Effect of Anthocyanin Extracts in Reproductive and Metabolic Parameters in Pregnant Rats ()
1. Introduction
Hibiscus sabdariffa (HS) or jamaica, as it is commonly known in Mexico, is an annual plant belonging to the Malvaceae family, which has been used in traditional medicine for its antihypertensive [1], anti-inflammatory [2], hypoglycemic [3] [4] and diuretic properties [5]. However, scientific evidence has also confirmed that HS has antioxidant [6] [7], phytoestrogenic [8], neuroprotective [9], hepatoprotective [10] and antibacterial effects [11]. These properties are generated by the broad phytochemical profile of HS, which includes fractions of phenolic acids, flavonoids, organic acids and particularly anthocyanins [12]-[14].
The consumption of bioactive plant-derived compounds has been associated with the control of gestational health [15]. For instance, the supplementation of products rich in polyphenols during pregnancy can prevent the development of gestational obesity and cardio-metabolic disorders in the mother [16]. Another example is the supplementation of anthocyanins to women with gestational diabetes, generating an insulin-mimetic effect and a decrease in pro-inflammatory signals [17]. On the other hand, models with pregnant rats show an increase in the body weight of fetuses with the consumption of anthocyanin extracts, which is associated with an enhanced glucogenic activity in the liver and improved placental efficiency [18]. Despite evidence demonstrating the health benefits of HS consumption, information on the effects generated by its consumption during pregnancy is limited and contradictory.
During pregnancy, women experience a series of physiological changes, including alterations in hormonal patterns, modifications in metabolic activity and nutritional requirements, as well as changes in body weight [19]. As such, the nutritional status of the mother is crucial for the optimal development of the physiological processes associated with pregnancy, as well as the correct consolidation of the physiology and morphology of the embryo. Therefore, the normal development of pregnancy depends on the precise regulation of these changes [20]. Several studies have shown that nutritional deficiencies during pregnancy are related to the induction of premature births, alterations in fetal development, malformations, and a high probability of developing chronic-degenerative diseases during the adult life of the offspring [16] [21] [22]. As a result, optimally maintaining nutritional and metabolic status of the mother during pregnancy is of great importance.
Some studies warn that the consumption of the phytochemicals from the HS plant during pregnancy may have potential adverse effects on the offspring; for example, a delay in puberty-related characteristics such as vaginal opening has been observed. [23] [24], while other studies suggest positive effects from its inclusion in the diet [25] [26], this has led to the fact that research on the effects of HS on gestational health remains debatable. For this reason, it is necessary that recommendations for the consumption of Hibiscus sabdariffa infusions or derivatives during pregnancy be established based on greater evidence, confirming that they do not cause metabolic alterations, modify the gestational health of the mother or alter the gestational development of the embryo. Thus, the main objective of this study was to analyze metabolic and reproductive parameters in rats that consumed HS or its extract of the anthocyanin fraction during gestation, to establish whether there is any risk by their consumption.
2. Materials and Methods
2.1. Preparation of the Anthocyanin Fraction and Hibiscus sabdariffa Calyx Infusion
The anthocyanin extract fraction derived from the calyces of the Nigerian variety of Hibiscus sabdariffa was prepared based on the recommendations of Farias-Cervantes et al. [27]. In brief, 2.0 kg of calyces of HS were processed; the sample was washed with distilled water and macerated. Once prepared, it was added to a 30% ethanolic solution for 7 days with occasional stirring, avoiding light and kept at room temperature. Following the resting period, the sample was filtered (150 µm diameter/pore) and the solution obtained was concentrated by rotary evaporation (BÜCHI®, USA) at a pressure of 170 bars, at 40˚C and 25 rpm. The pH of the concentrate was adjusted to 3.4 and kept refrigerated at 4˚C until it was freeze-dried (GEA Niro® Production mirror, Denmark). Maltodextrin (50:50) and agave fructans (Agave tequilana weber) were used as a vehicle, at a concentration of 0.5% (w/v).
The HS infusion was prepared according to the traditional method in Mexico for consumption. For this, 50 g of dried HS calyxes were weighed and processed by boiling in 1 L of purified water for 10 minutes. The infusion obtained was filtered (coarse pore, 20 µm) and stored at 4˚C for its later administration.
2.2. Quantification of Total Monomeric Anthocyanins by the Differential pH Method
Anthocyanin quantification was performed using the differential pH method [28]. From the infusion obtained, two dilutions were prepared, the first was made using a 0.025 M potassium chloride buffer solution pH 1 ± 0.05, the second was prepared using a 0.4 M sodium acetate buffer solution pH 4.5 ± 0.05. The dilution factor for the sample was 1:20. Both dilutions were centrifuged at 9500 rpm for 10 minutes at room temperature. The absorbances of both solutions were measured at 520 and 700 nm in a METASH UV-Vis spectrophotometer. The concentration of total monomeric anthocyanins was calculated with the following equation:
where:
TAC = Total monomeric anthocyanins (milliequivalents of cy-3-glu/100 g of sample).
Ab = pH 1.0 (absorbance 520 nm - absorbance 700 nm) - pH 4.5 (absorbance 520 - absorbance 700).
e = Molar absorptivity of cy-3-glu (26.900).
MW = Molecular weight of cy-3-glu (449.2 g/mol).
L = 1 (path length in cm).
D = Dilution factor.
V = Final volume of the solution.
G = Weight of the sample.
100 = Conversion factor.
2.3. Experimental Design
Sixty primiparous six month old female Wistar rats were used and randomly distributed into the following groups: non-pregnant females without treatment (Control, n = 12); non-pregnant females treated with the anthocyanin fraction of HS (Ext, n = 12); pregnant females without treatment (Preg, n = 12); pregnant females treated with the anthocyanin fraction (Preg/Ext, n = 12); and pregnant females treated with HS infusion (Preg/Inf, n = 12). During the entire experimental period, rats were housed in standard laboratory conditions at a temperature of 22˚C ± 1˚C and 60% relative humidity, with 12 h light/dark cycles. During the entire experimental period, they were allowed to consume commercial food (Nutricubes, Purina®) and handled according to the recommendations of the National Institute of Health Guide for the Care and Use of Laboratory Animals in its eighth edition and under the guidelines of the Mexican Official Standard NOM-062-ZOO-1999 for the use and management of experimental animals. On days 7, 14, and 19 of gestation, body weight and food consumption were recorded, and blood glucose was measured in all females from 7:00 to 9:00 a.m. using the Accu-Check® Active glucometer. Specifically, for blood glucose, capillary glucose in the tail was measured prior to the start of the different treatments and was considered as the baseline value.
2.4. Doses and Treatments
The anthocyanin extract fraction and the HS infusion were administered from day 1 to day 19 of gestation during the dark cycle to ensure daily consumption; meanwhile, during the light cycle, treatments were replaced with purified water. The anthocyanin fraction was prepared with purified water at a dose of 5 mg of anthocyanins per 100 g of body weight [8]. For the administration of the HS infusion, 30 mL of infusion was administered starting on day 1 of gestation. The quantification of polyphenols and anthocyanins was done for the infusion to determine daily consumption.
2.5. Acquisition of Pregnant Females
In females, staging of the estrous cycle was done, as well as identification of behavioral parameters such as acceptance toward the male, increased running activity, trembling of the ears and pronounced lordosis reflex after pelvic stimulation [29]. Once the estrous phase was confirmed, the female was housed with the male for 48 hours, monitoring for the presence of the vaginal plug. Once the vaginal plug was detected, this was deemed as day 1 of gestation [30]. Rats confirmed to be pregnant were separated from the males and randomly assigned to any of the groups of pregnant females.
2.6. Fetuses and Analysis of Somatometric Parameters
On day 19 of gestation, each pregnant female underwent thoracotomy to extract the uterine horns and fetuses; for this, they were anesthetized with sodium pentobarbital (50 mg/kg body weight, intraperitoneally Pisabental®, PiSA, Mex.). Body weight, height (cephalocaudal length) and head length (fronto-occipital length of the skull) were recorded, and morpho-anatomical abnormalities of each fetus were described.
2.7. Collection of Blood Samples and Quantification of Serum Lipids
During the thoracotomy, blood samples were obtained by direct intracardiac via. The samples were centrifuged at 7000 rpm for 10 minutes (DLab Mod. D1008) to obtain serum. To quantify the concentration of total cholesterol and triglycerides, the SPINREACT kit (Girona, Spain) was used.
2.8. Statistical Analysis
All data are presented as mean ± standard error and were analyzed using SPSS 21.0 software (IBM Corp.). Once the normality and homoscedasticity of the data were confirmed, the means were analyzed by one-way ANOVA and the Bonferroni pairwise comparison was used, using p < 0.05 to establish significant differences.
3. Results
3.1. Quantification and Consumption of Anthocyanins and Polyphenols from an Infusion Obtained with the
Calyces of Hibiscus sabdariffa
The chemical quantification analysis of the infusion revealed an anthocyanin concentration of 0.042 ± 0.000206 mg per milliliter of infusion, while the total polyphenol content was 1.988 ± 0.0017 mg per milliliter of infusion. Thus, the average daily consumption of anthocyanins was 0.4239 ± 0.049 mg per 100 g of body weight and 20.06 ± 2.32 mg of polyphenols per 100 g of body weight in the Inf group (Table 1). The consumption of anthocyanin fraction of HS in the Ext and Preg/Ext groups was kept constant by preparing their daily dose adjusted to body weight.
Table 1. Relationship between the concentration and consumption of polyphenols and anthocyanins in the Hibiscus sabdariffa infusion and the daily intake per rat.
|
Anthocyanins |
Polyphenols |
Concentration (mg/mL of sample) |
0.042 ± 0.000206 |
1.988 ± 0.0017 |
Mean daily consumption per rat
(mg/100 g body weight) |
0.4239 ± 0.049 |
20.06 ± 2.32 |
3.2. Analysis of Body Weight
Figure 1. Body weight gain in all groups. (A) Body weight during pregnancy, the bars represent mean ± SEM. (B) Body weight on days 7, 14, and 19 of the gestational period, each point represents mean weight ± SEM. Different letters indicate significant differences between groups (p < 0.05). *p < 0.05 compared to the same group on a different day. #p < 0.05 indicates significant difference with the other groups on the same day (p < 0.05).
The analysis of the body weight of the females (Figure 1(A)) revealed significant differences between the groups (F [4] = 23.510; p < 0.001). The Preg group showed the highest body weight (333.85 ± 53.42 g) compared to the Control (296.08 ± 29.13 g), Ext (268.79 ± 18.81 g) Preg/Ext (310.07 ± 34.53 g) and Preg/Inf (310.19 ± 37.51 g) groups. The weight of the Ext group was significantly lower than the average of all the groups (p < 0.05). When analyzing the variations in body weight throughout gestation (Figure 1(B)), it is observed that on day 7, there are no significant differences between the groups. On day 14, it was observed that only the rats in the Preg group had a significantly higher weight (324.77 ± 27.47 g) compared to day 7 (p < 0.05). Likewise, the Preg group had a higher body weight compared to the other experimental groups (p < 0.05). On day 19, the Preg (385.84 ± 51.0) and Preg/Ext (337.80 ± 37.98 g) groups had a higher body weight compared to their weight on days 7 and 14 (p < 0.05 respectively); also the Preg/Inf group had a higher body weight (331.69 ± 20.65 g) compared to day 7 (p < 0.05), and both groups had a higher weight compared to the Control and Ext groups (p < 0.05).
3.3. Food Consumption
Figure 2. Amount of food consumed in all groups. (A) Food consumption during pregnancy, the bars represent the mean ± S.E.M. (B) Amount of food consumed on different days of the gestational period, each point represents the mean food consumed ± S.E.M. Different letters indicate significant differences between groups (p < 0.05). *p < 0.05 compared to the same group on a different day. #p < 0.05 indicates significant difference with the other groups on the same day (p < 0.05).
Statistical analysis of the mean feed consumption of the groups (Figure 2(A)) showed significant differences (F [4] = 23.9; p < 0.001). The control group (131.29 ± 27.38) had a similar feed consumption with the Preg/Ext (120.59 ± 26.45) and Preg/Inf (138.84 ± 40.71) groups (p > 0.05); while the Preg group consumed less food (96.95 ± 52.15) than the control and Preg/Inf groups (p < 0.05), and the Ext group showed lower food consumption (61.75 ± 40.28) compared to the other groups during gestation (p < 0.05).
When analyzing the variations in food consumption on days 7, 14 and 19 of gestation in all groups (Figure 2(B)), it was observed that within each group the amount of food ingested did not change significantly compared to the same group. On day 7, the Control (138.37 ± 39.47), Preg/Ext (101.60 ± 34.63) and Preg/Inf (119.51 ± 16.18) groups consumed a greater amount of food than the Ext (64.45 ± 39.15) and Preg (101.60 ± 34.62) groups (p < 0.05). Thus, the Ext and Preg groups had a significantly lower consumption (p < 0.05) and remained on days 14 and 19.
3.4. Blood Glucose Analysis
The glycemic analysis on the baseline day showed similar glucose concentrations between the groups (normoglycemia) (Table 2). However, when analyzing the data obtained from the glycemic concentration, it was observed that significant differences between the groups (F [4] = 2.427, p < 0.05). On day 14, the Preg/Ext group presented a decrease in glycemia (70.67 ± 3.12 mg/dL) compared to the Control group (92.08 ± 3.70 mg/dL) (p < 0.05).
Table 2. Effect of Hibiscus sabdariffa consumption on blood glucose concentration.
|
Blood glucose concentration mg/dL |
|
Basal |
Day 7 |
Day 14 |
Day 19 |
Control |
83.17 ± 2.94 |
83.00 ± 4.40 |
92.08 ± 3.70 |
88.50 ± 4.38 |
Ext |
84.92 ± 3.66 |
85.25 ± 1.81 |
78.33 ± 4.64 |
83.75 ± 4.52 |
Preg |
89.69 ± 5.15 |
91.69 ± 3.38 |
83.15 ± 2.95 |
82.38 ± 3.47 |
Preg/Ex |
87.00 ± 3.97 |
86.58 ± 4.54 |
70.67 ± 3.12* |
79.50 ± 3.32 |
Preg/Inf |
87.90 ± 4.22 |
87.00 ± 3.06 |
78.25 ± 2.17 |
82.45 ± 3.31 |
*Represents significant differences (p < 0.05) with respect to the control group.
3.5. Serum Lipid Concentration
The analysis of serum triglycerides (Figure 3) did not show differences between groups (F [4] = 1.673; p = 0.172), the Preg/Inf group had a slightly higher concentration (139.8±72.8 mg/dL) compared to the Control (64.3 ± 18.9 mg/dL), Ext (76.5 ± 23.5 mg/dL), Preg (121.3 ± 33.9 mg/dL) and Preg/Ext (103.0 ± 19.6 mg/dL) groups (p > 0.05).
Figure 3. Serum triglyceride concentration in pregnant rats. Bars represent the mean ± S.E.M. of serum triglyceride concentration in different groups on days 7, 14 and 19. Different letters indicate significant differences between groups (p < 0.05).
Similar results were observed in the concentration of total serum cholesterol (Figure 4), with no differences between groups (F [4] = 1.368; p = 0.259). The Preg/Inf group exhibited a concentration (59.0 ± 9.4 mg/dL) similar to the Control (43.4 ± 12.2 mg/dL), Ext (34.6 ± 7.0 mg/dL), Preg (55.9 ± 25.9 mg/dL) and Preg/Ext (49.2 ± 12.2 mg/dL) groups.
Figure 4. Total cholesterol concentration in blood serum. Serum cholesterol levels of the different groups, the bars represent the mean ± S.E.M of days 7, 14 and 19. Different letters indicate significant differences between groups (p < 0.05).
3.6. Somatometric Analysis of Offspring
The analysis of body weight of the fetuses (Figure 5(A)) showed significant differences between the groups of pregnant animals (F [2] = 5.535; p < 0.05). Differences were only observed in the offspring of the Preg/Inf group with lower weight (4.63 ± 0.88 g), compared to the weight of the Preg group (5.02 ± 0.74 g; p < 0.05). Meanwhile, the weight of the litter of the Preg/Ext group (4.86 ± 0.91 g) was similar to the other groups (p > 0.05). Regarding the size of the fetuses (Figure 5(B)) significant differences were found between the groups (F [2] = 7.178; p < 0.05). Fetuses in the Preg/Inf group had a smaller size (3.87 ± 0.29 cm) compared to those in the Preg (4.00 ± 0.27 cm) and Preg/Ext (3.98 ± 0.26 cm) groups. Likewise, head length (Figure 5(C)) showed differences (F [2] = 10.022; p < 0.01), in fetuses of the Preg/Ext group which had the smallest length (1.56 ± 0.11 cm) compared to the Preg (1.61 ± 0.13 cm) and Preg/Inf (1.63 ± 0.10 cm) groups (p < 0.05).
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Figure 5. Somatometric analysis of fetuses on day 19 of gestation. (A) Average body weight of the fetuses. (B) Average size of the fetuses. (C) Average head circumference of the fetuses. Different letters indicate significant differences between groups (p < 0.05).
4. Discussion
Multiple studies have demonstrated the effects of polyphenols, such as anthocyanins, on metabolism [31]. This study evaluated the effects on body weight, food intake and glucose and lipid concentrations, as well as reproductive parameters of pregnant female rats after the ingestion of Polyphenols contained in Hibiscus sabdariffa, either in an infusion or from its extract with a high anthocyanin content. The results showed that the administration of the extract and the HS infusion modified the body weight of pregnant females, feed intake and the concentration of glucose in blood; on the other hand, blood lipid levels were not modified. Following the consumption of HS during gestation, the offspring showed a reduction of body weight and size; also, the anthocyanin extract significantly reduced their head circumference.
In this study, we observed a progressive increase in body weight that was significant in the Preg group compared to the Control group. In contrast, the Preg/Ant and Preg/Ext groups maintained body weights similar to the Control group and showed a reduction compared to the Preg group. These findings suggest that both the infusion and the anthocyanin fraction of Hibiscus sabdariffa (HS) may contain bioactive compounds associated with the regulation of weight gain during pregnancy, without inducing signs of malnutrition. The reduction in body weight may be attributed to the metabolic effects of HS polyphenols, as the anthocyanin extract administered to the Ext group also significantly decreased their body weight. In this sense, previous studies have shown that pregnancy induces chronic and systemic inflammation [32] [33], and sustained activation of the hypothalamic-pituitary-adrenal axis can alter glucose metabolism, reduce insulin sensitivity, and negatively affect the release of hormones involved in appetite and eating behavior [34] [35]. Possibly, the administration of both the infusion and the anthocyanin extract fraction of HS contributed to the attenuation of oxidative stress generated during gestation, accelerated lipid metabolism, prevented fat accumulation, and supported the regulation of intestinal microbiota and metabolic hormones in the mother [36] [37]. Furthermore, metabolic changes generated during pregnancy—such as excessive gestational weight gain—may imply an inappropriate development of pregnancy, increasing the risks of suffering from preeclampsia and post-pregnancy metabolic syndrome in the mother [38] [39]. Thus, the consumption of infusion or anthocyanin extract fraction of HS can prevent these effects due to the presence of its various polyphenol types: delphinidin 3,5-diglucoside, cyanidin-3-O-sambubioside, delphinidin-3-O-sambubioside, rutin, isoquercitrin, hyperoside and chlorogenic acid [40] [41]. Moreover, reports also indicate that the intake of Hibiscus sabdariffa infusion increases blood sodium levels, which could trigger an acute hypernatremia process, possibly caused by water imbalance due to the diuretic effect of HS. This condition may lead to a reduction in food intake [23], which could also contribute to the observed decrease in body weight.
Regarding blood glucose, only the Preg/Ext group reduced their blood glucose on day 14 compared to the control group. Golic et al. mention that, in pregnant rats, blood glucose concentration is reduced, particularly after 7 days of pregnancy until the last days of gestation, due to a circadian variation [42]. This phenomenon could be related to the decrease in sensitivity in insulin receptors, with greater release of insulin by the β-cells of the pancreas, increased postprandial blood glucose levels and decreased blood glucose during fasting. This generates changes in the plasma levels of circulating free fatty acids, triglycerides, cholesterol and phospholipids [43] [44]. Interestingly, the decrease in insulin receptor sensitivity during pregnancy is induced by the increase in circulating blood levels of placental lactogen, progesterone, corticosterone, prolactin and 17-β estradiol [39] [45]-[47]. Evidence indicates that elements contained in HS calyces, such as anthocyanins, can interact with hormones such as estradiol and glucocorticoids, regulating their activity, expression levels or competing with their receptors [48] [49], without excluding the possibility of interaction with other endocrine elements. It is possible that the glycemic results in our work are related to the intake of HS extract with high anthocyanin content, due to changes in the activity patterns of these hormones, modifying the allostatic mechanisms of sensitivity in insulin receptors and reestablishing pro-inflammatory signals.
During pregnancy, metabolic changes in cholesterol are essential for the correct development of the pregnancy and the fetus. The initial stages are characterised by an anabolic phase that enables the generation of energy reserves for future needs. In the final stage of pregnancy, lipid metabolism transitions to a catabolic phase that constantly generates a high concentration of cholesterol and triglyceride levels, which in non-pathological conditions initiates a process to reestablish plasma levels [50]. In our work, the quantification was carried out during the terminal phase of pregnancy, which could explain the fact that no differences were found between the groups, due to the restoration of basal levels. In addition, other studies have indicated that the consumption of anthocyanins can promote a decrease in serum lipid levels [51]. It should be noted that there are no reports describing the effect of anthocyanin extract consumption or HS infusion on total cholesterol or triglyceride concentration in a pregnancy model. Therefore, we assume that the trends observed in the Preg/Ext and Preg/Inf groups are related to the bioactive components present in HS. Among the proposed mechanisms for the allostatic maintenance of cholesterol levels during pregnancy is the regulation of metabolism by hormones such as estrogen and progesterone [52], since flavonoids such as anthocyanins have been observed to have a strong influence on estrogenic activity [8], and this can regulate lipid metabolic control through endocrine mediation.
Finally, we found changes in the somatometric measurements of the fetuses in the Preg/Inf and Preg/Ext groups. It is necessary to note that the changes in the offspring should not be considered a teratogenic effect or postnatal dysfunction; however, studies are needed to confirm whether the changes observed in fetuses are due to the interaction between the bioactive compounds and the hormonal and metabolic signaling of the pregnant woman. Although there are no long-term studies describing the effects of these changes in individuals, it has been reported that low birth weight in neonates is associated with cognitive and social developmental disorders [53]. In this regard, there is evidence that shows an interaction of flavonoids consumed from grape seeds with the fetus [54]. Although the placental transport of polyphenols may be limited and inefficient, these compounds probably have a direct effect on fetal cells. In conclusion, the consumption of HS modifies the amount of food consumed, generates metabolic changes in pregnant female rats and reductions in the somatometric proportions of the fetuses were observed. All of this generates the need to continue studying the mechanisms associated with somatometric characteristics and to determine whether there are long-term functional changes in neonates.
Acknowledgements
Deep gratitude to MVZ. Manuel Salas for his support in the acquisition of animals and training on rodent reproduction.
Funding
This work was funded by PRO-SNI 2019/248844 and 2020/254566.