Evaluation of Extract Fractions of Vernonia calvaona on Hormonal Parameters and Histopathological Changes in Male Albino Wistar Rats Exposed to Heavy Metals Laden Water

: Antioxidative potential of Vernonia calvaona on heavy metals induced toxicity in male albino wistar rats. thirty (30) albino rats weighting 90-120g were divided into six groups of five rats each. With the exception of the normal control (NC) group, all other groups were exposed to heavy metals laden water twice daily. Groups 1 and 2 served as normal (NC) and Positive control (PC) controls. Groups III were administered heavy metals laden water and vitamin C (0.0167g/g.b. w). Groups IV were administered heavy metals laden water and 400mg/kg b.w of aqueous crude extracts of Vernonia calvaona . Group V were administered the heavy metals laden water and 400mg/kg b.w of methanol extracts of Vernonia calvaona . Group VI were administered heavy metals laden water and 400mg/kg b.w of n-Hexane fraction of Vernonia calvaona . Administration of treatment was done onces daily for a period of 30 days. Rats were sacrificed and samples collected for analysis. There was increase (p<0 .05) in the concentration of testosterone in PC group (3.60mg/ml) when compared to NC (1.80mg/ml). Groups III, IV, VI, showed significant (P>0.05) decreases in serum testosterone concentration when compared to PC group. There was increase in the conc. Of PSA in the PC group II, when compared to the NC. FSH Conc in group II compared to NC groups show a significant decrease. Groups III showed an (P>0.05) increases in serum FSH concentration when compared to PC group. The PC group showed decreases in LH concentration compared to Normal Control. In conclusion, the changes observed in this study revealed that heavy metals adversely affected the gonads, causing alterations in the levels of major hormones. Furthermore, the extracts were able to exert varied degrees of ameliorative effects with the crude extract showing greatest potential at managing the heavy metals induced irregularities, and at reversing testicular damage.


Introduction
The physical, biological, and chemical components of the atmospheric system can be contaminated in such a way as to interfere with regular environmental processes.Additionally, it refers to environmental contamination that negatively impacts living https://doi.org/10.58614/jahsm364 things.The extraction of various important minerals from subsurface deposits is a time-honored technique that has been used for a very long time.Many quarried materials were employed by the ancient Egyptians and Romans to build the pyramids, temples, and monuments that are still standing today.Numerous other human activities also had a negative impact on the ecosystem.However, these activities produce a significant amount of particle pollution, which can be harmful to animals, plants, and water.The majority of these substances build up in living things' body tissues, and their concentration rises as they move from the lower tropics to the higher tropics (referred to as "bio magnifications") of the ecosystem (Langer and Bode, 2011).
During their production, transportation, and sale, heavy metal emissions from industry and vehicles may be deposited on the surfaces of plants.High quantities of heavy metals have also been found in plants, according to AL-Jassir et al. (2005) (Sharma et al. 2008).When heavy metal concentrations in plants and animals reach a particular level, they are considered hazardous.Despite playing vital roles in plant and animal cells, heavy metals like cobalt, copper, iron, molybdenum, nickel, and zinc can be extremely harmful when their concentrations surpass a predetermined level (WHO acceptable limit) (IOSHIC, 1999;Klaus, 2010).The amount of heavy metals in the soil varies according to its age, composition, and location, according to studies (Udosen et al. 1990;Haluschak et al. 1998;Odukoya et al. 2000).
Lead, zinc, and cadmium concentrations in all the vegetables analysed above the maximum allowable concentration, according to another study on the assessment of heavy metals concentration in urban-grown vegetables in Thikakown, Kenya.In accordance with the World Health Organization's acknowledged human health limit of 0.3 mg/kg, lead concentration in vegetables was reported to be over that level (WHO, 2004).In rural areas across Africa, Asia, and South America, medicinal plants are an essential part of the traditional healthcare systems.According to statistics, many people use medicinal plants, and around 80% of those in developing nations rely directly or indirectly on traditional medicine to treat the majority of their medical issues (Bodeker 1999;Joyce 1992;King 2000;WHO 2000).It is also well known that more than 75% of Nigerians living in rural areas rely on traditional medications made from herbal remedies for their medical requirements (Tolu, 2008).Traditional medicines based on herbal cures have long been a significant part of the healthcare systems of many African nations, including Nigeria, since the dawn of civilization.
The creation of medications in both poor and developed nations can benefit from research on medicinal plants.More than half of the population of Africa today relies on animal and plant-based medicine to meet their health needs (Ainslie, 1973).In complementary or alternative medicine, a variety of herbs with hypolipidemic, hypoglycemic, antiplatelet, antimalarial, antiviral, or immune stimulating and modifying activities may be helpful (Borchers et al. 1997).It is important to be aware that plants have long been thought of as sources of therapeutic substances for the treatment of infections and disorders like chicken pox and hepatitis (Bramstedt, 2006).The indigenous inhabitants of the Central Senatorial District of Cross River State, Nigeria, commonly refer to Vernonia calvaona as "ekeke leaves" (Igile et al. 2013).It is a little shrub that grows to less than one metre in height, with petiolate leaves that https://doi.org/10.58614/jahsm364are about 10mm wide.It is utilised both as an ethnomedical remedy and as a green leafy vegetable (Igile et al. 2013).With or without palm oil, it is a favourite local delicacy to be consumed raw or fresh.Among native customers, it also forms part of a classic salad.Additionally, it can be used to make native soups, stews, and porridge with potatoes, yams, and plantains (Igile et al. 2013).Its use is justified by the notion that the entire plant treats heart conditions, blindness, diabetes, malaria, and functions as an anti-helminthic agent.In South Western Cameroun and the South Eastern region of Nigeria, the plant is widely dispersed, just like its near cousins V. teniana and V. amygdalina.Although these three species share many morphological characteristics, it is possible to distinguish between them based on specific anatomical characteristics, such as the height and width of the leaves and the potency of the plant's bitter flavour.Vernonia calvaona is the least bitter and has the shortest height of the three species (Igile et al. 2013).In order to assess the biochemical safety and antioxidative potential of Vernonia calvaona's crude extract (CE) and extract fractions, as well as their impact on male sex hormones and histopathological changes in male albino wistar rats fed with heavy metal-polluted water, the study examined CE and extract fractions of Vernonia calvaona.

Materials and Methods
The following tools, supplies, and chemicals were needed for this study; all of the chemicals utilised were of analytical quality.Dimethyl Sulphur Oxide (DMSO), distil water, cheese cloth, water bath, syringes, EDTA bottles, cages, electronic weighing scale, wood shavings, glass beakers, and methanol were all obtained at a chemical store in Yeagona, Bayelsa State.Syringes (2ml and 5ml), containers for collecting blood samples, and all of these items were purchased from a chemical store in Yeagona, Bayelsa state.The United States' Sigma produced hematoxylin and eosin.FSH, LH, and testosterone hormone testing kits were bought from the mono-bind company in Lake Forest, USA.The Niger Delta University's department of pharmacology's animal house was where the experimental animals were purchased.The laboratory, Divic Medical Laboratory Choba, Port Harcourt, Rivers State, provided the micropipettes, centrifuge, pasteur pipette, refrigerator, atomic emission spectroscopy, dissecting equipment, dissecting board, aluminium foil, methylated spirit, dettol, and gloves that were used for the analysis.

Collection of Plant Sample
The Vernonia calvaona leaves were gathered from a nearby farm in Nigeria's Bayelsa state's Ogbia Local Government Area.A botanist from the Faculty of Science, University of Calabar, Cross Rivers State, Nigeria, validated the plant.Additionally, it matched a specimen that had already been deposited in the department's herbarium (BCH/VC/01).The plant portion (leaves) was thoroughly rinsed with distil water after being thoroughly washed with tap water.After that, the plant material was spread out to air dry for a week.https://doi.org/10.58614/jahsm364

Preparation of Material
Using an electronic blender, dry Vernonia calvaona leaves were reduced to a fine powder.The powder was measured using an electronic balance and then soaked in a mixture of 80% n-hexane and 20% methanol.For 48 hours, the mixture is stirred with a magnetic stirrer at a controlled temperature of 4-8 oC.Cheese cloth and then filter paper were used to double-filter the extract.A rotary evaporator was used to concentrate the filtrate at a controlled temperature of 40 to 50oC.Finally, the extract was evaporated in a water bath.
At the Niger Delta University in Bayelsa State, Nigeria, the Department of Pharmacology animal house provided thirty (30) albino Wistar rats with weights ranging from 90 to 120g.The Institute for Laboratory Animal Research (ILAR) established standard guidelines for the housing and care of the thirty (30) rats.
The purchased animals were given seven days to adjust at the animal house.They were housed in wire-mesh coverings over plastic cages to improve ventilation.The animals were kept in an environment with a controlled 28 2 °C temperature, 50 5% relative humidity, and a 12-hour light/dark cycle.The animals were housed in an appropriately ventilated animal facility, fed commercial rat pellets, and given unlimited access to water during the trial.

Experimental Design and Treatment of Animals
Orogastric intubation was used to administer the medication twice daily for a total of 30 days.Thirty (30) albino rats were used in the experiment.Six groups of five rats each were formed from the rat population.Rats in Group 1M, the usual control group, received placebo injections.Animals in Group II M, the positive control group, were given placebo water and water contaminated with heavy metals.Group III M received vitamin C and water contaminated with heavy metals.Treatment for Groups IV M included the administration of water contaminated with heavy metals and crude Vernonia calvaona preparations.The heavy metal-filled water and methanol extracts of Vernonia calvaona were given to Group V M. Group VI M received water contaminated with heavy metals as well as Vernonia calvaona's n-Hexane fraction.

Collection of Blood Samples for Analysis
The animals were killed 12 hours after the previous dose, and entire blood was extracted from the heart using a sterile syringe and needle during a cardiac puncture.The blood samples were placed in both standard tubes and tubes containing lithium heparin.The blood samples in the plain tubes were centrifuged with the blood samples in the lithium heparin tubes at 4000 rpm for 10 minutes to separate the serum from the red blood cells after being allowed to clot for two hours at room temperature.Using pasteur pipettes, serum was collected into another plain tube with the appropriate label from each centrifuged plain tube and lithium heparin tube.When required for various biochemical experiments, the distinct sera were then kept frozen in a refrigerator.

Serum Hormonal Assay
Estimation of the concentration of the Biochemical (hormonal) parameters were carried out by ELISA methods using analytical grade laboratory kits from ACCU-BIND as described below

Determination of Serum Testosterone Concentration
The determination of testosterone was carried out using a competitive enzyme immunoassay approach.An antibody, an enzyme-antigen conjugate, and a native antigen are needed for the determination.The native antigen and the enzyme antigen conjugate compete for a finite number of antibody binding sites when biotinylated antibody, enzyme antigen conjugate, and a serum containing the native antigen are combined.

Determination of Serum Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) Concentration
An immune-enzymometric assay was employed to measure the levels of serum FSH and LH, and for this purpose, antibodies with high affinity and specificity and separate and distinct epitopes to which an antigenic material binds were used.An immobilisation that occurs at the surface of a streptavidin-coated micro well with exogenously supplied antibody serves as the catalyst for the reaction.When monoclonal biotinylated antibody is combined with enzyme-labeled antibody and serum that contains native antigen and antibodies without steric or competitive interference, a sandwich complex is created.
The high affinity reaction between streptavidin and the biotinylated antibody produced a complex for each hormone that was determined in this study and was then deposited on the micro wells.The antibody-bound fraction is separated from unbound antigen by decantation or aspiration after equilibrium is reached.Each absorbance is evaluated by a microplate reader, and the enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration (Lashansky, 1991).Dose response curves were produced using a number of different serum references with known antigen concentrations, and from these curves extrapolation was used to calculate the antigen content of unknown samples (Odel, 1981).https://doi.org/10.58614/jahsm364

Data and Statistical Analysis
Data obtained were analyzed using SPSS package version 20 and statistical significance measured by One-way Analysis of variance (ANOVA) with a post hoc Donett value at P<0.05.Charts were plotted using Microsoft excel 13.All data were expressed as Mean ± SEM, n=5.

Acute toxicity test
The WHO permissible level was compared to the anticipated total mean of cadmium, lead, iron, nickel, and cobalt.Except for copper, mercury, and chromium, the concentration of heavy metals was substantially (p>0.05)below the WHO acceptable level.All other heavy metals concentrations were significantly (p<0.05) over the WHO allowable limit.

Effect of Treatment on Histology of Testes
Testis with dispersed seminiferous tubules and uneven basement membranes (BM) containing disorganised loosely atrophic spermatogonia cells were visible in histopathological photomicrographs of testes sections for the group exposed to heavy metal-laden water.Photomicrographs of the heavy metal exposed water exposed rats treated with n-hexane and crude extracts of vernonia calvaona showed partial recovery in spermatogenesis, but much better recovery was observed for the vitamin C and methanol group.Testes from normal control rats had closely packed seminiferous tubules lined by developing spermatogonia cells at various stages of development.Intact basement membranes (BM) are seen in well-packed seminiferous tubules in sections of the testis, which contain three to layers of spermatogonia in various stages of development that are held in place by sertoli cells.The cytoplasm is mild, and the cells feature round to oval basophilic nuclei.The leydig cells in the interstitial space between them are tightly packed and sparse.Spermatogonia A and B, primary spermatocyte (S1), secondary spermatocytes (S2), and spermatid (S3) made up the cell.Stem cells SA and SB; spermatogonia cells S1, S2, S3, S4; basement membrane BM; leydig cells LE; lumen.

Discussion
Heavy metal toxicity has been strongly connected with serious ailments such malignancies, immune system disorders, genotoxicity, skin and eye diseases, and infertility.Human exposure to heavy metals has primarily been by breathing.This investigation evaluated how well they could affect the amounts and operations of the reproductive hormones.(sometimes leading to infertility and erectile dysfunction in men).Plant decoctions have been investigated and found to have a tremendous potential for reversing or improving a variety of health issues over time.As a result of their consistent use for medicinal purposes, several plants are now regarded by traditional medical practitioners as being both safe and beneficial against specific illness conditions (Treasure, 2000).Unlike pharmaceutical medications, which only produce specific effects, medicinal plants produce both the individual and synergistic effects of their constituent phytochemicals, which are almost never harmful.They are known for their capacity to evoke a wide range of physiologic activities.2000'sTreasure.

Effect of Heavy Metal Toxicity on Reproductive Endocrinology
Heavy metal toxicity exposure has a major negative impact on health, including cancer (

Effect of Heavy Metals on the Plasma Concentration of Sex Hormones
The gonadotropic hormones (Testosterone, FSH, LH, and PSA) and PSA biochemical assay results significantly differed from those of the normal control (NC).LH and FSH levels were found to be significantly lower in the heavy metals exposed group (PC), and this could have an adverse effect on an individual's reproductive health because these hormones are crucial for the development of secondary sexual characteristics and the production of follicles in males.When compared to the heavy metals exposed group (PC), the groups treated with plant extracts exhibited a significant rise in the levels of sex hormones.
When compared to the normal control group, the heavy metals exposed group (PC) has considerably higher testosterone and PSA levels; a rise in PSA is a marker of posttreatment malignancies.The results of the study unmistakably demonstrated that the plant extract and its fractions may be to blame for the rise in FSH and LH hormone levels in the animal models, with the rise tending towards that of the normal control (NC).

Effect of Heavy Metals Exposure on histo-pathological architecture of the testis
The testis' histo-pathological sections revealed varying degrees of abnormalities in the groups exposed to heavy metals, with the heavy metals control group (PC) showing the most damage.Different levels of recovery were seen in the Vernonia calvaona extract-treated groups.In the n-hexane treated group, which shown a strong capacity for regeneration, exceptional outcomes were seen.
The study's histopathology findings demonstrated that heavy metals negatively impacted the gonads, altering the levels of two key hormones (FSH and testosterone), as well as the testis' histological architecture.These negatively affect hormone levels and reproductive functions, which leads to infertility in women and impotence in males.They also reduce sperm motility and count, hinder spermatogenesis, and destroy seminiferous epithelium.Additionally, different levels of ameliorative effects were demonstrated by extract fractions of the research plant, Vernonia calvaona, with the n-hexane fraction demonstrating the best potential for regulating the heavy metalsinduced abnormalities and for reversing testicular damage.

Conclusion
There are several ways that heavy metals enter the human body, including inhalation and ingestion.The most significant method of exposure to these substances in the population of humans is by ingestion.Normal hand-to-mouth activity with contaminated hands can expose children to dangerous amounts (Dupler, 2001;Sawicka-Kapusta, 2003).Another kind of exposure is by skin absorption.In industrial, agriculture, and the pharmaceutical industries, heavy metals are most frequently in contact with people (Roberts, 1999).Numerous health risks, such as tissue damage and abnormalities, have been connected to chronic exposure to hazardous heavy metals.The study went on to say that the formation of follicles in men and the development of secondary sexual characteristics in men depend on levels of key hormones that are significantly depleted in animals subjected to heavy metal exposure.The amounts of hormones rose in the groups given vitamin C and plant extracts, suggesting a potential reversal of the effects of the heavy metal.The research has also revealed associations between exposure to heavy metals and a number of health issues, including gonadotoxicity, haematological abnormalities, and possible infertility.

Recommendation
Occasion by our findings of this research reported above, the following are recommended: 1.
The proximate parameters of these plants could be used in identification and standardization of drug.

2.
Further purification of phytochemical constituents of Vernonia calvaona to produce pure compounds and elucidation of the chemistry of such phytochemicals.

3.
Evaluation of teratogenic effects of heavy metals and the ameliorative effects of Vernonia calvaona should be study.

4.
Isolation and structural elucidation of the components of the phytochemical fractions of Vernonia calvaona.

Figure 3 .
Figure 3. Photomicrographs (x 400) of Testis of rats exposed to heavy metal laden water (PC group) and given placebo treatment (Stained with Gomori Aldehyde Fuschin stain).

Figure 4 .
Figure 4. Photomicrographs (x 400) of Testis of rats exposed to heavy metal laden water and treated with Vitamin C. (Stained with Gomori Aldehyde Fuschin stain).

Figure 5 .
Figure 5. Photomicrographs (x 400) of Testis of rats exposed to heavy metal laden water and treated with Crude Extract of Vernonia calvaona.(Stained with Gomori Aldehyde Fuschin)

Figure 6 .
Figure 6.Photomicrographs (x 400) of Testis of rats exposed to heavy metal laden water and treated with Methanol fraction of Vernonia calvaona.(Stained with Gomori Aldehyde Fuschin)

Figure 7 .
Figure 7. Photomicrographs (x 400) of Testis of rats exposed to heavy metal laden water and treated with n-Hexane fraction of Vernonia calvaona.(Stained with Gomori Aldehyde Fuschin)

Table 2 .
Heavy Metals Analysis From Underground Water Sample (ppm) 50 of vernonia calvaona have been determined by previous studies to be greater than 5000mg/kg body weight (Iwara et al. 2015).4.

Effect of Heavy Metal Laden Water, Vitamin C, Crude Extract and Its Fractions on Serum Hormonal Parameters 4.1. Hormonal Profile Follicle Stimulating Hormone (FSH)
The serum testosterone concentration of animals in group PC (3.60±0.00)and n-Hexane (2.90±0.20)were significantly (P<0.05)higher compared with that of group NC (1.80±0.40)while other groups compare well with that of group NC.Crude extracts (CE) of Vernonia calvaona (VC) showed the best ameliorative potentials in normalizing the testosterone concentration levels.