Effects of rhaponticum carthamoides versus glycyrrhiza glabra and punica granatum extracts on metabolic syndrome signs in rats

Metabolic syndrome, a condition defined by a cluster of cardiometabolic risk factors including visceral adiposity, diabetes mellitus, dyslipidemia, and high blood pressure represents a significant global public health problem [1]. Among environmental factors, the high-fat diet and sedentary lifestyle common in the Western world are considered to be the major causes of obesity-associated impaired glucose tolerance and dyslipidemia [2]. Metabolic syndrome is also often characterized by chronic inflammation and hepatic steatosis [3]. Early treatment of people with metabolic syndrome may prevent the development of cardiovascular disease. Current treatment strategies include lifestyle modifications with pharmacological interventions targeted at complex manifestations of the metabolic syndrome as necessary [4]. Multiple biological targets controlling these manifestations will require a simultaneous application of different classes of drugs such as statins, thiazolidinediones, fibrates, biguanides, sulfonylureas, antihypertensive drugs, and many others for the attainment of beneficial effect. Although significant progress has been made in the development of therapeutic strategies for reducing risk factors for cardiovascular disease [5‐7], the integrated management of metabolic syndrome is often an elusive goal in practice. The problem of integrated management of metabolic syndrome arises due to the long-term use of expensive medications that sometimes may result in adverse side effects. Recently, a number of innovative nutritional strategies based on a long and successful practice have been proposed as a safe alternative treatments to reduce the morbidity as well as the cost of metabolic syndrome treatment [8‐10].

To date, more than 1000 officinal plant treatments for metabolic disorders have been reported, although only a small number of these have received scientific and medical evaluation to assess their comparative efficacy [11]. Therefore, more screening trials of commercial herbal products are needed to develop functional as well as analytical bases for standardization of dietary supplements [12]. Among herbs of this kind, nutritional ingredients of Glycyrrhiza glabra and Punica granatum are widely used in Indian and Chinese traditional medicine. The antidiabetic and anti-obesity effects of G. glabra[13, 14] and P. granatum[15, 16] are relatively well studied in animal models. In particular, the licorice flavonoids have recently been shown to suppress abdominal fat accumulation and increase in blood glucose level in obese rats [17] and mice [18]. High total polyphenol content is related to antidiabetic and antioxidant effects of Punica granatum extracts observed in mice [19] and rats [20].

Rhaponticum carthamoides (Willd) Iljin, commonly known as maral root or Russian leuzea, has been widely used in the traditional Siberian medicine, mostly to treat overstrain and common weakness after illnesses, as a stimulant, and a remedy against male sex dysfunction. The principal bioactive constituents of this plant are ecdysteroids, flavonoids, and phenolic acids. The extracts and preparations from this plant, which are practically safe, exhibited various additional antioxidant, immunomodulatory, antitumor, and antimicrobial effects [21]. However, relatively scarce literature is available on the medicinal properties of R. carthamoides as a treatment for metabolic disorders. In addition, the effect of R. carthamoides, especially on the high fat diet-induced metabolic syndrome development, remains to be clarified. The potential of R. carthamoides in the inhibition of the basic manifestations of metabolic syndrome is not sufficently studied. In the present study, we tested the effects of the ethanolic extract of R. carthamoides root versus the commercially available ethanolic extracts of G. glabra root and P. granatum peel with proclaimed antidiabetic and anti-obesity properties on the signs of metabolic syndrome in an obese rat model.

Feeding of rats on HFD is a useful tool for the induction of metabolic syndrome features including distinctive visceral adiposity, dyslipidemia, impaired glucose tolerance with diabetes type 2, and hepatic steatosis, which are typically associated with human obesity. This rat model of diet-induced obesity is often used to investigate the effects of metabolic syndrome ameliorating agents, including the medicinal plants, as possible sources of new drugs. Various types of extracts and individual compounds derived from R. carthamoides have been found to possess a broad spectrum of pharmacological effects [21, 24‐27]. However, the possibility that extracts and individual compounds derived from R. carthamoides may inhibit the manifestation of metabolic syndrome has been studied scarcely, unlike many other herbs such as G. glabra and P. granatum[13‐20]. Our comparative study seems to be the first evidence of the higher potential of the commercial ethanolic ERC extract, enriched in 20-hydroxyecdisone, to reduce the weight of epididymal adipose tissue and serum glucose level, to restore disturbances of serum lipids, corticosterone and inflammatory cytokine content in HFD-fed rats, compared to the commercial ethanolic EGG and EPG extracts, which enriched in glycyrrhizic acid and ellagic acid, respectively, and are believed to have antidiabetic and anti-obesity properties. This study also shows that ERC intake can ameliorate the hepatic steatosis that is associated with an increased expression of hepatic DNA binding activity of PPARα.

Supplementation of ERC, EGG and EPG to an HFD rat (300 mg/kg per day) did not change the body weight of rats in our experiment. However, the epididymal fat weight was significantly reduced by ERC only (Figure 1). In the present study, ERC normalized the plasma glucose level and improved glucose intolerance (Figure 2), decreased LDL-C, TC, TG, and FFA and increased HDL-C content (Table 1) to a significantly greater extent than EGG or EPG. In addition, ERC did not significantly change the systolic pressure of rats (Figure 3). ERC contains a relatively high concentration of phytoecdysteroids, mainly, 20-hydroxyecdysone (2.2% by HPLC data). Diverse extracts and ecdysteroids derived from R. carthamoides are widely used in sport medicine as anabolic substances strengthening biosynthesis in slow muscle fibers. The significant decrease of epididymal fat weight without change of body weight and improvement of glucose intolerance observed in rats receiving ERC (containing approximately 6.6 mg/kg per day 20-hydroxyecdysone) are most likely due to the activation of the pentose phosphate pathway and utilization of carbohydrates in protein synthesis stimulated by phytoecdysteroids. This assumption is consistent with the reduced hepatic glucose production and increased adiponectin production, as seen in mice fed with an HFD supplemented with 10 mg/kg per day 20-hydroxyecdisone [28]. Recent trials in ovariectomized rats also showed that modest doses of 20-hydroxyecdysone (18 mg/day per animal) increased muscle mass and reduced visceral fat mass, lowered serum LDL content, raised serum HDL content, and did not elevate TG content [29].

Although the hypocholesterolemic action of phytoecdysteroids has been demonstrated before [30], the molecular mechanisms underlying the effects of ERC in mammals are insufficiently studied. It is known that 20-hydroxyecdisone is the insect steroid hormone, which controls lipid metabolism through a specific receptor complex ecdysone receptor (EcR) [31]. EcRs are orthologs of farnesoid X receptors (FXR) and liver X receptors (LXR) [32], which play critical roles in the regulation of lipid metabolism in mammals. Keeping in mind that the EcR ligand binding domain is similar to the rat FXR and possesses high homology with LXR [33], it may be assumed that these receptors are the pharmacological targets of ERC phytoecdysteroids or their metabolites. Moreover, interaction of LXR with hormone responsive elements in the promoters of PPAR genes may provide for the mutual coordination of expression of these transcription factors.

Our finding of the significant beneficial effect of ERC on hepatic steatosis can be explained by an increase of PPARα DNA binding activity in liver (Figure 6). Mechanisms of this ERC action remain unclear. We can speculate that it is accomplished through direct interaction of phytosterols with FXR, LXR or other transcription factors, or indirectly (by changing blood hormonal levels). In the latter case, promotion of liver PPARα activity may be interconnected with reduction of insulin level. Indeed, insulin is known to strongly downregulate the PPARα mRNA level in rat hepatocytes [34]. Decrease of insulin level by the action of 20-hydroxyecdisone can therefore upregulate PPARα and promote its hepatic activity as observed in the present study. Also, one cannot exclude that demonstrated hypolipidemic effects (decrease of serum TG, serum FFA, and liver TG content) of ERC might be due not only to phytosterols, but also to the individual or synergistic action of flavonoids and other active plant phytochemicals.

Considerable evidence from human studies [35, 36] and HFD-fed rodent models [37, 38] suggests that increased blood or tissue levels of glucocorticoids and pro-inflammatory cytokines play a critical role in the development of metabolic syndrome. In the present study, we found that ERC exhibited a significant ability to reduce serum corticosterone content and restore its adrenal content (Figure 4), as well as to decrease the HFD-induced serum TNF-α and IL-6 levels (Figure 5), while EGG and EPG exhibited significantly less activity of that kind. Our observations agree with the data obtained in cultured HeLa cells in which ERC efficiently inhibited nuclear factor kappa B (NF-κB) [25] involved in cellular responses to inflammatory stimuli and stress. Interestingly, 20-hydroxyecdisone inhibited NF-κB activation less efficiently than ERC [25], indicating the presence of the other anti-inflammatory compounds in ERC, which are more active, than 20-hydroxyecdisone.

While R. carthamoides contains natural and low toxicity compounds [21], ERC elicited excellent outcomes without inducing side effects such as diarrhea, and in the present study, none of the animals died in the course of experiment (data not shown). In this emerging context, there are case reports in the literature that suggest that extensive intake of licorice may cause development of hypokalemia, edema, hypertension, and thrombocytopenia [39]. Ellagic acid (up to 40.0% in studied EPG) could induce a hypercoagulation state in mice, rats, and rabbits [40]. Moreover, the ellagic acid-rich pomegranate extracts have a serious drawback of being unstable in aqueous solutions [41].

Thus, complex action of ERC ethanolic extract exceeds the compensatory effects of EGG and EPG ethanolic extracts upon the metabolic syndrome cluster, and, thus, ERC can be proposed for the use as an efficient therapeutic option for the patients who refuse a low-fat diet for treatment of metabolic syndrome.

Our findings obtained with an HFD rat model suggest that ERC may prevent and ameliorate the signs of metabolic syndrome as a result of the complex beneficial effects on abdominal obesity, glucose intolerance, dyslipidemia, hepatosis, corticosterone and pro-inflammatory cytokine content with no visible signs or symptoms of toxicity in rats indicating a high margin of safety. The extract of R. carthamoides roots exhibited complex beneficial activity, which exceeded that of the commercial ethanolic extracts of G. glabra roots and P. granatum plant enriched in glycyrrhizic acid and ellagic acid, respectively, with proclaimed antidiabetic and anti-obesity properties. Accordingly, extract of R. carthamoides traditionally used mainly as a restorative substance is expanded by data from this study supporting evidence that natural ingredients of R. carthamoides are promising candidates for drug and food constituents to prevent or ameliorate the most prevalent manifestation of lifestyle-related cluster diseases caused by an extensively HFD.