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Epimedium

Nov. 27, 2024

Understanding Epimedium: A Traditional Herb with Multifaceted Benefits

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Thorough Exploration of Traditional Applications and Modern Investigations of Epimedium

Epimedium Folium has been a staple in traditional medicine in China for a considerable duration. This narrative review reviews literature on Epimedium and its various metabolites from the previous decade, harnessing resources like PubMed. By systematically organizing and synthesizing meaningful research outcomes, including disease models and pharmacological effects, it elucidates the primary pharmacological roles of Epimedium in enhancing reproductive health, supporting bone vitality, reducing inflammation, and combating cancers and viral diseases. This review thus enriches the understanding of advancements in Epimedium research.

1 Introduction

The herbal remedy Epimedium Folium, recognized by several names like Xian Lingpi and nine leaves on three stems, is sourced from the dehydrated foliage of Epimedium brevicornu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim., or Epimedium Koreanum Nakai. This traditional tonic has its roots chronicled in the Shennong Materia Medica Classic and is prevalently found across China. It flourishes in temperate and subtropical climates, favoring shaded and moist habitats. Common locales for Epimedium Folium include understory forests, shrubs by ditches, or humid zones on mountainous terrains.

This herbal remedy is extensively used in China, Japan, and South Korea due to its medicinal properties, which encompass kidney enhancement, aphrodisiac effects, and bolstering muscle and bone strength, while also dispelling wind and dampness. Recent investigations have isolated flavonoids, lignans, and alkaloids from Epimedium, with over 130 distinct metabolites identified, including various flavonoids and icarisides. Flavonoids, notably, possess significant pharmacological activities. The predominant pharmacologically active metabolites within Epimedium include Icariin, Epimedin A, Epimedin B, Epimedin C, Icariside II (Baohuoside II), and Icaritin (Chen et al.). According to the Chinese Pharmacopoeia (Edition), Icariin, Epimedin A, Epimedin B, and Epimedin C serve as quality control markers for Epimedium (The Free Encyclopedia) (Figure 1) (Table 1). Furthermore, polysaccharides also contribute to the active content in Epimedium, with the polysaccharides being complex carbohydrates primarily composed of mannose, rhamnose, galacturonic acid, glucose, and galactose (Ke et al.). Their pharmacological effects include immune regulation (He et al.) and antioxidant properties (He et al.).

Figure 1

Figure 1. Chemical structures: Icariin (A), Icariside II (B), Icariside I (C), Epimedin A (D), Epimedin B (E), Epimedin C (F), Wushanicaritin (G), Icaritin (H), Desmethylicaritin (I), Baohuoside II (J), Anhydroicaritin (K).

Table 1

Table 1. Key metabolites extracted from the Epimedium genus.

Numerous in vivo and in vitro studies, along with clinical applications, have substantiated that Epimedium Folium exhibits reproductive, bone-protective, anti-cancer, anti-inflammatory, and anti-oxidative stress activities. For example, it preserves bone integrity by balancing bone metabolism (Xu et al.) and also aids reproductive health by inhibiting PDE5 activity (Chen et al.) or repairing penile tissues, while demonstrating anti-cancer and anti-inflammatory effects by modulating pathways like PI3/AKT and oxidative stress. This paper will delve into the pharmacological actions and underlying mechanisms of Epimedium Folium.

2 Traditional Uses

According to traditional Chinese medicine principles, Epimedium Folium is considered spicy and sweet in flavor with mild properties. During the Northern and Southern Dynasties, it was reported that a shepherd noticed increased mating behavior in male sheep after they consumed a wild grass. This observation was relayed to the renowned medical professional Tao Hongjing, who verified the aphrodisiac effect of the wild grass, which was subsequently termed Epimedium Folium and included in the pharmacopoeia. Additionally, the illustrious poet Liu Zongyuan from the Tang and Song dynasties reportedly utilized Epimedium to address leg ailments caused by rheumatism. Thus, Epimedium Folium is recognized for its benefits of tonifying the kidneys and strengthening yang energy while dispelling wind and dampness. Traditionally, it can be combined with other kidney-tonifying and aphrodisiac herbs such as Rehmanniae Radix Praeparata, Lycii Fructus, and Curculigo orchioides (Ma et al., a). It can also be transformed into wine by soaking 0.5 kg of Epimedium Folium in 5 kg of alcohol for 10 days to treat conditions like bone pain, numbness, and impotence, as recorded in the Food Medicine Heart Mirror.

Growing interest in Epimedium Folium has arisen from its perceived effectiveness in treating bone and reproductive ailments, leading to meticulous scientific investigations surrounding its benefits. Epimedium Folium demonstrates potential in curtailing bone degeneration, enhancing bone density, and functioning as a natural PDE5 inhibitor. Its plant metabolites help maintain the equilibrium of human bone metabolism and regulate hormone levels critical for male erections while promoting cellular growth. Furthermore, ongoing studies have unveiled that Epimedium Folium and its metabolites possess notable anti-cancer (Sun et al.), anti-inflammatory (Huang et al.), and antiviral (Cho and Ma) activities.

3 Pharmacological Activity

3.1 Bone Protection

Healthy bones are dynamic tissues, and their functioning relies heavily on the balance of bone metabolism, involving osteoblasts and osteoclasts. Epimedium supports bone metabolism in various ways.

3.1.1 Bone Protection via Osteoporosis Models

Osteoporosis is a prevalent systemic skeletal condition typically treated with hormone therapies, anabolic agents, or bisphosphonates. Nonetheless, prolonged use of these medications can trigger significant adverse effects in patients. Hence, natural herbal options are being explored as alternatives (Gao and Zhang).

3.1.1.1 Estrogen Pathway

Imbalanced bone metabolism is among the leading causes of osteoporosis. Researchers identified through meta-analyses that Epimedium enhances bone mineral density (BMD) and alleviates discomfort in osteoporosis patients by modulating bone metabolism, while also significantly reducing alkaline phosphatase (ALP) levels, showcasing promising clinical outcomes (Shi et al.). Diminished estrogen levels disrupt bone metabolism, which is a principal factor in menopausal osteoporosis (Yong et al.), and Epimedium aids in the regulation (Li et al.). It achieves this by modulating bone metabolism proteins like TRAF6 through an estrogen-dependent mechanism.

Clinical studies affirm the safety of Icariin (ICA) in humans as supported by randomized, double-blinded investigations. Following the administration of ICA, postmenopausal women showed elevated serum levels of ICA metabolites, along with an increase in the bone synthesis marker BSAP and a decrease in TRAF6 (a crucial adaptor protein mediating RANKL/RANK signaling), indicating that ICA inhibits the enhancement of osteoclast function resulting from low estrogen concentrations (Yong et al.). Moreover, findings reveal that Icaritin (ICT) suppresses osteoclast formation in a dose-dependent fashion in an ovariectomized rat model. ICT undermines osteoclast activity by downregulating TRAF6 and inhibits bone resorption, counteracting changes in femoral mechanical properties provoked by OVX and terminating the acceleration of osteoclast formation and functioning (Tan et al.). The NF-kB and MAPK/AP-1 pathways have been highlighted as pivotal concerning ICT's restoration of bone metabolism disrupted by OVX (Indran et al.; Tan et al.). These studies imply Epimedium's ability to obstruct osteoclast activity by manipulating TRAF6, a core protein involved in bone metabolism, thereby exerting anti-osteoporosis benefits (Figure 2).

Figure 2

Figure 2. The traditional mechanism for bone metabolism.

One study indicates that ICA is more effective than the flavonoid compound genistein with respect to osteogenesis, potentially owed to the isoprene group present in the C-8 position of ICA, while genistein's efficacy is contingent upon its high affinity for estrogen receptor (ER) interactions. However, ICA shows a lower affinity than genistein for the ER spectrum. Therefore, it can be inferred that ICA's osteogenic impact may operate via non-estrogenic mechanisms (Ming et al.). Additionally, ICT, the enzymatically hydrolyzed derivative of ICA, exhibits a preventable impact on osteoclast formation, which persists even after administering estrogen receptor antagonists like ICI (Tan et al.). This suggests that the actions of Epimedium and its isolated extracts may not solely depend on estrogen receptor channeling.

3.1.1.2 Neuropeptide-Related Mechanisms

In studies using OVX models, serum levels of estrogen (E2), Vitamin K-dependent protein (BGP), and Osteoprotegerin (OPG) increased in rats treated with Epimedium (0.27, 0.81, 2.43 g/kg/day). The mRNA expression levels of related neuropeptides in the bone, brain, and spinal cord were significantly elevated, enhancing spinal CGRP signaling and promoting bone growth (Liu, H. et al.). Furthermore, neuropeptide Y (NPY) (Driessler and Baldock; Horsnell and Baldock) from the hypothalamus, calcitonin gene-related peptide (CGRP) (Liang et al.), substance P (SP) (Chen et al.), and vasoactive intestinal peptide (VIP) (Mukohyama et al.) found in the skeletal sympathetic nerve fibers, all contribute to maintaining equilibrium in bone metabolism. Hence, Epimedium may uphold the balance of bone metabolism via mechanisms associated with various neuropeptides involved in regulating the brain/spinal cord/bone axis.

3.1.1.3 Regulation of Glucocorticoid Release

Secondary osteoporosis is frequently instigated by glucocorticoids. Research utilizing glucocorticoid (GC)-induced osteoporosis (GIOP) rat models indicated that delivering Epimedium and Fructus Ligustri Lucidi (100, 200 mg/kg/day) amplified levels of bone-forming markers, including alkaline phosphatase (ALP), BGP, and bone mineral content (BMC), while concurrently lowering levels of tartrate-resistant acid phosphatase (TRACP) produced by osteoclasts. This remedy helped to abate glucocorticoid-induced bone atrophy and the decline in BMD. The preventive impact of Epimedium paired with Fructus Ligustri Lucidi against GIOP closely associates with the TGF-β1/Smads signaling pathways (Yang et al.). Furthermore, administration of Epimedium (10 mL/kg) in early SANFH rat models exhibited increased BMD, preventing osteoporosis-related collapses while curtailing cellular autophagy by diminishing the expression of relevant autophagy proteins, thereby showcasing bone protective properties (Liu, S. et al.). Moreover, studies have demonstrated that ICA (5 × 10-5, 1 × 10-4 M) facilitation improved EVs activity and repaired glucocorticoid-triggered injuries to bone microvascular endothelial cells (BMECs) (Zhang et al.). These findings propose that Epimedium may be a prospective candidate for the treatment of glucocorticoid-induced bone ailments.

3.1.1.4 Hypoxic Pathway

Bone hypoxia contributes to both primary and secondary osteoporosis. ICA (10-7, 10-6, 10-5 M) can advance the differentiation of bone marrow stem cells (BMSCs) into osteoblasts while alleviating oxidative stress and apoptosis of osteoblasts in hypoxic circumstances, preserving their functional capabilities (Ma et al.).

Extensive preclinical evaluations indicated that Epimedium and its key plant metabolites displayed anti-osteoporosis properties. While varied animal models were employed, they predominantly included rodents. To overcome this limitation, some researchers opted for a non-rodent species, Oryzias latipes, for constructing an osteoporosis model via RANKl induction, assessing the bone protective impact of ICA using designated technical procedures. The research disclosed that ICA (2.5, 5, 10, 15, 20, and 25 µM) mitigated matrix mineralization damage and exhibited therapeutic effects akin to standard control treatments (25 µg/mL, Alendronate), effectively curbing RANKL-induced bone loss (Pham et al.). This affirms that Epimedium reduces bone loss in non-rodent subjects and possesses anti-osteoporosis properties.

Nonetheless, the National Medical Products Administration of China has reported instances of the 'Zhuanggu Joint Pill' and 'Xianling Gubao Oral Preparation' resulting in hepatotoxicity in clinical settings. Notably, Epimedium is a principal plant metabolite within both formulations. It remains to be clarified whether studies researching the anti-osteoporosis effects of Epimedium meticulously consider and avoid hepatotoxic dosages within animalmodel design. Ensuring therapeutic efficacy while preventing hepatotoxicity is crucial in enhancing clinical integration.

3.1.2 Other Bone Protection

Beyond conventional osteoporosis, Epimedium and its extracts also hold therapeutic potential for bone impairment attributed to other causes. ICT serves as a prospective bone protector, reversing Pb-induced mineralization in bone nodules and fostering BMEC differentiation into osteoblasts. Following ICT (10 µM) treatment, Pb-induced drops in Wnt3a and β-catenin are notably mitigated, facilitating nuclear translocation of β-catenin (Sun et al.). The Wnt/β-catenin pathway is crucial for differentiating BMECs into osteoblasts (Fu Shuping; Oton-Gonzalez et al.). Accordingly, ICT can stabilize bone metabolism and counteract Pb-induced osteopathic damage via activation of the Wnt/β-catenin pathway. Additionally, Epimedium-derived flavonoids (EPF) can mitigate smoke-induced osteoclast activity by bolstering OPG and ALP, hence promoting bone formation and preventing smoke-induced bone depletion (Gao et al.). ICA potentially diminishes wear particle-induced bone absorption post-total joint replacement (Shao et al.). These findings indicate that Epimedium has potential therapeutic outcomes for various bone injuries, although further empirical investigation is warranted for clinical application.

3.2 Reproductive System Protection

3.2.1 Erectile Dysfunction (ED) Treatment

3.2.1.1 Inhibition of PDE5

Erectile dysfunction is a prevalent male sexual ailment characterized by insufficient penile erection to support satisfactory sexual engagement. Oral PDE5 inhibitors (PDE5Is) are the most favored treatment approach for ED. However, due to the side effects associated with PDE5Is, including headaches, visual disturbances, nausea, and diarrhea, seeking natural alternatives for ED management could mitigate these complications.

Epimedium, a classic herbal medicine, is often regarded as a viable choice for enhancing vitality (Figure 3). Recent research has identified that the isoflavones and biflavones present in Epimedium display robust PDE5 inhibitory properties (Dell'Agli et al.; Maschi et al.). This herbal preparation contains various flavonoid compounds, including Epimedium A, B, C, and rhamnosyl icariside II, ICA, and Icariside II (ICA II), which are isopentenyl flavonoid glycosides (Ma et al.). Accordingly, Epimedium exhibits in vitro effects on PDE5 inhibition, marking it as a natural PDE5I (Xin et al.; Zhaojian et al.) (Table 2).

Figure 3

Figure 3. Mechanism of reproductive protection exhibited by Epimedium.

Table 2

Table 2. Common dosages and experimental models in pharmacological research on the bone and reproductive protective effects of Epimedium.

Although ICA's inhibitory potency on PDE5 isoforms is below that of sildenafil (widely used clinical PDE5I), its metabolite (ICA II) exhibits inhibitory effects comparable to 50% of sildenafil's efficacy (Zhang et al.). Subsequent studies confirmed that rhamnose-based ICA II possesses substantial inhibition against PDE5, with 3D-QSAR analysis revealing that the C-8, C-3, C-7, and C-4 substitution sites dictate the levels of PDE5 inhibition (Li et al.). Consequently, a novel semi-synthetic derivative of ICA was developed by altering C-7 and C-8 positions in the ICA structure, yielding comparable inhibitory activities to conventional PDE5Is.

Cyclic guanosine monophosphate (cGMP), a critical element in ED treatment, can have its levels elevated by ICA. Concurrently, ICA effectively inhibits PDE5 mRNA expression (Zhaojian et al.). Additionally, flavonoids derived from Epimedium can activate the cGMP/PKG/Ca2+ pathway, facilitating smooth muscle relaxation in the corpus cavernosum and encouraging penile erection (Li et al.).

In summary, Epimedium and its extracts exhibit notable advantages in safety and effectiveness as PDE5Is. Future endeavors may position Epimedium preparations as viable replacements for existing ED treatments. Nonetheless, current research on Epimedium's SARS-CoV-2 regulation is still nascent, necessitating further experimental documentation to elucidate its mechanism of action.

3.2.1.2 Activation of the NOS/NO/cGMP Signalling Pathway

During the penile erection process, nitric oxide (NO) released from nerve terminals and endothelial cells within the corpus cavernosum plays a pivotal role (Burnett et al.). NO not only stimulates cGMP synthesis (Chen et al.) but also widens penile blood vessels, facilitating blood flow necessary for an erection. ICA promotes NO production (Zhang et al.) and achieves synergistic suppression of eNOS decoupling by enhancing BH4 output, thereby mitigating reductions in NO (Long et al.). Also, ICA regulates NO synthesis through interactions with eNOS and various co-factors like caveolin-1 and HSP90 (Liu et al.). This indicates the NO/cGMP pathway is a mechanism utilized by ICA to enhance endogenous NO generation.

3.2.1.3 Amelioration of Penile Pathological Changes

ICA can remediate damaged cavernous nerves by stimulating the proliferation and differentiation of neural stem cells (NSCs). After ICA treatment, typical neural spheres were observed in naturally aborted fetal striatal single cells, alongside the expression of NSC-associated markers and differentiation proteins (Yang et al.). Moreover, it has been noted that a new arrangement of ICA II could effectively treat neurogenic ED through activation of the Wnt signaling pathway (Gu et al.). Together, these findings indicate that ICA activates both Wnt and basic fibroblast growth factor (bFGF) pathways, resulting in NSC proliferation and differentiation alongside enhanced neuronal production. However, further investigation is essential to clarify the NSC action mechanisms of ICA.

The quality and quantity of endothelial cells, coupled with maintaining healthy and adequate smooth muscle cells (Wespes), are vital for proper penile function. Erection efficacy relies on the regulation of smooth muscle contraction and relaxation (Dean and Lue). Research indicates that both ICA and ICA II can reverse the depletion of smooth muscle cells by modulating TGF-β1 signaling pathways while promoting α-smooth muscle actin (α-SMA) upregulation (Liu et al.; Zhou et al.). Furthermore, they can amplify endothelial cell counts by downregulating TGF-β1, P-Smad2, and total Smad2 expressions in the corpus cavernosum (Liu et al.; Zhou et al.). Researchers hypothesized that the TGF-β1 pathway governs smooth muscle cell action by influencing endothelial progenitor cells (EPCs). Additionally, ICA can enhance endothelial function by adjusting the ERK1/2 signaling pools, counteracting oxidative stress-induced injuries to EPCs while promoting their growth and maturation (Tang et al.). Recent work has confirmed that ICA II can also alleviate excessive mitochondrial autophagy within smooth muscle cells, repairing resultant damage through the PI3K-AKT-mTOR pathway activation (Zhang et al.).

Erectile dysfunction frequently emerges as a complication among diabetes patients (AF et al.; Fedele). Comprehensive studies have verified ICA's role in mitigating EPC dysfunction caused by hyperglycemia by orchestrating p28/CREB and Akt/eNOS/NO pathways. These findings promote the notion that both ICA and ICA II could offer significant therapeutic potential for ED, requiring additional studies to corroborate these hypotheses.

Fibrosis in the corpus cavernosum significantly contributes to erectile dysfunction. Current research displays that ICA II can increase the smooth muscle-to-collagen fiber ratio, effectively ameliorating ED (Zhang et al.). Future investigations may explore the high expression levels of TGF-β1 and connective tissue growth factor (CTGF) (Moreland; Qabazard et al.) discovered in ED rat models, alongside the impact of lysyl oxidase activation resulting in penile fibrosis (Wan et al.).

3.2.1.4 Enhancement of Testosterone Synthesis

Decreased testosterone levels significantly impact ED. As a steroid hormone, testosterone secreted by Leydig cells is critical for male sexual health. Research indicates that Epimedium extracts augment testosterone release from Leydig cells (Sun et al.). ICA promotes testosterone production by invoking the Esr1/Src/Akt/Creb/Sf-1 signaling pathway (Sun et al.; Sun et al.). Additional studies reveal that the NOS/NO/cGMP pathway may serve as an autocrine route for testosterone synthesis, suggesting ICA may also stimulate testosterone secretion through this channel (Andric et al.).

3.2.1.5 Enhancement of Sperm Development and Recovery from Testicular Injury

Oxidative stress is a major contributor to male infertility (Smits et al.), making antioxidant treatment commonplace in clinical approaches. Investigations have revealed that Epimedium extracts restore sperm apoptosis induced by LHRH in male rats through the escalation of superoxide dismutase (SOD) levels, while lowering 8-OHdG (reflecting reactive oxygen species) levels (Park et al.; Zhao et al.). Through its antioxidant actions, Epimedium extracts also reverse testicular tissue wasting due to oxidative stress, while diminishing ROS levels and DNA oxidation (Khan and Ahmed; Munir et al.), regulating P16-CDK6 expression, and enhancing sperm morphology (Zhao et al.). In addition, researchers identified alternative mechanisms whereby Epimedium extracts mitigate oxidative testicular damage in aging male subjects, such as inhibiting the mitochondrial apoptosis pathway by inducing SRIT1-HIF-1α (He et al.) and down-regulating the p53-dependent pathways that handle DNA oxidative distress (Gambino et al.; Speidel; Zhao et al.).

Sertoli cells play an essential role in nurturing sperm growth. Research demonstrates that ICA and ICA II can replenish the decline in Sertoli cell populations caused by streptozotocin and rejuvenate sperm activity (He et al.; Xu et al.). ICA instigates improvements in sperm quantity and quality by triggering ERK1/2 signaling pathways (Nan et al.).

3.2.2 Female Reproductive System Protection

Premature ovarian failure (POF) symbolizes ovarian malfunctions. Women encountering POF commonly manifest with ineffectual ovarian functions and fertility challenges (Cong et al.). Epimedium has exhibited bone protective capabilities rooted in estrogen pathways. Subsequent studies have investigated the influences of Epimedium on ovarian functions through estrogen facilitation. Results confirmed Epimedium's ability to enhance mRNA and protein expressions of CYP17 and CYP19, agents responsible for estrogen synthesis, stimulating oocyte granule proliferation and augmentation of estrogen and progesterone secretion, thus boosting ovarian endocrine efficacy (Nie et al.).

Clinically, endometrial thinning stands as a key factor affecting female fertility (Bu et al.). Research revealed diminished levels of PI3K, AKT, and p-AKT proteins in thin endometrium compared to normal tissue (Le et al.). Prior findings indicated that ICA activates MEK/ERK and PI3K/Akt/eNOS pathways that are instrumental in angiogenesis (Chung et al.). Hence, researchers conjectured that ICA could stimulate this pathway, fostering endometrial vascularization and increasing endometrial thickness. Assessments on thin endometrial cells proposed that ICA indeed fulfills this hypothesis (Le et al.; Le et al.).

Though previous research showcases Epimedium's protective effects on the female reproductive system, further investigation is essential: Are there alternate mechanisms through which Epimedium enhances endometrial thickness apart from promoting vascular development? What are the mechanisms and targets associated with Epimedium in mitigating POF? Furthermore, can Epimedium replicate similar improvements in female oocyte development and tubal anomalies via its antioxidant attributes? These inquiries prompt greater scientific inquiry and clarification.

3.3 Anti-tumor Activities

3.3.1 Impact on Tumor Cell Proliferation and Apoptosis

Apoptosis is a regulated cell death process governed by genetic programming. Recently, numerous research initiatives have indicated that metabolites within Epimedium can trigger tumor cell apoptosis and suppress tumor growth through multiple signaling network modulations.

3.3.1.1 PI3/AKT/mTOR Pathway

Network pharmacology analysis has revealed that significant active components of Epimedium predominantly correlate with proteins associated with the PI3K/AKT signaling pathway. Consequently, in vitro studies have explored this pathway, indicating that ICA intensifies nucleus condensation and fragmentation in SKOV3 cells, suppressing their proliferation, migration, and invasiveness while amplifying cell apoptosis ratios by regulating the PI3K/AKT signaling cascade (Wang, S. et al.). Furthermore, ICA modulates the apoptosis and growth of ovarian cancer (OC) cells by targeting microRNA-21 and associated genes PTEN, RECK, and Bcl-2 (Li et al.). Thus, the PTEN/PI3K/AKT cascade could represent a pivotal pathway for utilizing Epimedium in OC therapies. Analysis showed that ICA activates caspase-dependent apoptosis through the mTOR apoptotic signaling pathway while impeding hepatocellular carcinoma (HCC) expansion via the inhibition of the NF-κB cascade (Guo et al.; Sun et al.).

Further research has demonstrated that ICT induces apoptosis within cisplatin-resistant OC cells by activating the p53 apoptotic pathway while inhibiting Akt/mTOR signaling (Gao et al.), underscoring ICT's potential to mitigate the resistance exhibited by cisplatin in clinical applications.

3.3.1.2 MAPK/ERK Pathway

A series of studies focusing on breast carcinoma cells revealed that ICT lowers ER-α36, a key tumorsurrogating protein involved in triple-negative breast cancer (TNBC), in addition to inhibiting epidermal growth factor receptor (EGFR) expression. It further obstructs ER-α36-induced stimulation of MAPK/ERK pathways and Cyclin D1, thereby curtailing cancer cell proliferation and stimulating apoptosis (Wang et al.). Additionally, ICT augments the radiosensitivity of cancer cells and collaborates synergistically with ionizing radiation (IR), a prevalent cancer therapy. ICT reduces tumor growth by curtailing IR-induced activation of ERK1/2 and AKT pathways while inhibiting G2/M cancer cell cycles (Hong et al.).

3.3.1.3 ROS Mediated Signaling Pathways

Reactive oxygen species accumulation is a marked feature across diverse cancer types (Moloney and Cotter). Evidence demonstrates that HCC cells subjected to prolonged ICT exposure exhibited notable ROS buildup, formation of ɣH2AX fluorescent aggregates, and significant cell cycle arrest in G0/G1 phases, coupled with declines in S and G2/M phases (Wang et al.). Thus, ICT promotes ROS production, leading to DNA injury and cellular aging in HCC.

In cervical cancer cells (CCA), ICT activates mitochondrial/Caspase apoptotic processes while inhibiting CCA growth through the AKT/Cyclin E/CDK2 pathway (Chen et al.; Sun et al., b). Conversely, in lung carcinoma cells, besides ICA's apoptosis-inducing effect through MAPK/ERK pathways (as previously noted) (Zheng et al.), further studies reveal ICA's apoptosis effect on cancer cells requiring the activation of JNK and p38MAPK downstream of ROS. Employing JNK and p38MAPK inhibitors significantly abated ICA's apoptotic capabilities towards tumor cells (Song et al.). Therefore, the ROS/MAPK/JNK signaling cascades reflect another mechanism by which Epimedium combats lung cancer.

3.3.2 Impediment of Tumor Cell Metastasis

Evident concentrations of ICA II can hinder tumor cell motility by modulating the MMP2/9 signaling pathways through JNK (Sun et al., a). In addition, Epimedium extracts and ICA II manipulate tumor development, survivability, and migration by blunting NF-κB signaling pathways (Kim and Park; Lee et al.; Shi et al.).

Epithelial-mesenchymal transition (EMT) stands as a marker of tumor metastasis (Talmadge and Fidler). ICT obstructs the invasion of Glioblastoma multiforme (GBM) cells by downregulating EMMPRIN expression. Follow-up analyses indicated that ICT modulates EMMPRIN (matrix metalloproteinase inducer) via the PTEN/Akt/HIF-1α cascade (Xu et al.).

Moreover, ICT encourages HCC apoptosis through the p53/alpha-fetoprotein (AFP) axis and indirectly impacts protein expressions linked to tumor development, apoptosis, and metastasis by targeting HMG-box transcription factor 1 (HBP1) and AFP (Cao et al.; Li et al.).

In summary, Epimedium and its derived extracts can foster cancer cell apoptosis and inhibit cell growth across various tumor types through multiple signaling pathways, generally characterized by mitochondrial/Caspase, MAPK/ERK, PI3K/AKT, and ROS pathways (Table 3).

Table 3

Table 3. Selected monomeric plant metabolites alongside relevant pathways pertaining to Epimedium Folium’s anti-cancer effects.

3.3.3 Auxiliary Mechanisms

Targeting cancer stem cells (CSCs) and immune modulation presents new strategies for cancer therapy. CSCs are neoplastic cells with stem-like properties, which may enhance resistance, spread, and recurrence of cancer post-treatment (Najafi et al.). Recent evidence suggests ICT fosters non-breast cancer stem cell (BCSC) apoptosis through the ERK pathway while suppressing BCSC proliferation characterized by elevated ALDH (Aldehyde Dehydrogenase, a BCSC marker) (Guo et al.). For hepatocellular carcinoma initiating cells (HCICs), ICT inhibits their malignant growth via the IL-6/Jak2/Stat3 pathway, diminishing the proportion of EpCAM (HCIC marker) positive cells (Zhao et al.). These studies suggest Epimedium and its derivatives possess potential to address cancer via their antiproliferative actions on CSCs. Nonetheless, available data remains limited, and most findings come from in vitro assessments. Hence, expansive in vivo and in vitro research is necessary for further evaluation.

During tumor progression, malignant cells can circumvent host immune defenses, thus leading to the exploration of tumor immunology as a cancer treatment avenue. Notably, Epimedium has shown immune regulatory effects. ICA is found to elevate the quantity of CD3+/CD69+ and CD69+/NKG2D+ lymphocytes in murine spleen samples, alongside enhancing cytotoxic lymphocyte (CTL) activity. Consequently, the body's immune defense against malignancies is bolstered while the immune evasion of cancerous cells is curtailed (Zhang et al.). Further, ICT may induce anti-tumor immunity by inhibiting extramedullary hematopoiesis (EMH) in the spleen, consequently suppressing myeloid-derived suppressor cell (MDSC) activities vital for facilitating cancer cell immune evasion (Tao et al.). Recently, findings indicate ICA II and ICA I can invoke immune-mediated drug-induced liver injury (IDILI), enhancing overall immune functions by activating the NLRP3 inflammasome, which can lead to hepatic injury (Gao et al.; Wang Z. et al.). In clinical settings, attention should turn to Epimedium and its related preparations, assessing potential toxic repercussions arising from varying applications. The above implies ICA and ICT could serve potential roles in cancer therapies by modulating immune responses.

Moreover, the pathway linking Epimedium induction of apoptosis with inhibiting bladder cancer proliferation diverges from that of other cancers. Findings confirm ICT reduces ATP essential for sustaining tumor survival by diminishing the electron transport system (MMP) in cancer cells, culminating in suppressed tumor invasiveness. This approach does not involve ROS/JNK and other associated pathways (Pan et al.).

To summarize, Epimedium's anti-cancer effects may engage diverse targets and mechanisms, with individual components exhibiting specificity towards various tumors. For instance, ICT addresses OC by adjusting the p53/AKT/mTOR axis (Gao et al.), impedes breast carcinoma cell proliferation through MAPK/ERK regulation (Wang et al.), and impacts HCC inducing ROS generation and cell cycle cessation (Wang et al.). ICT also tangentially engages host immune systems to facilitate tumor eradication (Tao et al.).

Current anti-tumor research remains predominantly based on in vitro frameworks, revealing notable divergences from pharmaceuticals' mechanisms in vivo, with significant gaps in experimental designs devoid of positive control comparisons. Accordingly, in vivo investigations are essential to validate conclusions derived from in vitro research and enhance substantiated findings with adequate experimental comparisons.

3.4 Anti-inflammatory Effects

The inflammatory process is a natural defense response by the human body. However, surges in inflammatory mediators can lead to immune and inflammatory disorders. The active metabolites of Epimedium demonstrate varying degrees of anti-inflammatory properties. Water extracts of Epimedium suppress inflammatory mediators like NO, IL-6, and IL-1 prompted by lipopolysaccharides (LPS) in macrophages via inhibition of the NF-kB/MAPK pathway. Furthermore, Epimedium water extracts alleviate xylene-induced ear edema in mouse models (Oh et al.). ICA reduces NF-kB/MAPK signaling by targeting ERK and p38 phosphorylation while decreasing immune-inflammatory factors via promoting glucocorticoid receptor (GR) α nuclear translocation and enhancing GR binding to NF-kB, c-Jun, and Stat3, thus boosting GR functionality and fostering anti-inflammatory actions (Sun et al.). Additionally, ICA mitigates TNF-α and IL-8 serum numbers in cigarette-smoke-induced mouse models and A549 cells, inhibiting NF-kB p65 phosphorylation whilst preventing IκB-α degradation, effectively minimizing CS-triggered inflammatory responses. Meanwhile, ICA reinstates GR protein expression, contributing to inflammation mitigation (Li et al.). Furthermore, ICA has also shown improvement in CYP-induced acute cystitis and LPS-mediated endometritis/peritonitis in rodent models (Amanat et al.; Huang et al.; Shaukat et al.).

In addressing neurological disorders, total flavonoids from Epimedium display positive biological activities concerning central nervous system demyelination, curtailing neuroinflammatory reactions to safeguard the myelin sheath (Meng-Ru et al.). ICA II presents neuroprotective properties amidst cerebral ischemia/reperfusion by counteracting inflammation and inciting apoptotic pathways (Deng et al.). As a response to cognitive deficits linked with Alzheimer’s disease, ICA II reverses cognitive impairments attributed to β-Amyloid protein by lessening neuroinflammation, restoring neural integrity, and promoting cell apoptosis (Deng et al.). Follow-up exploration of Epimedium's anti-inflammatory applications underscores its promise across diverse pathologies, making it a promising contender for managing inflammation-related conditions.

3.5 Antiviral Properties

Studies have confirmed that Epimedium water extracts inhibit influenza A viral activity by reducing the protein expression in a dose-responsive manner, thereby mitigating cellular damage caused by H1N1, H3N2, and Influenza B strains. Specifically, at a 100 µg/mL concentration of Epimedium water extracts, there exists a 90% decrease in influenza virus expression while simultaneously hindering hemagglutinin (HA) and neuraminidase (NA) proteins of H1N1 and blocking viral cell adhesion (Cho and Ma). Moreover, Epimedium appears to exhibit broad-spectrum antiviral efficacy, both in vivo and in vitro. It significantly diminishes the replication of various strains including the influenza virus PR/8, vesicular stomatitis virus (VSV), herpes simplex virus (HSV), and Newcastle disease virus (NDV) cells, activating the type I interferon (IFN-I) pathway by enhancing IRF-3, STAT1, and TBK1 phosphorylation, leading to increased IFN-I and pro-inflammatory factors release. Mice orally administered Epimedium were shown to combat several influenza A subtypes such as H1N1, H5N2, H7N3, and H9N2 (Cho et al.). In a 2020 study, chicken embryo fibroblasts co-administered with Epimedium Flavones, Newcastle disease virus (NDV), and ND vaccine demonstrated that Epimedium Flavones notably elevated cellular survival rates, curtailing NDV infectivity and improving the ND vaccine's protective effects (Yuqing et al.). These findings advocate that Epimedium merits exploration as a natural antiviral agent.

4 Safety Concerns

Epimedium is a traditionally termed 'non-toxic' herbal medicine extensively applied in treating conditions like osteoporosis and ED. In recent years, however, clinical formulations containing Epimedium, including 'Zhuanggu Joint Pill' and 'Xianling Gubao Oral Preparation,' have prompted adverse reaction reports from the National Medical Products Administration of China. Notably, some cases involved the 'Xianling Gubao Oral Preparation' resulting in liver damage and gastrointestinal issues among patients. This indicates an oversight surrounding the safety assessment of Epimedium and the identification of potential toxic compounds.

In previous studies, in vitro tests indicated that the flavonoid compounds derived from ICA within Epimedium exhibited substantial cytotoxicity (Zhang et al.). Another subchronic toxicity experiment lasted 13 weeks on SD rats, using Epimedium water extract (7.5, 15, or 30 g/kg). Results showed undefined no-observed-adverse-effect levels but indicated liver and adrenal damages in rats post-treatment (Song et al.). Zebrafish embryos exposed to Epimedium's flavonoid-ICA (0, 2.5, 10, and 40 µM) revealed diminished hatching rates and disrupted thyroid function after receiving 10 µM and 40 µM ICA, confirming ICA's developmentally toxic properties impacting thyroid growth and hormone synthesis (Wu et al.). Furthermore, in an analysis of the detoxifying mechanisms concerning the combination of Epimedium and Ligustri lucidi fructus, the latter was found to significantly decrease concentrations of flavonoids from Epimedium in rat systems, alleviating their toxic effects (Wang et al.).

In conclusion, while Epimedium is historically regarded as benign, its photochemical compounds may carry potential toxicity. The contraindications for using Epimedium remain ill-defined, along with the absence of established clinical safe dosages. While its toxicity might not be immediately observable in traditional models, modern operating organisms like zebrafish, organoids, or organ-on-chip systems could be more sensitive to its toxic exposure. Consequently, in-depth evaluations are needed to ascertain Epimedium's toxic constituents and safe dosages.

5 Discussion

Traditional Chinese medicine perceives the kidneys as repositories of essence, which mediate bone generation. This perspective positions the study of Epimedium’s protective roles in reproductive health, bone vitality, and anti-cancer pharmacology as crucial research avenues. This article synthesizes findings on the compounds, experimental models, and potential study limitations associated with Epimedium (Table 4). In the sphere of bone protection, this article delineates in detail the anti-osteoporosis capacity exhibited by Epimedium, analyzing bone metabolism as a focal point. Epimedium accomplishes osteoclast functionality inhibition by modulating estrogen levels while also supporting osteoblast health through antioxidative processes. In conjunction, it maintains bone metabolism equilibrium via non-estrogenic pathways and neuropeptide mechanisms while showcasing robust protection across vertebrate and invertebrate models. Currently marketed Epimedium formulations, such as 'Xianling Gubao Oral Preparation' and 'Zhuanggu Joint Pill,' have been linked to liver toxicity in users. Thus, investigations on Epimedium’s bone-preserving attributes must strictly adhere to safe dosing standards while clarifying its toxic components and potential reductions of hepatotoxic manifestations through conjunction with additional herbal remedies.

In reproductive concerns, recently prevalent PDE5Is like sildenafil, tadalafil, and vardenafil are frequently utilized in ED treatments, but can provoke headaches, visual irregularities, and gastrointestinal discomforts. Given the challenges posed by synthetic PDE5Is, natural alternatives like Epimedium denote promising opportunities for effective treatment. Active metabolites from Epimedium exhibit varying mechanisms for alleviating reproductive system ailments in both genders. Epimedium enhances penile tissue repairs, testosterone biosynthesis, sperm maturation, and promotes estrogen and progesterone release to aid ovarian functions. Despite its potential, more intensive research is mandated on the protections offered to the female reproductive system by Epimedium. Given the reports of toxic side effects from Epimedium products targeting osteoporotic conditions, it's prudent to scrutinize Epimedium's potential toxicities or assess if reactions stem from other ornamental components within the preparation or combine with traditional herbal agents. These concerns warrant further exploration and thorough evaluation by clinical researchers. While debate surrounds Epimedium’s adverse side effects, substantial research data is available supporting its use in treating ED and osteoporosis, highlighting its metabolites as catalysts in advancing new therapeutics for treating these ailments through ongoing clinical studies.

Table 4

Table 4. Experimental models and limitations in select publications.

In anti-cancer research, Epimedium's active metabolites target an array of pathways. Notably, the apoptosis pathways including the ERK, JNK, and p38 MAPK subfamily are thoroughly studied and widely engaged. Epimedium Folium induces not only endogenous apoptosis but also promotes a variety of extrinsic apoptotic modalities in malignant cells. Besides traditional therapeutic pathways against cancer, Epimedium's active components may also inhibit cancerous cells via immune regulation and suppression of CSCs. As a commonly employed herbal medicine in clinical settings, it remains to be evaluated whether Epimedium achieves its anti-cancer potential by enhancing immune functionalities or inadvertently inflicting immune disruptions, whose verification awaits substantial data. Furthermore, other monomeric Epimedium components convey risks of hepatotoxicity, necessitating exploration to determine whether Epimedium can combat tumors through inherent toxicity. With respect to its anti-inflammatory efficacy, Epimedium can alleviate conditions related to central nervous system demyelination and neurological disorders, covering a broad scope of ailments. On the topic of antiviral efficacy, specifics regarding the Epimedium extract composition necessitate clarification, and further investigations into active constituents should be prioritized.

6 Conclusion

Epimedium, recognized as a traditional Chinese medicinal agent, boasts extracts and associated compounds exhibiting diverse biological activities including anti-osteoporosis, enhancement of erectile function, anti-tumor, and anti-inflammatory effects. The flavonoid content in Epimedium predominantly drives these pharmacological activities, thereby substantiating the viability of Epimedium flavonoids for development into clinical therapies targeting various diseases.

Recent endeavors have initiated clinical applications for Epimedium within reproductive health and bone preservation; however, due to the risk of potential toxicities, comprehensive investigations into its toxicological properties are warranted. While Epimedium's anti-cancer capabilities show considerable promise, most existing studies are predisposed to in vitro examination, necessitating in vivo experiments to substantiate its anti-tumor efficacy. Presently, empirical studies examining the anti-inflammatory actions of Epimedium are insubstantial, thus necessitating investigations of its efficacy under pathological circumstances.

This paper encapsulates the core pharmacological benefits and relevant mechanisms of Epimedium, marking significant strides in our understanding over the past decade, and provides essential reference points for future pharmacological inquiries connected to Epimedium.

Author Contributions

JC: Drafting the original manuscript, reviewing, and editing. LL: Resources, software contributions, reviewing, and editing. FH: Reviewing and editing. ZS: Reviewing and editing. YG: Resource management and reviewing. TY: Resource management and reviewing. CY: Resource management and reviewing. XW: Resource management and reviewing. RG: Resource management and reviewing. YR: Resource management and reviewing. FL: Resource management and reviewing. CX: Resource management and reviewing. YG: Funding acquisition, resources, oversight, reviewing, and editing. YW: Funding acquisition, resources, oversight, reviewing, and editing.

Funding Acknowledgment

The author(s) confirm the receipt of funding supporting the research, authorship, and/or publication of this paper. This manuscript was funded by the National Key Research and Development Program (YFC) and the Innovation Team and Talents Cultivation Program from the National Administration of Traditional Chinese Medicine (Nos ZYYCXTD-C- and ZYYCXTD-D-).

Appreciation

The illustrations presented in this paper (Figures 2, 3) were produced by Figdraw.

Conflict of Interest Disclosure

The authors affirm that the research was executed devoid of any commercial or financial entanglements that might present a conflict of interest.

Publisher's Note

All claims articulated within this article representatives are solely those of the authors. These do not necessarily align with the positions of their affiliated organizations, nor do they represent the views of the publisher, editors, or reviewers. Any products presented in this manuscript or claims made by their manufacturers are not guaranteed or endorsed by the publisher.

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