{"id":28400,"date":"2019-09-25T11:00:17","date_gmt":"2019-09-25T11:00:17","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=28400"},"modified":"2020-04-22T10:20:25","modified_gmt":"2020-04-22T10:20:25","slug":"comparative-efficacy-of-syzygium-cumini-seed-extracts-in-alleviating-arsenic-induced-hepatotoxicity-and-blood-cell-genotoxicity-in-wistar-albino-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol12no3\/comparative-efficacy-of-syzygium-cumini-seed-extracts-in-alleviating-arsenic-induced-hepatotoxicity-and-blood-cell-genotoxicity-in-wistar-albino-rats\/","title":{"rendered":"Comparative Efficacy of Syzygium Cumini Seed Extracts in Alleviating Arsenic-Induced Hepatotoxicity and Blood Cell Genotoxicity in Wistar Albino Rats"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Syzygium cumini<\/em> (L.) Skeels (Myrtaceae) is a tropical plant\u00a0widely\u00a0distributed in different countries of the world such as India, Sri Lanka, Australia and Malaysia1<\/sup>. It has been attributed in the Indian\u00a0folklore\u00a0system\u00a0of medicine to possess various medicinal properties2<\/sup>. Different parts of this plant, such as fruits, seeds and leaves are reported to have antidiarrheal3,4<\/sup>, hypoglycemic, antipyretic, anti-inflammatory5,6<\/sup>, and antibacterial7<\/sup> properties. It has been\u00a0valued\u00a0in\u00a0Ayurveda for possessing astringent, digestive, acrid and wound healing properties2<\/sup>. The leaves are used to strengthen gums and teeth, to treat stomachalgia, leucorrhoea, fever, strangury, dermopathy, constipation and gastropathy. The seeds and fruits are also used to treat pharyngitis, urethrorrhea, spleenopathy and ringworm infections2,8<\/sup>. Different parts of this plant are known to possess various bioactive compounds having free radical scavenging and antioxidant activities1,51<\/sup>.<\/p>\n

Arsenic is a widespread pollutant in several parts of the world. Arsenic contaminated water is reported in more than 30 countries worldwide. However, the major affected areas are in the basins of rivers Brahmaputra, Ganga, and Meghna in India and Bangladesh and some parts of China9,10<\/sup>. In Taiwan, Argentina, Mexico, and the Indo-Bangladesh regions, drinking water concentrations of arsenic have been reported to be much above the standard (10 \u03bcg\/l) adopted by the US Environmental Protection Agency (USEPA) in 200111<\/sup>. Arsenic exists in both organic and inorganic forms in the environment. Inorganic arsenic compounds include trivalent (arsenite or As III) and pentavalent (arsenate or As V) species which are highly toxic for humans and animals, and are considered as class I carcinogens12<\/sup>. Chronic human exposure to inorganic arsenicals is associated with various toxic effects including liver injury, peripheral neuropathy and increased incidences of skin, lung, liver, and bladder cancers9,13<\/sup>.<\/p>\n

As for other heavy metals, chelation therapy is the mainstream treatment for\u00a0arsenic poisoning also. In chelation therapy, drugs such as British anti-Lewisite (BAL) and dimercaptosuccinic acid (DMSA) are used; however, these compounds are associated with several moderate to severe side effects including nausea, hypertension, itching, abdominal pain and changes in body temperature14-16<\/sup>. Administration of antioxidants from plant sources is reported to be highly effective in reducing arsenic toxicity12,17,18<\/sup>. Some studies reported that combined administration of antioxidants and chelating agents is also beneficial against arsenic poisoning-induced toxicity14,19<\/sup>. The recent trend is to exploit the therapeutic value of medicinal and\/or dietary plants with\u00a0antioxidative\u00a0potential to alleviate the arsenic toxicity.<\/p>\n

Owing to the notable antioxidative properties of Syzygium cumini<\/em>, the present study was planned to determine the effect of various seed extracts of this plant on arsenic-induced hepatotoxicity and blood cell genotoxicity in Wistar albino rats.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Plant Material<\/strong><\/p>\n

Seeds of Syzygium cumini <\/em>were purchased from the local market and authenticated by Raw Materials Herbarium & Museum, NISCAIR (National Institute of Science Communication and Information Resources), New Delhi.<\/p>\n

Seed Extract Preparation<\/strong><\/p>\n

After removing pericarps, seeds were dried at room temperature and then finely powdered. The dried seed powder was extracted with different solvents (methanol, ethanol and water) in a Soxhlet\u00a0apparatus. The extracts were concentrated in a rotary vacuum evaporator and then freeze dried. The yield of SCM, SCE and SCA were, respectively, 10.8%, 10.2% and 9.8% of the dried powdered seeds. The seed extracts were stored at -20 \u00b0C until further use.<\/p>\n

Experimental Animals<\/strong><\/p>\n

Wistar albino rats of either sex (100 – 125 g) were obtained from DFSAH (Disease Free Small Animal House), LUVAS, Hisar. Rats were kept under standard laboratory conditions with dark and light cycle (12\/12 hr) and fed on a normal balanced rat diet. The studies were approved by the Institutional Animals Ethics Committee (CPCSEA\/0436) of Guru Jambheshwar University and all animal experiments were performed in accordance with the guidelines of the same on animal experimentation. Animals were acclimatized for a week prior to the experiment.<\/p>\n

Experimental Design<\/strong><\/p>\n

Rats were divided into five groups of 6 animals each and treated as follows.<\/p>\n\n\n\n\n\n\n\n\n
\n

Groups <\/strong><\/p>\n<\/td>\n

\n

Treatment<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/p>\n<\/td>\n

\n

Normal drinking water<\/p>\n<\/td>\n<\/tr>\n

Arsenic<\/td>\nArsenic in drinking water (100 ppm) ad libitum<\/em><\/td>\n<\/tr>\n
\n

SCM<\/p>\n<\/td>\n

\n

SCM (400 mg\/kg\/day) along with arsenic water (100 ppm) ad libitum<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

SCE<\/p>\n<\/td>\n

\n

SCE (400 mg\/kg\/day) along with arsenic water (100 ppm) ad libitum<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

SCA<\/p>\n<\/td>\n

\n

SCA (400 mg\/kg\/day) along with arsenic water (100 ppm) ad libitum<\/em><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Seed extracts were administered by\u00a0oral\u00a0gavage. Body weight of animals, and their\u00a0food\u00a0and\u00a0water\u00a0intake were monitored throughout the treatment period of 60 days. At the end of treatment period the rats were weighed and blood was collected from the retro-orbital plexus of the eye under ether anaesthesia.<\/p>\n

Lymphocyte Isolation<\/strong><\/p>\n

Freshly collected blood samples were diluted (1:1 ratio) with PBS (phosphate buffered saline) and carefully layered on the top of lymphocyte separation medium (LSM 1084) and centrifuged\u00a0for\u00a030\u00a0minutes\u00a0at 400 x g. The buffy coat interface, which represented the lymphocytes, was aspirated and washed with PBS twice by centrifugation for 10 minutes at 250 x g. The supernatant was discarded and lymphocytes (pellet) were used immediately for the comet assay.<\/p>\n

Comet Assay <\/strong><\/p>\n

The comet assay was performed according to Singh et al.20<\/sup>, with slight modifications. 150 \u00b5l of 0.5% NMA (normal melting agarose) was layered on to precleaned\u00a0microscope slides and dried at 65 \u00b0C for 10 min. A second layer containing isolated lymphocytes resuspended in 75 \u00b5l of 0.5% LMA, was placed on the NMA precoated slides and solidified at 4 \u00b0C for 10 min. The slides were covered with 0.5% LMA and stored at 4 \u00b0C for 15-20 min. Afterwards the slides were placed in freshly prepared lysing solution (2.5 M NaCl , 100 mM Na2<\/sub>EDTA, 10 mM Tris, 1% Triton X-100, 10% DMSO and pH 10-10.5) at 4 \u00b0C for 2 h in the dark. Following lysis, the slides were immersed in an electrophoretic buffer (300 mM NaOH , 1 mM Na2<\/sub>EDTA, pH 13.5) for 25 min at 0 \u00b0C and electrophoresed in the same buffer for next 20 min (24 volts, 300 mA). Electrophoresis was conducted under dim light to prevent additional DNA damage. After that, slides were rinsed with 0.4 M Tris (pH 7.5) twice for 5 min, fixed for 3 min in absolute ethanol and stained with 0.4 \u00b5g\/ml ethidium bromide. Comet\u00a0images\u00a0were observed at 400\u00d7 magni\ufb01cation with a fluorescence
\nmicroscope (Olympus CX 41). For each sample, images of randomly\u00a0selected\u00a050\u00a0cells were examined. Open Comet software was used for\u00a0DNA\u00a0damage quantification by analysis of the tail percent DNA, tail moments and tail lengths.<\/p>\n

Activities of Serum Markers <\/strong><\/p>\n

Commercially available diagnostic kits were used for assaying the activities of serum ALT, ALP, AST and total proteins.<\/p>\n

Statistical Analysis<\/strong><\/p>\n

Data analysis was performed using One-way ANOVA followed\u00a0by post-hoc\u00a0Tukey\u2019s test. The differences were considered statistically significant at P < 0.05.<\/p>\n

Results<\/strong><\/p>\n

The effects of arsenic on body weight gain in control and experimental rats are depicted in Fig. 1. In arsenic-only treated rats, body weight gain percent was lower than control, SCM, SCE and SCA groups. Among experimental groups, body weight gain percent was highest in SCM group.<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 1: Changes in body weight gain in different experimental groups;\u00a0<\/strong>Values are expressed as means with n = 6 rats per group.<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Activities of serum ALP, AST and ALT were significantly higher in arsenic-only group than in controls, while the same activities were all less in groups treated with Syzygium cumini <\/em>seed extracts along with arsenic. Activities of ALP and AST were significantly lower in SCM, SCE and SCA groups compared to arsenic-only group. Serum total protein was significantly lower in the arsenic-only group than in control group and significantly greater in SCM, SCE and SCA groups than in arsenic-only group (Fig. 2).<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 2:\u00a0<\/strong>Serum biomarkers activity\u00a0<\/strong>in different treatment groups<\/strong>. # p<0.05 compared with the arsenic group, * p<0.05 compared with controls; Data are expressed as means \u00b1 SDs, n = 6 rats per group.<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

DNA damage in individual blood lymphocytes was assessed by measuring various comet assay parameters viz.; tail percent DNA, tail moments and tail lengths. Fig. 3 depicts the value\u00a0of\u00a0percent\u00a0tail\u00a0DNA in different treatment groups. Tail percent DNA was significantly greater in arsenic group than in control. Co-administration of S. cumini <\/em>seed extracts (SCM, SCE, and SCA) significantly reduced the tail percent DNA. A significant increase in\u00a0tail\u00a0moment\u00a0was observed in arsenic intoxicated rats as compared to control rats. However, tail moment values were significantly reduced in SCM, SCE and SCA group rats (Fig. 4). Similarly, the mean\u00a0comet\u00a0tail\u00a0length\u00a0was\u00a0significantly\u00a0higher\u00a0in the arsenic-treated rats than in untreated rats, while the same values were significantly lower in SCM, SCE, and SCA rats than in arsenic-only exposed rats (Fig. 5). Among experimental groups treated with both arsenic and S. cumini <\/em>seed extract, the mean values of tail percent DNA, tail moments and tail lengths were lowest in SCM, followed by SCE and SCA groups. Fig. 6 illustrates the representative comet assay images obtained by fluorescent\u00a0microscopy.<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 3: Values\u00a0of tail percent\u00a0DNA in different treatment groups; <\/strong># p<0.05 compared with the arsenic group, * p<0.05 compared with controls; Data are expressed as means \u00b1 SDs, n = 6 rats per group<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 4: Tail moment values in different treatment groups; <\/strong># p<0.05 compared with the arsenic group, * p<0.05 compared with controls; Data are expressed as means \u00b1 SDs, n = 6 rats per group<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 5: Tail length values in different treatment groups; <\/strong># p<0.05 compared with the arsenic group, * p<0.05 compared with controls; Data are expressed as means \u00b1 SDs, n = 6 rats per group<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 6: Representative comet assay images obtained by fluorescent\u00a0microscopy; Groups: (A) Control (B) Arsenic (C) SCM (D) SCE (E) SCA<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Discussion<\/strong><\/p>\n

The present\u00a0study\u00a0reveals\u00a0substantial amelioration of arsenic-induced blood cell genotoxicity and hepatotoxicity by various seed extracts of Syzygium cumini.<\/em> Our\u00a0results\u00a0are\u00a0corroborated\u00a0by\u00a0previous studies that have reported protective activity of antioxidant-rich plants such as Emblica officinalis<\/em>, Camellia sinensis<\/em> and Trichosanthes dioica<\/em> against arsenic toxicity21-23<\/sup>. The present findings also\u00a0support\u00a0the modulatory effects of Syzygium cumini <\/em>on genomic damage and oxidative stress induced by various agents such as radiations24<\/sup>, DMBA (7,12-dimethyl benz(a<\/em>)anthracene) and urethane25<\/sup>, among few others.<\/p>\n

Arsenic\u00a0is\u00a0an established carcinogen present ubiquitously in the environment. In addition to its carcinogenic effects, long-term arsenic exposure is associated with hyperpigmentation, hyperkeratosis, type II diabetes mellitus, neurological damage, and black foot and cardiovascular diseases26-30<\/sup>. Chronic arsenic exposure leads to accumulation of arsenic in various body organs, primarily the kidneys, liver, lungs and skin, which adversely\u00a0affect\u00a0these organs31-35<\/sup>. Reduction in body weight is also observed in arsenic exposed animals which is supposed to be caused by oxidative damage to\u00a0body\u00a0cells\u00a0and\u00a0tissues23,36<\/sup>. Our results, too, revealed\u00a0a\u00a0decline\u00a0in\u00a0the\u00a0body weight\u00a0of arsenic challenged rats compared with
\ncontrols. However, the combined treatment of arsenic and Syzygium cumini<\/em> seed extracts resulted in body weight recovery towards control\u00a0levels. Body weight gain was maximum in SCM, followed by SCE and SCA groups which indicates that methanolic seed extract was most effective in maintaining general body weight and thereby reducing arsenic-induced toxicity in rats with maximum\u00a0potential\u00a0among\u00a0all extracts.<\/p>\n

Liver is a potential target organ of arsenic toxicity13,22<\/sup>. Arsenic-mediated increase in activities of serum ALT, AST and ALP indicates hepatic toxicity and these results agree with previous findings6,22<\/sup>. Administration of Syzygium cumini<\/em> seed extracts in arsenic exposed rats significantly restored the activities of these biochemical variables. Serum total protein levels were significantly less in arsenic exposed rats than the controls and this difference might be due to damaging effect of arsenic on hepatic cells or alterations in protein synthesis and\/or metabolism6<\/sup>. Treatment with Syzygium cumini<\/em> seed extracts significantly increased serum total protein levels towards normal in arsenic treated rats. Methanolic seed extract was most effective in decreasing arsenic-induced hepatic toxicity followed by ethanolic and aqueous extracts.<\/p>\n

The\u00a0comet\u00a0assay is a simple, fast and reliable method for detection of DNA strand breaks in individual cells20,37<\/sup>. The present results from\u00a0comet assay show that arsenic exposed rats exhibited\u00a0significant lymphocyte DNA\u00a0damage when compared to the controls. Our\u00a0results\u00a0are\u00a0in\u00a0line\u00a0with earlier reports demonstrating considerable DNA damage in arsenic intoxicated animals and humans. Balakumar et al. observed increased DNA strand breaks in liver, blood, bone marrow and kidney cells of rats challenged by sodium arsenite38<\/sup>. Elevated\u00a0levels\u00a0of\u00a0DNA\u00a0damage were detected by DNA fragmentation assay\u00a0in hepatic cells of experimental rats subchronicaly\u00a0exposed to arsenic39<\/sup>. In\u00a0another\u00a0study, significant DNA damage in peripheral blood lymphocytes was reported in a population exposed to chronic arsenic through contaminated drinking water in West Bengal, India40<\/sup>. The increased lymphocytic DNA damage in arsenic intoxicated rats, as observed in our experiment, was markedly decreased in rats treated with Syzygium<\/em> cumini <\/em>seed extracts along with arsenic. Methanolic seed extract provided the maximum protection followed by ethanolic and aqueous seed extracts against the arsenic-induced DNA damage.<\/p>\n

The exact mechanism of arsenic toxicity is not\u00a0yet\u00a0fully\u00a0understood;\u00a0however, the evidences suggest that oxidative stress is involved in arsenic-induced DNA damage and toxicity of various organs. Arsenic can disturb the natural oxidation\/reduction balance through several mechanisms involved in intricate redox reactions with endogenous oxidants and antioxidant systems of cells41<\/sup>. Arsenic metabolism generates free radicals and reactive oxygen species (ROS) which induce cell signaling and transcription factor activation eventually leading to gene mutations, DNA strand breakage, sister chromatid exchange, generation of micronuclei and chromosomal aberrations12,42<\/sup>. It is suggested that superoxide anion (O2<\/sub>\u2022<\/sup>\u207b) is the primary ROS induced by arsenic in various cellular systems; which triggers\u00a0formation\u00a0of other ROS such as hydrogen peroxide (H2<\/sub>O2<\/sub>) and hydroxyl radical (\u2022<\/sup>OH). H2<\/sub>O2 <\/sub>is converted to highly reactive \u2022<\/sup>OH radicals via Fenton reaction. \u2022<\/sup>OH radicals formed in vicinity of DNA may react with deoxyribose backbone of DNA or with DNA bases causing DNA strand breaks or producing damaged bases42,43<\/sup>. Enhanced nitric oxide (NO) production induced by arsenic also plays an important role in oxidative damage to DNA44,45<\/sup>. 8-hydroxy-2\u2032-deoxyguanosine (8-OHdG) is a sensitive biomarker of ROS induced oxidative DNA damage and its elevated\u00a0levels\u00a0have been reported in various biological systems exposed to arsenic42,46<\/sup>. Arsenic inhibits DNA repair processes which potentiate the genotoxicity of other DNA damaging agents such as UV radiation, X-rays and benzo[a]pyrene47-49<\/sup>. Arsenic-induced oxidative stress may lead to altered DNA methylation and genomic instability resulting in a\u00a0higher\u00a0risk\u00a0of\u00a0carcinogenesis36,50<\/sup>.<\/p>\n

The protective action of Syzygium<\/em> cumini <\/em>on arsenic-induced blood cell genotoxicity and hepatotoxicity may be attributed to the presence of various active phytochemicals such as triterpenoids, kaempferol, ellagic acid, myricetin, quercetin and acetyl oleanolic acid in the seeds of this plant1,6<\/sup>. Most of these compounds are reported to exhibit free radical scavenging and antioxidant properties51-54<\/sup>, which might have protected the animals against arsenic toxicity, probably by augmenting endogenous antioxidants12,55<\/sup>, and\/or by altering apoptotic pathways12<\/sup>, and\/or by directly scavenging DNA-damaging free radicals. The polyphenol ellagic acid is reported to possess antioxidant, antimutagenic and chemopreventive\u00a0activities52,56<\/sup>. In a previous study, ellagic acid potentially inhibited the lipid peroxidation induced by radiation in the liver of mice57<\/sup>. The \ufb02avonoids kaempferol, quercetin and myricetin are\u00a0potent\u00a0antioxidants which protect cells by scavenging \u2022<\/sup>OH radicals, nitric oxide and superoxide anion, and by inhibiting lipid peroxidation53,56,58-61<\/sup>. They possess reactive hydroxyl groups and stabilize various ROS by donating hydrogen atom53<\/sup>. Free radical scavenging by flavonoids decreases production of highly damaging peroxynitrite by preventing reaction of nitric oxide with free radicals54<\/sup>. Myricetin has\u00a0been\u00a0reported\u00a0to\u00a0have even higher antioxidant capacity than Vitamin E (D-\u03b1-tocopherol)62<\/sup>. Our\u00a0results\u00a0are\u00a0in\u00a0consonance\u00a0with\u00a0previous reports indicating ameliorative effects of antioxidants such as tetrahydrocurcumin, resveratrol, and vitamins C and E on arsenic-induced toxicity either in vivo<\/em>36,38<\/sup> or in vitro<\/em>63<\/sup>. We\u00a0also have\u00a0reviewed the therapeutic potential of various plant-based antioxidants in arsenic genotoxicity, which further supports\u00a0the\u00a0results\u00a0of this\u00a0study12<\/sup>.<\/p>\n

Conclusions<\/strong><\/p>\n

From the observations, we conclude that methanol, ethanol and aqueous seed extracts of Syzygium cumini <\/em>mitigated arsenic-induced blood cell genotoxicity and hepatotoxicity in Wistar albino rats. Among all, methanol extract was the most effective in alleviating arsenic toxicity. The findings here support the growing evidence that antioxidant-rich plant sources exhibit protective effects against oxidative damage to DNA and other cellular components.<\/p>\n

Acknowledgements<\/strong><\/p>\n

We acknowledge the funding provided by the Council of Scientific & Industrial Research (CSIR), India. LUVAS, Hisar for providing experimental animals and NISCAIR, New Delhi, for authenticating Syzygium cumini <\/em>seeds. We thank Mr. Ravi Kumar, GJUS&T, Hisar, for assisting with animal handling and blood sampling.<\/p>\n

Compliance With Ethical Standards<\/strong><\/p>\n

All applicable international, national, and\/or institutional guidelines for the care and use of animals were followed.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

The authors declare that they have no conflict of interest.<\/p>\n

Funding Source<\/strong><\/p>\n

Council of Scientific and Industrial Research (CSIR)(09\/752 (0043)\/2012-EMR-1).<\/p>\n

References<\/strong><\/p>\n

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