Chemical Profile and Hepatoprotective Activity of Ethyl Acetate Extracts of Euphorbia paralias and Euphorbia geniculata (Euphorbiaceae) from Egypt

Plants have used as an essential source of drugs and remedies on treatment of diseases and health disorders since ancient times1. Flavonoids and phenolic compounds containing plants which are common among medicinal plants were reported for its various health benefits and applications. They have a wide spectrum of pharmacological activities including antiinflammatory, antioxidant, hepatoprotective, anticancer and antimicrobial activities. They also decrease the risk of cardiovascular diseases, enhance regeneration of the liver and increase life expectancy2,3. As liver damage can be life threatening and its damage is caused by several factors such as alcohol, viruses, organic chemicals, metabolic and genetic abnormalities4. Liver transplantation was improved survival rate of patients in some cases only and is limited to a small number of patients due to non-availability of suitable donors. And so, finding new drugs that are able to enhance liver regeneration and prevent liver failure is a very important need. Natural products as plant extracts exhibiting antioxidant and hepatoprotective activities can be useful in these needs2. The aim of the current study was to identify the polyphenolics and flavonoids in the ethyl acetate (EA) fractions of the aerial parts of Ep and Eg using UPLC-ESI-MS/MS and to investigate the possible hepatoprotective activities of the studied fractions.


INTRODUCTION
Plants have used as an essential source of drugs and remedies on treatment of diseases and health disorders since ancient times 1 . Flavonoids and phenolic compounds containing plants which are common among medicinal plants were reported for its various health benefits and applications. They have a wide spectrum of pharmacological activities including antiinflammatory, antioxidant, hepatoprotective, anticancer and antimicrobial activities. They also decrease the risk of cardiovascular diseases, enhance regeneration of the liver and increase life expectancy 2,3 . As liver damage can be life threatening and its damage is caused by several factors such as alcohol, viruses, organic chemicals, metabolic and genetic abnormalities 4 . Liver transplantation was improved survival rate of patients in some cases only and is limited to a small number of patients due to non-availability of suitable donors. And so, finding new drugs that are able to enhance liver regeneration and prevent liver failure is a very important need. Natural products as plant extracts exhibiting antioxidant and hepatoprotective activities can be useful in these needs 2 . The aim of the current study was to identify the polyphenolics and flavonoids in the ethyl acetate (EA) fractions of the aerial parts of Ep and Eg using UPLC-ESI-MS/MS and to investigate the possible hepatoprotective activities of the studied fractions.

Plant material and extraction
Aerial parts of E. paralias L. and E. geniculata Ortega were collected in the flowering stage on May and August 2015, respectively. E. paralias was collected from the North beach of Alexandria, Egypt. While E. geniculata was collected from roadsides in the vicinity of Banha, Qalubya, Egypt. The identification was kindly verified by Dr. Ahmed Abd El-Razik Lecturer of Plant Taxonomy, Department of Botany, Faculty of Science, Banha University, Egypt. The vouchers specimens (no. S303 and S304) were deposited in National Research Centre, Dokki, Cairo, Egypt. The air-dried powdered plant materials Ep and Eg (500 g of each plant) were extracted by cold maceration with 70 % methanol until complete exhaustion. The methanolic extracts were evaporated under reduced pressure at 45 o C. The greenish brown viscous residues (105.0 and 100.5 gm respectively) were separately dissolved in MeOH-H 2 O mixture (500 ml, 1:9 v/v) and subjected to fractionation with dichloromethane then by ethyl acetate to afford ethyl acetate fractions (18.5 and 9.2 gm) of Ep and Eg, respectively.

UPLC-ESI-MS/MS
The sample (100 μg/ml) solution of each fraction (ethyl acetate fractions of E. p. and E. g.) were prepared using high performance liquid chromatography (HPLC) analytical grade solvent of MeOH, filtered using a membrane disc filter (0.2 μm) then subjected to LC-ESI-MS analysis. Samples injection volumes (10 μl) were injected into the UPLC instrument equipped with reverse phase C-18 column (ACQUITY UPLC-BEH C18 1.7 μm particle size-2.1×50 mm column). Mobile phase elution was made with the flow rate of 0.2 ml/min using gradient mobile phase comprising two eluents: eluent A is H 2 O acidified with 0.1 % formic acid and eluent B is MeOH acidified with 0.1 % formic acid. Elution was performed using the following gradient: 20 % B, 0-1 min; 20-90 % B, 1-18 min; 20 % B, 18-20 min. Mass spectra were detected in the ESI negative ion mode between m/z 50-900 at 30 ev capillary conc. and capillary voltage 3 KV. The peaks and spectra were processed using the Maslynx 4.1 software and tentatively identified by comparing its retention time (Rt) and mass spectrum with reported data. For fragmentation collision energy 40 eV was used.

Animals
Male Sprague Dawley rats weighting (220±5 g) and Balb C mice (20-25 g) were purchased from Theodor Bilharz Institute (Giza, Egypt). The animals were housed in the animal facility of Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, Egypt. They were fed with standard diet pellets (El-Nasr Company, Abou-Zaabal, Cairo, Egypt), and allowed free access to water. The animals were kept at room temperature (25°C±2.0) and natural humidity (it was 555) with 12 h-light/12 h dark cycle. The experiments were conducted in accordance with the ethical guidelines for investigations in laboratory animals and comply with the guidelines for the care and use of laboratory animals. The approval committee was given by ZU-IACUC committee with approval number ZU-IACUC/3/F/145/2019.

Acute toxicity or lethality (LD 50 ) test
To assess for Ep and Eg extracts safety margin, the lethality test (LD 50 ) was carried out by estimation of acute toxicity monitored in animals. Ethyl acetate fractions of Ep and Eg were dissolved in dist. water at the mentioned doses below. The (LD 50 ) was estimated in mice by oral intake according to OECD guidelines No. 420. At a preliminary test, the mice were divided into three groups each of 12 animals and each animal received one dose of 5000 mg/kg body weight of the mice. Animals were kept under observation for 24 h for any signs of toxicity or cases of deaths. Control animals received the vehicle (normal saline) and were kept under the same conditions without any treatment. Toxicity signs and number of deaths for each extract during 24 h were recorded and the LD 50 was calculated as the geometric mean of the dose. The results obtained showed no lethality.

Antifibrotic effect
Ethyl acetate fractions of Ep and Eg were dissolved in dist. water at the required doses, forty eight rats were divided into eight groups, six animals each, and the following schedule of treatment was adopted: Group 1 (Control group): rats were given normal saline daily (2 ml/kg b.w., orally) for 4 consecutive weeks and served as negative control group. Group 2 (TAA): rats were injected intra-peritoneal with TAA (200 mg/ kg b.w.) dissolved in saline three times weekly for 4 consecutive weeks. Group 3 (Sil): rats were treated with silymarin orally (50 mg/kg b.w.) dissolved in normal saline daily for 4 consecutive weeks. Group 4 (Ep): rats were administered Ep fraction orally (200 mg/kg b.w.) dissolved in normal saline daily for 4 consecutive weeks. Group 5 (Eg): rats were treated with Eg ethyl acetate fraction orally (200 mg/kg b.w.) dissolved in normal saline daily for 4 consecutive weeks. Group 6 (Sil+TAA): rats were pretreated orally with silymarin then with thioacetamide daily for 4 consecutive weeks. Group 7 (Ep+TAA): rats were pretreated orally with Ep ethyl acetate fraction then with thioacetamide daily for 4 consecutive weeks using the same dose schedules as mentioned above. Group 8 (Eg+TAA): rats were pretreated orally with Eg ethyl acetate fraction then with TAA for 4 consecutive weeks using the same dose schedules as mentioned above, plants fractions and silymarin were administered to the animals orally by gastric intubation for 4 weeks following the procedure of (Dutta et al., 2013) 5 .

Serum and tissue preparations
Samples of blood were collected retro-orbital venous plexus of rats (under light ether anesthesia) in non-heparinized tubes and for measuring biochemical parameters; the sera were separated. Later the animals were sacrificed; liver was dissected, washed in saline, blotted between dry filter papers and kept until antioxidants and histopathological examinations.

Biochemical analysis
Serum separated from blood samples was used for the determination of liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol (CH), triglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and total bilirubin 6 . Part of liver tissue was homogenated and centrifuged at 5000 rpm for 10 min and the resulting supernatant was used for lipid peroxides malondialdehyde (MDA) contents, determination of oxidative enzymes; superoxide dismutase (SOD), catalase (CAT) activities, and reduced glutathione (GSH) 7,8 . All tests were carried out using colorimetric spectrum Biodiagnostics TM and Diamond TM kits (Cairo, Egypt),

Histopathological examination
Autopsy samples were taken from the rats livers in the different groups and fixed in 10 % neutral buffered formalin for 24 h. Then serial dilutions of alcohol (ethyl, absolute ethyl and methyl) were used in specimens' dehydration. The specimens were cleaned by xylene and embedded in paraffin in a hot air oven at 56°C for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 4 µm thicknesses by sledge microtone. The obtained sections of tissue were embedded between glass slides, deparaffinized, stained by hematoxylin and eosin stain, and another slides from the same samples stained with a specific stain (Masson ‫׳‬ s trichrom) then all were examined using the light electric microscope 9 .

Statistical analysis of data
All data are presented as mean±SEM. Statistical analysis was performed using GraphPad prisim version 7 (GraphPad, San Diego, CA). Group differences were analyzed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer for multiple comparison tests. The difference was considered significant at P ≤ 0.05.

UPLC-ESI-MS/MS identification of secondary metabolites
Structural analysis of different compounds found in the aerial parts of Ep and Eg ethyl acetate fractions resulted in the separation and tentative identification of 32 compounds using ULPC-ESI-MS/MS. Identification of compounds was performed using (M-1) + /MS 2 and comparison with reported data. Ellagitannins and phenolic acids (22.00 and 11.48 %), flavonoids such as quercetin glycosides (35.00 and 39.97 %) and kaempferol glycosides (20.00 and 3.44 %) were found to be the major components in Ep and Eg, respectively; as ellagitannins (tetragalloyl hexoside, ellagic acid, gallic acid) and flavonoids (kaempferol-3-Oβ-(6''-galloyl-O-glucopyranoside, quercetin glycosides (glucoside and arabinoside) were found to be the major components in Ep while quercetin rutinoside and other quercetin glycosides (glucoside, arabinoside and rhamnoside) were highly abundant in Eg. As observed; molecular and fragment ions were listed in Table 1. LC-MS/MS profiles for Ep and Eg ethyl acetate fractions in the negative ion mode are shown in (Figure 1).     13 . Peak 32 identified as dimethoxy apigenin due to molecular ion peak at 329 m/z and daughter ion at 269 amu 13 .

Hepatoprotective
Thioacetamide is hepatotoxic agent known to induce acute or chronic liver disease (fibrosis and cirrhosis) in the experimental animal model 17 .
In the present work, TAA is used as potent hepatotoxic agent in rats. A dose of 200 mg/kg ip TAA administration is reported to be the cause of hepatic toxicity. Its effect is due to increased oxidative stress 18 .

Acute toxicity or lethality (LD 50 ) test
The results showed that the animals survived during the 24 h observation and no visible signs of toxicity were observed. According to Hodge and Sterner toxicity scale 19 , the LD 50 values of the two fractions were in the practically non-toxic categories.

Evaluation of liver biochemical parameters
Exposure of animals with the hepatotoxic agent, TAA , resulted in significant (p ≤ 0.05) increase in the liver enzymes (ALT and AST) and total bilirubin in serum, lipid profiles (CH, TG and LDL) and lipid peroxidation (MDA) while significant decrease in HDL, GSH,

Histopathological results
Histopathological examinations of the sections of rat liver exposed to TAA showed (in H and E staining) severe tissue damage and hepatocytes degeneration. Ep and Eg pre-treatment attenuated the hepatic injury and showed significant protection of the hepatic cells from damage. There were no such alterations in Ep and Eg groups in compared to normal and silymarin treated groups (Figures 5 and 6).

DISCUSSION
The liver is the largest gland in the human body and susceptible to almost many different diseases including hepatitis, cirrhosis, alcohol related disorders and liver cancer. A major cause of these disorders is due to exposure to different environmental pollutants and xenobiotics 20 . Also, the exposure to a lot of chemicals, such as carbon tetrachloride, bromobenzene, ethanol, thioacetamide and polycyclic aromatic hydrocarbons have been implicated in the etiology of liver diseases 21 . It is fundamentally known that the regulation of apoptosis is a potential mechanism through which many agents such as polyphenolic compounds; can prevent toxicity and carcinogenesis 22 .
Silymarin is a mixture of natural flavanolignans contains at least seven compounds 23 . The hepatoprotective and antioxidant activities of silymarin were attributed to control free radicals (FR), produced by the hepatic metabolism of toxic substances 24 . The present study revealed that both Ep and Eg contain several types of gallotannins, phenolic  . These compounds were known to have high antioxidant activity that attributed to their ability to control FR demonstrated by high hepatoprotective activity in comparison with standard drug silymarin 25,26 . That was evidenced by the significant improvement of liver enzymes (ALT and AST), total bilirubin, lipid peroxidation (MDA), oxidative stress related parameters (CAT, GSH and SOD) and lipid profile (CH, LDL, TG and HDL). According to the study; the potency strength of the fractions and silymarin on the liver enzymes differs from one to other; Ep+TAA gp show more improvement in ALT and CH, than Eg+TAA gp but nearly equal to Sil+TAA gp, while Eg+TAA gp show more improvement in GSH and CAT in comparison with Sil-gp but the both plant fractions were equally in improvement degree in the rest of liver enzymes. Histopathologicaly, parallel structural improvement was elicited by either plants extracts as compared to silymarin. This is evidenced by the views of rat's liver tissues treated with both plants as showing no collagen aggregation around central veins, no inflammatory infiltrate and no fibrosis. The results of the study on liver were indicating the promising values of these plants as hepatoprotective herbs in the future with more flow up.

CONCLUSIONS
This study revealed the identification of 32 polyphenolic compounds in the ethyl acetate fractions of the Ep and Eg using UPLC-ESI-MS/ MS analysis; mainly tannins and flavonoid glycosides. Hepatoprotective activity exhibited by the studied extracts might be attributed to the high content of these compounds. These findings need more explored and investigated through further set of experiments to recommend the ethyl acetate extracts of the two plants as hepatoprotective drugs of natural origin.