In vivo antioxidant and lipid peroxidation effect of various extracts from aerial parts of Chomelia asiatica ( Linn ) in rat fed with high fat diet

The present investigation was to examine the in vivo antioxidant activity and lipid peroxidation activities of different extracts of aerial parts of Chomelia asiatica (Linn). High fat diet rats demonstrated fundamentally decreased the levels of tissues enzymatic antioxidant and non-enzymatic antioxidant (Glutathione). The level of thiobarbuturic acid reactive substance (TBARS) is reduced in high-fat diet (HFD) rats when compared and control group. Administration of ethyl acetate extract of Chomelia asiatica in high fat diet rats were indicated altogether (p<0.001) increased the levels of antioxidant enzymes, for example, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and level of non enzymatic antioxidant glutathione (GSH) when contrasted and HFD rats (Group II). The ethyl acetate extract of C. asiatica in high fat diet rats were discovered lowered the concentration of TBARS when contrasted and HFD rats. In comparison of all the three extracts treated group with standard group, the ethyl acetate extract of C. asiatica showed significant (p<0.001) result than that of other groups. Taking into account the outcomes, we concluded that the ethyl acetate extract of C. asiatica is a significant source of antioxidant, which may be useful in keeping the advancement of different oxidative stresses.


INTRODUCTION
Oxidation is essential in many living organisms for the production of energy to fuel biological processes.However, the uncontrolled production of oxygen derived free radicals is involved in the onset of many diseases such as atherosclerosis, rheumatoid arthritis, and cancer as well as in degenerative processes associated with aging (Halliwell et al 1984).Almost all organisms are well protected against free radical damage by enzymes such as superoxide dismutase and catalase, or compounds such as ascorbic acid, tocopherols and glutathione (Mau et al., 2002).When the mechanism of antioxidant protection becomes unbalanced by factors such as aging, deterioration of physiological functions may occur resulting in diseases and accelerated aging (Israel, 2012).
However, the antioxidants present in human diet are of great interest as possible protective agents to help the human bodies reduce oxidative damage.Antioxidants are often used in oils and fatty foods to retard their autoxidation.Synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), have restricted use in foods as they are suspected to be carcinogenic.Therefore, the importance of search for natural antioxidants has greatly increased in the recent years (Jayaprakasha et al., 2003).Ethnomedical literature contains a large number of plants that can be used against diseases, in which reactive oxygen species and free radical play important role.There is a plethora of plants that have been found to possess strong antioxidant activity (Badami et al., 2003).Recent reports indicate that there is an inverse relationship between the dietary intake of antioxidant-rich foods and the incidence of human diseases (Halliwell, 1997).So, many researchers have focused on natural antioxidants and in the plant kingdom numerous crude extracts and pure natural compounds were previously reported to have antioxidant properties.However, no data are available in the literature on the antioxidant activity of aerial parts of C. asiatica.Therefore we undertook the present investigation to examine the in vivo antioxidant and lipid peroxidation activities of different extract of aerial parts of C. asiatica (Linn) in rat fed with high fat diet.

Chemicals and reagents
Unless stated, general chemicals and reagents were purchased from Sigma, Cadila.Fisher chemicals were of analytical grade or equivalent.

The plant materials (Study species)
C. asiatica (Linn) belongs to the family Rubiaceae, commonly known as Tharani in tami: Kuppipoovu, Tharana in makayalam.Chomelia Gaertn is a genus of about 370 species distributed in tropical and subtropical Africa, Asia, Madagascar and pacific islands.Chomelia asiatica (Linn) is a common species which occurs in india, Srilanka and China.The leaves or powder extracts of C. asiatica are used as antimicrobial activities (Jayasinghe et al., 2002).It had been accounted for pain relieving and mitigating activities (Amutha et al., 2012).The parts of Tarenna asiatica (Rubiaceae) plants are customarily used to advance suppuration (Anonymous, 1976), as anthelmintic (Ramarao and Henry, 1996) and antiulcer operator (Rao et al., 2006).The phytochemical constituents of it are accounted for to be antiseptic (Vinothkumar et al., 2011), injury healing (Anjanadevi and Menaga, 2013) and antioxidant (Ramabarathi et al., 2014).Moreover, the extract of shoots, leaves and fruits are purportedly dynamic against Mycobacter phlei (Rajakaruna et al., 2002).

Collection and identification of plant materials
The aerial parts of C. asiatica (Linn), were collected form Thayyil et al. 811 Shencottai, Tirunelveli District, Tamil Nadu, India.Taxonomic identification (Plant deposition no is Au/CA/810) was made from Botanical Survey of Medical Plants Unit Siddha, Government of India.Palayamkottai, Tirunelveli.The aerial parts of C. asiatica (Linn), were dried under shade, separated, pulverized by a mechanical processor and went through a 40 mesh sieve.

Preparation of extracts
The above powered materials were successively extracted with petroleum ether (40-60°C) by hot continuous percolation technique in Soxhlet apparatus (Harborne JB, 1984) for one day.Then the marc was subjected to ethyl acetate (76-78°C) for one day and then mark was subjected to methanol for one day.The extracts were extracted by utilizing a rotary evaporator and subjected to freeze drying in a lyophilizer till dry powder was obtained.

Animals and treatment
Male

Assessment of in vivo antioxidant and lipid peroxidation
Rats of III, IV and V groups were orally fed with the different extracts of C. asiatica (Linn) and rats of VI group were fed with standard drug atorvastatin.Both the extract and atorvastatin were suspended in 2% tween 80 (Waynforth BH et al., 1980) separately and fed to the respective rats by oral intubation.At the end of sixty three days all the animals were sacrificed by cervical dislocation  1.

Statistical analysis
Results were communicated as mean ± SE of 6 rats in every group.
The statistical significance between the groups was analyzed by utilizing one way analysis of variance (ANOVA), followed by Dunnet's multiple correlation test.Significance level was fixed at 0.05.

RESULTS
As shown in Tables 1 and 2. The TBARS and conjugated dienes levels were increment in liver, heart and aorta in II group rats are a clear reasonable sign of excessive formation of free radical and initiation of lipid peroxidation.The significantly (p<0.001)decreased the levels of TBARS and conjugated dienes, in rats regulated with ethyl acetate extract of C. asiatica along with HF diet (IV group) when contrasted to HF diet rats (II group).Tables 3 and 4 demonstrates that the effect of various extracts of C. asiatica on tissues SOD and CAT enzyme levels in HF diet rats.The activities of SOD and CAT in the tissue like liver, heart and aorta were significantly (P<0.001)brought down in rats fed with high fat diet (II group) than that of control group .After treatment of ethyl acetate extract of C. asiatica along with HF diet significantly expands the activities of SOD and CAT in tissues of rats when compared with other extracts.
The activities of tissues glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-Stransferese in HF diet rats were presented in Tables 5 to  7. Tissues glutathione peroxidase and glutathione reductase levels were significantly (p<0.001) lowered in rats sustained with HF diet (II group) when compared with the control rats (I group).Administration of ethyl acetate of C. asiatica alongside the HF diet significantly (p<0.001) increase the levels of glutathione peroxidase, glutathione reductase and glutathione-S-transferese in every one of the tissues as contrasted and HF diet rats.A standard drug atorvastatin regulated rats likewise demonstrated raised level of glutathione peroxidase, glutathione reductase and glutathione-S-transferese.
As shown in Table 8.The significant (p<0.001)fall in the levels of tissues glutathione (GSH) were seen in high fat diet rats (II group) when compared with the control rats (I group).Administration of ethyl acetate extract of C. asiatica alongside HF diet rats significantly (p<0.001) increase the levels of glutathione when contrasted and HF diet rats (II group).Similarly the ethyl acetate extract of C. asiatica was indicated significant result than that of other two treated groups.

DISCUSSION
The elevated levels of TBARS and conjugated denies were observed in aorta, heart and liver of rats fed with high fat diet (group II).The high fat diet is known to induce oxidative stress in the cells by producing reactive oxygen species (ROS) (Khan et al., 2004).This results in increased lipid peroxidation leading to elevated concentration of TBARS and conjugated dienes (Boccio et al., 1990).The significant decrease in the level of TBARS and conjugated dienes in rats administered with ethyl acetate extract of C. asiatica .This effect may be due to phytoconstituents, flavonoids present in the c. asiatica.
The activities of SOD and CAT in the tissue like liver, heart and aorta significantly (P<0.001) lowered in rats fed with high fat diet (group II).High fat diet can cause the formation of toxic intermediates that can inhibit the activity of antioxidant enzymes (Thampi et al., 1991) and the accumulation of O 2 -and H 2 O 2 which in turn forms hydroxyl radicals (Batra et al., 1989).Catalase decomposes hydrogen peroxide and helps to protect the tissues from highly reactive hydroxyl radicals.Administration of with ethyl acetate extract of C. asiatica along with high fat diet significantly increased the activities of SOD and CAT in tissues of rats when compared to other extracts treated groups.
The results indicated that the concentration of glutathione peroxidase (GPX), glutathione reductase and glutathione-s transferase significantly decreased in tissues (aorta, heart and liver) of rats fed with high fat diet.High fat diet decreased the ratio of oxidized glutathione/reduced glutathione in tissue (De La Cruz et al., 2000).Administration of acetate extract of C. asiatica along with the high fat diet increased the activities of glutathione peroxidase, glutathione reductase and glutathione S-transferase in all the tissues.It might be due to help to propagation of biological membranes found to be associated with increase in the activities of GPX.Glutathione peroxidase (GPX) mainly detoxifies H 2 O 2 (Izawa et al., 1996) and is involved in the reduction of a variety of hydroperoxides such as phospholipid hydroperoxides, fatty acid hydroperoxides.
Glutathione (GSH), a tripeptide which is present in all the cells is an important antioxidant (Chance, 1992).GSH also functions as free radical scavenger in the repair of radical caused biological damage (Meister, 1984).The reduced levels may be an attempt by the tissue to counteract the increased formation of lipid peroxides that are handled by antioxidant enzymes such as Glutathione peroxidase which scavenges H 2 O 2 utilizing GSH as substrate (Rajashree et al., 1998).Increase in glutathione concentration in C. asiatica extract treated rats with high fat diet might be due to the increase in the activity of the enzyme glutathione reductase which catalyses the conversion of oxidized glutathione to reduced glutathione in liver (or) might be due to enhanced synthesis of GSH (Sethupathy et al., 2002).

Conclusion
The results of the above study clearly indicated that the ethyl acetate extract of aerial parts C. asiatica had significant in vivo antioxidant and lipid peroxidation action.The phytoconstituents might be responsible for the inhibition of lipid peroxidation and enhance the antioxidant activities of ethyl acetate extract of C. asiatica.

Table 1 .
Effect of various extracts of aerial parts of Chomelia asiatica (Linn) on tissues TBARS in HF diet rats.Values are mean  SE of 6 rats.P values: * < 0.001, ** < 0.05; a → groups II, III, IV, V and VI compared with group I (control).b → groups I, III, IV, V and VI compared with group II (High fat diet).I group: standard chow pellet.(Control); II group: High Fat Diet.III group: High fat diet + Petroleum ether extract of C. asiatica (200mg/kg B.wt); IV group: High fat diet + Ethyl acetate extract of C.asiatica (200 mg/kg B.wt);V group: High fat diet + Methanolic extract of C. asiatica (200mg/kg B.wt); Group VI: High fat diet + standard drug atorvastatin (1.2 mg/kg B.wt).

Table 2 .
Effect of various extract of aerial parts of C. asiatica (Linn)on tissues conjugated diene in HF diet rats.
Values are mean  SE of 6 rats.P values: * < 0.001, ** < 0.05; a → groups II, III, IV, V and VI compared with group I (control) .b →groups I, III, IV, V and VI compared with group II (High fat diet).Group I-VI details are same as in Table

Table 3 .
Effect of various extracts aerial parts of C. asiatica (Linn) on tissues SOD in HF diet rats.III, IV, V and VI compared with group II (High fat diet).Group I-VI details are same as in Table1.

Table 4 .
Effect of various s from aerial parts of C. asiatica (Linn) on tissues CAT in HF diet rats.
Values are mean  SE of 6 rats.P values: * < 0.001, ** < 0.05; a → groups II, III, IV, V and VI compared with group I (control).b→groupsI,III, IV, V and VI compared with group II (High fat diet).Group I-VI details are same as in Table1.

Table 5 .
Effect of various extracts of aerial parts of C. asiatica (Linn) on tissues glutathione peroxidase in HF diet rats.Values are mean  SE of 6 rats.P values: * < 0.001, ** < 0.05; a → groups II, III, IV, V and VI compared with group I (control).b→groupsI,III, IV, V and VI compared with group II (High fat diet).Group I-VI details are same as in Table1.

Table 6 .
Effect of various extracts of aerial parts of C. asiatica (Linn) on tissues Glutathione Reductase in HF diet rats.
Values are mean  SE of 6 rats.P values: * < 0.001, ** < 0.05; a → groups II, III, IV, V and VI compared with group I (control).b→groupsI,III, IV, V and VI compared with group II (High fat diet).Group I-VI details are same as in Table1.

Table 7 .
Effect of various extracts of aerial parts of C. asiatica (Linn) on tissues Glutathione S-Transferase in HF diet rats.Values are mean  SE of 6 rats.P values: * < 0.001, ** < 0.05; a → groups II, III, IV, V and VI compared with group I (control).b→groupsI,III, IV, V and VI compared with group II (High fat diet).Group I-VI details are same as in Table1.

Table 8 .
Effect of various extracts of aerial parts of Chomelia asiatica (Linn) on tissues glutathione in HF diet rats.Values are mean  SE of 6 rats.P values: * < 0.001, ** < 0.05; a → groups II, III, IV, V and VI compared with group I (control).b→groupsI,III, IV, V and VI compared with group II (High fat diet).Group I-VI details are same as in Table1.