INTRODUCTION:

Lead is a hazardous element that is commonly found in the environment and can induce histological, physiological, biochemical, and behavioral problems in people and animals¹. As a result of unavoidable environmental and occupational exposure, lead poisoning is a highly widespread concern in all countries ². The production of oxidative stress by lead (Pb) poisoning is the mechanism of toxicity³. Lead-induced oxidative stress is caused by an imbalance in the creation and elimination of reactive oxygen species (ROS) in cellular components, resulting in DNA, protein, and membrane damage⁴. The capacity of lead to produce reactive oxygen species (ROS) like superoxide radicals, hydrogen peroxide, and hydroxyl radicals, as well as lipid peroxides, results in oxidative stress⁵. Lead increases lipid peroxidation and nitric oxide production in testes which result in reduction of antioxidant enzymes such as catalase and superoxide dismutase⁶.

The therapeutic qualities of plants have been investigated⁷. The majority of fruits and vegetables are vital sources of nutrition and a well-balanced diet⁸. Cyperuse sculentus is a 90-centimeter-tall annual or perennial crop plant⁹. It gets its solitary stems from a tuber. Cyperus esculentus phytochemical screening revealed a significant concentration of alkaloids, saponins, and tannins^10,11. According to¹², Cyperus esculentus is healthy and aids in the prevention of numerous ailments such as cardiac thrombosis, blood circulation, and the prevention and treatment of urinary tract infections and other bacterial infections. It has also been stated that Cyperus esculentus oil is used in the cosmetic sector¹³. It helps slow down the ageing of body cells since it is antioxidant (due to its high vitamin E concentration). Chufa sedge, Nut grass, Yellow nut sedge, Tigernut sedge, or Earth almond are some of the local names for Cyperus esculentus. The Hausa, Igbo, and Yoruba tribes of Nigeria refer to it as Aya, Ofio, and Aki hausa, respectively¹⁰.

Tannins are polyphenolic chemicals with a high molecular weight that are water soluble.

They have the ability to attach to proteins and precipitate them¹⁴.

Tannins can be found in a variety of plant parts, including the leaf, root, wood, bark, and fruit, and are classified as either condensed tannins (CT) or hydrolysable tannins (HT) (HT).

Tannins are phenolic chemicals that, according to several studies, impair the digestibility and nutritional quality of herbivore meals¹⁵.

The presence of tannins, which may be the result of a complex development between tannins and proteins, protects the plant from predators and diseases¹⁶.

Many studies have been done on the antioxidant¹⁷, anti-inflammatory¹⁸, anticancer¹⁹, and antibacterial^20,21 properties of tannins from diverse sources.

The goal of this work was to evaluate the protective role of tannins from Cyperus esculentus on lead-induced testicular damage in male Wistar rats.

MATERIALS AND METHODS:

Procurement of Cyperus esculentus and Extraction and Isolation of tannins from Cyperus esculentus:

In January 2021, Cyperus esculentus was purchased at Ogbete Market in Enugu, Enugu State, Nigeria. The tuber was recognized and authenticated at the Department of Plant Science, University of Nigeria Nsukka, Nsukka, Nigeria, using authentication and identity number UNN131. Fresh Cyperus esculentus was air-dried at room temperature before being processed into a fine powder with a grinding mill. In a mechanical shaker, around 80 g of dried and powdered Cyperuse sculentus was defatted with petroleum ether for 48 hours at room temperature. It was then extracted with aqueous acetone (70 percent acetone) in a water bath for 60 minutes at 60°C with continual stirring. After that, the mixture was filtered and centrifuged for 10 minutes at 3000rpm. The supernatant was allowed to evaporate at room temperature. The extract was lyophilized to remove the acetone (Lark, Penguin Classic Plus, Chennai). The powder was collected, weighed, and the % yield calculated before being stored in sterile bottles in the refrigerator at 4°C.

Evaluation of antioxidant activity:

The Phosphomolybdenum technique, as described by ²² was used to measure the total antioxidant activity of the tannins with minor changes. 1ml of the reagent solution was combined with 100g of extract (0.6M sulphuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The tubes were capped and incubated for 60 minutes at 100 degrees Celsius.After cooling the samples to room temperature, the absorbance was measured at 695nm against a blank in a UV-Vis spectrophotometer (Hitachi U-500).

As a standard, 5-30g scorbic acid was utilized. On a dry weight basis, the antioxidant activity was measured in mg ascorbic acid equivalents per gram of material.

Experimental animals:

Twenty-five (25) male Wistar rats with mean weight of 120g were used for the experiment. The rats were caged and fed with grower’s mash. They were kept for a period of two weeks for acclimatization prior to the experiment.

Dose Determination: Doses were determined based on the results of acute toxicity test according to method described by Ghosh M. N.²³

Ethical Approval:

Ethical approval was obtained from the research and ethics committee of the Faculty of Basic Medical Science, University of Nigeria, Enugu Campus.

Experimental Designs and Experimental Animals:

Twenty-five male Wistar rats (n=5) were randomly divided into five groups. Normal saline was given to group NS (negative control). Only 30mg/kg BW of lead was given to the PBO group. The other three groups (LDTPB, MDTPB, and HDTPB) received 30mg/kg BW of lead with 50mg/kg, 100mg/kg, and 150mg/kg BW of Tannin, respectively. For the next twenty-eight days, the administration was completed.

Determination of hormone level:

Before the animals were slaughtered, blood samples were taken by capillary tube via retro orbital puncture and maintained in a non-heparinised vacutainer that was spun at 2500rpm for 10 minutes using a bio-centrifuge after 24 hours (MSE, O-5122A, Germany).

The ECOBAS-6000 hormone analysis machine was used to measure the levels of free serum testosterone, LH, and FSH in serum.

HISTOLOGICAL OF THE TESTIS:

All of the animals were sacrificed using the anesthetic thiopental at a dose of 50mg/kg. The reproductive organs (testes) were exposed when the abdominal cavities were opened up with a midline incision.

The testes of the rats in each group were then removed immediately and weights of the testes were recorded with electronic beam balance.

These samples were obtained for histological investigation and stored in Bouin's fliud for further analysis ²⁴.

Epididymis sperm count and motility:

By cutting into 2ml of medium (Hams F10) containing 0.5 percent bovine serum albumin, spermatozoa were extracted from the cauda epididymis²⁵. After 5 minutes of incubation at 37°C, the sperm reserves of the cauda epididymis were measured using a hemocytometer (with 5 percent CO₂). Sperm motility was measured using a microscope (Leica DM750) in the department of anatomy, faculty of Basic Medical Sciences, UNEC, and reported as the mean number of motile sperm using the WHO method²⁶.

Biochemical estimations:

Lipid peroxidation products estimated by measuring TBARS and Nonenzymatic antioxidants such as glutathione (GSH)and catalase (CAT) and SOD activity in the testes were also determined as described by method of Wheel et al.²⁷.

Data presentation and statistical analysis:

Data expressed as Mean ± SEM. Statistical differences between the groups were evaluated by one-way ANOVA. Differences with p<0.05 considered statistically significant.

RESULTS:

Table 1: The Effects of tannins and lead on body and testicular weights

Groups	Initial BW (g)	Final BW (g)	% Change in BW	Relative Testicular weight
NS	144.20±0.86	245.53±0.40	41%	1.66±.05
PBO	112.83±0.57	241.13±0.57	53%	1.15±.05^a,b.c
LDTPB	92.23±0.57	201.53±0.57	54%	1.34±.05
MDTPB	103.83±0.57	173.23±0.57	40%	1.29±.05^a
HDTPB	87.83±0.57	218.23±0.57	60%	1.30±.05

The group the received the highest dose of tannin showed the highest percentage increase in body weight. With regards to testicular weight, there was a significant (p˂0.05) decrease in the relative testicular weight of rats administered with lead only when compared to the group administered with normal saline (negative control). However, the groups that were treated with tannins had a significant increase in testicular weight when compared to the rats administered with only lead.

As shown in table 2, Lead treatment significantly decreased sperm count, motility, and viability in the lead group (PBO) compared with negative control (NS) and other experimental groups. All the treatment groups had a significant (p˂0.05) increase in sperm count when compared to the positive control group (PBO). The sperm motility also increased in treatment groups when compared with positive control, PBO.

RESULTOF HORMONAL ASSAY:

The groups LDTPB, MDTPB and HDTPB had significant increase in level of serum testosterone with p<0.05 when compared with positive control group. There was also increase in level of FSH in groups 3, 4 and 5 when compared with positive control group. There was significant decreased in LH in groups 3 and 4 compared with negative control (group 1).

Table 4: The Effects of tannins and lead on Oxidative stress Makers

Groups	SOD (U/mg pro)	CAT (U/mg pro)	MDA (nmol/mg pro	GSH (nmol/mg protein
NS	26.10±.00	13.30±.00	0.52±.00	2.30±.00
PBO	12.60±.00^a	8.30±.00^a	3.40±.00^a	1.10±.00^a
LDTPB	18.20±.00^a,b,d	14.20±.00^b	1.20±.00^b	2.90±.00
MDTPB	14.20±.00^a,c,d	10.20±.00	0.90±.00^b,c,d	1.40±.00
HDTPB	23.30±.00^b,d	9.30±.00^a	0.90±.00^b,c,d	2.40±.00

Oxidative Markers (SOD, GSH, and CAT and MDA) levels:

As shown in table 4, result showed a significant (p˂0.05) decrease in the activity level of SOD, CAT, GSH and a significant (p˂0.05) increase in the level of MDA in rats treated with Lead-alone (group 2) when compared to the negative control group (group 1). All the groups that had tannins + lead treatment had a significant (p˂0.05) improvement in the activity level of SOD, CAT, MDA but not GSH.

Figure 1: histological sections of testis of experimental rats: NS: seminiferous tubules were circular in outline with normal seminiferous epithelium and numerous spermatozoa radiating towards and within the lumen (star). PBO: showing loss of spermtogenic cells, reduced density of mature spermtozoa within the lumen and absence of sperm bundles in most tubules. LDTPB: Showing marked increase in the germinal epithelium and interstitial tissue as compared to PBO group. MDTPB: Showing a more pronounced increase in the number and volume of germinal epithelium, the interstitial cells of leydig and the sizes of the seminiferous tubules. HDTPB: Seminiferous tubules were circular in outline with normal seminiferous epithelium and numerous spermatozoa radiating towards and within the lumen Stain: haematoxylin and eosin; Magnification: *200

Figure 2: Micrograph of a cross-section of testis of rat treated with normal saline (0.5ml/kg, gastric gavage) (Group 1). VVG revealed normal basement membrane with reticular fibres with normal spermatogenic epithelium. Stain: VVG; Magnification: *200

DISCUSSION:

Plant parts have been employed and used in the treatment and prevention of a variety of ailments over time ²⁸. Ancient people were aware of the use of herbal medicine to cure a variety of diseases, including infertility, increased sexual performance, sexual desire, and pleasure ²⁹. Herbal remedies are non-prescription treatments made from plants and plant parts that are used to cure a variety of illnesses and ailments. Herbal medicines have been used for a long time and are considered forerunners of modern medicine³⁰. This herbal medicine is very available, affordable and less harmful ³⁰. Herbal treatments can be made from a variety of plant parts, including leaves, bark, flowers, and roots ³¹.

Tannins are said to have antioxidant properties ⁽¹⁷⁾. According to the findings of this investigation, lead ingestion lowered the relative weights of the testes and the body weight of the rats. Lead exposure dramatically reduced sperm count, motility, and viability in this investigation. This was caused by oxidative stress, which was caused by the presence of significant amounts of polyunsaturated fatty acids (PUFAs) in plasma membranes^32,33. Reduced sperm motility caused by be lead is linked to increased production of reactive oxygen species (ROS)^34,35. The association between reactive oxygen species and sperm immobility could be due to events that cause a rapid depletion of intracellular ATP, resulting in axonemal damage and sperm immobilization^36,32. Lead exposure was also linked to lower testosterone levels³⁷. The administration of tannins fraction of Cyperus esculentus boosted testosterone levels, indicating a beneficial effect of Cyperus esculentus tannins fraction on the hypothalamic-pituitary-testicular axis.

Tannins protected the testes from the effects of lead exposure. The competitive mode of interaction is a potential method by which tannins protect against lead toxicity. The production of oxidative stress is the cause of this impact. The activities of GSH, CAT, and SOD were shown to be significantly reduced in the testes of lead-exposed rats. The results showed tannins boosted testicular antioxidant enzymes and lowered MDA levels in this investigation. Lead's devastating effects on sperm parameters and testicular antioxidant enzymes were similarly inhibited by the tannins fraction of Cyperus esculentus.

Cyperus esculentus contains substantial levels of antioxidants, as we previously reported ¹⁰. As a result of this research, it was discovered that tannins are one of the phytochemicals in Cyperus esculentus that is responsible for the plant's antioxidant qualities Furthermore, the testicular tissue of rats administered lead only revealed markedly depleted and atrophied seminiferous tubules, interstitial oedema, deteriorated and vacuolated germinal epithelium, absence of late stage germ cells, and degeneration spermatogenic cells in this study. This is consistent with a recent study ^{38, 39}, which found that lead caused germ cell sloughing in the seminiferous tubules and visible elevations in histological lesions in the seminiferous tubules and epithelial lining of the testes in rats. In addition, Lead injection resulted in the absence of Sertoli cells in the seminiferous lumen, which was not detected in the Lead-only group. For decades, it has been known that testosterone produced in Leydig's interstitial cells is a powerful hormone⁴⁰. There was a decrease in interstitial cells in the testes of rats treated with lead alone, which could have resulted in a drop in testosterone and, as a result, impaired spermatogenesis. When compared to a positive control, the Tannins fraction of Cyperus esculentus preserved the histological architecture of the testes, boosted the proliferative activity of spermatogonia, and maintained cells of the spermatogenic series. Tannins protected the testes from the detrimental effects of lead, according to the findings of this study. According to our findings, antioxidants found in tannin fractions protected testes from the harmful effects of lead.

CONCLUSION:

Finally, it was discovered that the tannins fraction of Cyperus esculentus effectively reduced Lead-induced oxidative stress by lowering MDA levels, alleviated the negative effects of Lead on serum testosterone and FSH, depleted the germinal epithelium, and caused hypocellularity and interstitium widening, and depleted the germinal epithelium. Tannins from Cyperus esculentus promoted germinal epithelial development and protected the testes' cytoarchitecture from the harmful effects of lead. Through an antioxidant system of activities, the tannins component of Cyperus esculentus protected testes from lead-induced testicular damage in male Wistar rats.

COMPETING INTERESTS DISCLAIMER:

There is absolutely no conflict of interest

REFERENCES:

1. El-Tantawy, W.H. Antioxidant effects of Spirulina supplement against lead acetate-induced hepatic injury in rats. J. Tradit. Complement. Med. 2016; 6: 327–331.

2. Abdel-Zaher, A.O.; Abd-Ellatief, R.B.; Aboulhagag, N.A.; Farghaly, H.S.M.; Al-Wasei, F.M.M. The interrelationship between gasotransmitters and lead-induced renal toxicity in rats. Toxicol. Lett. 2019; 310: 39–50.

3. Flora, G.; Gupta, D.; Tiwari, A. Toxicity of lead: A review with recent updates. Interdiscip. Toxicol. 2012; 5: 47–58.

4. Patra RC, Swarup D, Dwivedi SK. Antioxidant effects of αtocopherol, ascorbic acid and L-methionine on lead-induced oxidative stress to the liver, kidney and brain in rats. Toxicology. 2001; 162(2): 81-88.

5. E-Nekeety AA, El-Kady AA, Soliman MS, Hassan NS, Abdel-Wahhab MA. Protective effect of Aquilegia vulgaris (L.) against lead acetate-induced oxidative stress in rats. Food Chem Toxicol. 2009; 47: 2209–2215.

6. AbdelMoniem AE, Dkhil MA, Al-Quraishy S. Protective role of flaxseed oil against lead acetate induced oxidative stress in testes of adult rats. Afr J Biotechnol. 2010; 9(42): 7216–7223.

7. Tamunotonye W, Jacks SA, Asala JP. Testicular enhancement activity of Aqueous extract of Pausinystalis macroceras stem bark in Wistar rats. J Anat Sci. 2007; 1(1): 3-6.

8. Ibrahim TA, Jude BS, Giwa EO, and Adebote VT. Rea search Journal of Agric and Biological Sciences, 2009; 5(6): 1143-1145.

9. Vilmorin A. The Vegetable Garden. Ten Speed Press ISBN 0-89815-041-8.

10. Hassan LA, Anyanwu GE, Nto JN, Abireh IE, Akunna GG. Protective Effect of Aqueous Extract of Cyperus esculentus on Flutamide-Induced Testicular Defect of Male Wistar Rats. Scholar Journal of Applied Medical Sciences. 2018; 6(6): 2391-2395.

11. Ekeanyanwu RC, Njoku O and Ononogbu IC. The Phytochemical Composition and Some Biochemical Effect of Cyperus esculentus. Pakistan Journal of Nutrition. 9(7): 709-715.2010.

12. Adejuyitan JA, Otunola ET, Akande EA, Bolarinwa IF and Oladokun FM. Some Physicochemical properties of Flour obtained from fermentation of tiger nut (Cyperusesculentus)sourced from a market in Ogbomoso, Nigeria. Afric. J. of Food Science. 2009; 3: 51-55.

13. Abano EE, Amoah KK. Effect of moisture content on the physical properties of tiger nut (Cyperusesculentus). Asian Journal of Agricultural Research. 2011; 5(1): 56-66.

14. Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 2010; 15: 7313-52.

15. Rothman JM, Dusinberre K, Pell AN. Condensed tannins in the diets of primates: a matter of methods. Am J Primatol. 2009; 71: 70-76.

16. Scalbert A. Tannins in woods and their contribution to microbial decay prevention. In: Hemingway RW. Laks PE. editors. Plant Polyphenols. New York: Plenum Press; 1992.

17. Ujwala W, Vijender S, Mohammad A. In vitro antioxidant activity of isolated tannins of alcoholic extract of dried leaves of Phyllanthus Amarusschonn and Thonn. Int J Drug Dev Res. 2012; 4: 274-85.

18. Mota ML, Thomas G, Barbosa FM, Antiinflammatory actions of tannins isolated from the bark of Anacardium occidentale L. J. Ethnopharmaco. 1985; 13: 289-300.

19. Chung KT, Wong TY, Wei CI, Huang YW, Lin Y. Tannins and human health: a review. Crit Rev Food Sci. 1998; 38: 421-64.

20. Doss A, Mubarack HM, Dhanabalan R. Antibacterial activity of tannins from the leaves of Solanumtrilobatum Linn. Indian J Sci Technol. 2009; 2: 41-3.

21. Oyaizu M. Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine. Jap J Nutr.1986; 44: 307-15.

22. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal Biochem 1999; 269: 337–41.

23. Ghosh M. N., Toxicity studies. In Fundamentals of Experimental Pharmacology, Scientific Book Agency, Culcutta. 1984; 153-158.

24. Avwioro OG. Histology and Tissue Pathology. Principles and Techniques. 2nd ed. Ibadan: Claverianum Press; 2010.

25. Feng R, He W, Ochi H. A new murine oxidative stress model associated with senescence. Mech Ageing Dev. 2001; 122: 547-59. PMID: 11295171 DOI: 10.1016/S0047-6374(01)00232-9.

26. World Health Organization (WHO) “WHO laboratory manual for the examination of human semen and sperm cervical mucus interaction” 5th edition. WHO Cambridge University press. 2010; 5: 332-345.

27. Wheel C. R., Salzman J. A., Elsayed N. M., Omaye S. T., Korte D. W. Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Anal Biochem .1990. 184: 193-199.

28. Ayyanar M, Ignacimuthu S. Herbal medicines for wound healing among tribal people in Southern India: Ethnobotanical and Scientific evidences. International Journal of Applied Research in Natural Products. 2009; Sep 15; 2(3): 29-42.

29. Malviya NE, Jain SA, Gupta VB, Vyas S. Recent studies on aphrodisiac herbs for the management of male sexual dysfunction--a review. Acta Pol Pharm. 2011; Jan 1; 68(1): 3-8.

30. Burke A, Smyth A, Fitz Gerald GA. Analgesic antipyretic agents. In: Goodman LS, Gilman A, Brunton LL, eds. The pharmacological basis of therapeutics. 11th ed. New York: McGraw-Hill; 2006. p. 637-731

31. Khaki A, Fathiazad F, Nouri M, Khaki AA, Ghanbari Z, Ghan-bari M, Ouladsahebmadarek E, Javadi L, Farzadi L. Anti-ox-idative effects of citro flavonoids on spermatogenesis in rat. Afr J Pharm Pharmacol. 2011; 5: 721-5.

32. Bansal AK, Bilaspuri GS. Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int. 2010; 2010. pii: 686137.

33. Alvarez C., Castilla J. A., Martinez L., Ramire Z. J. P. (2007) Biological variation of seminal parameters in healthy subjects. Human Reproduction 18: 2082 - 2088.

34. Aitken, R. J. and Krause, C. Oxidative stress, DNA damage and the Y-chromosome. Reproduction. 2001; 122: 497-506.

35. Armstrong JS, Rajasekaran M, Chamulitrat W, Gatti P, Hellstrom WJ, Sikka SC. Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Rad Biol Med. 1999; 26: 869-80.

36. Anyanwu GE &Agbor CA. Assessment of Testicular Histomorphometric Parameters and Reticular Fibres Density on Testicular Tissue of Diabetic Wistar Rat Placed on Auricularia Polytricha. Jordan Journal of Biological Sciences. 2020; 13 (Supplementary), 709 – 714.

37. Anyanwu GE, Hassan LA, Abireh IE, Nto JN. The Effects of Alkaloids Fractions Cyperus esculentus on Some Biochemical Parameters and Histopathology of the Testis of Lead-Induced Toxicity. Journal of Pharmaceutical Research International. 2021: 33; 55-62.

38. Olufunke O, Dosumu, Oluwole B. Akinola, Edidiong N. Akang. Alcoho-induced testicular oxidative stress and cholesterol homeostasis in rats- The therapeutical potential of virgin coconut oil. Middle East Fertility Society Journal. 2012: 17; 122-128.

39. Chowdhury A. R. recent advances in heavy metals induced effect on male reproductive function. Aretrospective, Al Ameen J. Med. Sci.2009; 2:) 37-42.

40. Zirkin BR. Spermatogenesis: its regulation by testoster-one and FSH. Semin Cell Dev Biol. 1998; 9: 417-21.

Received on 18.03.2024 Modified on 30.05.2024

Res. J. Pharmacology and Pharmacodynamics.2024;16(3):137-142.

DOI: 10.52711/2321-5836.2024.00024

Groups	Sperm count (x10^-6)	Sluggish motility (%)	Non-Motile Sperm (%)	Active Motility %
NS	190.33±.57	19.66±.57	15.0±.00	65.33±.58
PBO	80.33±.57^a	0.00±.00^a	100.00±.00^a	0.03±.05^a
LDTPB	130.36±6.31^a	3.33±5.77^d	15.03±.00^a,c	21.66±7.52^b,c
MDTPB	160.00±.00^a,b,c	0.00±.00^a,d	10.00±.00^a	60.00±.00^a,d
HDTPB	180.00±.00^a,b,c	10.00±.00^a,c,d	10.00±.00^a	70.00±.00^a,d

Hormone	NS	PBO	LDTPB	MDTPB	HDTPB
Testosterone (ng/ml)	5.04±.00	2.04±.00^a	4.04±.00^b	4.03±.00^b	5.02±.00^b
Follicle stimulating hormone(ng/ml)	0.80±.00	0.30±.00	0.60±.00	0.60±.00	0.60±.00
Luteinizing hormone(ng/ml)	8.20±.00	7.70±.00	6.20±.00	6.30±.00	7.20±.00