Focus on phytosubstances – amazing properties of epimedium and icariin

By Vince Giuliano

Icariin is the active flavinoid substance in the traditional Chinese medicinal herb Epimedium brevicornum Maxim.  Icariin can be derived from several species of plants in the Epimedium family.  These plants are known most popularly as Horny Goat Weed or Yin Yang Huo.^[1]”  Although known and marketed widely in the United States as an aphrodisiac(ref), icariin and its sister epimedium-derived flavinoids have been subect to extensive research, mainly in China and elsewhere in Asia, and have been shown to exhibit amazing health-producing properties. A series of in-vitro and animal model studies have shown that icariin can promote the differentiation and proliferation of cardiomyocyte s and otherstem cells in multiple organ systems,  act as an antidepressant, be protective of neural cells, inhibit the breakdown of bone tissue, stimulate the development of new bone tissue, inhibit the actions of several toxic substances, attenuate unwanted microglial activation, stimulate angiogenesis, have a powerful effect in regulating the immune response, inhibit the inflammatory response in arthritis and other inflammatory disease conditions, and reduce or reverse bone loss due to injury or arthritis.  Icariin administration extends the lifespan and healthspan of nematodes.  It affects expression in numerous signaling pathways including MAPK, IGF-1, BMP, AMPK, NF-kappaB, MEK/ERK- and PI3K/Akt/eNOS, and, potentially could be the basis for new treatments addressing cancers, arthritis, osteoarthritis, asthma, acne, Alzheimer’s disease — and the list goes on.  Finally, yes: older rats systematically administered icariin do exhibit accelerated sexual activity.

Image source Northern Shade Gardening

Other than for its sexual effects, icariin and epimedium are not well known in the US.  The Pumbed datbase shows 241 research publications related to icariin and 344 related to epimedium, some of which overlap.  Almost all of this research is conducted in China, a little elsewhere in Asia and Europe and almost none in the US.  My purpose in this blog entry is to review this research and highlight what is known about icariin. 

I am grateful to my reader Louis who pointed me in the direction of epimedium in several comments to the blog entry In-vivo cell reprogramming for longer lives.

“Epimedium, also known as Rowdy Lamb Herb, Barrenwort, Bishop’s Hat, Fairy Wings, Horny Goat Weed, or Yin Yang Huo (Chinese: 淫羊藿), is a genus of about 60 or more species of herbaceousflowering plants in the family Berberidaceae. The large majority are endemic to southern China, with further outposts in Europe,^[1] and central, southern and eastern Asia(ref).”

Icariin is neuroprotective due to several different actions.

Perhaps a good way to start is with the 2010 publication Icariin attenuates lipopolysaccharide-induced microglial activation and resultant death of neurons by inhibiting TAK1/IKK/NF-kappaB and JNK/p38 MAPK pathways.  Several important recurrent themes are introduced: 1. icariin inhibition of the NF-kappaB and P38 MAPK pathways, 2. icariin promotion of release of nitric oxide, 3. Neuroprotection due to icariin, and 3.  icariin-induced reduction of microglial activation.  “Microglia in the central nervous system (CNS) play an important role in the initiation of neuroinflammatory response. Icariin, a compound from Epimedium brevicornum Maxim, has been reported to have anti-inflammatory effect on the macrophage cell line RAW264.7. However, it is currently unknown what anti-inflammatory role icariin may play in the CNS. Here, we reported the discovery that icariin significantly inhibited the release of nitric oxide (NO), prostaglandin E (PGE)-2, reactive oxygen species (ROS) and mRNA expression of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in lipopolysaccharide (LPS)-activated microglia. — Icariin also inhibited the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in a dose-dependent manner. Further mechanism studies revealed that icariin blocked TAK1/IKK/NF-kappaB and JNK/p38 MAPK pathways. It was also found that icariin reduced the degeneration of cortical neurons induced by LPS-activated microglia in neuron-microglia co-culture system. Taken together these findings provide mechanistic insights into the suppressive effect of icariin on LPS-induced neuroinflammatory response in microglia, and emphasize the neuroprotective effect and therapeutic potential of icariin in neuroinflammatory diseases.”

The 2010 publication way Neuroprotective effects of icariin on corticosterone-induced apoptosis in primary cultured rat hippocampal neurons expands on another aspect of neuroprotection:  “Neurons are damaged following prolonged

tissue showed that IL-4 expression was significantly reduced (P < 0.05), while the exposure to high concentrations of corticosterone, particularly during chronic inflammatory and immune diseases. One of the main mechanisms underlying neuronal injury is apoptosis. In the present study the neuroprotective effects of icariin, an active natural ingredient from the Chinese plant Epimedium sagittatum maxim against corticosterone-induced apoptosis were examined in primary cultured rat hippocampal neuronal cells. Pre-treatment of neuronal cells with icariin suppressed corticosterone-induced cytotoxicity in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling assay (TUNEL) labeling demonstrated that icariin significantly reduced TUNEL-positive cell numbers induced by exposure of cultured neurons to corticosterone. Moreover, icariin markedly inhibited corticosterone-induced mitochondrial dysfunction, including improved mitochondrial membrane potential and inhibition of caspase-3 activation. Using western blot analysis, corticosterone activated p38MAPK, extracellular regulated kinase 1/2(ERK1/2) ,and c-jun N-terminal protein kinase 1(JNK1) ,while icariin blocked p38 MAPK, but not JNK1 or ERK1/2. Pharmacological approaches showed that the activation of p38 MAPK plays a critical role in corticosterone-induced mitochondrial dysfunction and apoptosis. Taken together, the present results suggest that the protective effects of icariin on apoptosis in hippocampal neuronal cells are potentially mediated through blockade of p38 MAPK phosphorylation.”  Again, the role of inhibiting the P38 MAPK pathway is emphasized.

The March 2011 publication Icariin inhibits hydrogen peroxide-induced toxicity through inhibition of phosphorylation of JNK/p38 MAPK and p53 activity reports: “Oxidative stress caused by hydrogen peroxide (H(2)O(2)) plays an important role in the pathogenesis of Alzheimer’s disease (AD). The prominent damages caused by H(2)O(2) include the ruin of membrane integrity, loss of intracellular neuronal glutathione (GSH), oxidative damage to DNA as well as the subsequent caspase-3 and p53 activation. — Icariin is a flavonoid extracted from the traditional Chinese herb Epimedium brevicornum Maxim. We have previously reported that icariin has a good curative effect on patients with mild cognitive impairment (MCI), AD animal and cell models. However, the molecular mechanism of how icariin exerts neuroprotective effects is still not well understood. To address this question, we exposed undifferentiated neuronal cell lines (PC12 cells) to hydrogen peroxide (H(2)O(2)) and investigated the possible neuroprotective mechanisms of icariin. Vitamin E was used as a positive control. We observed that H(2)O(2) activated the JNK/p38 mitogen-activated protein kinase (MAPK) and induced PC12 cells apoptosis in a concentration-dependent manner. More over, we demonstrated that icariin protected PC12 cells by attenuating LDH leakage, reducing GSH depletion, preventing DNA oxidation damage and inhibiting subsequent activation of caspase-3 and p53, which are the main targets of H(2)O(2)-induced cell damage. In addition, we also found that icariin’s neuroprotective effect may partly correlate with its inhibitory effect on JNK/p38 MAPK pathways. Therefore, our findings suggest that icariin is a candidate for a novel neuroprotective drug to against oxidative-stress induced neurodegeneration.”

The 2009 publication Icariin enhances neuronal survival after oxygen and glucose deprivation by increasing SIRT1reports: “It has been reported that icariin protects neurons against ischemia/reperfusion injury. In this study, we found that icariin could enhance neuronal viability and suppress neuronal death after oxygen and glucose deprivation (OGD). Further study showed that neuroprotection by icariin was through the induction of Sirtuin type 1 (SIRT1), an effect that was reversed by SIRT1 inhibitor III and P38 inhibitor SB203580. SIRT1 is an endogenous gene of longevity, which increased neuronal viability and could be activated by stimulating the mitogen-activated protein kinase (MAPK) pathway. However, this study found that icariin activated the MAPK/P38 pathway, not the extracellular signal-regulated kinase (MAPK/ERK) or c-Jun N-terminal protein kinase (MAPK/JNK) to regulate SIRT1 expression. The results suggest that icariin may be developed into a neuroprotectant for ischemia-related brain injury.”

Besides providing neuroprotection, icariin may promote neurogenesis in humans.

The 2010 publication Effects of Epimedium flavonoids on proliferation and differentiation of neural stem cells in vitro reported: “Objective: The purpose of this study is to investigate the effects of Epimedium flavonoids (EF), which is extracted from a traditional Chinese Epimedium herb, and its effect on the proliferation and differentiation of neural stem cells (NSCs) in vitro.  Methods: The single cells isolated from the hippocampi of 1 day old neonatal rats were cultured in a serum-free condition medium DMEM/F12 (1 : 1) with different concentrations of EF or 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml basic fibroblast growth factor (bFGF). After 7 and 28 days, the neurospheres’ diameters were measured. The formed neurospheres were cultured in the differentiation medium containing EF or 10% fetal bovine serum (FBS). After 12 hours and 7 days, immunofluorescent studies for nestin, Musashi-1, BrdU, beta-III-tubulin, NF-200 and GFAP were performed. The number and lengths of 10-15 axons of NF-200 immunopositive cells were measured.  Results: The results showed that the isolated cells had the ability to propagate as neurospheres in the medium with 200 and 400 m g/ml EF, but without any EGF or bFGF, and the volume of neurospheres increase gradually from 7 to 28 days. In comparison with FBS control, the number of NF-200 positive neurons had significantly increased in the EF groups where the newborn neurons were morphologically more mature and able to migrate farther away from neurospheres than in the FBS control.  Discussion: The results demonstrate that EF effectively promotes the proliferation and differentiation of NSCs in vitro, suggesting that EF may have new properties of regulating central nervous system function by neurogenesis.”

Icariin has an effect on regulating the innate imune system.

The November 2011 publication Icariin induces the expression of toll-like receptor 9 in ana-1 murine macrophages reports: “Icariin is the major pharmacologically active compound of Herba epimedii which has been used as a tonic, aphrodisiac and an antirheumatic in traditional Chinese medicine. This study analysed the effect of icariin on the expression of Toll-like receptor 9 (TLR9) which plays an important role in regulation of the innate immune response. Stimulation of Ana-1 murine macrophages with icariin induced a significant dose-dependent expression of TLR9, and its mRNA expression which increased from 3 h post-treatment was approximately five-fold that of DMSO-treated cells. Several molecules, such as myeloid differentiation factor 88, tumor necrosis factor-α and interleukin 6, which are involved in the TLR9 downstream signaling pathway, were also significantly up-regulated in response to icariin stimulation. Our findings demonstrated that icariin is able to induce the expression of TLR9.”

Icariin may be immunoregulatory for patients with allergic rhinitis.

The December 2011 publication [Immunoregulatory mechanisms of an optimal Chinese herbal monomer compound in mice with allergic rhinitis] concluded as a result of a mouse model study: “The Chinese herbal monomer compound can inhibit the proliferation of cultured splenic lymphocytes of mice with allergic rhinitis. The effects of the compound of lowering intracellular calcium concentration and arresting cell cycle at G(0)/G(1) phases from entering into S and G(2)/M phases are responsible for its antiproliferation activity.”

Icariin limits inflammation.

The 2010 publication Icariin attenuates LPS-induced acute inflammatory responses: involvement of PI3K/Akt and NF-kappaB signaling pathway reports: “This study aimed to investigate the mechanism underlying the attenuation of LPS-induced lung inflammation by icariin in vivo and in vitro. The anti-inflammatory effects of icariin on LPS-induced acute inflammatory and the molecular mechanism were investigated. Pretreatment with icarrin (20mg/kg) could attenuate acute lung inflammation by inhibiting mRNA expressions of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), metalloproteinase cycloxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) in the lung of LPS-treated mice.  In addition, icariin suppressed the secretion of TNF-alpha, prostaglandin E2 (PGE(2)) and nitric oxide (NO) as well as NF-kappaB p65 activation. Furthermore, decreased myeloperoxidase (MPO) activity was observed in the lung tissue and LPS-induced cytotoxicity in the RAW 264.7 macrophages cells was also markedly attenuated by icariin. Western blotting analysis and confocal microscopy showed that icariin pretreatment reduced the nucleus transportation and constant level of NF-kappaB p65 in the RAW 264.7 macrophage cells. However, the protective effects of icariin were reversed by a PI3K/Akt inhibitor (wortmannin). Our in vitro and in vivo results suggested that activation of the PI3K/Akt pathway and the inhibition of NF-kappaB were involved in the protective effects of icariin on LPS-induced acute inflammatory responses.”

The January 2012 publication Attenuation of LPS-induced inflammation by ICT, a derivate of icariin, via inhibition of the CD14/TLR4 signaling pathway in human monocytes reports: “Objective: To evaluate the anti-inflammatory potential of ICT in LPS stimulated human innate immune cells.  Background: 3, 5, 7-Trihydroxy-4′-methoxy-8-(3-hydroxy-3- methylbutyl)-flavone (ICT) is a novel derivative of icariin, the major active ingredient of Herba Epimedii, an herb used in traditional Chinese medicine. We previously demonstrated its anti-inflammatory potential in a murine macrophage cell line as well as in mouse models.  Methods: We measured TNF-α production by ELISA, TLR4/CD14 expression by flow cytometry, and NF-κB and MAPK activation by western blot all in LPS-stimulated PBMC, human monocytes, or THP-1 cells after treatment with ICT.  Results: ICT inhibited LPS-induced TNF-α production in THP-1 cells, PBMCs and human monocytes in a dose-dependent manner. ICT treatment resulted in down-regulation of the expression of CD14/TLR4 and attenuated NF-κB and MAPK activation induced by LPS.  Conclusion: We illustrate the anti-inflammatory property of ICT in human immune cells, especially in monocytes. These effects were mediated, at least partially, via inhibition of the CD14/TLR4 signaling pathway.”

Inflammation of airways is an important aspect of asthma pathology.  The September 2011 publication Molecular mechanism of icariin on rat asthmatic model reports: “Background: Effects of icariin on airway inflammation in asthmatic rats and the intervention of LPS induced inflammation are interfered with the machanism of icariin. Our study aimed to observe the effect of icariin on ovalbumin-induced imbalance of Th1/Th2 cytokine expression and its mechanism.  Methods: Sixty male SD rats were randomly divided into control group (PBS), asthma group (ovalbumin (OVA)-induced), dexamethasone group, and OVA+icariin low, medium and high dose groups (5, 10, 20 mg/kg, respectively). Each group had ten rats. The model of OVA sensitization was a rat asthma model. Enzyme-linked immunosorbent assay (ELISA) method was used to observe the effects of icariin on interleukin-4 (IL-4) and inerferon γ (IFN-γ) in rats’ lung tissue. Immunohistochemical staining was applied to detect the intervention effects of icariin on T cells (T-bet) and gatabinding protein 3 (GATA-3) in rat pulmonary tissue. Realtime RT-PCR was used to observe the intervention effects of icariin on T-bet and GATA-3 mRNA expression in rat pulmonary tissue and spleen lymphocytes. Western blotting was used to observe the icariin intervention effects on T-bet, GATA-3 and nuclear factor-Kappa B (NF-κB) p65 protein expressions in rat pulmonary tissue.  Results: The ELISA results from pulmonary IFN-γ expression increased but not significantly when we compared OVA+icariin medium and high dose groups with the asthma group. Immunohistochemical staining of pulmonary tissue showed that the GATA-3 decreased significantly while the T-bet staining did not change in the OVA+icariin high dose group. In pulmonary tissue and spleen lymphocytes T-bet and GATA-3 mRNA expressions were significantly reduced (P < 0.05) in icariin treatment groups compared with the asthma model group. GATA-3 and T-bet mRNA in rat spleen lymphocytes in the asthma group were higher than in the control group. GATA-3 mRNA expression in pulmonary tissue significantly decreased (P < 0.05) while T-bet mRNA expression decreased but not significantly in the icariin treatment group compared with the asthma group. T-bet and GATA-3 protein expressions in pulmonary tissue increased significantly compared with the asthma group, which meant that icariin could inhibit the increase of GATA-3 protein, but not of T-bet. The bronchus, blood vessels and periphery pulmonary tissue had infiltration of inflammatory cells in the OVA+icariin high dose group while NF-κB p65 cells were reduced, and expression of NF-κB p65 in this group was less than in the asthma group. The expression of total p65 protein decreased with icariin treatment while the expression of cytoplasmic p65 protein increased.  Conclusions: Icariin could regulate the imbalance of Th1/Th2 cytokines in asthmatic rat pulmonary tissue. Icariin could regulate the imbalance of Th1/Th2 associated transcription factors T-bet and GATA-3 in asthmatic rat pulmonary tissue and spleen lymphocytes. Icariin could inhibit the activation of NF-κB p65 protein in asthmatic rat pulmonary tissue.”

Icariin may delay aging or postpone the onset of age-related diseases.

At least, the substance appears capable of doing that in nematode C.-elegans worms.  The December 2011 publication Icariin and its derivative icariside II extend healthspan via insulin/IGF-1 pathway in C. elegansreported: “Compounds that delay aging might also postpone age-related diseases and extend healthspan in humans. Icariin is a flavonol extracted from several plant species of the Epimedium family. The icariin and its metabolic derivatives have been shown to exert wide protective effects in age-related diseases. However, whether icariin and its derivatives have the potency of delaying aging remains unclear. Here, we report that icariin and its derivative icariside II extend C. elegans lifespan. Using HPLC, we found high level of icariside II in the animals treated with icariin, suggesting icariside II is the bioactive form in vivo of icariin. Icariside II also increased the thermo and oxidative stress tolerance, slowed locomotion decline in late adulthood and delayed the onset of paralysis mediated by polyQ and Aβ(1-42) proteotoxicity. The lifespan extension effect of icariside II is dependent on the insulin/IGF-1 signaling (IIS) since the daf-16(mu86) and daf-2(e1370) failed to show any lifespan extension upon icariside II treatment. Consistently, icariside II treatment upregulates the expression of DAF-16 targets in the wild-type. Moreover, our data suggests that the heat shock transcription factor HSF-1 has a role in icariside II-dependent lifespan extension further implicating the IIS pathway. In conclusion, we demonstrate a novel natural compound, icariside II as the bioactive form of icariin, extends the healthspan via IIS pathway in C. elegans.”—“ Icariside II increases the mRNA expression of FOXO/DAF-16 targets hsp12.3 and sod3 significantly.”—“ Icariside II ameliorates protein aggregation and protetoxicity-mediated paralysis phenotype.” – “Icariside II promotes stress resistance and slows age related decline in movement in C. elegans.” 

So, the IGF1, IIS and FOXO/DAF-16, pathways are also affected by icariin administration,.  This is not surprising given the known relationships of these pathways to longevity.

Icariin promotes the directed differentiation of embryonic stem cells into cardiomyocytes.

The 2005 publication Inducible effects of icariin, icaritin, and desmethylicaritin on directional differentiation of embryonic stem cells into cardiomyocytes in vitroreported:  “Aim: To investigate the possible inducible effects of icariin, icaritin, and desmethylicaritin on the directional differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro.  Results: The total percentage of beating EBs treated with 10(-7) mol/L icariin, icaritin, or desmethylicaritin was 87% (P<0.01), 59% (P<0.01), and 49%, respectively. All the beating cardiomyocytes derived from the ES cells expressed cardiac-specific proteins for a-actinin and troponin T. Among them, 10(-7) mol/L icariin treatment resulted in a significantly advanced and increased mRNA level of a-cardiac major histocompatibility complex (MHC) and myosin light chain 2v (MLC-2v) in EBs in the early cardiac developmental stage. Before shifting to the cardiomyocyte phenotype, icariin could evoke the accumulation of ES cells in G0/G1 and accelerate apoptosis of the cell population (P<0.05).  Conclusion: Icariin facilitated the directional differentiation of ES cells into cardiomyocytes at a concentration of 10(-7) mol/L. The promoting effect of icariin on cardiac differentiation was related to increasing and accelerating gene expression of a-cardiac MHC and MLC-2v, as well as regulating the cell cycles and inducing apoptosis.”

The 2007 publication Icariin-mediated expression of cardiac genes and modulation of nitric oxide signaling pathway during differentiation of mouse embryonic stem cells into cardiomyocytes in vitro reports: Aim: To investigate effects of icariin on cardiac gene expression and the modulation of nitric oxide (NO) signal transduction during the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro.  Methods: The expression levels of cardiac developmental-dependent genes were measured using reverse transcription-polymerase chain reaction (RT-PCR). The chronotropic responses of cardiomyocytes to b-adrenoceptor stimulation were determined. The levels of cAMP and cGMP in ES cells were measured using radioimmunoassay. Endogenous NO levels were measured by using the Griess reaction. Aminoguanidine (AG) was used to confirm the influence of icariin on the endogenous NO signal pathway.  Results: Icariin significantly elevated mRNA levels of cardiac transcription factors GATA4 and Nkx2.5, and cardiac-specific alpha-MHC, MLC-2v and beta-AR genes in a concentration- and time-dependent manner (P<0.05). Cardiomyocytes derived from embryoid body (EB) treated with icariin were more sensitive to isoprenaline (P<0.01). Treatment of ES cells with icariin resulted in a continued elevation in the cAMP/cGMP ratio before a shift to the cardiomyocyte phenotype (P<0.05). AG decreased the NO level, and delayed and decreased the incidence of contracting EB to only approximately 35% on d 5+11, an effect that could be rescued by icariin. When cells were cocultured with icariin and AG, the percentage of beating EB reached a peak level of 73% on d 5+11 (P<0.05).  Conclusion: The inducible effects of icariin were partly related to increase in the expression of cardiac developmental-dependent genes, and elevation of the cAMP/cGMP ratio in ES cells, as well as upregulation of endogenous NO generation during the early stages of cardiac development.”

The March 2010 publication Icariin induces mouse embryonic stem cell differentiation into beating functional cardiomyocytes reports: “Icariin, the primary active component of Epimedium extracts, has recently been shown to induce cardiomyocyte differentiation of murine embryonic stem (mES) cells in vitro. However, as these cardiomyocytes were not functionally characterized, the potential application of icariin-induced cardiomyocytes in clinical practice remains unclear. Therefore, in this study, we characterized the structure and function of icariin-induced cardiomyocytes to evaluate their potential application in transplantation for cardiac failure treatment. mES cells were cultured as embryoid bodies (EBs) via the direct suspension method in the presence of icariin. The protein expression profiles and ultrastructural characteristics of mES cell-derived cardiomyocytes were then characterized by immunofluorescence and transmission electron microscopy, respectively. In addition, the expression of cardiac-specific and calcium handling genes was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Cardiomyocytes induced by icariin treatment expressed the cardiac-specific proteins myosin light chain-1v (MLC1v), atrial natriuretic polypeptide (ANP), and cardiac troponin I (cTnI). Furthermore, these cells appeared to possess myofibrils organized into mature sarcomeres that had formed A and I bands. In addition, icariin treatment upregulated the mRNA levels of MLC1v, ANP, cTnI, calsequestrin (CSQ), and sodium-calcium exchanger (NCX) in these cells. Icariin induces the differentiation of mES cells into beating cardiomyocytes with normal structure and function. Therefore, these cells may have promising applications in cardiac cell therapy or tissue engineering.”

The November 2010 publication Icariin-mediated differentiation of mouse adipose-derived stem cells into cardiomyocytes reported: “In this study, we investigated the ability of mouse adipose-derived stem cells (ADSCs) to differentiate into a cardiac phenotype in vitro. Icariin (ICA) has previously been shown to induce cardiomyocyte (CM) differentiation of murine embryonic stem cells in vitro, but its effect on ADSCs remains unclear. We isolated ADSCs from white adipose tissue and analyzed selected surface antigens using flow cytometry. ADSCs and CMs were co-cultured in transwell plates, with or without the addition of either ICA or ICA plus the extracellular signal-regulated kinase (ERK) inhibitor PD98059. Cardiac-specific gene expression was examined by reverse transcription-polymerase chain reaction and western blotting. ICA facilitated differentiation of ADSCs into CMs that expressed cardiac-specific genes, including the transcription factors NKX-2.5, GATA-4, MLC-2v, α-actinin, and cardiac troponin-T. Expression of α-actinin, the Z band-constituting protein, was promoted by ICA in a dose- and time-dependent manner. ICA can induce ERK activation and cardiac-specific gene expression was partially inhibited by PD98059 after treatment with ICA. These results suggest that ICA-stimulated CM differentiation of ADSCs, and that it acted partially by activating ERK-dependent signaling pathways in vitro.”

A number of proteins and pathways play key roles in icariin-induced cardiomyocyte differentiation of embryonic stem cells.   For example, P53 plays an important role as described in the 2005 publication Icariin-mediated modulation of cell cycle and p53 during cardiomyocyte differentiation in embryonic stem cells. Inhibitionof NF-kappaB expression is involved as pointed out in the 2008 publication Involvement of NF-kappaB and AP-1 activation in icariin promoted cardiac differentiation of mouse embryonic stem cells.  “IkappaBalpha phosphorylation and NF-kappaB p65 translocation to the nucleus appeared rapidly when embryoid bodies exposed to icariin, and the expression of IkappaBalpha or NF-kappaB p65 in cytoplasm was decreased concomitantly. Moreover, icariin increased c-jun and c-fos mRNA and protein expression. Either SB203580 or U0126 displayed inhibitory effect on icariin induced NF-kappaB and AP-1 activation. It could be concluded that p38 and ERK1, 2 are activated in a coordinated manner, which in turn contribute to NF-kappaB and AP-1 activation in icariin induced cardiomyogenic cell lineage differentiation of mouse ES cells.” 

And, a 2011 publication Involvement of ubiquitin-proteasome system in icariin-induced cardiomyocyte differentiation of embryonic stem cells using two-dimensional gel electrophoresis relates: “MALDI-TOF/MS showed that icariin treatment resulted in the induction of five ubiquitin-proteasome system (UPS)-related proteins, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), ubiquitin-conjugating enzyme E2N, proteasome 26S, proteasome subunit-alpha type 6, and proteasome subunit-alpha type 2 in the differentiated cardiomyocytes. These results implied that UPS might play an important role in the control of cardiomyocyte differentiation.” The 2008 publication Involvement of p38MAPK and reactive oxygen species in icariin-induced cardiomyocyte differentiation of murine embryonic stem cells in vitro reports “Taken together, these results suggest that ROS generation and the subsequent activation of p38MAPK are essential for the inducible function of icariin on cardiomyocyte differentiation of murine embryonic stem cells in vitro.”

Epimedium flavonoids (EF) can promote the proliferation and migration of adrenocortical stem cells in certain disease-model rats.

The 2009 publication [Activating effect and mechanism of epimedium on endogenous stem cells] reports: “Stem cells are the cells with capacities of self-renovation, multiplication and differentiation. By activating endogenous stem cells to promote regeneration response has provided a new thinking for the treatment of degenerative diseases. The authors found that epimedium flavonoids (EF) can promote the proliferation and migration of adrenocortical stem cells in corticosterone-treated rats (as a model of Shen-yang deficiency); and through gene-chip test it was showed that EF could significantly up-regulate the growth hormone (GH), growth hormone releasing hormone (GHRH) and other growth factors such as insulin-like growth factor binding protein (IGFBP) and nerve growth factor (NGF) in the model rats. — In natural aging rats (as model for Shen deficiency), EF could make the gene expression of multiple tissues youthening, and up-regulate the lowered expressions of GH, GHRH, IGFBP and NGF, etc. Further study on the in vitro isolated and cultivated neuro-stem cells proved that EF and its components have direct promoting actions on stem cell proliferation. All the above-mentioned outcomes indicated that the actions of EF and its extracts on stem cells are possibly the cytological basis for their effects on counteracting the suppression of glucocorticoids on hypothalamus-pituitary-adrenal (HPA) axis and retarding aging; also illustrated that TCM could treat diseases by a way of activating endogenous stem cells through mobilizing and elevating hormones and cytokines levels, and bringing the reserved potential of organism into full play.”

Icariin promotes expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine ES cells in vitro.

The 2007 publication Icariin promotes expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine embryonic stem cells in vitro reports:  “Aim: To investigate the effect of icariin on the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alpha), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear respiratory factor 1 (NRF-1) on cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro.  Methods: The cardiomyocytes derived from murine ES cells were verified by immunocytochemistry using confocal laser scanning microscopy. Cardiac-specific sarcomeric proteins (ie alpha-actinin, troponin T) were evaluated when embryoid bodies (EB) were treated with icariin or retinoid acid. The expression of PGC-1alpha, PPARalpha, and NRF-1 were analyzed using both semiquantitative RT-PCR and Western blotting in cardiomyocyte differentiation. The phosphorylation of the p38 mitogen-activated protein kinase (MAPK) was studied in the differentiation process, and its specific inhibitor SB203580 was employed to confirm the function of the p38 MAPK on icariin-induced cardiac differentiation.  Results: The application of icariin significantly induced the cardiomyocyte differentiation of EB as indicated by the promoted expression of alpha-actinin and troponin T. The expression of PGC-1alpha, PPARalpha, and NRF-1 increased coincidently in early differentiation and the increase was dose-dependently upregulated by icariin treatment. The phosphorylation of the p38 MAPK peaked on d 6 and decreased after d 8, and the activation was further enhanced and prolonged when the EB were subjected to icariin, which was concurrent with the elevation of PGC-1alpha, PPARalpha, and NRF-1. Moreover, the inhibition of the p38 MAPK pathway by SB203580 efficiently abolished icariin-stimulated cardiomyocyte differentiation and resulted in the capture of the upregulation of PGC-1alpha, PPARalpha, and NRF-1.  Conclusion: Taken together, icariin promoted the expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine ES cells in vitro and the effect was partly responsible for the activation of the p38 MAPK.”

Several blog entries have discussed PGC-1alpha, PPAR alpha and the NRF family.of proteins.  From Victor’s blog entry Mechanisms and Effects of Dietary Restriction:  ““PPAR-Gamma Coactivator-1” refers to a family of proteins which regulate the transcriptional activity of many nuclear receptors including the PPARs. The subtypes are PGC-1alpha, PGC-1beta, and PRC (PGC-1 related coactivator). These regulatory proteins are highly responsive to changes in metabolic signals; hence, they are able to directly regulate the transcription of specific genes in response to cues such as nutrient status, oxidative and inflammatory stress, and energy requirements. They even respond to ambient temperature; in fact, PGC-1alpha was originally discovered to promote uncoupled thermogenesis in BAT in response to cold temperatures. The PGC1 family plays a central role in mitochondrial function and the regulation of energy homeostasis. PGC-1alpha induces expression of Sirt3, a sirtuin known to improve mitochondrial function(ref).  Relationships among PGC-1alpha and Sirt3 are discussed in the blog entry SIRT3 research– tying together knowledge of aging. PGC-1alpha deficiency is associated with metabolic and neurodegenerative disorders such as Parkinson’s and Huntington’s disease.(ref) Like PPAR, PGC-1alpha is known to decline with age, but is increased by DR.(ref) A recent rodent study found that DR increased PGC-1alpha up to 5-fold. “CR mice exhibited a significant increase in PGC-1alpha level in the heart (5.13-fold), kidney (3.57-fold), skeletal muscle (3.02-fold), liver (2.60-fold), small intestine (2.45-fold) and brain (2.05-fold), compared to normal (ad libitum) fed. The elevation in PGC-1alpha level, especially in highly oxidative tissues such as heart, kidney and skeletal muscle of CR mice might synergistically up-regulate genes that require PGC-1alpha co-activation.”(ref) Overexpression of PGC-1alpha, also further contributes to the metabolic shift towards fat oxidation.(ref)” 

The blog entry PQQ – activator of PGC-1alpha, SIRT3 and mitochondrial biogenesis is about PGC-1alpha, the role of exercise in enhancing its expression and PQQ, a dietary supplement that enhances the expression of PGC-1alpha.

Epimedium-derived flavonoids down-regulate bone tissue breakdown and resorption (osteolysis) and upregulates bone tissue buildup (osteogenesis).

An important use of horny goat weed in traditional Chinese medicine is the treatment of degenerative bone-related conditions.   A substantial number of publications relate to the actions of icariin and other epimedium-derived flavonoids on bone tissue destruction/regeneration and the potential role of epimedium flavonoids for treatment of osteoarthritis, rheumatoid arthritis, and bone fractures.  As a reminder, “An osteoclast (from the Greek words for “bone” (Οστό) and “broken” (κλαστός)) is a type of bone cell that removes bone tissue by removing its mineralized matrix and breaking up the organic bone (organic dry weight is 90% collagen). This process is known as bone resorption(ref).”  An osteoblast is a  mononucleate cell  that is “responsible for bone formation; in essence, osteoblasts are specialized fibroblasts that in addition to fibroblastic products, express bone sialoprotein and osteocalcin(ref).^[1]”

To start, the 2005 publication Icariin, a flavonoid from the herb Epimedium enhances the osteogenic differentiation of rat primary bone marrow stromal cells reports: “The herb Epimedium has long been used in Traditional Chinese Medicine to treat bone fracture and prevent osteoporosis. Researchers believe that the flavonoids contained in the herb are the effective component for this activity. However, no single flavonoid has been studied for its effect on bone-related cells. In the present study, icariin, one of the major flavonoids of the herb, supplemented the primary culture medium of rat bone marrow stromal cells (rMSCs) at 0.1 microM , 1 microM and 10 microM respectively. It was found that icariin stimulated the proliferation of rMSCs and increased the number of CFU-F stained positive for alkaline phosphatase in a dose-dependent manner. Icariin also dose-dependently increased the alkaline phosphatase activity, osteoalcin secretion and calcium deposition level of rMSCs during osteogenic induction. The addition of 10 microM icariin caused four times more mineralized bone nodules to be formed by rMSCs than in the control. The results demonstrated that icariin should be an effective component for bone-strengthening activity, and one of the mechanisms is to stimulate the proliferation and enhance the osteogenic differentiation of MSCs.”

The 2010 publication Icariin protects murine chondrocytes from lipopolysaccharide-induced inflammatory responses and extracellular matrix degradation reports: “Septic arthritis is an inflammatory arthropathy characterized by degeneration of articular cartilage. Icariin, the main active flavonoid glucoside isolated from Epimedium pubescens, is used as antirheumatics (or antiinflammatory), tonics, and aphrodisiacs in traditional Chinese medicine. In this study, we used lipopolysaccharide (LPS) to simulate the in vitro inflammatory response of chondrocytes during septic arthritis. Our hypothesis is that the icariin can protect chondrocytes from LPS-induced inflammation and extracellular matrix degradation. — The inflammation of neonatal mice chondrocytes was induced by LPS and the antiinflammatory effects were examined. The synthesis of nitric oxide was analyzed, whereas the titer of glycosaminoglycan and total collagen were measured and the gene expressions (including inducible nitric oxide synthase [iNOS], matrix metalloproteinase [MMP]-1, MMP-3, and MMP-13) were evaluated. The results showed that the viability of chondrocytes, extracellular matrix synthesis, was significantly decreased, whereas nitric oxide synthesis was significantly increased in the presence of 10(-5) g/mL LPS. Icariin pretreatment can partially reverse these effects. The up-regulated expressions of MMP-1, 3, 13, cyclooxygenase-2 (COX-2), and iNOS genes by LPS treatment were also significantly down-regulated by the pretreatment of icariin to 1.8%, 0.056%, 7.7%, 3.1%, and 5.3% of the LPS-positive control sample, respectively. Our results demonstrate that icariin is a safe anabolic agent of chondrocytes. Icariin may exert its protective effects through inhibition of nitric oxide and MMP synthesis, and may then reduce the extracellular matrix destruction.”

The 2010 publication Flavonoids of Herba Epimedii regulate osteogenesis of human mesenchymal stem cells through BMP and Wnt/beta-catenin signaling pathway reports: “Herba Epimedii is one of the most commonly used Chinese herbs for treating osteoporosis. In the present study, the flavonoids of Herba Epimedii (HEF) have shown to promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. They were noted to enhance the mRNA expression of BMP-2, BMP-4, Runx2, beta-catenin and cyclinD1, all of which are BMP or Wnt-signaling pathway related regulators. The osteogenic effect was inhibited by the introduction of noggin and DKK-1, which is classical inhibitor of BMP and Wnt/beta-catenin signaling, respectively. These results suggest that HEF exerts promoting effect on osteogenic differentiation, which plausibly functions via the BMP and Wnt/beta-catenin signaling pathways. Considering the therapeutic efficiency and economical issues, HEF may be a potential candidate for promoting bone regeneration. On the other hand, osteogenic differentiation of MSCs may also be a promising and attractive tool to apply in bone repair.”

The 2011 publication Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesisrelates: “Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the detail molecular mechanisms of icariin on lipopolysaccharide (LPS)-induced osteolysis. Our hypothesis is that icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis.  — After treatment with icariin, the activity of osteoclasts differentiation maker, tatrate resistances acid phosphatease (TRAP), significantly decreased at the concentration of 10(-8)M. Icariin (10(-8)M) reduced the size of LPS-induced osteoclasts formation, and diminished their TRAP and acid phosphatease (ACP) activity without inhibition of cell viability. Icariin also inhibited LPS-induced bone resorption and interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) expression. — The gene expression of osteoprotegerin (OPG) was up-regulated, while receptor activator of NF-κB ligand (RANKL) was down-regulated. Icariin also inhibited the synthesis of cyclo-oxygenase type-2 (COX-2) and prostaglandin E(2) (PGE(2)). In addition, icariin had a dominant repression effect on LPS-induced hypoxia inducible factor-1α (HIF-1α) expression of osteoclasts. On osteoclasts, icariin suppresses LPS-mediated activation of the p38 and JNK; while on the osteoblasts, icariin reduced the LPS-induced activation of ERK1/2 and I-kappa-B-alpha (IκBα), but increased the activation of p38. In conclusion, we demonstrated that icariin has an in vitro inhibitory effects on osteoclasts differentiation that can prevent inflammatory bone loss. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway.”

The August 2011 publication [Comparative study on effect of icariin and genistein on proliferation and mineralization of osteoblasts in vitro]concludes “When the final concentration of icariin and genistein is 1 x 10(-5) mol x L(-1), they can significantly promoted ROB maturation. And on the level of osteoblasts, the activity of icariin is stronger than that of genistein.”

The new (this-week) publication Icariin Promotes Extracellular Matrix Synthesis and Gene Expression of Chondrocytes In Vitro reports: “To effectively treat articular cartilage defect with tissue engineering there is an urgent need to develop safe and cheap drugs that can substitute or cooperate with growth factors for chondrogenesis promotion. Here, we demonstrate the chondrogenic effect of icariin, the major pharmacological active constituent of Herb Epimedium (HEP).  Rabbit chondrocytes were isolated from articular cartilage and cultured in vitro with different concentrations of icariin.  Icariin at concentrations under 1 × 10(-5)   m showed low cytotoxicity toward chondrocytes, but icariin at 5 × 10(-5)   m inhibited the proliferation of chondrocytes. Icariin hardly affected the cell morphology with concentrations ranging from 1 × 10(-7)   m to 5 × 10(-5)   m. However, the higher concentration of icariin produced more extracellular matrix (ECM) synthesis and expression of chondrogenesis genes of chondrocytes. Indeed, the promotion of icariin on the synthesis of glycosaminoglycans (GAGs) and collagen of chondrocytes, and finally exerting a potent chondrogenic effect, might be due to its ability to up-regulate the expression of aggrecan, collagen II and Sox9 genes and to down-regulate the expression of the collagen I gene of chondrocytes. — These preliminary results imply that icariin might be an effective accelerant for chondrogenesis and that icariin-loaded biomaterials might have the potential for cartilage tissue engineering.”

Another new this-week publication is Icariin stimulates MC3T3-E1 cell proliferation and differentiation through up-regulation of bone morphogenetic protein-2 which reports: “Previous studies suggest that icariin has anabolic effects on bone, but the mechanisms are unknown. We aimed to investigate the osteogenic effects of icariin in an undifferentiated osteoblast cell line by detecting cell morphology, viability, cell cycling and bone morphogenetic protein-2 (BMP-2) expression. We treated pre-osteoblastic MC3T3-E1 cells with different concentrations of icariin [0 (as a control), 10, 20 and 40 ng/ml] for 48, 72 and 96 h. Cell morphology, viability and the cell cycle were examined and measured using microscopy, the MTT assay or flow cytometry, respectively. BMP-2-positive cells and BMP-2 protein expression levels in icariin-treated MC3T3-E1 cells were examined using immunohistochemistry staining with fluorescence optical density analysis and Western blotting. MC3T3-E1 cells showed typical characteristics of osteoblasts in response to treatment with icariin. Cells treated with all concentrations of icariin had increased percentages of S-phase cells and decreased percentages of G1-phase cells, especially in the 10 and 20 ng/ml icariin groups. The number of BMP-2-positive cells and BMP-2 protein expression levels in the 10 and 20 ng/ml icariin treatment groups were greater compared to the 0 and 40 ng/ml groups. Treatment of icariin promotes osteoblast MC3T3-E1 proliferation and differentiation in vitro, potentially owing to its role in increasing BMP-2 protein expression. Icariin potentially can be used as a drug in clinical settings to treat osteoporosis.”

The December 2011 publication Maohuoside A promotes osteogenesis of rat mesenchymal stem cells via BMP and MAPK signaling pathwaysrelates to maohuoside, another compound derived from epimedium. “Osteoporosis is becoming a more prevalent health problem with the aging of the population around the world. Epimedium koreanum Nakai is one of the most used herbs in East Asia for curing osteoporosis, with its major ingredient, icariin, mostly explored by researchers. In this article, maohuoside A (MHA), a single isolated compound from the herb, was identified to be more potent than icariin in promoting osteogenesis of rat bone marrow-derived mesenchymal stem cells (rMSCs) (increasing by 16.6, 33.3, and 15.8% on D3, D7, and D11, respectively). Alkaline phosphatase (ALP) assay and calcium content measurement were assigned to quantify the promoted osteogenesis and alizarin red S (ARS) staining was conducted to visualize it. Quantitative real-time PCR (Q-PCR) was assayed to evaluate the mRNA expression of marker genes in osteogenesis and master regulators in BMP pathway. Moreover, PD98059 (PD) and SB203580 (SB), inhibitor of ERK1/2 and p38 MAPK pathway, were administered to assess the involvement of MAPK pathway in the promotion process. In conclusion, MHA pronouncedly enhanced the osteogenesis of rMSC, plausibly via the BMP and MAPK signaling pathways.”

Finally, the December 2011 publication The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells reported: “Icariin had been reported as a potential agent for osteogenesis, but the dose-effect relationship needed further research to realize the clinical application of icariin. We isolated and purified human bone mesenchymal stem cells (hBMSCs) and stimulated them with different concentrations of icariin. The cytotoxicity of icariin was evaluated by the methylthiazolytetrazolium (MTT) assay method. The proliferation and osteogenic differentiation of such hBMSCs were investigated for different concentrations of icariin. We found that icariin had a dose-dependent effect on the proliferation and osteogenic differentiation of hBMSCs in a suitable concentration range from 10(-9) M to 10(-6) M, but at concentrations above 10(-5) M, the cytotoxicity limited its use. The extremely low cost of icariin and its high abundance make it appealing for bone regeneration.”

Icariin has antidepressant and stress-resistance properties.

The 2005 publication Antidepressant-like effect of icariin and its possible mechanism in mice reports: “The behavioral, neurochemical and neuroendocrine effects of icariin isolated from Epimedium brevicornum were investigated in behavioral despair models of KunMing strain of male mice. Icariin was found to significantly shorten immobility time in the forced swimming test (FST) after orally administration for 21 consecutive days. Icarrin also produced a marked reduction in immobility time in the tail suspension test (TST) when administered for at least 7 consecutive days. The preferable antidepressant action by icariin was obtained at 17.5 and 35 mg/kg in the present study. Moreover, it was observed that the stress of FST exposure induced increases in brain monoamine oxidase (MAO) A and B activities, serum corticotropin-releasing factor (CRF) levels, as well as decreases in brain monoamine neurotransmitter levels. Treatment of icariin for 21 consecutive days mainly reversed the above effects in the mouse FST. These results suggested that icarrin possessed potent antidepressant-like properties that were mediated via neurochemical and neuroendocrine systems.”

The 2006 publication Effects of icariin on hypothalamic-pituitary-adrenal axis action and cytokine levels in stressed Sprague-Dawley rats  reported: “Icariin is one of the major active flavonoids constituents of Epimedium brevicornum MAXIM (Berberidaceae). Icariin and E. brevicornum have a wide range of pharmacological activities. Abnormality in the hypothalamic-pituitary-adrenal (HPA) axis is considered to be a key neurobilogical factor in major depression, and cytokines have a close relationship with the activation of the HPA axis. In the present study, the aim was to determine whether icariin possesses an antidepressant-like activity, and to explore the effects of icariin on the HPA axis and cytokine levels in chronic mild stress (CMS) model of depression in Sprague-Dawley rats. Icariin significantly increased the sucrose intake of CMS-treated rats from week 3. It not only attenuated the CMS-induced increases in serum corticotropin-releasing factor (CRF) and cortisol levels, but also reversed the abnormal levels of serum interleukin-6 (IL-6) and tumor-necrosis-factor alpha (TNF-alpha) to the normal in the stressed rats. These results suggested that icariin possessed an antidepressant-like property that was at least in part mediated by neuroendocrine and immune systems.”

The 2007 publication Icariin from Epimedium brevicornum attenuates chronic mild stress-induced behavioral and neuroendocrinological alterations in male Wistar rats reports: “Chronic mild stress (CMS) is suggested to produce abnormalities in the hypothalamic-pituitary-adrenal (HPA) axis and hypothalamus-pituitary-thyroid (HPT) axis. Therefore, compound that attenuates the neuroendocrinological alterations may have potential as antidepressant. The behavioral and neuroendocrinological effects of icariin, a major constituent of flavonoids isolated from Epimedium brevicornum, were investigated in the CMS model of depression in male Wistar rats. CMS procedure caused an anhedonic state in rats resulted in increased corticotropin-releasing factor (CRF) concentrations in dissected brain regions and serum, decreased total triiodothyronine (tT3) in serum with no significant changes in serum adrenocorticotrophic hormone (ACTH) and thyroxine (tT4). Administration of icariin reversed CMS-induced sucrose intake reduction and CRF elevation. These results suggested that icariin possessed potent antidepressant-like activities which were at least in part mediated by improving the abnormalities in the HPA axis functions. However, we did not find a clear correlation between the HPT axis and icariin treatment in the CMS-treated rats.”

The 2009 publication Icariin attenuates chronic mild stress-induced dysregulation of the LHPA stress circuit in rats reports: “Chronic mild stress (CMS) is suggested to develop dysregulation of the limbic-hypothalamic-pituitary-adrenal (LHPA) stress circuit. Icariin, a major constituent of flavonoids isolated from Epimedium brevicornum, has been previously confirmed to rescue the HPA axis abnormalities in animal models of depression. However, antidepressant treatment of icariin on corticotropin-releasing factor (CRF) system within the LHPA stress circuit and its interaction with serotonergic receptor are still seldom studied in CMS model of animals. The present study further investigated the effects of CMS procedure and subsequent icariin treatment on mRNA and protein levels of CRF, CRF receptor 1 (CRFR1) and CRF binding protein (CRFBP), as well as sucrose intake in rats. Moreover, the levels of cyclic adenosine 3′,5′-monophosphate (cAMP) response element binding protein (CREB), glucocorticoid receptor (GR) and 5-hydroxytryptamine 1A receptor (5-HTR1A) in hypothalamus, hippocampus and frontal cortex were simultaneously evaluated for their participations in CRF system in this model. We found that CMS procedure significantly increased CRF expression levels in the brain regions, and decreased GR and 5-HTR1A in hippocampus and frontal cortex, with sucrose intake reduction representing the hedonic deficit in rats. Icariin restored these alterations in CMS rats. These results confirmed the hypothesis that icariin exerted antidepressant-like effect via its regulation of central CRF system. And hippocampus was suggested as an important neural area controlling the LHPA stress circuit in icariin-treated CMS rats. These findings for the first time proved that the potential molecular mechanism of antidepressant action of icariin was targeted on the interaction of the LHPA stress circuit and serotonergic function in CMS rats.”

Icariin-based therapy may possibly be effective in combating some forms of cancer.

The 2009 publication Anti-proliferative efficacy of icariin on HepG2 hepatoma and its possible mechanism of action relates soecifically to hepatomas.  “The aim of the present work was to explore the anti-hepatoma effects of icariin both in vitro and in vivo and to elucidate its potential mechanism of action. The MTT assay was applied to test the anti-proliferative effects of icariin in vitro. HepG2 bearing NMRI nu/nu mice were used to test the anticancer effects of icariin in vivo. Immunohistochemical assay and flow cytometry assay (FACS) were applied to detect the possible mechanisms of action of icariin. MTT assay illustrated that icariin inhibited the proliferation of HepG2 cells in a concentration dependent manner; meanwhile, icariin inhibited the tumor growth in HepG2 bearing NMRI nu/nu mice. The tumor weight was inhibited by 55.6% and tumor volume was inhibited by 47.2%. Icariin did not influence the spleen and body weights or blood parameters. Immunohistochemical analysis indicated that the expressions of both CD31 and Ki67 in the icariin treated group were significantly lower than those in the control group (p < 0.01). FACS assay showed that icariin dramatically decreased the percentage of CD4+ and CD8+ cells in bone marrow and CD19+ cells in blood on day 8. On day 17, the percentage of CD8+ cells in blood was lower than those in the control group. CD4/CD8 ratio in icariin group was significantly elevated in bone marrow on day 17. Icariin showed anticancer efficacy both in vitro and in vivo. The possible mechanism of action could be related to its anti-angiogenesis and anti-proliferative effects in tumors.”  Of course there is a long path from an in-vitro study like this one to establishing a safe and efficacious clinical anti-cancer treatment.

Icaritin is another compound found in epimedium  The May 2011 publication An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells reports:  “Icaritin, a prenylflavonoid derivative from Epimedium Genus, regulates many cellular processes. However, the function and the underlying mechanisms of icaritin in breast cancer cell growth have not been well established. Here, we report that icaritin strongly inhibited the growth of breast cancer MDA-MB-453 and MCF7 cells. At concentrations of 2-3 μM, icaritin induced cell cycle arrest at the G(2)/M phase accompanied by a down-regulation of the expression levels of the G(2)/M regulatory proteins such as cyclinB, cdc2 and cdc25C. Icaritin at concentrations of 4-5 μM, however, induced apoptotic cell death characterized by the accumulation of the annexin V- and propidium iodide-positive cells, cleavage of poly ADP-ribose polymerase (PARP) and down-regulation of the Bcl-2 expression. In addition, icaritin also induced a sustained phosphorylation of extracellular signal-regulated kinase (ERK) in these breast cancer cells. U0126, a specific ERK activation inhibitor, abrogated icaritin-induced G2/M cell cycle arrest and cell apoptosis. Icaritin more potently inhibited growth of the breast cancer stem/progenitor cells compared to anti-estrogen tamoxifen. Our results indicate that icaritin is a potent growth inhibitor for breast cancer cells and provide a rationale for preclinical and clinical evaluations of icaritin for breast cancer therapy.”

Icariin stimulates in-vitro angiogenesis.

The 2008 publication Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells reports: “We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.”

Icariin could be the basis for an acne treatment

The new February 2012 publication Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds is written by researchers in Belgium, one of the few non-Asian publication mentioned here.  It reports: “Propionibacterium acnes is a Gram-positive bacterium that plays an important role in the pathogenesis of acne vulgaris. This organism is capable of biofilm formation and the decreased antimicrobial susceptibility of biofilm-associated cells may hamper efficient treatment. In addition, the prolonged use of systemic antibiotic therapy is likely to lead to the development and spread of antimicrobial resistance. In the present study we investigated whether P. acnes biofilms could be eradicated by plant extracts or their active compounds, and whether other mechanisms besides killing of biofilm cells could be involved. Out of 119 plant extracts investigated, we identified five with potent antibiofilm activity against P. acnes (extracts from Epimedium brevicornum, Malus pumila, Polygonum cuspidatum, Rhodiola crenulata and Dolichos lablab). We subsequently identified icariin, resveratrol and salidroside as active compounds in three of these extracts. Extracts from E. brevicornum and P. cuspidatum, as well as their active compounds (icariin and resveratrol, respectively) showed marked antibiofilm activity when used in subinhibitory concentrations, indicating that killing of microbial cells is not their only mode of action.”

Along with quercetin, astragaloside IV, and ginsenoside, icariin may produce Cytochrome P450 drug interactions in a dose-dependent manner.

The November 2011 publication Effects of natural products on the function of human organic anion transporting polypeptide 1B1 reports: “In this study, the effects of 136 naturally occurring products, which have been reported to play important roles in modification of Cytochrome P450 (CYP450) activities, on the uptake of estrone-3-sulfate (E3S), a typical OATP1B1 substrate, were evaluated using human embryonic kidney 293 cells stably expressing OATP1B1. At a concentration of 100 μM, 42 natural products inhibited OATP1B1-mediated [(3)H]E3S uptake by more than 50%, and five of them significantly inhibited OATP1B1-mediated [(3)H]E3S by more than 80% with the following rank order of potency: quercetin > astragaloside IV > icariin > glycyrrhizic acid > ginsenoside Rc. Inhibitory effects of these natural products on OATP1B1 activity were in a concentration-dependent manner. 11 natural compounds were found exhibiting greater than 50% inhibition at 30 μM with IC(50) values ranging from 14.6 ± 3.3 to 28.5 ± 3.0 μM. In conclusion, our data suggest that modification of OATP1B1 transport activity by these natural occurring products may be a mechanism for natural product-drug interactions in humans.”

The Cytochrome P450 superfamily (officially abbreviated as CYP) is a large and diverse group of enzymes involved in drug bioactivation, metabolism, transport and clearance.  P450 interactions may be among drugs or, as discussed here, among drugs and natural substances, or among natural substances themselves.

Epimedium may provide a means for overcoming male erectile dysfunction exploiting the same biological mechanism of action used in popular drugs like Viagra^® and Cialis^®.

One of the important applications of epimedium in traditional Chinese medicine is penis erection enhancement.  There appears to be a biological basis for this.  Viagra^® (Sildenafil citrate) is a drug used to treat erectile dysfunction and pulmonary arterial hypertension (PAH), and so is Cialis^®. (Tadalafil).  Both work by inhibiting PDE5. “Penile erection during sexual stimulation is caused by increased penile blood flow resulting from the relaxation of penile arteries and the smooth muscle of the corpus cavernosum. This response is mediated by the release of nitric oxide (NO) from nerve terminals and endothelial cells, which stimulates the synthesis of cGMP in smooth muscle cells. Cyclic GMP relaxes smooth muscle and increases blood flow to the corpus cavernosum. The inhibition of phosphodiesterase type 5 (PDE5) enhances erectile function by increasing the amount of cGMP. Tadalafil (and sildenafil and vardenafil) inhibits PDE5 (Wikipedia).” 

It turns out that icariin is also a PDE5 inhibitor; this has been known for some time.  The 2003 publication Effects of icariin on cGMP-specific PDE5 and cAMP-specific PDE4 activities reported: “Aim: To clarify the mechanism of the therapeutic action of icariin on erectile dysfunction (ED).  Methods: PDE5 was isolated from the human platelet and PDE4 from the rat liver tissue using the FPLC system (Pharmacia, Milton Keynes, UK) and the Mono Q column. The inhibitory effects of icariin on PDE5 and PDE4 activities were investigated by the two-step radioisotope procedure with [(3)H]-cGMP/[(3)H]-cAMP. Papaverine served as the control drug.  Results: Icariin and papaverine showed dose-dependent inhibitory effects on PDE5 and PDE4 activities. The IC(50) of Icariin and papaverine on PDE5 were 0.432 micromol/L and 0.680 micromol/L, respectively and those on PDE4, 73.50 micromol/L and 3.07 micromol/L, respectively. The potencies of selectivity of icariin and papaverine on PDE5 (PDE4/PDE5 of IC(50)) were 167.67 times and 4.54 times, respectively.  Conclusion: Icariin is a cGMP-specific PDE5 inhibitor that may be developed into an oral effective agent for the treatment of ED.”

A Wikipedia discussion of icariin as an Aphrodisiac has to say “Like sildenafil (the erectile dysfunction drug commonly sold as Viagra), icariin, the active compound in Epimedium, inhibits the activity of PDE-5. In vitro assays have demonstrated that icariin weakly inhibits PDE-5 with an IC₅₀ of around 1 μM,^[6][7] while sildenafil has an IC₅₀ of about 6.6 nM (.0066 μM) and vardenafil (Levitra) has an IC₅₀ of about 0.7 nM (.0007 μM).^[8] Measured differently, the EC₅₀ of icariin is approximately 4.62 μM, while sildenafil’s is .42 μM.^[9] With the weak potency of Epimedium, and its unknown oral bioavailability, the amount of Epidemium extract necessary to have any effect is unclear from the literature.”

The literature, on the other hand, suggests that long-term administration of icariin may have a positive effect on erectile functioning, different than the one-shot effect of taking Viagra^® or Cialis^®.  The 2004 publication [Effects of icariin on the erectile function and expression of nitrogen oxide synthase isoforms in corpus cavernosum of arterigenic erectile dysfunction rat model] reports: “Objective: The study the effects of oral administered icariin on intracavernosal pressure (ICP) and on expression of the nitrogen oxide synthase (NOS) isoforms in corpus cavernosum (CC) of arteriogenic erectile dysfunction (A-ED) rat model.  Methods: Forty adult male Wistar rats were randomly divided into 4 groups of 10 rats: shame operated group (group A) and three A-ED model groups (group B, C and D). The internal pudendal arteries were isolated and ligated with 7-O nylon thread at both the main trunk and the penile branches to establish the A-ED model. ICP were tested after the operation to make sure the successful model establishment. The groups A and B were treated with saline: and the groups C and D were treated with icariin (5 mg/kg/day and 10mg/kg/day respectively) orally for 30 days. Then the ICP was measured again. The tissues of corpus cavernosum were taken and RT-PCR was used to detect the mRNA expression of nNOS, iNOS and eNOS in CC, and Western-blot was used to detect the protein expression of these NOS isoforms.  Results: The ICP in the group B was significantly decreased compared to the group A (P < 0.01), but the ICP values in the groups C and D were both increased compared to those in the group B (both P < 0.01). The expressions of the mRNA and protein of nNOS, iNOS, and eNOS were all decreased in the group B, however, the mRNA and protein expressions of eNOS were increased a in the groups C and D. In the group C, iNOS also increased. The expression of nNOS showed no obvious changes in the group C and group D.  Conclusion: Chronic oral treatment with Icariin increases the erectile function (ICP) and restores the eNOS expression in CC of A-ED rats. Icariin may have a long-term therapeutic effect on ischemia/hypoxia induced ED.”

The December 2007 publication Effect of lipid-based suspension of Epimedium koreanum Nakai extract on sexual behavior in rats is one of the very few publications authored by non-Chinese.  It is by a group of Russians in St.-Petersburg.  Ethnopharmacological Relevance: Herba of Epimedium koreanum is used in traditional Chinese and Korean herbal medicine as a potent enhancer of erectile function. Icariin, the main active component of Epimedium koreanum, possesses many biological effects, such as improving cardiovascular function, hormone regulation, immunological function modulation, and anti-tumor activity.  Aim Of The Study: This study supports the traditional use of extracts from Epimedium species in erectile dysfunction.  Materials and methods: The Epimedium koreanum dry extract was suspended in wheat germ oil using lecithin and bee wax for oral administration. The effect of oral administration of two compositions (E-01 and E-02) standardized by their icariin content on the number of complete intromissions, the number of ejaculations, and the latent period of ejaculation (LPE) in rats were evaluated. E-01 and E-02 were administered orally for 10 days to the experimental animals. The control animals received olive oil for 10 days. On day 10, 0.5h after the dose was administered to male rats, one virgin female rat was placed with one male rat.  Results: The number of complete intromissions increased to 23.3+/-2.6 in the E-01 and E-02 group (dose 300 mg/kg body weight) (b.wt) and to 20.1+/-2.3 in the E-02 group (dose 750 mg/kg b.wt) compared with 15.2+/-2.4 in the control group of aged rats.  The number of ejaculations increased from 1.1+/-0.3 in the control-aged group to 2.6+/-0.4 in the E-01 group. The LPE of male rats was 14.2+/-1.8 min in the control-aged group. The LPE of the aged group was reduced to 9.8+/-1.5 min, 9.8+/-1.6 min, and 11.4+/-1.8 min when treated with E-01 at a dose of 300 mg/kg b.wt, and E-02 at a dose of 300 mg/kg b.wt and 750 mg/kg b.wt, respectively.  Conclusion: It was established that oral administration of lipid-based suspension of dry extract of Epimedium koreanum in wheat germ oil improved erectile function of aged rats.” 

The 2010 publication Erectogenic and neurotrophic effects of icariin, a purified extract of horny goat weed (Epimedium spp.) in vitro and in vivo reports: “Aim: To evaluate the penile hemodynamic and tissue effects of ICA in cavernous nerve injured rats. We also studied the in vitro effects of ICA on cultured pelvic ganglia.  Methods: Rats were subjected to cavernous nerve injury and subsequently treated for 4 weeks with daily gavage feedings of a placebo solution of normal saline and Dimethyl sulfoxide (DMSO) vs. ICA dissolved in DMSO at doses of 1, 5, and 10 mg/kg. A separate group underwent a single dose of ICA 10 mg/kg 2 hours prior to functional testing. Functional testing with cavernous nerve stimulation and real-time assessment of intracavernous pressure (ICP) was performed at 4 weeks. After functional testing, penile tissue was procured for immunohistochemistry and molecular studies. In separate experiments, pelvic ganglia were excised from healthy rats and cultured in the presence of ICA, sildenafil, or placebo culture media.  Main Outcome Measure: Ratio of ICP and area under the curve (AUC) to mean arterial pressure (MAP) during cavernous nerve stimulation of subject rodents. We also assayed tissue expression of neuronal nitric oxide synthase (nNOS), eNOS: endothelial nitric oxide synthase (eNOS), calponin, and apoptosis via immunohistochemistry and Western blot. Serum testosterone and luteinizing hormone (LH) were assayed using enzyme-linked immunosorbant assay (ELISA). Differential length of neurite outgrowth was assessed in cultured pelvic ganglia.  Results: Rats treated with low-dose ICA demonstrated significantly higher ICP/MAP and AUC/MAP ratios compared with control and single-dose ICA animals. Immunohistochemistry and Western blot were revealing of significantly greater positivity for nNOS and calponin in penile tissues of all rats treated with ICA. ICA led to significantly greater neurite length in cultured specimens of pelvic ganglia. Conclusion: ICA may have neurotrophic effects in addition to known phosphodiesterase type 5 inhibiting effects.

So there we have it: icariin significantly increased sexual activity by the rats who consumed icariin and it may work through mechanisms in addition to inhibition of PDE5.

Additional publications of relevance include:

• Constitutional flavonoids derived from Epimedium dose-dependently reduce incidence of steroid-associated osteonecrosis not via direct action by themselves on potential cellular targets (2009)
• Different molecular targets of Icariin on bMSCs in CORT and OVX -rats (Jan 2012)
• [Study of molecular mechanisms of fuyuan capsule, icariin and arasaponin R1 in treatment of osteoarthritis] (Aug 2011)
• [Icariin and its pharmaceutical efficacy: research progress of molecular mechanism] (Nov 2011)