INTRODUCTION:

Nature has always been helpful to us in providing herbs of medicinal importance¹. Phytoconstituents of the herbs such as flavonoids, alkaloids, glycosides and lignans have been found to possess medicinal benefits in the prevention of liver disorders and cancer².

The utilization of herbal medicines and herbal therapy by number of patients is increasing exponentially³. Human usage of herbal medicines for the treatment of diseases has a long history since from Neanderthal ages⁴. With the above informative background the present research was Designed to extract and evaluate the hepato protective and cytotoxic potentials of Chrozophora plicata leaves. The leaves of the plant are rich in flavonoids^{5, 6}. Chrozophora plicata leaves are known to possess medicinal properties such as anthelmentic, antiulcer, anti-inflammatory, antiasthamatic, hepato protective and also used in skin diseases⁷. There are no documented evidence regarding the hepatoprotective and anticancer activity of the Chrozophora leaves. Hence the present research is required and is justifiable.

MATERIALS AND METHODS:

Fresh leaves of Chrozophora plicata plant was collected from the fields of the Gajwel (mandal), Medak dist, Telangana, India. The sample specimen is kept in fresh condition by adding few drops of disinfectant formalin (2%). Leaves were identified and authenticated by a Senior Scientist i.e Dr. N. Sivaraj, National Bureau of Plant Genetic Resources, Rajendanagar, Hyderabad. Chrozophora plicata leaves were shade dried and mechanically pulverized to a coarse powder and weighed. The coarse powder weight was found to be 1500g. The powder was subjected to hot continuous successive extraction in a Soxhlet apparatus with solvents in the increasing order of polarityusing petroleum ether, chloroform, hexane, ethyl acetate, methanol and water under controlled temperature (50-60 °C) ⁸. Extractives were concentrated below 40 °C and further drying was carried out under reduced pressure. The six dried extractives were stored in a dessicator for further evaluation⁹. The obtained extracts were subjected to phytochemical tests for detection of phytoconstituents present in it viz. alkaloids, carbohydrates, glycosides, phytosterols, fixed oils and fats, phenolic compounds and tannins, proteins and free amino acids, gums and mucilages, flavanoids, lignins and saponins¹⁰. The methanolic extract was found to contain a large amount of flavonoids, alkaloids, glycosides and lignans. Hence methanolic extract of Chrozophora plicata leaves was selected for further acute toxicity studies and pharmacological investigations.

Acute toxicity studies:^{11, 12}

The Acute toxicity study was done in accordance to OECD guidelines 423 with fixed dose studies where the limit dose is 2000 mg/kg body weight of test rats. The Procedure was divided in to 2 phases, phase I (Day 1 observation) and phase II (observation of rats for the next 14 days). The rats were dosed at 100, 250, 500, 700, 1000 and 2000 mg/kg of methanolic extract of Chrozophora plicata leaves. Individual rats were observed for 4 hrs for behavior, autonomic and neurological symptoms or mortality. Body weights were recorded 6 hrs post dosing. From next day onwards, each day every 1 hour the behavioral change, toxic signs or mortality was observed in the same animals for next 14 days and body weights were recorded on 8^th and 14^th day post dosing. In the absence of lethality, 1/10^th and 1/5^th of the higher dose was selected as therapeutic dose.

Hepatoprotective activity:

CCl₄ induced hepatotoxicity:¹³

Albino rats were divided in to 5 groups each group consisting of 6 animals.

Group I:

Untreated control group (Normal control) (1% liq. Paraffin, 1 ml/kg, s.c.)

Group II:

Positive control group (hepatotoxin control) (Vehicle once daily for 7 days (s.c) followed by 1 ml/kg b.w CCl₄:liq.paraffin (1:1) (s.c) on 7^th day)

Group III:

Standard group (100 mg silymarin once daily for 7 days (p.o) followed by 1ml/kg b.w CCl₄: liq. paraffin (1:1) s.c on 7^th day).

Group IV:

Methanolic extract of Chrozophora plicata leaves, MECP (200 mg/kg b.w dose daily for 7 days (p.o) followed by 1 ml/kg b.w CCl₄:liq.paraffin (1:1) s.c on 7^th day)

Group V:

Methanolic extract of Chrozophora plicata leaves, MECP (400 mg/kg b.w dose daily for 7 days (p.o) followed by 1 ml/kg b.w CCl₄:liq.paraffin (1:1) s.c on 7^th day)

Experimental Details:

All groups of rats were treated as shown above. On 8^th day, 18 h after the administration of CCl₄, all the animals were anesthetized with anesthetic ether for the collection of blood from retro-orbital plexus by using heparinized capillary tube. The collected blood was centrifuged at 12000 rpm for 10 minutes and the serum was separated. The separated serum was used for analyzing biochemical parameters such as SGPT, SGOT, ALP, total proteins, total bilirubin and triglycerides.

Paracetamol Induced Hepatotoxicity:¹⁴

Albino rats were divided in to 5 groups containing six animals in each group.

Group I:

Normal control group (2% w/v acacia suspension)

Group II:

Toxin control group (vehicle for 7 days + paracetamol 2 g/kg b.w (p.o) on 5^th day)

Group III:

Standard group (silymarin 100 mg/kg b.w daily for 7 days and paracetamol 2g/kg b.w on 5 th day)

Group IV:

MECP (200 mg/kg b.w dose daily for 7 days (p.o) and paracetamol 2g/kg b.w (p.o) on 5^th day).

Group V:

MECP (400 mg/kg b.w dose daily for 7 days (p.o) and paracetamol 2g/kg b.w (p.o) on 5^th day).

Experimental details:

All groups of rats were treated as shown above. On 8^th day, all the rats were anesthetized with anesthetic ether and the blood was collected from retro-orbital plexus with the help of heparinized capillary tube. The collected blood was centrifuged at 12000 rpm for 10 minutes and the serum was separated. The separated serum was further used for the estimation of biochemical markers such as SGPT, SGOT, ALP, total proteins, total bilirubin and triglycerides.

Thioacetamide – induced hepatotoxicity:¹⁵

Rats were divided in to 5 groups of each containing 6 rats.

Group I:

Normal control group (2% w/v acacia suspension, p.o)

Group II: Toxin control group (Vehicle for 9 days, p.o + thioacetamide 100 mg/kg, s.c prepared in distilled water on 7^th day)

Group III:

Standard group (Silymarin 100 mg/kg b.w daily for 9 days, p.o and thioacetamide 100 mg/kg s.c on 7^th day)

Group IV:

MECP (200 mg/kg b.w dose daily for 9 days, p.o and thioacetamide 100 mg/kg s.c on 7^th day)

Group V:

MECP (400 mg/kg b.w dose daily for 9 days, p.o and thioacetamide 100 mg/kg s.c on 7^th day)

Experimental Details:

All groups of rats were treated as shown above. After the treatment on 9^th day, all the rats were anesthetized with anesthetic ether and the blood was collected from retro-orbital plexus with the help of heparinized capillary tube. The collected blood was centrifuged at 12000 rpm for 10 minutes and the serum was separated. The separated serum was further used for the estimation of biochemical markers such as SGPT, SGOT, ALP, total proteins, total bilirubin and triglycerides.

Isolation of Liver:

After sacrificing the rats, liver was dissected out and washed with ice cold saline solution. The weight of the liver was noted after pressing between filter paper pads. A small portion of liver was suspended in 10% formaldehyde for histopathological studies.

Invitro cytotoxic activity of methanolic extract of Chrozophora plicata leaves:

Dalton’s Lymphoma Ascites (DAL) cell lines were used for the present cytotoxic assessment. These cell lines were procured from Amala Cancer Research Center, Thrissur, Kerala, India.

Dose Selection for cytotoxic evaluation:

The following doses of methanolic extract of Chrozophora plicata leaves i.e. 10mg/ml, 25 mg/ml; 50 mg/ml 100 mg/ml and 200 mg/ ml were selected.

Preparation of 5- Fluorouracil (5-FU) dose:

5mg/ml stock solution of 5-FU was prepared and from the stock solution dilution was made to 100µg/ml with sterile water for injection.

Counting of cells:

The live cells were counted by using haemocytometer by diluting the stock solution to 1x10⁶ cells and then transferred cells in to the 96 well plates in tryphan blue dye exclusion method. Tryphan blue dye assay involves addition of 0.5ml Trypan Blue (0.4 %) to 0.5ml cell suspension and the cells were loaded on to a haemocytometer after 5mins of incubation. The number of dead cells was counted and the percentage of dead cells was calculated. Viable cells exclude the dye while non-viable cells take up the dye and appear blue in colour.

In vitro cytotoxic assays:

1. Tryphan Blue dye exclusion test:

The cytotoxic activity of methanolic extract of chrozophora plicata leaves was evaluated by in vitro method on Dalton’s ascitic lymphoma (DAL) cell lines using Tryphan Blue dye exclusion assay. Short term cytotoxicity studies were done on DAL cells by tryphan blue exclusion method. The Tryphan blue dye exclusion assay is used to find out the number of viable cells in cell suspension. The principle involved in this test is that viable cells contain intact cell membrane that excludes certain dyes like tryphan blue, eosin or propidium whereas dead cells take up the stain. One million cells were taken for cell cytotoxicity studies. Different concentrations of methanolic extract of Chrozophora plicata leaves 10mg/ml, 25 mg/ml; 50 mg/ml, 100 mg/ml and 200 mg/ ml were added to the cells and volume was made up to 1 ml with demineralized water. DAL cells suspension was incubated at 37⁰C in Co₂incubator with continuous flow of CO₂. After incubation, 0.2 ml of cell suspension was taken and diluted to 0.5 ml with phosphate-buffered saline, PBS (0.3 ml) and tryphan blue (0.2 ml), mixed well and kept aside for 5 mins. The number of dead cells was counted and the percentage of dead cells was calculated. Viable cells exclude the dye while non-viable cells take up the dye and appear blue in colour.

% of Dead cells = Dead cells/total cells x 100.

2. Cell Viability Assay by MTT:

This colorimetric assay measures the reduction of yellow MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] by mitochondrial succinate dehydrogenase in viable cells into an insoluble blue colored product known as formazan which is measured spectroscopically. Since the reduction of MTT can only occur in metabolically active cells the level of activity is a measure of viability of the cells.

Procedure:

MTT solution preparation:

Add 5mg/ml MTT in PBS (stock solution)

MTT working solution:

1:10 dilution of stock in PBS

DAL cell lines were used for the MTT assay. The cell lines were cultured in tissue culture bottles containing minimum essential media (MEM) supplemented with 10% heat inactivated fetal calf serum (FCS) containing 5% of mixture of Gentamycin, Penicillin (100Units/ml) and Streptomycin (100µg/ml) in presence of 5% Co₂ at 37ºC for 3-4 days. After 3 days the supernatant media was removed and replaced with Dulbecco’s modified eagle medium (DMEM) media with 5% Gentamycin, Penicillin and Streptomycin. Incubate overnight. The growth of DALL cell lines were observed microscopically and after sufficient growth of cell lines in the DMEM media, the supernatant media was discarded and the cell lines were obtained as individual cell suspension. The cell suspension was treated with different concentrations of chrozophora plicata leaf extract diluted with demineralized water and the cell lines diluted with DMEM medium. Control group contains only DMEM media and cell lines. DAL cell lines were plated in 96 wells microlitre plate and incubated for 24 hrs at 37⁰ C in a humidified atmosphere of 5% CO₂ , after the medium was replaced with MTT solution (20µl) and further incubated for 4 hrs. The supernatant carefully aspirated and the precipitated crystals of Formazan blue’ were solubilized by adding DMSO (200µl) and optical density was measured at wavelength of 570nm

The result was represented as the mean of three readings.

Mean OD of test compound X 100

(%) Surviving cells = ____________________

Mean OD of control

RESULTS AND DISCUSSION:

Chrozophora plicata which is also known as Chrozophora rottleri in India is a plant belonging to euphorbiaceae family. It is an annual herb and is used traditionally for various skin and blood disorders. Chrozophora plicata plant leaves are subjected to shade drying followed by pulverization in to coarse powder and then subjected to soxhlation with polar and nonpolar solvents like petroleum ether, chloroform, hexane, ethyl acetate, methanol and water. Methanolic extract revealed the presence of high amount of flavonoids by producing intense pink-red colour with shinoda test. Hence methanolic extract was selected for further investigations. Acute toxicity studies of methanolic extract of Chrozophora plicata leaves was done in accordance to OECD guidelines 423. The methanolic extract was treated to female rats at doses 100, 250, 500, 700, 1000 and 2000 mg/kg by using oral gavage did not exhibited any signs of toxicity or adverse effects. The rats were observed twice daily for fourteen days did not produced any drug related toxic signs or mortality. Hence the acute oral LD₅₀ of the methanolic extract was concluded to exceed 2000 mg/kg body weight. Therefore 2000 mg/kg was considered as safest higher dose and 1/10^th i.e 200 mg/kg and 1/5^th of 2000 mg/kg i.e 400 mg/kg was selected for the further studies. The hepato protective potential of methanolic extract of Chrozophora plicata leaves was assessed against CCl₄, paracetamol and thioacetamide induced hepatotoxicity. The serum levels of SGPT, SGOT, and ALP, total protein, total bilirubin, triglycerides and liver weight were analyzed and compared with CCl₄, paracetamol and thioacetamidetreated toxicant groups. The weight of the liver and the levels of SGPT, SGOT, ALP, bilirubin and triglycerides were significantly increased while the protein levels were significantly decreased in the positive control group. Methanolic extract of Chrozophora plicata leaves (MECP) significantly altered serum parameters thus exhibiting hepatoprotective activity of the plant leaves. SGPT levels were significantly increased to five fold in the toxicant group. However, it was significantly decreased in the MECP (p<0.01 for 200 mg/kg and p<0.001 for 400 mg/kg) treated rats. Similarly SGOT levels were increased to an extent of 3 fold in hepatotoxic control rats. This was significantly decreased in MECP treated rats with p<0.001 for 200 and 400 mg/kg. Alkaline phosphatase levels were nearly doubled in the hepatotoxic group and were significantly reduced in MECP treated rats (p<0.001 for 200 and 400 mg/kg). Total protein levels in the serum were decreased in the positive control group. MECP at 200 and 400 mg/kg significantly increased the levels of total proteins. Total bilirubin levels were elevated to 5 folds in the positive control group and significantly decreased with MECP leaves treated groups at 200 and 400 mg/kg (p<0.001).There was a 3 fold increase in serum triglyceride levels in the hepatotoxin control group. Whereas with MECP 200 mg/kg the triglyceride levels was not significantly reduced, but with MECP 400 mg/kg the elevated triglycerides were significantly reduced (p<0.001). There was a significant increase in the liver weight with toxicant group. However MECP at 400 mg/kg significantly decreased liver weight. The standard hepatoprotective silymarin reduced the levels of serum enzymes, total bilirubin, triglycerides and increased the total protein levels significantly (p<0.001). However silymarin exhibited moderate effect on liver weight (p<0.05). The results are shown in the table no.1, 2 and3 and figure No. 1,2 and 3.

Histopathological studies:

Normal control group liver revealed normal architecture. The hepatocytes, sinusoids, central veins and portal tracts appear within normal limits. Positive control (CCl_4, paracetamol and thioacetamide treated) groups revealed loss of normal liver architecture with fibrosis, congested sinusoids, ballooning degeneration, lymphocytic infiltration, hemorrhage and centrilobular necrosis ( fatty liver). In standard silymarin treated rats, liver architecture was preserved with minimal fibrosis, portal tract and lesser extent of hepatic focal degeneration. In case of methanolic extract of Chrozophora plicata leaves (200 mg/ml) treated rats, the liver architecture was normal with moderate lymphocytic infiltration, light regeneration of hepatocytes and light ballooning degeneration associated with haemorrhage. Methanolic extract of Chrozophora plicata leaves (400 mg/ml) treated rats showed mild lymphocytic infiltration, more regeneration of hepatocytes and mild ballooning degeneration associated with haemorrhage. The liver photomicrographs are shown in the figure no. 4. In tryphan blue dye exclusion test for invitro cytotoxic activity it was found that the methanolic extract of Chrozophora plicata leaves at dose 200 mg/ml exhibited significant anticancer activity by increasing the percentage of dead DAL cells to 94 %. Whereas with MTT cell viability assay it was found that Chrozophora plicata leaves at dose 200 mg/ml exhibited significant anticancer activity by decreasing percentage of survival of DAL cells to 0.951%. The results are depicted in the table no. 4 and 5 and figure no. 5 and 6.

Table No.1: Effect of methanolic extract of Chrozophora plicata leaves (MECP) on serum parameters and liver weight in CCl₄ induced hepatotoxicity

Groups

Liver weight (g/100g b.w)

SGPT (U/I)

SGOT (U/I)

ALP (U/I)

Total Proteins (g/dl)

Total bilirubin (mg/dl)

Triglycerides (mg/dl)

Normal control

3.642± 0.17

51.72

±5.03

98.95

±7.14

154.63

±5.04

9.317

±0.615

0.415

±0.153

84.36

±5.65

Positive control

4.281 ±0.32

286.76

±8.25

328.6

±27.3

345.82

±15.4

6.318

±0.715

2.5

±0.168

196.82

±15.6

Standard silymarin

3.61± 0.71

125.24

±8.19***

128.04 ±11.7***

145.6

±11.3***

8.914

±0.16***

0.564

±0.18***

95.116

±1.6***

MECP (200 mg/kg)

4.354± 0.65

218.91

±13.6**

211.16

±8.15***

205.6

±6.7**

7.651

±0.11**

1.356

±0.16**

148.1

±1.34

MECP (400 mg/kg)

3.96± 0.08

165.43

±11.5***

171.3

±15.2***

158.14

±8.21***

8.416

±0.92***

0.71

±0.05***

106.26

±6.94**

Values are expressed as Mean ± SEM. Data compared against positive control group. One way analysis of variance (ANOVA). *p<0.05, **p<0.01, ***p<0.001. Turkey-kramer multiple comparision test.

Figure No. 1: Effect of methanolic extract of Chrozophora plicata leaves (MECP) on SGPT, SGOT and ALP in CCl₄ induced hepatotoxicity

Table No. 2: Effect of methanolic extract of Chrozophora plicata leaves (MECP) on serum parameters and liver weight in paracetamol induced hepatotoxicity

Groups

Liver weight (g/100g b.w)

SGPT (U/I)

SGOT (U/I)

ALP (U/I)

Total Proteins (g/dl)

Total bilirubin (mg/dl)

Triglycerides (mg/dl)

Normal control

3.632±

0.164

51.13±

4.18

99.35±

5.12

151.34±

5.07

9.712±

0.217

0.492±

6.216

84.15±

4.216

Positive control

5.716±

0.127

196.36±

5.85

291.17±

7.16

315.6±

7.5

4.912±

0.241

2.568±

0.215

183.19±

3.15

Standard silymarin

4.326±

0.6***

102.54±

4.16***

135.1±

4.21***

111.19±

9.1***

7.315±

0.15***

0.9815±

0.011***

95.12±

3.2***

MECP (200 mg/kg)

4.94±

0.015

154.32±

4.04***

215.17±

5.4***

224.15±

6.1***

5.652±

0.715*

1.483±

0.215***

146.13±

3.1***

MECP (400 mg/kg)

4.563±

0.17**

122.15±

4.91***

152.16±

6.1***

156.72±

4.5***

6.934±

0.18***

1.079±

0.026***

105.81±

3.2***

Values are expressed as Mean ± SEM. Data compared against positive control group. One wayanalysis of variance (ANOVA). *p<0.05, **p<0.01, ***p<0.001. Turkey-kramer multiple comparision test.

Table No. 3: Effect of methanolic extract of Chrozophora plicata leaves (MECP) on serum parameters and liver weight in thioacetamide induced hepatotoxicity

Groups

Liver weight (g/100g b.w)

SGPT (U/I)

SGOT (U/I)

ALP (U/I)

Total Proteins (g/dl)

Total bilirubin (mg/dl)

Triglycerides (mg/dl)

Normal control

2.952±

0.016

37.6±

4.12

83.19±

7.62

148.16±

7.33

8.415±

0.415

0.491±

0.121

80.7±

4.75

Positive control

4.781±

0.316

195.72±

8.125

240.18±

15.4

285.14±

9.35

5.316±

0.514

2.267±

0.071

165.12±

3.14

Standard silymarin

3.512±

0.75***

81.14±

7.3***

113.68±

7.15***

169.4±

6.5***

7.821±

0.15***

0.751±

0.11***

86.61±

4.12***

MECP (200 mg/kg)

4.061±

0.03***

142.35±

3.1***

188.63±

7.05***

223.5±

7.6***

7.152±

0.37***

1.31±

0.53***

128.86±

2.15

MECP (400 mg/kg)

3.561±

0.05***

106.9±

3.6***

144.26±

7.15***

188.11±

3.7***

7.145±

0.17***

0.885±

0.02***

97.46±

5.17***

Values are expressed as Mean ± SEM. Data compared against positive control group. One way analysis of variance (ANOVA). *p<0.05, **p<0.01, ***p<0.001. Turkey-kramer multiple comparison test.

Figure No. 6: Chromatogram representing cytotoxic activity of methanolic extract of Chrozophora plicata leaves by MTT assay

CONCLUSION:

Phytochemical screening of the methanolic extract of leaves of Chrozophora plicata has confirmed the presence of flavonoids, alkaloids, glycosides and lignans. With acute toxicity studies of plant leaves on rats, therapeutically safest doses were selected i.e 200 mg/kg and 400 mg/kg. Further hepatoprotective and invitro anticancer activities has shown that the methanolic extract of Chrozophora plicata leaves possesses significant hepatoprotective and cytotoxic potentials at both the selected doses. Hence it can be hypothesized that the above mentioned active principles in the plant leaves may be responsible for hepatoprotection and cytotoxic functions.

CONFLICT OF INTEREST:

This is to inform that the authors declare that they have no conflicts of interest regarding this article.

ACKNOWLEDGEMENTS:

The authors are grateful to the management of Vijaya College of Pharmacy and Bhaskar Pharmacy College, Hyderabad for providing facilities to carry out the present research. Dr. Avanapu Srinivasa Rao helped in designing the experiment and writing this research article.

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Received on 09.02.2017 Modified on 15.02.2017

Res. J. Pharmacology and Pharmacodynamics.2017; 9(1): 19-26.

DOI: 10.5958/2321-5836.2017.00004.0

Groups	Drug concentrations in mg/ml	Percentage (%) of dead DAL cells
Control	-	0
Methanolic extract of Chrozophora plicata leaves	10	13 ± 0.172
	25	35 ± 1.286
	50	57 ± 0.914
	100	81 ± 1.731
	200	94 ± 0.495**
Standard 5-FU	5	100 ± 1.335***

SL.No	Concentration (mg/ml)	Mean OD *Chrozophora plicata*	Mean OD Standard (5-FU)	% of survival cells *Chrozophora plicata*	% of survival cells Standard (5-FU)
1.	0	0.487	0.491	100± 0.532	100±0.531
2.	10	0.421	0.441	89.89± 1.643	85.42± 3.745
3.	25	0.309	0.372	75.76± 0.747	62.40±0.147
4.	50	0.202	0.230	46.89± 1.392*	41.55± 1.832*
5.	100	0.033	0.060	12.35± 2.721**	5.915± 0.487***
6.	200	0.004	0.003	0.951± 0.945***	0.812±1.925***