Myrciaria cauliflora Peel Flour Had a Hypolipidemic Effect in Rats Fed a Moderately High-Fat Diet

Abstract

The aim of this study was to evaluate the hypolipidemic effects of Myrciaria cauliflora peel flour (MPF) in rats. Previously, it was determined the proximate composition, total phenolics, and anthocyanins of MPF. Then, groups of male rats were randomly assigned to one of the five treatment groups. The standard group received a basal diet as recommended by the American Institute of Nutrition (AIN93M). The control group (CTRL) received the AIN93M diet, which added 7% of pork lard. The jaboticaba groups had their diets similar to CTRL, but added MPF at 7 (JAB1), 10 (JAB2), or 15% (JAB3). Serum and liver cholesterol as well as serum levels of high-density lipoprotein (HDL), triglycerides (TG), and glucose were evaluated. Fecal output of lipids was also measured. MPF had expressive amounts of fibers (25.28±2.52 g/100 g), total phenolics (18.95±5.27 mg/100 g), and anthocyanins (6.82±0.18 mg/100 g). The inclusion of the MPF at three ratios, reduced serum cholesterol and TG compared with CTRL. JAB3 raised serum HDL and reduced liver cholesterol compared with CTRL. JAB3 and JAB2 reduced serum glucose compared with CTRL. JAB groups eliminated more feces and had pH values lower than CTRL. However, there was no difference in lipid fecal output between all groups. MPF has a hypolipidemic potential, especially on reducing serum cholesterol and TG. These effects can be attributed, at least in part, to its fiber and phenolic composition.

Introduction

Dyslipidemia, a well-known risk factor for cardiovascular disease (CVD),¹ is characterized by elevated serum cholesterol and low-density lipoprotein, and decreased concentrations of high-density lipoprotein (HDL).² Accordingly, lowering blood lipids, especially triglycerides (TG) and total cholesterol (TC) levels, and/or rising HDL levels can help prevent the occurrence and progression of these diseases.¹

It is known that high intake of fruits and vegetables is protective against CVD and its risk factors, since these foods show in their composition, several compounds, such as fibers and phytochemical, which have been associated with these effects.³

Dietary fiber (DF) is a broad category of nondigestible food ingredients that includes nonstarch polysaccharides, oligosaccharides, lignin, and analogous polysaccharides with an associated health benefit.⁴ The physiologic effects of a particular DF are attributed to its degree of viscosity and fermentation.⁵ It has been reported that DFs reduce the risk of hyperlipidemia, hypercholesterolemia, and hyperglycemia by modulating food ingestion, digestion, absorption, and metabolism.⁶

Polyphenols are a large family of phytochemicals, which are the most abundant antioxidants in our diet.⁷ Despite their antioxidant properties, according to the recent literature, the mechanisms by which polyphenols express their beneficial properties on CVD appear to involve their interaction with molecular signaling pathways and related machinery that regulate cellular processes, such as inflammation.^7
–9

In this context, fruit residues stand out. Studies have shown that some of those exhibit high biologic potential, since they have higher contents of fibers and antioxidants, such as several phenolic compounds and vitamins.^10,11 In addition, fruit residues often have higher contents of these compounds compared with any other plant parts.¹²

Jaboticaba (Myrciaria cauliflora), a native fruit from the Brazilian Atlantic Forest, has a peel with interesting nutritional properties. It is a good source of minerals, fibers, and phenolics, especially anthocyanins.^13
–15 Jaboticaba is known as a Brazilian berry, due to its high concentrations of anthocyanins, like cyanidin-3-O-glucoside and delphinidin-3-O-glucoside found in its peel.¹⁵

Despite its nutritional composition, studies regarding jaboticaba peel use as a functional food are poor in the literature. Reynertson et al. ¹⁶ reported that jaboticaba peel is cited in popular medicine as an astringent, useful against diarrhea, skin irritations, gut inflammation, and hemoptysis. In a recent article, Lenquiste et al. ¹⁷ showed that freeze-dried jaboticaba peel powder increased HDL cholesterol and improved insulin resistance in rats in a diet-induced obesity model. Dragano et al. ¹⁸ also showed improvements on insulin sensitivity in high-fat–fed mice after intake of the same freeze-dried jaboticaba peel powder.

However, these recent studies did not evaluate the preventive effects of jaboticaba peel intake regarding risk factors for CVD. They induced obesity and insulin resistance by means of a high-fat diet (60% of calories from fat), and then treated with jaboticaba peel, with focus on its anthocyanin content, so that lower quantities were used in the experimental diets. In addition, freeze-drying is a process that is not available to the population in general. Also, dyslipidemia is not always associated with obesity.

Thus, the preventive effects of jaboticaba peel, under light conditions that also stimulate dyslipidemia, should be addressed. The intake of higher amounts of this product or its use as an ingredient can improve the intake of fibers, beyond phenolic compounds, and bring health benefits.

Still, considering the potential use of the jaboticaba peel as a food ingredient, the aim of this study was to determinate the hypolipidemic effects of M. cauliflora peel flour (MPF) intake in rats fed a moderately high-fat diet.

Materials and Methods

Samples

Ripe M. cauliflora fruits (jaboticaba) were collected in Diamantina city, located in the northwestern region of Minas Gerais State, Brazil, at an 18°24′47″S latitude and 43°60′03″ longitude west of Greenwich. The fruits were taken from the ground or from the tree tops at different heights and from all sides (north, south, east, and west). The fruits were gathered manually.

M. cauliflora peel flour preparation

The jaboticaba fruits were soaked for 10 min in a sodium hypochlorite solution (200 mg/L), washed with tap water and subsequently with distilled water. Afterwards they were manually separated from the pulps and seeds. Then, peels were placed on trays and dried at 40°C±5°C for 5 days into an air circulation oven. After drying, the material was grounded and sieved at 80 mesh to obtain homogeneous flour. The MPF was wrapped in a plastic bag, covered with brown paper, labeled, and stored at −18°C until its utilization.

Chemical analysis in MPF

Protein was determined by the semimicro Kjeldahl method and ashes were quantified by means of sample incineration in a muffle furnace. Total lipids were extracted with ethyl ether, in a Soxhlet apparatus, and moisture in an oven at 105°C±1°C. These analyses were performed as described by the Association of Official Analytical Chemists.¹⁹ Total DFs were quantified by the gravimetric nonenzymatic method according to the Association of Official Analytical Chemists.²⁰ Carbohydrates were obtained by the difference.

The total phenolic content was determined according to Zielinski and Kozlowska²¹ by spectrophotometry at 725 nm and anthocyanins by means of the pH differential method.²²

Rat study

Thirty-five male rats (Federal University of Viçosa, Viçosa, Minas Gerais, Brazil) weighing 123.34±5.55 g, were housed in individual stainless steel cages and maintained in a room with controlled temperature (22°C±2°C) and a 12-h light/12-h dark cycle, with free access to food and water. Animal care, research, and euthanasia protocols were in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA) and EC Directive 86/609/EEC for animal experiments. The study lasted for 4 weeks.

At the beginning, animals were acclimatized to the experimental conditions for 5 days, and then randomly assigned to one of the five treatment groups, according to each experimental diet. The standard group (SG) received a basal diet as recommended by the American Institute of Nutrition, AIN93M.²³ The control group (CTRL) received the basal diet, added 7% of pork lard.²⁴ The jaboticaba groups had their diets similar to CTRL, but added 7 (JAB1), 10 (JAB2), or 15% (JAB3) of MPF (Table 1).

Table 1.

Composition and Energy Density of the Experimental Diets

	Experimental diets ^*
	SG	CTRL	JAB1	JAB2	JAB3
Ingredients (g/kg)
Casein	164.7	164.7	164.7	164.7	164.7
Cornstarch	441.0	371.0	301.0	271.0	221.0
Dextrose	155.0	155.0	155.0	155.0	155.0
Sucrose	100.0	100.0	100.0	100.0	100.0
Cellulose	50.0	50.0	50.0	50.0	50.0
Soybean oil	40.0	40.0	40.0	40.0	40.0
Mineral mix	35.0	35.0	35.0	35.0	35.0
Vitamin mix	10.0	10.0	10.0	10.0	10.0
L-cystine	1.8	1.8	1.8	1.8	1.8
Choline bitartrate	2.5	2.5	2.5	2.5	2.5
Pork lard	0.0	70.0	70.0	70.0	70.0
MPF	0.0	0.0	70.0	100.0	150.0
Energy density (kcal/kg)	3802.8	4152.9	4076.2	4043.4	3988.7

Composition based on AIN93M diet. SG=AIN93M diet; CTRL=AIN93M diet+7% pork lard; JAB1=CTRL+7% MPF; JAB2=CTRL+10% MPF; JAB3=CTRL+15% MPF.

MPF, Myrciaria cauliflora peel flour.

The weight gain and food intake were monitored throughout the experimental period. The feed efficiency ratio (FER=g weight gain/g food intake) was calculated.²⁵

Feces were collected for 72 h before the end of the experiment to determine weight, total lipids,¹⁹ and pH. The fecal pH was determined in a solution of 1 g of oven-dried feces (60°C/48 h), which were homogenized in 10 mL of distilled water.²⁶

At the end of the experiment, all rats were euthanized, and blood samples were collected by means of cardiac puncture. Samples were centrifuged at 2000 g for 5 min to remove serum. The TC, high-density lipoprotein (HDL), triglyceride (TG), and glucose were determined using commercial kits from LabTest^® according to the specifications of the manufacturer. These analyses were performed in samples from all seven animals of each experimental group.

Animal livers were removed, submerged in a saline solution, dried with filter paper, and weighed in an analytical balance (Shimadzu AX 200), to determine their relative weights (LRW=[wet liver weight (g)×100]/final body weight [g]). Next, they were oven-dried (60°C±5°C/72 h), grounded, and their lipids were extracted.²⁷ Then, TC was determined in samples obtained from all seven animals of each experimental group, using the enzymatic LabTest^® kit, according to the specifications of the manufacturer.

Statistics

All chemical analyses in MPF were performed in three repetitions, and results are expressed as mean and standard deviation. Data from the rat study were analyzed by one-way analysis of variance and Tukey test, à posteriori, and expressed as mean and standard deviation, using the Statistica^® 10.0²⁸ software at a significance level of P<.05.

Results

According to the proximate composition of MPF, the flour constituted mostly of fibers and carbohydrates (Table 2). The anthocyanin content corresponded to almost 36% of the total phenolic compounds (Table 2).

Table 2.

Proximate Composition, Total Phenolics, and Anthocyanins of Myrciaria cauliflora Peel Flour

Composition (g/100 g)
Lipids	0.72±0.23
Protein	1.26±0.30
Total fiber	25.28±2.52
Carbohydrates	51.61±1.29
Moisture	14.45±0.63
Ash	4.26±0.16
Total phenolics	18.95±5.27
Anthocyanin (mg/g)	6.82±0.18

Values expressed as mean of three repetitions±standard deviation.

Adding fat and MPF did not affect the animal weight gain. Food intake in JAB groups was similar to SG and lower than control ones (P<.05). FER was lower in CTRL compared with JAB groups (P<.05), which were all fat-enriched diets (Table 3).

Table 3.

Weight Gain, Food Intake, and Feed Efficiency Ratio of Rats Fed a Moderately High-Fat Diet, Adding Myrciaria cauliflora Peel Flour at Different Proportions

Diets	Weight gain (g)	Food intake (g)	Feed efficiency ratio (%)
SG	125.58±43.16^a	537.27±90.06^b	23.37±0.05^a
CTRL	132.93±21.24^a	624.29±56.67^a	21.30±0.02^b
JAB1	121.04±19.76^a	500.90±95.00^b	24.16±0.04^a
JAB2	120.16±15.07^a	522.13±71.17^b	23.01±0.04^a
JAB3	131.93±20.85^a	565.75±63.83^b	23.31±0.04^a

Values expressed as mean±standard deviation.

Different letters within the same column indicate significant difference between groups (P<.05) by the Tukey test.

SG=AIN93M diet; CTRL=AIN93M diet+7% pork lard; JAB1=CTRL+7% MPF; JAB2=CTRL+10% MPF; JAB3=CTRL+15% MPF.

Animals fed the CTRL diet had their serum TC, TG, and glucose levels increased compared with SG (P<.05). MPF diets reduced serum cholesterol and TG compared with CTRL (P<.05) and those levels were not different from SG (Table 4).

Table 4.

Serum Levels of Total Cholesterol, Triglycerides, High-Density Lipoprotein, and Glucose of Rats Fed a Moderately High-Fat Diet, Adding Myrciaria cauliflora Peel Flour at Different Proportions

Diets	TC	TG	HDL	Glucose
SG	75.23±5.83^b	72.79±15.53^b	35.87±5.37^c	117.07±14.29^c
CTRL	107.38±14.7^a	139.52±10.11^a	31.14±8.22^c	125.12±14.86^a
JAB1	79.00±6.54^b	69.81±9.66^b	33.28±7.10^c	121.81±8.71^ab
JAB2	68.99±4.65^b	63.01±8.98^b	43.55±1.32^b	120.96±14.33^b
JAB3	71.60±2.94^b	61.88±3.56^b	55.13±2.93^a	114.71±22.85^c

Values expressed in mg/dL as mean±standard deviation.

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Different letters within the same column indicate significant difference between groups (P<.05) by the Tukey test.

TG, triglycerides; TC, total cholesterol; HDL, high-density lipoprotein.

HDL levels were not altered by the CTRL diet compared with SG or JAB1. JAB3 led to a significant increase (∼77%) in the HDL levels, followed by JAB2 (∼40%) (P<.05) compared with CTRL (Table 4).

JAB2 animals had similar levels of serum glucose compared with JAB1 ones and lower (P<.05) than CTRL. However, the JAB1 group did not differ from CTRL. The JAB3 group had lower glucose levels (−8.8%) compared with CTRL (P<.05) and similar to SG (Table 4).

Adding both fat and MPF (7, 10, and 15%) to the diets, did not affect the, absolute, or relative liver weights of the animals. In the JAB groups, JAB3 showed the lowest level of liver cholesterol (P<.05), also being lower than CTRL and SG (P<.05). JAB2 had lower levels than JAB1 (P<.05) and similar to SG. On the other hand, JAB1 animals had liver cholesterol levels similar to CTRL (Table 5).

Table 5.

Liver Weight, Liver Relative Weight, and Liver Cholesterol of Rats Fed a Moderately High-Fat Diet, Adding Myrciaria cauliflora Peel Flour at Different Proportions

Diets	Liver weight (g)	Liver relative weight (g)	Liver cholesterol (mg/dL)
SG	8.80±1.12^a	3.54±0.21^a	161.90±36.46^b
CTRL	8.41±2.36^a	3.37±0.32^a	220.53±50.07^a
JAB1	8.78±0.69^a	3.29±0.12^a	193.90±52.22^a
JAB2	8.05±0.57^a	3.37±0.15^a	170.20±53.04^b
JAB3	8.27±0.79^a	3.33±0.19^a	140.45±44.12^c

Values expressed as mean±standard deviation.

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Different letters within the same column indicate significant difference between goups (P<.05) by the Tukey test.

Wet and dry feces weights were higher for all groups fed MPF compared with CTRL and SG (P<.05), JAB3 had the highest weight. There was no difference in the fecal lipids among the experimental groups. Feces pH was lower for JAB1, JAB2, and JAB3, compared with SG and CTRL (P<.05), JAB3 being lower than JAB2 and JAB1 (P<.05) (Table 6).

Table 6.

Dry and Wet Weight, Lipids, and pH of Feces from Rats Fed a Moderately High-Fat Diet, Adding Myrciaria cauliflora Peel Flour at Different Proportions

Diets	Dry weight (g)	Wet weight (g)	Lipids (g/100 g)	pH
SG	11.03±0.89^c	17.07±2.61^c	4.53±2.04^a	7.65±0.20^a
CTRL	9.39±2.32^c	14.39±4.50^c	5.05±0.93^a	7.27±0.22^a
JAB1	14.65±1.40^b	23.57±3.09^b	3.48±0.72^a	6.67±0.12^b
JAB2	15.12±1.58^b	22.87±2.57^b	4.91±3.88^a	6.57±0.06^b
JAB3	18.48±1.80^a	32.48±4.78^a	5.27±1.21^a	6.32±0.05^c

Values expressed as mean±standard deviation.

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Different letters within the same column indicate significant difference between groups (P<.05) by the Tukey test.

Discussion

The American Dietetic Association²⁹ recommends a daily intake of fibers ranging from 20 to 30 g for adults and a high-complex carbohydrate and low-fat diet. Based on these recommendations, 100.0 g of MPF provides nearly 76.00% of the daily intake of fiber. This can account for the effects of fibers in health and disease. It is postulated that some kind of fibers have the ability to absorb nutrients from the diet and form a viscous environment, which retards or reduces their absorption. Others promote motility, reducing the contact of nutrients with the intestinal walls and, thus, reducing or preventing their absorption.³⁰

MPF showed a phenolic content higher than other exotic fruits, such as Copaifera langsdorffii aril flour (4.53 mg/g),³¹ Swartzia langsdorfii fruit peel (3.18 mg/g), and Eugenia dysenterica (11.90 mg/g) seeds.³² Also, MPF had anthocyanin levels higher than other known sources such as Vitis labrusca (grape) peel (0.21 g/100 g).³³ Then, maybe the incorporation of MPF into food preparation can account for the antioxidant intake from the diet.

Adding fat and MPF reduced food intake compared with CTRL. According to Valeille et al.,³⁴ increasing the fat content of a diet leads to higher energy density, which reduces food intake. Moreover, the higher fiber intake from MPF also could have affected the appetite, contributing for reducing the food intake.³⁵

Adding MPF to the diet promoted benefic changes, since it did not allow rising serum cholesterol and TG compared with CTRL. Moreover, JAB2 and JAB3 diets promoted an increase in HDL levels. Among MPF chemical constituents related to its cholesterol and triglyceride-lowering effects, stand fibers and phenolics.

Fibers can link irreversibly to ileal bile acids carrying them to the feces, so that they will not be reabsorbed and the liver will have to resynthesize them, using blood cholesterol, which leads to lower levels.³⁶ They also can increase the transit time, reducing the nutrient contact with intestinal walls, increase the viscosity, preventing or lowering their absorption.^30,37 When fermented by the gut microbiota, it produced short-chain fatty acids, such as acetic, propionic, and butyric.³⁸ The propionic acid, for example, inhibits cholesterol endogenous synthesis in the liver by lowering the 3-hydroxy-3-methyl-glutaryl–coenzyme A reductase (HMGR) activity.³⁹

Polyphenols, especially anthocyanins, can increase the lecithin cholesterol acil transferase (LCAT) activity, which esterifies cholesterol, allowing it to be selectively captured by the liver, and the lipase lipoprotein (LpL) activity, which catalyzes the hydrolysis of TG from chylomicrons.⁴⁰ According to Leite-Legatti et al.,¹⁵ jaboticaba peel has expressive amounts of anthocyanins, especially cyanidin-3-O-glucoside and delphinidin-3-O-glucoside, which also have been associated to lipid-lowering effects.

The lack of difference in the total and relative liver weights for JAB 1, 2, and 3 points out there was no lipid accumulation in this organ. In addition, liver cholesterol was also lower for those groups, similar to SG. Then, we can infer that MPF, besides reducing TC, prevented its liver accumulation. This result also can be attributed, at least in part, to the fiber physiologic effects because these nutrients are present in expressive amounts in the MPF.

In addition, MPF fiber content can explain the higher fecal output for JAB groups, as well as their humidity, since these nutrients can influence fecal moisture and promote laxation.^41,42

Another possible effect attributed to MPF fibers was the lower pH, especially for JAB3. High amounts of these nutrients can favor the acidophilus bacteria activity, leading to greater fatty acid production, mainly in the cecum colic region. This effect is useful to maintain the intestinal epithelium integrity, since it favors the proliferation of beneficial bacteria and/or prevents the proliferation of pathogenic and putrefactive ones.⁴³

Therefore, MPF showed a hypolipidemic effect, probably due its fiber content. Its phenolic composition may have also influenced on those effects. Further studies should be accomplished to know its fiber compounds, phytochemical composition, and toxicity. This information could be used to better understand MPF metabolic effects and to ensure the safety of its intake as a food.

Footnotes

Acknowledgments

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).

Author Disclosure Statement

None of the authors has financial or proprietary interest in any of the materials mentioned.

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