١ Effect of different garlic preparations on testosterone, thyroid hormones

٢ and some serum trace elements in rats

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٤ Aliaa Ezz El Arab, Osama Ahmed Abbas, Mohamad Taha Abdelrahman*
٥ Radioisotopes Department, Egyptian Atomic Energy Authority, Giza, Egypt
٦ *Corresponding author: [email protected]
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٥١ Abstract
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٥٣ Garlic is a house-available vegetable which is widely used for its spicy and medicinal
٥٤ benefits. Impact of different preparations on testosterone, thyroid hormones and blood
٥٥ micro and trace elements were studied. Eight groups of male albino rats were selected
٥٦ including control group and other seven groups administered different doses of different
٥٧ garlic preparations by oral gavages for one month. At the end of the experiment, blood
٥٨ samples were collected for determination of serum hormones by radioimmunoassay,
٥٩ serum micro and trace elements by ICP and testes tissues for histological examination.
٦٠ All treated groups with different garlic preparations revealed a highly significant decrease of
٦١ testosterone level in rats as compared to control which confirmed with histological changes.
٦٢ Increase of thyroid hormones in some groups was seen. Hypokalaemia and hyponatriemia
٦٣ effect was recorded due to garlic treatments. Ca, Mg, Se, Zn, Mn, Fe. Cd, Pb, Si, Mo, Ge, Ba,
٦٤ B, Nb and Al levels showed alterations in different preparations groups. On the other hand,
٦٥ insignificant changes of Sr, Cr, Cs and the Ni serum levels were noted.
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٦٧ Conclusion: Interestingly, although all garlic preparations have negative effects on serum
٦٨ testosterone level and testicular tissues, some garlic preparations have different effects on
٦٩ blood elements. Consequently, it infers that the usage of different garlic preparations must
٧٠ abide benefit/risk assessment to avoid unexpected health issues.
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٧٢ Key words: Garlic -thyroid hormones - blood micro and trace elements

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٨٥ Introduction
٨٦
٨٧ Garlic (Allium sativum) is a member of Liliaceae family that also includes onions, shallot

٨٨ and leeks. Allium sativum is a habitually used plant in Mediterranean cuisine. It is

٨٩ regularly consumed at various doses whether crude or cooked and the therapeutic uses of

٩٠ this plant are many (Agarwal 1996). An underground bulb (head) composed of fragment

٩١ lobules ordinarily called cloves (Harris 2016) is used as a vegetable spice and medicinal

٩٢ herb. Recent studies on garlic have used it in the form of tablets, fresh, dried raw and the

٩٣ cooked preparations (Gorinstein et al. 2006). Thus, scientists have been trying to

٩٤ authenticate garlic properties, especially in terms of the identification of the active

٩٥ constituents, their mechanisms of action and explore the potential benefits as diet

٩٦ supplements (Verma et al. 2008) .

٩٧

٩٨ Garlic contains over 2000 biologically active substances such as volatile, water-soluble

٩٩ and oil-soluble sulfur organic compounds together with essential oils, dietary fiber,

١٠٠ sugars, flavonoids and pectin (Swiderski et al. 2007). Crushing, mincing, or otherwise

١٠١ processing garlic bulb, allow alliin to be released from compartments and interacts with

١٠٢ the enzyme alliinase. Upon hydrolysis and instant condensation of the reactive

١٠٣ intermediate allylsulfenic acid, allicin (diallyl thiosulfinate) is formed; a substance

١٠٤ possesses a stronger antibiotic activity than penicillin or tetracycline. Allicin itself is also

١٠٥ an unstable product and can go through further reactions to form new derivatives,

١٠٦ depending on environmental and processing settings(Yi and Su 2013). Garlic exhibits a

١٠٧ wide range of properties including immunomodulatory, hepatoprotective, anti-mutagenic,

١٠٨ anti-carcinogenic and antioxidant effects (Horie et al. 1989). However, its impact on the

١٠٩ male reproductive system has been not clearly defined.

3
١١٠

١١١ Different garlic products are available to consumers. Commercial garlic preparations in

١١٢ the form of garlic oil, powder and pills are also widely used for therapeutic purposes

١١٣ (Alkreathy et al. 2010). Aged garlic extract (AGE) is an odorless garlic preparation made

١١٤ by prolonged extraction of fresh garlic at room temperature for up to 20 months

١١٥ (Amagase et al. 2001). It has been shown to be the most useful garlic product as

١١٦ antioxidant (Borek 2001). As a result to the increased the industrial activity over the past

١١٧ few decades, metals have been accumulating in the environment. Although some metals

١١٨ are essential to human health, they may act as toxicants when humans or animals are

١١٩ exposed to high concentration of them. Various types of heavy metals exist on the earth

١٢٠ including some toxic metals like Ni, Cr, As, Pb, Hg and Cd (Guthrie and Perry 1980).

١٢١

١٢٢ One of the most broadly spread metals in the environs is Aluminum (Al), which is

١٢٣ extensively used in daily life offering easy exposure to human beings. It exists in many

١٢٤ factory-made foods and medications (Yousef and Salama 2009). Lead (Pb) toxicity is

١٢٥ probably the most common form of heavy metal intoxication. It is well-documented as

١٢٦ one of the most dangerous and insidious poisons (Ashry et al. 2010). Chronic lead

١٢٧ poisoning is responsible for renal failure in experimental animals (Ahmed et al. 2012)

١٢٨ The global annual cadmium consumption exceeds 20000 tones. Cadmium was reported,

١٢٩ by numerous studies for heavy metals poisoning, to causes damage to liver and tests. The

١٣٠ human body is limited in processing and disposing of the heavy metal, therefore the

١٣١ metal ions would deposit in different internal organs. Heavy metals have the ability to

١٣٢ form compounds that can be carcinogenic and mutagenic within the body even at very

١٣٣ low levels. It is, therefore, essential for people to avoid unnecessary exposure of heavy

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١٣٤ metals (Songsungkan and Chanthai 2014). Chelation of the heavy metals by natural

١٣٥ ligands like allicin and/or organosulfurs can be a promising option of elimination of the

١٣٦ trace metals (Songsungkan and Chanthai 2014).

١٣٧ According to the proposed mechanism of toxicity, heavy metals favourably combine with

١٣٨ –SH radicals of protein and enzymes and inhibit their functions. Active research on the

١٣٩ protective effect of chelating agents containing –SH radicals is being conducted for

١٤٠ prevention and the treatment of heavy metal poisoning. -SH compounds like

١٤١ pencillamine where developed as cures for heavy metal poisoning later this was followed

١٤٢ by the development of compounds with less toxicity and side effects such as 2-,3

١٤٣ dimercapto succinic acid. considering the pharmacological effects of these SH

١٤٤ compounds, it is expected that garlic which is widely used as a central spices in cooking

١٤٥ contains an abundant quantity of S-S compounds such as dially disulphide, prolylally

١٤٦ disulphide, disulfide and -SH compounds such as glutathione , thiolactic acid, cysteine,

١٤٧ homocystine and vitamin B can provide a protective effect and human body against

١٤٨ heavy metal poisoning by reacting with heavy metals and producing sulphur compounds

١٤٩ (Cha 1987). It was proposed that different types of garlic preparations have different

١٥٠ pharmacologic effects, and AGE could be the most beneficial garlic preparation (Perello

١٥١ et al. 2008).

١٥٢ So the current study aimed at studying the versatile effects of different garlic extracts and

١٥٣ preparations on metals and hormones in male albino rats.

١٥٤

١٥٥ Materials and methods

١٥٦ Garlic extracts preparations:

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١٥٧ Tomex: Tomex tablet is a garlic powder coated with sugar, each tablet contains 200 mg

١٥٨ of garlic powder. It was supplied from SEKEM, Egypt. The tablets were crushed and

١٥٩ suspended in water to prepare daily oral doses of 5 and 10 mg of garlic powder/kg of rat

١٦٠ body weight. Cooked garlic: Raw garlic bulbs were from the Giza region and purchased

١٦١ at the peak of their maturity at the local market. Bulbs of garlic were smashed with

١٦٢ blender then fried in some corn oil and then left aside to dry. Finally, it was grinded to

١٦٣ powder from which the water suspension was prepared at a dose of 5 and 10 mg/kg/body

١٦٤ weight. Dried garlic powder: Dried garlic powder was purchased from local market and

١٦٥ suspended in water to prepare the oral doses of 5 and 10 mg/kg/body weight. Kyolic or

١٦٦ aged garlic extract (AGE): The Aged Garlic Extract (Age-Kyolic, Wakunaga

١٦٧ Pharmaceutical co., USA) used in this study is a commercially available product as

١٦٨ capsules which was evacuated in water to prepare a dose of 5 mg/kg/body weight.

١٦٩

١٧٠ Experimental animals

١٧١ Forty male albino rats weighing 200–240 g were used in this study from nuclear research

١٧٢ centre animal house. The animals were provided with water and standard food ad libitum

١٧٣ while housed in plastic cages in ventilated rooms maintained under animal house with 12

١٧٤ h light/dark cycles. The study was conducted in accordance with the Guide for the Care

١٧٥ and Use of Laboratory Animals (Institute for Laboratory Animal Research 2011). The

١٧٦ rats were maintained for one week for acclimatization. To evaluate the effect of different

١٧٧ garlic preparations on parameters studied, animals were divided into eight groups. Group

١٧٨ 1: rats administered orally vehicle as control animals for one month. Group 2: rats

١٧٩ administered orally tomex (fresh garlic powder extract) at a dose of 5 mg/Kg body weight

١٨٠ for one month (bw/1m). Group3: rats administered orally tomex (fresh garlic powder

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١٨١ extract) at a dose of 10 mg/Kg/bw/1m. Group 4: rats administered orally cooked garlic

١٨٢ at a dose of 5 mg/Kg/bw/1m. Group 5: rats administered orally cooked garlic at a dose of

١٨٣ 10 mg/Kg/bw/1m. Group 6: rats administered orally dried garlic powder at a dose of 5

١٨٤ mg/Kg/bw/1m. Group 7: rats administered orally dried garlic powder at a dose of 5

١٨٥ mg/Kg/ bw/1m. Group 8: rats administered orally Kyolic (aged garlic powder extract) at

١٨٦ a dose of 5 mg/Kg/ bw/1m. Twenty four hours after the last dose of treatment, blood

١٨٧ samples were collected through heart puncture after the diethyl ether light anesthesia to

١٨٨ separate serum samples. Animals were euthanized and testes from the different groups of

١٨٩ rats were saved in 10% formalin for histological examination.

١٩٠

١٩١ Biochemical analysis:

١٩٢ A microwave digestion oven instrument model: MLS 1200 Mega (Milestone

١٩٣ Corporation, Sorisole, Italy) was used for the digestion of serum samples. Serum (0.5 ml)

١٩٤ samples digestion with 2 mL of concentrated HNO3 and 1 mL of H2O2 30% (v/v) were

١٩٥ heated at 250oC in microwave. Determination of trace elements in serum samples (Na, K,

١٩٦ Ca, Mg, Se, Mn, Se, Zn, Fe, Cu, Ag, Al, Pb, Cd, As, Cr, Ni, Cs, Sr, Co, Mo, Ba, V, Ti, Ge,

١٩٧ B, Nb and Ta) was carried out by Prodigy axial high dispersion inductively coupled

١٩٨ plasma- optical emission spectrometer (ICP-QES) in the central lab for elemental and

١٩٩ Isotopic analysis NRC-EAEA.

٢٠٠ Serum total testosterone (T), Triiodothyronine (T3) and thyroxine (T4) hormones were

٢٠١ measured following the manufacturer's protocol (Hollander et al. 1974) and using

٢٠٢ commercial solid-phase radio-immunoassay kits purchased from DiaSource, Korea.

٢٠٣ Histopathology

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٢٠٤ Fixed testes samples in formalin were washed with tap water, and then serial dilutions of

٢٠٥ alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. The specimens

٢٠٦ were cleared in xylene and embedded in paraffin at 56 °C in a hot air oven for 24 h.

٢٠٧ Paraffin – bees wax tissue blocks were prepared for sectioning at 4 μm with a sliding

٢٠٨ microtome. The tissue sections were collected on glass slides, de-paraffinized, and then

٢٠٩ stained with hematoxylin and eosin and examined using light microscopy (Bancroft and

٢١٠ Layton 2013).

٢١١ Statistical analysis

٢١٢ Values of parameters are expressed as means ± standard deviation. Analysis of variance

٢١٣ (ANOVA) test was performed to compare means of experimental groups against controls

٢١٤ at confidence level 95% (p < 0.05). Post-hoc analysis; Duncan’s analysis was performed

٢١٥ when needed. The procedure was performed by using the Statistical Package for the

٢١٦ Social Sciences (SPSS 17) statistical package (SPSS, Inc., Chicago, IL, USA).

٢١٧ Results

٢١٨ As shown in table (1), administration of different garlic preparations in rats significantly

٢١٩ decreased sodium levels in sera compared to control group. The most decreased groups

٢٢٠ were kyolic dried 5% and 10%. The lesser change was noted in tomex 5%. In respect to

٢٢١ potassium, all garlic preparations significantly decreased its level in rats except animals

٢٢٢ given tomex 10% which showed insignificant change compared to control group. Dried

٢٢٣ garlic 5%, tomex 5%, and 10% in addition to the cooked 10% showed an increase of

٢٢٤ calcium level compared to control. Tomex10% revealed significant increase of calcium

٢٢٥ when compared to all other animal groups. Except tomex 5%, all gavaged groups with

٢٢٦ different garlic preparations showed a significant decrease of magnesium level in serum

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٢٢٧ compared to the control. Kyolic, either cooked 5% or 10% and dried 5% showed a

٢٢٨ significant decrease of manganese level compared to control animals.

٢٢٩ While in table (2), cooked garlic 5%, dried 5% & 10% and kyolic showed a significant

٢٣٠ decrease of selenium when compared to control animals. The potent reducing effect on

٢٣١ Se was in appeared in case of cooked 5%. Rats administered kyolic, tomex 10% and

٢٣٢ cooked 10% showed insignificant change of zinc level. Tomex 5% cooked 5% dried 5%

٢٣٣ and the 10% revealed a significant decrease of zinc compared to control. Cooked 5%

٢٣٤ treatment resulted in a significant decrease of zinc compared to all groups. For serum

٢٣٥ iron, kyolic, dried 5% & 10% and cooked 5% showed a significant decrease of serum Fe

٢٣٦ level compared to control. While cooked 10%, tomex 5% and tomex 10% revealed

٢٣٧ insignificant changes of Fe. In respect to copper (Cu), all treated groups revealed

٢٣٨ insignificant changes compared to control. Noticeably, all treated groups with different

٢٣٩ garlic preparations showed a significant decrease of silver level compared control.

٢٤٠ Kyolic, cooked 5 & 10% and dried 5 & 10% showed a significant decrease of aluminium,

٢٤١ while tomex 10% revealed a significant increase of its level as compared to control group.

٢٤٢ Both tomex 10% and Cooked 10% showed a significant decrease of lead (Pb) level

٢٤٣ compared to control. Insignificant changes were noted in serum cadmium (Cd) in all

٢٤٤ animal groups. Cooked garlic 5% and 10% revealed increase of arsenic (As) level as

٢٤٥ shown in table (3).

٢٤٦ Table (4) exhibited that all treated groups showing insignificant changes in the levels of

٢٤٧ chromium (Cr), caesium (Cs) nickel (Ni) and the strontium (Sr), when compared to

٢٤٨ control levels. Cooked garlic 5% showed a significant decrease of silicone (Si) level

٢٤٩ compared to control and other treated groups. While tomex 5% revealed a significant

٢٥٠ increase of Si compared to control and the other groups.

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٢٥١ All treated garlic groups showed insignificant change of cobalt (Co) compared to control.

٢٥٢ Only, kyolic treated animals showed a significant decrease of serum cobalt compared to

٢٥٣ control. Animals treated with kyolic, cooked 5&10%, dried 5&10% and tomex 10% showed

٢٥٤ significantly decreased serum molybdenum (Mo) level compared to control. Regarding

٢٥٥ barium (Ba) cooked 10%, dried 5& 10% and kyolic showed a significant increase of

٢٥٦ barium level compared to control. Vanadium (V) and Titanium (Ti) were insignificantly

٢٥٧ changed as shown in (table 5).

٢٥٨ Table (6) present that groups treated with tomex 10%, cooked 5% and dried 5 &10%

٢٥٩ showed a significant decrease of germanium (Ge) level when compared to control. All

٢٦٠ treated groups revealed a significant decrease of boron (B) level compared to control. On

٢٦١ another case, niobium (Nb) was decreased in sera of tomex 10%, cooked 5&10%, dried 5

٢٦٢ & 10% and kyolic treated groups compared to its level in control animals. All treated

٢٦٣ groups resulted in a significant increase of tantalum (Ta) compared to control.

٢٦٤ As shown in figure (1), all treated groups with different garlic preparations highly significant

٢٦٥ decreased of testosterone level in rats as compared to control. Tomex 10%, cooked 10%,

٢٦٦ dried 10%, kyolic and tomex 5% are the highest effective on testosterone respectively. The

٢٦٧ lesser effective is dried garlic 5%. In figure (2), dried 5% & 10% and cooked 5% &10%

٢٦٨ groups showed insignificant change of T3 hormone level compared to control while Kyolic,

٢٦٩ tomex 5% and 10% groups revealed a significant increase as compared to control level.

٢٧٠ While figure (11) depicts the insignificant change of the serum T4 level caused by dried 5%&

٢٧١ 10%, cooked 10% and tomex5% compared to control. On the other hand in figure (3), kyolic,

٢٧٢ cooked 5% and tomex10% 3 treatments significantly increased T4 level compared to control.

٢٧٣ 05).

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٢٧٥ Histological results:

٢٧٦ Testis of rat from control group (fig 4) is showing normal histological structure of

٢٧٧ seminiferous tubules with normal spermatogoneal cell lining. Testis of rat from tomex

٢٧٨ 5% group (fig 5) showed congestion of interstitial blood vessel and slight degeneration of

٢٧٩ spermatogoneal cells lining seminiferous tubules. Testis of rat administered tomex 10%

٢٨٠ (Fig 6) is showing marked vacuolations, degeneration and necrosis of spermatogoneal

٢٨١ cells lining seminiferous tubules together with interstitial edema. Testis from animals of

٢٨٢ cooked garlic 5% group (Fig 7) exhibited necrosis and desquamation of germ cells in the

٢٨٣ lumen of seminiferous tubules. Testis of rat from cooked garlic 10% group (Fig 8)

٢٨٤ revealed slight degeneration of spermatogoneal cells associated with interstitial edema.

٢٨٥ Testis tissues from rats given dried garlic 5% (Fig 9) showed marked necrosis with

٢٨٦ desquamation of spermatogoneal cells lining seminiferous tubules. Vacuolations,

٢٨٧ degeneration and necrosis of spermatogoneal cells lining seminiferous tubules together

٢٨٨ with interstitial edema and a notice of incomplete spermatogenesis were seen in dried

٢٨٩ garlic 10% group (Fig 10). Testis of rat from kyolic group (Fig 11) showed marked

٢٩٠ degeneration and necrosis of spermatogoneal cells lining seminiferous tubules, small

٢٩١ atrophied seminiferous tubules and congestion of interstitial blood vessel

٢٩٢

٢٩٣ Discussion:

٢٩٤ Herbal detoxication regimes are attracting interest with their bench accessibility and

٢٩٥ poorly investigated adverse effects. Prospective health benefits of allium plants,

٢٩٦ particularly garlic (Allium sativum) has its foundation in ancient times but still there is a

٢٩٧ need to unveil the details of these benefits (Lewis and Elvin-Lewis 2003). The present

٢٩٨ study investigated the effect of different garlic preparations on trace elements and some

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٢٩٩ hormones in rat model. A significant decrease in both Na and K serum levels due to

٣٠٠ garlic administration was seen. However, acute severe hyponatremia appears as a serious

٣٠١ health implication of herbal detox regimens (Soliman et al. 2018). This marked decrease

٣٠٢ in serum sodium concentration may be due to a change in glomerular filtration rate and

٣٠٣ renal blood flow or interference with aldosterone action on the distal tubules interference

٣٠٤ with adrenergic sodium handling caused by garlic administration (Zanchetti and Leonetti

٣٠٥ 1985). This finding is suggestive that garlic preparations have a mild hypokalaemic and

٣٠٦ hyponatriemic effects. On the other hand, this finding disagreed with a study (Oluwole

٣٠٧ 2010) who reported a mild increase of serum Na and K with the higher doses of garlic

٣٠٨ used, which may be by increasing sodium and potassium renal reabsorption and a

٣٠٩ possible increase in renal blood flow. The significant increase in serum calcium due to

٣١٠ different garlic preparations agreed with the work of MuKharejee and his colleagues

٣١١ (Mukherjee et al. 2006). They attributed this rise to the action of garlic extract on

٣١٢ enhancing intestinal transfer of calcium by moderating the activities of both intestinal

٣١٣ alkaline phosphatase and calcium activated ATPase. However, this result was

٣١٤ inconsistent with the finding of (Ghalehkandi et al. 2012) who reported insignificant

٣١٥ change upon prolonged intake of garlic aqueous extract. Magnesium availability and

٣١٦ absorption are prone to alteration by dietary levels of both calcium and

٣١٧ phosphorus which appear to cause a decrease in magnesium absorption

٣١٨ (O’Donnell and Smith 1973). Several studies have suggested that calcium

٣١٩ and magnesium may share a common transport mechanism (Bronner and Coburn

٣٢٠ 2014). Except tomex 5%, all gavaged groups with different garlic showed

٣٢١ a significant decrease of magnesium level at the same time they witness

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٣٢٢ a significant increase in Ca levels. Our results come in agreement with

٣٢٣ literature (O’Donnell and Smith 1973), which can be attributed to the

٣٢٤ competitive absorption between calcium and magnesium.

٣٢٥ Manganese is a vital trace element for the human body due to its role in metabolism,

٣٢٦ bone formation and inflammation (Deng et al. 2013). The Kyolic, Cooked 5% & 10%

٣٢٧ and dried 5% showed a significant decrease of manganese level compared to control

٣٢٨ animals suggesting the possibility of using garlic for Mn de-toxication programs.

٣٢٩ Copper is a vital metal for viable systems and its transport is tightly regulated. A number

٣٣٠ of metals, including zinc, cadmium, and molybdenum, has been reported to affect copper

٣٣١ transport or availability in biological systems. It has also been shown in rodents that lead

٣٣٢ can alter copper metabolism, leading to decreases in plasma copper levels (Skoczyńska et

٣٣٣ al. 1994). The copper serum level result that showed insignificant change, disagreed with

٣٣٤ the finding of (Metwally 2009). Whilst the cooked 5% showed the highest reduction

٣٣٥ compared to all groups inferring an impact of cooking on Zn level in contrast with these

٣٣٦ studies that reported that cooking has limited effect on metals. With regard to serum Zn

٣٣٧ level, only tomex 5% , cooked 5%, dried 5% and 10% showed a significant decrease in

٣٣٨ agreement with (Metwally 2009). Selenium is an indispensible trace element of central

٣٣٩ biological significance. Selenium is integrated into selenoproteins to employ its

٣٤٠ pleiotropic effects, such as antioxidative, anti-inflammatory effects, and the production of

٣٤١ active thyroid hormones (Arthur et al. 1992).

٣٤٢ The different garlic preparations were able to significantly reduce the serum levels of

٣٤٣ titanium and niobium. This finding is of importance to children and patients who use

٣٤٤ implants of titanium where elevated serum titanium and niobium is a major concern after

٣٤٥ implanting the alloy i.e. metal debris release in serum (Cundy et al. 2014). In the current
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٣٤٦ study, all treated groups resulted in a significant increase of tantalum which may be

٣٤٧ attributed to its storage in garlic bulbs due to soil contamination/richness with tantalum.

٣٤٨ Tantalum is frequently used among the most corrosion-resistant metals at the present time

٣٤٩ (Davis 2006). Limited number of studies has addressed the issue of the potential side

٣٥٠ effects of tantalum in human tissues. Tantalum is not reactive or irritant to body fluids

٣٥١ and its biocompatibility in vivo is excellent (Lide 2001).

٣٥٢ Aluminum (Al) is a strong neurotoxin and has been linked to Alzheimer's disease

٣٥٣ causality for 10s of years. The significant decrease of aluminum observed in Kyolic,

٣٥٤ cooked 5 & 10% and dried 5 & 10% showed agreed with the finding that garlic extracts

٣٥٥ detoxifies Al and reduce its concentration (Shrivastava 2012).

٣٥٦ In the current study all groups revealed insignificant changes in serum As levels

٣٥٧ compared to control. Arsenic is ubiquitous in the environs so chronic or acute exposure

٣٥٨ through food and water is guaranteed (Chung et al. 2014). (Chowdhury et al. 2008)

٣٥٩ reported that garlic extract can be a potential protective regimen for arsenic mediated

٣٦٠ toxicity due to its chelating efficacy. Disagreement with their findings, only both groups

٣٦١ treated with cooked garlic showed a significant increase of As, it might be attributed to

٣٦٢ leakage from cooking vessels during cooking process.

٣٦٣ There is a growing evidence of literature that Garlic administration (Allium sativum) has

٣٦٤ some beneficial effects in preventing heavy metal toxicity(Chowdhury and Chaudhuri

٣٦٥ 2010). Some results suggest that different types of garlic preparations have different

٣٦٦ pharmacologic properties, and among the four garlic preparations studied, AGE could be

٣٦٧ the most useful garlic preparation. It was found by (Perello et al. 2008) that the cooking

٣٦٨ process is only of a very limited impact on decreasing metal (As, Cd and Pb)

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٣٦٩ concentrations This proposed declining is governed by cooking conditions (time,

٣٧٠ temperature, and medium of cooking) (Perello et al. 2008).

٣٧١ Lead (Pb) toxicity is probably the most common form of heavy metal intoxication. It is

٣٧٢ well-documented as one of the most dangerous and insidious poisons (Ashry et al. 2009).

٣٧٣ Lead exerts toxic effects by multiple modes of action, including inhibition of enzymatic

٣٧٤ function, generation of oxidative stress, intervention with the action of essential cations,

٣٧٥ particularly calcium, zinc and iron (Katzung 2017). In the current work, only tomex 10%

٣٧٦ and cooked 10% showed a significant decrease of Pb level compared to control. This

٣٧٧ ability of the oral feeding of minced fresh garlic (tomex 10%) to reduce lead in serum

٣٧٨ agreed with previous studies (Tandon et al. 2001; Ayoka et al. 2016). It was noted that

٣٧٩ the co-existence of calcium can affect lead’s intestinal absorption (Hernberg 2000), it

٣٨٠ was suggested that higher Ca directly drops Pb absorption by making competition for its

٣٨١ transfer over the gastro-intestinal phospholipidic barrier (Barton et al. 1978). Meanwhile

٣٨٢ lead competes with calcium on transporters, lead can likewise substitute calcium, with

٣٨٣ perturbing effects on bone formation, kidney function and, most importantly, neural

٣٨٤ function (calcium is indispensable for processes like learning and memory)(Papanikolaou

٣٨٥ et al. 2005). Here, in the groups that recorded the significant decrease in Pb level (tomex

٣٨٦ 10% and cooked 10%), also recorded an increase of calcium which agree with this body

٣٨٧ of accumulated studies.

٣٨٨ The reducing effect of garlic on cadmium agreed with study by Cha (1987) (Cha 1987).

٣٨٩ Strontium has a chemical likeness to calcium and exchanges calcium as the mineral

٣٩٠ in bone. Because strontium atoms are heavier than calcium atoms, swapping some of

٣٩١ the calcium atoms with strontium atoms makes the bone mineral density act to increase

٣٩٢ (S Mirza 2016). In the current study all garlic preparations showed non-significant

15
٣٩٣ changes in serum Sr level which may due to increase of calcium level. Barium is a soft

٣٩٤ alkaline terrestrial metal. Owing to the fact of its high chemical reactivity, it is never

٣٩٥ found in nature in the form of a free element. The toxicity of barium compounds is

٣٩٦ influenced by its solubility, which increases as the acidity of medium increases. The

٣٩٧ soluble salts, such as acetate, chloride, oxide, hydroxide, nitrate and sulfide, are very

٣٩٨ toxic to humans (Oskarsson and Reeves 2007). Barium ion brings severe hypokalemia

٣٩٩ due to its ability to block potassium channels. As a result, it interfere with passive K+

٤٠٠ diffusion and blocks the intracellular pathway of potassium and decreases its level in the

٤٠١ blood (Rhyee and Heard 2009; Joshi et al. 2012). This agrees with our finding where the

٤٠٢ Kyolic and dried groups exhibited a significant increase in serum barium level, witnessed

٤٠٣ the lowest level of K level (hypokalemic effect).

٤٠٤ Whereas chromium is an important element (Ghalehkandi et al. 2012), chromium

٤٠٥ compounds are known to be mutagenic and carcinogenic. on the other hand, Ni is known

٤٠٦ to cause cancer, it also causes haemorrhages and renal problems and frank haematuria in

٤٠٧ experimental animals (Kasprzak et al. 1980). It disrupts the endocrine system and has

٤٠٨ severe adverse effects on the hypothalamic-pituitary-gonadal axis (Das and Dasgupta

٤٠٩ 2002). In the current study, all garlic preparations showed non-significant changes in

٤١٠ serum Cr and Ni levels.

٤١١ Molybdenum is an essential trace element for plants and mammals. It is a constituent of

٤١٢ three mammalian enzymes, xanthine oxidase, aldehyde oxidase, and sulfite oxidase.

٤١٣ These enzymes all comprise the molybdenum cofactor, a complex of molybdenum and

٤١٤ pterin; molybdopterin (Sigel and Sigel 2002). Molybdnum is quickly absorbed, however,

٤١٥ its metabolism is affected by copper and sulfur intake in some species (Turnlund and

٤١٦ Friberg 2007). It was found in sheep that sulfur intake drives the formation of Mo-

16
٤١٧ complexes, possibly thiomolybdates which are both poorly absorbed and poorly excreted

٤١٨ (Grace and Suttle 1979). Garlic is rich in sulfur compounds that might interfere in Mo

٤١٩ metabolism. It may be a cause for resulted significant serum molybdenum decrease in all

٤٢٠ garlic-treated groups except tomex 5%.

٤٢١ Germanium is a member of ultra-trace elements group; it has a silicon-like behavior in a

٤٢٢ sum of biological routes (Azam et al. 1973). Data from experimental animal studies

٤٢٣ showed that both inorganic and organic germanium compounds, are swiftly and just

٤٢٤ about completely absorbed by the lungs and the gastrointestinal tract (Faroon et al. 2007).

٤٢٥ Garlic is a rich source for germanium (Morioka et al. 1993); however, in the current work

٤٢٦ there was a significant decrease in sera of groups treated with cooked 5%, dried 5 &10%

٤٢٧ and tomex 10% in comparison to control. This observation may infer a secondary reason

٤٢٨ for this decrease like antagonism with another component during absorption.

٤٢٩ Boron (B) is a vibrant trace element, within the physiological limits,

٤٣٠ can affect the metabolism or utilization of numerous other substances

٤٣١ involved in biological events including macrominerals, energy substrates

٤٣٢ like triglycerides, glucose, nitrogen compounds such as amino acids and

٤٣٣ proteins, reactive oxygen species, and estrogen (Nielsen 1997). However,

٤٣٤ boron deficiency in humans is not well recognized or may be a concern in

٤٣٥ some special cases like arthritis (Shah and Vohora 1990; Newnham 1994).

٤٣٦ The result of current study, where all treated groups recorded a significant decline

٤٣٧ of boron level compared to control, infers that garlic treatments can be used in boron toxicity.

٤٣٨ Cobalt gets its importance for humans by involvement in cobalamin (Cbl, vitamin

٤٣٩ B12) (Yamada 2013). In the current study, only, Kyolic treated animals showed a

17
٤٤٠ significant decrease of serum cobalt compared to control. It may bring attention to using the

٤٤١ kyolic preparation of garlic in special cases with B12 deficiency. Silver is a rare but

٤٤٢ naturally occurring element. Its soluble compounds are rapidly absorbed compared to

٤٤٣ metallic or insoluble ones (Rosenman et al. 1987) and therefore have the possibility to

٤٤٤ cause adverse effects to the human body (Weir 1979). All treated rats with different

٤٤٥ garlic preparations showed a significant decrease of serum silver level suggesting the

٤٤٦ possibility to use garlic based regimes for silver reduction/de-toxication in mammals.

٤٤٧ Fetal and adult Leydig cells have at least four steroidogenic enzymes that contribute to

٤٤٨ testosterone (T) biosynthesis (Huhtaniemi and Pelliniemi 1992). Disruption any step in

٤٤٩ the steroidogenesis process, has the potential to cause reproductive problems, diminished

٤٥٠ fertility and hypogonadism (Moline et al. 2000). T biosynthesis commences with the

٤٥١ substrate cholesterol. The first steroidogenic enzyme is cholesterol side chain cleavage

٤٥٢ enzyme (CYP11A1). The enzyme has two binding sites for heme and Fe. Iron is an

٤٥٣ important factor for building heme which is a cofactor for the enzyme CYP11A1 in

٤٥٤ mitochondria that catalyse the 1st sequential step in testosterone synthesis (conversion of

٤٥٥ cholesterol to pregnenolone by working as monooxygenase). A subsequent enzyme

٤٥٦ CYP17A1 also requires Iron for heme as a prophetic group required for function on

٤٥٧ substrate (Akhtar et al. 2005). (Dixit and Joshi 1982) found that garlic powder arrested

٤٥٨ spermatogenesis in rats. This agree with our finding that garlic preparations led to a

٤٥٩ significant decrease in serum Fe levels so testosterone synthesis was affected too. These

٤٦٠ results of decreased serum testosterone were confirmed by hitological alterations found in

٤٦١ tests of all garlic treated rats. Alterations included marked vacuolations, degeneration

٤٦٢ and necrosis of spermatogoneal cells lining seminiferous tubules, together with interstitial

18
٤٦٣ edema and a notice of incomplete spermatogenesis. Marked necrosis with desquamation

٤٦٤ of spermatogoneal cells lining seminiferous tubules were observed.

٤٦٥ Fabrication of thyroid hormones is conducted by the enzyme thyroid peroxidase, an

٤٦٦ integral membrane protein present in the apical (colloid-facing) plasma membrane of

٤٦٧ thyroid epithelial cells. Thyroid peroxidase catalyzes two sequential reactions. To

٤٦٨ perform its function, thyroid peroxidase, requires calcium and heme. The significant and

٤٦٩ insignificant increase in T3 and T4 levels can be understood in the light of calcium and

٤٧٠ Fe results where garlic preparations led to a significant increase in serum Ca and

٤٧١ significant decrease in Fe levels. Thyroid hormone biosynthesis by the thyroid gland

٤٧٢ requires adequate supplies of selenium homeostasis (Zhang et al. 2018).

٤٧٣ Selenium shortage weakens thyroid hormone metabolism by inhibiting the synthesis and

٤٧٤ activity of the iodothyroninedeiodinases, that catalyse thyroxine (T4) conversion to the

٤٧٥ extra metabolically active 3,3'-5 triiodothyronine (T3) (Arthur et al. 1992). In the present

٤٧٦ study, Selenium levels fluctuated from significant decrease to insignificant changes

٤٧٧ among groups.

٤٧٨ Conclusion:
٤٧٩ Garlic has beneficial effects can vary from preparation to another., in the current study

٤٨٠ different prepations of garlic revealed Hypokalaemia and hyponatriemia effects .In

٤٨١ addition, testosterone and testis have been adversely affected. Those findings suggest

٤٨٢ more wise use of garlic preparations in dietary and health enrichment regimes.

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