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Synergistic inhibitory effect of Icariside II with Icaritin from Herba Epimedii on pre-osteoclastic RAW264.7 cell growth.


Increasing evidence shows the therapeutic superiority of herbal extracts in comparison to isolated single constituents. One of the reasons may be attributed to the synergy effect of compound combinations. Flavonoids from Herba Epimedii have been shown to have therapeutic effect against bone loss. Our previous study showed that Icariside II inhibited pre-osteoclast RAW264.7 growth. The aim of this study was to investigate whether the activity of Icariside II is synergized by other components of Herba Epimedii. The inhibitory activity of Icariside II was significantly enhanced in the presence of the extract of Herba Epimedii (EHE) at the ratio of 1:1.1:5 and 1:10. Icaritin, another flavonoid constituent, was shown here to inhibit RAW264.7 growth in a dose-dependent manner. Further, we found that Icariside II, together with Icaritin, synergistically inhibited RAW264.7 growth. The synergistic effect is significant when the ratio of Icariside Hand Icaritin was 10:1,5:1,1:1,1:2, and 1:5, respectively. In conclusion, Icaritin were an active component. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as osteoporosis


Herba Epimedii


Icarlside II




The efficacy of plant extracts used for centuries has been verified in many cases by clinical studies. It is believed that synergy effect of the mixture of bioactive constituents from either several types of herbal extracts or mono-extract preparations could be responsible for the improved therapeutic efficacies. However, most herbal extracts contain many chemical constituents, resulting in the synergistic action of the mixture remaining to be addressed. Herba Epimedii is a commonly used Chinese medicine for "strengthening kidney" for thousands of years. It contains active components such as flavonoids and phytosteroids (Wu et al., 2003; Zhao et al., 2008). Total flavonoids of Herba Epimedii are suggested to be potential candidates for treating osteoporosis (Zhang et al., 2009; Zhang et al., 2010). It includes Icariin, Epimedin A, Epimedin B, Epimedin C, Icariside II, Icaritin, etc. The anti-osteoportic effect of total flavonoids is shown to be more potent than its single compound (Meng et al., 2005; Zhang et al., 2008). Although Icariin is the principal flavonoid glycoside in Herba Epimedii, Icariside II exerted higher bone resorption activity of osteoclasts (Huang et al., 2007). Our previous study showed that Icariside II inhibited pre-osteoclast RAW264.6 growth (Yang et al., 2012). In the present study, we investigate whether the active component Icariside II exerts synergistic effect with other flavonoids from Herba Epimedii on osteoclastic growth.

Osteoclasts, originated from monocyte/macrophage precursors, mediate bone resorption of old bone tissue, which was coupled by osteoblast-mediated bone formation of new bone tissue. This process, called bone remodeling, maintains bone homeostasis (Matsuo and trie, 2008). However, under certain pathological conditions, such as osteoporosis, there is an abnormally high bone turnover, with enhanced osteoclastic bone resorption (Ralston and Uitterlinden, 2010; Ohlsson 2013). Therefore, inhibition of osteoclast proliferation and activation is the target for the therapeutic intervention of pathological bone loss. Previously, Herba Epimedii extract and its component including icariin were shown to inhibit osteoclast differentiation (Huang et al. 2007). In this study, we investigated the inhibitory effect of Icariside II and Icaritin on preosteoclastic RAW264.7 cell growth. We further investigate whether the inhibitory action of Icariside II is synergized by other components in the Herba Epimedii extract.

Materials and methods


The extracts of Epimediums (EHE) were prepared and characterized by HPLC analysis as our previously reported (Yang et al., 2012). Icariside II was purchased from Shanghai Winherb Medical Science Co., Ltd. (Shanghai, China). The purity of Icariside II is 99%. Icaritin was prepared in our lab. The purity is above 98%. The chemical structures of Icariside II and Icaritin are shown as Fig. 1. Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco (Grand Island, NY, U.S.A.). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, USA). Fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Dimethyl sulfoxide (DMSO) was from Ameresco (U.S.A.).

Cell culture

Murine preosteoclast RAW264.7 cells were obtained from the Chinese Academy of Sciences (Shanghai, China). The RAW 264.7 cell line was established from the ascites of a tumor induced in a male BAB/14 mouse by the intraperitoneal injection of A-MuLV (Raschke et al., 1978; Ralph and Nakoinz, 1977). The cells were cultured in DMEM supplemented with 10% FBS, penicillin (100U/ml) and streptomycin (100 [micro]g/ml). The cells were maintained at 37[degrees]C with 5% C[O.sub.2] in a humidified atmosphere.

MTT assay

RAW264.7 cells were seeded into 96-well plates at 1 x [10.sup.5]/ml density. After overnight incubation with 10% FCS, various concentrations of extract, Icaritin and Icariside II were added to the plates. Following incubation, cell growth was measured at different time points by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as previously described with a plate reader (Tecan, Switzerland). The percentage of inhibition was calculated as follows:

Inhibitory effect (%) = [A.sub.492(control)] - [A.sub.492(sample)]] x 100%

Drug assays

To test the synergistic activity of Icariside II with EHE, Icariside II was added at the concentrations of 0.1,1,10,20 [micro]g/ml. According to 10-fold concentration of Icariside II, the combined concentrations of EHE were 1,10,100,200 [micro]g/ml. According to five-fold concentrations of Icariside II, The combined concentrations of EAE were 0.5, 5, 50,100 [micro]g/ml. According to equal concentrations of Icariside II, the combined concentrations of EAE were 0.1,1,10, 20 [micro]g/ml. The concentrations of Icaritin were determined according to the ratio of Icaritin and Icariside II, which was chosen to be 10:1, 5:1, 2:1, 1:1, and 1:2, respectively.

Dose-inhibitory curve methods were used to evaluate the synergistic activities of Icariside II alone or in combination with EHE or Icaritin. Synergistic activity of Icariside II with Icaritin was evaluated according to the "isobol method" reported by Berenbaum (1989). Synergy was defined when combined index (CI) was less than 1; while additive in which CI was equal to 1; and antagonistic in cases which CI was greater than 1.

Statistical analysis

Assays were set up in triplicate. Statistical analysis and significance were performed by using CalcuSyn software (Biosoft, Cambridge, UK).

Results and discussion

The growth inhibitory effect of Icariside II alone was moderate at 10 [micro]g/ml, but significant synergistic activities against RAW264.7 cell growth were observed when combinations of 10 [micro]g/ml Icariside II with 10 [micro]g/ml EHE (Fig. 2a). The inhibitory rate reached 47.2%, which was higher than the inhibitory rate (23.8%) of 10 [micro]g/ml Icariside II treatment alone. EHE was ineffective in inhibiting the growth of RAW264.7 cells below the dose of 200 [micro]g/ml. The increased inhibitory rate was more potent when 20 [micro]g/ml Icariside II and 20 [micro]g/ml EHE were combinated, in contrast with 20 [micro]g/ml Icariside II treatment alone. To further confirm the synergistic activity of Icariside II with EHE, the different combinated ratios of Icariside II to EHE were used. When the ratio of Icariside II to EHE is 1:5, the inhibitory effect of Icariside II at 10p.g/ml combined with EHE at 50 [micro]g/ml was significantly enhanced, in relevant to Icariside II alone, with the increased inhibitory rate from 23.8% to 47% after 24 h incubation (Fig. 2b). Moreover, the synergism of Icariside II with EHE at the ratio 1:10 against RAW264.7 cell growth was stronger than that of the combination at the ratio 1:5. The inhibitory rate of combination of 10 [micro]g/ml Icariside II and 100ptg/ml EHE is three-fold more than 10 [micro]g/ml Icariside II alone (Fig. 2c).

The major components in Epimediums included Icariin, Epimedin A, Epimedin B, and Epimedin C. Icaritin is one of minor components in Epimediums (Zhao, et al., 2008). Icaritin inhibited RAW264.7 cell growth in a dose-dependent manner with the inhibitory activity of 40%, 54%, and 60%, observed at 5, 10, and 20 [micro]g/ml, respectively (Fig. 3a). Compared with Icariside II, the inhibitory action of Icaritin on RAW264.7 cell growth was stronger from 2.5 to 10 [micro]g/ml. To determine whether Icaritin could exhibit synergistically inhibitory effect with Icariside II on RAW264.7 cell growth, the inhibitory effect of Icaritin alone and combination with Icariside II was tested. When Icaritin was combined with Icariside II according to the 1:1 ratio, the inhibitory effect was further enhanced (Fig. 3a). The CI value of Icariside II and Icaritin was smaller than 1, and therefore corresponds to the isobole that is concavely curved toward the zero point (Fig. 3f), suggesting that they acted synergistically to inhibit RAW264.7 cell growth. When the ratio was changed to 1:2 (Fig. 3b) or 2:1 (Fig. 3c), the synergistic effect was still significant. Although the curve fitting for Icaritin is not convincing at the low doses, the significant synergistic effect of Icariside II with Icaritin was observed at the ratio of 10:1 (Fig. 3d) and 5:1 (Fig. 3e). The IC50 values were determined and the according concentrations of Icariside II and Icaritin were given. The combination effect was described by the means of the isobol curve for 50% inhibition of Icariside II and Icaritin combination. As shown in Fig. 3f, Icariside II and Icaritin at concentrations ranging from 0.25 to 40 [micro]g/ml had CI values < 1, indicating synergistic effect between Icariside II and Icaritin.

In the past few years, the pharmaceutical industry has seen a shift from the search for "one gene, one drug, one target" to the pursuit of combination therapies that comprise more than one active compound (Hopkins, 2008). TCM has been commonly used in extract forms for >2500 years. The efficiency of extract may be attributed to synergistic effect between its active ingredients (Williamson, 2001). Herba Epimedii is a commonly used Chinese medicine for "strengthening kidney" for thousands of years. Its extract reduced the occurrence of osteoporosis in experimental models (Qian et al., 2006) and a clinical study (An et al., 2000; Xie et al., 2005). Although Icariin is a principal flavonoid glycoside in Herba Epimedii, Icariside II and Icaritin, two hydrolytic metabolites, showed stronger inhibitory activity against osteoclast differentiation. Icariside II and Icaritin were also found in EAE (data not shown). To investigate whether active ingredients from EAE exhibit synergistic effect, combination of Icariside II with EHE was performed. Synergistic inhibitory action of Icariside II with EAE on RAW264.7 cell growth was observed, indicating that synergistic components with Icariside II maybe exist in EHE. With the ratio of EAE to Icariside II increasing, the synergistic effect of Icariside II with EHE became more significant, indicating that certain minor components could synergize with Icariside II. Additionally, we confirmed that the major components including Icariin, epidemin A, epidemin B, and epidemin C did not show synergistic effect with Icariside II (data not shown).

Icaritin is one of minor components in Herba Epimedii (Zhao, et al., 2008). We found that Icaritin exist in EAE by HPLC analysis (data not shown). Similar to the activity of Icariside II, Icaritin has been shown to suppress osteoclastic differentiation and activity in vitro. In this study, Icaritin was demonstrated to inhibit preosteoclastic RAW264.7 proliferation. When Icaritin was combined with Icariside II at the ratio of 1:1, the synergistically inhibitory effect was observed. Further, various Icaritin and Icariside II mixtures were used for this experiment and IC50 values were determined in the RAW264.7 growth inhibition test. According to the means of isobol equation, the CI is < 1, and therefore corresponds to a concave curve. In this way, it clearly signalized a synergistic effect. Moreover, this proven synergy effect may have impact on the therapeutic use of Herba Epimedii, because Icariin and its glycoside epimedin A, epimedin B, or epimedin C were metabolized to Icariside II and then to Icaritin (Zhao et al., 2008), which could result in synergy in vivo. Although the synergy effect of Icariside II and Icaritin was not stronger, relative to their individual high activity, the fact of this synergy helps to explain efficiency of extract in comparison of a single compound. It is interesting that Icariside II and Icaritin with similar chemical structures generate synergistic effect. Similarly, Ginkgolide A and Ginkgolide B are reported to synergistically inhibit PAF-induced thrombocyte aggregation (Wagner and Steinke, 2005). The synergistic mechanism of Icariside II and Icaritin could be attributed to the multi-target effect. Thus, it is interesting for study of molecular biological mechanism of synergistic action of Icariside II with Icaritin in the future.

Conflict of interest

The author declares no competing financial interest.


This work was supported by grants from National Natural Science Foundation of China (81102739).


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Article history:

Received 16 April 2014

Received in revised form 18 June 2014

Accepted 29 July 2014

Yan-Qiu Liu (a,b), Qian-Xu Yang (b), Meng-Chun Cheng (b), Hong-Bin Xiao (b,c,*)

(a) College (Institue) of Integrative Medicine, Dalian Medical University, Dalian 116044, China

(b) Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

(c) Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

* Corresponding author at: Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Key Laboratory of Separation Science for Analytical Chemistry, Dalian 116023, China. Tel: +86 411 8437 9756; fax: +86 411 8437 9907.

E-mail addresses:, (H.-B. Xiao).
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Title Annotation:Short communication
Author:Liu, Yan-Qiu; Yang, Qian-Xu; Cheng, Meng-Chun; Xiao, Hong-Bin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Oct 15, 2014
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