Effect of Hibiscus sabdariffa L. Dried Calyx Ethanol Extract on Fat Absorption-Excretion, and Body Weight Implication in Rats

The effect of Hibiscus sabdariffa L. (Hs) calyx extract on fat absorption-excretion and body weight in rats, was investigated. Rats were fed with either a basal diet (SDC = Control diet) or the same diet supplemented with Hs extracts at 5%, 10% and 15% (SD5, SD10 and SD15). Only SD5 did not show significant increases in weight, food consumption and efficiency compared to SDC. The opposite occurred in SD15 group which showed a significant decrease for these three parameters. The SD10 responses were similar to SD15, with the exception of food consumption. In both SDC and SD5 groups, no body weight loss was observed; however, only in the latter group was there a significantly greater amount of fatty acids found in feces. A collateral effect emerging from the study is that components of Hs extract at the intermediate and greater concentrations used in this experiment could be considered possible antiobesity agents.

Hs calyx extract contains hibiscus acid, or (+)-hydroxycitric acid, known as (+)-HCA [21][22][23]. Its isomer, (−)-hydroxycitric acid or (−)-HCA, the active ingredient principle present in Garcinia indica and Garcinia cambogia fruits, is an inhibitor of citrate lyase [24,25], and because of this, it has been proposed as an anti-obesity agent [25][26][27][28][29][30]. Tee et al. [31] reported that hydroxycitric acid (no isomer specified), present in Hs calyx extract, inhibits fat production from carbohydrates in experiments carried out on rats. In addition, Carvajal-Zarrabal et al. [6] suggested that racemization of (+)-HCA to (−)-HCA by the intestinal flora may be a possible explanation to warrant the significant decrease in triacylglycerols in the experiment carried out on rats supplemented with Hs extract. This proposal agrees with the generalization made by Borriello et al. [32], which establishes that phytochemicals absorbed in the intestine can be transformed by colonic bacteria, resulting in serum components different from the original phytochemicals.
Therapeutic effects reported in studies carried out on animal models supplemented with Roselle, Hibiscus sabdariffa L. extracts include its influence on lipid metabolism as well as its antihypertensive and apoptotic actions. The aim of the present study was to research the action of Hs calyx extract on fat absorption, excretion, and body weight, as no information was encountered referring specifically to these aspects of lipid metabolism. were purchased from Harlan Teklad, Co. (Mexico City) and individually housed in stainless steel mesh cages in a temperature-controlled room (22-25 • C regulated by an electronic timer) with a 12-hours light/dark cycle. They had free access to food and non-ionized water throughout the feeding period. The basal diet was prepared according to the American Institute of Nutrition [33] and is shown in Table 1. Lard (10 g/100 g diet) was employed as the source of dietary fat, and cholesterol and cholic acid were added at 1 and 0.25 g/100 g diet, respectively. The experimental diet is the basal diet plus ethanol dried extract of Hibiscus sabdariffa calyces at levels of 5, 10, and 15 g of extract/100 g diet. Animals were fed the basal diet for one week in order to develop an atherogenic condition (cholesterol ≥ 220 mg/dL, atherogenic index defined as total cholesterol, cHDL/cHDL ≥ 2.5). Thereafter, they were divided into four groups (10 rats each). The control group (SD C ) was maintained on the basal diet and three groups of rats, designated as SD 5 , SD 10 , SD 15 , received the respective experimental diet for 4 weeks. The parameters that were quantified are directly related to the digestive process and its effects; as this lasts approximately 3 hours, its effect on body weight can be measured with confidence and reproducibility within an experimental period of 4 weeks. Diets were prepared once a week and stored in powdered form at 4 • C until feeding. Body weight and food intake were measured daily. Feces were collected during the last 5 days and freeze-dried. At the end of the experimental period, diets were withdrawn for at least four hours.  [35]. Fatty acids were determined by gas chromatography (Hewlett Packard 5890, Palo Alto, CA.) with pentadecanoic acid as an internal standard. All chemicals used were analytical grade.

Statistical Analysis.
The obtained data were expressed as mean ± standard deviation of means (x ± SD). A oneway analysis of variance (ANOVA) was used to compare the means of the studied groups with post hoc Duncan multiple range tests at 5% and 1% for those results where a significant difference was indicated. Minitab version 12 statistical software was used.

Atherogenic Condition, Growth, and Diet Consumption Parameters.
Animals fed the basal diet attained an atherogenic condition, their levels of cholesterol and their atherogenic index being 481 ± 82 mg/dL and 6.1, respectively. Table 2 shows growth, food consumption, and weight parameters for both the control group SD C and the three  experimental groups supplemented with Hs extract (SD 5 , SD 10 , and SD 15 ). No significant differences were observed between the experimental groups and control as regards body weight; however, body weight gain in SD 10 and SD 15 groups was significantly less (P < .01) than in control group SD C . Food consumption in the experimental groups (SD 5 , SD 10 , SD 15 ) decreased with Hs extract dose, but this only became significant (P < .05) in the SD 15 group. Food efficiency for the SD 5 group was the same as for control SD C ; however, for SD 10 and SD 15 groups, a significant decrease in this parameter was observed (P < .05 and P < .01, resp.) compared to control. Feces weight (g/d) in all experimental groups varied significantly compared to the control, with a significant increase (P < .05) observed in the SD 5 treated group and a significant decrease (P < .01) in the SD 10 and SD 15 treated groups. Table 3 reflect the effect of the diet the animals were subjected to in this study, concerning fat absorption and excretion. In the case of SD 5 , fat absorption was significantly lower (P < .01) compared to control group SD C . A tendency toward higher excretion was observed in the fecal fatty acid profiles of experimental groups, as compared to control. Specifically, SD 5 group showed an increase for all fatty acids in feces, significantly so for 16:0 (palmitic), 18 : 0 (stearic), 18 : 1 (oleic), and 18 : 2 (n-6) (linoleic). Fatty acids measured in groups SD 10 and SD 15 showed an increase in four cases: 16 : 0 (palmitic), 16 : 1 (palmitoleic), 18 : 1 (oleic), and 18 : 2 (n-6) (linoleic); only for palmitoleic was the increase not significant.

Discussion
Both experimental (SD 5 , SD 10 , and SD 15 ) and control (SD C ) groups were fed ad libitum. Only SD 5 group did not show a significant difference in the three parameters studied: weight gain, food consumption, and efficiency, as compared to control. These results were different between groups SD 5 and SD 15 , SD 5 showing behavior similar to control, while in SD 15 a significant decrease in all three parameters was observed. SD 10 was similar to SD 15 , except in the case of food consumption. Considering food efficiency comprises both weight gain and food consumption, and that, as a result, dietary components and their effect on body weight are related; it is evident that Hs extracts, at intermediate and greater concentrations used in these experiments (SD 10 and SD 15 ), by not increasing body weight, reveal themselves as potential antiobesity agents.
On the other hand, the SD 5 group absorbed the least amount of fat, exhibited an increase in all fatty acids in feces resulting from fat hydrolysis, and did not lose weight, the latter behavior being similar to control. Fatty acid excretion in SD C , however, was less than in SD 5 group. One possible explanation for this behavior in control and SD 5 groups, where weight gain was similar though with differential lipid excretion, could be due to weight gain in SD 5 group basically through carbohydrate absorption; this assumes that Hs extract components at this level of concentration do not exert an inhibitory effect on pancreatic amylase.
Lower weight gain in SD 10 and SD 15 groups, added to their similar total lipid absorption, though different in excreted fatty acid type (greater palmitic, oleic, and linoleic and lower stearic acids as compared to control) seems to indicate that at these concentrations Hs extract components could inhibit pancreatic amylase, as reported by Hansawasdi et al. [36,37], who identified Hibiscus acid, or (+)-HCA, as responsible for this action. This would consequently prevent polysaccharide unfolding and absorption. Mention should be made of the significant decrease in food consumption observed in the SD 15 group, possibly related to a dietary palatability problem when Hs extract concentration was increased. Significantly increased C16:0 excretion, observed in all groups, is attributable to Hs extract chemical components. It is relevant to recall that the fat administered in this experiment was lard, rich in triacylglicerols with C16:0 in sn-2 position. Renaud et al. [38] observed that C16:0 interesterification in fat triacylglycerols results in a greater fat secretion in experimental animal feces accompanied by a decrease in TAG and cholesterol levels, including HDL cholesterol. We consider that this result is due to, amongst other factors, the specificity of lingual and gastric lipases which hydrolyse esters in positions 1 and 3, the latter being twice as susceptible as the former. In order to explain the effect of Hs extract chemical components, in their original state or modified by the intestinal flora, on increased C16:0 concentration (20, 7, and 4.5 times in SD 5 , SD 10 , and SD 15 , respectively, compared to SD C ), as observed in the present study of fecal fat, the following hypotheses are proposed: inactivation of lipases; impediment of 2-monoacylglycerol uptake into the enterocyte, due to a competitive saturation of the specific transport system [39], or interesterification of the C16:0 from sn-2 to sn-1 or sn-3. Validation of these hypotheses requires further study.
It can be concluded that animals kept on a diet supplemented with Hs calyx ethanol extract showed significant C16:0 excretion in feces. The three hypotheses proposed to explain this excretion need subsequent testing and validation. Besides, a collateral effect emerging from the study is that Hs extract components at the intermediate and greater concentrations used in this experiment could be considered possible anti-obesity agents, through their tendency to inhibit α-amylase.