Biochemical and histopathological effects on liver due to acute oral toxicity of aqueous leaf extract of Ecliptaalba on female Swiss albino mice.

BACKGROUND: Limited data is available about the toxicity of herbal remedies used for self-medication. Since a popular medicinal plant Ecliptaalba contains various bioactive molecules, the present study aimed to observe the biochemical and histological changes in liver associated with acute oral toxicity (LD50) of aqueous extract of E. alba (L.) Hassk. in female Swiss albino mice.
MATERIALS AND METHODS: For the acute oral toxicity study, the animals were divided into six groups of 6 mice each. Group- I was normal control and the treatment groups were administered aqueous leaf extract of E. alba orally at different doses of 500 mg (group - I),1750 mg (group-III), 2000 mg (group- IV), 2500 mg (group- V) and 3000 mg/ kg/b.wt.(group- VI) for seven consecutive days. The mice were sacrificed on the eighth day and blood was collected for the analysis of ALP (alkaline phosphatase), SGPT (serum glutamic pyruvic transferase), total protein and albumin. The liver was dissected, weighed, and processed for histopathological analysis.
RESULTS: The LD50 was found to be 2316.626 mg/kg /body weight in female mice. Serum SGPT, total protein and albumin increased in treated group- IV (P < 0.05), V (P < 0.01), and VI (P < 0.01) as compared to the control (group- I). ALP level significantly decreased in the treated group- IV (P < 0.05), V (P < 0.01) and VI (P < 0.01). Histopathological changes were observed at dose of 2000 mg (group- IV), 2500 mg (group- V) and 3000 mg (group- VI).
CONCLUSION: It was concluded that oral administration of aqueous leaf extract of E. alba had detrimental effects on biochemical parameters and induced histopathological alterations in liver of female Swiss albino mice at doses higher than 2000 mg/kg/day indicating that its indiscriminate use should be avoided.

Introduction

Medicinal plants constitute an effective source of both traditional and modern medicines and about 80% of rural populations depend on it for their primary health care.[1] Toxicity from botanical compounds has been underestimated due to the perception that drugs made from plants are absolutely safe. However, severe liver injury has been described after the ingestion of a large variety of different herbal preparations.[2] Determination of efficacy and safety of herbal remedies is necessary as many people use them for self-medication and little data is available about the pharmacology and toxicology for most of the common herbal remedies.[3]

Ecliptaalba (L.) Hassk. (Asteraceae) commonly known as Bhringaraja, is a popular plant used for the therapeutic purpose in traditional systems of medicine. Its leaves contain beta- amyrin, Wedelolacetone, triterpenoids, flavonoids, luteolin-7-O-glucoside, L-terthienyl methanol and stigmasterol.[4] It is used in treating enlargement of the liver and spleen and various chronic diseases traditionally.[5] In vivo hepatoprotective activity,[6] immunomodulatory and free radical scavenging activity have also been reported.[7]

Due to the wide spread use of E. alba and possible toxicity of various bioactive alkaloids, flavonoids etc, it was considered necessary to study its biochemical and histological toxicity. Liver has a pivotal role in regulation of physiological processes and is also involved in detoxification of a variety of drugs and xenobiotics.[8] Therefore, this study aimed to observe the biochemical and histopathological changes in liver associated with acute oral toxicity (LD50) of aqueous extract of E. alba (L.) Hassk.in female Swiss albino mice.

Material and Methods

Plant Material

E. alba (L.) was collected from the campus of B.M.D College, taxonomically identified by Dr. S. Bedi, (Associate Professor, Department of Botany, PWC, Patna University, Patna) and kept in the herbarium of the laboratory under the voucher specimen number: B.M.D/BOT/03/10. The leaves were shade dried at room temperature (25°C) for 10 days, powdered and stored. Primary phytochemical investigation was conducted by the methods of Kokate.[9]

Female Swiss albino mice (Musmusculus) weighing 30- 35gm (age 6-8 weeks) were housed in stainless-steel wire cages in a well-ventilated room at temperature 25 ± 1°C, humidity 56 ± 5% and under 12-h light/dark cycle. Food (Amrut Laboratory Animal Feed, Mysore Feed Limited, Banglore, India) and tap water were given ad libitum. All animal experiments were carried out as per CPCSEA guidelines (Approval No. -1129/bc/07/CPCSEA).

Acute Toxicity Study and Dose Selection

After acclimatization, the animals were divided into 6 groups of 6 mice each. The control (group- I) received food and tap water ad libitum, while the experimental groups in addition received aqueous leaf extract of E. alba orally at different doses of 500 mg/ kg (group- II), 1750 mg/kg (group- III), 2000 mg/kg (group- IV), 2500 mg/kg (group- V) and 3000 mg/kg (group- VI). The extract was prepared by dissolving 500 mg-3000 mg of dried powder of E. alba leaves in 10 ml of distilled water. The volume of aqueous extract to be administered was determined based on body weight and given to the mice for seven days. The toxicological effects were observed in terms of mortality expressed as LD50. Based on the experimental observations, the acute oral LD50 of the extract was calculated by the use of software for probit analysis (Environmental Protection Agency PROBIT ANALYSIS PROGRAM, used for calculating LC/EC value, Version 1.5).

Biochemical Study

The serum was obtained from the blood (orbital sinus puncture) by centrifugation (3000 rpm for 15 minutes).Liver function test parameters like ALP (alkaline phosphatase), SGPT (serum glutamate pyruvate transaminase), total protein and albumin were determined by the use of standard kit methods[10] using fully Automated Biochemistry Analyzer (Model No-SELECTRA-"E",VITALAB BY MERCK) in the Biochemistry Department of Mahavir Cancer Sansthan and Research Centre, Patna.

Histological Examination

Mice were sacrificed and liver was dissected out, washed thoroughly in normal saline, trimmed, processed, embedded in paraffin, sectioned at a thickness of 4-5 μm, stained with haematoxylin and eosin and observed under light microscope.

Statistical Analysis

Results were presented as mean and standard error (Mean ± S.E). The statistical significance between the control and each of the treated groups were determined by Dunnett's test after one-way ANOVA. The level of significance was set at P < 0.05.

Results

Phytochemical analysis of aqueous leaf extract of E. alba showed presence of alkaloid, phytosterols, triterpenoid and flavanoids (Coumastone), saponins, tannins, sugar, gum and mucilage. We observed that groups V and VI, who were administrated E. alba aqueous leaf extract above 2000 mg/kg b.w. showed a highly significant decrease (P < 0.01) in both body and liver weight when compared to control group- I, while a significant decrease (P < 0.05) in liver weight was observed in group- IV [Table 1]. The LD50 of E. alba in mice was found to be 2316.626 mg/kg/body weight in mice using EPA PROBIT analysis software program (version 1.5) as shown in Table 2 and 3000 mg/kg was the observed acute lethal dose. When compared to the liver function test parameters of control group- I, the increase in serum SGPT was not significant in group- II and group- III, it was statistically significant (P < 0.05) in group- IV and highly significant (P < 0.01) for group- V and group–VI [Table 3]. The decreasein serum ALP level was also statistically highly significant (P < 0.01) in group–IV, group- V and group–VI. Level of total protein was significantly higher (P < 0.01) for the treated group- III, IV, V and VI [Table 3] as compared to the control groupI. A significant increase was observed even in serum albumin level in treated group–III (P < 0.05), group–IV, group–V and group–VI (P < 0.01) [Table 3].

Table 1

Changes in body weight and organ weight of control and female mice treated with aqueous leaf extract of E. alba for seven days exposure period

Table 2

Environmental Protection Agency PROBIT analysis program used for calculating LC/EC values (Version 1.5) for E. alba

Table 3

Biochemical changes in liver of female Swiss albino after administration of different concentration of aqueous leaf extract of E. alba for seven days exposure period

Acute doses of aqueous leaf extract of E. alba induced discrete pathological changes in the liver tissue of female Swiss albino mice in doses more than 2000 mg/kg b.w. In group- II and III (Figure 1b and 1c respectively), hepatocytes are distinct and relatively normal, no fatty changes, dilation of blood vessels and necrosis were observed. In group- IV [Figure 1d], doubling of nuclei and dilated blood vessels were observed. In groups- V [Figure 1e] and VI [Figure 1f] damage to hepatic cell architecture were more pronounced when compared to control group- I [Figure 1a] and binucleated hepatocytes, pleomorphic nuclei, bile ducts and ductules surrounded by collar of lymphocytes denoting the chronic inflammatory changes in the liver cell [Figures 1e and f] were observed.

Figure 1

(a) Microphotographs of liver of Gr-I showing round polygonal cells with spherical nucleus (arrow) H and E (400 X). (b) Microphotographs of liver of Gr-II showing hepatocytes are distinct and relatively normal, no fatty change, cytoplasm not vacuolated. (arrow) H and E (400 X). (c) Microphotographs of liver of Gr-III showing no area of necrosis, no fatty degeneration and change only slight dilation of blood vessels.(arrow) H and E (400 X). (d) Microphotographs of liver of Gr-IV showingdoubling of nuclei and dilated blood vessel (arrow). (e) Microphotographs of liver of Gr-V showing fatty degenaration, and pleiomorphic nuclei. (arrow) H and E (400 X). (f) Microphotographs of liver of Gr-VI showing pleiomorphicnulei, dilated blood vessels and Lymphocytes surrounding ducts and ductules. (arrow). H and E (400X)

Discussion

There is growing concern about the toxicity of herbal remedies as they contain substantial amounts of pharmaceutically active ingredients whose mechanisms of actions and adverse effects are mostly unknown.[11] Severe liver injury, including acute and chronic abnormalities and even cirrhotic transformation and liver failure, have been described after the ingestion of a wide range of herbal products such as mushrooms, germander (Teucriumchamaedrys), chaparral (Larrea tridentate) etc.[2] Hence, investigations on biochemical and histopathological alterations associated with acute oral toxicity of aqueous extract of E. alba was conducted.

Acute toxicity (LD50) test gives a clue on the range of doses that could be used in subsequent toxicity testing and estimating the therapeutic index of drugs and xenobiotics.[12] In our study the LD50 value of the aqueous leaf extract of E. alba on female mice was found to be 2316.6 mg/kg. b.wt. which was similar to previous reportswhereit was observed that the plant did not show any signs of toxicity up to 2g/kg and the minimum lethal dose was greater than 2g/kg when given orally and intraperitonially in mice.[13] The calculated LD50value of aqueous extract of E. alba on female mice is slight less than our earlier reported acute oral toxicity value (LD50) of aqueous leaf extract of E. alba on male Swiss albino mice which was 2413.407 mg/ kg b.wt.[14] This difference may involve factors which account for gender-related pharmacokinetic differences. However in a report of Qadri et al.,2001[15] LD50 values of aqueous extract of E. alba is calculated for oral, intravenous and intraperitoneal routes were 7.841 g kg-1, 302.8 and 328.3 mg kg-1 respectively in albino mice and in an another study of Uddinet al., 2011[5] intraperitonial injection of crude ethanolic extract of this plant at various dose levels (500: 4000 mg/kg) did not show any abnormalities or toxicity after ten days of administration. These variations in the acute toxicity study may be due to difference in route of entry, duration and frequency of exposure, age, sex, variations between different species (interspecies) and variations among members of the same species (intraspecies).[16]

Organ weights are widely accepted for the evaluation of test article-associated toxicities.[17] Hence the decrease in body and organ weight in the present investigation at high dose of aqueous extract of E. alba indicates its toxic potential. Alterations in liver weight may suggest treatment-related changes including hepatocellular hypertrophy.[18]

Liver function tests conducted through blood assays give information about the state of the liver, describing its functionality (albumin), cellular integrity (transaminases) and its link with the biliary tract (alkaline phosphatase).[19] SGPT is the enzyme produced within the cells of the liver and the serum levels of this enzyme increases after liver damage due to increased membrane permeability or liver cell necrosis and cytosol leakage into the serum.[20] The results of present study showed that SGPT level was elevated in the treated mice which indicates hepatocellular damage or injury caused by high doses of aqueous extract of E. alba.

Serum ALP originates mostly from liver and bone. The significant reduction in ALP levels by E. alba aqueous extract shows that no possible cholestasis occurred at the dose levels tested since a rise in plasma alkaline phosphatase (ALP) level is usually a characteristic finding in cholestatic liver disease.[21] The exact cause of significant decrease in ALP level at higher doses [Table 3], is not known but it may be inferred that high dose E. alba may be adversely affecting liver function. There are several reports that document a decrease in the level of serum alkaline phosphatase due to liver damage associated with pernicious anemia, zinc deficiency and hypophosphatasia.[22]

The liver is the major source of most of serum proteins and it is also the only site of synthesis of albumin.[21] Total protein and albumin levels increased in treated group- III, group- IV, group- V and group- VI as compared to control group- I. An increase in total protein and albumin level may be due to conditions of severe or acute dehydration or their increase in synthesis in liver.[23] This is also reported that in various liver dysfunctions, generally the protein content increases so as to maintain the protein concentration in liver.[24] Corticosteroids and thyroid hormone also stimulate albumin synthesis in hepatocytes[22] and in our study, phytochemical analysis of aqueous leaf extract of E. alba showed positive tests for alkaloid, phytosterols, triterpenoid and flavanoids (Coumastone). It has also been reported by Prakash et al.,2011[4] that E. alba contains mainly coumestans polypeptides, polyacetylenes, steroids, triterpenes and flavonoids that may be responsible for increase in albumin levels of treated groups.

The functional studies in toxicology should be coupled with the appropriate histological studies, because appropriate morphological studies are useful for the anatomical localization of action of toxin.[24] In our study, the findings observed at the higher dose of 2000 mg/kg/day in female mice, were well corroborated by histopathological outcomes of liver. In normal liver the hepatic cells are round, polygonal and contain clear spherical nucleus [Figure 1]. Changes in liver architecture at high doses of 2000 mg, 2500 mg and 3000 mg/kg b.w./day of aqueous extract of E. alba were observed indicating toxicity and adverse effects on liver. The toxic effect of aqueous extract of E. alba on liver may be due to anyone or more of the phytochemicals present in the extract.

It is thus clear from this study that this plant has toxic potential and the median lethal dose (LD50) was found to be 2316.626 mg/kg/body weight in female mice. It was also observed that oral administration of aqueous leaf extract of E. alba had adverse biochemical and histological effects on 2000 mg/kg b. wt and at higher doses of 2500 mg and 3000 mg/ kg b.wt/day in liver of female Swiss albino mice. In conclusion, these results provide evidence for the toxicity profile of the aqueous leaf extract of E. alba at high doses and therefore, it should be ingested with caution. This result can also form the basis for clinical trials in human.