Protective effect of aqueous extract of Alhagi maurorum in spermatogenesis and antioxidant status of adult rats exposed to carbon tetrachloride

This study aimed to investigate the efficiency of aqueous extraction of Alhagi maurorum leaves against oxidative stress induced by carbon tetrachloride (CCl4) on spermatogenesis and the level of glutathione, superoxide dismutase, malondialdehyde in adult rats. Plant Leaf's dried and then extracted. Experiment included 24 male rats divided into 4 groups 6 subjects in each group. Groups treated orally for 30 days as following: first was control group which administered with 1 ml of physiological saline 0.9%, second group administered once with CCl4 3 ml/Kg, third and fourth groups administered with aqueous extract 300 mg/kg and aqueous extract together with CCl4 respectively. The results showed that CCl4 caused a significant decrease in sperm count, sperm vitality, normality, glutathione (GSH) and superoxide dismutase (SOD), significant increase in sperm mortality, abnormality and malondialdehyde (MDA) compared with control group. While aqueous extract treatment caused no significant difference in compare to control group. Groups treated with aqueous extract together with CCl4 showed a significant increase in sperm count, vitality, normality and GSH and decreasing in mortality, abnormality and MDA in compare to CCl4 group. It could be concluded that the aqueous extract of Alhagi maurorum have a positive effect on male reproduction and antioxidants in rats exposed to oxidative stress.


Introduction
Male fertility correlated with a proper function of organs and hormones, most of male infertility cases because of sperm abnormalities which categorized by whether they affect sperm count, sperm movement, or sperm shape (1).Sperm abnormalities caused by lots of factors, including, disease, lifestyle and chemical exposure, but many causes of sperm abnormalities are still unknown (2).Previous studies (3,4) approved that CCl 4 is a chemical exposure cause oxidative stress due inducing free radical production which cause oxidation of germ cells in the testes.Free radicals contribute to male infertility, damaging cells, tissues and organs (5,6).To maintain and protect normal cell function, excess of free radicals must be inactivated continuously by antioxidants in seminal plasma and block the formation of new reactive oxygen species ROS or keep removing generated ROS (3).
Herbal is the oldest form of medicine, it is used widely in practiced form of medicine (7).Alhagi, is a genus from family Fabaceae, Its distributed in many countries of Asia, Europe and Australia.Commonly it has lots of names like Camel thorn or Aqool (8).The previous studies showed that Alhagi maurorum plant contained many secondary metabolites, it exerted antibacterial, anti-inflammatory, antipyretic, analgesic, antioxidant, gastrointestinal, cardiovascular, diuretic, and dermatological, and many other effects (9)(10)(11).Very rare studies demonstrated the effect of aqueous extract of alhagi plant in fertility of males therefore the present study was designed to explore the protective effect of Alhagi maurorum on testicular dysfunction-induced by CCl 4 in rats.

Experiment design
Twenty-four adult male rat average weight is 200-250 g housed at the experimental animal unit, College of Veterinary Medicine, University of Tikrit.Feed and water were provided all time.Rats divided randomly into four groups, first was control group which administered with physiological saline 0.9% and the other three groups administered once with CCl 4 3 ml /kg B.W. (5,6), aqueous extract of Alhagi maurorum 300 mg/kg B.W (12) and aqueous extract together with CCl 4 respectively.The animals were dosed orally once daily for 30 days using gavage needle.

Preparation of plant material
Aerial parts of Alhagi maurorum were collected from Alalam desert, Sallahidin province in the middle of Iraq.After scientific approval by botanical experts, leaves boiled in 3 L of distilled water for 1 hour then dried by reduced pressure.Powder kept in sterile container until use (7).

Active phytochemical compounds
Active phytochemical compounds (phenolic acids and flavonoids) determined in aqueous extracts (AE) of leaves using method of high-performance liquid chromatography (HPLC).Plant extract were performed on Shimadzu HPLC system (Fig. 1 and 2).Phenolic compounds separated on a Thermo Scientific Hypersil Gold reverse phase (RP-18) column, 250 mm x 4.6 mm C18 is the stationary phase.The gradient maintained at flow rate 1-2 ml/min, the time of run was over 10 min.The binary mobile phase consisted of a solvent A (water: acetic acid; 99:1; v/v) and solvent B (acetonitrile).The gradient elution from the column was achieved with 13% of solvent B until 10 min.The phenolic acids and flavonoids were detected by a UV detector at wavelength 200-500 nm.The chromatographic peaks were identified by comparing retention time of analytics with that of reference compounds (Fig. 1) (13,14).

Blood samples
At the end of the experiment, blood samples were taken by retro-orbital vein placed in a test tube free of anticoagulant then placed in the incubator at 37 °C then centrifuged at 3000 rpm for 15 minutes.Serum were kept at -20 °C until biochemical tests were carried out.

Histological preparation
Testes tissue were immediately put in formalin 10% for 24 hours.Dehydrated by graded alcohol series then embedded in paraffin.Samples cut into 5µm sections, stained using hematoxylin and eosin (H&E).Slides examined using light microscopy under 40x magnification (15).

Sperm count assay
Male rats were killed and the cauda epididymis were excised and then placed in petri dish containing 9.8 of formalin buffer 10%.Cauda epididymis was then cut using a fine scissors to make sperm suspension.Eosin stain added to suspension, the sperm counted using hemocytometer (improved Neubauer, Deep 1/10 mm, LABART, Germany).Using following equation, total sperm number = (N/80) x 400 x 1000 x 10 x 10 (16).

Abnormality and viability assay
Cauda epididymis was excised and then placed in petri dish containing physiological solution 0.9%.Cauda epididymis was then cut a fine scissors.Smears prepared with stains (0.2 g of Eosin and 0.6 g of Fast green) dissolved in distilled water and ethanol in ratio 2:1.Abnormality detected according to the head, neck and tail of sperm.Live/dead ratio determined using the stains 1% Eosin and 5% Nigrosine in 3% sodium citrate dehydrate solution according to (17).

Glutathione assay
The test was performed by using the laboratory analysis kit (OxiselectTM Total Glutathione (GSSG / GSH) assay kit) prepared by Cell Biolabs Inc., according to the company's approved mechanism and the tools required for the method of work (18).Figure 3 shows the standard curve of GSSG.

Superoxide dismutase assay
The SOD Assay kit -WST, manufactured by Dojindo, Japan and using ELISA, was used to estimate the level of the enzyme and according to the manufacturer's method of operation (19).

Malondialdehyde assay
The NWLSSTM Malondialdehyde Assay was used by Northwest Life Science Specialists, LLC to measure the concentration of MDA in the serum and tissue extract, according to the work mechanism and materials required and prepared by the company (19).

Statistical Analysis
Data were recorded and analyzed using one-way analysis of variance (one-way ANOVA) test.Group differences were determined using Duncan multiple range test.P<0.01 was considered statistically significant value (20).

Results
Active phytochemical compounds (phenolic acids and flavonoids) have been detected in Alhagi marourum leaves extract (Table 1) and (Table 2).The results referred that treatment with CCl 4 caused a significant decrease (P<0.01) in sperm count (Oligospermia), normal sperm, live sperm (Teratospermia), GSH, SOD levels and significant increase in dead and abnormal sperm ratio and MDA level compared to control group (Fig. 4-9).While the treatment with aqueous extract AE plus CCl 4 showed significant increase (P<0.01) in sperm counting, live and normal sperm and GSH level and a significant decrease in the percentage of dead and abnormal sperm and the level of MDA compared to CCl 4 group.The treatment with aqueous extract showed a significant increase (P<0.01) in GSH level and no significant difference in the rest of the studied variables compared to the control group in significant value (P<0.01) (Fig. 4-9).Testes tubules of CCl 4 group are almost empty of sperm (Fig. 10-B) in compare to control(Fig 10-A), most of cells show damages in vital cell components.In CCl 4 plus aqueous extract group(Fig.10-C) , majority of sperm cells in the stages of morphological transition and look similar to normal but, less dense in compare to the normal state (Fig. 10-D).Male germ cell through stages of differentiation generate reactive oxygen species ROS in low levels to regulate sperm differentiation.ROS level maintained by antioxidant (21).Sperm plasma membrane contain unsaturated fatty acid and these attacked by ROS leads to chemical reaction that called lipid peroxidation (22), and that reduces membrane enzymes activity, ion channels and the membrane fluidity, resulting inhibition in required mechanisms for production and fertilization of sperm (23) (Fig. 10-B).Excess ROS overwhelm antioxidant defense called oxidative stress (22,23).Treatment using CCl 4 cause oxidative stress of male reproductive system (Fig. 5, 6 and  7) which linked negatively with sperm production, Normality and sperm vitality.(Fig. 4, 5 and 6) in compare to control(Fig.10-A) and that combined with (3,4).ROS promote apoptosis leading to decreased sperm concentration and increased level of caspases and proteases which increase apoptosis in mature spermatozoa (24).Researchers mentioned that the sperm is more susceptible to oxidative stress than other cells because of the limited amount of cytoplasm and the concentration of ROS suppressing antioxidants in the mature sperm in addition to high levels of unsaturated fatty acids in the sperm structure (11).The health and fertility of sperm dependent on the antioxidants to protect plasma membrane surrounding acrosome and tail (25).The leaves extract of Alhagi maurorum contain polyphenols(Table 2) (Fig. 3) and flavonoids (Table 1) that has antioxidant potential activity (26,27), most flavonoids and phenols are metabolized in vivo, which can effectuate antioxidant capacity.The main phytochemical classes that comprised are phenolic acids and flavonoids.Flavonoids include several subclasses (24) (Table 1).Flavonoid-rich foods, correlated with a lower risk of many diseases (28).the aqueous extract increased glutathione GSH, SOD levels and decreased MDA level in serum in subject's exposure to oxidative stress (Fig. 7, 8 and 9).MDA is produced due to the degradation of the peroxides of unsaturated fatty acids.It is considered biomarker to determine the rate of oxidative damage (24).Testicular tissue is susceptible to oxidative stress (29), so testes protect itself from damage using antioxidants and free radical scavengers to maintain that steroidogenic and spermatogenic functions are not impacted by oxidative stress in healthy rats (25) (Fig. 8, 9 and (Fig. 10-A).Histological changes had been noticed in subject's exposure to oxidative stress induced by CCl 4 (Fig. 7, 8, 9 and 10-B) while maintain antioxidant and free radical scavengers in subject's exposure to oxidative stress give a positive result (Fig. 7, 8, 9 and 10-D).antioxidants have the ability to avoid damage by counteracting free radicals or preventing their formation in the testicular cells and prevent oxidation chain by finding primer free radicals, preventing various diseases (30,31).It could be concluded that the aqueous extract of Alhagi maurorum have a positive effect on male reproduction and antioxidants in rats exposed to oxidative stress.(Fig. 10-C).

Figure 3 :
Figure 3: The standard curve for GSSG.

Figure 4 :
Figure 4: Effect of aqueous extract of Alhagi maurorum on sperm count in rats exposed to CCl 4 .Value were expressed as means ± SE from 6 rats per group.Significant value P≤0.01.

Figure 5 :
Figure 5: Effect of aqueous extract of Alhagi maurorum on ratio of live/dead sperm in rats exposed to CCl 4 .Value were expressed as means ± SE from 6 rats per group.Significant value P≤0.01.

Figure 6 :
Figure 6: Effect of aqueous extract of Alhagi maurorum on sperm morphology in rats exposed to CCl 4 .Value were expressed as means ± SE from 6 rats per group.Significant value P≤0.01.

Figure 7 :
Figure 7: Effect of aqueous extract of Alhagi maurorum on GSH level in serum n in rats exposed to CCl 4 .Value were expressed as means ± SE from 6 rats per group.Significant value P≤0.01.

Figure 8 :
Figure 8: Effect of aqueous extract of Alhagi maurorum on Super oxide dismutase level in serum n in rats exposed to CCl 4 .Value were expressed as means ± SE from 6 rats per group.Significant value P≤0.01.

Figure 9 :
Figure 9: Effect of aqueous extract of Alhagi maurorum on MDA level in serum n in rats exposed to CCl 4 .Value were expressed as means ± SE from 6 rats per group.Significant value P≤0.01.

Table 1 :
Detecting flavonoid in aqueous extract of Alhagi maurorum using HPLC method (table show retention time, peak area and flavonoid concentration)

Table 2 :
Detecting phenols in aqueous extract of Alhagi maurorum using HPLC method (table show retention time, peak area and phenol concentration)