Wattakaka volubilis leaf Extract Regulates the Oxidative ...joics.org/gallery/ics-2237.pdf · They act as reducing agents (free radical terminators) , hydrogen donars, singlet oxygen

Wattakaka volubilis leaf Extract Regulates the Oxidative

Status and Antioxidant Gene Transcripts in aluminium

sulphate Induced Hepatotoxicity

S. Usharani, R.Abinaya, M.Arulmozhi, R.Devika, P.Priyanga

Department of Biochemistry, Idhaya College for Women- Sarugani.

Corresponding author :

S. Usharani, Department of Biochemistry, Idhaya College for Women- Sarugani.

Email: [email protected]

Abstract

The ability of Wattakaka volubilis to protect against aluminium sulphate induced

oxidative stress and hepatotoxicity was evaluated in male albino rats. (n=6) and the rats treated

with saline throughout the study period, served as control (group I). Rats were treated

aluminium sulphate (50mg/kg b.w.; i.p.) for a period of 14 days (group II). Group III rats were

treated with Aluminium sulphate+ MEWV (200 mg/kg/b.w) dissolved in normal saline (1ml/kg

b.wt) orally for 14days. Group IV rats treated Aluminiumsulphate + Treated with silymarin (25

mg/kg/b.w) dissolved in normal saline (1ml/kg b.wt) orally for 14days. Group V

Wattakakavolubilis(200 mg/kg/b.w) dissolved in normal saline (1ml/kg b.wt) orally for

14days.The levels of hepatic lipid peroxidation, antioxidants, and molecular biomarkers were

estimated twenty-four hours after the lastAluminium sulphate injection. Pretreatment with

wattakakavolubilis leaf extract significantly reduced aluminium sulphate -induced elevationof

malondialdehyde levels and nearly normalized levels of glutathione and activity of glutathione S-

transferase, glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) in the liver.

Wattakakavolubilis leaf extract also attenuatedAluminium sulphate -induced downregulation of

hepatic mRNA expression levels of MMP – 2 Results of DNA fragmentation support the ability of

W.volubilis leaf extract to ameliorate aluminium sulphate -induced liver toxicity. Taken together,

our results demonstrate that W.Volubilis leaf extractis rich in natural antioxidants and able to

attenuate Aluminium sulphate -induced hepatocellular injury, likelyby scavenging reactive free

radicals, boosting the endogenous antioxidant defense system,and overexpressing genes

encoding antioxidant enzymes.

Journal of Information and Computational Science

Volume 10 Issue 1 - 2020

ISSN: 1548-7741

www.joics.org1065

Keywords : Aluminium sulphate, Wattakaka volubilis, Antioxidant enzymes TNF – alpha,

SDS PAGE , Wattakakavolubilis.

Introduction

Reactive oxygen species (ROS), include free radicals such as superoxide (O2) hydroxyl

radicles (OH), Peroxyl radicle (ROO) as well as non-radical species such as hydrogen peroxide

(H2O2) [1]. Invivo, such species are securely coupled at their site of generation or are detoxified

by endogenous antioxtative defenses, so as to preserve optimal cellular function. In pathological

condition, however, the detoxifying mechanism are often inadequate as excessive quantities of

ROS are generated. This resulting pro-oxidant shift, a process known as oxidative stress can

result in the degradation of cellular components viz., DNA, carbohydrates, polyunsaturated lipids

and proteins or precipitate enzyme inactivation, irreversible cellular dysfunction and ultimately

cell death, if the pro-oxidant-antioxidant balance is not restored. Furthermore, ROS play a

cardinal role in the a etiology of numerous diseases [2]. In recent years, there is an increasing

interest in finding antioxidant phytochemicals, because they can inhibit the propagation of free

radical reactions, protect the human body from disease[3].

Polyphenols constitute a large group of naturally occurring substances in the plant

kingdom, which include the flavonoids. The plant phenolics are commonly present in fruits,

vegetables, leaves, nuts, seeds, barks, roots and in other plant parts. These substances have

considerable interest in the field of food chemistry, pharmacy and medicine due to wide range of

favorable biological effects including antioxidant properties. The antioxidant property of

phenolics is mainly due to their redox properties. They act as reducing agents (free radical

terminators) , hydrogen donars, singlet oxygen quenchers and metal chelators [4]. The alteration

of gene expression is the most fundamental and effective way for the cells to respond to

extracellular signals and changes in their environment (D’Angio and Finkelstein 2000). Cellular

response to oxidative stress includes changes in the pattern of gene expression through

regulatory factors. One of the superoxide-radical-sensitive and redox-sensitive transcription

factors is the nuclear factor-kappaB (NF-κB) which is required for the expression of many

cytokines involved in the pathogenesis of diverse injuries. TNF-α is responsible for hepatic

injury as well as for complications after liver transplantation.[5]. TNF-α effects are antagonized

by interleukin-10 (IL 10). It protects the liver against pro-inflammatory cytokines, at least in part

by counteracting their pro-apoptotic effects [6]. On the other hand, TNF-α has not only negative

effects on the liver, but it also plays an important role in liver regeneration [7]. MMP-2 and

MMP-9 over-expression has been reported in cardiac, lung [9] and brain ischemia-reperfusion

injury. [8] Similarly to TNF-α, MMP-9 was found to be an important mediator of liver

regeneration [9]. Since ischemia-reperfusion injury is an example of oxidative stress, it is

important to investigate the expression of MMP-2 andMMP-9 in a model of metal-induced

oxidative stress



ISSN: 1548-7741

www.joics.org1066

Aluminium is the third most abundant element of the Earth’s crust, is a non essential and

toxic metal in humans [10]. Aluminium and its salts are commonly used in daily life, a

widespread use that was enhanced by the belief that it is non -toxic and is quickly excreted from

the body in the urine. However, this element has a negative impact on human health [11]. Due to

its abundance, every organism contains small quantities of aluminium,[12] and it can be found in

practically all of the tissues of mammals, including the brain, liver, heart, kidney, blood and

bones [13].Aluminium accumulation in the liver leads to cholestasis [14]. There has been

considerable debate as to whether chronic exposure to aluminium is involved in neuro-

degenerative disorders, such as Alzheimer’s disease [15] dialysis, Parkinson’s dementia, [16, 17]

and hepatotoxicity [18, 19]. The toxic effects of aluminium appear to be mediated, at least in

part, by free -radical generation [20, 21] The treatment commonly used in aluminium disorders is

desferrioxamine [22].

Wattakaka volubilis belongs to the family (Asclepiadaceae). The plants are distributed

along subtropical of Malaysia and India, South China, Taiwan and Srilanka. The flora is

important in the Indian traditional system of medicine and is utilized to treat several diseases.

[23]Wattakaka volubilis widely practiced in Indian traditional medicines and the leaf paste to

treat rheumatic pain, cough, fever and wicked cold [24]. Leaf paste is removed along with pepper

to treat dyspepsia [25]. Bark paste, mixed with hot milk is utilized internally for treating urinary

troubles [26] , and leaf powder is taken orally along with cow’s milk have antidiabetic activity

[27]. In present study the Wattakakavolubilis leaf extract regulates the oxidative status and

antioxidant gene transcripts in aluminium sulphate inducedhepatotoxicity.

MATERIALS AND METHODS

Collection & Authentication of Plant

The fresh leaves of Wattakakavolubilis were collected from Trichirapalli August, 2012.

The plant was compared with voucher specimen (voucher specimen No. 001) deposited

andidentified by Dr. John Britto Rapinet Herbanium. St. Joseph College, Trichy. The leaves were

washed thoroughly with tap water, shade dried, homogenized to fine powder and stored in

airtight bottles.

Preparation of the extract

The leaves of Wattakakavolubilis were dried in shady condition and powdered. About200

gof powdered material was dissolved with 250 ml of methanol and theextract was prepared by

using soxhlet apparatus for about48hr. The extract was filtered and concentrated inrotary

evaporator at 35-40⁰C under reduced pressure and was stored in refrigerated condition for further

use.



ISSN: 1548-7741

www.joics.org1067

Drugs and chemicals

Aluminium sulphate and silymarin were purchased from sigma-aldrich chemical

company .The diagnostic kits required for enzymatic assays were purchased from Span

Diagnostics, India

Maintenance of animals

Adult male Albino Wister rats weighing 150-200 g were used for the present

investigation. They were housed in a clean polypropylene cage and maintained under standard

laboratory conditions (temperature 25±2⁰C with dark/light cycle 12/12h).They were fed with

standard pellet diet (Hindustan lever, Kolkata, india) and water adlibitum. The animals were

acclimatized to laboratory conditions for one month prior to experiment. All procedures

described were reviewed and approved by IAEC NO: 02/005/2014.

Experimental Design

Animals were randomized and divided into six groups (n = 6)

Group I : Control rat (saline treated)

Group II : Aluminium sulphate (50mg/kg/day) control and received normal saline

(Domingo,1995)[12]

Group III : Methanol Extract of Wattakakavolubilis (200mg/kgb.w) + Aluminium

sulphate respectively daily for 14 days.

Group IV : Aluminium sulphate + Silymarin(25mg/kg b.w;p.o.)daily for 14 days.

Group V : Wattakaka volubilis (200 mg/kg/b.w) daily for 14 days.

Antioxidant Parameters

Lipid peroxidation assay

Lipid peroxidation (LPO) was measured following the method of Fraga etal., [28]. Acetic

acid 1.5mL (20%; pH 3.5), 1.5 of TBA (0.8%), and 0.2mL of sodium dodecyl sulfate (8.1%)

were added to 0.1ml of supernatant and heated at 100°C cooled for 60 min. The mixture was

cooled, and 5mL of n-butanol: pyridine (15 : 1) mixture and 1mL of distilled water were added

and vortexed vigorously. After centrifugation at 1200g for 10min, the organic layer was

separated and the absorbance was measured at 532nm using a spectrophotometer.

Malonyldialdehyde (MDA) is an end product of LPO, which reacts with TBA to form pink

chromogen–TBA reactive substance. It was calculated using a molar extinction coefficient of

1.56 X 105M-1 cm-1 and expressed as nanomoles of TBARS mg-1 of protein.



ISSN: 1548-7741

www.joics.org1068

Superoxide dismutase assay

Superoxide dismutase (SOD) activity was analyzed following the method described by

[29] . Assay mixture contained 0.1mL of supernatant, 1.2mL of sodium pyrophosphate buffer

(pH 8.3; 0.052M), 0.1mL of phenazine methosulfate (186 mM), 0.3mL of nitroblue tetrazolium

(300 mM), and 0.2mL of NADH (750 mM). Reaction was started by the addition of NADH.

After Incubation at 30o°C for 90s, the reaction was stopped by the addition of 0.1mL of glacial

acetic acid. Reaction mixture was stirred vigorously with 4.0mL of n-butanol. Colour intensity of

the chromogen in the butanol was measured spectrophotometrically at 560nm and the

concentration of SOD was expressed as Umg-1 of protein.

Reduced glutathione assay

Reduced glutathione (GSH) was measured according to the method of Ellman [30] .The

equal quantity of homogenate was mixed with 10% trichloroacetic acid and centrifuged to

separate the proteins. To 0.01 ml of this supernatant, 2ml of phosphate buffer (pH 8.4), 0.5 ml of

5’5-dithio, bis (2-nitrobenzoic acid) and 0.4ml double distilled water were added. The mixture

was vortexed and the absorbance to be read at 412nm within 15 min. The concentration of

glutathione was expressed as μ g/mg of protein.

Catalase assay

Catalase activity (CAT) was measured following the method of Sinha [31] . A 0.1mL of

supernatant was added to the cuvette containing 1.9mL of 50mM phosphate buffer (pH 7.0).

Reaction was started by the addition of 1.0mL of freshly prepared 30mM H2O2. The rate of the

decomposition of H2O2 was measured spectrophotometrically at 240 nm. Activity of CAT was

expressed as Umg-1 of protein.

Glutathione peroxidase assay

Glutathione peroxidase (GPx) activity was determined following the method [32]. The

reaction mixture consist of 400μL of 0.25M potassium phosphate buffer (pH- 7.0), 200 mL

supernatant, 100 μL GSH (10 mM), 100 μL NADPH (2.5mM), and 100μL GRD (6UmL-1).

Reaction was started by adding 100μL hydrogen peroxide (12mM) and absorbance was

measured at 366nm at 1min intervals for 5 min using a molar extinction coefficient of 6.22X

103M-1cm-1. Data are expressed as mU mg-1 of protein.

Immunohistochemistry of TNF-alpha

Tumor tissue had been routinely 10% formalin fixed (24–72 hr) and paraffin embedded.

Four-µm-thick sections were cut using microtome and dried for 60 min at 65C. Sections were

dewaxed in Xylene Substitute and rehydrated through graded series of ethanol and rinsed in

water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase for 10 min.

Heat-induced antigen retrieval was performed in a microwave oven in 10 mM Tris/EGTA (pH =



ISSN: 1548-7741

www.joics.org1069

9) by heating for 10 min followed by 15 min of boiling. Sections were cooled for 15 min at room

temperature and rinsed in Tris-buffered saline (TBS) with 0.05% Tween 20 for at least 5 min.

Sections to be incubated in anti TNF-alpha were additionally treated with 0.5% casein in TBS for

10 min to block non-specific binding sites. Section treated with polymer-HRP for 30 min

followed by visualization with DAB chromogen for 12 min. Sections were rinsed in water, and

0.5% cobber sulfate in TBS was added to enhance the staining intensity. Between incubations the

sections were washed several times in TBS buffer.

RESULTS

The result showed the activities of TBARS, SOD, CAT, GPx, and non enzymatic

antioxidants (GSH) levels in liver. Aluminium sulphate induced rats had shown decreased

activities of antioxidant enzymes when compared to control animals. Oral administration of

W.volubilis and kampferol to aluminium sulphate administered rats showed a significant

reduction in LPO levels and significant increase in the activities of antioxidant enzymes when

compared to aluminium sulphate induced rats. (Table.1)

Table : 1Effect of MEWV on LPO and antioxidant enzymes in Al2(SO4)3 induced rats

Parameters

(U/L)

Group I Group II Group III Group IV Group V

MDA 2.31±0.16 4.17±0.49* 3.84±0.21 a 4.05±0.27 a 2.27±0.19

GSH 47.6±1.9 15.5±0.11* 31.6±1.2 a 18.7 ± 1.1 a 46.5±3.2

SOD 7.41±0.15 3.3±0.18* 5.1±0.43 a 21.4 ± 1.7 a 7.39±0.68

CAT 6.6±1.5 3.2±0.16* 6.4±0.2 a 15.6± 1.2 a 7.3±0.41

GPx 48.6±2.5 19.8±1.4* 26.4±2.7 a 28.2 ± 1.6 a 47.3±0.41

Results are expressed as mean± S.E.M, n= 6 *P˂0.001, statistically significant as compared with

Al2(SO4)3 induced group. Values are expressed as SOD- mµ of epinephrine oxidized /min/mg

protein; GPx- mg of GSH reduced /g tissue; GSH- mg/g/ tissue.

Immunohistochemistry-TNF-alpha

Immuno-stained liver of control and MEWV groups for TNF – alpha expression showed

normal hepatic architecture. Liver of aluminium sulphate intoxicated group showed an increase

in the expression of TNF – alpha in the cytoplasm in the area of coagulative necrosis surrounding

hepatic central vein compared with that of negative control. The liver of the protective group

administered MEW plus aluminium sulphate showed no expression for TNF- α supporting the

protective effect of aluminium sulphate on hepatic toxicity (Figure 1).



ISSN: 1548-7741

www.joics.org1070

Figure: 1 Immunohistochemical changes of

Tumor Necrosis Factor – alpha

Group I- Normal Control Group II- Al2 (SO4)3 (50 mg/kg/b.wt)

Group III- Al2(SO4)3 Group IV- Al2(SO4)3

+MEWV (200 mg/kg/b.wt) +Silymarin (25 mg/kg/b.wt)

Group V-MEWV (200 mg/kg/b.w)

MMP-2 protein expression by SDS PAGE

The expression of matrix metalloproteinase in control and experimental group of rat was

evaluated. A substantial increase in MMP was observed following treatment with Al2 (SO4)3

compared with control group of rats. No significant changes were observed in methanolic extract

of W.volubilis administered group of rats for a period of 30 days compared to that of control



ISSN: 1548-7741

www.joics.org1071

group of rats. However, the expression of matrix metalloproteinase gradually decreased by the

methanolicextractof W.volubilis treatment as compared to the Al2(SO4)3 induced group of rats.

Similarly, silymarin treatment to Al2(SO4)3 induced mice showed significant reduction in

matrix metalloproteinase expression (Figure 2). So these results confirmed that W.volubilis

andsilymarinhave the ability to reduce the expression of matrix metalloproteinase during free

radical produced oxidative damage in liver tissues.

Fig : 2 MMP-2 protein expression by SDS PAGE

Lane M - Marker Lane

Lane 1 – Normal rats showed normal expression of MMP-2

Lane 2 –Group II liver tissue showed elevated expression of MMP-2

Lane 3 –Extract treated hepatotoxicity induced rats showed very mild expression of MMP-2

Lna e 4 – Silymarin treated hepatotoxicity induced rats down regulated the expression of MMP-2

Lane 5 –Extract treated rats showed normal expression of MMP-2.

DISCUSSION

Aluminium toxicity cause oxidative damage to the plant system by activating the

production of reactive oxygen species [33] . These ROS like superoxide radical (O2-), hydroxyl

radicals (OH-), singlet oxygen (1O2) and hydrogen peroxide (H2O2) generally are detoxified by

enzymatic antioxidant system. ROS if not detoxified causes serious damage to macro molecules

such as proteins, lipids and nucleic acids. In order to scavenge ROS and to combat oxidative

stress.

In vivo antioxidant defence mechanism fight againstfree radicals and reactive oxygen

species induceddamage, in which the endogenous enzymatic andnon-enzymatic antioxidants

such as glutathione, lipid peroxidase and superoxide dismutase [34] .Glutathione is as an

essential intracellular reducingsubstance for the maintenance of thiol groups onintracellular

M 1 2 3 4 5

Kda 94.0

66.2

71.8

26.0

33.0

MMP-2



ISSN: 1548-7741

www.joics.org1072

proteins and antioxidant molecules inliving organisms. Perturbation ofglutathione status in a

biological system has beenreported to lead serious consequences [35]. Glutathione conjugatewith

free radicals directly, marking them ready forrenal excretion, which is especially important

fordealing with the products of hepatic cytochrome P 450 enzyme activity. The sulf-hydryl

portion of theglutathione can be used to reduce a variety of freeradicals in a reaction catalyzed by

the antioxidantenzyme, glutathione peroxidase [36]. Lipid peroxidase, superoxide dismutase

andother antioxidant enzymes constitute a mutuallysupportive team of defense against reactive

oxygenspecies. Superoxide dismutase is a metalloproteinaseto detoxify superoxide anions as an

efficientdismutative mechanism and is the first enzymeinvolved in the antioxidant defense[37].

However, once the balance between reactive oxygen species production and antioxidant

defensesis lost, oxidative stress consequently occurs, whichthrough a series of biological events

deregulates thecellular functions leading to various pathologicalconditions [38] .The results of

the treatment of kaempferol effectively blocked the aluminium sulphatecaused abnormal changes

in the level of glutathione,indicated that kaempferol has a potent antioxidantproperty towards

chemical induced hepatic injury[39]. Elevated level of MDA in aluminium sulphatetreated rats

indicates excessive formation of freeradicals and activation of lipid peroxidation systemresulting

in hepatic damage. The significant decline inthe concentration of TBARS in the rat’s liver tissues

of rats,treated with aluminium sulphate and MEWV indicates anti-lipid peroxidative effect of

watakaka volubilis.

In the present study, the activities of antioxidant enzymes like SOD, CAT and GPx, in rat

liver were dramatically decreased by the treatment of aluminium sulphate. This decrease could

be due to a feed back inhibition or oxidative inactivation of enzyme protein due to excess ROS

generation. The generation of H2 O2 may also lead to inactivation of this enzyme [40] .SOD

requires copper and zinc for its activity. The reduced activity of SOD in the presence of

aluminium sulphate may cause accumulation of O2∙-, H2O2 or the products of its

decomposition. The SOD activity was elevated in rats dosed with MEWV with aluminium

sulphate. This elevation may be due to the presence of antioxidant bioactive compound such as

phenolic compound which responsible for scavenging the super oxide anion radicals which was

evident by similar findings of [41]. Catalase is one of the important enzymes in the supportive

team of defense against reactive oxygen species (ROS). Catalase is a haemoprotein containing

four haem groups, that catalyses the decomposition of H2O2 to water and O2 and thus protects

the cell from oxidative damage by H2O2 and OH– [42].

Glutathione peroxide (GPx) has a well established role in protecting cells against

oxidative injury. GPx is a selenium containing metalloenzyme, partially located within cellular

membranes, which can remove hydrogen peroxide by converting reduced glutathione into

oxidized glutathione. GPx can also terminate the chain reaction of lipid peroxidation by

removing lipid hydroperoxides and H2O2 from the cell membrane. The decreased activity of

GPx in aluminium sulphate intoxicated group might be correlated to the decreased availability of



ISSN: 1548-7741

www.joics.org1073

its substrate GSH. After the treatment with a MEWV improved the GPx levels significantly to

near normal [43].

Immunohistochemistry-TNF-α

TNF – α is a central pro-inflammatory cytokines. Activated kupffer cells produce

various mediators, including cytokines, proteases and oxygen radicals that participate in

inflammation, immune responses and modulation of hepatocyte metabolism.

Authors defend the use of molecular methods in clinical practice to perform differential

diagnosis; and to study biological targets for novel therapies [44]. Oxidative stress is one of the

responses for the production of pro -inflammatory cytokines, among which TNF-α, transforming

growth factors alpha (TGF-α) and beta (TGF-β), interleukins 6 (IL-6) and 8 (IL-8), nuclear

factor-kappa B (NFκB) and adiponectin stand out. These cytokines are produced by lymphocytes

and Kupffer cells, through free radical-mediated mechanisms, by altering mitochondrial

membrane permeability and inhibiting the respiratory chain [45,46].

Increased expression of TNF- α in aluminium sulphate rats may be due to inflammation,

necrosis and oxidative stress. Supplementation of Wattakakavolubilis effectively decreased TNF-

α expression in hepatic aluminium sulphate rats. Decreased TNF- α expression may be due to

attenuated inflammation, necrosis, and reduce the oxidative stress.

MMPs are important in many normal biological processes including embryonic

development, angiogenesis, and wound healing, as well as in pathological processes such as

inflammation, cancer and tissue destruction. The Lane 1 which consists of Normal rats with

normal expression of MMP-2. The Lane 2 which includes Group II liver tissue showed elevated

expression of MMP-2. The lane 3 Extract treated hepatotoxicity induced rats showed very mild

expression of MMP-2. The lane 4 Silymarin treated hepatotoxicity induced rats down regulated

the expression of MMP-2. From this study it cames to know that, MMP2 get altered after the

ingestion of Aluminium sulphate the MMP2 returns to normal level after herbal treatment. This

indicates the MMP2 involves in inflammation of liver injury.

CONCLUSION

It has been concluded that kaempferol is a tremendous bioactive flavanoid with potent

hepatoprotective activity. It proves the antioxidant enzymes and tumour necrosis factor.The

present study clearly reflected the effectiveness of the extract in ‘in vivo’ in terms of lipid

peroxidation inhibitory capacity and further confirmed the significant hepatoprotective activity

of its antioxidant mechanisms of action.

REFERENCES

1. Osinska E, Kanoniuk D, Kusiak A. Aluminium hemotoxicity mechanisms. Ann Univ

Mariae Curie Sklodowska [Med]. 2004; 59: 411-6.



ISSN: 1548-7741

www.joics.org1074

2. Williams RJP. What is wrong with aluminium? The J.D. Birchall Memorial Lecture. J

Inorg Biochem. 1999; 76: 81-8.

3. Yokel RA. Brain uptake retention and efflux of aluminum and manganese. Coordination.

Chem Rev. 2002; 228: 97-113.

4. Perl DP, Bondy AR. Alzheimer’s disease: X-ray spectrometric evidence of aluminium

accumulation in neuro fibrillary tangle bearing neurons. Science. 1980; 208: 297-9.

5. Droge, W. 2002. Free radicals in the physiological control of cell function. Physiol. Rev.

82: 47–95.

6. Halliwell, B, Gutteridge, J.M.C. 1999. Free radicals in biology and medicine, 3 rd

edition, Oxford University press, London, UK.

7. Haddad, J.J, Olver, R.E. & Land S.C. 2000. Antioxidant/prooxidant equilibrium

regulates HIF-1alpha and NF-kappa B redox sensitivity. Evidence for inhibition by

glutathione oxidation in alveolar epithelial cells. J. Biol. Chem. 275: 21130–21139.

8. Suzuki, S, Nakamura, S, Sakaguchi, T, Ochiai, H, Konno, H, Baba, S. & Baba S. 1997.

Alteration of reticuloendothelial phagocytic function and tumor necrosis factor-alpha

production after total hepatic ischemia. Transplantation 64: 821–827.

9. Flach, R, Speidel, N, Flohe, S, Borgermann, J, Dresen, I.G, Erhard J. & Schade F.U.

1998. Analysis of intragraft cytokine expression during early reperfusion after liver

transplantation using semi-quantitative RT-PCR. Cytokine 10: 445–451.

10. Zhong. J, Deaciuc, I.V, Burikhanov, R. & de Villiers, W.J. 2006. Lipopolysaccharide-

induced liver apoptosis is increased in interleukin-10 knockout mice. Biochim. Biophys.

Acta 1762: 468–477.

11. Schwabe, R.F. & Brenner D.A. 2006. Mechanisms of liver injury. I. TNF-alpha-induced

liver injury: role of IKK, JNK, and ROS pathways. Am. J. Physiol. Gastrointest. Liver

Physiol. 290: G583–G589.

12. Oyaizu M., studies on product of browning reaction prepared from glucose amine. J.

Nutr., 1986; 44: 307-315.

13. Halliwell B. and Gutteridge J.M.C., Freeradicals, other reactive species and

disease; Clarendon Press, Oxford, 1989.

14. Kinsella J.E., Frankel E., German B. and Kanner J., Possible mechanisms for the

protective role of antioxidants in wineand plant foods, Food Technol., 1993; 47:85-89.

15. Good PF, Perl DP, Bierer LM, Schmeidler J. Selective accumulation of aluminum and

iron in the neurofibrillary tangles of Alzheimer’s disease: a laser microprobe (LAMMA)

study. Ann Neurol 1992; 31: 286-92.

16. Bjertness E, Candy JM, Torvik A, Ince P, McArthur F, Taylor GA. Content of brain

aluminum is not elevated in Alzheimer disease. Alzheimer Dis Assoc Disord, 1996 ;10:

171-4.

17. Flaten TP.Aluminium as a risk factor in Alzheimer’s disease, with emphasis on drinking

water. Brain Res Bull. 2001; 55: 187-96.



ISSN: 1548-7741

www.joics.org1075

18. Hirsch EC, Brandel JP, Galle P, Javoyagid F, Agid Y. Iron and aluminum increase in the

substantia nigra of patients with Parkinson’s disease: an x-ray microanalysis. J

Neurochem , 1991 ; 56: 446-51.

19. Erasmus RT, Kusnir J, Stevenson WC, Lobo P, Herman MM, Wills MR.

Hyperalbuminemia associated with liver transplantation and acute renal failure. Clin

Transplant. 1995; 9: 307-11.

20. Yumoto S, Ohashi H, Nagai HS, Kakimi A, Ishikawa K, Kobayashi. Aluminum toxicity in

the rat liver and brain. Nucl Instrum Methods Phys Res B. 1993;75: 188-90.

21. Chinoy, NJ, Patel TN. Reversible toxicity of fluoride and aluminium in liver and

gastrocnemius muscle of female mice. Fluoride. 1999; 32: 215-29.

22. Moumen R, Ait-Oukhatar N, Bureau F, Fleury C, Bougle D, Arhan P. Aluminium

increases xanthine oxidase activity and disturbs antioxidant status in the rat. J Trace

Elem Med Biol. 2001; 15: 89-93.

23. Anane R, Creppy EE. Lipid peroxidation as a pathway to aluminium cytotoxicity in

human skin fibroblast cultures: prevention by superoxide dismutase plus catalase and

vitamins E and C. Hum Exp Toxicol. 2001; 20: 477-81.

24. Missel JR, Schetinger MR, Gioda CR, Bohrer DN, Packolski IL, Zanatta N. Chelating

effects of novel pyrimidines in a model of aluminum intoxication. J Inorg Biochem. 2005;

99: 1853-57.

25. Koperuncholan M and Ahmed John S. Biosynthesis of Silver and Gold Nanoparticles and

Antimicrobial Studies of Some Ethno medicinal Plants in South-Eastern Slope of Western

Ghats, IJPI’s- Journal of Pharmacognosy and Herbal Formulations. 2011; 1: 10-5.

26. Rajadurai M, Vidhya VG, Ramya M and Bhaskar A . Ethno-Medicinal plants used by the

Traditional Healers of Pacchamalai Hills, Tamil Nadu, India. Ethnomedicine. 2009; 3:

39-41.

27. Muthu C, Ayyanar M, Raja N and Ignacimuthu S. Medicinal plants used by traditional

healers in Kancheepuram District of Tamil Nadu, India. Journal of Ethnobiology and

ethnomedicine. 2006; 2: 43-52.

28. Pandikumar P, Ayyanar M and Ignacimuthu S. Medicinal plants used by Malasar tribes

of Coimbatore district, Tamil Nadu. Indian Journal of Traditional Knowledge. 2007; 6:

579-82.

29. Ayyanar M, Sankara Sivaraman K and Ignacimuthu S. Traditional Herbal Medicines

used for the treatment of Diabetes among two major tribal groups in South Tamil Nadu,

India.Ethno Botanical Leaflet. 2008; 47: 389-394.

30. Fraga CG, Oteiza PI, Golub MS, Gershwin ME, Keen CL. Effects of aluminum on brain

lipid peroxidation. Toxicol Lett. 1990; 51: 213-19.

31. Kakkar P, Das B, Viswanathan PN. Indian J Biochem Biophys. 1984; 2: 130–2

32. Ellman GL. Tissue sulfhydryl Groups: Archives of Biochemistry and Biophysics. 82,

33. Sinha KA. Colorimetric Assay of Catalase. Analytical Biochemistry. 1972; 47: 389-94.



ISSN: 1548-7741

www.joics.org1076

34. Mohandas J, Marshall JJ, Duggin, GG, Horvath JS, Tiller D. Differential distribution of

glutathione and glutathione related enzymes in rabbit kidneys: possible implication in

analgesic neuropathy. Cancer Research. 1984; 44: 5086–91.

35. Shah K, Kumar RG, Verma S, Dubey RS. Effect of cadmium on lipid peroxidation,

superoxide anion generation and activities of antioxidant enzymes in growing rice

seedlings. Plant Science. 2001; 161: 1135–44.

36. Chow CK. Nutritional influence on cellularantioxidant defence systems. American J.

ofClinical Nutrition. 1979; 32: 1066–81.

37. Bandyopadhyay U, Das D and Banerjee RK. Reactiveoxygen species: Oxidative damage

and pathogenesis.Current Science. 1999; 77: 658–66.

38. Webb C and Twedt D. Oxidative stress and liverdisease. Veterinary Clinics of North

America: SmallAnimal Practice. 2008; 38: 125–135.

39. Salvemini D, Riley DP and Cuzzocrea S. SOD mimetic are coming of age. Nature Review

DrugDiscovery. 2002; 367–74.

40. Sahu SC and Gray GC. Pro-oxidant activity offlavonoids: effects on glutathione and

glutathione S-transferase in isolated rat liver nuclei. Cancer Letters. 1996; 104: 193–6.

41. Chen CJ, Deng AJ, Liu C, Shi R, Qin HL and WangAP. Hepatoprotective activity of

Cichorium endivia L.extract and its chemical constituents. Molecules. 2011; 16:9049–66.

42. Ohata H, Kitauchi S, Yoshimura N, Mugitani K, Iwane M. Progression of chronic

atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric

cancer. J. Cancer. 2004; 109: 138-43.

43. Cao G, Alessio HM, and Cutler RG. Oxygen-radical absorbance capacity assay for

antioxidants. Free Radical Biol. Med. 1993; 14: 303–11.

44. Tolbert NE. Metabolic pathways in peroxisomes and glyoxysomes. Annual Review of

Biochemistry. 1981; 50: 133–57.

45. Roberta JW, Timothy JP. Free radicals. Clinical bio-chemistry metabolic and clinical

aspects, edited by Williams J. Marshall, 1995; 765-77.

46. Kang, M, Oh, J.W, Lee, H.K, Chung, H.S, Lee, S.M, Kim, C, Lee, H.J, Yoon, D.W, Choi

H, Kim H, Shin M, Hong, M, Bae H. 2004. Anti-obesity effect of PM-F2-OB, an anti-

obesity herbal formulation, on rats fed a high-fat diet. Biol Pharm Bull, 27(8): 1251-

1256.

47. Paolo Zatta, T.K, Mario Suwalsky, Guy Berthon, 2002. Aluminum (III) as a promoter of

cellular oxidation, Coord. Chem. Rev. 228: 271–284.

48. Brahmachari, G. 2009. Mother nature: An inexhaustible source of drugs and lead

molecules. In Brahmachari G, editor. Chemistry, Biochemistry and Pharmacology (1st

edn). Narosa Publishing House Pvt. Ltd: New Delhi. 1-20.



ISSN: 1548-7741

www.joics.org1077

Documents

Wattakaka volubilis leaf Extract Regulates the Oxidative ...joics.org/gallery/ics-2237.pdf · They act as reducing agents (free radical terminators) , hydrogen donars, singlet oxygen