What Is the Benefit of Icariin Powder?

Icariin is the main active ingredient of total flavonoids (total flavonoids) extracted from the stems and leaves of Epimedium plants in the Epimedium genus of the Berberidaceae family. Modern pharmacological studies have shown that icariin has multiple pharmacological effects, such as anti-tumor, immune enhancement, improvement of cardiovascular function, and endocrine regulation. It is one of the most researched single Chinese herbal ingredients in recent years by scholars at home and abroad.

1 In vivo metabolic processes and products

Ye Lika et al. [1] administered injection solutions containing different concentrations of icariin to rats by gavage and intravenous injection, and used high-performance liquid chromatography to determine the distribution of icariin in rats. The results showed that icariin is not easily accumulated in the body of rats, is eliminated quickly, and is widely distributed. After 2 hours of intravenous injection of the drug, icariin was found in high concentrations in the lungs and plasma, and the drug in its original form could be measured in brain tissue, indicating that icariin can enter brain tissue through the blood-brain barrier.

Zou Jieming [2] used radioisotope tracing technology to compare the dynamic distribution of icariin in mice in a single state and a compound state, and found that the kinetic behavior of icariin in both states in animals conformed to an open two-compartment model. In addition, in the compound state, other ingredients in the compound have a synergistic effect on icariin, promoting absorption and distribution, delaying elimination, maintaining a high level of drug in the body for a long time, and increasing its bioavailability. Icaritin is highly concentrated in the adrenal gland, and at 48 hours after administration, its concentration is still higher than in any other organ, suggesting that the adrenal gland is the most sensitive target organ for Icaritin. In vitro conditions, icariin can be metabolized by intestinal bacteria, and the main metabolite is the aglycone of icariin. After icariin is orally administered to rats, the main metabolite absorbed into the blood is icariside I [3].

2 Pharmacological effects

In recent years, pharmacological research on icariin has focused on the mechanisms of action of icariin on bone tissue, the cardiovascular and nervous systems, and the reproductive system.

2.1 Effect on the immune system

Icariin has a definite regulatory effect on the immune system, which can increase the mass of the thymus and spleen, the immune organs of mice, significantly enhance the phagocytic ability of macrophages, and significantly increase the percentage of rosette formation of red blood cells and the serum TNF-α level [4].

2.1.1 Effect on nonspecific immunity

Icariin can improve the phagocytic function of peritoneal macrophages in mice and restore the phagocytic function of peritoneal macrophages damaged by cyclophosphamide and ionizing radiation to normal levels [5]. It can also regulate immune function by affecting the secretion of macrophage cytokines [6].

Icariin at certain concentrations can enhance the activity of lymphokine-activated killer (LAK) cells and natural killer (NK) cells from healthy individuals and tumor patients. Icaritin can also stimulate the production of tumor necrosis factor-alpha (TNF-α) from mononuclear cells from monitoring donors. These findings suggest that icariin can be used in adoptive immunotherapy, because LAK cells treated with a suitable dose of icariin in combination with IL-2 are more active than LAK cells treated with IL-2 alone [7], suggesting that icariin and IL-2 have a synergistic effect.

2.1.2 Effect on specific immunity

Li Shutong et al. [8] used the 3H-TdR incorporation method to observe the effect of icariin on the proliferative response of mouse splenic lymphocytes induced by Con A and the effect of icariin on the colony-stimulating factor (CSF)-like activity of mouse splenic lymphocytes induced by Con A or lipopolysaccharide. Icariside can significantly promote the proliferation of mouse spleen lymphocytes induced by C on A in vitro and promote the production of CSF-like activity by mouse spleen lymphocytes. Icariside V and Panax notoginseng saponins (PNS) in drug-containing serum can significantly promote C on A -induced mouse splenocyte proliferation and IL-2 production. The combined effect of icariside V and PNS is stronger than that of either alone, suggesting that icariside V and PNS have a certain selective regulatory effect on the mouse immune system, mainly enhancing T cell function [9]. For T lymphocyte subsets T s cells, icariin has an inhibitory effect, thereby enhancing humoral immunity. Epimedium also promotes the proliferation and transformation of B lymphocytes and increases antibody production levels.

2.1.3 Modulatory effect on cytokines

Icaritin can significantly increase the production of IL-2 by thymus and spleen cells in mice. It has also been reported [10] that icaritin can synergistically induce the production of IL-2, IL-3, and IL-6 by tonsil mononuclear cells in combination with PHA. The mechanism may be related to icaritin's upregulation of IL-3 mRNA and IL-6 mRNA expression [11]. Liu Tiehan et al. [3] used a radioimmunoassay to compare the effects of icariin and its intestinal metabolites (baohuoside I and icariigenin) on cytokine secretion by human tissue cell tumor THP-1 cells. The results showed that under different experimental conditions, icariin and its intestinal metabolites have a specific regulatory effect on the production of various inflammatory cytokines. The exact mechanism remains to be explored in further work.

2.2 Antitumor effect

2.2.1 Icariin induces apoptosis and differentiation of tumor cells

2.2.1.1 Inducing apoptosis of tumor cells

Li Guixin et al. [12] found that icariin induces apoptosis of leukemia cells in vitro, with typical morphological and biochemical characteristics, and is time- and dose-dependent. Icariin induces apoptosis of leukemia cells, which can affect the changes in the distribution of each phase of the cell cycle. The increase in the apoptosis rate is related to the decrease in S phase cells and the increase in G0/G1 phase cells. Icaritin can down-regulate the mRNA and protein expression levels of apoptosis-related genes bcl-2 and c-myc, suggesting that these may be the mechanisms by which icaritin induces apoptosis in leukemia cells.

2.2.1.2 Induced differentiation of tumors by icariin

Icaritin has a significant inhibitory effect on the proliferation of acute promyelocytic leukemia cells HL-60 cells [13] or all-trans retinoic acid (ATRA)-resistant HL-60 cells, induces differentiation and apoptosis, and there is no significant difference between drug-resistant and non-drug-resistant cells. The expression of differentiation antigens CD11 b and CD15 on the surface of both cell types increased, further confirming the differentiation-inducing effect [14]. Icariin has a significant synergistic effect with ATRA, increasing the number of cells in the interphase, and Icariin can significantly inhibit the telomerase activity of promyelocytic leukemia or HL-60 cells after 72h [15]; moreover, the decrease in telomerase activity is negatively correlated with the expression rate of the granulocyte differentiation antigen CD11b on the cell surface, inducing HL-60 cells to differentiate in the direction of granulocytes [16].

2.2.2 Mechanism of icariin inhibiting tumor cell invasion and metastasis

2.2.2.1 Icariin inhibits tumor cell adhesion to the extracellular matrix and invasion

The main lethal harm of malignant tumors to the human body lies in the infiltration of tumor cells into surrounding cells and distant metastasis. The ability to adhere, move and invade is a key step in the infiltration and metastasis of tumor cells. Icariin can inhibit the adhesion of tumor cells and reduce their motility and invasiveness. After treatment with icariin at 200 mg·L-1, the adhesion rate of human giant cell lung cancer PG cell line cells on laminin matrix was significantly reduced after 40 min of adhesion, and this effect was time-dependent. PG cells with invasive ability, the ability to pass through artificial basement membranes, motility, and the ability to pass through fibronectin are reduced compared to controls [17-18]. During this process, adhesion molecules containing the mutated exon 6 (CD44V6) and LN-R are key factors in the metastasis of tumor cells across basement membranes. Icariin affects the adhesion rate of PG cells on the LN matrix by downregulating the expression levels of CD44V6 and LN-R on the surface of PG cells [19].

2.2.2.2 Icariin affects the expression levels of invasion-inducing genes and metastasis-related genes

The metastasis-related metastasis genes that are important include invasion-inducing genes Tiam-1 and c-myc and metastasis-inhibiting gene Nm23-H1. The Tiam-1 gene encodes a GDS protein that can act on Rho or Rac proteins, affecting the organization of the cytoskeleton, cell adhesion and movement by participating in the Tiam-1-Rac signaling pathway. The protein product expressed by Nm23 is involved in the transmembrane information transmission mechanism regulated by G protein by regulating GTP synthesis, thereby affecting the polymerization state of microtubules in cells and changing the adhesion and movement capabilities of cells.

After 48 hours of treatment with icariin, the mRNA levels of the PG cell Tiam-1 and c-myc genes were significantly reduced, while the mRNA levels of Nm23 were significantly increased. At this time, no changes in protein levels had yet occurred. After 96 hours of drug treatment, the changes at the gene level have been reflected in the protein level of its expression product. The rising and falling trends of the c-myc and Nm23 gene proteins are consistent with their mRNA levels [20]. It is speculated that icariin affects the organization of the cytoskeleton by downregulating the mRNA levels of the Tiam-1 and c-myc genes, and reduces the expression of CK18 in the cytoplasm. At the same time, it upregulates the mRNA level of the metastasis suppressor gene Nm23, promotes the polymerization of microtubules and the formation of spindles, and inhibits the movement ability of cells, thereby inhibiting the invasion and metastasis of tumor cells. Therefore, icariin is a substance with great potential for anti-tumor or anti-leukemia activity, and is worthy of further development and research.

2.3 Effect on bone tissue

2.3.1 Effect on osteoblasts

Osteoblasts are protein-secreting cells that can produce type I collagen, synthesize and secrete bone matrix, have high alkaline phosphatase (ALP) activity, and can absorb and transport calcium ions. They are important functional cells for bone formation and bone reconstruction. It is currently believed that ALP activity is an indicator of osteoblast function and degree of differentiation. The higher the ALP activity, the more mature the cells become, while a decrease in ALP activity indicates that the cells tend to proliferate. Icariin can significantly promote osteoblast proliferation.

Wang Junqin et al. [21] studied the effect of icariin on rat osteoblasts cultured in vitro and found that icariin can promote osteoblast proliferation, significantly inhibit the ALP activity of early osteoblast differentiation, while it has a promoting effect on the ALP activity of late-stage differentiated osteoblasts, indicating that icariin has different regulatory effects on the proliferation and differentiation of osteoblasts. Yin Xiaoxue et al. [22] directedly induced the differentiation of human bone marrow stromal stem cells into osteoblasts.

MTT assay showed that icariin significantly promoted the proliferation of human osteoblasts and significantly increased their ALP activity, i.e., promoted osteoblast differentiation. This effect may be related to its upregulation of human osteoblast BMP-2 mRNA expression. The osteoblast cell line UMR106 is widely used as a model of developing osteoblasts to study the mechanism of action of anti-osteoporosis drugs. M eng [23] and others used icariin to co-culture with the UMR106 cell line and found that icariin had a very significant effect on promoting the proliferation of UMR106.

The mechanism of action of icariin on osteoblasts is not fully understood, but existing research suggests that icariin is related to the promotion of intracellular signal transduction factors, cytokines and certain transcription factors in osteoblasts. The transforming growth factor (TGF) superfamily plays an important role in the morphogenesis, proliferation, differentiation, and apoptosis of osteoblasts. Smad4 is the necessary pathway for the intracellular transmission of TGF-β family members. SMAD s molecules are also involved in the transmission of various membrane protein receptors. The level of Smad4 mRNA is directly related to the functional state of osteoblasts.

Icariside can stimulate the increase in Smad4 mRNA in MCT3T-E1 cells [24]; icariside promotes the secretion of cytokines TGF-β1 mRNA expression in osteoblasts, inhibits TNF-α, IL-6 mRNA expression, regulates osteogenic proliferation and differentiation, promotes matrix synthesis and secretion, and promotes bone formation; and regulates osteoclast function to reduce bone resorption [25]. Osx is a transcription factor discovered in 2002 that is only expressed specifically in developing bone tissue and is a key substance necessary for OB differentiation and bone formation. Icariin has a promoting effect on OsxmRNA expression in osteoblast cells cultured in vitro, and is concentration-dependent [26].

2.3.2 Effect on bone tissue of ovariectomized rats

For the establishment of an osteoporosis model by removing the ovaries of rats, icariin can increase the bone density, maximum load and flexural rigidity of ovariectomized rats, reduce the serum TRACP and BALP activities, enhance the ability of ovariectomized osteoporosis rats to resist external force impact, effectively inhibit bone mass loss in ovariectomized rats, prevent the occurrence of osteoporosis [27].

2.4 Effect on the reproductive system

2.4.1 Male

effect on the reproductive system is an important aspect of the pharmacological effects of Epimedium. Roman [28] used a glycoside compound extracted from Epimedium sagittatum to feed young Dutch guinea pigs, revealing that the compound can strongly stimulate sensory nerves and have the special physiological effect of producing sexual arousal. Icariside can significantly promote the development of the epididymis and seminal vesicles in young mice. In rat testicular interstitial cells cultured in vitro, icariside can significantly promote basal secretion of testosterone and cAMP production.

Japanese scholars have confirmed that icariside has the effect of promoting sperm production and improving sexual behavior. In a rat model of subacute aging caused by D-galactose, icariin not only reduces germ cell apoptosis by increasing serum SOD activity and androgen levels, and improves degenerative changes in testicular tissue, but also delays gonadal aging by inhibiting the expression of the germ cell aging gene P16 protein [29]. Fu Jie [30] found that icariin can increase the concentration of cGMP in the corpus cavernosum, and suggested that the mechanism of action of icariin on penile erection is related to its ability to increase the concentration of cGMP in corpus cavernosum smooth muscle, thereby enhancing the relaxation of corpus cavernosum smooth muscle. Xin Zhongcheng [31] studied the NO-cGMP pathway in the smooth muscles of the corpora cavernosa, which plays an important regulatory role in the erection process. It was revealed that NO activates guanylate cyclase to synthesize cGMR, and the specific hydrolysis of cGMP by phosphodiesterase (PDE5) inactivates it to participate in regulating penile erection. Icariin can inhibit PDES activity and enhance the activity of the NO-cGMP pathway.

2.4.2 Female

Sexually mature female rats were given different doses of icariin to observe the effect on the weight of the ovaries and uteri, and the effect on follicle development and uterine development was observed by histological sections. The results showed that the ovarian index of rats in the low-dose and high-dose groups of icariin was significantly higher than that of the control group. Tissue sections showed that icariin could significantly promote follicular development in rats, and the number of follicles with lumens was significantly higher than that of the control group. Although there was no significant difference between the uterine index of the icariin drug group and the control group, there was a tendency to increase. Suggestion: Icariin can promote the development of the rat ovary and uterus [32].

Qiao Liang [33] found that PDE5 is expressed in the clitoral sponge tissue, and icariin can significantly increase the concentration of cGMP in the clitoral sponge, with a significant concentration dependence. This shows that icariin's effect on the intracellular cGMP level of clitoral sponge smooth muscle cells may also be through PDE5, which is related to the NO-cGMP signaling pathway.

2.5 Effect on the endocrine system

Icaritin has a significant effect on the endocrine system, which is related to its hormone-like effect. The experimental results of Li Fangfang et al. show that icariin has a direct stimulating effect on the secretion of estradiol by follicular granulosa cells [34]. Icaritin can also increase the levels of FSH and LH in the serum of female rats. This effect is related to the direct stimulation of pituitary cells by icariin to promote gonadotropin GTH [35]. Icariin can increase the basal secretion of testosterone and the production of cyclic adenosine monophosphate (cAMP) in testicular mesenchymal cells from male rats cultured in vitro.

Qin Lu Ping et al. [36] used propylthiouracil (PTU) to establish a mouse model of hypothyroidism with “kidney yang deficiency” and observed the effect of icariin on serum thyroid hormones in mice with the hypothyroidism “kidney yang deficiency” model. The results showed that model mice serum triiodothyronine (T3), reverse triiodothyronine (rT3) and thyroxine (T4) concentrations were all reduced, and the mortality rate of mice increased. During the modeling process, if mice were simultaneously given icariin, it could effectively inhibit the decrease in T3, rT3 and T4 concentrations, and significantly reduce the mortality rate of model mice. At the same time, after treatment with icariin, the concentrations of T3, rT3 and T4 in the model mice were significantly increased. It is suggested that icariin can not only antagonize the thyroid suppressive effect of PTU and prevent the occurrence of “kidney yang deficiency”, but also promote the increase of thyroid hormone levels in mice with hypothyroidism caused by PTU and “kidney yang deficiency”, which has the effect of tonifying the kidney and strengthening yang.

2.6 Effects on the cardiovascular system

2.6.1 Effects on hemodynamics

Icariside can significantly inhibit myocardial contractility and reduce the rate of increase in left ventricular pressure, indicating that it can reduce myocardial oxygen consumption, while also reducing peripheral resistance, alleviating cardiac afterload, and improving cardiovascular diseases such as myocardial ischemia and arrhythmias. In addition, icariin can increase cerebral blood flow, reduce cerebral vascular pressure, and improve microcirculation in experimental animals by dilating vascular smooth muscle, thereby protecting against cerebral ischemia and hypoxia.

Guan Lixin et al. [37] discussed the mechanism of the vasodilatory effect of icariin. The results showed that icariin had a non-competitive antagonistic effect on the dose of 20, 40 mg·L-1 of norepinephrine (NE), potassium chloride (KCl) and calcium chloride (CaCl2) contraction of rabbit aortic strips showed a non-competitive antagonistic effect on the dose of 20.40 mg·L-1; when the dose was 30 mg·L-1, it could significantly inhibit the NE-induced contraction of rabbit aortic strips that depended on extracellular calcium, but had no effect on the contraction response that depended on intracellular calcium. At this dose, its relaxing effect on the aortic strip was independent of blocking α receptors or agonizing β receptors. Tip: The mechanism of the vasodilatory effect of icariin may be related to its blocking effect on calcium channels. The effect of icariin on increasing cerebral blood flow may not be the result of its systemic hemodynamic effects, but is mainly related to its direct dilating effect on cerebral blood vessels [38].

2.6.2 Effect on cardiovascular tissue

Pretreatment with icariin on cardiomyocytes, vascular endothelial cells, smooth muscle cells, etc., can repair damaged cells and thus protect the body.

Isoproterenol treatment of cardiomyocytes for 72 hours can reduce the survival rate of cardiomyocytes, induce apoptosis in cardiomyocytes, with an apoptosis rate of 35%, and significantly reduce mitochondrial membrane potential. Pretreatment with icariin can increase the survival rate of cardiomyocytes, improve mitochondrial membrane potential, and reduce the apoptosis rate of cardiomyocytes. Icaritin has a significant protective effect on ISO-induced injury to primary cultured cardiomyocytes, and this effect is closely related to its protective effect on mitochondria [39].

Both H2O2 and hypoxia can cause damage to the umbilical vein endothelial cell line ECV-304: both can induce apoptosis in endothelial cells and increase the release of MDA and LDH. Icariside pretreatment can inhibit the reduction of vascular endothelial cells caused by hypoxia and H2O2 oxidative damage, reduce LDH activity, inhibit MDA production, increase SOD activity, and significantly inhibit endothelial cell apoptosis. Icaritin also reduces the nitric oxide (NO) content of H2O2-damaged endothelial cell models and reduces the expression of the apoptotic gene caspase-3 caused by H2O2 [40-41]. Icaritin's protective effect on endothelial cells is mainly achieved through a common anti-oxidative damage pathway.

Cardiovascular endothelial cell damage leads to a decrease in NO production, which plays an important role in the pathogenesis of coronary heart disease and acute myocardial infarction. Experiments have shown that icariin can significantly increase NO activity in short-term (5 min) culture with umbilical vein endothelial cells. Long-term culture was found to significantly upregulate the expression of NO synthase mRNA and protein, and this upregulation was proportional to time. L ou [43] and others found that icariin significantly up-regulates the mRNA levels of cardiac transcription factors GATA4 and N kx2∙5, which are related to the NO signaling pathway, as well as the cardiovascular-specific genes α- MHC, MLC-2v and β-AR.

Vascular smooth muscle cells (VSMC) play a very important role in the pathological changes of atherosclerosis and hypertension, and the excessive proliferation of VSMC is one of the main links. Icariin can promote VSMC apoptosis at low concentrations (≤ 10-7 mol·L-1), and the effect is not greatly affected by concentration. It is positively correlated with time, and may have a synergistic effect on promoting VSMC apoptosis when combined with puerarin [44].

2.7 Effects on the nervous system

2.7.1 Effects on the β-amyloid model

Normal rats were injected with Aβ25–35 into the lateral ventricle to create a rat model of Alzheimer's disease. Before and after modelling, Icariside V was continuously gavaged with PNS for 21 days. The spatial learning and memory abilities of rats before and after administration were determined using the jumping platform method and the eight-arm electric maze method, and the acetylcholinesterase (AChE) activity in brain tissue was measured. The number of errors in the eight-arm electric maze of the model rat was significantly increased, and the number of errors in the jumping platform learning and memory was significantly increased. The behavioral indicators of the drug-treated rats were significantly improved, and the AChE activity in brain tissue was reduced. Icariin has a significant preventive and therapeutic effect on spatial learning and memory disorders in rats caused by intraventricular injection of βAP25~35, and this effect is correlated with the reduction of AChE activity in brain tissue [45].

2.7.2 Effect on oxidative damage model

Neuronal hypoxia-induced injury is related to free radical damage to mitochondria. An in vitro model of oxygen free radical damage to mitochondria was established using Fe2+ and vitamin C (VitC) as an oxygen free radical generation system. The effects of icariin on mitochondrial swelling, respiratory chain complex enzyme I~IV activity, and malondialdehyde (MDA) content were observed. F e2+ /VitC can significantly increase mitochondrial swelling and MDA content, and respiratory chain complex enzyme II ~Ⅳ activity decreases to varying degrees. Pre-addition of icariin can significantly inhibit mitochondrial swelling, reduce MDA content, and increase respiratory chain complex enzyme II ~Ⅳ activity. Icariin has a protective effect on the respiratory chain of rat brain mitochondria damaged by oxygen free radicals [46].

2.7.3 Effect on learning and memory in rats with vascular dementia (VD)

A VD rat model was created using the method of permanently ligating the bilateral common carotid arteries and the method of cerebral ischemia-reperfusion. The rats in the icariin administration group performed better than the model group in each indicator in the M orris water maze experiment; the results were similar in the cerebral ischemia-reperfusion experiment. It also increased the activity of SOD and the content of AChE in the cerebral cortex and hippocampus of rats, and decreased the content of MDA. The dose-dependent effect increased the expression of C hAT and AChE in the hippocampus. Observation under a light microscope of the hippocampal CA1 region of the model rat showed that a large number of neurons had degenerated, necrotized, or disappeared, mitochondria were swollen and vacuolated, and synapses were degenerated. In the treatment group, however, nuclear shrinkage was significantly reduced, the cell morphology in the CA1 region was normal, the cells were neatly arranged, normal mitochondria were more common, synapse degeneration was rare and the structure was intact. Icariin can reduce the apoptosis of cortical and hippocampal nerve cells [47].

Cytochrome C oxidase (CO) is a key enzyme in the respiratory chain, and cytochrome C oxidase sub-unit II (COII) plays an important role in maintaining the activity and function of CO. A mouse model of cerebral ischemia-reperfusion injury was established using the 2-VO method combined with blood pressure reduction and reperfusion. The expression of COⅡ subunit mRNA in the brain of each group of mice was detected at different time points using semi-quantitative RT-PCR. The amount of COⅡ mRNA in the model group increased significantly 72 h after cerebral ischemia-reperfusion, and icariin significantly prevented the increase in expression. After 14 days of cerebral ischemia and reperfusion, the amount of COⅡ mRNA decreased again, and the icariin group could prevent the decrease in COⅡ mRNA expression. Icariin has a certain effect on maintaining the normal expression of COⅡ, suggesting that it may be one of the mechanisms by which it exerts its cerebral protective effect [48].

Icariside has very strong biological activity and has a significant effect on many organs and tissues, especially in terms of endocrine, immune system and anti-tumor effects, and there have been many related studies. Icariside also has a good effect on neurological diseases, such as dementia models, but there are not many studies on this at present, and the research on the mechanism of action is also limited to aspects such as anti-oxidation. Pharmacokinetic studies have shown that icariin can pass through the blood-brain barrier, so further research is needed to determine whether icariin has other effects on the nervous system.

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