Synergic effect of pomegranate and maca extracts against the decrease in sperm concentration caused by lead acetate

Peralta FJ, Rubio J, Gonzales GF

Faculty of Science and Phylosophy. Department of Biological and Physiological Sciences. Laboratory of Endocrinology and Reproduction. Universidad Peruana Cayetano Heredia. Lima, Peru

Purpose

The goal of this research was to determine a synergic effect of the ethanolic extract of pomegranate (Punica granatum) and the aqueous extract of black maca (Lepidium meyenii) alone or in combination (50%-50%).

We assessed the ability of both extracts (alone and combined) to reverse the damage caused by the administration of lead acetate (LA) in sperm concentration at testis and epididymis level.

Materials and Methods

Animals Thirty six healthy adult male Holtzman rats, at reproductive age (10 weeks), obtained from the animal house of Universidad Peruana Cayetano Heredia were used in this study. Animals were housed six per group and maintained in polycarbonate cages at environmental temperature (2022C), humidity between 80% and 85%, and a 12 h light/dark cycle. Rats were fed Purina laboratory chow and tap water ad libitum.

Materials and Methods

Preparation of aqueous extract of Maca

Next, the preparation was left standing to cool and filtered.

Finally, the filtrate containing dry Maca hypocotyls was placed in small vials and kept in 4 C refrigerator.

First, 500 g of the dried hypocotyls were pulverized and placed in a container with 1500 ml of water, and boiled for 120 min.

Materials and Methods

Preparation of ethanolic extract of pomegranate
First, seeds were removed from the fruit and discarded. Then, the pericarp was cut and dried at open-air shade for 10 days. After that, it was pulverized and macerated in ethanol 96C for 8 days in darkness with constant agitation.

The obtained extract was kept at 4C.

The extract was filtered, concentrated, and evaporated to constant weight.

Materials and Methods

Study design Group 1: H2Od Group 2: Lead acetate (LA) 8mg/kg Group 3: LA (8mg/kg) + Ascorbic acid (140mg/Kg) Group 4: LA (8mg/kg) + Maca (1g/kg) Group 5: LA (8mg/kg) + Pomegranate (500mg/kg) Group 6: LA (8mg/kg) and Combined extract (50%-50%)

Materials and Methods

Treatment Animals were treated with LA for 35 days. The ascorbic acid and the extracts were administered from day 18 by gavage with an intubation needle N18. Outcomes Sperm concentration in epididymis and daily sperm production were determined. Statistical analyses STATA V.11 was used. If the distribution was normal, differences between groups were assessed by analysis of variance (ANOVA). We used the Bonferroni test to determine the differences between pair of means. Differences were considered statistically significant when P-value was <0.05 (P < 0.05).

Results
Table 1.- Percentage of body weight increased in all groups. GROUPS Control Lead Acetate (LA) LA + Ascorbic Acid LA + Maca LA + Pomegrante LA + Combined extract

BODY WEIGHT INCREASED 14% 8%* 16%+ 15%+ 11% 17%+c

Percentage obtained by the difference between final weight and initial weight divided by the initial weight. *p<0.05 compared to Control ;+p<0.05 compared to LA; cp<0.05 compared to LA+Pomegranate.

Results
Table 2.- Organs weight (g) in all groups.
GROUPS Control Lead (LA) RE 0.580.02 LE 0.560.02 0.540.01 RT 1.760.03 1.750.03 LT 1.760.03 1.750.03 LV 10.460.6 9.740.11 SV 1.010.01 1.050.06 PR 0.410.04 0.450.02

Acetate 0.490.01*

LA+Ascorbic Acid LA+Maca LA+Pomegrante LA+Combined Extract

0.580.02+ 0.510.01 0.540.01 0.540.01

0.500.01 0.510.02 0.510.01 0.550.02

1.530.05 1.700.03 1.690.03 1.660.08

1.550.07 1.640.05 1.680.05 1.700.09

11.370.42 11.540.15 11.410.11 11.691.34

1.070.05 1.050.06 1.000.06 1.050.09

0.430.04 0.400.03 0.450.04 0.410.04

Data are presented as Mean Standard Error. RE: right epididymis, LE: left epididymis, RT: right testicle, LT: left testicle, LV: liver, SV: seminal vesicle, PR: prostate.*p<0.05 compared to Control; +p<0.05 compared to LA.

Results
Table 3.- Epididymal count (106) in all groups. GROUPS Control Lead Acetate (LA) LA + Ascorbic Acid LA + Maca LA + Pomegrante LA + Combined extract EPIDDYMIS CAPUT/CORPUS CAUDA 261.66 3.16 278.42 7.37 208.63 8.97* 221.85 8.73 247.75 2.70+ 301.56 13.76*+ 234.65 8.74 208.32 14.9* 209.33 4.5* 271.33 8.39+ 287.45 19.77+a 319.05 18.86+a

Data are presented as Mean Estndar Error. *p<0.05 compared to Control; +p<0.05 compared to LA; ap<0.05 compared to LA+Ascorbic Acid.

Results
Table 4.- Daily Sperm Production (DSP) (106/testicle) in all groups

GROUPS Control Lead Acetate (LA) LA + Ascorbic Acid LA + Maca LA + Pomegrante LA + Combined extract

DSP 18.00 0.45 8.23 0.97* 18.94 1.60+ 17.57 0.53+ 19.35 0.61+ 22.96 0.20*+abc

Data are presented as Mean Standard Error. . *p<0.05 compared to Control; +p<0.05 compared to LA; ap<0.05 compared to LA+Ascorbic Acid; bp<0.05 compared to LA+Maca; cp=0.08 compared to LA+Pomegranate

Conclusions
The use of maca and pomegranate separately reversed deleterious effects of lead acetate in epididymal sperm concentration and daily sperm production. A combined extract of those plants seems to have a better effect in reversing the decrease in sperm concentration caused by lead acetate.

This study show evidence of better results by using more than one medicinal plant in one extract.