EFFICIENT USE OF TRADITIONAL MEDICINE TO DETOXIFY UPPER REPRODUCTIVE ORGANS TOXICITY THAT MAY LEAD TO INFERTILITY

EFFICIENT USE OF TRADITIONAL MEDICINE TO DETOXIFY UPPER REPRODUCTIVE ORGANS TOXICITY THAT MAY LEAD TO INFERTILITY

Joseph O. Ezugworie ¹ and Obinna O. Uchewa ^{* 2}

Department of Anatomy ¹, Faculty of Basic Medical Sciences, University of Nigeria Enugu Campus (UNEC), Enugu State, Nigeria.

Department of Anatomy ², Faculty of Basic Medical Sciences, College of Medicine, Alex Ekwueme Federal University Ndufu-Alike Ikwo (AE-FUNAI), PMB 1010, Ebonyi State, Nigeria.

ABSTRACT: This study aims at investigating the use of herbal medicine (F. vogelii) as a natural antioxidant to detoxify reproductive toxicity. Twenty-five (25) female Wistar rats used in this research were divided into five groups of five (5) rats per group. Group A served as negative control and received feed and water ad libitum. It also received formal saline. B and C served as the low (100 mg/kg) and high (300 mg/kg) dose extract groups. They received lead acetate for 14 days after the extract has been administered for first 7 days. Groups D served as the positive control and received only lead acetate 14 days too while E received only the extract for 14 days. The experiment lasted for 28 days including an acclimatization period of 7 days. The positive control group showed a significant increase in SOD activity at P ≤ 0.01 while that of group E rats had ovarian SOD closer to group A. Group B and C showed reduced SOD level as well as significant weight gain. The extract presented chemicals capable of removing free radicals generated by toxins in the body. The ovarian and uterine weights were significantly reduced in the positive control group (group D). The histological changes include edema, necrosis, empty optical spaces, denudations, and fatty changes. The extract had a positive effect on the organs against the lead acetate. The results of this study suggest F. vogelii leaves can be used as an antioxidant to treat lead reproductive toxicity.

Reprotoxins, Traditional, Infertility, Antioxidant, Toxicity, Ogbu

INTRODUCTION: Women participation in the menial jobs is on the increase especially in the mining sites, and there is evidence showing the health implications they suffer from occupational exposure to reprotoxins ^{1, 2, 3}. Therefore, the European Commission presented an analysis of the impacts of a possible amendment of the Carcinogens and Mutagens Directive 2004/37/EC to include reprotoxic substances ⁴.

Lead is a toxic metal not essential for bodybuilding, and it is deleterious to every part of the body especially the reproductive systems of both male and female ^{5, 6}. Lead has been implicated in the reduction of sperm count in males, and it also causes other changes in the volume of sperm when blood lead levels exceed 40 μg/dL ⁷.

At this level, motility and morphology of sperm are also affected ⁸. The problems caused by lead exposure in the female reproductive organs are more severe and complicated than their male counterparts. Increase in lead toxicity in the body can lead to miscarriages, premature delivery ⁹, low birth weight and developmental problems ^{10, 11}. Blood lead levels in mothers and infants are usually similar to the lead present in mothers’ blood passes into the fetus through the placenta and also through breast milk ¹². Increased calcium intake during pregnancy can help mitigate this phenomenon) ^{6, 13}.

Ficus is a genus of about 850 species of woody trees, shrubs, vines and epiphytes in the family of Moraceae ¹⁴. Collectively known as fig trees or figs, they are native throughout the tropics with a few species extending into the semi-warm temperate zone ¹⁵. The common fig (F. carica) is a temperate species native to southwest Asia and the Mediterranean region (from Afghanistan to Portugal), which has been widely cultivated from ancient times for its fruit.

The fruit of most other species is also edible though they are usually of only local economic importance or eaten as bush food ¹⁵. The leaves of F. vogelii are used locally as vegetables. However, they are extremely important food resources for wildlife and are of considerable cultural importance throughout the tropics as an object of worship. The Igbos refers to F. vogelii as ogbu.

Since this herb serves as a delicacy in Ikwo and there is a large lead mining site in this same place, we decided in this research to check the efficacy of the leave in the detoxification of the reproductive organs (ovary and uterus) toxicities as a natural antioxidant.

­MATERIALS AND METHODS:

Collection of Plant Material: F. vogelii leaves were taken from Ndufu-Alike in Ikwo Local Government Area of Ebonyi State and were authenticated in Botany Department of the University of Nigeria Nsukka (UNN).

Extract Preparation: The leaves were dried in a ventilated room for two weeks and grinded to get a fine powder. The powder was soaked in a container with ethanolic were kept in a refrigerator for 48 h. The mixtures were filtered, first, with cheesecloth, and then re-filtered with Whatman no. 1 filter paper (24 cm). The filtrates were separated and concentrated in vacuum using Rotary Evaporator to 10% of their original volumes at 37 ºC - 40 ºC. These were concentrated using a water bath until a powder was gotten. The extracts were stored in a refrigerator at a very low temperature until it is required for use.

Ethical Approval: The ethical clearance for this work was obtained from the Ethics Committee of the Faculty of Basic Medical Sciences, University of Nigeria Nsukka, Enugu Campus (UNEC). There was a strict adherence to International Guidelines for the use of animals in research studies based on European Commission Directive on Animals use ⁴.

Animal Housing: Twenty five (25) adult female rats were procured from the animal house of the Department of Pharmacology, University of Nigeria Enugu Campus (UNEC) and maintained in the Animal House of Anatomy Department of Faculty of Basic Medical Sciences of the same University. The animals were housed in netted cages, fed with grower's mash and allowed water ad libitum with acclimatization period of one week (7 days) was observed.

Experimental Design: The animals were grouped as follows and received standard rat's diet containing 0.5% NaCl, 16.8% protein and 46.5% dietary fat, 35.7%, vitamins, other micronutrients and tap water ad libitum ¹⁶, throughout the study.

Group A: Control Group (Negative): This group received feed as stated above with water ad libitum. The rats also received normal saline to expose them to the sane stress as the other groups.

Group B and C: (Protective Effects of Ethanolic Extract): The rats in these groups received an ethanolic extract of F. vogelii for 7 days and 24 hours later exposed to lead acetate (1.5 mg/kg) for the remaining 7 days.

Group D: Lead Acetate Solution (Positive): The rats in this group received 1.5 mg/kg of Lead acetate solution daily for 14 days.

Group E: (Effect of Ethanolic Extract FV): The rats in this group received 300 mg/kg ethanolic extract of F. vogelii only for 14 days.

Drug Administration: The animals were randomly assigned into seven groups (A, B, C, D, and E) after they have been weighed. Groups A and D contained 4 animals per group and received normal saline and lead acetate solution respectively. Group A and C received ethanolic extract for 14 days and lead acetate solution for 7 days. In both groups, B and C were dose-dependent.

Group E received ethanolic extract. The extracts and lead acetate solution were administered by oral intubation using oral gavage (cannula). Administration lasted for 2 weeks (14 days) and 24 h after the last administration the animals were sacrificed, and the uterus and ovaries were removed and preserved for histological studies.

Phytochemical Screening: The phytochemical screening was performed using 500 g of the extract. This portion was subjected to quantitive phytochemical screening using the method described by Prashant, et al., ¹⁷ and Ene-Obong et al. ¹⁸

Ovarian SOD Activities: During the sacrifice, one of the ovaries was removed and homogenized with mortar and pestles ¹⁹ and then preserved with phosphate buffer. The preserved homogenate was centrifuged and kept under refrigeration before it was sent to a laboratory for the antioxidant check. Superoxide dismutase activity was measured according to the method of Winterbourn et al., ²⁰ as described by Rukmini et al. ²¹ The principle of the assay was based on the ability of SOD to inhibit the reduction of nitro-blue tetrazolium (NBT) ^{22, 23}. Briefly, the reaction mixture contained 2.7 ml of 0.067M phosphate buffer, pH 7.8, 0.05 ml of 0.12mM riboflavin, 0.1 ml of 1.5mM NBT, 0.05 ml of 0.01M methionine and 0.1 ml of enzyme samples.

We made sure that the tubes were uniformly illuminated by placing it in air aluminium foil in a box with a 15W fluorescent lamp for 10 min. Control without the enzyme source was included. The absorbance was measured at 560 nm ^{24, 25, 26}. One unit of SOD was defined as the amount of enzyme required to inhibit the reduction of NBT by 50% under the specific conditions.

Histological Study: At the end of 28 days, the animals were starved overnight and anesthetized before decapitating and sacrificing them ²⁷. After sacrificing and dissecting the animals, the uterus and ovaries were removed, weighed and immediately fixed in bouin’s fluid for routine histological procedures. These tissues were processed and embedded in paraffin wax. The thin sections (5-6 μm) were obtained and stained using hematoxylin and eosin (H and E) and were examined under a light microscope to determine the histological changes caused by the toxicity.

Statistical Analysis: Data from the study were analyzed using descriptive statistics and presented as Mean ± Standard Error of Mean (SEM). The statistical significance level was established at a value of P ≤ 0.05 (*) or P ≤ 0.01 (**) by comparing the data with those for negative control and positive control groups. Inferential statistics of paired student’s t-Test was adopted with the aid of the Statistical Software Package (SPSS) version 20.0.

RESULTS:

TABLE 1: TABLE SHOWING THE WEIGHT (g) OF ANIMALS THAT RECEIVED ETHANOLIC EXTRACT OF F. VOGELII AND LEAD ACETATE SOLUTION DURING THE PERIOD OF THE RESEARCH

* Significant increase in weight at P≤0.05. ** Significant increase in weight at P≤0.01. + Significant reduction in weight at P≤0.01

TABLE 2: MEAN WEIGHT (mg) OF UTERUS OF CONTROL AND EXPERIMENTAL GROUPS OF ADULT FEMALE WISTAR RATS

** Significant reduction in uterine weight when compared to A (P≤0.01 and P≤0.05). * Significant uterine weight gain when compared to D (P≤0.01). *** Significant uterine weight gain when compared to D (P≤0.01). **** Significant uterine weight gain when compared to A (P≤0.01)

FIG. 1: THE CHART SHOWING THE MEAN WEIGHT OF THE ANIMALS DURING THE EXPERIMENT

 TABLE 3: MEAN WEIGHT (mg) OF OVARIES OF CONTROL AND EXPERIMENTAL GROUPS OF ADULT FEMALE WISTAR RATS

*** Significant increase in ovarian weight when compared to group A (P≤0.01). **Significant reduction in ovarian weight when compared to group A (P≤0.01). A significant increase in ovarian weight when compared to group D (P≤0.01)

Phytochemical Screening: Using standard method as recorded in Sahiru and Catherine ²⁸, the following chemicals below screened for in F. vogelii.

TABLE 4: PHYTOCHEMICAL COMPOSITIONS OF ETHANOLIC EXTRACTS OF DRIED LEAVE OF F. VOGELII

* = Indicates the presence of phytochemicals. - = Indicates the absence of phytochemicals. ** = Shows moderate concentration level of phytochemicals. *** = Shows a high concentration level of phytochemicals

Ovarian SOD Activities: The results of the SOD are presented in Table 5 and Fig. 4 below.

TABLE 5: RESULTS OF THE ACTIVITIES OF OVARIAN ENZYME SUPEROXIDE DISMUTASE (SOD) OF THE EXPERIMENTAL

*** Significant increase in SOD activity when compared to group A (P≤0.01 and P≤0.05). ** Significant reduction in SOD activity when compared to group D (P≤0.01)

FIG. 4: CHART SHOWING THE MEAN SOD LEVELS OF THE OVARIES OF THE ANIMALS

 It indicates that the positive control (group D) has a significant increase in SOD activity as compared to the negative control (group A).  

The ovaries of B and C showed a significantly decreased ovarian SOD activity when compared to that of the positive control (group D). There was a significant decreased in SOD level were the protective groups of both aqueous and ethanolic extracts.

Histological Studies: The tissues were processed and stained with hematoxylin and eosin (H and E) stain and view with a microscope under the magnification of 200X. The alterations an observed microscopically is presented as photo micro-graphical plate below.

FIG. 5: NEGATIVE CONTROL UTERUS REVEALS THE PRESENCE OF N- NORMAL MUCOSA STRUCTURE, M-MYOMETRIUM, AND UG- UTERINE GLANDS) AND OVARY SHOWING PRF- PRIMARY FOLLICLE AND SF- SECONDARY FOLLICLE. H AND E 200X

FIG. 6: GROUP B UTERUS SHOWING DO-DIFFUSE OEDEMA, UG-UTERINE GLAND, AND OES-OPTICAL EMPTY SPACE AND OVARY SHOWING NZ-NECROTIC ZONE AND IC- INTERSTITIAL CELLS H AND E STAINS, 200X

FIG. 7: GROUP C UTERUS SHOWING OES-OPTICAL EMPTY SPACES AND UG-UTERINE GLAND AND OVARY SHOWING AC-ANTRAL CAVITIES OF SECONDARY FOLLICLES, OES-OPTICAL EMPTY SPACES AND NZ-NECROTIC ZONE, H & E STAINS, 200X

FIG. 8: GROUP D NZ-NECROTIC ZONE, UG-UTERINE GLANDS, UGN-UTERINE GLANDS NECROSIS, AND ENDOMETRIAL DENUDATION AND OVARY SHOWING OES-OPTICAL EMPTY SPACES, PF-PRIMORDIAL OVARIAN FOLLICLES AND DO-DIFFUSE OEDEMA, H AND E 200X

FIG. 9: GROUP E UTERUS UG-UTERINE GLANDS, M-MYOMETRIUM AND N-NORMAL MUCOSA, AND OVARY PRF-PRIMARY FOLLICLE, PF-PRIMORDIAL FOLLICLE, AND IC- INTERSTITIAL CELLS, H AND E STAINS, 200X

DISCUSSION: The female reproductive system is adversely affected by exposure to environmental toxicants, and only a few researchers’ attention has been drawn to toxic environmental factors that cause uterine and ovarian toxicity ^{29, 30}. The uterus is a thick-walled, hollow, pear-shaped muscular organ responsible for the reception, implantation, and nutrition of the fertilized ovum ^31-33. The ovaries produce and periodically release ova, secrete estrogen, testosterone and progesterone ^34-36. Much attention has been given for the approval of herbal medicine in the new millennium, and F. vogelii is a vegetable that has been used locally to treat some illnesses and also implicated by some researchers as an herbal remedy for some human diseases ³⁷.

According to OECD ³⁸, and Ali et al., ³⁹ weight determination is considered as a constituent in the study of the safety of any therapeutic agent. The body weight of the negative control group increased significantly throughout the research (P≤0.01 and P≤0.05) as recorded in Table 1, while the lead acetate group recorded an insignificant body weight increase first before recording a significant weight loss (P≤0.01). This reduction in weight might be due to the deleterious effect of lead on the organs at this stage. This increase in weight is one of the major pointers that the leave of F. vogelii has a nutritive value as a vegetable ³⁹. There was a significant decrease in the uterine and ovarian weights of positive control at P≤0.01 and P≤0.05. This agrees with the research conducted by Durgesh and Lata ⁴⁰ which stated that “the animals that received only lead acetate experienced a significant reduction in uterine weight.” The experimental groups showed ovarian and uterine weights that were not affected much which might be due to the protective effects of the extract as shown in Tables 2 and 3 above. The above findings are in agreement with the findings of Durgesh and Lata in 2014. The higher the dose, the higher the weight gained by the organs signifying their role in restoring the normal physiology of the organs of the body which may be dose-dependent ⁴¹. The groups that received only the extract recorded a significant uterine and ovarian weight gain at P≤0.01. This weight gain could be an insight into the therapeutic benefits of the leaves.

Natural antioxidants are very effective in removing free radicals released in the body during reactive oxygen species (ROS) as a result of various disease conditions, and it has been an active area of research ⁴². Various researches have revealed over a long period that these antioxidants are capable of either preventing or curing the damages caused by the presence of these free radicals in the body ⁴² and antioxidants such as SOD, flavonoids, etc. are compositions of many vegetables like F. vogelii ⁴³. Toxicity causes an increment in the concentration of free radicals leading to an imbalance between the free radicals and antioxidants which cause oxidative stress ⁴⁴.

F. vogelii as a vegetable contains an antioxidant which can be taken through diet to maintain the homeostasis between free radicals and antioxidants and thus prevent various deleterious effects, like lead toxicity, inflammation, cancer, aging, cardiovascular and brain disorders ^{45, 46}. According to Flora et al., ⁴² and Ene-Obong et al., ¹⁸ “those consume antioxidant-rich diet are better positioned to reaping various health benefits and boost their antioxidant levels; therefore, food is always favored over supplements mainly because it contains thousands of antioxidants in contrast to supplements which are generally rich in single or a few antioxidants”.

As shown in Table 5, there was a significant increase in ovarian SOD in the positive control group while the experimental groups showed a significant decrease when compared to the positive control. These reductions in SOD could be due to the role of the extract as an antioxidant. The extracted group showed SOD level closer to the negative control which is suspected to be a result of the extract in removing free radicals produced due to inflammation caused by the presence of toxins that cause it to rise above normal ^{47, 48}.

On screening, the following chemicals were seen; saponins, flavonoids, alkaloids, triterpenoids, polyphenols, coumarins, steroids, carotenoids and cardiac glycosides. Various phytosteroids on plants have been shown to promote fertility ⁴⁹.  Flavonoids present in the leave of plants have been shown to possess many pharmacological properties such as anti-oxidant activities, anti-inflammatory activities, anti-cancer activities and anti-microbial effects ^{51, 52}. Flavonoids might have contributed to the extracts healing (anti-inflammatory) properties as observed from the histological studies ⁵². The extract is very rich in triterpenoids, a compound which plays a very active role in wound healing ⁵³. According to Krishnaiah et al., ⁵⁴ triterpenoids are shown to strengthen the skin, increase the concentration of antioxidants in wounds and restore inflamed tissues by increasing blood supply.

In this experiment, we also considered the role of the extract in restoring the normal histological of the ovaries and uterus after some alterations. The examination of the ovary microscopically showed damages and reduction in the formation of follicles with a marked increase in the number of atretic follicles which is in agreement with the work done by Patrick ⁵⁵, Dumitrescu et al., ⁵⁶ and Igile et al., ⁵⁷. According to Taupeau et al., 2001, low lead in the ovary caused dysfunction of folliculogenesis, with fewer primordial follicles and increased atretic antral follicles ⁴³. The organs also presented diffuse edema, necrosis, empty optical spaces, denudations and atretic follicles ⁵⁷.

The organs looked well vascularized in the groups that received the extracts indicating restoration of the blood supply to the organs that were lost due to the lead-induced toxicity. According to Shah et al., ⁵⁸ oral administration of lead causes reduced ovarian follicles and increased atretic follicles as observed in this study.

The organs (uterus and ovaries) belonging to the animals in the negative control group presented a normal histoarchitecture ⁵⁶. The organs of the animals that received only lead acetate (positive control) presented a fatty change (a reversible change that occurs in organs due to toxicity) appearance, focal loss of tissue (empty optical spaces) and endometrial denudation with polypoid appearance. This fatty change may have occurred due to toxicity caused by loss of blood supply leading to tissue necrosis. Lead also inhibits the body's ability to making hemoglobin by interfering with several enzymatic steps in the haem pathway ⁵⁹. The uterine glands were seriously affected by the lead acetate toxicity. The structural changes observed as a direct effect of lead exposure on the ovaries and uteri in this research were also reported by Dumitrescu et al. ⁵⁶

The uterus of low dose (100 mg/kg) group showed the efficacy of the extract in protecting the uterus from histological alterations caused by the induced lead acetate toxicity although there were still some areas of edema Fig. 7. The high dose (300 mg/kg) group showed tiny uterine glands with little damages caused by the lead toxicity. This agrees with the report of Dumitrescu et al. ⁵⁶ The uterine glands edema was observed to be far smaller than edema seen in the low dose group. The above may be a pointer that the extract is dose-dependent, that is, the higher the dose, the more efficacious it is. The ovaries of this group showed a multiple ovarian follicular developments. It also showed ovarian follicles protected from the induced lead toxicity by the extract. The extracts group only, the uterus and ovaries looked well perfused with blood and also healthier than the positive control group. In this group, an improvement was observed in the organ perfusion signifying the nutritional values of the extracts.

CONCLUSION: In summary, our results demonstrated that F. vogelii leave extract is a very good natural antioxidant that could be used as alternative herbal medicine. Our results also showed that the herb is good in fighting reactive oxygen species (ROG) production and also can resist lead reproductive toxicity.

ACKNOWLEDGEMENT: We want to appreciate Mr. Nwajagu G. I., Chief Technologist Gross unit of Anatomy Department Dr. G. G. Akunna and Dr. Ogugua Egwu an Associate Professor of Anatomy and Dean Faculty of Basic Medical Sciences, Alex Ekwueme Federal University Ndufu-Alike (AE-FUNAI), Ikwo for their advice and assistance all through the study period. We equally appreciate all the lecturers and Staff of Anatomy Department, Faculty of Basic Medical Sciences, University of Nigeria Nsukka Enugu Campus (UNEC).

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Ezugworie JO and Uchewa OO: Efficient use of traditional medicine to detoxify upper reproductive organs toxicity that may lead to infertility. Int J Pharm Sci & Res 2019; 10(1): 347-55. doi: 10.13040/IJPSR.0975-8232.10(1).347-55.

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