In vitro and in vivo effects of Tribulus terrestris on immunological parameters, lymphocyte proliferation, and DNA integrity in sheep

Accepted Manuscript Title: In vitro and In vivo Effects of Tribulus terrestris on Some Immunological Parameters, Lymphocyte Proliferation, and DNA Integrity in Sheep Authors: Heba M.A. Abdelrazek, Rania A. Elgawish, Eman A. Ahmed, Hoda I. Bahr PII: DOI: Reference:

S0921-4488(18)30675-8 https://doi.org/10.1016/j.smallrumres.2018.10.014 RUMIN 5779

To appear in:

Small Ruminant Research

Received date: Revised date: Accepted date:

28-7-2018 22-10-2018 23-10-2018

Please cite this article as: Abdelrazek HMA, Elgawish RA, Ahmed EA, Bahr HI, In vitro and In vivo Effects of Tribulus terrestris on Some Immunological Parameters, Lymphocyte Proliferation, and DNA Integrity in Sheep, Small Ruminant Research (2018), https://doi.org/10.1016/j.smallrumres.2018.10.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

In vitro and In vivo Effects of Tribulus terrestris on Some Immunological Parameters, Lymphocyte Proliferation, and DNA Integrity in Sheep

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Heba M. A. Abdelrazeka, Rania A. Elgawishb, Eman A. Ahmedc*, Hoda I. Bahrd

Department of Physiology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia,

Egypt. b

Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Suez Canal

Department of Pharmacology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia,

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University, Ismailia, Egypt.

Department of Biochemistry, Faculty of Veterinary Medicine, Suez Canal University, Ismailia,

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Egypt.

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Egypt.

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Running title: Immunological changes after exposure to Tribulus terrestris in sheep

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Corresponding author: Eman A. Ahmed E-mail address: [email protected],edu.eg

Mobile: +201010253404

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Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt.

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Highlights The study assessed the immunomodulatory effect of TTE either in vitro or in vivo.

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TTE declines lipid peroxidation and improves antioxidant capacity in rams.

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TTE modulates lymphocyte proliferative ability, DNA integrity, and pro-inflammatory

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cytokines production.

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Abstract

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There are limited studies in which effects of Tribulus terrestris extract (TTE) on the

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immune system of small ruminants have been investigated. This research was carried out on 20

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rams divided into control and treated groups (n=10 each). The treated rams received TTE in a dose of 1.5 g/ head/ day for consecutive 45 days. The body weights and blood samples of rams

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were measured and collected before and after the end of the experiment. Meanwhile, in vitro

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lymphocyte culturing was carried out using peripheral blood mononuclear cells (PBMCs). In

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vivo and in vitro evaluations of lymphocyte transformation, comet assay, IL-6 and TNF-α were performed, as well as in vivo serum MDA and GSH. The body weight of rams treated with TTE

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was significantly (P<0.05) higher than that in control. Lymphocyte transformation was significantly (P<0.05) induced in rams treated in vivo with TTE. Interestingly, the 0.5 µl/mL of

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TTE induced (P<0.05) the lymphocyte transformation in PBMC cultured in vitro compared to that in control and 1 µl/mL of TTE treated cells. A dose of 0.5 µl/mL of TTE significantly (P<0.05) increased the level of IL-6 in vitro compared to that in control and 1 µl/mL of TTE treated cells. TNF-α didn’t show any significant changes in vivo and in vitro. The in vivo

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treatment with TTE declined the percentage of lymphocyte DNA in the comet tail in treated rams compared to control. However, in vitro addition of TTE either in 0.5 or 1 µl/mL to the PBMC culture significantly (P<0.01) affected the DNA integrity. Serum MDA was significantly

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(P<0.05) reduced, while GSH were significantly (P<0.05) increased in TTE treated rams than control. In conclusion, TTE could modulate the immune functions and induce lymphocyte transformation in rams.

Keywords: comet assay, interleukin, lymphocyte transformation, rams, Tribulus terrestris.

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1. Introduction

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Immunomodulation can generate systemic effects that regulate several physiological

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processes through feedback loops. These feedback loops are possibly represented by the

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expression of receptors for hormones, neurotransmitters, and cytokines on leukocytes, which

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modulate key cellular functions like cellular proliferation, differentiation, and their secretion

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profile. Those physiological effects depend on the amount of cytokines and the other inflammatory mediators (Pavón et al., 2013). Several promising biological and phytogenic

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immunomodulating substances have been tested to improve immune response that reflected positively on the animal weight and production (Abdelrazek et al., 2015; Devasagayam and

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Sainis, 2002; dos Santos and Carvalho, 2014).

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Many plants extracts have been considered as immunomodulator in a number of

ayurvedic formulations either alone or in a group (Carrasco et al., 2009; Chen et al., 2009; Latorre et al., 2009; Nfambi et al., 2015). The genus Tribulus has 25 species and many of them are classified as noxious weeds. Because they are hazard to grazing animals due to the spiny structure of the fruits (Aslani et al., 2004). Moreover, sheep stagger is recorded to be as a result 3

of beta-carboline alkaloids contained in the fruit (Bourke et al., 1992). Tribulus terrestris (T. terrestris) commonly known as ‘Gokhru’, is a shrub belonging to the family Zygophyllaceae used as rejuvenation tonic and for various health conditions affecting the immune system,

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cardiovascular, liver, and kidney (Adaikan et al., 2000; Gauthaman and Ganesan, 2008; ZhelevaDimitrova et al., 2012). T. terrestris is of pharmaceutical and medicinal interest as it extremely and considerably rich in components having potential biological effect, including flavonoids, saponins, alkaloids, and other nutrients (Hashim et al., 2014; Wang et al., 1997). Nikoo et al. (2015) indicated that T. terrestris extract (TTE) has a proliferative impact on PBMCs but not on

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cancer cells. Subsequently, herbal medicine with proliferating potential, anti-cancer effect, and

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low toxicity are promising for adjustment of the defense system facing different diseases

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condition (Garodia et al., 2007).

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Since many of T. terrestris uses have not been fully validated on a scientific basis, it is

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natural to have doubts when classified as herb purportedly useful in so many illnesses. Moreover,

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knowledge on the influence of TEE on immune functions and DNA integrity in sheep is scarce. In this vein, the in vivo and in vitro effects of TTE on immune functions were investigated

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through evaluation of lymphocyte proliferation, tumor necrosis factor (TNF-α), interleukin 6 (IL6), and in rams as well as influence of TTE on serum lipid peroxidation and reduced glutathione

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(GSH) with respect to immunological function in rams.

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2. Material and methods

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2.1. Animals and treatment

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This study was designed on 20 rams weighting 39-42 kg in a private farm. They were

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divided into control and treated groups (10 animals each). The treated rams were received TTE in

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a dose of 1.5 g/ head/ day dissolved in 100 mL distilled water (Kistanova, 2005). Treatment was

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continued for consecutive 45 days. T. terrestris seed extract containing 80% steroidal saponins was purchased from Nate Biological technology Co., China. The animals were free from internal

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and external parasites and were kept under the same environmental conditions and allowed to free access to water and feed. The study protocol were carried out according to the ethical

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guidelines for the use of animals in laboratory experiments of the Faculty of Veterinary

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Medicine, Suez Canal University, Egypt. 2.2. Body weight The body weights of rams of both groups were evaluated before and after the end of experiment.

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2.3. Blood samples Three different jugular blood samples were collected from each ram in control and treated groups. The samples collected before starting of the TTE administration and after the end

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of treatment. The first sample was collected in lithium heparinized tubes for comet assay and lymphocyte transformation test (LTT). The second samples were collected in a plain tube for sera separation. The sera were used for malonaldehyde (MDA), reduced glutathione (GSH), IL6, and TNF-α assay. The third sample was collected in EDTA containing tubes for total and differential leukocytes counts (TLC & DLC) according to Pierson (2000).

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2.4. In vitro experiment

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An in vitro study was carried out by using blood from healthy sheep. The peripheral

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blood mononuclear cells (PBMCs) was separated and lymphocyte culture was carried out. 2.4.1. PBMC separation

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In lithium heparinized tubes, jugular blood from healthy and disease free sheep. The

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PBMCs were prepared from buffy coats using Ficoll-Hypaque density gradient centrifugation according to the manufacturers’ guidelines (Sigma-Aldrich Co., USA). Cells were washed 3

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times with cold Roswell Park Memorial Institute (RPMI)-1640 media. Washed cells re-

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suspended in 1 ml RPMI-1640 supplemented with 10% foetal calf serum (FCS) (Sigma-Aldrich Co., USA), 100 U/mL penicillin, and 100 pg/mL streptomycin (Böyum, 1968). Lymphocytes

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were counted and re-suspended at 2 x l07 cells/mL in RPMI-1640 medium with the previously mentioned enrichments. The cells were stimulated by addition of Phytohaemagglutinin (PHA) in a rate of 15µl/mL. 2.4.2. Lymphocyte culture

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The PBMCs were placed in 96 well flat bottom microplate (100 µl). The wells were sectioned equally into 3 groups: Group I; considered as control. Group II, treated with 0.5 µl/mL TTE (1% in saline). While, group III, treated with 1 µl/mL TTE (1% in saline) (Bedir et al.,

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2002). The final volume of each well should be completed up to 150 µl by RPMI-1640 medium. Each treatment was run in triplicate. The plates were incubated at 37°C in a 5% CO2 incubator. Finally, lymphocyte transformation, comet assay, IL-6, and TNF-α were assayed after 72 hr of

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incubation.

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2.4.3. Lymphocyte transformation assay

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The in vivo blood samples that were collected from control and TTE treated rams before

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and after the end of treatment were immediately transferred on ice to the laboratory for

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lymphocyte transformation assay. The separated buffy coat was washed with RPMI-1640

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medium (Sigma-Aldrich Co., USA). The washed lymphocytes were re-suspended in RPMI-1640 medium containing 10% of FCS (Sigma-Aldrich Co., USA) (Böyum, 1968; Burrells and Wells,

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1977). The number of viable lymphocytes / mL was calculated (Hudson and Hay, 1980). The in vitro lymphocytic transformation assay was carried out using 3-(4,5-dimethythiazol-2-yl)-2,5-

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diphenyl tetrazolium bromide (MTT) assay staining procedures (Bounous et al., 1992; Gao et al.,

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2008).

2.5. Comet assay (Single cell gel electrophoresis) The comet assay was evaluated the effect of TTE on DNA integrity on isolated

lymphocytes from blood of each ram per group in vivo before and after the treatment, as well as the in vitro cultured PBMCs that were treated with 0.5 µl/mL and 1 µl/mL of TTE. Experiments 7

were started by incubating lymphocytes for 2 hr. Negative controls were carried out by incubating lymphocytes with 1% DMSO. aliquots of treated and untreated lymphocytes (10 μL) were mixed with low-melting agarose (120μL, 0.5%) and layered on 1% normal-melting agarose

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coated glass slides and cover slipped. The slides were allowed to gel at 4 °C for 20 min. Then carefully take off the cover slips, another layer of 0.5% agarose was pipetted onto the slides and left at 4 °C for 20 min. Slides without cover slips were immersed in cold lysing solution 12hr at 4 °C (Singh et al., 1988). Before electrophoresis, the slides were equilibrated for 20 min in alkaline electrophoresis solution (300 mM sodium hydroxide and 1 mM disodium salt of

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ethylene diamine tetra acetic acid, pH >13). Electrophoresis was performed using the previous

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buffer for 25 min. Slides were rinsed with 0.4 M Tris buffer (pH 7.5) for 2–3 times, 5 min each.

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Then the slides were rinsed with tap water and air-dried. Slides were stained using 0.2 mg/mL

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silver nitrate solution (Kizilian et al., 1999). The procedures were performed in a dim light in order to minimize DNA damage artifacts. Nikon Light microscope was used to analyze the slides

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(×100). At least 100 cells/slide were screened. 2.6. IL-6 and TNF-α assay

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Blood samples that were obtained from rams of both groups before and after the treatment were centrifuged at 3000 rpm for 15 minutes. Sera were keept at -20°C. IL-6 and TNF-

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α were assessed in serum as well as the supernatant of cultured PBMC incubated in vitro for 72 hr using sheep commercial ELISA kits (Genorise Scientific Inc., USA). All procedures were

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performed according to manufacturer protocol. 2.7. MDA and GSH The MDA and GSH in serum were assayed using kinetic enzymatic assay (BioVision, USA) according to manufacturer instructions (Ohkawa et al., 1979; Tietze, 1969). 8

2.8. Statistical analysis All values were expressed as mean ± standard error of the mean. Differences of in vivo experiment were evaluated by Mann-Whitney nonparametric analysis. Kruskal-Wallis followed

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by Dunn's test analysis was used for examining differences among groups of in vitro experiment. Inter-group comparisons were made by Dunn's test. A value of P < 0.05 was considered to indicate significance. All the analyses were done using GraphPad Prism (Version 7.0, San Diego,

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USA).

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3. Results

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3.1. Body weight

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The body weights of rams treated with TTE for 45 days was significantly (P<0.05) higher

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than that in non-treated rams, while the body weights of both groups were nearly same before the

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starting of in vivo experiment (Fig. 1).

3.2. Total and differential leukocytic count (TLC & DLC)

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There were no significant changes observed in TLC or DLC in control and treated rams

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with TTE before and after the end of in vivo experiment, although a numerically low eosinophil and basophils were counted in rams treated with TTE (Fig. 3).

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3.3. Lymphocyte transformation Lymphocyte transformation was significantly (P<0.05) induced in rams treated for 45

days in vivo with TTE compared to that in control rams, while there was no differences in lymphocyte transformation in rams in both groups before the starting of treatment. Interestingly,

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the 0.5 µl/mL of TTE induced (P<0.05) the lymphocyte transformation in PBMC cultured in vitro compared to that in control and 1 µl/mL of TTE treated cells (Fig. 2). 3.4. Comet assay

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The in vivo treatment with TTE for 45 days significantly (P<0.01) improved the integrity of DNA in lymphocytes of rams compared to non-treated rams (Table 1). On the same trend, the in vitro addition of TTE either in 0.5 or 1 µl/mL to the PBMCs culture significantly(P<0.05) altered the head diameter, tail length, and tail moment of DNA compared to control PBMCs

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culture (Table 2).

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3.5. IL-6 and TNF-α

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IL-6 was not altered in control or treated rams with the TTE in vivo, while the dose of 0.5 µl/mL of TTE significantly (P<0.05) increased the level of IL-6 in PBMC cultured in vitro

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compared to that in control and 1 µl/mL of TTE treated cells. However, the level of TNF-α did

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in vitro (Fig. 2).

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not show any significant changes in control or treated rams in vivo as well as in PBMC cultured

3.6. MDA and GSH

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The level of MDA was declined in sera after TTE treatment with higher significance

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(P<0.01) than control rams. The activity of GSH was significantly (P<0.05) improved after TTE administration to rams than the non-treated ones.

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4. Discussion

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Several immunomodulatory and therapeutic agents have been obtained from medicinal plants

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(Citarasu, 2010). Additionally, herbal extracts have received increased attention as antibiotic

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growth promoter substitutions. They have been accepted by consumers as healthy natural additives (Chakraborty et al., 2014). Therefore, it is of considerable interest to carry out a

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screening and examination of these plants constituents in order to validate their benefit or toxicity in traditional medicine.

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In the current study, the body weight of rams treated with TTE for 45 days was significantly

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higher than that in non-treated rams. These results were in agreement with the findings of Dimitrov et al. (1986) who reported the TTE promoting activity to rams' daily weight gain and sexual activity. The possible attribution to these results is the presence of protodioscin, a steroidal saponin compound in TTE (Dinchev et al., 2008; Nassiri and Hosseinzadeh, 2008). Protodioscin was recorded to increase the levels of various androgens in mammals (Gauthaman 11

and Ganesan, 2008). These steroidal hormones may have rendered anabolic effects leading to a growth increase as well as enhancement of muscle strength and mass (Griggs et al., 1989; Yazdi et al., 2014). Moreover, the body weight enhancement in TTE treated rams may reflect the ability

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of the herb to reduce microbes, stress, increase protein synthesis (Mothana and Lindequist, 2005). Thus may confer a growth increase due to increase efficiency in feed digestibility and utilization causing an improvement in feed conversion ratio (Bedford, 2000).

TTE didn’t alter TLC or DLC in control and treated rams although a numerically low eosinophil and basophils were observed in TTE treated rams. The current results were

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harmonized with those obtained in sheep by Aslani et al. (2003).

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The lymphocytes proliferation activity is a pivotal indicator of their blastogenic activity

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during the emergence of adaptive immune responses versus any invading pathogen. Moreover,

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this activity permits the further allocation of antigen-specific lymphocytes throughout the

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infection course (Miszczyk et al., 2014). The herby study, the lymphocyte transformation was

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significantly induced in rams treated in vivo with TTE compared to that in control rams. Interestingly, the 0.5/mL µl of TTE induced (P<0.05) the lymphocyte transformation in PBMCs

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cultured in vitro compared to that in control and 1/mL µl of TTE treated cells. These results

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augment the immunomodulatory effect of TTE on cell-mediated immunity where lymphocytes play a pivotal role. Lymphocytes secrete numbers of cytokines (Stanford et al., 2002). This gives

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host defense the ability to counter the infectious diseases and pathological conditions. These results could lead us to the beneficial effects of TTE on a delayed type of hypersensitivity (DTH) that were observed by Tilwari et al. (2011) and (2013). The promoting effect of TTE on lymphocytes is necessary for the manifestation of the reaction is showing higher DTH response (Luster et al., 1982). DTH needs the specific recognition of the antigen by the activated T12

lymphocytes that proliferate and produce their cytokines. In turn, these increase vascular permeability, enhance macrophage accumulation and activation with promoting phagocytic activity and increase lytic enzymes concentrations for more effective killing.

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Current results proved the antioxidant effect of TTE where it produced significant decline in serum MDA, as an indication for lipid peroxidation, and increment in GSH. In normal cellular metabolism, reactive oxygen species (ROS) are liberated as a by-product (Ahmed et al., 2017). They are beneficial for some physiological processes, such as activation of some transcription factors, phosphorylation, apoptosis, cellular competing to microorganisms, and steroidogenesis

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(Agarwal et al., 2005; Celi, 2010). Although they are beneficial in low amount, they are harmful

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when produced in surplus amount. As they can deteriorate cellular lipids, DNA, and proteins

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(Celi, 2010). Intracellular GSH contents usually constitute a corner stone in lymphocytes

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apoptosis via caspase-3 induction (Franco et al., 2008). The presence of hydroxyl groups in the

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structure of TTE saponins and phenolic compounds especially di-p-coumaroylquinic acid

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derivatives that possesses potent antioxidant activity (Hammoda et al., 2013). These groups can affect electron transferring system and/or enzymes regulating oxidative reactions and lipid

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peroxidation (Colegate and Molyneux, 1993). Thus they limit the generation of free radicals and subsequent lipid peroxidation that keeps the antioxidant reserve of GSH. Moreover, maintenance

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of antioxidant reserve with minimizing oxidative stress positively influence immune system through maintenance of the DNA integrity and proliferation ability of T-lymphocytes (Rahal et

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al., 2014).

The innate immune system is involved in pathogen recognition through toll-like receptors (TLRs) as well as the production of pro-inflammatory cytokines like TNF-α and interleukins (IL6 and IL-8). TNF-α is a cytokine, which contributes to both physiological and pathological 13

processes. It is involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction (Chu, 2013). In the present research, the immunostimulant activity of TTE in sheep was investigated for the first time. A dose of 0.5/mL µl of TTE significantly

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(P<0.05) increased the level of IL-6 in PBMCs cultured in vitro compared to that in control and 1/mL µl of TTE treated cells. However, the level of TNF-α did not show any significant changes in vivo as well as in PBMCs cultured in vitro. These results were suggestive of the protecting promoting role of TTE to PHA stimulated T- lymphocytes to produce IL-6 that seemed to be dose-dependent. IL-6 plays a pivotal role in the communication between immune

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neuroendocrine systems via influencing hypothalamic-pituitary-adrenal axis (Liu et al., 2007).

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Moreover, it is responsible for induction of inflammatory cytokine cascade production through

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intracellular signaling via TLRs. The absence of in vivo IL-6 level changes among TTE and

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control groups may be attributed to the absence of a mitogen as stimulator for T-lymphocytes

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function that existed by PHA at the in vitro experiment.

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In the current study, the in vivo treatment with TTE declined the DNA % in the comet tail in lymphocytes of rams compared to non-treated rams. However, the in vitro addition of TTE either

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in 0.5 or 1 µl/mL to the PBMCs culture significantly affected the DNA integrity. This is indicative that TTE may protect DNA of lymphocytes from deterioration. These results could

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explain the promotion of ram lymphocytes transformation as comet assay usually used as a tool for representing cellular apoptosis. The maintaining effect of DNA integrity of T-lymphocytes

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could be attributed to the antioxidant effect of TTE and its lipid peroxidation limiting activity observed in current work. The effect of TTE on DNA integrity is controversial, some authors clarified that TTE is safe on DNA molecule of cultured human lymphocytes at lower doses (Manish et al., 2009; Qari and El-Assouli, 2017). Another study clarified the ability of the extract

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of T. terrestris fruits to do lymphocytes damage in the target DNA at the higher concentrations (40–80 mg/L) not with the low concentration of the extract (10–20 mg/L). Thus, it could be considered that the aqueous extracts of the T. terrestris fruits have genotoxic effect in the

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therapeutic protocols if it used in high doses (Qari and El-Assouli, 2017).

Conclusion

In conclusion, the results of the present study provide an evidenced reference for the traditional use of TTE, in rams, as potent in vitro and in vivo immunostimulant. The plant extract

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prevented lymphocytes DNA degradation and supported their ability for blastogenesis through

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its antioxidant effect that inferred on their ability for cytokines production. These

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immunostimulant effects beneficially reflected on rams' performance and profitability. Further

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studies in the light are necessary to elucidate complete mechanisms behind its traditional effects.

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Further investigations should be addressed toward that topic taking in consideration the effect of

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inflammation.

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TTE on rams' immune performance during the course of experimental infection and/ or

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Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or

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financial relationships that could be considered as a potential conflict of interest.

Sincerely yours,

Eman Ahmed, DVM, PhD Assistant Professor

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Department of Pharmacology

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Faculty of Veterinary Medicine

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Suez Canal University, Ismailia, Egypt.

Mail: [email protected]

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6. References

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[email protected]

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Mobile phone: +20-1010253404

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Garodia, P., Ichikawa, H., Malani, N., Sethi, G., Aggarwal, B.B., 2007. From ancient medicine to modern medicine: ayurvedic concepts of health and their role in inflammation and cancer. J Soc Integr Oncol 5, 25-37. Gauthaman, K., Ganesan, A.P., 2008. The hormonal effects of Tribulus terrestris and its role in the management of male erectile dysfunction – an evaluation using primates, rabbit and rat. Phytomedicine 15, 44-54. Griggs, R.C., Kingston, W., Jozefowicz, R.F., Herr, B.E., Forbes, G., Halliday, D., 1989. Effect of testosterone on muscle mass and muscle protein synthesis. Journal of Applied Physiology 66, 498-503. Hammoda, H.M., Ghazy, N.M., Harraz, F.M., Radwan, M.M., ElSohly, M.A., Abdallah, I.I., 2013. Chemical constituents from Tribulus terrestris and screening of their antioxidant activity. Phytochemistry 92, 153159. Hashim, S., Bakht, T., Marwat, K.B., Jan, A., 2014. Medicinal properties, phyto-chemistry and pharmacology of tribulus terrestris L.(Zygophyllaceae). Pak. J. Bot 46, 399-404. Hudson, L., Hay, F., 1980. Isolation and structure of immunoglobulins. Practical immunology 3. Kistanova, E., 2005. Improvement of the reproductive performances of rams by the biological active substances-plant extract and probiotic. Biotechnology in Animal Husbandry 21, 69-72. Kizilian, N., Wilkins, R., Reinhardt, P., Ferrarotto, C., McLean, J., McNamee, J., 1999. Silver-stained comet assay for detection of apoptosis. Biotechniques 27, 926-930. Latorre, A.O., Furlan, M.S., Sakai, M., Fukumasu, H., Hueza, I.M., Haraguchi, M., Gorniak, S.L., 2009. Immunomodulatory effects of Pteridium aquilinum on natural killer cell activity and select aspects of the cellular immune response of mice. Journal of immunotoxicology 6, 104-114. Liu, Y.-L., Bi, H., Chi, S.-M., Fan, R., Wang, Y.-M., Ma, X.-L., Chen, Y.-M., Luo, W.-J., Pei, J.-M., Chen, J.-Y., 2007. The effect of compound nutrients on stress-induced changes in serum IL-2, IL-6 and TNF-α levels in rats. Cytokine 37, 14-21. Luster, M.I., Dean, J.H., Boorman, G.A., 1982. Cell-mediated immunity and its application in toxicology. Environmental health perspectives 43, 31. Manish, K., Meenakshi, P., Ravindra, S., Ashok, K., 2009. Evaluation of radiomodulatory influence of Tribulus terrestris Root extract against gamma radiation: Hematological, Biochemical and cytogenetic alterations in swiss albino mice. Pharmacologyonline 1, 1214-1228. Miszczyk, E., Walencka, M., Rudnicka, K., Matusiak, A., Rudnicka, W., Chmiela, M., 2014. Antigen-specific lymphocyte proliferation as a marker of immune response in guinea pigs with sustained Helicobacter pylori infection. Acta Biochim Pol 61, 295-303. Mothana, R.A., Lindequist, U., 2005. Antimicrobial activity of some medicinal plants of the island Soqotra. Journal of ethnopharmacology 96, 177-181. Nassiri, M., Hosseinzadeh, H., 2008. Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytotherapy research 22, 709-724. Nfambi, J., Bbosa, G.S., Sembajwe, L.F., Gakunga, J., Kasolo, J.N., 2015. Immunomodulatory activity of methanolic leaf extract of Moringa oleifera in Wistar albino rats. Journal of basic and clinical physiology and pharmacology 26, 603-611. Nikoo, M., Hasanpoor, Z., Mostafaei, A., 2015. Effect of Tribulus Terrestris Aqueous Extract on Survival and Growth of Human Peripheral Blood Mononuclear Cells (Hpbmc) and Several Cancerous Cell Lines. Journal of Reports in Pharmaceutical Sciences (J. Rep. Pharm. Sci.) 4, 24-29. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry 95, 351-358. Pavón, L., Besedosky, H., Bottasso, O., Hernández, R., Velasco, M., Loria, R., 2013. Clinical and Experimental Immunomodulation. Clinical and Developmental Immunology 2013.

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Pierson, F., 2000. Laboratory techniques for avian hematology. Schlam’s veterinary hematology, BF Feldman, JG Zinkl and NC Jain (eds.). Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, 11451146. Qari, S.H., El-Assouli, S.M., 2017. Evaluation of cytological and genetic effects of Tribulus terrestris fruit aqueous extract on cultured human lymphocytes. Saudi Journal of Biological Sciences. Rahal, A., Kumar, A., Singh, V., Yadav, B., Tiwari, R., Chakraborty, S., Dhama, K., 2014. Oxidative Stress, Prooxidants, and Antioxidants: The Interplay. BioMed Research International 2014, 761264. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental cell research 175, 184-191. Tietze, F., 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Analytical biochemistry 27, 502-522. Tilwari, A., Shukla, N., Devi, P., 2013. Comparative study of alcoholic and aqueous extracts oftribulus terrestris on specific and non specific immune responses in wistar rats: An in vivo study. Tilwari, A., Shukla, N., Devi, P.U., 2011. Effect of five medicinal plants used in Indian system of medicines on immune function in Wistar rats. African Journal of Biotechnology 10, 16637-16645. Wang, Y., Ohtani, K., Kasai, R., Yamasaki, K., 1997. Steroidal saponins from fruits of Tribulus terrestris. Phytochemistry 45, 811-817. Yazdi, F.F., Ghalamkari, G., Toghyani, M., Modaresi, M., Landy, N., 2014. Efficiency of Tribulus terrestris L. as an antibiotic growth promoter substitute on performance and immune responses in broiler chicks. Asian Pacific Journal of Tropical Disease 4, S1014-S1018. Zheleva-Dimitrova, D., Obreshkova, D., Nedialkov, P., 2012. Antioxidant activity of tribulus terrestris—a natural product in infertility therapy. Int J Pharm Pharm Sci 4, 508-511.

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Figures legends

EP

Fig.1. In vivo effect of TTE treatment for 45 days on rams’ body weight and total and

A

CC

differential leukocytes counts (TLC & DLC). * Significantly different (P˂0.05).

20

21

D

TE

EP

CC

A

SC RI PT

U

N

A

M

Fig. 2. In vivo and in vitro effects of TTE treatment on lymphocytic proliferation assay, interleukin 6 (IL-6) and tumor necrosis factor (TNF-α). * Significantly different (P˂0.05) when compared with the control and # Significantly different (P˂0.05) when compared with the treated

M

A

N

U

SC RI PT

group.

Fig. 3. In vivo effect of TTE treatment for 45 days on rams’ serum malonaldehyde (MDA) and

A

CC

EP

TE

D

reduced glutathione (GSH) assay. * Significantly different (P˂0.05).

22

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Table 1: In vivo effect of TTE treatment (1.5 g/ head/ day) for 45 days on the DNA integrity in rams’ lymphocytes (Comet assay) In vivo experiment Tailed Intact

Head diameter (px)

A

Treatment

Tail length

80.40

before

±2.08

Head DNA %

Tail moment

(px)

%

122.2

95.33

18.71

2.72

0.486

±1.25

±3.98

±3.05

±0.76

±0.116

±0.014

22.05

114.4

95.71

18.12

2.78

0.498

±.66

±0.95

PT

±4.92

±1.63

±1.29

±0.037

±0.028

CC E

76.78

18.37

ED

Control

M

%

Tail DNA

80.23

18.33

115.6

94.31

19.99

2.73

0.502

±2.05

±1.16

±4.03

±3.46

±2.03

±0.061

±0.034

85.18

15.66

121.9

101.5

17.02

1.43**

0.453

±2.08

±1.72

±3.06

±1.86

±1.881

±0.08

±0.069

0.181

0.108

0.389

0.0815

0.364

< 0.001

0.983

Before TTE

A

Control after

After TTE

P value

* Significantly different (P˂0.05) when compared with the control group. 23

N U SC RI PT

Table 2: In vitro effect of TTE treatment (0.5 µl/mL and 1 µl/mL) on DNA integrity of cultured PBMCs (Comet assay) In vitro experiment Tailed Treatment

Intact

Head diameter (px)

Tail moment

93.44

9.34

2.400

0.136

±1.03

±0.58

±2.59

±2.26

±0.66

±0.10

±0.01

13.93

76.35**

94.28

25.81**

3.610

1.291**

±1.94

±1.65

±0.60

±3.41

±0.50

±0.05

9.100

39.00**

98.59

7.59

1.477*

0.140

±0.92

±0.67

±1.01

±0.32

±0.38

±0.17

±0.001

0.0003

0.072

< 0.001

0.0757

0.0013

0.045

< 0.001

ED

M

114.9

PT

80.59**±0.89

CC E P value

Tail DNA %

10.23

92.77*

TTE 1 µl/ mL

(px)

88.09 Control

TTE 0.5µl/ mL

Tail length

A

%

Head DNA %

A

* Significantly different (P˂0.05) when compared with the control cells.

24