Assessment of Garcinia pedunculata Roxb. Role on Biological Markers in Carbon Tetra Chloride Induced Rat Model

Abstract

Garcinia pedunculata Roxb. is an important medicinal plant of North Eastern (NE) region of India, having number of medicinal properties and used against various diseases in folk medicine. An empirical research was designed to carry out evaluation of hepatoprotective activity of the leaves of G. pedunculata with special reference to its putative protective role. Methanolic extract from the dried leaf powder of G. pedunculata Roxb. was prepared by hot continuous extraction method. The prepared extract was investigated at different dose levels for its hepatoprotective nature and further histopathological study was carried out to ascertain the degree of reversing the hepatotoxic manifestation induced by CCl₄ (Carbon tetrachloride). LD₅₀ values of the G. pedunculata Roxb. extract was found to be safe up to 2000 mg. The in vivo biological studies on serum and tissues of male Wister rats at the doses of 100 mg, 300 mg and 600 mg/kg body weight respectively was carried out taking Silymarin as standard. The methanolic extract of G. pedunculata Roxb, improved the cholesterol level along with significant improvement of SGPT (Serum Glutamate Pyruvate Transaminase), SGOT (Serum Glutamate Oxaloacetate Transaminase), ALP (Alkaline Phosphatase) and total protein in respect to Silymarin group. The test extract at the dose 600 mg/kg body weight was found to significantly reverse the elevated marker enzymes i.e. SGOT, SGPT, ALP indicating its hepatoprotective role. The higher dose extracts were also found to have pronounced effect on oxidative stress parameters such as GSH and catalase on CCl₄ induced rats. The histopathological studies further augmented the protective activity of G. pedunculata leaf extract thereby endorsing the traditional usage of the plant.

Introduction

With the growing relation between mankind and nature, people have discovered remedies for various diseases along with other ailments from natural products. Exploration of these secrets of nature continues with advancement of knowledge, skills and techniques. It has been estimated that out of 4, 22,000 of flowering plants available in this world, approximately 50,000 are used for medicinal purpose of which India represents 43% [1].

Liver being one of the most overburdened organ, liver associated complexities are more common among the population hence posing as major health issue challenging health care personnel. Phytochemical research in this field reported isolation of nearly 170 phytoconstituents having hepatoprotective activity [2].

Garcinia pedunculata is an important predominant fruit of North Eastern (NE) region of India, having numbers of medicinal properties and used against various diseases in folk medicine. It is locally known as Bor-thekera in Assam [3]. Garcinia pedunculata is a large evergreen tree with fluted trunk with short spreading branches. Branchlets slightly four angled. It can grow up to 20 m tall. Leaves are thick obovate or oblanceolate, rigid sub-coriaceous and 13–30 × 15–21 cm in size. The mature fruit is eaten cooked or raw; generally it is mixed with other vegetables, especially with pulses [4].

The fruit is used in gastric, dysentery, diarrhoea and cooling [5, 6]. The fruit is also used for treatment of jaundice [7].

The aim of this study is to carry out evaluation of hepatoprotective activity of the leaves of G. pedunculata with special reference to its putative protective role in carbon tetrachloride induced liver toxicity on Wister albino rats.

Materials and Methods

Collection and Authentication of Plant Material

The leaves of G. pedunculata were collected from Dibrugarh district, Assam of NE India. The plant is identified and authenticated taxonomically by Dr. S.K. Singh, Scientist-D, BSI, Shillong, Meghalaya vide letter No. BSI/ERC/2015/Plant identification/01 Dt. 06.03.2015. The voucher specimen of the collected sample was deposited in the departmental museum for future reference.

Preparation of Leaf Powder

The leaves were cleaned and cut into small pieces allowed for shed dry. The dried leaves were grinded and passed through mesh 40 and stored in an airtight container for further studies.

Extraction of Powdered Leaves

The powdered shade dried leaves were packed in Soxhlet extractor for extraction with methanol. The extract was concentrated by distilling off the solvent and evaporated to dryness in rotary vacuum evaporator. Further the methanolic extract of the leaves of G. pedunculata Roxb. was subjected to fractionation using dichloromethane (DCM) to remove chlorophyll like substances. The fractionated extract labeled as FEGP, was used for further study.

In vivo Hepatoprotective Study

Selection of animals Male Wister rats weighing about 100–200 g were used for the study. All studies were performed in accordance with the guidance for the care and use of laboratory animals, as adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, India. Regd. No.—1576/GO/a/11/CPCSE Dated: 17/2/2012.

Maintenance and acclimatization of animals The animal house was well ventilated and animals were kept under conditions of 24 ± 20 °C and RH 44–56% and 12 h light: 12 h dark cycles each day for 1 week before and during the experiments. Animals described as fasted were deprived of food for 16 h but allowed free access to water.

Acute Toxicity Studies

Oral acute toxicity study was performed according to OECD guideline to reduce both the number of animals and the level of pain associated with acute toxicity testing.

Female albino rats were kept fasting for overnight providing only water, after which the extract was administered orally at various dose level (200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight) dissolved in 0.3% w/v carboxymethylcellulose suspension [8, 9]. Continuous observation for the initial couple of hours followed by intermittent behavioral study for another 4 h was done. Finally any mortality along with other toxic manifestation was recorded up till 72 h. The animals were furthermore observed for 2 weeks.

Experimental Design for Study of Hepatoprotective Activity

The animals are divided randomly into six groups of six rats each as shown in the Table 1.

Table 1.

Experimental design for in vivo hepatoprotective activity (n = 6)

Group	Induction of hepatotoxicity	Treatment	Name of group
I	Vehicle control	0.3% CMCa	Control
II	CCl4 (i.p.)	0.3% CMCa	Negative control
III	CCl4 (i.p.)	Silymarin (100 mg/kg b.wt./day)	Reference standard
IV	CCl4 (i.p.)	Plant extract (FEGP) p.o. (100 mg/kg b.wt./day)	Test 1
V	CCl4 (i.p.)	Plant extract (FEGP) p.o. (300 mg/kg b.wt./day)	Test 2
VI	CCl4 (i.p.)	Plant extract (FEGP) p.o. (600 mg/kg b.wt./day)	Test 3

^aCMC, carboxymethylcellulose

Group I served as the control, and received the vehicle (0.3% CMC in distilled water) at a dose of 1 ml/kg b.wt./day/i.p. for 14 days [10]. Group II served as the Negative Control group, and received carbon tetrachloride (CCl₄) prepared in the vehicle, at a dose of 1 ml/kg body weight, i.p., (1:1 v/v mixture of CCl₄ and 0.3% w/v CMC in distilled water) twice a week. The second dose of the toxicant was given 36 h after administering the first dose [10, 11]. Group III served as the Reference Standard group, received CCl₄ (1 ml/kg b.wt./twice a week/i.p.) followed by silymarin at a dose of 100 mg/kg b.wt./day for 14 days/p.o [12]. Group IV served as the Test 1 group, received CCl₄ (1 ml/kg b.wt./twice a week/i.p.) followed by test drug at a dose of 100 mg/kg b.wt./day for 14 days/p.o [12]..Group V served as the Test 2 group, received CCl₄ (1 ml/kg b.wt./twice a week/i.p.) followed by test drug at a dose of 300 mg/kg b.wt./day for 14 days/p.o. [12]. Finally, Group VI served as the Test 3 group, received CCl₄ (1 ml/kg b.wt./twice a week/i.p.) followed by test drug at a dose of 600 mg/kg b.wt./day for 14 days/p.o. [12].

Bio-Chemical Parameter Investigation

Blood samples from each group were collected through retro-orbital route. The collected blood samples were allowed to stand for 20 min, and then centrifuged for 15–20 min at 2000 rpm to separate the serum and the later was used for biochemical estimations of parameters viz. Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP), Total Cholesterol, Total Protein, Bilirubin (Total and Direct) [13].

Estimation of Glutathione (GSH)

Liver tissues of the animals sacrificed by cervical dislocation were collected, washed in 0.9% saline, soaked in filter paper, weighed and stored at − 20 °C. Tissue fragments (200 mg) were thawed and homogenized on ice in 1 ml of 250 Mm sucrose, 20 mg Tris-HCl, 1 mg dithiothreitol, pH 7.4, using glass-Teflon homogenizers. The homogenates were centrifuged at 75,000 rpm at 4 °C for 2 h. Supernatants were stored at − 20 °C. Reduced glutathione was estimated by determination of dithiobis (2-nitro)-benzoic acid (DTNB) reduced by SH-groups, as described by Ellman 1959 and experessed as mole/mg protein [14].

Estimation of Catalase

10 µl samples were taken in tube containing 3.0 ml of H₂O₂ in phosphate buffer. Time required for 0.05 optical density changes was observed at 240 nm against a blank containing the enzyme source in H₂O₂ free phosphate buffer diluted to 100 ml [15].

Histopathological Examination

Following method was followed to prepare the liver slides for observation:

Stage 1: Tissue Processing

Tissues were at first cleared by washing with acetone. Tissue samples were kept in acetone for half hour. The whole process is repeated for three times.

In this step the tissues were subjected to dehydration. Samples were kept in benzene for about half hour. It is also repeated for three times.

Stage 2: Embedding

Paraffin was melted in oven for about 2 h. Tissue samples were embedded in the molten paraffin and made into blocks. Then the blocks were allowed to cool. After that the blocks were placed in microtome and ribbon strips were sectioned with a thickness of few millimeters. The slides were then incubated overnight at a temperature of 37 °C.

Stage 3: Staining

Slides were melted in spirit lamp and were washed with xylene for 3–5 min, repeated twice. Then washed again with absolute, 90% and 70% alcohol for 3–5 min repeating each washing twice. After that the slides are washed under running water for about 20 min.

In this step the slides were stained with haematoxylin, the latter was kept for about 3–5 min for proper tissue penetration. The slide appeared bluish in colour. After that the excess stain was washed off under running water.

The slides were then treated with acid-alcohol (1% HCl in absolute alcohol). The slide now appears violet. Then the slides were washed under running water for about 2–3 min. Then these are treated with eosin for 30 s.

Stage 4: Dehydration and Fixing

Dehydration was done with the help of alcohol treatment followed by xylene. Xylene treatment was done for about 3–5 min and the process was repeated thrice. After that these were mounted in DPX and covered with cover slip. The slides were then observed under microscope.

On the last day, 1 h after the treatment animals were sacrificed followed by isolation of liver for histopathological examination as per above mentioned method and observed under microscope (Leica Photomicroscope DM1000) [16, 17].

Results and Discussion

Study of Non-invasive Biomarkers

The body temperature and body weight of the experimental animals was recorded which served as non-invasive biomarkers of disease progression and overall health. As depicted in Fig. 1 the body temperature of all the Wister rats in each group was found to be within normal range, however incase of body weight screening it was found that except the test groups, the negative control group showed a decrease in body weight and standard group witnessed increment in their body weight (Fig. 2).

Fig. 1.

Body temperature variation of the animals in different groups

Fig. 2.

Body weight variation of the animals in different groups

In Vivo Hepatoprotective Study

Bio-Chemical Parameter Investigation

The effects of different doses of FEGP on the SGPT, SGOT, ALP, cholesterol, total and direct bilirubin and total protein levels are summarized in Tables 2 and 3. FEGP at doses of 100, 300 and 600 mg/kg body weight showed varied effect on biochemical parameters. Altered liver function and/or liver mitochondrial injury were evident from the data, indicating an increment in serum concentration of enzymes such as SGPT, SGOT and ALP. Liver injury contributes to increased serum level of transaminase enzymes due to easy availability of amino acids. After treating the test group in different dose level, significant reversal of elevated marker enzymes were observed in dose level of 600 mg/kg body weight (Table 2).

Table 2.

Effects of FEGP on SGOT, SGPT, ALP and Cholesterol level on experimental animals

Groups	SGOT (U/L)*	SGPT (U/L)*	ALP (U/L)*	Cholesterol (mmol/L)*
G-I vehicle control	75.67 ± 2.90	47.58 ± 2.77	99.26 ± 3.79	41.54 ± 2.19
G-II negative control	261.65 ± 3.04#	221.81 ± 3.71#	310.95 ± 3.79#	93.16 ± 2.30#
G-III standard drug	63.56 ± 2.94#	54.22 ± 2.97#	118.25 ± 3.02#	51.59 ± 2.91#
G-IV test 1 (100 mg/kg)	122.35 ± 2.96	102.63 ± 3.65	267.73 ± 3.33	87.97 ± 2.58
G-V test 2 (300 mg/kg)	107.15 ± 3.51	90.56 ± 2.98	184.989 ± 3.37	76.59 ± 2.30
G-VI test 3 (600 mg/kg)	95.04 ± 2.95#	74.61 ± 2.85#	135.37 ± 3.08#	62.01 ± 2.95#

*Values are mean ± SEM (n = 6)

#Values are statistically significant at p < 0.05

Table 3.

Effects of FEGP on direct bilirubin, total bilirubin and total protein level on experimental animals

Groups	Direct bilirubin (mg/dl)*	Total bilirubin (mg/dl)*	Total protein (mg/dl)*
G-I vehicle control	0.46 ± 0.07	1.31 ± 0.36	8.14 ± 0.68
G-II negative control	1.26 ± 0.22#	4.13 ± 0.22#	4.27 ± 0.44#
G-III standard Drug	0.56 ± 0.03#	1.67 ± 0.19#	7.65 ± 0.48#
G-IV test 1 (100 mg/kg)	0.78 ± 0.04	3.15 ± 0.25	6.63 ± 0.49
G-V test 2 (300 mg/kg)	0.69 ± 0.03	2.39 ± 0.31	5.74 ± 0.53
G-VI test 3 (600 mg/kg)	0.61 ± 0.05#	1.83 ± 0.12#	5.04 ± 0.43#

*Values are mean ± SEM (n = 6)

#Values are statistically significant at p < 0.05

The lipid profile gives an important indication of metabolic disturbance including hepatic disease. The higher concentration of serum cholesterol, serum bilirubin and lower concentration of total protein may be attributed to liver disease such as hepatitis, cirrhosis, obstructive jaundice etc. The treatment with FEGP at the dose level of 600 mg/kg body weight showed marked declination in the levels of markers as compared to negative control along with moderate declination in markers with lower test dose as summarized in Table 3.

Liver Enzyme Activity Determination

The effects of different doses of FEGP on the oxidative stress were determined in the CCl₄ induced Wister rats using Silymarin as standard drugs. The results of oxidative stress marker status are summarized in Table 4. A decreased level of GSH (Glutathione) was observed in the negative control group indicating reduction in GSH synthesis or degradation of GSH by oxidative stress. The treatment with FEGP at the dose level of 600 mg/kg body weight showed prominent increase of reductase power with reductase activity of 12.36 ± 0.58 as compared to the negative control group 5.72 ± 0.38 and standard group with 14.62 ± 0.45.

Table 4.

Effects of FEGP on glutathione and catalase activity level on experimental animals

Groups	Glutathione (U/mg protein)	Catalase (U/mg protein)
G-I vehicle control	16.74 ± 0.73	21.89 ± 0.83
G-II negative control	5.72 ± 0.38	9.44 ± 0.69
G-III standard Drug	14.62 ± 0.45	19.27 ± 0.75
G-IV test 1 (100 mg/kg)	8.11 ± 0.55	11.96 ± 0.77
G-V test 2 (300 mg/kg)	10.58 ± 0.62	13.53 ± 0.84
G-VI test 3 (600 mg/kg)	12.36 ± 0.58	16.68 ± 0.56

*Values are mean ± SEM (n = 6). Values are statistically significant at p < 0.05

Antioxidants may have a role in the prevention of hepatic disease. Catalase is a haem containing enzyme which detoxify the H₂O₂ into water and oxygen. The level of catalase in liver was improved significantly by FEGP in a dose dependent manner in the test group indicating its potentiality to detoxify the reactive oxidation species.

Histopathological Examination

The liver section slides of different groups are examined for the study of histopathological changes occurring due to administration of various doses of test extracts, and are compared with that of standard and negative control groups (Fig. 3; H&E).

Fig. 3.

Comparative changes in histo-architecture of liver with different doses of test (FEGP) and standard drug as observed under Leica Photomicroscope DM1000 at × 100 and × 400 magnification

The microanatomy of the positive control group reveals the normal hepatocellular cords with the all the three zones i.e. periportal, mid and centrilobular zones. Portal tract and central vein or terminal hepatic venules are intact and normal (Fig. 3a, g; H&E). Sinusoids are observed to be lined by the endothelial cells and Kupffer cells without any sinusoidal dialations. Stellate (Ito) cells are seen to be present in the space of Disse (Fig. 3g; H&E). Histoarchitecture analysis of negative control group reveals large lipid vacuoles within the hepatocytes which are suggestive of macrovesicular steatosis (fatty liver). Liver parenchymal injury was clearly revealed with the observation of swelling hepatocytes, bleb formation and cell rupture (Fig. 3c, i; H&E). This maybe the resultant of lipoprotein transport disruption due to interference with mitochondrial and microsomal function by CCl₄ leading to the accumulation of lipids in the hepatocytes. Widespread parenchymal loss including zonal loss of hepatocytes indicating confluent necrosis is observed. Collapsed histoarchitecture, inflammed portal tract, ductular cholestasis are prominently revealed (Fig. 3i; H&E). The standard drug treated section revealed areas of regenerative changes characterized by mostly smaller hepatocytes that are seen to be arranged in cords which are found to be separated by sinusoids. The space of Disse is found to be expanded (Fig. 3b, h; H&E). The liver sections of lower dose drug treated group (dose 100 mg/kg body wt.) revealed significant hepatocyte necrosis, ballooning degeneration and Kupffer cell hyperplasia with negligible restoration of cellular integrity as observed by scattered blood clotting (Fig. 3d, j; H&E). The 300 mg/kg bw receiving group histopathology slide observation revealed focal necrosis, portal triditis along with parenchymal injury with slight signs of regeneration of hepatocytes (Fig. 3e, k; H&E).The group receiving dose of 600 mg/kg bw, showed cells integrity reversal to nearly normal. Lobules are clear, the portal triads are distinctly visible. Sinusoids lined by fenestrated endothelial cells are clearly observed. Kupffer cells present in the luminal side of sinusoids are seen. Marked change in sinusoidal dilation and congestion are clearly observed and is comparable with that of the standard (Fig. 3f, l; H&E).

Conclusion

From the above study, it can be stated that the hepatoprotective activity of FEGP, particularly the higher doses were significantly able to reverse and restore the serum and histopathological imbalances induced by CCl₄ in the experimental animals. The study thereby justifies the traditional usage of the plant in treating jaundice hence, fully supporting the in vivo hepatoprotective status. Further study is warranted with regard to elucidation and characterization of active bimolecules from the extract so as to augment the traditional practices.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving animals were in accordance with the ethical standards promulgated by the Institutional Animal Care Committee, CPCSEA, India. The institutional registration number is 1576/GO/a/11/CPCSE dated: 17/2/2012. The present study is approved vide Approval No. IACE/DU/99 Dated 27/3/2015.