الفهرس 
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Abstract
Male infertility is a multifactorial disease that can be due to a variety of genetic and acquired (environmental and lifestyle-related) factors as nutritional imbalances and exposure to chemicals. These factors increase the levels of oxidative stress in spermatozoa and could produce pituitary-hypothalamic or sex hormonal effects which in turn could affect spermatogenesis or cause abnormalities in seminal fluid resulting in functional or structural impairment of the sperm cells of test organisms.
Calcium Carbide is a chemical compound with the formula (CaC2), used as artificial fruits ripening agent because once it is dissolved in water the carbide produces acetylene gas that quickens the ripening process.
CaC2 + 2H2O → Ca(OH)2 + C2H2
Consumption of carbide-ripened fruits is extremely hazardous for health because it contains traces of toxic metals as arsenic and phosphorous. Arsenic, phosphorous and acetylene gas may affect the different body organs, increasing oxidative stress level and causing various health problems.
Herbal plants, Echinacea (Echinacea purpurea L.) and Chamomile (Matricaria chamomilla L.), have different functions due to the presence of numerous active compounds. Antioxidants are one of the main parts of the herbal structure with a high content of bioactive compounds which shown to have the ability to inhibit reactive oxygen species (ROS) generation and to scavenge free radicals. Carotenoids, flavonoids, tocopherols, cinnamic acid, folic acid and ascorbic acid are the most prominent antioxidants produced by plants.
For that the present study was conducted to investigate the effect of feeding CaC2 containing diet to male rats for 6 weeks and the impact of Echinacea and Chamomile extracts consumption on preventing or improving the side effects of CaC2 containing diet in male rats.
Throughout this study a number of 50 healthy male albino rats Sprague-Dawley strain weighing 160±20g were available for experimentation. All rats were offered the balanced diet with drinking water ad libitum for 7 days for adaptation (acclimatization period). The animals were divided as following (10rats/group); group (1): Healthy rats fed on balanced diet served as healthy control group. group (2): Rats were fed on CaC2-intoxicated diet at a dose of 25g CaC2/kg diet. group (3): Rats were fed on CaC2-intoxicated diet + 200 mg/kg body weight of Echinacea ethanol extract given by intra-gastric tube daily. group (4): Rats were fed on CaC2-intoxicated diet + 200 mg/kg body weight of Chamomile ethanol extract given by intra-gastric tube daily. group (5): Rats were fed on CaC2-intoxicated diet + mix of (100mg/kg body weight of Echinacea ethanol extract and 100mg/kg body weight of Chamomile ethanol extract) given by intra-gastric tube daily.
The study included:
Firstly, identification of total phenolic and total flavonoids content and also the active constituents of Echinacea and Chamomile ethanol extracts by HPLC analysis. The results revealed that each 1 g of Echinacea extract contained 67.9±0.360 mg GAE as total phenols and 63.15±0.350 mg CE as flavonoids. While each 1 g of Chamomile extract contained 58.50±0.953 mg GAE as total phenols and 31.68±1.265 mg CE as flavonoids. The HPLC analysis showed that the highest bioactive compound in both extracts was cinnamic acid recording 1492.24 mg and 315.75 mg per 100 g Echinacea and Chamomile extracts respectively.
Considering the antioxidant properties of the tested extracts, the present results indicated higher content of polyphenols and flavonoids present in E. purpurea extract than in M. chamomilla extract. The content of polyphenols and flavonoids in both plant extracts might indicate higher free radical scavenging propriety (antioxidant) and anti-inflammatory effects of the tested plants.
Secondly, determination of the impact of Echinacea and Chamomile ethanolic extracts administration on (1) Biological evaluations which included feed intake, change in body weight, FER, FCR and relative testis weight, (2) Biochemical measurements included; oxidative stress marker; MDA, 4HNE, AGEs and PCC levels; antioxidant as CAT activity and GSH level; inflammatory markers; IL-6, IL-1β and CRP levels; sex hormones; LH, FSH and T levels; as well as sperms count; immunoglobulis; IgG and IgM levels, (3) hematological measurments included; Hb level, RBC’s, WBC’s and platelets counts; (4) Microscopic examinations of testis tissue and epididymis.
1) Biological evaluations
It is clear from the results that there were statistically significant (P≤0.05) decrease in feed intake and FCR and increase in body weight, FER and relative weight of testis in CaC2-intoxicated rats group in comparing with the healthy control rats. On the other hand, the oral administration of Echinacea and Chamomile extracts each one alone or in mix improved the biological indices and the relative weight of testis by causing significant (P≤0.05) decrease in body weight, FER and relative weight of testis and increase in FCR with non- significant effect on feed intake.
2) Biochemical and sperms count measurements
With respect to oxidative stress condition, feeding CaC2-intoxicated diet to male rats showed a significant (P≤0.05) elevation in MDA, 4-HNE, AGEs and PCC levels by percentage of increment reached 732%, 467.9%, 132.04% and 215.93 % respectively, and depletion in CAT activity and GSH level by percentage of decrement reached 67.82 % and 69.16% respectively when compared with healthy control group. On the other hand, adminstration of Echinacea and Chamomile extracts either alone or in compination to CaC2-intoxicated rats showed a significant (P≤0.05) decrease in serum MDA level by percentage of decrement reached 78.43%, 63.08% and 82.63% respectively, 4-HNE level by 56.45%, 46.45% and 63.47% respectively, AGEs level by 42.14%, 39.05% and 44.59% respectively and PCC level by 54.39%, 50.98% and 55.58% respectively, in addition to a significant (P≤0.05) increase in plasma CAT activity by percentage of increment reached 109.2%, 98.11% and 138.69% respectively and blood GSH level by 312.34%, 111.72% and 312.71% respectively from the value of CaC2-intoxicated rats group. The mix of both extracts was the effective in improving oxidative stress state.
In regard to inflammatory condition, feeding CaC2-intoxicated diet to male rats showed a significant (P≤0.05) elevation in IL-6, IL-1β and CRP levels by percentage of 361.07%, 206.23% and 557.29% respectively. In contrast, adminstration of Echinacea and Chamomile extracts either alone or in a mix to CaC2-intoxicated rats showed a significant (P≤0.05) decrease in serum IL-6 level by percentage of 63.33%, 55.27% and 64.63% respectively, IL-1β level by 51.16%, 46.5% and 58.25% respectively and CRP level by 64.19%, 62.17% and 81.07% respectively. The mix of both extracts was the effective in improving inflammatory markers level.
The statistical analysis exhibited a significant (P≤0.05) depletion in serum LH, FSH and T levels by percentage of decrement reached 55.29 %, 55.5 % and 80.4% respectively, as well as a significant (P≤0.05) depletion in sperms count by 40.9% in CaC2-intoxicated rats group when compared with healthy control group. On the other hand a significant (P≤0.05) elevation in LH level by percentage of increment reached 37.03%, 20.74% and 51.85% respectively, FSH level by 41.57%, 19.10% and 53.93% respectively and T level by 167.34%, 134.69% and 253.06% respectively in CaC2-intoxicated rats supplemented with Echinacea and Chamomile extracts each one alone or in mix, as well as a significant (P≤0.05) elevation in the sperms count by 29.8%, 20.19% and 65.38% respectively from the value of CaC2-intoxicated rats group. Supplementation of the two extracts in a mix was the effective in elevation of sex hormones level and sperms count.
The results revealed that there was significant (P≤0.05) enhancment in the serum IgG and IgM levels by percentage of 55.52% and 26.88% respectively in CaC2-intoxicated rats group (2) when compared with healthy control group. However, a significant (P≤0.05) improvement in IgG level by percentage of 25.92%, 17.04% and 32.89% respectively and IgM level by percentage of 16.39%, 15.32% and 20.74% respectively in CaC2-intoxicated rats supplemented with Echinacea extract group (3) and Chamomile extract group (4) alone or in combination group (5) from the value of CaC2-intoxicated rats group (2). Administration of the plant extracts in compination was effective in improving immunoglobulins level.
3) Hematological measurements
Considering the hematological parameters in blood; feeding the animals with CaC2-intoxicated diet resulted in a statistically significant (P≤0.05) reduction in level of Hb by percentage of decrement 16.76% as well as decreased RBC’s, WBC’s and platelets counts by percentage of 29.52%, 43.99% and 17.62% respectively as compared with healthy control group. However, a significant (P≤0.05) increase in level of Hb in groups (3), (4) and (5) reached to 11.4%, 6.03% and 15.57% respectively as well as a significant (P≤0.05) increase in RBC’s counts by percentage of 30.18%, 17.23% and 37.73% respectively, WBC’s counts by percentage of 37.34%, 23.23% and 45.19% respectively and platelets counts by percentage of 16.29%, 15.05% and 21.18% respectively in the same groups in coparing to the values of CaC2-intoxicated rats. The mixture of Echinacea and Chamomile extracts in group (5) showed the best results in improving the hematological profile.
4) Microscopic examinations in the testis tissue and epididymis
Microscopic examination of testis tissue and epididymis in the tested groups showed that feeding CaC2-containing diet to male rats resulted in degeneration and complete loss of spermatogenic series from the lumen of most of the seminiferous tubules and also irregular arrangement of the spermatogonial cells in the tubular lumen of testis. As well as most of the tubular lumen in epididymis was free from spermatozoa. However, supplementation with Echinacea and Chamomile extracts together with CaC2-intoxicated diet resulted in that degeneration and loss of spermatogenic series only in some few or individual seminiferous tubules and only few of the tubular lumen free from spermatozoa compared with CaC2-intoxicated rats. The mixture of Echinacea and Chamomile ethanolic extracts showed the most significant improvement in the microscopic examinations of testicular tissues and also the same detected in epididymis.
In general, the results obtained in this study showed that oral administration of CaC2 in diet can induce sex hormonal disturbances, impaired spermatogenesis and finally infertility in male rats. CaC2 altered biochemical tests, sperm counts, hematological profile and microscopic examinations of testis. However, supplementation with E. purpurea and M. chamomilla extracts attenuated the pathogenic effects induced by CaC2, these extracts can improve altered symptoms due to their high content of polyphenols and flavonoids that have strong antioxidant propertiest. The improvement was more pronounced in the mix group than other remaining groups, this might be due to their high content of strong antioxidants.