Essay: The Abundance of Phytochemicals

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Ethnopharmacological relevance: Ardisia colorata (AC) is being used as a folk medicine to treat liver diseases, cough, diarrhoea and diabetes mellitus in Asia especially in North-eastern India.
Materials and Methods: In this study, we investigated (1) the abundance of phytochemicals present in the methanol extract of AC (ACME) by GC-MS analysis, and (2) gonado modulatory effects of ACME on diabetes mediated testicular dysfunction in streptozotocin-induced diabetes in Wistar albino rats in relation to spermatogenesis, testis histopathology and histomorphometrics, sperm quality, antioxidant enzymes defence and germ cell proliferation. Diabetic male rats were orally administered with ACME (100, 200 and 400 mg/kg) for 30 days along with normal (0.5% carboxymethyl cellulose), diabetic (streptozotocin, 60 mg/kg) and glibenclamide (0.1 mg/kg) treatment groups. Reproductive organs and serum were collected after 30 days from each treatment groups for histological, biochemical and sperm analysis. Phytochemicals of ACME and their ameliorative effect on diabetes induced testicular dysfunction were computed using univariate and multivariate analyses.
Results: GC-MS analysis revealed that ACME is abundant with polyphenols, quinones and fatty acids (bergenin, embelin, quercetin, myricetin, myricetic acid, kaempferol, gallic acid, and resorcinol). In vivo acute toxicity test revealed ACME is safe for consumption and no signs of mortality or toxicity were observed up to a dose of 3000 mg/kg. Polyphenols rich ACME significantly reverses diabetes mediated testicular dysfunction via increasing serum testosterone concentrations, upsurging germ cells proliferation (proliferating cell nuclear antigen), improving histopathological and histomorphometric changes, modulating spermatogenic events, restoring sperm quality, reducing sperm DNA damage and balancing the antioxidant enzymes levels (superoxide dismutase and glutathione –S- transferase). Univariate and multivariate analyses illustrate the relationship (1) between streptozotocin induced diabetes mellitus testicular complications and their magnitude in testis damage, suppression of spermatogenic process, impairment in sperm quality; and (2) the ameliorative role of ACME supplementation on restoration of testis damage and preservation of sperm quality.
Conclusion: ACME fraction, rich with polyphenols, quinones and fatty acids, is significantly modulating the spermatogenic events and reverses the diabetes induced testicular dysfunction in rats. Hence, ACME could be used as a viable source of dietary antioxidants and a phytotherapeutic agent in animals to treat diabetes-induced testicular dysfunction.
Key words: Diabetes induced testicular dysfunction; Ardisia colorata polyphenols; sperm quality;
cell proliferation marker PCNA; antioxidant enzymes; histopathology.

1. Introduction
Diabetes mellitus (DM) is a metabolic disorder alters protein, carbohydrate and lipid homeostasis, induces oxidative stress in the organ systems and mediates changes in numerous intracellular signalling pathways (Kilarkaje et al., 2014). Sustained hyperglycemia with poorly managed DM results in the generation of reactive oxygen species (ROS) and attenuates antioxidant defense systems leading to oxidative stress in a variety of tissues causing diabetic complications in due course (Kilarkaje and Al-Bader, 2015; Rashid and Sil, 2015). Increased oxidative stress in DM adversely affects male reproductive function at multiple levels particularly spermatogenesis, histology of testes, steroidogenesis, altered pituitary-testicular hormonal axis, sperm quality and fertility both in diabetic men and experimental diabetic animals (Kanter et al., 2013; Heeba and Hamza, 2015; Faid et al., 2015).
The genus Ardisia (coral berry/marlberry, Myrsinaceae, 500 species) is found in tropical and subtropical regions (North and south America, Asia, Australia, and the Pacific Islands) and have been used as food and medicines in different parts of the world because of its rich source of polyphenols, triterpenoids, saponins, isocoumarins, quinines and alkyl phenols (Sumino et al., 2001; Sumino et al., 2002; Kobayashi and de Mejia, 2005; Newell et al., 2010). Traditional medicinal uses attributed to Ardisia include all liver cancer, rheumatism, cough, fever, diarrhea, dysmenorrheal, respiratory tract infections, inflammation, pain, snake and insect bites, birth complications and to improve general blood circulation (Khan and Yadava, 2010). A. squamulosa, A. pyramidalis, A. colorata, A. solanacea leaves, flowers and fruits are eaten as vegetable, used as greens for salad, or cooked with meat or fish (Raga and Pocsidio, 2011; Deb et al., 2015). Boiled leaf extract have been used for treatment of DM by Meitei and Meitei-pangal communities in Manipur, Northeast India (Khan and Yadava, 2010). The health benefits of A. colorata or their phytochemical constituents have not been completely investigated, resulting in under exploitation of their uses. The wide spread consumption and availability of A. colorata throughout the northeastern India creates the possibility of exploiting their properties as an anti-hyperglycemic agent. Therefore, it has become essential to exemplify the actual magnitude of health benefits of A. colorata to modulate metabolic events linked to diabetic complications especially spermatogenesis, sperm quality and reproduction and explicate the potential mechanisms of action and its phytochemical constituents, particularly those of polyphenols.

2. MATERIALS AND METHODS
2.1. Plant collection, identification, processing and extraction
The leaves of AC (Myrsinaceae) were collected from Nambol Maibam Chingning, Bistmupur District, Manipur (February – March, 2016), identified, authenticated by a taxonomist and deposited a voucher sample (MZU/ZOO/MYR/L/2016/3011) in the Department of Zoology, Mizoram University, Aizawl, Mizoram. AC leaves were washed, air dried, powdered with a mechanical grinder and stored in an airtight container for further use. Cold percolation method (Roy et al., 2015) was followed to extract the pharmacologically active phytochemicals from the ACME. The powdered leaves of AC (3 kg) were taken in a rectangular polypropylene vessel containing methanol solvent (99.8%, Merck grade, 6L) sealed and kept for 7 days in a shaker incubator (model: SLM-INC-OS-250, Shalimar Specialty Chemical Ltd., Bangalore, Karnataka, India) at 25 °C and 500 rpm. After 7 days, the extract obtained was filtered twice through Whatman no.1 filter paper, concentrated to dryness using a rotary evaporator (Buchi, Germany) under reduced pressure at 40 °C for 4 h to obtain a dry mass. Then, the dark greenish brown semi-solid residue was further dried under a vacuum oven drier at 25 °C for 7-12 days to give 26.2 g (w/w) solid residue and stored at 4 °C in an air tight container until further use. This solid residue was then reconstituted in distilled water to give the required doses of 100, 200 and 400 mg/kg body weight that was used for all the bioassays.

2.2. Gas chromatography–mass spectroscopic analysis of ACME
The active phyto-constituents of methanol soluble fraction of AC leaves were determined qualitatively using gas chromatography system (Agilent system 6890N) that was interfaced with a mass selective detector (model 5973N, EIMS, electron energy, 70eV), and an Agilent ChemStation data system. The GC column was a DB-5ms fused silica capillary with a 5% phenyl methyl poly siloxane (PNMPS) stationary phase, film thickness of 0.5 μm, a length of 30 m, and an internal diameter of 0.25 mm (Agilent J&W DB-5ms Ultra Inert). The initial oven temperature was held at 60 °C for1min and increased at10-180 °C/min held for 1 min, and then increased at 20 °C/min to 300 °C and held for 15min. The injector temperature was maintained at 250 °C. The sample (1 μL, diluted 10:1 in acetone) was injected, with a split ratio of 1:10. The carrier gas was helium at a flow rate of 1.5mL/min. Spectra were scanned by Electron ionization (EI) scan mode from 25 to 800m/z at 2 scans/s. Total GC running time was 32 min. Qualitative identification of the different constituents was performed by composition of the relative retention times and mass spectra with those of authentic reference compounds by retention indices (RI) and mass spectra. Interpretation on mass spectrum of GC-MS was done using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The mass spectrum of the unidentified component was compared with the spectrum of the identified components stored in the NIST08 and Wiley275 library. The name, molecular weight, structure of the components detected, chemical nature and their biological activity were ascertained (Table 1).
2.3. Animal maintenance and ethical permit
Colony bred male albino rats of Wistar strain (150–200 g, 3 months old) were maintained under controlled standard animal house conditions (temperature: 25–27°C; photoperiod: 12 h natural light and 12 h dark; humidity: 80–90%) with access to food and water ad libitum (standard pellet diet; Pranav Agro Industries, Maharashtra, India) at Animal Care Facility at the Department of Zoology, Mizoram University, Aizawl, Mizoram, India. The pellet diet consisted of 23% protein, 5% lipids, 4% crude fiber, 8% ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, and 55% nitrogen-free extract (carbohydrates). This study was carried out following approval from the Committee on the Ethics of Animal Experiments of the Mizoram University Animal Ethical Committee (MZUAEC), Mizoram University, Aizawl, Mizoram, India (Permit Number: MZU/IAEC/15-16/11) on the use and care of experimental animals.
2.4. Acute toxicity
Acute toxicity was carried out as described in ‘Guideline for Testing of Chemicals – Acute Oral Toxicity – Fixed Dose Procedure’ (OECD, 2001). A total of 20 male rats were equally divided into 4 groups that administered with the vehicle (0.5% carboxy methyl cellulose, 5 mL/kg), 750, 1500, and 3000 mg/kg of ACME (5 mL/kg) to determine a safe dose. The animals were fasted overnight (but allowed water) prior to dosing. Food was withheld for a further 3 to 4 h after dosing. The animals were monitored for 48 h after the administration of the ACME for the onset of clinical or toxicological symptoms and further observations were continued up to 15 days. On the 15th day, the rats were sacrificed by an overdose of ketamine anesthesia and histological and serum biochemical parameters (hepatic total proteins, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), renal urea and renal creatinine) were determined according to standard methods (Cheesbrough, 2009). The liver and kidney were excised for histology study.

2.5. Induction of DM
DM was induced by a single intraperitoneal (i.p.) injection of freshly prepared saline solution (0.9% saline) of streptozotocin (STZ) at a dosage of 60 mg/kg b.w. dissolved in 0.1 M cold citrate buffer of pH 4.5 immediately before injection (n=5 per treatment group). One week after STZ injection, rats showing blood glucose levels > 350 mg/dl were considered to be diabetic and were used for the experiment (Gurusubramanian and Roy, 2014). Body weight and blood glucose concentration was checked before and after STZ injection.

2.6. Dose and experimental design
After the induction of experimental DM by STZ, the rats were divided into six groups (n = 5 rats/group) as C: healthy untreated normal control (0.5% carboxymethyl cellulose), DC: diabetic control (0.5% carboxymethyl cellulose), GC: diabetic rats treated with glibenclamide (0.1 mg/kg), and A100, A200 and A400: diabetic rats treated with 100, 200 and 400 mg/kg of ACME, respectively. The vehicle, GC and ACME were orally administered once a day for 30 days. Body weight and blood glucose levels were measured periodically. After 30 days, all rats were anesthetized (90 mg/kg ketamine) and were sacrificed by cervical dislocation. The blood was collected, centrifuged at 1500 g for 15 min at -4 ºC and serum was separated and stored at -80 ºC. Reproductive organs were collected and weighed for histopathological and biochemical analysis.

2.7. Sperm quality, sperm production, transit time and DNA damage
2.7.1. Sperm motility, viability, morphology
The reproductive organs (testis parenchyma, cauda and caput epididymis) of each rat were separated immediately after euthanasia, cleaned and minced in ice-cold phosphate buffered saline (PBS, 1:2 w/v, pH 7.4; 37 ºC) and squeezing it gently to obtain the fresh undiluted semen in a clean Petri dish and incubated at 37 °C for half an hour for liquefaction to proceed to observe sperm quality (motility, viability, morphology) according to standard procedures (World Health Organization, 1999; Filler, 1993)
Sperm motility (40 magnification, scored from 50 different fields) was calculated as: motility (%) = {[No. of motile spermatozoa/Total number of spermatozoa]  100}. The viability of sperm (40 magnification, counted from 200 spermatozoa/group) was computed as viability (%) = {[No. of viable unstained spermatozoa/Total No. of unstained live and pinkish stained dead spermatozoa]  100}. Sperm abnormalities were categorized as percentage of head (detached, amorphous, globose, banana) and tail (coiled, folded, broken, separated) abnormalities and computed as percent abnormality = {[No. of abnormal spermatozoa/Total no. of spermatozoa]  100}.

2.7.2. Daily sperm production (DSP), sperm production efficiency (DSPr) and sperm transit time in the epididymis
Homogenization-resistant step 19 spermatids and spermatozoa from both caput/corpus and cauda epididymis were scored as previously described (Robb et al., 1978). The number of homogenization-resistant spermatids obtained was divided by 6.1 to calculate the DSP, which refers to the number of days that these spermatids are present in the seminiferous epithelium (×106 sperms/testis/day). The daily sperm production relative to testis weight (DSPr) or sperm production efficiency was calculated by dividing DSP with the testis weight and expressed as ×106 sperms /testis/day/g. The sperm transit time through the epididymis (cauda and caput) was determined by dividing the number of spermatozoa in each portion by the DSP (Robb et al., 1978).

2.7.3. Sperm DNA integrity and DNA fragmentation index
DNA integrity and chromatin condensation assessments were assessed by standard cytochemical technique including acridine orange test. Sperm with normal DNA fluoresce green and those abnormal DNA fluoresce yellow or red (Tejeda et al., 1984). DNA fragmentation index (DFI), which is the ratio of abnormally denatured single-stranded DNA (orange/red fluorescent, AO+) over the sum of double-stranded DNA (green fluorescent, AO-) and single-stranded DNA (orange/red fluorescent, AO+). The results of the DFI were expressed as percentage and considered normal when they were below 30% (Sergerie et al., 2005).

2.8. Testis histopathology and histomorphometrics
2.8.1. Histology – hematoxylin and eosin staining
Left testis tissues were fixed in Bouin’s fixative for 24 h. Bouin’s fixative was removed by using lithium carbonate in 70% ethanol after 24 h. Gastric tissues were trimmed, dehydrated in a series of graded ethanol for about 1 h each for two times followed by xylene treatment and embedded in the paraffin blocks after they had been dehydrated in xylene. Sections of 5 µm thickness were obtained by using a rotary microtome (Leica, model RM2125 RTS), deparaffinized in xylene and stained with hematoxylin and eosin for histopathological alterations under standard light microscope (Leica DM 2500) with a digital camera (model-DFC 450C) (Leica Microsystems, Wetzlar, Germany) and photographed (Bancroft and Gamble, 2002).

2.8.2. Seminiferous tubule damage scoring and evaluation of spermatogenic recovery

Histopathological alterations in the seminiferous tubules were randomly sampled in one hundred tubular sections per treatment group. The level of damage was categorized by a crescent score between 1 and 4 and expressed in per cent score as: 1 = 0-25%; 2 = 26-50%; 3 = 51-75%; 4 = 76-100% with some modifications (Cabral et al. 2014). The recovery of spermatogenesis was evaluated based on differentiation of tubules which contained three or more cells that had attained the type B spermatogonia (dense clumps of heterochromatin around the periphery of the nucleus, while type A characterized and differentiated by pale staining nucleus with a fine dusty distribution of heterochromatin) stage or later. The tubule differentiation index (TDI) was computed as the per cent of tubules showing differentiation using 100 seminiferous tubules in one section from each rat, thus 600 tubules for each treatment group (Meistrich and van Beek 1993).

2.8.3. Mean seminiferous tubule diameter (MSTD), Johnsen’s testicular biopsy score (JTBS) and quantification of germ cells and Sertoli cells
Nine slides (3 parts × 3 slides) from upper, mid, and lower parts of right testis were prepared to calculate MSTD, JTBS, and number of spermatogenic cells per tubule. All the observations were made at random order in 100 tubules of the right testis–tissue sections under blindfold conditions. Only cells with whole nuclei were considered for counting. Same testis tissue sections were used to compute MSTD, JTBSs, and number of germ cells and Sertoli cells per tubule. MSTD was measured on H&E stained paraffin sections at 100 × magnification under light microscope (Olympus CX41) using a micrometric ocular lens. Testis injury and disorder in spermatogenesis were appraised histopathologically using JTBS criteria (Johnsen, 1970). The scoring grades for JTBS were as follows: Score 10: complete spermatogenesis with regular tubules; Score 9: many sperms, irregular germinal epithelium; Score 8: few sperms; Score 7: no sperms, many spermatids; Score 6: few spermatids; Score 5: no sperm or spermatids; Score 4: few spermatocytes; Score 3: presence of spermatogonia; Score 2: presence of Sertoli cells; Score 1: no cells. JTBS was computed as sum of all scores in each treatment group/ total number of seminiferous tubules. A decrease in the average value of JTBS was assessed as an impairment of spermatogenesis. In addition, average number of germ cells (spermatogonia, spermatocytes, and spermatids) and Sertoli cells/tubule were examined and quantified. Only Sertoli cells exhibiting distinctive morphologic features and clear nucleolus were quantified. Since the number of germ cells in tubular section at stages I–VIII is different from those at stages IX–XIV, fifty tubules at stage I–VIII and fifty tubules at stage IX–XIV per rat were counted (Roy et al., 2017).

2.9. Immunohistochemical evaluation of PCNA
Immunohistochemical staining for PCNA proteins was performed according to the avidin–biotin-peroxidase complex method with slight modifications as per the instructions of the manufacturer. Xylene and graded alcohol were used to de-paraffinize the slides heated in a hot air oven. Then, 10 mM boiled sodium cit¬rate buffer was used for antigen retrieval. The slides were then incubated for 15 minutes with biotinylated primary antibodies, namely PCNA (1:200), and then secondary labelling with streptavidin conjugated to horserad¬ish peroxidase was performed. The slides were soaked with 3,3′-diaminobenzidine substrate chromogen and then washed and stained with hematoxylin (Roy et al., 2015). PCNA index was calculated as the ratio of stained cells to the total number of germ cells (Altay et al., 2003).

2.10. Serum testosterone
The serum testosterone was measured by using human enzyme-linked immuno sorbant assay (ELISA) kit (cat# AA E-1300, Labor Diagnostika Nord, GmbH, Am Eichenhain, Nodhorn, Germany) as per the instructions of the manufacturer.

2.11. Lipid peroxidation and antioxidant enzymes activity
The testis tissue was homogenized with ice cold Tris EDTA suspension buffer (10% w/v, 50 mmol, pH 7.8) using a homogenizer. The homogenates were filtered and centrifuged for 30 min at 10,000 × g at 4 ºC and the supernatants were frozen at -80 ºC in aliquots until used for biochemical assays. The protein content of the supernatant was determined using the Lowry method (Lowry et al., 1951). Lipid peroxidation was assessed by measuring the thiobarbituric acid reactive substances and quantified as malondialdehyde equivalents (MDA, nmol/mg protein) concentrations by using 1,1,3,3 tetramethoxypropane as the standard (Ohkawa et al., 1979). Superoxide dismutase (SOD) activity was measured by the nitro blue tetrazolium reduction method (Asada et al., 1974). Glutathione S-transferase (GST) activity was computed at 340 nm using 1-chloro-2,4-dinitrobenzene as a substrate (Habig et al., 1974).

2.11. Univariate and multivariate statistical analyses
All data were expressed as mean ± SEM. Normality distribution of the variables was tested using one sample Kolmogrov-Simirnov test. Differences in measured parameters among the groups were analyzed by one-way analysis of variance (ANOVA) test due to normal distribution. The results were analyzed by one-way ANOVA followed by Tukey’s test for post hoc comparisons using SPSS for Windows (SPSS, Inc. Chicago, IL, USA, ver. 20.0). Correlation and regression analysis were made to find the relationship (i) between lipid peroxidation and serum testosterone, DSP, sperm number in cauda, sperm head and tail abnormalities and no. of PCNA positive cells, respectively.

Multivariate analysis of variance (MANOVA) including principal component analysis (PCA), hierarchical cluster analysis (Euclidean distance measure) and detrended correspondence analysis (DCA) was performed using PAST (version 1.86b) software (Hammer et al., 2001). PCA was performed to investigate and ordinate the relationship between supplementation of ACME and their remedial measures of diabetes mediated complications in the rat testis. Thus, the axes derived correspond to gradients of modulatory action of ACME and diabetes caused impairment in testes and dysfunction of spermatogenesis in relation to morphological, histopathological, histomorphometric, hormonal, biochemical, immunohistochemical and sperm quality assessments. Hierarchical cluster analysis was carried to classify the uniqueness and differences of the ACME treatment groups and their impact on restoration of diabetes mediated testicular toxicity using Bray-Curtis distance measure. In addition, ACME treatment groups were classified according to dose and their modulation effects on revival of diabetes mediated testicular toxicity by detrended correspondence analysis (DCA) (Hammer et al., 2001).

3. RESULTS
3.1. GC-MS analysis of ACME
GC-MS results showed that ACME fractions are abundant with polyphenols and fatty acids and comprising of mixture of pharmacologically active compounds possessing antioxidant, anti-inflammatory, anti-diabetic and cytotoxic properties. The major phytocompounds obtained from ACME were identified as embelin, gallic acid, myricetin, bergenin, kaempferol, myricetic acid, quercetin, resorcinol and fatty acids (Fig. A.1 and Table 1).
3.2. Acute toxicity of ACME
The rats treated with ACME at 750, 1500, and 3000 mg/kg doses throughout 15 days exhibited no signs of mortality, hepatic or renal toxicity symptoms and abnormal physiological and behavioural changes based on clinical and histopathological observations. No significant alterations observed in the body weight and blood glucose levels. Renal and liver function tests of rats after 14 days of ACME acute toxicity study showed a normal range of serum alanine aminotransferase (52.47 – 53.40 IU/L), aspartate aminotransferase (151.60 – 153.01 IU/L), urea (5.02 – 5.41 µmol/L) and creatinine (51.33 – 53.17 µmol/L) levels (Table A.1).
3.3. Body and reproductive organs weight
A significant reduction (p < 0.05) in body weight as well as absolute and relative reproductive organs weight (testes, epididymis, full and empty seminal vesicles, ventral prostate and vas deferens) was observed in DC group in comparison with the control indicating testicular atrophy and damage. While ACME treated rats (A100, A200 and A400) as well as GC administered rats showed no statistically (p > 0.05) significant alterations in the organs weights in comparison with the controls indicating the rejuvenation and restoration ability of ACME supplement (Table A.2).
3.4. Sperm quality
Motile sperms of the epididymal contents were observed to be high in the control group rats (59.36%) while a significant reduction was noticed (p < 0.05) in diabetic rats (6.52%). Most of the spermatozoa were observed to be dead in DC. But, significantly high number of motile sperms was seen in ACME (49.46 – 81.9%) and GC (66.7%) treated groups in comparison with DC indicating the protective and restoring role of polyphenol rich ACME supplement on sperm quality. The frequency of number of motile sperms in ACME and GC supplemented groups was at par with the C group (Fig. 1A). Percent live viable sperms (21.35%) were observed to be significantly decreased (p < 0.05) in DC compared to the C group (93.25%). Sperm viability was found to high in GC (91.26%) and ACME groups (85.82 – 92.35%) compared to DC group. Sperm number in the testis, caput and cauda was significantly decreased in the DC rats (testis: 43.12, caput: 3.57, and cauda: 1.37 ×106/mL) in comparison with the controls (testis: 168.87, caput: 6.8, and cauda: 9.85 ×106/mL) supported the detrimental effects of hyperglycaemic condition on the sperm number (Fig. 1B, D). However, ACME administration significantly reinstates sperm counts toward the normal untreated control group rats. Moreover, the treatment of ACME demonstrated a significant higher count of epididymal (cauda and caput) sperms when compared with the DC rats, which confirmed the additional protective role of ACME on diabetes induced testicular sperm degeneration (Fig. 1B, D).

3.5. DSP, DSPr and sperm transit time
DSP per testis (the number of homogenization-resistant step 19 spermatids) and sperm production efficiency (DSPr – DSP relative to testis weight) were significantly lower (DSP: 7.06 ×106 sperms/mL; DSPr: 15.69 ×106/g) in DC rats (Fig. 1C) in comparison with those from the control groups (DSP: 27.68 ×106 sperms/mL; DSPr: 43.6 ×106/g). On the other hand, ACME supplementation demonstrated a significant increase in DSP (18.21 – 40.47 ×106 sperms/mL) as well as DSPr (29.36 – 43.84 ×106/g) toward the control group level which confirmed its effectiveness in restoring the sperm quality (Fig. 1C). In addition, the sperm transit time in the epididymal cauda and caput/corpus regions were significantly lower in the DC group rats (cauda: 5.6 days; caput: 4.1 days) than in the control group (cauda: 9.1 days; caput: 8.3 days) while no significant alterations were found in the ACME supplemented group rats (cauda: 8.1 – 9.8 days; caput: 6.3 – 7.4 days) (Fig. 1C).

3.6. Sperm abnormality and DNA damage
The sperm head and tail abnormalities were increased significantly in DC group when compared with the controls (Table 2). The normal sperm phenotypes were significantly decreased to 51.45% in DC treatment and most of the sperms were observed to be dead (Fig. A.2F). While ACME groups significantly restored (92.95 – 96.60%) this parameter toward the control level (95.65%). The common abnormalities encountered during the morphological examination of the sperms were banana heads, two heads, small/big heads, untied heads, amorphous heads, lack of usual hook, separated flagellum, coiled tails, broken tails and spiral twisted tails (Fig. S.2E-H). Within head abnormalities observed in DC rats, banana head were the predominant form of sperms (14.82%) followed by detached head (8.16%) and amorphous head (3.63%). Regarding tail abnormalities in DC treatment group, bent tail forms were observed mostly (13.91%) followed by broken tail sperms (2.11%). All together DM condition significantly damaged the sperm morphology and in turn noticed more number of morphologically (26.62% total head abnormalities and 16.02% total tail abnormalities) abnormal spermatozoa (Table 2). ACME supplementation significantly protected the spermatozoa completely from the detrimental effects of DM as a lower percentage of sperm head and tail abnormalities were evident (Table 2).

In acridine orange staining, the presence of normal native double-stranded DNA (green fluorescent, AO-) was observed to be high in the treatment groups of untreated control (Fig. A.2A), and ACME (Fig. S.2C, D) while reverse trend was noticed in DC rats (Fig. S.2B), i.e. detection of high number of abnormally denatured single-stranded DNA (orange/red fluorescent, AO+). Significant increments in DNA fragmentation index (DFI, %) were observed in DC group (73.18%) revealed the influence of DM mediated complications on sperm DNA integrity and chromatin condensation. While, rats in the ACME treatment (11.48 – 11.76%) showed a significant decrease (p < 0.05) in DFI signifying the protective role of ACME on sperm integrity and damage (Table 2).

3.7. Histopathology of testis, damage scoring and spermatogenic recovery
Similar to the control group (Fig. 2A, B), nearly normal histological features of rat testis were observed in the GC (Fig. 2D, E) and ACME treatment groups wherein (i) the seminiferous tubules were normal and lined with germinal epithelium, (ii) Leydig cells were observed in the interlobular spaces, and (iii) all sequence of spermatogenesis were present along with all types of spermatogenic cells, spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and mature sperm were present (Fig. 2G-L). In contrast to untreated control, the profile of seminiferous tubules in DC group observed as irregular with wide spaces in between them, marked disorganization and depletion of germ cells, sloughing and desquamation of germinal cells, arrest of spermatogenesis, splitting of spermatogonia from the intact membrane and were scant, separation and lesser number of spermatogenic cells, no mature sperms in the lumen, vacuolization of tubules, low number of Leydig’s and germ cells, lumen was empty or filled with debris and the stages of spermatogenesis were interrupted and atrophy of seminiferous tubules due to the effect of diabetes mediated reproductive complications (Fig. 2B, C). The untreated control, GC and ACME treatment groups exhibited normal histological characteristics of the seminiferous epithelium and scaled the level of damage as score 1, i.e. nil germ cell depletion, 4.08 – 7.75% vacuolization and < 8% sloughing of germ cells (Table 3). In the DC group, a vast quantity of sloughed cells (72.38%, score 4), vacuolization (69.30%, score 4) and depletion of germ cells (46.55% – 56.88%) along with degenerative characteristics was noticed in the tubular lumen of many seminiferous tubule sections. Thus, ACME treatment significantly (p < 0.05) reduced the seminiferous tubules damage as well as protecting the tubules effectively. The TDI reduced significantly in the DC treatment (12.16%) whereas treatment with the ACME significantly (p < 0.05) restored the ability of the testis to support stem cell differentiation to 50 – 60% (intensifying the TDI) at 30 days post treatment, respectively (Table 3).

3.8. MSTD, JTBS, quantification of germ cells and Sertoli cells and PCNA expression
Histomorphometric analyses of DC rat testis showed a significant (p < 0.0001) decrease in the MSTD, JTBS, number of germ cells and Sertoli cells which exemplify the impairment of spermatogenesis and dysfunction of testicular activity in diabetic rats while these parameters restored significantly with ACME supplementation showing its protective role on recovery of spermatogenesis (Fig. A.3A-C). The number of PCNA-positive cells and PCNA index were significantly decreased (down regulated) in the DC group while the same parameters observed to be high (up regulated) in C, GC and ACME groups (Fig. 3D and Fig. 4).

3.9. Serum testosterone, lipid peroxidation and antioxidant enzymes
Serum testosterone concentrations were decreased significantly in DC rats (2.26 ng/mL) than the control rats (6.97 ng/mL), suggesting the inhibitory role of DM complications on testicular androgenesis. However, ACME administration (3.43 – 3.68 ng/mL) significantly restored the serum testosterone concentrations to the control level. Moreover, significant higher values of serum testosterone concentrations were noted in the ACME (4.27 – 6.85 ng/mL) when compared with the control groups, suggesting that ACME at a high dose of 400 mg/kg has a stronger influence on testicular androgenesis than the lower dose of 100 mg/kg (Fig. 1F). MDA content (a product of lipid peroxidation of the polyunsaturated fatty acid present in cell membrane) of the reproductive tissues (testis, epididymis, seminal vesicle and prostate) was significantly (p < 0.05) increased in DC group with respect to the controls, indicating ROS generations in the testis and accessory organs and induction of oxidative stress. ACME treatment significantly restored these parameters toward the level of control (Fig. 5A-D). A severe inhibitory response on the testis as well as accessory organs antioxidant status (SOD and GST) and protein levels was observed in DC rats than the controls signifying the suppressed testicular and accessory organs antioxidant defense against ROS, which assisted the generation of oxidative stress. While ACME treatment significantly recuperated these testicular and accessory organs antioxidant enzymes activities (Fig. 5E-L).and protein levels (Fig. 5M-P) toward the control levels.

3.10. Univariate and multivariate analyses
3.10.1 Interactions between lipid peroxidation and serum testosterone, sperm production, sperm quality and germ cell proliferation
Generation of lipid peroxidation radicals due to diabetic complications significantly disrupted the serum testosterone levels, daily sperm production, sperm concentration in cauda and germ cell proliferation and found an inverse relationship between them. While, a strong positive correlation and regression relationships were computed between lipid peroxidation and head and tail sperm abnormalities (Fig. A.3). Correlation and regression analysis inferring the magnitude of diabetic complications and their influence on serum testosterone levels, spermatogenesis and sperm production and how these physiological conditions were restored by ACME supplementation.

3.10.2. MANOVA – PCA, cluster analysis and DCA
MANOVA data matrix included of six treatment groups and as independent variables and their effect on forty morphological, physiological and metabolic variables as dependent variables to illustrate the relationship (1) between DM complications and their magnitude in testis damage, suppression of spermatogenic process, impairment in sperm quality; and (2) the ameliorative role of ACME supplementation on restoration of testis damage and preservation of sperm quality (Fig. A.4). The PCA, DCA and Euclidean distance measure loading plot diagram indicate that the chosen forty variables generated two principal component axes based on testicular impairment due to DM as well as restoration activity ACME supplementation on recovery of spermatogenesis, germ cell proliferation, sperm quality and reproductive function. The distant and isolated variables in the plot, i.e. treatment groups indicate a strict and extremely significant correlation between the variables. The ACME treatment groups were separated in the PCA plot completely from the DC groups showing the modulating activity of ACME against DM mediated testicular dysfunction in rats and this effect is exerted by ACME could be due to the broad array of biological activities including male gamete maturation, production of energy for sperm motility, DNA repair, germ cell recovery and Sertoli cell metabolism (Fig. A.4). MANOVA analyses distinctly showed a very good separation and there was a significant dose dependant variation between ACME supplementation and their antioxidant and gonadoprotective properties in comparison with the DC group. The results indicated that the DC group showed variation in biochemical, histopathological, immunohistochemical and metabolic profiles than ACME supplemented groups and these profiles were not affected and back to normal as a result of gonadoprotective action of ACME (Fig. A.4).

4. Discussion
Diabetes-induced glucotoxicity and oxidative stress in the testes and accessory organs are the key factors responsible for reproductive disorders and male infertility (Kilarkaje et al., 2014; Alves et al., 2013). This is the first report to examine the ameliorative potential of polyphenols rich ACME on restoration of DM mediated testicular dysfunction in rats. The diabetic state results in up regulation of oxidative stress, decreased body, testis and accessory organs weight, induces dramatic changes in the testicular morphology, decreased concentrations of testosterone, abnormal spermatogenesis and alterations in sperm quality (Aybek et al., 2008). These testicular impairments were alleviated by the polyphenolic antioxidants (bergenin, embelin, gallic acid, myricetin, kaempferol, myricetic acid, quercetin and resorcinol) present in the ACME (Kilarkaje and Al-Bader, 2015; Rashid and Sil, 2015). Sumino et al. (Sumino et al., 2001, 2002) reported the antioxidant and cytotoxic properties of A. colorata and isolated bergenin, norbergenin, demethoxy bergenin and alkyl resorcinols from the fruits. A significant decrease in testis and epididymis weights in DC rats appear to be due to inhibited spermatogenesis following germ cell death (Kilarkaje and Al-Bader, 2015). In support of this inference, the testes of DC rats showed sloughing and depletion of germ cells, vacuolization, degeneration, lesser number of Leydig’s cells, Sertoli cells and spermatogenic cells and no mature sperms in the lumen. ACME supplementation normalizes the decreased testis and epididymis weights in diabetic rats, may be by stimulating cell survival and spermatogenesis due to polyphenols rich ACME-mediated alleviation of oxidative stress (Faid et al., 2015). In support of this, the testes of ACME supplemented diabetic rats showed normal histomorphology and histomorphometric values (MSTD, JTBS and TDI). Sperm quality in both diabetic men and animal models is severely affected due to increased oxidative stress, apoptosis of germ cells, inhibition of seminiferous epithelial cell proliferation, and alterations in pituitary–testicular hormonal axis (Jangir and Jain, 2014; Ko et al., 2014). The decrease in sperm quality (motility, viability, concentration, morphology and integrity) may be due to structural and functional changes during sperm morphogenesis as DM is well-known to promote sperm head and tail abnormalities and affect the sperm DNA integrity (Kilarkaje et al., 2014). The recovery of sperm quality and DNA integrity to the control levels in ACME treated diabetic rats can be attributed to antioxidant properties of ACME. The sperm head and tail abnormalities designate the effects of hyperglycemia on spermatogenesis as well as on DNA damage or metamorphosis of germ cells (Rama Raju et al., 2012). The DNA damage correlates with the levels of advanced glycation end products and their receptors and decreased sperm count in our study was probably due to fewer spermatids entering into spermiogenesis and acceleration of sperm transit time as diabetes is known to cause apoptosis of germ cells and mature sperm (La Vignera et al., 2012). Reduction in sperm motility may be caused by elevated abnormalities and impairment of structure and function of sperm tails due to sperm DNA damage and disruptions in transmembrane mitochondrial potential (Roessner et al., 2012). Optimal sperm transit time (8-15 days) through epididymis is required for spermatozoa to attain maturation and motility which is vital for sperms to fertilize ova (Bellentani et al., 2011). The sperm transit time in caput and cauda was accelerated in DC rats which may decrease the sperm reserves in the caudal portion of epididymis and interfere with the reproductive process of rats. PCNA immunoreactivity was observed to be strong in the seminiferous tubules of the ACME groups as observed in controls and especially spermatogonia, while moderate reactivity was observed in spermatocyte-I and spermatocyte-II inferring the synthesis of PCNA required for DNA replication or repair in testicular germ cells. PCNA was nonreactive in Sertoli cells. In the DC group, moderate immunoreactivity was observed in a few germ cells and generally mild reactivity was observed. In parallel, Altay et al. (Altay et al., 2003) and Donmez et al. (Donmez et al., 2014) reported similar results in their study conducted with type I diabetic rats.
Antioxidant enzymes are particularly abundant in testis and relevant for the maintenance of testicular physiology. The decreases in SOD and GST levels also probably led to accumulation of hydrogen peroxide in the testes of diabetic rats. Thus, the decreased antioxidant levels in the DC rat testes favored lipid peroxidation as indicated by an increase in MDA levels. In accordance with previous reports (Kilarkaje et al., 2014; Jaganjac et al., 2013) diabetic testicular damage in the current study was associated with enhanced lipid peroxidation product (MDA levels) and depletion of antioxidant defenses including SOD and GST activitities in testicular and other accessory reproductive tissues. Thus, mitigation of ROS is important for treatment of testicular damage in diabetic patients. In further support of this notion, the present work demonstrated that treatment with ACME attenuated testicular and accessory organs MDA together with protected testicular GST and SOD activities in DC rats, illustrating the antioxidative actions of ACME.
5. Conclusion
streptozotocin induced DM in rats severely affected the spermatogenesis (histological alterations), testicular function and caused gonadotoxicity. The body and reproductive organs weights, serum biochemical profiles (ALT, AST, Urea and creatinine), serum testosterone levels and antioxidant enzymes (SOD and GST) were significantly disturbed and altered. Sperm quality (motility, viability, DSP, DSPr, number, DNA integrity and DFI) was significantly affected and observed defective sperms with head and tail abnormalities. The sperm transit time in caput and cauda was accelerated and resulted in incomplete spermatogenic process and formation of immature spermatozoa leading to infertility. ACME supplementation significantly improved the physiological and metabolic processes and in turn recovered the spermatogenic process, germ cell proliferation, DNA repair and sperm quality (Fig. 6). Based on these findings, ACME could be used as a therapeutic as well as protective agent for DM mediated testicular complications in rats.

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