Comparative evaluation of anti-obesity effect of Aloe vera and Gymnema sylvestre supplementation in high-fat diet fed C57BL/6J mice

INTRODUCTION

Now-a-days, obesity is considered to be epidemic disease which leads toward a positive energy balance. It is associated with many health-related complications such as insulin resistance, Type-2 diabetes mellitus (T2DM), and other metabolic disorders. In obese individuals, the accumulation of more fat occurs in the adipose tissue and later stage fat deposition takes place other than non-adipose tissue (e.g., muscle and liver) [1]. Due to the accumulation of triglycerides (TG), the body does not respond to the action of insulin and results in insulin resistance, a hallmark of Type-2 diabetes [2-4]. Moreover, both familial and environmental factors play an significant part in obesity. The consumption of high-fat diet (HFD), an environmental factor is associated with the development of obesity and other metabolic diseases. Diet control, physical exercise, liposuction, and bariatric surgery are available to curb the obesity, but these are not effective with prolonged treatment duration. Hence, people are looking for alternative therapies such as herbal medicines and functional foods which are gaining the importance in treatment of obesity and its associated diseases. Nearly, 1200 plant species have been examined for their use in obesity and diabetes [5].

Herbs such as Aloe vera (AV) and Gymnema sylvestre (GS) showed antihyperglycemic and hypolipidemic effect in animal studies and clinical studies when administered through intragastric gavage [6,7]. Aloebarbadensis Miller (AV) belongs to the Liliaceae family, of which there are about 360 species and widely used in the manufacture of food and beverages, pharmaceuticals and cosmetics [8]. It has been used for centuries, treating obesity, diabetes, immunomodulator, anti-inflammatory, antiseptic, healing, and anti-tumor activities. Aloe gel contains predominantly polysaccharides such as glucomannan and acemannan possessing several medicinal values [9]. In a clinical trial, AV juice showed anti-diabetic activity performed by Mahidol University Research Group [10,11]. In a recent study, we demonstrated that AV gel powder (0.5% and 1% w/v) showed an increase in proteolysis and angiotensin I converting enzyme inhibitory activity in milk fermented with probiotics [12]. GS is another medicinal plant belonging to the Asclepiadaceaea family. In the Ayurvedic system, it is referred as “Meshasringa,” and it has potent anti-obesity and anti-diabetic activities. It is also used in the treatment of asthma, eye complaints, inflammation, family planning, and snake bite [13]. Its leaves contain gymnemic acids which are a potent inhibitor of glucose absorption in the intestine [14]. In addition, gymnemic acids bind to the receptors present in the tongue that prevent the glucose intake [15-18]. Studies showed that GS have been found to increase insulin secretion from the pancreatic β-cells and causes lowering of blood glucose levels in animals and T2DM patients [19,20]. No reports are available with the addition of whole extracts of AV and GS herbs to the HFD for studying its anti-obesity effects. In this study, we investigated the comparative evaluation of AV and GS whole extract powders role in anti-hyperglycemic, hypolipidemic effect, and obesity-related gene expression analysis in HFD fed C57BL/6J mice.

MATERIALS AND METHODS

RESULTS

Effect of AV and GS on Body Weight Gain

At the end of experiment, no statistically significant difference was observed in the body weight gain between control and HFD fed groups. However, supplementation of both herbal ingredients (AV and GS extracts) showed reduction in the body weight gain but not statistically significant (P > 0.05, Table 1).

Table 1

Body weight, plasma lipids, and insulin levels

Effect of AV and GS on Glucose and Insulin Levels

As shown in Figure 1, fasting blood glucose levels were determined at 0, 6, and 12 weeks. High-fat fed obese mice showed increase in glucose levels at 6 weeks (153 ± 8.00 mg/dl) and 12 weeks (168.5 ± 7.90 mg/dl), respectively. Administration of both herbs such as AV (122.0 ± 7.79 and 124.0 ± 10.36 mg/dl, P < 0.05) and GS (131.3 ± 7.34, P < 0.05 and 104.2 ± 10.36 mg/dl, P < 0.001) to HFD fed mice, displayed a statistically lowering effect of blood glucose levels at the end of both 6 and 12 weeks, respectively. In OGTT levels were higher in HFD treatment and these glucose levels were lowered by both herbal extract supplementations without a significant difference (data not shown). On the other hand, HFD fed mice had higher levels of plasma insulin levels at the end of experiment. However, both AV and GS supplementation displayed lowering of plasma insulin concentrations without significant difference (Table 1). HFD fed mice exhibited increased fasting blood glucose and plasma insulin levels (P < 0.05). Both herbs showed significant decrease in the glucose levels; however, no effect was observed within insulin levels.

Figure 1

Effect of Aloe vera (AV) and Gymnema sylvestre (GS) on fasting blood glucose levels in mice fed with high-fat diet (HFD). Values are expressed as mean±standard error of mean (n=6). The bar over the histogram shows the significant difference of HFD+AV and HFD+GS with HFD group (*P<0.05, ***P<0.001)

Effect of AV and GS on Expression Analysis

Expression analysis of genes such as adiponectin, leptin, and proinflammatory markers (interleukin 6 [IL-6] and tumor necrosis factor-a [TNF-a]) was done in E. fat tissue (Table 2). HFD fed mice showed downregulation of adiponectin expression levels. These adiponectin levels were significantly up-regulated by AV supplementation (4-fold change). No significant effect was observed in HFD + GS fed group. In addition, leptin and proinflammatory cytokines expression did not show any significant difference with the herbal ingredients.

Table 2

Effect of Aloe vera and Gymnema sylvestre on analysis of gene expression in epididymal fat mass of mice fed with HFD

DISCUSSION

In the present investigation, comparative evaluation of both herbal ingredients was studied for their role in anti-obesity effect in HFD fed C57BL6/J mice for 12 weeks. Surprisingly, we did not observe any significant difference between control and HFD diet groups. The reason might be that the lard which is used in HFD preparation obtained from the local supplier. At the end of experiment, oral administration of AV and GS powders resulted in no significant difference in body weight and cumulative feed intake in comparison with the HFD fed group of mice (data not shown). Our results were consistently similar with Chihara et al. [22] who reported that AV gel extract did not show any significant difference in the reduction of body weight and food consumption after 7 weeks experimental period in HFD fed mice. Similarly, HFD supplementation of aloe formula such as processed AV gel (PAG), Aloe QDM, and Aloesin had no effect on body weight [23]. Administration of dried AV gel powder to diet-induced obesity rats did not display any significant effect on reduction in the body weight and food intake [24]. Misawa et al. [8,25] studied the oral ingestion of AV phytosterols (cycloartanol-Cy and lophenol-Lo) to Zucker diabetic fatty (ZDF) rats for 6 weeks and reported that no significant difference on body weight reduction and food intake. In contrary, administration of AV phytosterols (Cy and Lo) significantly reduced the body weight in diet induced obese (DIO) mice [26]. Few reports are available in relation to body weight and food intake by GS treatment. Our results are consistent with the observations of Shigematsu et al. [27] who reported that no significant effect on reduction in the body weight and food intake when GS administration to HFD fed Wistar rats. In contrary to our results, Kumar et al. [28] reported that GS extract in the HFD-induced obese Wistar rats showed a significant decrease in the body weight while no effect on food intake for 28 days. On the other hand, saponins rich aqueous extract of GS also showed reduce gain in body weight, and no effect in food consumption in HFD fed obese rats [29]. The different AV and GS extract preparations used by the researchers had different effects on reduction in the body weight gain and food intake. In our study, we speculated that the less body weight in herbal fed groups might be due to the satiety effect that results reduction in fat tissue (E. fat) weight which is related to body weight gain. However, we could not measure the fat and lean mass parameters in all treatment groups.

Fasting blood glucose results of the present study were significantly reduced at both 6 and 12 weeks, respectively by both herbal supplementations in comparison to HFD fed group. Our results were corroborated by Shin et al. [23] demonstrating that dietary aloe components showed significant reduction in the blood glucose levels in obese mice. Kim et al. [6] observed that the fasting blood glucose and insulin levels were significantly reduced in a dose-dependent manner when PAG was orally administered. On the other hand, AV phytosterols (Cy and Lo) administration to ZDF rats showed no effect on fasting blood glucose and serum insulin levels after 5 weeks [25]. Similarly, Pérez et al. [30] observed that polyphenol-rich extract from AV gel did not show any effect on insulin levels in insulin resistance mice. However, GS administration of HFD fed Wistar rats showed a significant reduction in the blood glucose and serum insulin levels [28]. The possible mechanism underlying the lowering of blood glucose levels might be due to inhibition of its absorption in the intestine. Hypoglycemic effect of AV might be associated with pancreatic insulin synthesis and its secretion responsible for the lowering of circulatory glucose levels [31] whereas, gymnemic acids present in the GS binds to the glucose receptors to prevent the intestinal glucose absorption [14].

Herbal administration showed no effect on organ weights (liver, kidney, and spleen) whereas, E. fat weight was decreased significantly in case of GS fed group alone as compared to HFD fed obese mice. Our results are in agreement with several reports which showed no significant effect on liver and E. fat mass on AV treatment [8,24,25,32]. Reddy et al. [29] reported that the weights of liver, kidney, heart, and adipose tissue (peritoneal and perirenal fat) were lowered upon GS supplementation in HFD fed Wistar rats. In contrast, Shigematsu et al. [27] reported that epididymal and mesentery fat weight was not decreased by GS administration. In obesity, the pattern of body fat accumulation is thought to play a part in disease risk and body fat accumulation was closely associated with E. fat mass. In our study, gymnemic acids present in the GS might have been involved in increasing the energy expenditure through uncoupling protein-2 (UCP-2-involved in thermogenesis) expression. Nevertheless, we could not the measure the expression of UCP-2 in adipose tissue and subcutaneous, mesenteric, retroperitoneal, and brown adipose tissue weights were also could not evaluate.

At the end of 12 weeks experimental period, plasma lipids were analyzed in HFD fed mice. Oral administration of both herbs showed a significant decrease in the plasma TC levels alone. Different results were observed by the researchers with the herbal extracts. Chihara et al. [22] reported that no significant effect on plasma TC and TG levels. Similarly, Misawa et al. [24] studied the oral administration of dried aloe gel (20 mg/kg body weight) and reported no effect on serum TC and TG levels in Sprague-Dawley rats. In contrary, serum TG levels were lowered by AV phytosterols (Cy and Lo) while no effect was observed in TC levels of ZDF rats [25]. Nomaguchi et al. [26] also reported that both serum TG and TC levels were significantly reduced by AV phytosterols in DIO C57BL/6J mice. In another study, reduction in the serum TC, TG, and LDL-C levels by GS administration in diabetic rats [7]. Reddy et al. [29] observed that decrease in the TG, LDL-C, VLDL-C, and increased in the HDL-C levels in HFD fed rats. Kumar et al. observed that GS extract showed a significant reduction in the serum total lipids (TC, TG, LDL-C, and VLDL-C) in HFD fed Wistar rats [28]. By considering above studies, it can be concluded that the differences in lipids levels depends on the type of animal models and extract preparations used. However, in our study, phytosterols present in the whole herbal extracts do not extensively absorb from the intestine rather it can bind to the cholesterol and prevent its absorption to show a hypolipidemic effect [33]. Furthermore, GS contains flavonoids, phenols, saponins, phenolic, and terpenoids might be responsible for lowering effect on lipids in high cholesterol fed rats [34].

Finally, expression analysis of adiponectin (role in regulating energy homeostasis) leptin, IL-6, and TNF-a genes were analyzed by qRT-PCR. In HFD fed mice, AV supplementation alone displayed up regulation of adiponectin mRNA levels. However, no significant difference was observed with the remaining genes. To the best of our knowledge, there are no such expression study reports available in this regard. However, further experiments are necessary to study the effect of AV and GS on the expression analysis of other obesity-related genes in animal models.

CONCLUSIONS

In conclusion, this study was carried out for comparative evaluation of anti-obesity effect and expression analysis of adiposity genes by AV gel and GS powders in high-fat fed C57BL/6J mice for 12 weeks. Both herbal extracts did not show any significant effect on body weight and food intake and further they displayed anti-hyperglycemic and hypolipidemic effects in HFD fed mice. In addition, E. fat pad was significantly decreased by GS administration alone. AV fed group alone showed a significant up regulation of adiponectin levels. Moreover, supplementation of both powder extracts did not show any effect on other genes. This study is the only preliminary experiment where we used 1% whole extract of herbs added to the HFD instead of active components. However, several researchers they did not show any significant effect on the supplementation of herbs using active component. For the better anti-obesity effects, using type of diet, herbal extraction, treatment length, and animal models may be play crucial role.