The liver is one of the most important organs and mainly involves the metabolism of biomolecules, protein synthesis, production of various biochemical compounds, detoxification and regulating homeostasis in the body (Rose, 2001; Udomsinprasert & Jittikoon, 2019). In addition, aids in the bile secretion and the storage of minerals as well as vitamins (Ahsan, Islam, & Bulbul, 2009; Tavakoli, Rostami, & Avan, 2019). The liver has been exposed to substances from exogenous origin like environmental toxins, drugs and alcohol, which may lead to complications in the liver, generally presenting as a distinct pattern of diseases such as cirrhosis, hepatitis, haemochromatosis, cholestatic, coronavirus (Dinesh, Sivakumar, & Selvapriya, 2014; Li & Fan, 2020). In the case of disturbed liver functions due to toxins, may alter the chemical composition of the liver and its subcellular organelles (Figure 1). The modification in the structure of the liver and its function may result in jaundice, increased bleeding, portal hypertension and causes multiple metabolic changes which may affect the functions of the other organs (Ibrahim, Ishizuka, & Soliman, 2008).

Figure 1

Carbon tetrachloride-induced membrane damage.

Synthetic antioxidants like butylated hydroxyl anisole and butylated hydroxyl toluene are having various toxic effects in animals, including human beings (Madhavi & Salunkhe, 1995). The CCl4-toxicity, which increases the cytochrome P450 system, induces free radical formation, affects the liver microsomes and consequently causes lipid peroxidation in the liver (Hamed, Bellassoued, Feki, Gargouri, & A, 2019). Due to the adverse effect of allopathic drugs, research is interested to find safer drugs from medicinal plants. Medicinal herbs and their extracts are widely used for the identification and development of new therapeutic agents for treating liver complications including hepatitis, cirrhosis, and loss of appetite (Lee, Cho, & Kim, 2018; Recknagel, 1983). Euphorbia thymifolia Linn. and Euphorbia hirta Linn. plants belong to the family of Euphorbia are widely used in their general medicinal activities.

The E. thymifolia is found in tropical regions. The traditional use of this E. thymifolia is mainly due to its actions involving laxative, aromatic, sedative, blood purification, anti-viral, antihelminthic, anti-inflammatory, anti-spasmodic, anti-fungal, anti-bacterial, anti-microbial, diuretic properties (Mani, Anand, Manikandan, & R, 2013; Mani, Hedina, Kausar, Anand, & Pushpa, 2016). The E. hirta has been commonly used as a medicinal plant for treating diseases including gastrointestinal disorders, inflammations of the skin and mucous membranes and respiratory system disorders by the use of whole plant and weed extract. The plant is generally found in India, China, Malaysia, the Philippines, Australia and Africa (Kausar, Mani, G, & Hedina, 2016; Mani et al., 2016). Hence, the present study focuses on the protective effect of the membrane-bound, mitochondrial enzyme activity and lipid profile levels of the ethanolic leaf extract of E. thymifolia Linn. and E. hirta Linn. on hepatotoxic rats induced by carbon tetrachloride (CCl4).


Plant materials collection and extracts preparation

The E. thymifolia and E. hirta were collected from Kanchipuram, India and the species were identified by a botanist from St. Joseph’s College, Tiruchirappalli, India and the same were deposited at the Rapinet Herbarium with voucher numbers GDMM 001 and GDMM 002. The shade dried E. thymifolia and E. hirta leaves were powdered mechanically and then extracted using ethanol as the solvent by hot continuous percolation method using Soxhlet apparatus for 24 h. The extracts were filtered and evaporated on a water bath followed by drying in a vacuum. The prepared extracts were stored under the refrigerator until further analysis. All other chemicals mentioned in this study were analytical grades obtained from Hi-Media (Mumbai, India).

Animal housing and experimental design

The eight weeks old Albino Wistar rats (150-165 g) were procured from the Biogen Laboratory Animal Facility, Bengaluru, India. The same was maintained at 25 ± 1˚C with a 12 h light/dark cycle. The animals were fed with a standard pellet diet which is obtained from Amrut Laboratory Animal Feed, Bangalore, India. The diet consists of 22.21 per cent of protein, 3.32 per cent of fat, 3.11 per cent fiber, which is balanced with 67% of carbohydrates, minerals and vitamins as well as water ad libitum. The study and experimental procedures were approved by the Ethical Committee of the Srimad Andavan College of Arts and Science (Registration Number: 790/03/ac/CPCSEA), Tiruchirappalli, India. The rats have cared as per the guidelines provided by the board for control and supervision of experimental animals (CPCSEA, 2004).

Ethanolic leaf extract of E. thymifolia and E. hirta (300 mg/kg b.w.) were freshly suspended in sterile water and the same was administered to the animals orally by intubation early morning every day till the end of the study period. The rats were arbitrarily divided into 7 groups with 6 rats in each group and the same were housed individually in the ventilated cages. Animal groups were categorized as control (basal diet, G1) were given saline water, CCl4-induced single cardiac dose (1.5 mL/kg, b.w., i.p.) as negative control (G2), G1 were given 300 mg/kg b.w., of ethanol extract of E. thymifolia (G3) and E. hirta (G4), G2 supplemented with 300 mg/kg b.w., of ethanolic extract of E. thymifolia (G5), E. hirta (G6), and 25 mg/kg b.w., of silymarin (G7) for the period of 21 days.

Biochemical evaluation

All the rats were sacrificed by decapitation at the end of the study period and the blood was taken from the jugular vein. It has been centrifuged at 3000 rpm for 20 min and the serum samples were stored under a refrigerator until used. The following parameters were analyzed for the ATPase including calcium Ca2+)-ATPase and sodium/potassium (Na+/K+)-ATPase (Hjerten and Pan, 1983), magnesium (Mg2+)-ATPase (Ohnishi, Suzuki, & Suzuki, 1982), and mitochondrial enzymes like ICH (isocitrate dehydrogenase) (Bell & Baron, 1960) , KDH (α-ketoglutarate dehydrogenase) (Reed & Mukherjee, 1969), SDH (succinate dehydrogenase) (Slater & Bonner, 1952), MDH (malate dehydrogenase) (Mehler, Kornberg, & Grisolia, 1948), cytochrome-C-oxidase (Pearl, Cascarano, & Zweifach, 1963) and nicotinamide adenine dinucleotide dehydrogenase (NADPH) (Minakami, Ringler, & Singer, 1962). The lipid profiles including TC (total cholesterol) (Allain, Poon, & Chan, 1974), TG (triglycerides) (Mcgowan, Artiss, & Strandbergh, 1983), PL (phospholipids) (Zilversmit & Davis, 1950), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol and very-low-density lipoprotein (VLDL) cholesterol by the calculating method described by (Friedewald, Levy, & Fredrickson, 1972). All the biochemical parameters are done using commercial available kits and was determined by fully automated biochemical anlyzer (Turbo Chem 100, CPC Diagnositics).

Statistical analysis

The data were analyzed using the commercial statistical software (SPSS for Windows, with version 17.0, Chicago, USA) for one-way analysis of variance (ANOVA) which is followed by Duncan’s multiple range tests (DMRT) analysis. The results were expressed as means±SD (standard deviation). In the present study, P values with <0.05 were considered significant.


The different membrane-bound enzymes such as Na+/K+ATPase, Mg2+ATPase and Ca2+ATPase of the control and treatment groups were seen in Table 1. These enzyme activities were significantly reduced at P<0.05 when the administration of CCl4. After, the treatment of silymarin and ethanolic extracts of leaves of the E. thymifolia and E. hirta significantly (P<0.05) restored the levels of the altered enzyme when compared with the control group. There was no adverse effect was observed in plant extracts alone groups of both plants. Table 2 depicts the mitochondrial enzymes (ICH, KDH, SDH, MDH, cytochrome-C-oxidase and NADPH dehydrogenase) in the control and treated groups. Toxication with CCl4 reduced the levels of the mitochondrial enzymes significantly (P<0.05) when compared to the control group. The altered activities of the above enzymes were restored normalcy significantly (P<0.05) when the administration of ethanolic extract of both plant extracts and silymarin. Table 3 showed that the serum lipid profile of TC, TG, HDL, LDL, VLDL and PL in the normal control group and experimental groups. The administration of CCl4 disturbed the lipid metabolism. The ethanolic extract of E. thymifolia and E. hirta (300 mg/kg) and silymarin (25 mg/kg) normalize the altered lipid profile level significantly at P<0.05. In the present study, the plant extracts group did not show any side effect in all the studied parameters.

Table 1

Activities of ethanolic extract of E. thymifolia and E. hirta on the membrane-bound enzymes.

Animal Group
































[i] G1 - Control, G2 – Negative control – CCl4-induced, 1.5 mL/kg, G3 – G1 + E. thymifolia 300 mg/kg, G4 - G1 + E. hirta, 300 mg/kg, G5 – CCl4 + E. thymifolia, 300 mg/kg, G6 – CCl4 + E. hirta, 300 mg/kg, G7 – CCl4 + silymarin, 25 mg/kg.

[ii] All the values are mentioned as means ± SD, six rats in each group

[iii] The different superscripts significantly differ at p ≤ 0.05

Table 2

Activities of ethanolic extract of E. thymifolia and E. hirta on mitochondrial enzymes.

Animal Group

ICH(U/mg protein)

KDH(U/mg protein)

SDH(U/mg Protein)

MDH(U/mg protein)

NADPH(U/mg protein)

CYT-C(U/mg protein)


















































[i] G1 - Control, G2 – Negative control – CCl4-induced, 1.5 mL/kg, G3 – G1 + E. thymifolia 300 mg/kg, G4 - G1 + E. hirta, 300 mg/kg, G5 – CCl4 + E. thymifolia, 300 mg/kg, G6 – CCl4 + E. hirta, 300 mg/kg, G7 – CCl4 + silymarin, 25 mg/kg.

[ii] All the values are mentioned as means ± SD, six rats in each group. The different superscripts significantly differ at p ≤ 0.05

Table 3

Effect of ethanolic extract of E. thymifolia and E. hirta on Lipid profiles.

Animal Group

TC (mg/dl)

TG (mg/dl)

PL (mg/dl)

HDL (mg/dl)

VLDL (mg/dl)

LDL (mg/dl)


















































[i] G1 - Control, G2 – Negative control – CCl4-induced, 1.5 mL/kg, G3 – G1 + E. thymifolia 300 mg/kg, G4 - G1 + E. hirta, 300 mg/kg, G5 – CCl4 + E. thymifolia, 300 mg/kg, G6 – CCl4 + E. hirta, 300 mg/kg, G7 – CCl4 + silymarin, 25 mg/kg.

[ii] All the values are mentioned as means ± SD, six rats in each group. The different superscripts significantly differ at p ≤ 0.05


The ATPase is related to the cell membrane and it helps for the translocation of ions which includes magnesium, calcium, sodium and potassium, and. It requires energy for the translocation process. The ATPase regulates the concentration of cellular electrolytes and transmembrane electrolytes. In hepatotoxic conditions, the subcellular metabolism and structural alterations are formed in the cell membrane (Premalatha & Sachidanandam, 1998), which are in agreement with the same in the present CCl4-induced hepatotoxic animals. Among the ATPase, the Na+/K+ATPase regulates the concentration of intracellular Na+ at a low level and it also maintains the water content in the cell (Chandramohan, Ebrahim, & Babu, 1996). Inhibition of this Na+/K+ATPase has minimized the cellular metabolism. This ATPase is inhibited by increasing the concentration of cholesterol (Yeagle, 1983). The statement has been proved in this study, because the increasing concentration of cholesterol may be there as on for the decreasing concentration of the Na+/K+ATPase level are noted in the present findings.

Figure 2

Protective effect of Euphorbia thymifolia and Euphorbia hirta leaf on carbon tetrachloride-induced membrane damage.

The Ca2+ATPase regulate the Ca2+ pump activity, and it regulates the cellular processes. (Alkon & Rasmussen, 1988) proves that the the Ca2+ATPase are involved in the muscle concentration and neurosecretion process. The protein depletion has been associated with the diminished level of Ca2+ATPase in CCl4-administrated animals. In CCl4-induced animals, the level of Ca2+ATPase decreased due to the H2O2 present in the toxic condition. This may be evidence of the previous study (Monte, Ross, & Bellomo, 1984). The increasing concentrations of lipid peroxide decrease the Ca2+ATPase level due to the inhibition of thiol oxidation. The treatment of plant extracts may increase the Ca2+ATPase level. After the treatment, the level of lipid peroxide increases and favours the thiol oxidation and increases the Ca2+ATPase level. The Mg2+ATPase plays an important role in the electrolyte transport across the biological membrane. The administration of CCl4 alters the membrane permeability, may be due to the decreasing concentrations of Mg2+ATPase. In the present findings, the level of Mg2+ATPase has been noted to be decreased in CCl4-induced animals. The restoration of the normal levels of the Mg2+ATPase is achieved may be due to the treatment of E. thymifolia and E. hirta treated animals. The decreasing concentration of lipid peroxide may protect the membrane-bound enzymes from oxidative damage and this is the reason for the reducing levels of Mg2+ATPase has noted in the plant extract-treated animals. This is evident in cold-pressed Coriandrum sativum oil have potential antioxidant properties (El-Hadary & Hussanien, 2016). Similarly, Physalis peruviana juice acts as a modulate the apoptosis and cell cycle arrest linked to hepatocellular carcinoma.

The viability of the cell is mainly determined by the functions of the mitochondria, the cell energy obtained from the mitochondria by β-oxidation of fatty acids, Kreb’s cycle and oxidative phosphorylation. Some of the exogenous toxins like juice act inhibit the above pathway and the events in the respiratory chain (Pessayre, Mansouri, & Haouzi, 1999; Vijayakumar, Anand, & Manikandan, 2020). In CCl4-treatment induces oxidative stress, thereby it reduces the formation by reducing equivalents (Vijayakumar, Rengarajan, & Radhakrishnan, 2019). The mitochondria is one of the important intracellular targets of CCl4. Hence, Kreb’s cycle enzymes and respiratory chain enzymes are easily affected by CCl4. The ICH is an important mediator enzyme used to supply the NADPH is essential for the production of GSH, which is used to protect the mitochondria from cellular damage. In the present findings, the decline of ICH was noted in CCl4-induced animals due to oxidative damage of the liver (Al-Assaf, 2014).

The liver is the main site for metabolic processes including lipogenesis and lipoprotein synthesis. In the present findings, CCl4 damages the liver and causes hepatic necrosis. It leads to the accumulation of fat in the liver (Becker, Messiner, & Berndt, 1987). (Roullier, 1964), proves that the fat molecules are deposited in adipose tissue in the liver during the CCl4-induced toxicity. In the disease condition, the production and the metabolism of cholesterol were impaired. Due to the fat accumulation in the liver the cholesterol level is increased in the bloodstream as well as significant changes are noted TG and hepatic PL is seen in the CCl4-induced animals in the present investigation. The elevated serum TG is an independent and important risk factor for the pathophysiology of cardiovascular disease (Anand, Chenniappan, & Kalavathy, 2008). The PL, an important component in a cell membrane, and responsible for cell integrity. This compound regulates the permeability of cells because it involves signal transduction activity. In the present investigation, the PL level has been raised in the CCl4-induced animals, due to the cell damage by the toxin. This statement has been proved in the earlier study of (Kaffarnik, Schneider, & Eimer-Brede, 1975). After the treatment of E. thymifolia and E. hirta decreases the PL level. This may be due to the protective activity of plant extracts on the cell membrane (Figure 2).

The present study demonstrated that the LDL and VLDL were increased and the level of HDL has decreased in the CCl4-treated animals. The HDL plays an important role in the removal of excess cholesterol in the liver via the bile (Dietschy, 1997). The increases in cholesterol level may result from the decline of HDL level (or) the increased fatty acid synthesis in the liver as well as the accumulation of TG suppress the secretion of lysosomal acid triacylglycerol lipase activity (Gans, 1973). Due to the liver damage, the LDL receptor defect occurs at the site of the liver and fails to perform its function, this may increase the LDL level in the blood (Bharathi, Rengarajan, & Radhakrishnan, 2018; Vijayakumar, Rengarajan, & Radhakrishnan, 2018). The HDL level is indirectly proportional to the concentration of LDL and VLDL. After the treatment of E. thymifolia and E. hirta alters the levels lipid profile including TC, TG, PL, LDL, HDL and VLDL to near normal. Treatment with 200 mg/kg of cold-pressed Syzygium aromaticum reduced the levels of lipid profile in CCl4-induced hepatotoxicity (El-Hadary & Hassanien, 2016). In the present study, there is observable change noted in the plant only treated group when compared with the control group. This was evidenced in the safety of the plants. The maximum dose of 300 mg of both plant extract values is like the standard drug silymarin treated group.


From the present findings, the extracts of E. thymifolia and E. hirta enhances the activities of mitochondrial enzymes and reduce the membrane-bound enzymes, thereby it improves the anti-oxidant mechanism of mitochondria, as well as it, restored the lipid profile levels. Hence, further molecular studies are needed for the supporting evidence and it may be used as a drug soon for various liver-related diseases.


The data set for the present study is available from the corresponding author upon request.

Conflicts of interest

Given his role as Associate Editor, Balamuralikrishnan Balasubramanian has not been involved and has no access to information regarding the peer review of this article. Full responsibility for the editorial process for this article was delegated to Associate Editor Onur Bender. There is no conflict of interest.

Author contributions

BB, VAA - Research concept and design; DMMG, MR, SP - Collection and/or assembly of data; BB, MR, SP, AM, RLR, WL, VAA - Data analysis and interpretation; DMMG - Writing the article; BB, SP, AM, RLR, WL, VAA - Critical revision of the article. All authors approved final version of the article for publication.