The Protective Effect Of Herba Cistanches On Statin-induced Myotoxicity in Vitro
Mar 24, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Elaine Wat a,b,1, Chun Fai Ng a,b,1, Chi Man Koon a,b, Eric Chun Wai Wong a,b, Brian Tomlinson c, Clara Bik San Lau a,b,n
an Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
b State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
c Division of Clinical Pharmacology, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
Abstract:
Ethnopharmacological relevance: Herba Cistanches (HC, Cistanche deserticola, or Cistanche tubulosa) is a Chinese herb traditionally used for muscle problems. Previous studies demonstrated that HC extract could reduce muscle damage and improve ATP storage in post-exercise rats. However, its effect on statin-induced muscle toxicity has never been investigated.
Aim: The objective of this study was to determine if the aqueous extract of HC (HCE) could prevent simvastatin-induced toxicity in L6 rat skeletal muscle cells and whether verbascoside is the major bioactive constituent that contributes to the effects
Materials and methods: MTT was performed to determine the effects of HCE (0–2000 mg/ml) or verbascoside (0–160 mM) on simvastatin (10 mM)-treated L6 cells. Annexin V-FITC/PI apoptosis assay and Caspase 3 assay were performed to determine the protective role of HCE on simvastatin-induced cell death, and to evaluate if HCE exerted its protective effect through the caspase pathway. ATP production was measured to investigate if HCE could prevent simvastatin-induced reduction in ATP production in vitro.
Results: Simvastatin significantly increased apoptotic cell death in L6 cells. HCE significantly exerted a dose-dependent reduction on simvastatin-induced apoptotic cells, possibly via a caspase-3 pathway. Simvastatin reduced the ATP production in L6 cells, which was dose-dependently prevented by HCE. There was only a trend but not significant effect (except at high dose) of verbascoside on the protection of simvastatin-induced muscle toxicity.
Conclusions: In conclusion, we demonstrated for the first time that HCE could exert a dose-dependent protective effect on simvastatin-induced toxicity in vitro, which was unlikely due to the presence of verbascoside. Our study suggested the potential use of HC under the situation of simvastatin-induced muscle toxicity.
Keywords: Herba Cistanches, StatinCholesterol, Hyperlipidemia, Muscle toxicity, Verbascoside, Caspase-3
1. Introduction
HMG-CoA reductase inhibitors (statins), being the best-selling prescription drug class in the world in history, are well documented to be beneficial to patients of both genders at different ages with moderate and high cardiovascular disease (CVD) risk (Golomb and Evans, 2008). Different statins were then developed and approved clinically including pravastatin, atorvastatin, fluvastatin, lovastatin, pitavastatin, rosuvastatin, and simvastatin. In general, all statins act via inhibiting the reduction of HMG-CoA to mevalonic acid during the early stage of the mevalonate pathway to reduce cholesterol production (Kromer and Moosmann, 2009; Thompson et al., 2003). Although statins are usually well-tolerated, one of the most important and well-known clinical adverse effects is skeletal muscle toxicity (rhabdomyolysis) (Kobayashi et al., 2008). Skeletal muscle abnormalities can range from benign myalgia to severe myopathy. While a medley of mechanisms are proposed and could be responsible for the muscle adverse effects of statin, mitochondrial mechanisms are believed to play an important role (Golomb and Evans, 2008). Mevalonate is not only a precursor of cholesterol, but also for other compounds including selenoproteins, dolichol, and ubiquinone (Kaufmann et al., 2006; Vaklavas et al., 2009). The ability of statins to inhibit mevalonate would therefore also inhibit the production of selenoproteins such as glutathione peroxidase (GPx), which are important enzymes in maintaining the anti-oxidative defense mechanism (Kromer and Moosmann, 2009). Statins could also lead to ubiquinone depletion, causing a reduction of oxygen consumption and ATP synthesis (Beltowski et al., 2009). Furthermore, increasing in vitro and in vivo studies demonstrated that statin could also act directly on tissue mitochondria to induce Ca2þ-dependent membrane permeability transition (MPT) in a dose-dependent manner (Beltowski et al., 2009; Velho et al., 2006). It is also associated with increased reactive oxygen species (ROS) and mitochondrial oxidative stress, leading to cell death and therefore contributing to liver and muscle injury (Beltowski et al., 2009; Velho et al., 2006).
Herba Cistanches, the whole dried plant of Cistanche deserticola Y.C. Ma, or Cistanche tubulosa (Schrenk) Wight (Orobanchaceae family), are parasitic plants that predominantly grow in the desert areas of north and northeast China (Siu and Ko, 2010). Herba Cistanches is a Yang-invigorating Chinese tonic herb that is primarily used to treat kidney deficiency with symptoms such as impotence, infertility, premature ejaculation. The heavy and cloying nature of the herb also moistens the intestines and helps to ease constipation. It is also a Chinese herb that is traditionally prescribed to patients for pain in the loins and knees and is a herb commonly used in Chinese formulations for the treatment of muscle problems (Siu and Ko, 2010; Xiong et al., 1998). Interestingly, this is also consistent with the modern scientific studies which demonstrated the anti-fatigue activities of a polysaccharide-rich and phenylethanoid-rich extract of Herba Cistanches in rats post-exercising by decreasing muscle damage and improving ATP storage (Cai et al., 2010). Recent work demonstrated that a methanol extract of Herba Cistanches could enhance mitochondrial ATP generation (Leung and Ko, 2008). Siu and Ko (2010) demonstrated that Herba Cistanches could also enhance mitochondrial glutathione status by mediating levels of glutathione synthesis and GPx, thus protecting tissues from oxidative stress in rats’ hearts. Siu and Ko (2010) also showed that Herba Cistanches could decrease mitochondrial Ca2þ content, thereby reducing Ca2þ-dependent MPT. Furthermore, Herba Cistanches was also proven to be a strong antioxidant and free radical scavenger in various organs, reducing the oxidative stress and ROS activities in vivo studies (Lu et al., 1995; Sui et al., 2011). More interestingly, in an attempt to determine the active fraction which contributes to the anti-fatigue activity of Herba Cistanches, it was found that verbascoside was the major constituent in the active fraction (Cai et al., 2010). Verbascoside had also been demonstrated to reduce muscle fatigue in toads, possibly due to its antioxidative activities (Liao et al., 1999), suggesting verbascoside could be the bioactive component contributing to the beneficial effects of Herba Cistanches.
The aim of this study was to determine whether the use of herbal Cistanches water extract (HCE) could reduce the muscle toxicity induced by simvastatin in vitro. It was hypothesized that the use of HCE could correct for the side effects of muscle toxicity caused by simvastatin. An attempt was also made to determine if the beneficial effects of Herba Cistanches are attributed to the presence of verbascoside.
2. Materials and methods
2.1. Herbal materials authentication and preparation
Raw herbal material of Herba Cistanches was purchased from a renowned supplier in Guangzhou, China. Herba Cistanches was chemically authenticated using thin-layer chromatography (TLC) in accordance with Chinese Pharmacopoeia (CP), 2010, with verbascoside and echinacoside as the chemical markers (data not shown). Upon chemical authentication, herbarium voucher specimen of Herba Cistanches was deposited at the Museum of the Institute of Chinese Medicine at the Chinese University of Hong Kong (CUHK), with voucher specimen number 2014–3434.
Briefly, 1 kg of the raw herb was soaked for 1 h, followed by extraction twice by heating for 1 h under reflux at 100 °C using 10 distilled water for each extraction. The aqueous extracts (HCE)were combined and filtered. The filtrate was concentrated under reduced pressure at 60 °C. The concentrated extract was lyophilized to dryness. The percentage of yield was 50.1% w/w. A small amount was used to determine the quantity of verbascoside and echinacoside using ultra-performance liquid chromatography(UPLC). All extracted powder was vacuum packed and stored until use.
2.2. UPLC analysis of the aqueous extract
UPLC analyses were performed using the Waters Acquity Ultra Performance LC System (Waters, USA). All solvents required for UPLC analyses were purchased from the Department of Chemistry at the Chinese University of Hong Kong. Verbascoside and echinacoside were purchased from the National Institute for the Control of Pharmaceutical and Biological Products, China. The sample solution was injected into a Waters Acquity UPLC BEH C18 column (100 2.1 mm2 i.d., particle size 1.7 mm) with Waters ACQUITY UPLC BEH C18 VanGuard Pre-Column (5-2.1 mm2 i.d., particle size 1.7 mm). All solvents were pre-filtered with 0.45 mm Millipore filter disk (Millipore) and de-gassed. Gradient elution was carried out using the following solvent systems: mobile phase A – acetonitrile; mobile phase B – double distilled water/formic acid (99.9/ 0.1; v/v). The elution was performed with a gradient procedure as follows: 0–5 min, 88% B; 5–10 min, from 88% B to 83% B. The flow rate used was 0.4 ml/min and detection was performed at 331 nm in accordance with CP (2010). Each sample (5 μl) was injected into the column after filtration through a 0.2 mm filter disk. Identifies- cation of the chemical markers was carried out by comparing the retention times and the UV absorbance of the unknown peaks to those of the standards. A calibration curve was plotted by a series of concentrations of verbascoside and echinacoside (40, 20, 10, 5, 2.5, and 1.25 mg/ml) for quantification. Quantification of verbascoside and echinacoside within the herbal extract was performed in triplicates. The system was monitored by a computer equipped with the Waters MassLynx Software for data collection, integration, and analysis.
2.3. Cell culture
The L6 rat skeletal muscle cell line was purchased from the American Type Culture Collection (ATCC, Manassa, VA, USA). The cells were maintained at subconfluent density in Dulbecco’sModifified Eagle’s Medium (DMEM, ATCC, Manassas, VA, USA)supplemented with 10% v/v fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S), and incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air.
To determine the effects of HCE on statin-induced muscle toxicity, L6 cells were treated with simvastatin at 10 μM for 48 h to induce toxicity. HCE (at concentrations 0, 250, 500, 1000, and2000 μg/ml) or verbascoside (at concentrations 0, 20, 40, 80, and160 μM) were added as co-treatment with a statin to determine if the herbal extract could prevent the toxicity of statin-induced in skeletal muscle cells and that if verbascoside could exert similar protective effects as the herbal aqueous extract.
Herba Cistanche extract protective effect on simvastatin-induced toxicity in vitro
2.4. In vitro cytotoxicity
L6 cells (1x105/well) were seeded in 96-well flat-bottom culture plates (Iwaki, Japan) overnight, followed by treatment with10 μM of simvastatin, together with or without various doses of HCE (0–2000 μg/ml) or verbascoside (0–160 μM), for 48 h. Plain medium (DMEM) was added to the control wells. The herbal extract and compound were dissolved in DMEM.
Following the 48 h treatment, the medium from all cells was discarded and 30 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma, USA) in phosphate-buffered saline (PBS) were added to each well and the plates were further incubated for 3 h at 37 °C. The supernatant was then removed and 100 μl of DMSO was added to each well to dissolve the purple formazan crystals (Sigma-Aldrich, St. Louis, MO. USA). The absorbance of each sample was read at 540 nm using a microplate reader (Biotek μ-Quant, USA). Results were expressed as the percentage of MTT absorbance with respect to control cells.
2.5. Annexin-V FITC/PI staining assay
L6 cells (3 x105/well) were treated with simvastatin, together with or without various doses of HCE or verbascoside. Cells werethen collected, washed, and stained with annexin-V FITC kit according to the manufacturer's protocol (Trevigen, MD, USA). Briefly, cells were washed twice with 1 x binding buffer and incubated in 100 μl labeling solution containing 1 μl annexin-V FITCconjugate and 10 μl PI in the dark for 15 min at room temperature. The fluorescence of the samples was detected by flow cytometry(Becton Dickinson FACSCanto II, USA).
2.6. Caspase 3 activity assay
Caspase 3 activity was determined using a fluorometric, immunosorbent enzyme assay (Sigma) according to the manufacturer's instruction. Briefly, L6 cells (3 105/well) treated with simvastatin, together with or without various doses of HCE verbascoside were lysed using the lysis buffer. Free 7-amino-4-methylcoumarin (AMC) generated through the proteolytic cleavage of the substrate by Caspase 3 were measured and quantified fluorometrically with the excitation wavelength at 360 nm, and emission wavelength at 460 nm with a BMG FLUOstarOptimamicroplate reader (BMG LABTECH GmbH, Germany).
2.7. Cellular ATP assay
L6 cells (3x105/well) were treated with simvastatin, together with or without various doses of HCE or verbascoside. Following treatment, cells were collected and the ATP content was measured using the commercially available colorimetric kit (Abcam, UK) by measurement of the glycerol phosphate content of the lysed cells and reading at 570 nm using a microplate reader (Biotek μ-Quant, USA). Measurement of ATP production was expressed as a percentage relative to the control group.
2.8. Statistical analysis
Data were presented as means7SD, Prism 5 for Window(version 5.0c, GraphPad Software, Inc., USA) was used for statistical analysis. Significant differences among all groups were assessed by one-way ANOVA, followed by Bonferroni's MultipleComparison Test. A probability of po0.05 was considered statistically significant.
3. Results
3.1. UPLC analysis of Herba Cistanches aqueous extract
Fig. S1A showed the UPLC profile of a standard mixture containing echinacoside and verbascoside. The retention time points of each of these markers were compared to the UPLC profile of Herba Cistanches aqueous extract (Fig. S1B). The amount of echinacoside and verbascoside within Herba Cistanches aqueous extract was found to be 0.45% w/w and 0.085% w/w, respectively.
3.2. Effect of HCE and verbascoside on the in vitro cytotoxicity of simvastatin in L6 skeletal muscle cells
As shown in Fig. 1A, simvastatin caused significant cytotoxicity to L6 cells as reflected by the significant reduction in L6 cells viability at 10 mM simvastatin. HCE treatment dose-dependently and significantly prevented the simvastatin-induced cytotoxicity at 500, 1000, and 2000 mg/ml. Verbascoside exerted a trend to improve the viability of L6 cells co-treated with simvastatin. However, none of the concentrations had reached statistical significance (Fig. 1B).
Fig. 1. Protective effect of (a) HCE; and (b) verbascoside on simvastatin-induced cytotoxicity in L6 cells. Values represent means7SD (n¼3). A significant difference between simvastatin treatment alone and the control group using Student's t-test: ### po0.001. Significant difference among all simvastatin treated groups with or without HCE verbascoside co-treatment using one-way ANOVA: ** po0.01,*** po0.001.
3.3. Effect of HCE and verbascoside on simvastatin-induced cell death in L6 skeletal muscle cells
To examine the mode of cell death induced by simvastatin and determine the protective role of HCE or verbascoside on the simvastatin-induced cell death, the proportion of apoptotic cells were detected by propidium iodide (PI) staining. The results showed that simvastatin induced a significant increase in the proportion of early apoptotic cells (Annexin V-FITC positive and PI negative, Q4–2), and late apoptotic/dead cells (Annexin V-FITC positive and PI-positive, Q2–2) as compared to the control group. Co-treatment with HCE reduced the proportion of early apoptotic, and late apoptotic/dead cells in a dose-dependent manner up to 1000 mg/ ml (Fig. 2A). This protective effect was also observed in verbascoside co-treatment groups. However, none of the concentrations tested had reached statistical significance (Fig. 2B).
Fig. 2. Effect of simvastatin with or without HCE or verbascoside co-treatment on phosphatidylserine externalization in L6 cells. Cells were stained with Annexin-V FITC and I and detected by flow cytometry
3.4. Effect of HCE and verbascoside on caspase 3 activity in L6 skeletal muscle cells treated with simvastatin
As shown in Fig. 3A, 10 mM simvastatin significantly induced caspase 3 activity in L6 cells, suggesting simvastatin induced apoptosis to L6 cells through the caspase cascade. However, in L6 cells co-treated with simvastatin and HCE, HCE dose-dependently and significantly reduced the caspase 3 activity, suggesting the ability of HCE to prevent simvastatin-induced apoptosis through the caspase cascade. On the other hand, although verbascoside also exerted a trend to reduce the simvastatin-induced increase in caspase 3 enzyme activity, only the concentration at 160 mM reached statistical significance (Fig. 3B).
Fig. 3. Protective effect of (a) HCE; and (b) verbascoside on simvastatin-induced caspase 3 enzymatic activity in L6 cells. Values represent means7SD (n¼3–5).A significant difference between simvastatin treatment alone and control groups using Student's t-test: ###po0.001. Significant difference among all simvastatin treated groups with or without HCE or verbascoside co-treatment using one-way ANOVA: *po0.05, **p o0.01, ***po0.001.
3.5. Effect of HCE on simvastatin-induced reduction in ATP production in L6 skeletal muscle cells
Simvastatin (10 mM) significantly reduced ATP production in L6 cells by about 60% compared to the control group (Fig. 4A). HCE co-treatment dose-dependently attenuated the reduction in ATP production in simvastatin-treated cells, of which only the concentrations at 500, 1000, and 2000 mg/ml had reached statistical significance. On the other hand, although verbascoside exerted a trend to restore ATP production at 50 mg/ml, it did not reach statistical significance (Fig. 4B).
Fig. 4. Protective effect of (a) HCE and (b) verbascoside on simvastatin-induced reduction in ATP production in L6 cells. Values represent means7SD (n¼3–5).A significant difference between simvastatin treatment alone and control groups using Student's t-test: ###po0.001. Significant difference among all simvastatin treated groups with or without HCE or verbascoside co-treatment using one-way ANOVA: *po0.05, **po0.01.
4. Discussion
With the increasing prevalence of cardiovascular disease (CVD) and patients diagnosed with hypercholesterolemia globally, the use of statins has become more common. Although statins are in general very well tolerated, muscle injury is a frequently reported side effect associated with statin use which can occur acutely at weeks or months after the drug has been initiated (Fine, 2003). Muscle symptoms may vary from mild problems, for example, pain, tenderness, or weakness with or without creatine kinase (CK) elevation, to the most severe condition of rhabdomyolysis (Armitage, 2007; Hu et al., 2012). Due to the occurrence of these side effects, and the that there exists no effective treatment for statin-induced muscle toxicity, statins remain underused. In a wide survey on 10,409 French subjects conducted through a telephone interview, 10% of the patients receiving statin treatment reported muscular symptoms, from which 30% of these symptomatic patients resulted in treatment discontinuation (Rosenbaum et al., 2013).
We have for the first time demonstrated that the water extract of Herba Cistanches, a commonly used traditional Chinese herb, could exert a protective effect against simvastatin-induced toxicity in vitro in L6 skeletal muscle cells, suggesting the potential of Herba Cistanches aqueous extract (HCE) for its protective role statin-induced muscle toxicity, which is a recognized common side effect in patients taking statins. The potent ability of HCE to protect against statin-induced muscle toxicity would therefore be of great potential.
According to the traditional theory in TCM, Yang is related to mitochondrial energy metabolism in the body, and the prescription of Yang-invigorating herbs was found to enhance mitochondrial ATP generation (Ko and Leung, 2007). Interestingly, we observed a reduction in ATP production in simvastatin-treated L6 skeletal muscle cells, and this reduction in ATP production was significantly improved with HCE co-treatment. In our study, using L6 rat skeletal muscle cells, we demonstrated a reduction in cell viability caused by simvastatin. This reduction in cell viability was significantly improved with the co-treatment of Herba Cistanches in a dose-dependent manner. Although the exact mechanism of statin-induced muscle toxicity has not been fully elucidated, statin-induced apoptosis of healthy skeletal myocytes was suggested to be a contributing factor causing myopathy, the most common side effect experienced by statin users (Dirks and Jones, 2006). In our study, we observed a significant increase in apoptotic cells induced by simvastatin, which was associated with significantly elevated caspase 3 activity. These data are also consistent with previous studies, which demonstrated that various statins could induce apoptosis in skeletal myoblasts, myotubes, and in differentiated primary human skeletal muscle cells through activation of caspase-9 and caspase-3 activities (Dirks and Jones, 2006). Our HCE was able to prevent this simvastatin-induced increase in caspase-3 activated apoptosis in a dose-dependent manner.
Herba Cistanches
Besides, previous literature suggested that verbascoside is one of the major active constituents within Herba Cistanches. Verbascoside, also known as acteoside, belongs to the member of the phenylethanoid glycosides, a naturally occurring group of polyphenolic compounds that is soluble in water (Liao et al., 1999). Previously, it has been shown that verbascoside is a potent antioxidant that could exhibit strong ROS-scavenging and antioxidative activities (Liao et al., 1999). In our study, to determine if verbascoside is the major component within our HCE that contributes to the observed beneficial effects, we tested the effects of verbascoside at the concentrations of 0–160 mM. Since we have demonstrated that our HCE is active at the concentration range of 250–2000 mg/ml, based on our HPLC results which suggested our HCE contained verbascoside at the concentration of 0.085% wt/wt, the active concentration of verbascoside should range between 0.2125–1.7 mg/ml (i.e. 0.34–2.72 mM), should verbascoside be the active constituent? Nevertheless, we failed to observe a significant beneficial effect of verbascoside with the concentration range tested, although we did observe a dose-dependent trend for a beneficial effect of verbascoside on L6 skeletal muscle cells. These data suggested that the beneficial effect of our HCE observed in L6 skeletal cells is unlikely to be attributed to the effect of verbascoside alone, suggesting components other than verbascoside could be responsible for the observed beneficial effects. It is also possible that verbascoside might interact with other bioactive compounds within the extract to contribute to the beneficial effects. Additional experiments using bioassay-guided fractionation will be useful in providing further insight on discovering the bioactive components responsible for contributing to the observed protective effects of HCE on simvastatin-induced muscle toxicity.
In conclusion, the present study indicates that HCE could exert potent protective effects on simvastatin-induced reduction in muscle cell viability in vitro. These results provide the preliminary evidence suggesting that our HC aqueous extract might be of therapeutic value as a functional food or nutraceutical to hyperlipidemic patients who are required to withdraw from statin treatment due to statin-induced myotoxicity.
AcknowledgementThis study was financially supported by Food and Health Bureau HKSAR, Health, and Medical Research Fund no. 11120831.
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