Hala Salah Eldin, Haidar Abdal Gadir*, Abdel Wahab Hassan
Medicinal and Aromatic Plants Research Institute (MAPRI), National center for Research, Khartoum, Sudan.
Received: 17/06/2015 Accepted: 02/09/2015 Published: 12/09/2015
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Fagonia cretica L. (Zygophyllaceae) herb is widely distributed in Sudan and used in Sudanese traditional medicine for the treatment of various disorders. The present study was designed to assess the hepatoprotective activity and cytotoxicity of methanolic and water extracts of F. cretica L. These extracts were studied for their hepatoprotective activity using CCl4 induced liver damage in wistar albino rats. The methanolic and water extracts of F. cretica L. at oral dose of (400 mg/kg) showed highly significant (p<0.01) hepatoprotective effect. These biochemical observations wereconfirmed by histopathological examinations of liver sections comparable with the standard hepatoprotective drug Silymarin (100 mg/kg) which served as a positive control.
Fagonia cretica, Hepatoprotective, Histopathological, Silymarin
Traditional medicine has a long history. It is the sum of knowledge, skill, and practices based on the theories, beliefs, and experience indigenous to different cultures, used in the maintenance of health as well as in the prevention . Sudan is one of the largest countries in Africa with a diverse flora. Most of Sudanese people in rural areas rely on traditional medicine for the treatment of many infectious diseases. Sudanese traditional medicine is a unique combination of knowledge and practices of Arabic, Islamic and African cultures [2,3]. Medicinal plants have a promising future because there are about half million plants around the world, and most of their medical benefits have not been investigated yet, and their medical activities could be decisive in the treatment of present or future studies . More than 80% of the world population in developing countries uses plant derived medicines and about half of the population in industrialized countries also are using traditional medicines practices to meet their healthcare needs [5,6]. In Sudan, people dependant on medicinal plants for health care is estimated at over 90% of the population, these plants and derived products play an important role in the primary health care of Sudan .
Liver is vital organ that play a major role in metabolism, energy production and bile production, storage of iron, vitamins and trace elements, detoxification and conversion of waste products for excretion by the kidneys . Liver injury or liver dysfunction is a major health problem that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies. Liver cell injury caused by various toxicants (certain anti-biotic, chemotherapeutic agents, carbon tetrachloride (CCL4), thioacetamide (TAA) etc.), D-galactosamine, Paracetamol, excessive alcohol consumption and microbes [9,10]. A large number of plants and formulations have been claimed to posses hepatoprotective activity.
Fagonia cretica L.
Is a small spiny under shrub mostly found in dry calcareous rocks, belongs to family Zygophyllaceae , It is locally known as (Shokat algemal, Umm Shawaika and Al agoul), and found in the West, Northern and Central Sudan  (Figure 1).
In traditional medicine, Fagonia cretica L. is used as astringent, febrifuge, liver trouble and prophylactic against small-pox. An aqueous decoction of the plant is used to induce abortion, a popular remedy for cancer in the indigenous system of medicine  and in the treatment of snake bite .
Fagonia cretica L. exhibited antimicrobial activity against bacterial strains . It also reported that the plant aqueous extract has an anti breast cancer effect without exhibiting common side effects of standard cytotoxic therapy . The methanolic extract possesses potentials as haemorrhagic inhibitor against snake venom compared to the standard antiserum . Antitumor, antioxidant activities were also reported .
The Plant materials were collected from their natural habitat from different localities in Sudan. Plants were taxonomically identified and authenticated by Dr. Haidar Abd Algadir at the Herbarium of Medicinal and Aromatic Plants and Traditional Medicine Research Institute, where the voucher specimens have been deposited for future references
Preparation of plant extracts
The shade dried materials were ground, powdered and divided into two portions. One portion was extracted with 100% methanol at room temperature, and then the obtained residue was evaporated to dryness using rotatory evaporator to afford methanolic extracts. Whereas, other portion of the powdered material was extracted using boiling water then filtered, the obtained filtrate was dried using a freeze dryer to afford aqueous extract or water extract.
Phytochemical screening of the studied extracts gives information about the nature of the constituents found in the plants. (Tannins, Triterpenes, Alkaloids, Flavonoids, Saponins, Coumarins and Anthraquinone glycosides).
Assessment of hepatoprotective activity
Methanolic and water extracts of Fagonia cretica L. were evaluated for their hepatoprotective activity. Wistar albino rats weighing 90-100 g of either sex maintained under standard husbandry conditions (temp 23 ± 2°C, relative humidity 55 ± 10% and 12 hours light dark cycle) were used for the screening(as models ). Animals were fed with standard laboratory food and ad libitum during the study period. Rats were divided into seven groups of five animals each, for each extract. Normal control rats ( group I) received three doses of 5% gum acacia mucilage (1 ml/kg) orally at 12 hours intervals (0 hour, 12 hours and 24 hours) negative control rats (group II) received three doses of vehicle at 12 hours intervals and a single dose of Carbon tetrachloride (1.25 ml/kg i.p.) diluted in liquid paraffin (1:1) subcutaneous injection.30 minutes after the administration of first dose of vehicle, rats of standard (group III) received three doses of Silymarin, (100 mg/kg) at 0 hour, 12 hours and 24 hours. Carbon tetrachloride was administered (1.25 ml/kg i.p.) 30 minutes before the first dose of Silymarin which represents positive control. On the other hand, rats of test( group IV),and (V), received two doses of methanolic extracts 200 and 400 mg/kg body weight, while ( group VI), (VII) received two doses of water extracts at dose of 200 and 400 mg/kg body weight respectively at 0 hour, 12 hours and 24 hours. Carbon tetrachloride was administered (1.25 ml/kg i.p.) 30 minutes before the first dose of test extracts , after 36 hours of administration of Carbon tetrachloride, animals were sacrificed and blood sample for Hematological and Biochemical Analysis were collected into test tubes with and without EDTA, respectively. Hemoglobin (HB), Haematocrit(HCT), Red Blood Cell count (RBC), White Blood Cell count(WBC), Mean Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV) and Platelet Count were determined using Hematology Analyser (Humacount Plus, Germany). Biochemical analysis was performed on serum obtained after centrifugation of total blood (without anticoagulant) at 2500 rpm for 15 min. automated system (COBAS Plus Integra 400, Germany) was used for determination of the biochemical parameters (Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Creatinine, Alkaline Phosphatase (ALP), Bilurubin, Albumin, Total Protein, Urea, Creatinine,and Cholestrol).
Liver sample from each rat were removed after dissection and preserved in 10% formalin. Using the standard micro technique . Sections were taken at (5 μm) thickness and stained with hemotoxylin and eosin dye, the sections were then observed under microscope for histopathological changes in liver architecture, and their photomicrographs were taken using micro imaging microscope (Olympus BX 51, Japan).
Statistical Package for Social Science (SPSS) was used for the analysis of the data. The significance of differences between means was compared among the groups using Independent-sample T-test. For brine shrimp test data was analyzed with Finney computer program to determine LD50 values with 95% confidence intervals.
Phytochemical screening was performed for the extracts of the studied plant. In the present study, water extracts of Fagonia cretica L. revealed presence of high content of saponin compared to the methanolic extracts, same content of flavonoids. Tannins, coumarin and triterpens were also detected
There were no significant changes in the hematological parameters of both methanolic and water extracts of Fagonia cretica L. compared to Silymarin and CCl4 treated groups of rats. These results were shown in Table 1 and Figure 2.
Biochemical Parameters of Fagonia cretica L.
At dose of 400 mg/ml methanolic extract represented highly significant changes (P<0.01) on the enzyme levels alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphotase (ALP) compared to Silymarin and CCl4 treated groups of rats.
The methanolic and water extracts of Fagonia cretica L. exhibited remarkable effect in lowering the high level of liver enzymes that elevated by CCl4 induced cell damage. These results were shown in Table 2 and Figure 3.
Histopathological changes occurred in liver after 48 hrs of CCl4-injection, these changes were shown in Figure 4 which indicated that the water extract slightly preserved the normal hepatocellular architecture from the damaging effects of CCl4 as compared to methanolic extract. The effects of Silymarin doses on liver sections showed multifocal tissue loss, perilobular necrosis and portal thrombosis, while, CCl4 doses showed inflammatory cells, necrosis, thrombosis in both central and portal vein and lobucentral necrosis (Figure 5).
Liver diseases remain one of the serious health problems. In the absence of reliable liver protective drugs in allopathic medical practices, herbs play a role in the management of various liver disorders in ethnomedical practices as well as in traditional systems of medicine . Liver damage induced by CCl4 is commonly used as model for the screening of hepatoprotective activity. The rise in serum levels of SGPT, SGOT and ALP, has been attributed to the damaged structural integrity of the liver, because they are cytoplasmic in location and released into circulation after cellular damages. Carbon tetrachloride induces hepatotoxicity by metabolic activation; therefore, it selectively causes toxicity in liver cells maintaining semi normal metabolic functions. CCl4 metabolically activated by Cytochromes P450 (CYPs) in the endoplasmic reticulum to form a trichloromethyl free radical (CCl3), which combined with cellular lipids and proteins in presence of oxygen to induce lipid peroxidation . Highly reactive trichloro free radicals formation directly attacks the poly unsaturated fatty acids of the endoplasmic reticulum and thus cause over production of SGOT, SGPT and ALP. In the present study, pre-treatment with methanolic and water extracts of Fagonia cretica L. (200 mg/kg and 400 mg/kg) attenuated the increases in the activities of SGOT, SGPT and ALP levels in blood serum of CCl4 induced hepatotoxicity. Silymarin is a known hepatoprotective compound obtained from Silybum marianum family (Asteraceae) is reported to have a protective effect on hepatic plasma membrane and possess multiple mechanisms of actions against different hepatotoxic agents. The study shows that the methanolic and water extracts of Fagonia cretica L. at higher dose (400 mg/ kg) exhibited remarkable effect in lowering the high level of liver enzymes that occurred by CCl4 compared to standard drug Silymarin, But the histopathological findings showed that the methanolic extract was superior to the water extract in protecting the hepatocellular architecture against CCl4 induced toxicity.
On the other hand, cytotoxicity was evaluated using Brine shrimp Lethality Test (BSLT), the Fagonia cretica L. extracts revealed significant cytotoxic activity against brine shrimps . The LD50 ranged from 0 to 100 μg/ml and those with <20 μg/ml were considered to be very active. Methanolic extract of Fagonia cretica L. showed LD50=77.2849 μg/ml. Therefore, the water extract which showed (LD50=1.7 μg/ml) was found to be very active against brine shrimp [21,22].
In conclusion, preliminary phytochemical screening of extracts of Fagonia cretica L. showed the presence of some major bioactive compounds such as saponin, flavonoids and tannin, that can protect the liver. Fagonia cretica L. extracts showed cytotoxic effects. The methanolic and water extracts of F. cretica L. showed hepatoprotective activity. There is no previous study for hepatoprotective activity for F. cretica L. This plant can be considered as new biosource of hepatoprotective drugs