spermatogenesis and semen quality, improved sperm motility, and antioxidant capacity in semen and blood, and prevented embryonic mortality, suggesting benefits for reproductive health and performance due to its antioxidant effects [143] Table 2 (continued)

Acacetin
To evaluate the effect of acacetin on cisplatin-induced reproductive and renal toxicity in mice The study revealed that acacetin, a natural flavonoid, protects against cisplatininduced inflammation, oxidative stress, and damage to the male reproductive system and kidneys.Acacetin therapy improved sperm quality, testicular function, and kidney health, suggesting its potential as a therapeutic agent to mitigate these adverse effects [144] Tangeretin To evaluate the effect of tangeretin on erectile and testicular dysfunction in hypertensive rats The study demonstrated that tangeretin improved erectile function and testicular health in hypertensive rats by reducing oxidative stress and inflammation, increasing nitric oxide availability, and regulating the renin-angiotensin system, indicating its therapeutic benefits in managing hypertension-induced reproductive dysfunction [145] Hesperidin To evaluate the effect of hesperidin on varicocele-induced testicular damage in rats The research showed that hesperidin decreased pro-apoptotic gene expression and increased anti-apoptotic gene expression in the testicles of male rats with varicocele, suggesting its potential as a medication to protect against testicular injury and improve fertility in varicocele cases [146] Genistein To evaluate the effect of genistein on mono-(2-ethylhexyl) phthalate (MEHP)induced testicular injury in embryonic rats The study demonstrated that a modest dosage of genistein reduced oxidative stress and apoptosis in embryonic testis tissues, alleviating testicular injury caused by MEHP by activating the Nrf2/HO-1 signaling pathway, enhancing the cellular antioxidant response and mitigating MEHP's harmful effects [147] Soy isoflavones To evaluate the effect of soy isoflavones on spermatogenesis in diet-induced obese mice The research discovered that soy isoflavones significantly improved sperm quality and quantity by activating the Nrf2/HO-1 signaling pathway, enhancing antioxidant defenses, and reducing oxidative stress in the testes, suggesting their benefit for improving reproductive health in cases of obesity-induced spermatogenic abnormalities [148] Vitexin To evaluate the effect of vitexin on diabetes-associated infertility and sexual dysfunction in male mice The study found that vitexin improved sexual dysfunction and reproductive deficits caused by diabetes by regulating the hypothalamus-pituitary-gonadal (HPG) axis, increasing testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), enhancing sperm count, motility, and testicular health by boosting antioxidant activity and reducing inflammation [118] EGCG: Epigallocatechin-3-gallate, ER: Endoplasmic Reticulum, NOS/NO: Nitric Oxide Synthase/Nitric Oxide, ROCK: Rho-Associated Coiled-Coil Kinase, TGF-β: Transforming Growth Factor Beta, H2S: Hydrogen Sulfide, Nrf2/ HO-1: Nuclear Factor Erythroid 2-Related Factor 2/Heme Oxygenase-1, HPG: Hypothalamus-Pituitary-Gonadal.FSH: follicle-stimulating hormone, LH: luteinizing hormone become known as viable treatment choices in recent years for problems with the male reproductive system [78].
Oxidative imbalance plays a major part in apoptosis, lipid peroxidation, DNA damage, and decreased sperm motility [79].Flavonoids exhibit antioxidant activity [80].Flavonoids can penetrate lipid membranes being amphipathic molecules and prevent oxidative damage.This helps to protect the acrosome and spermatozoa membrane to ensure the sperm acrosome reaction essential for fertilization [81].Studies reveal that increasing antioxidant enzymatic activity in rooster semen and boar sperm with 1.0 mM rutin and 0.010 mg/mL quercetin, respectively, reduced reactive oxygen species (ROS) buildup and malondialdehyde generation [82].In a study, it was found that the negative effects of hydrogen peroxide on total antioxidant activity, semen motility, viability, malondialdehyde, and nitric oxide were reversed on the administration of 40 and 80 μM quercetin in addition to hydrogen peroxide [83].Furthermore, the motility of spermatozoa in swine semen experiences a significant decrease when exposed to concentrations of quercetin as high as 0.50 and 0.75 mM.This is likely a result of the compound's ability to heighten the accumulation of calcium by reducing the function of the enzyme known as 'Ca 2+' adenosine triphosphate enzyme ('Ca 2+ ATPase') [84].Flavonoids possess antioxidative properties that can effectively safeguard the proper functioning of the male reproductive system.This protection extends beyond laboratory studies to in vivo conditions.According to one study, the toxicity caused by acetonitrile to the male reproductive system can be mitigated by providing 468 mg/kg of apigenin for 12 weeks.Glutathione to glutathiol (GSH/ GSSG) ratio levels are aided by this supplementation in returning to levels that were able to compare with the control group [85].ACP (Alkaline phosphatase), TCP (Testicular acid phosphatase), and lactate dehydrogenase (LDH) all showed significant increases in rats given 30 mg/kg rutin for five weeks following cadmium exposure.These enzymes are closely associated with the process of spermatogenesis.A study highlighted that rutin effectively protected against abnormalities in testicular maturation and spermatogenesis [86].In accordance with the previous studies, superoxide dismutase 1 (SOD1), SOD2, and SOD3 are essential for scavenging superoxide anions under normal physiological conditions.This process produces hydrogen peroxide (H 2 O 2 ), which is a relatively stable form of active oxygen [87].It may be possible to reverse the reduction in SOD3 and SOD1 mRNA expression levels brought on by nicotine administration with a 35-day course of 75 mg/kg icariin [69].Even though numerous studies have shown the advantages of flavonoids, some have questioned their use in vivo [88].
Research conducted by Lotito SB and Frei et al. has concluded that flavonoids' in vivo absorption is restricted and that their antioxidant action appears to be minimal [70].Their limited ability to act as antioxidants within the body does not affect their usefulness as an alternative to the culture medium during cryopreservation of sperm [89].It is recommended that in future research the challenges of limited in vivo bioavailability of flavonoids can be addressed.
Spermatogenesis depends on the accurate control of cell death in testicular cells, which is advantageous to the organism as a whole.Rutin at 10, 20, and 40 mmol/L may be able to successfully shield Leydig cells from H 2 O 2 -induced cell death, according to recent research [90,159].The germinal cells in the testis that are TUNEL positive got reduced on administration of 50 mg/kg of hesperetin to diabetic rats for 46 days [91].The research highlights the significance of mitochondria in controlling cell death in the testes.Any alteration in metabolism or mitochondrial structure can cause the death of germ cells via the intrinsic apoptosis pathway dependent on mitochondria [92].There is encouraging evidence that hesperetin, rutin, and morin can stop testicular apoptosis by lowering caspase 3(cysteine aspartic acid-specific protease 3) levels and Bax (BCL2-associated X) and raising Bcl-2 (B-cell leukemia/lymphoma 2) protein levels [93][94][95][96][97][98].Furthermore, giving rats with diabetes 50 mg/ kg of hesperetin for 46 days may increase mitochondria membrane potential (MMP) and decrease DNA fragmentation.These results imply that the medication may have subcellular level anti-apoptotic and protective qualities [99].The PI3K/Akt pathway, also known as the phosphoinositide 3 kinase/Akt/protein kinase B pathway, has the function of promoting cell survival and preventing cell death.Rutin at 40 mmol/L activates the PI3K/Akt signaling pathways and inhibits cell death in Leydig cells induced by H 2 O 2 [100].It has been demonstrated that rutin offers protection against apoptosis brought on by restraint stress and damage to the testicles.Moreover, it has been noted that giving mice 200 mg/ kg of rutin for 15 days inhibits the breakdown of caspase 3 and PARP-1in the mice testes [101].According to studies, DNA may be protected by deoxyribonuclease I (DNase I) inhibitors.But as of right now, there are not many organic DNase I inhibitors, either synthetic or natural [102].Rutin, a flavonoid, had inhibitory concentration (IC 50 = 137.17± 16.65 μM), effectively inhibits testicular cell death.Its ability to inhibit DNase I, along with ascorbic acid, makes it a valuable dietary antioxidant.In addition, rutin seems to inhibit DNase I activity more successfully than ascorbic acid.This implies that rutin, which inhibits DNase I and preserves sperm DNA, might be a dietary element that helps prevent male infertility [103,104,160,161].Furthermore, a lower percentage of high-quality embryos and a higher incidence of miscarriages have been connected to damage to sperm DNA [105,[162][163][164].Rats receiving a 13-day therapy of 75 mg/kg of rutin were able to reverse the increased comet count, tail lengths, tail moment, and tail percentage induced by cisplatin.ATM (ataxia telangiectasia mutated) activation plays a role in the checkpoint mechanism of cell cycle and DNA damage repair.In zygotes fertilized with H 2 O 2 -treated sperm, Epigallocatechin gallate (EGCG) (10 μg/mL) reduces ATM expression and prevents DNA damage by controlling the cell cycle [106].Moreover, testicular cell death is the result of endoplasmic reticulum stress (ERS), which can cause pro-death or pro-survival reactions in response to stress [107].Sustained endoplasmic reticulum stress can mainly cause the death of germ cells via the signaling pathways Inositol-requiring enzyme type 1(IRE1)/Junction N-terminal kinases (JNK) and protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor-2α (eIF-2α).Junction N-terminal kinases (JNK) activation is one of these pathways that, in testicular injury, activates the mitochondrial-dependent intrinsic apoptotic mechanism.MAS is a blend of monotropein and two flavonoid compounds, Spiraeoside and astragalin.When varicocele animals are administered 200 mg/kg of MAS, the components of the IRE1 pathway, p-JNK, p-IRE1, and glucose-regulated protein 78 (GRP78), are downregulated.Furthermore, MAS prevents mitochondrial-dependent intrinsic apoptosis, which in turn reduces the levels of cleaved caspase 3 and Bax/Bcl-2 ratio in germ cell death [108].Astragalin in MAS could be able to prevent mitochondrial apoptosis caused by ERS.Studies like the ones stated above have shown that flavonoids can dramatically lower apoptosis.It suggests that it has preventive and subcellular level anti-apoptotic properties.

The role of flavonoids on Sertoli cells and bloodbrain barrier
Blood-testis barrier (BTB) is a structure produced by Sertoli cells (SCs) to protect germ cells from toxic chemicals and provide the ideal environment for spermatogenesis.The sperm production process depends on the BTB's integrity.When certain proteins associated with the blood-testis barrier (BTB), such as claudin-11, occludin, connexin 43 (Cx43), N-cadherin, and occludens-1 (ZO-1), are downregulated, the BTB can become damaged.This can lead to limitations or disruptions in spermatogenesis.However, a flavonoid called luteolin has been shown to protect Sertoli cells (SCs) and the integrity of the BTB by increasing the expression of several downstream antioxidant genes and the protein expression of Cx43occludin, ZO-1, and claudin-11.A concentration of 10 µM of luteolin has been found to be effective for this purpose.Icaridin, a flavonoid, affects male rats' reproductive systems in a variety of ways.It can affect spermatogenesis at a dose of 100 mg/kg by regulating the expression of claudin-11 mRNA in SCs.However, taking too much icariin might have negative consequences such as oxidative damage to organs and tissue, which can then affect reproductive processes.Research has indicated that Cx43 is a new avenue for reversing malfunction in the male reproductive system.More specifically, Cx43 is essential for controlling oxidative stress, spermatogenesis, signal transmission, and BTB integrity.Sperm motility decreases when Cx43 is lost in SCs [109].It is essential for the prevention and treatment of male reproductive disorders and to find an effective and safe medication that can restore BTB's function, 10 µM luteolin restored the BTB's integrity and increased the expression of Cx43 in the triptolide-induced reproductive damage model Flavonoids are the most effective natural substances for treating abnormalities in spermatogenesis and problems related to the male reproductive system.

Role of flavonoids on Leydig cells and testosterone
The cAMP/PKA signaling pathway, also known as cyclic adenosine 3' ,5'-monophosphate/cAMP-dependent Protein Kinase-A, has been extensively researched for its role in regulating testosterone release.To this end, several flavonoids have shown the capacity to inhibit signal transduction mediated by Cyclooxygenase-2(COX-2), including 10 μM of luteolin, apigenin, and quercetin.It has been demonstrated that this inhibition efficiently increases the cAMP/PKA signaling pathway-dependent processes of steroidogenesis and expression of the StAR gene in Leydig cells as indicated in Fig. 1 representing the target pathway of flavonoids.Moreover, Li et al. explored that adding 10 μM apigenin may prevent Leydig cells of mice from exhibiting COX-2-dependent signaling.This is accomplished by inhibiting the thromboxane A2 receptor and suppressing gene-1 (DAX-1).As a result, in intending cells of aged mice, this inhibition causes an increase in the expression of the StAR gene and the steroidogenesis process overall.It should be mentioned that DAX-1 inhibits the StAR transcription, which reduces cAMP activation [110].Furthermore, Andric et al. suggested that the regulation of steroidogenic activity in the testis interstitial cells that produce testosterone may be influenced by the NO/cyclic guanosine monophosphate (NO/ cGMP) signaling pathway.Saraiva et al. revealed that Leydig cells have demonstrated the presence of phosphodiesterase type 5 (PDE5), indicating that PDE5 inhibitors (PDE5-Is) might play a role in enhancing testosterone secretion and steroidogenic pathways [111].A10 μM concentration of icariin, a PDE5-I, has the ability to raise nitric oxide synthase in the corpus cavernosum tissue of diabetic rats that are experiencing erectile dysfunction (ED), which may increase cellular cGMP levels [112].

Testicular inflammation and flavonoids
Male infertility can be effectively linked to inflammation as the primary cause, despite the immune privilege typically enjoyed by the testis.Numerous studies have established a connection between the start of pathways that trigger inflammation and signal transduction factors activation, such as nuclear factor kappa (NF-κB) is a transcription factor that regulates the expression of specific genes that promote inflammation Several genes involved in inflammation development such as interleukin-6, inducible nitric oxide synthase, and tumor necrosis factor alpha, can be regulated by activated NF-κB.Compared to other NO synthases, an increase in iNOS may result in the production of more toxic nitric oxide, which may harm testicular tissues or cells TNF-α is important in regulating spermatogenesis under normal conditions and also stimulates the production of other inflammatory mediators [113].Moreover, Wang et al. found that TNF-α has the potential to exacerbate testicular inflammation by promoting the synthesis of IL-6.Han et al. explored that astragalin (30 mg/kg) was given to diabetic mice over the course of an eight-week study.Astragalin is well recognized for its capacity to increase antioxidant activity and lower inflammatory cytokine levels.Additionally, it suppresses NF-κB expression, which lowers NO levels in the testicles and reduces the generation of iNOS.Moreover, Skondras et al. found that apigenin at 10 mg/ kg dramatically decreases TNF-α levels and especially IL-10 in terms of immunoreactivity in rats suffering from testicular ischemia-reperfusion injury.The important endogenous anti-inflammatory cytokine IL-10 is essential for regulating and inhibiting the production of TNFα.In testicular cells, hesperetin has the ability to inhibit caspase 3. Four distinct signal transduction pathways i.e., ERK, inhibitory-κB kinase (IKK) α and Nuclear Factor-κB (NF-κB)-inducing kinase (NIK), JNK, and p38 mitogenactivated protein kinases (p38MAPK) are used to reduce the expression of NF-κB in order to produce this inhibitory effect.To achieve this, doxorubicin-treated rats were administered with 100 mg/kg hesperetin for a duration of five weeks.Furthermore, research investigations have shown that COX-2 can increase the production of TNFα [114] 0.100 mg/kg Rutin was used to lower the COX-2 and TNF-α expression in rats whose reproductive systems had been damaged by HgCl 2 [115].This implies that by lowering oxidative stress, rutin may have anti-inflammatory properties as a result, consuming these flavonoids can aid in the reduction of testicle inflammation and help shield male reproductive problems.
Ijaz et al. studied a natural dietary flavonoid, eriodictyol, and its role in inhibiting reproductive dysfunctions which was induced by furan.The forty-eight male rats were divided into four groups: furan (10 mg/kg), eriodictyol (20 mg/kg), furan (10 mg/kg) + eriodictyol (20 mg/ kg), and untreated/control.The results on the 56th day were evaluated and it was found that eriodictyol reduced testicular toxicity by improving sperm count, viability, and mobility.Therefore, the study concluded the essential role of eriodictyol in increasing anti-apoptotic markers of testicles, LH, and FSH levels which in turn proved as a potential candidate for clinical trials in the treatment of testicular damage [116,117].
One of the recent studies conducted by Amevor et al. revealed that a combination of vitamin E and quercetin led to a synergistic effect on antioxidant, reproductive, and immune capabilities in aged male breeder chickens as quercetin protects the plasma membrane of spermatozoa.One hundred and sixty breeder male chickens of the Tianfu breed, aged sixty-five weeks, were divided randomly into four groups of ten birds each.For a period of eleven weeks, the birds were fed with basal diet, Q (0.4 g/ kg), VE (0.2 g/kg), and Q + VE (0.4 g/kg + 0.2 g/kg) diets.The results concluded that altogether intake of vitamin E and quercetin was responsible for higher biosynthesis and metabolism of testosterone resulting into increased spermatogenesis, reproductive hormones, high libido, and improved sperm quality [139].

Bioavailability challenges of flavonoids
Manach et al. discuss apigenin's bioavailability challenges (30%), highlighting its potential to protect sperm from oxidative stress-induced damage, which could enhance male fertility.However, its therapeutic application is hindered by poor absorption, prompting research into advanced delivery systems to improve its efficacy [119].Similarly, Manach et al. review the bioavailability issues of quercetin (< 10%), noting its benefits but limited therapeutic use due to rapid metabolism and low absorption.Strategies such as co-administration with synergistic compounds and nano-formulations are being explored to enhance its bioavailability.Quercetin has demonstrated potential in treating testicular toxicity by enhancing testicular histology and decreasing germ cell apoptosis [119].Xiao et al. emphasize luteolin's potential to manage inflammation and strengthen male reproductive health by protecting Sertoli cells but cite its limited bioavailability due to rapid breakdown and poor solubility.It could increase its clinical effectiveness by improving its stability and solubility through advanced drug delivery methods [120].Tang et al. discuss astragalin's anti-inflammatory and antioxidant effects but note its poor bioavailability due to rapid metabolism and low solubility.Liposomal and nanoparticle-based delivery systems aim to overcome these challenges [121].Riva et al. highlight rutin's pharmacological activities, including spermatogenesis and anti-inflammatory effects, while addressing its bioavailability limitations (20%) arising from rapid metabolism and poor absorption.Formulation strategies such as phospholipid complexes and cyclodextrins are being investigated to enhance their bioavailability [122].Munnangi et al. review various drug complexes and highlight advancements in enhancing the bioavailability of poorly soluble compounds like rutin.The study discusses using lipid-based formulations, such as phospholipid complexes and nanostructured lipid carriers, which significantly improve solubility and absorption.Wang et al. emphasize myricetin's neuroprotective and `reproductive health benefits but acknowledge its limited therapeutic use due to weak solubility (9.70%) and substantial first-pass metabolism.Research into myricetin-loaded nanoparticles aims to improve its bioavailability and therapeutic benefits [123][124][125].Li et al. ( 2018) review Icariin's efficacy in bone health and sexual dysfunction, noting challenges in bioavailability (30.4%) due to fast metabolism and low solubility.Techniques such as polymeric nanoparticles and lipid-based formulations are explored to enhance their bioavailability [125][126][127][128]. Chen et al. discuss hesperetin testicular and anti-inflammatory benefits but identify its low bioavailability (20%) as a limitation due to weak solubility and rapid metabolism.The development of hesperetin-loaded nanoparticles aims to enhance its therapeutic potential [126,129].Lambert et al. discuss EGCG's potent antioxidant and anticancer properties but highlight its poor bioavailability (< 10%) due to low stability and rapid metabolism.Investigation into prodrugs and lipid-based carriers seeks to improve their bioavailability [127,165].Kim et al. review menotropin's anti-inflammatory potential but point out its poor bioavailability (10%) due to low solubility and rapid metabolism, necessitating advanced delivery methods for effective therapeutic use [128,166].Chen et al. discuss spiraeoside's antioxidant and anti-inflammatory properties but note its bioavailability challenges due to limited solubility and gastrointestinal instability, hindering its absorption and effectiveness [129,[167][168][169].
The reviewed literature highlights that many natural compounds-such as apigenin, quercetin, luteolin, astragalin, rutin, myricetin, icariin, hesperetin, EGCG, monotropin, and spiraeoside-have significant therapeutic potential for conditions like oxidative stress, inflammation, and reproductive health.However, due to their quick metabolism and low solubility, their poor bioavailability limits their clinical utility.Research is concentrating on developing new drug delivery methods, such as phospholipid complexes, liposomal carriers, and nanoparticles, to improve medication absorption, stability, and efficacy to solve these problems.

Future perspectives
The potential for flavonoids to be a significant player in the treatment of male reproductive dysfunction is indeed promising.To truly unlock their full therapeutic potential, further investigation is required to uncover the intricate molecular mechanisms and pathways by which flavonoids bring about their effects.By enhancing the bioavailability of these compounds through innovative delivery systems like nanoparticles, we can see a substantial improvement in their efficacy.However, thorough clinical trials involving human participants are essential to definitively establish the safety and efficacy of flavonoids for this specific application.Investigating the potential synergistic effects of flavonoids when combined with other treatments, as well as delving into personalized medicine approaches, holds the key to optimizing therapeutic outcomes.Moreover, incorporating a diet rich in flavonoids may offer preventive benefits against reproductive disorders.By exploring a variety of sources for these beneficial compounds, the scope of natural therapeutics can be broadened.Ongoing research and innovation are crucial in fully harnessing the potential of flavonoids within the realm of mainstream medical practice, especially in the treatment and prevention of male infertility.

Conclusion
A number of factors, such as oxidative stress, excessive sperm, and testicular cell apoptosis, compromised Leydig cell and stem cell function, and inflammation within the testicles, can lead to dysfunction in the male reproductive system.These elements lower the quality of sperm, interfere with testosterone synthesis, and have a detrimental effect on the environment that promotes sperm production.A class of molecules called flavonoids is present in many fruits and vegetables and has a variety of pharmacological effects.Numerous studies reported the potential effects of flavonoids for the treatment of male reproductive system disorders.Nrf2, a transcription factor, is also essential for controlling cellular defense systems against oxidative damage.Two essential genes are involved in this regulatory mechanism: NAD(P)H quinine oxidoreductase 1 and heme oxygenase 1. Notably, Cx43, a protein closely associated with the above two genes in somatic cells, can be activated by flavonoids due to its antioxidative properties.Therefore, further research is imperative to fully comprehend the molecular processes underlying the interaction of Cx43, flavonoids, and Nrf2 in somatic cells.Due to their therapeutic potential, flavonoids could be developed into future medications for treating male reproductive system dysfunction.One limitation of flavonoids is their limited bioavailability, as they are rapidly metabolized in the liver and intestinal mucosa.Thus, further research should focus on overcoming these limitations pertaining to solubility and stability.The design and development of novel formulations containing flavonoids can be a more effective strategy to treat male reproductive system disorders.

Fig. 1
Fig. 1 Schematic representation of the pathway target of flavonoids

Table 1
Flavonoids and their mechanisms of action in male reproductive health