Effect of different levels of hydrolysable tannin intake on the reproductive hormones and serum biochemical indices in healthy female rats

The present work aimed to find out the effect of different levels of hydrolysable tannin (HT) on serum hormonal profile, biochemical indices, lipid profile, apparent digestibility of nutrients and body weight gain in healthy female rats. Forty five adult healthy female rats of 8 weeks old were randomly divided into five equal groups. Different doses of HT 0, 0.5, 1, 1.5 and 2% were administered daily to each rats group on the body weight basis for 6 weeks. The results had shown the reduction trend (p < 0.05) in the blood glucose, serum cholesterol, low density lipoprotein, testosterone, prolactin, ghrelin, total oxidative stress and serum iron levels; while an improvement (p < 0.05) was seen in serum follicle stimulating hormone, progesterone, luteinizing hormone, high density lipoprotein, IgM and total antioxidant capacity. However, no effect (p > 0.05) was noticed in serum IgG, protein, estrogen and calcium levels. A significant reduction (p < 0.05) was seen in the apparent nutrient digestibility and body weight gain. The results had shown improvement in the feed conversion ratio (p < 0.05) but non-significant decrease (p > 0.05) in the feed intake. The findings showed that HT had healthy effects on the serum biochemical indices and reproductive hormonal profile but had a negative impact on the nutrient digestibility. Thus, the study concluded that HT could be used as an herbal medicine for the treatment of leading metabolic and infertility diseases like obesity and polycystic ovarian syndrome in females.

Scientific Reports | (2020) 10:20600 | https://doi.org/10.1038/s41598-020-77672-0 www.nature.com/scientificreports/ was observed when used in different quantities; fewer intakes have shown no significant impact on digestibility of crude protein, organic matter and ash in animals' trials 8 . Higher intake of HT decreases the daily feed intake, gain in weight 9 and final body weight 10 . So, the previous studies proved that HT had shown both healthy as well as detrimental effects. However, no such study was planned which aimed to find out the healthy as well as the anti-nutritional effects of HT on the blood biochemical indices in healthy female rats which might be useful for the treatment of metabolic disorders. Secondly, this study was also aimed to rationalize the use of HT for the management of female hormonal and infertility disorders in relation to their secondary metabolite profiles so that some natural therapeutic alternatives might be discovered.

Materials and methods
Animals. Forty five adult female Wistar Albino rats of 8 weeks old were selected in this trial with an average body weight of 135 ± 5 g. They were kept at 25 ± 1 °C with 45 to 55% of humidity with light and dark cycle of 12-h. The rats were housed in metabolic cages for the collection of feces. The total duration of this trial was 57 days, out of which 15 days were for diet adjustment and 42 days were of HT intake period. The rats were completely randomized into five groups (n = 9), named as per HT dose levels i.e. Ht0: group with 0% HT; Ht0.5: group with 0.5% HT; Ht1: group with 1% HT; Ht1.5: group with 1.5% HT; Ht2: group with 2% HT. Each rat was given an identification mark using permanent ink markers of different colors on their tail 11 . All procedures were performed according to the guide lines for the management of the Laboratory Animal 12 , approved by the Animal Ethical Committee of Government College University, Faisalabad, Pakistan.
Dose preparation. The HT powder was sponsored by Diversified Marketing Group (DMG, Rawalpindi).
The different doses of HT 0, 0.5, 1, 1.5 and 2% per kg body weight were calculated for each rat by using the formula 13 : Dosage in mg = Body weight of the rat (g)/1000 g × dose (mg). The calculated doses of HT/rat/day/kg of body weight were dissolved in warm water of required volume 14 . The groups of female rats were offered the prepared solutions once in a day between 10 to 11 am.
Chemical composition of diet. All experimental female rats were fed with iso-caloric and iso-nitrogenous diet as shown in Table 1. The standard diet contained all the required vitamins and minerals as per AIN-93 guidelines 12,15 . Rats were fed food and water ad libitum. The blood glucose (Accu-Check Glucometer, Bayer) and body weight (Weighing Balance) of each rat were monitored on weekly basis.
Feed intake (g). The feed given to each group was weighed and recorded on daily basis. Split and leftover feed were also measured in order to get the exact estimation of daily feed intake and feed conversion ratio 16 .
Gain in Body weight (BWG) was measured by using formula 17 : Final weight (g) -Initial weight (g).

Apparnt nutrient digestibility (%).
In the last 7 days of the trial, the daily feces were collected, weighed, pooled and stored at − 20 °C to determine the apparent digestibility of nutrients 18 .
The Official Methods of Analysis 19 were used for the estimation of dry matter, crude fat, protein, fiber and ash in feed and feces. The dry matter (DM) was determined when the samples were dried at 65 °C for 48 h by using a hot air oven. The ash contents were determined by incineration at 550 °C for 4 h in a Muffle Furnace and Petroleum Ether Extraction method was used for the estimation of crude fat (EE). The crude fiber (CF) Feed conversion ratio(%FCR) = Consumption of Feed g /Body weight gain(g) Apparent Nutrient Digestibility(%) = Nutrient Intake g/day − Nutrient in Feces g/day × 100 Nutrient Intake g/day For the detection of total antioxidant capacity (TAC; mmol of Trolox equiv/L ) Erel Method was used 22 . The standard curve was made with standard 0.1, 0.3, 0.6, 0.9, 1.2 and 1.5 mmol/L from Trolox (Vitamin E analog; 30mM stock ). Minimum detectable range 0.18 mmol/L and was up to 6 mmol of Trolox equiv/L with intra-assay CV under 3%. Total antioxidant status (TOS; µmol of H 2 O 2equiv .•L −1 ) was measured in serum samples by Erel Method 23 in which the TOS was determined with the equivalent standards of H 2 O 2 (12.50, 6.25, 3.12, and 1.56 μM/L) from the standard curve. Minimum range was calculated from standard curve 0.13 µmol of H 2 O 2equiv/L and linearity up to 200 µmol of H 2 O 2equiv/L with intra-assay CV which was kept under 10%. Serum IgG (g/l) and IgM (g/l) were assessed by Bindarid Radial Immune-Diffusion (RID) kit method in which antigen diffused radially into the agarose gel that contained appropriate mono specific antibody. Antigen-antibody complexes formed a precipitin ring. The ring size was increased until an equilibrium was reached between the formation and breakdown of these complexes. A linear relationship existed between the square of the ring diameter and the antigen concentration. By measuring the ring diameter, a calibration curve was constructed (The Binding Site Ltd., Birmingham, UK). Serum protein was analyzed spectrometrically with Biuret Method 24 . Serum iron (µg/ dl) was determined by performing Photometric Colorimetric Test with iron lipid clearing factor (LCF) by using commercially available kit (Human Diagnostic worldwide, Netherland). Serum calcium (mg/dl) was determined by Arsenazo III Colorimetric Method 25 .
Serum hormonal profile. Serum follicular stimulating hormone (FSH; mlµ/ml), prolactin (ng/ml), progesterone (ng/dl) and testosterone (ng/dl) were determined by Radioimmunoassay (RIA; Gamma Counter) using the kits of Beckman Coulter, Inc. USA. The Enzyme-Linked Immune-sorbent Essay (ELIZA) Method was used for the determination of serum luteinizing hormone (LH mlµ/ml) using the kit of Pointe Scientific Inc. USA; serum insulin (µlU/ml) was determined by using kit of Calbiotech Inc. USA; serum estrogen (pg/ml) was determined by using kit of ALPCO, USA; serum leptin (ng/ml) and ghrelin (ng/ml) were determined by using kits of Elabscience Biotechnology Inc. Corporate, USA (Eliza Reader).
Statistical analysis. Data expressed in Mean ± SEM and statistically significant differences were calculated by Analysis of Variance (ANOVA) using IBM SPSS Statistics 21(USA) at p value < 0.05 and < 0.01.

Discussion
Hydrolysable tannin (HT) is made up of carbohydrates, mainly D-glucose in their central core, which is used for the farm animals' production and improves their health status. Previous studies also proved that HT had shown anti-diabetic, anti-obesity, anti-inflammatory, antiviral and antioxidants activity in various animal models. The present study was aimed to find out the protective and anti-nutritional effects of HT on healthy female rats. The first finding in present study was that HT had decreased significantly the digestibility of protein and weight gain. The present data was also co-related with previous results that the utilization of tannin had significantly reduced the protein digestion and weight gain as compared to low tannin diet 39 . The previous studies reported that tannin intake had reduced the protein digestion by forming the tannin-protein complexes which were not easily absorbed and eventually resulted weight loss 40 . The FCR was also increased in the present trial which might be due the anti-nutritional property of the HT which was responsible for poor feed utilization due to remarkably low weight gain 41 . Different studies reported that the consumption of various kinds of tannin had also reduced the feed intake or had shown no effect on it 42 . The HT had shown non-significant decrease in feed intake which might be due to the poor digestibility of the dry matter and decreased serum ghrelin level which helped to reduce feed intake; while on the other hand the decreased fiber digestibility was observed in the present trial which helped to improve feed intake, so the effect on intake was non-significant in the present study. However, the serum ghrelin level might be reduced due to the poor fiber digestibility as reported previously 43 .
The first biochemical results after oral administration of HT had shown a significant decrease in blood glucose while no effect was observed on insulin level when compared with control, which was also reported in previous studies 44 .The possible mechanism through which HT had decreased the glucose level might be the activation of the insulin signaling pathway in adipocytes 45 or it might have enhanced the insulin activity 46 . Another possible reason of the decreased glucose level might be the decline in the glucose absorption from intestine 46 due to the decrease in the digestibility of carbohydrates which was also observed in the present trial. As reported previously 44,47 in different research trials that poor digestibility of the carbohydrate might be due to the formation of complexes of tannin with starch digestive enzymes which further inhibited the process of digestion; while no change in insulin level was observed in the present trial due to the extra pancreatic effect of tannin which was independent of insulin 46 or the HT had insulin like activity that maintained the antioxidant environment of the pancreatic β-cells 48 . Another statistical finding of present trial was the decreased levels of serum LDL, cholesterol and triglycerides and increased level of serum HDL which was also reported previously 47 . The intake of HT had also improved serum lipid profile which might possibly be due to the binding of tannin with fat excreted in the form of fecal bile and by increasing the cholesterol catabolism greater than that of its synthesis in the liver 49 or it might be decreased due to the poor digestibility of fat which was also another finding of this study. A decrease was noticed in nutrient digestibility of crude fiber and ash content which was also reported previously 40 .
The digestive tract was one of the main sites for the anti-nutritional effect of the tannin 38 and possible mechanism of the decreased nutrient digestibility might be due to the changes involved in the permeability of the intestinal wall. As a result of this reaction between HT and the membrane protein of the intestinal mucosal cells, there appeared less nutrient absorption 42 . In the present trial the serum leptin had shown non-significant effects which might be due the fact that the experimental rats were healthy. As previously mentioned 6 that HT was a biological antioxidant which helped to reduce the production of free radicals which were present in excess quantity into the reproductive tracts of males and females subjects and caused OS and further enhanced the infertility problems 50 . So the decreased level of OS was required to maintain the reproductive health and hormonal balance which was another finding of the present trial and a significant increase in TAC and significant decrease in TOS were also observed in experimental rats after six weeks of tannin intake and it was also reported in previous study 51 . So an improvement was observed in the reproductive hormones like LH, FSH and progesterone of female rats whereas a significant decrease was observed in serum testosterone and prolactin levels in the present trial, which might be due to the reduction in TOS that further improved the reproductive hormones. However, no effect was observed on serum estrogen which might be due to the fact that the rats were healthy and there was no disturbance present in the estrous cycle of female rats. The HT possessed antiviral and anti-microbial properties which improved the body immune system 52 . The present study had also found a significant increase in the immunoglobulin IgM which was correlated with the previous works that ellagic acid, a form of HT, had improved the serum IgG and IgM in infected mice 53 . But in another study, the intake of tannin had decreased the level of IgG and IgM in growing chicks 54 . However, no change in IgG level was observed in present study due to the healthy experimental rats. In addition, the increased IgM level might be due to the reason that HT had acted as an immune-modulator substance which enhanced the serum globulin level 55 and this increase might contribute to further increase in serum immunoglobulin IgM level. Serum protein was the bio-marker of the nutritional status while no change was observed in serum protein levels with different levels of tannin intake as previously reported in many studies 40 . Hence, it meant that tannin might not have a toxic effect on the liver protein synthesis as well as on the metabolism of dietary protein. Another finding of present study was the decreased serum iron level which was also previously reported in many studies and the possible mechanism of this decrease was due to the binding of tannin with iron 56 .

Conclusion
The results proved that 1.5% and 2% levels of HT had shown strong antioxidant potential which produced a healthy and beneficial impact on the serum biochemcal indices and also improved the reproductive hormonal profile like FSH, LH, progesteron, prolactin and testosterone. Therefore, HT had the theraputic potential for the management of the metabolic and hormonal disorders like diabeties, cardiovascular risk factors, obesity and might be used for the treatment of polycystic ovarian syndrome.