Ellagic Acid as a Multi-target Antifertility Agent: Integration of Network Pharmacology Analysis with In vivo Validation of Steroidogenic and Reproductive Effects

Previous studies from our laboratory have suggested that, in addition to an effect on spermatozoa in the testis, cyclophosphamide may have an adverse effect on spermatozoa after they leave the testis, during epididymal transit. To elaborate on this post-testicular effect on germ cells and to determine at which site(s) in the epididymis germ cells are most sensitive to cyclophosphamide treatment, three experiments were undertaken. First, the time course of the effect of treatment of male rats with cyclophosphamide on the outcome of their progeny was determined. Male rats were treated daily by gavage with saline or one of two doses of cyclophosphamide (6.8 mg/kg or 10.0 mg/kg) for 1, 4, or 7 days. At the end of each treatment period, males were mated to assess the effect on pregnancy outcome. No effect was observed on pre-implantation loss at any time among any of the groups, but there was a time-dependent and dose-related increase in post-implantation loss. Post-implantation loss was significantly increased after 4 days of treatment and reached nearly 40% after 7 days of drug exposure (10.0 mg/kg). Second, the effect of treatment with single high doses of cyclophosphamide was studied. Male rats were treated with a single dose of cyclophosphamide (10, 30, or 70 mg/kg) and bred 1 day and 4 days post-treatment. No significant change in pre-implantation loss was observed at either time point; no change in post-implantation loss was found after 1 day post-treatment. However, a significant increase in post-implantation loss was observed in the two high-dose groups 4 days post-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of nonselective cyclo-oxygenase (COX) inhibitor and selective COX-2 inhibitors on preimplantation loss, postimplantation loss and duration of gestation: an experimental study

During this study, topographic changes in healthy and atretic granulosa cells have been investigated during follicular atresia in goat ovary. Under scanning electron microscopy atresia was marked by asymmetrical shrinkage and vacuolization of cytoplasm. The specific topographical alterations observed in atretic cells were loss of micro extensions, disruption of cell-cell interaction, and smooth-textured membrane with a number of uneven depressions and ruffles. Some portions of the cell membrane were marked by extensive shrinkage due to condensation of cytosol. Irregular membrane at occasions was studded with blunt microextensions. The findings of present investigation will help in understanding the cellular changes in granulosa cells during follicular atresia and will find applications in screening of follicles for in vitro culture, in vitro fertilization and Embryo transfer technology.

Context Follicular development plays an important role in the growth and reproduction of female mammals. Ellagic acid (EA), as a natural antioxidant, has been used in freezing protection of pig semen. However, the effects of EA on immunity and the anti-apoptotic ability of ovarian granulosa cells (GCs) are still unclear. Aims The aim of this study was to analyse the effects of different concentrations of EA on the immune and anti-apoptotic ability of ovarian GCs of Guizhou black goats. Methods In this study, different concentrations of EA (0, 50, 100, 150, 200 μmol/L) were added to the culture of ovarian GCs in vitro, and Cell-Counting Kit 8 (CCK8) assay, cell wound scratch assay, and real-time fluorescence quantitative polymerase chain reaction (RT–qPCR) assay were used to detect the effects of different concentrations of EA on the proliferation, migration, and reproductive marker genes of ovarian GCs. Then the optimal addition concentration of EA was selected and the effects of EA supplementation on immune factors, cytochrome P450 family 19 subfamily A member 1 gene (CYP19A1), estradiol concentrations, intracellular reactive oxygen species concentrations, and apoptosis-related protein expression were detected by RT–qPCR, enzyme-linked immunosorbent assay (ELISA), ROS, and western blotting on the basis of the optimal addition concentration. Key results The CCK8 test and cell scratch test showed that the addition of EA could significantly inhibit the proliferation and migration ability of ovarian GCs compared with the control group, and a dose effect was observed with the increase in concentration. RT–qPCR results showed that different concentrations of EA significantly increased the expression of genes associated with reproduction, including bone morphogenetic protein 15 (BMP15), bone morphogenetic protein receptor 1B (BMPR-1B), growth differentiation fFactor 9 (GDF9), and follicle-stimulating hormone β subunit (FSHβ), and the maximum increase was observed at 150 μmol/L EA. Further analyses using 150 μmol/L EA as the optimal concentration showed significantly increased expressions of CYP19A1, interleukin-10 (IL-10), and superoxide dismutase (SOD2) after EA supplementation, while the expression of IL-8 was significantly decreased compared with those of the control group. ELISA and ROS showed that both intracellular and extracellular estradiol concentrations were higher, while ROS concentrations were significantly lower than those in the control group. Western blotting results showed that 150 μmol/L EA significantly decreased the expression of Caspase-3 and Caspase-9 and the ratio of BCL2-associated X:B-cell lymphoma-2. Conclusions The supplementation of 150 μmol/L EA had significant effects on improving GC immunity and delaying GC apoptosis in goats. The addition of EA also increased the expression of BMP15, BMPR-1B, GDF9, FSHβ, and CYP19A1 and promoted the secretion of estradiol in GCs. Implications These results provided a preliminary lead for further research on the effect of EA on the maturation and development of goat oocytes in vitro.

During follicle maturation, oxygen levels continuously decrease in the follicular fluid and reach lowest levels in the preovulatory follicle. The current study was designed to comprehensively understand effects of low oxygen levels on bovine granulosa cells (GC) using our established estrogen active GC culture model. As evident from flow cytometry analysis the viability of GC was not found to be affected at severely low oxygen condition (1% O2) compared to normal (atmospheric) oxygen condition (21% O2). Estimations of hormone concentrations using competitive radioimmunoassay revealed that the production of estradiol and progesterone was significantly reduced at low oxygen condition. To understand the genome-wide changes of gene expression, mRNA microarray analysis was performed using Affymetrix’s Bovine Gene 1.0 ST Arrays. This resulted in the identification of 1104 differentially regulated genes of which 505 were up- and 599 down-regulated under low oxygen conditions. Pathway analysis using Ingenuity pathway analyzer (IPA) identified 36 significantly affected (p < 0.05) canonical pathways. Importantly, pathways like “Estrogen-mediated S-phase Entry” and “Cyclins and Cell Cycle Regulation” were found to be greatly down-regulated at low oxygen levels. This was experimentally validated using flow cytometry based cell cycle analysis. Up-regulation of critical genes associated with angiogenesis, inflammation, and glucose metabolism, and down-regulation of FSH signaling, steroidogenesis and cell proliferation indicated that low oxygen levels induced early luteinization associated changes in granulosa cells. Identification of unmethylated CpG sites in the CYP19A1 promoter region suggests that granulosa cells were not completely transformed into luteal cells under the present low oxygen in vitro condition. In addition, the comparison with earlier published in vivo microarray data indicated that 1107 genes showed a similar expression pattern in granulosa cells at low oxygen levels (in vitro) as found in preovulatory follicles after the LH surge (in vivo). Overall, our findings demonstrate for the first time that low oxygen levels in preovulatory follicles may play an important role in supporting early events of luteinization in granulosa cells.

Fajriaty I, Hariyanto I.H, Haryanto Y. 2017. Anti-fertility effect of ethanol extract of lerak (Sapindus rarak DC) fruits in female Sprague Dawley Rats. Nusantara Bioscience 9: 102-106. This research aims to observe the antifertility effect of ethanol extract of Lerak fruits (SFEE) in female sprague dawley rats. Group of rats are divided into four groups, the first group is given carboxymethyl cellulose (CMC) as negative control (KN), the second group is given ethinylestradiol as positive control (KP), the third group is given SFEE with dose 50 mg/kg BW, and the fourth group is given SFEE with dose 100 mg/kg BW. The female rats in estrus phase is mated with male rats. In plug vaginal checking and smear vaginal there is sperm that signify if female rats have experienced copulation and currently on the day of the zero pregnancy. All treatment are given orally from 0 day of pregnancy to 10th day of pregnancy. Antifertility effect on treatment in rats is shown from laparotomy in the 20th day of pregnancy. Determining of the antifertility effect that to do include pre-implantation loss effect and post implementation loss effect. The result of this research shows that SFEE with dose 50 mg/kg BW has antifertility effect that is pre-implantation loss as big as 0% and post-implantation loss as big as 10.53% and SFEE with dose 100 mg/kg BW shows antifertility effect is pre-implantation loss and post implantation loss in each of 47.50% and 28.57%. This observation shows that SFEE has antifertility effect.

Maternal metabolic disorders associated with the consumption of an obesogenic (high fat/high sugar, HF/HS) diet are strongly linked with reduced fertility in women. The impact of obesity and an obesogenic diet on the oviductal microenvironment is much less characterized compared to the impact on the follicular environment and oocyte quality. Furthermore, the onset and duration of changes after the start of an obesogenic diet were unclear. In addition, the sensitivity to obesogenic diet-induced obesity and its influence on the metabolic profile and oocyte quality have been shown to be dependent on the genetic background of the mouse model. Therefore, the impact of feeding an obesogenic diet for different time periods on the transcriptome of oviductal epithelial cells (OECs) in Swiss compared to C57BL/6N mice, specifically focusing on genes involved in oxidative and cellular stress levels and inflammatory responses, was tested. Our findings demonstrated, for the first time, that feeding an obesogenic diet for a short period of only three days resulted in acute systemic changes and acute local oxidative stress or mitochondrial stress effects on OECs, evident already before the development of an obese phenotype. The acute effects in the OECs initiated a cascade of transcriptomic changes to control mitochondrial ROS production and endoplasmic reticulum (ER)-stress. However, prolonged, long-term feeding resulted in a persistent upregulation of (mitochondrial) oxidative stress and ER-stress, with ultimate signs of local and systemic inflammation. Furthermore, different responses to the obesogenic diet were observed between Swiss and C57BL/6N mice. Since fatty acids play an important role as structural membrane components, in cell signalling and cell-to-cell interactions, alterations to the lipid composition of the oviduct may reduce its ability to support early embryo development, which may have long-lasting consequences. Therefore, the dynamics and the nature of changes in the lipidomic profile of the oviductal epithelium upon and during obesogenic diet feeding over time in an outbred Swiss mouse model was studied. It was demonstrated that feeding the obesogenic diet resulted in acute changes in the lipid profile in the oviductal epithelium already after 3 days on the obesogenic diet. The changes in the lipid profile progressively increased and became more persistent after long-term obesogenic diet feeding. Functional annotation revealed a differential abundance of phospholipids, sphingomyelins and lysophospholipids in particular. The different durations of feeding used in this PhD research, allowed a clear distinction of acute responses and later changes after long-term feeding and after the development of the obese phenotype, and the associated unhealthy metabolic phenotype. Our findings in the oviduct illustrate that the mechanism by which the obesogenic diet impacts fertility is not only mediated through reduced oocyte quality but might also directly impact early embryo development in the oviduct, potentially leading to long-term effects on foetal development, pregnancy success and postnatal health through epigenetic alterations. In the ovary, it is known that the impact of lipotoxicity, caused by an obesogenic diet, on oocyte quality is mediated by oxidative stress, ER stress and mitochondrial dysfunction. However, the nature and magnitude of these changes may be dependent on the duration of feeding (and thus the development of the obesogenic phenotype). Therefore the impact of feeding an obesogenic diet for different time periods on the ovary and ovarian cells of outbred Swiss mice was tested, with a focus on dynamic changes in granulosa cell transcriptomics and oocyte quality. The results demonstrated that feeding an obesogenic diet resulted in acute changes in the lipid content of oocytes and impacted endoplasmic reticulum and mitochondrial functions in granulosa cells already after short-term feeding of only 24 hours till 1 week. The effects progressively increased over time after prolonged obesogenic diet feeding, with increased mitochondrial abnormalities in oocytes and different affected pathways, linked to DNA repair, cell signalling, UPRs and many other GO annotation biological terms in the granulosa cells. Exposure to an obesogenic diet thus resulted in cell specific changes in oocytes and granulosa cells, already after 24 hours to 1w, which can directly affect oocyte quality and early embryo development, eventually leading to reduced fertility. The deep analysis and novel insights of this PhD thesis can be useful for many researchers and form an important fundament for further studies that focus on protecting and enhancing oocyte and embryo development during the very early stages under diet-induced metabolic stressed conditions. Understanding the magnitude and nature of reproductive cell dysfunction after different exposure periods to a HF/HS diet is crucial to develop more efficient preconception care intervention strategies to enhance fertility under diet-induced metabolic stress conditions.

DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on >95% inhibition of 8-oxodeoxyguosine (8-oxodG) and other unidentified oxidative DNA adducts induced by 4-hydroxy-17ß-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 μM) than that for 8-oxodG (100 μM). In the in vivo study, female CD-1 mice (n=6) were fed either a control diet or diet supplemented with ellagic acid (400 ppm) and dehydrated berries (5% w/w) with varying ellagic acid contents – blueberry (low), strawberry (medium) and red raspberry (high), for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%). However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001) and 48% (p < 0.01), respectively. Both diets also resulted in a 3–8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA), DNA excision repair protein (ERCC5) and DNA ligase III (DNL3). These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

Colorectal carcinoma (CRC) is the third most commonly diagnosed cancer in the world. Phytochemicals have become a research hotspot in recent years as cancer prevention and treatment agents due to their low toxicity and limited side-effects. Ellagic acid (EA), a natural phenolic constituent, displays various biological activities, including anticancer effects. However, the detailed anticancer mechanisms of EA remain unclear. In the present study, we found that EA inhibited the growth of HCT-116 colon cancer cells. Moreover, we identified differentially expressed genes (DEGs) by microarray profiling of HCT-116 cells treated with EA. A total of 857 DEGs (363 upregulated and 494 downregulated) were identified with a >1.5-fold change in expression after treatment with EA for 72h. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that a large number of cellular functions were modified by EA including proliferation, apoptosis, cell cycle and angiogenesis. Interaction network analysis using DEGs provided details of their interactions and predicted the key target pathways of EA. To verify the result of cDNA microarray, 10 selected DEGs related to proliferation, apoptosis or cell cycle were further confirmed by real-time RT-PCR. Based on microarray data, we identified several crucial functions of EA. These results provide important new data for EA in anti-CRC research.

The aim of this study is to analyze the chemical composition of raspberry using liquid chromatography-mass spectrometry (LC-MS) technology, predict the potential effects of raspberry in treating type 2 diabetes through network pharmacology, and conduct preliminary validation through invitro experiments. A Waters CORTECS C18 column (3.0 mm × 100 mm, 2.7 μm) was used; mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile. Gradient elution was performed with full-scan mode in both positive and negative ion modes, covering a mass range of m/z 100-1500. The chemical components of raspberry were analyzed and identified based on secondary spectra from databases and relevant literature. The disease targets related to type 2 diabetes were searched, and protein-protein interaction network analysis as well as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted on the intersecting targets of the active components of raspberry and the disease. HepG2 cells were used for experimental validation, with high glucose-induced insulin resistance models established. The CCK-8 method was employed to assess the effects of raspberry on cell proliferation, while Western blotting was used to measure the expression of proteins related to the AGE/RAGE signaling pathway. A total of 47 components were identified, including 10 organic acids, 15 flavonoids, 12 phenols, 2 alkaloids, 4 terpenoids, 1 miscellaneous compound, 1 stilbene, 1 steroid and its derivatives, and 1 diterpenoid. Through database screening, seven active components were identified: kaempferol, epicatechin, ellagic acid, crocetin, stigmasterol, fisetin, and isorhamnetin. KEGG and GO results indicated that the therapeutic effects of raspberry on type 2 diabetes may be related to the advanced glycation end product (AGE)- receptor for advanced glycation end product (RAGE) signaling pathway. Establishment of an insulin resistance model in HepG2 cells demonstrated that, compared to the control group, the raspberry treatment group upregulated p53 protein expression while downregulating the expression of RAGE, Akt1, and Caspase-3 proteins. This study preliminarily elucidates that the therapeutic effects of raspberry in treating type 2 diabetes may be mediated through the inhibition of the AGE-RAGE signaling pathway, providing important references for the study of the pharmacological basis and clinical application of raspberry.

Epidemiological studies suggest that the incidence of CVD and postmenopausal osteoporosis is low in the Mediterranean area, where herbs and nuts, among others, play an important role in nutrition. In the present study, we sought a role of walnuts (Juglans regia L.) in endothelial and bone-cell function. As the endothelial cell expression of adhesion molecules has been recognised as an early step in inflammation and atherogenesis, we examined the effect of walnut methanolic extract and ellagic acid, one of its major polyphenolic components (as shown by HPLC analysis), on the expression of vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1 in human aortic endothelial cells. After incubating the cells with TNF-alpha (1 ng/ml) in the absence and in the presence of walnut extract (10-200 microg/ml) or ellagic acid (10- 7-10- 5 m), the VCAM-1 and ICAM-1 expression was quantified by cell-ELISA. We further evaluated the effect of walnut extract (10-50 microg/ml), in comparison with ellagic acid (10- 9-10- 6m), on nodule formation in the osteoblastic cell line KS483. Walnut extract and ellagic acid decreased significantly the TNF-alpha-induced endothelial expression of both VCAM-1 and ICAM-1 (P < 0.01; P < 0.001). Both walnut extract (at 10-25 microg/ml) and ellagic acid (at 10- 9-10- 8 m) induced nodule formation in KS483 osteoblasts. The present results suggest that the walnut extract has a high anti-atherogenic potential and a remarkable osteoblastic activity, an effect mediated, at least in part, by its major component ellagic acid. Such findings implicate the beneficial effect of a walnut-enriched diet on cardioprotection and bone loss.

Chymotrypsin inhibitor isolated from Ascaris suum (ACHI) was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ACHI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Two groups of adult BALB/c males received 24 or 40 mg per kilogram body weight (BW) per day intraperitoneal (IP) injection of ACHI in sterile phosphate-buffered saline (PBS) for five consecutive days (subacute exposure). Males from a third group were administered single IP injections of ACHI-60 mg/kg BW (acute exposure). The control group received concurrent injections of PBS for five successive days. After the last dose, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, spermatogonia), every second week, two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ACHI did not impair mating activity of males. Fertility index was reduced (P < 0.05) only for females mated at the third week with males exposed to the highest dose of ACHI. In the females bred to ACHI-treated males, significant (P < 0.05) increase in preimplantation loss was observed at postinjection weeks 1 (reflecting exposure to spermatozoa after single treatment and to spermatozoa or late spermatids after subacute dosing) and 3 (reflecting exposure to mid and early spermatids for acute dosing and to mid and early spermatids or late spermatocytes following acute treatment), regardless of dose and length of exposure to the inhibitor. At the 60-mg/kg-BW group, a significant increase of this parameter was also noted at week 5 (reflecting exposure to early spermatocytes). During mating days 15-21, a significant (P < 0.05) increase in postimplantation loss and dominant lethal effects were observed for all doses of ACHI. Acute ACHI exposure 5 weeks prior to mating resulted in dominant lethal effects in early spermatocytes. These preliminary data suggest that ACHI induces dominant lethal mutations at postmeiotic and meiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ACHI. These results show that ACHI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.

Induction of dominant lethal mutations by Ascaris trypsin inhibitor in male mice

The objective of the present study was to determine if periods of exposure would modify the developmental toxicity of butyl benzyl phthalate (BBP). Pregnant Wistar rats were given BBP at a dose of 2.0% in the diet on days 0-20, days 0-7, days 7-16 or days 16-20 of pregnancy. Food consumption and body weight gain were decreased in the pregnant rats given BBP. All dams given BBP on days 0-20 exhibited complete resorption of all the implanted embryos. Post-implantation loss in the pregnant rats given BBP on days 0-7 or 7-16 was higher than that in the control and pair-fed pregnant rats. No increase in post-implantation loss was found in the pregnant rats given BBP on days 16-20. Pre-implantation loss in the BBP-treated groups was comparable to the control and pair-fed groups. Striking teratogenicity was detected in fetuses of the dams given BBP on days 7-16. Cleft palate and fusion of the sternebrae were predominantly observed. About 95% of the fetuses in this group had cleft palate. The incidence of malformations in this group was significantly and markedly higher than that in the control and pair-fed groups.

The content of ellagic acid was determined from the berries of the family Rosaceae (strawberry, red raspberry, cloudberry, arctic bramble). Extraction and hydrolysis procedures were optimized and analysis was done with an HPLC method and UV detection. The influence of processing on ellagic acid content was studied in strawberry jam. Strawberries, red raspberries, and strawberry jam were analyzed fresh and after 3, 6, and 9 months of storage in a domestic freezer or refrigerator. Ellagic acid contents after 3 months of storage at −20 °C varied between 31.5 (strawberry ‘Senga Sengana’) and 68.6 mg/100 g f.w. (arctic bramble). Ellagic acid content in strawberry jam (23.8 mg/100 g f.w.) was 80% of that in unprocessed strawberries. The content of ellagic acid in strawberries and red raspberries was reduced by 40% and 30%, respectively, during the 9 months of storage at −20°C. The unprocessed berries studied, together with nuts, make the main contribution to the total dietary intake of ellagic acid in Finland.