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RESEARCH ARTICLE

 

Evaluation of Antibacterial action and Hepatoprotective efficiency of Solanum nigrum leaves extract on acetaminophen induced hepatotoxicity.

 

R. Bhavani1, G. Geetha1, J. Santhoshkumar2 and S Rajeshkumar1*

1PG and Research Department of Biochemistry,  Adhiparasakthi College of Arts and Science, Kalavai – 632506, Vellore District, TN, India

2 Departmant of Biochemistry, Periyar University, Salem -  636011, TN, India

*Corresponding Author E-mail: ssrajeshkumar@hotmail.com, j3ssrajesh@gmail.com

 

ABSTRACT:

Solanum nigrum leaf is one of the most important greens in throughout the world. Plants are playing a vital role in hepatoprotective activity. In this present investigation we used Solanum nigrum for the antimicrobial activity and hepatoprotective activity against acetaminophen induced liver toxicity in male albino rats. The important chemical groups present in the aqueous extract canbe determined using Fourier transform infrared spectroscopy (FT-IR). Zone of inhibition in bacterial spread plate showing a antimicrobial property of the plant extract. The aqueous extract of Solanum nigrum significantly decreases the serum enzyme alanine amino transferase (ALT), asparate amino transferase (AST), triglycerides (TGL), total cholesterol (TC) and significantly increased the total protein level. Silymarin (100 mg/kg), a known hepatoprotective drug used for comparison exhibited significant activity. The morphology of liver cells clearly indicating the treatment of Solanum nigrum against acetaminophen induced liver damage near to normal cells. 

 

KEYWORDS:

 

 


INTRODUCTION:

The liver is the largest and special organ inside the body because it has the wonderful capability to create new liver tissues from health liver tissues and an important organ that we can survive only one or two days if it shuts down. The liver play an vital role in many functions such as storage of nutrients, carbohydrate metabolism, breakdown of erythrocytes, bile Secretion, synthesis of plasma proteins, synthesis of cholesterol and etc.

 

 

 

 

 

 

 

 

 

 

 

 

Received on 01.07.2015          Modified on 20.07.2015

Accepted on 28.07.2015        © RJPT All right reserved

Research J. Pharm. and Tech. 8(7): July, 2015; Page 893-900

DOI: 10.5958/0974-360X.2015.00145.6

 

Figure 1: Solanum nigrum fresh plant

 

Solanum nigrum is such as amazing medicinal plant belonging to the family Solanaceae. It is traditionally used as medicine for various health implications (fig 1). The aqueous juice extract extracts of this leaves are very famous for its antiulcer activity [1] used for the treatment of pain, inflammation fever [2,3] and enteric diseases. [4].  The phytochemicals such as glucoalkaloids, glycol proteins, polysaccharides and polyphenolic compounds such as catechin, gallicacid, protocatechuic acid, caffeic acid, rutin, and naringenin are major compounds (fig 2) of S. nigrum extract responsible for pharmacological applications [5].

 

Scientific classification

Kingdom: Plantae

Order: Solanales

Family: Solanales

Genus: Solanum

Species: nigrum

 

 

Figure 2: Polyphenols of Solanum nigrum (Ravi et al., 2009) [5]

 

 


Table 1: Pharmacological applications of  S. nigrum

S. No

Applications

Part Used / Phytochemcilas / Nature of extract

References

1

Hypoglycemic or Antidiabetic activity

Aqueous and Hydro-alcoholic extract of leaf, fruit and stem

Ethanolic crude extact

(Akubugwo et al., 2008). [6]

 

(Ali et al., 2010) [7]

2

Antiulcerogenic Actvity on aspirin induced ulceration

 

 

methanolic extract of Solanum nigrum berries

 

(Jainu and Devi  2004) [1]

3

Immunostimulant activity

for preventing fish diseases

 

Ethanol, Methanol, chloroform, acetone and tolune extracts of S. nigrum

(Hanifa 2011) [8]

4

Protective effect on lead acetate induced toxicity in brains of albino mice

Aqueous leaf extract

(Chinthana and Ananthi  2012) [9]

5

Antioxidant and free radical scavenging activity

 

Methanolic extract of berries

Ethanolic extract of the dried fruit

(Jainu M and Devi 2004) [1]

( Rawani et al., 2010) [10]

 

6

Cytotoxic activity

Ethanolic extract of the dried fruit

( Rawani et al., 2010) [10]

 

7

Cardioprotective activity

 

Methanolic extract of berries

(Balaji et al., 2011) [11]

 

8

Antidiarrhoeal activity on

castor oil induce diarrhoeain

ethanolic extract of the dried fruit

(Bhatia et al., 2011) [12]

 

9

Antimicrobial activity Escherichia coli, Staphylococcus aureus, Enterobacter aerogenes and Pseudomonas aeruginosa

methanol and water extracts of

leaves

(Kavishankar et al., 2011) [13]

10

gram negative bacteria namely Xanthomonas campestris (plant pathogen) and Aeromonas hydrophila (animal pathogen)

methanol and aqueous extracts of leaves

(Britto AJD et al., 2011) [14]

11

Escherichia coli, Citrobacter, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia aldovae

Fungal: Saccharomycescereviciae, Aspergillus parasiticus, Trichophyton rubrum, Macrophomina, Fusarium solani and Candida albicans

Methanolic extracts of leaves and seeds of black and red

Varieties

 

(Sridhar TM et al., 2011) [15]

12

Penicillium notatum, Aspergillus niger, Fuserium oxisporium and Trichoderma viridae

Ethanol methanol and ethylacetate extracts of Solanum nigrum leaf, seed and root

Sridhar et al., 2011 [15]

13

S. aureus and  B. sublitis

ethanolic extract of the dried fruit

(Kaushik et al., 2009) [16]

14

Anti-HCV activity

 

Methanol and chloroform extracts of

seeds

 

(Javed et al., 2011)

[17]

15

Analgesic activity

 

ethanolic extract of the dried fruit

(Bhatia et al., 2011) [12]

16

Carbon tetrachloride induced hepatoprotective activity

aqueous and methanolic extracts

(Elhag  et al., 2011) [18]

                   


With these points under consideration, the present study was carried out to isolate the aqueous extracts of leaf of Solanum nigrum and to study their phytochemical constitution by FT-IR studies, to investigate the antimicrobial activity against E.coli, B. subtilis, K. pneumoniae, B. cereus and S. aureus, assess the hepatoprotective activity of the aqueous extracts of leaf of Solanum nigrum and by studying the parameters such as changes in the activity of AST, ALT, and the level of bilirubin, triglycerides, total cholesterol, and protein. Finally the histological changes in normal and experimental rats were compared.

 

MATERIALS AND METHODS:

Collection of plant

The plant Solanum nigrum leaves were collected from Walaja, Vellore district, Tamil nadu.

 

Preparation of plant extract

Fresh leaves were collected and dried under shade. The dried leaves were powdered by mixer grinder. 10 g of Solanum nigrum powdered was taken and added 100 ml of distilled water in a beaker and boiled for 5 to 10 minutes and are filtered through filter paper whatmann no 1. The extracts were allowed to store and are used for experimental animals.

 

Chemicals

Acetaminophen and bacterial media was purchased from M/s. Himedia Ltd., Mumbai. The rest of the chemicals and biochemicals utilized were obtained from local firms and were of analytical grade.

 

Antibacterial Assay

Antibacterial activity of aqueous leaves extracts of solanum nigrum was tested against Gram negative and Gram positive strains such as E. coli, B. subtilis, K. pneumoniae, B. cereus and S. aureus. By agar well diffusion method, significant activity of the leaves extract was observed against the tested bacterial strain.

 

Animals

Adult male Wistar albino rats maintained at the college weighing 150-180 g were used for the hepatoprotective studies.  The laboratory animal protocol used for this study was approved by the Institutional Animals Ethics Committee.

 

Hepatoprotective studies of S. nigrum aqueous extract

 

Experimental Design

The rats were randomly divided into five groups of 6 animals each.

 

 

Group I (Control)

:

Control rats, received orally distilled water.

Group II (Induced)

:

Induced rats, orally received paracetamol (2 g/kg body weight) dissolved in water for 7 days.

Group III (Paracetamol + Silymarin)

:

Standard rats, orally received paracetamol (2 g/kg body weight), followed by silymarin (100 mg/kg body weight) dissolved in water for 7 days.

Group IV (Paracetamol + ASN)

:

Treated rats, orally received paracetamol (2 g/kg body weight), followed by aqueous extract of Solanum nigrumleaf (300 mg/kg body weight) dissolved in water for 7 days.

Group V (Paracetamol + ASN)

:

Treated rats, orally received paracetamol (2 g/kg body weight), followed by aqueous extract of Solanum nigrum leaf  (600 mg/kg body weight) dissolved in water for 7 days.

 

 

Collection of blood

Animals of all the groups were sacrificed by cervical decapitation on the 8th day. Blood samples of each group were collected separately into sterilized dry centrifuge tubes, and allowed to coagulate for 30 min at 37 ºC.  The clear serum obtained after centrifugation was used for the estimation of serum alanine amino transferase, serum aspartate amino transferase, alkaline phosphatase, lactate dehydrogenase, serum bilirubin, serum protein, cholesterol and triglycerides.

 

Biochemical analysis

Assay of AST and ALT was performed according to the method of Reitmann and Frankel. Alkaline phosphatase was assayed by the method of King and Armstrong. The Serum total bilirubin was estimated according to the method of Malloy and Evelyn. The serum total protein was estimated as per the method of Lowry. Serum total cholesterol was determined in serum by the method of Parekh and Jung. Serum triglycerides were estimated by the method of Foster and Dunn.

 

Histopathology

A portion of liver tissue in each group was fixed in 10% formalin (formalin diluted to 10% with normal saline) and processed for histopathology. After paraffin embedding, and block marking, serial section of 5 μm thicknesses were made, stained with haematoxylin and eosin and examined under microscope.

 

Statistical Analysis

The statistical significance was assessed using one-way analysis of variance (ANOVA) using SPSS 16 software. The values are expressed as Mean ± SD and P < 0.05 was considered significant.

 

 

 

 

 


Figure 3: FT-IR spectrum of Solanum nigrum plant leaves extract

 

 

 


RESULTS AND DISCUSSION:

FT-IR Analysis

The figure 3 shows the FT-IR spectrum of aqueous extract of S. nigrum. The peaks of 3282.13,  2918.33 and 1613.47  corresponds to the functional groups of C-H Stretch of alkynes, C≡C Stretch 0f alkynes and c-c=c symmetric stretch respectively.

 

Antibacterial Assay

Antibacterial activity (figure-4) of aqueous leaves extracts of Solanum nigrum was tested against Gram negative and Gram positive strains such as E. coli, B. subtilis, K. pneumoniae, B. cereus and S. aureus shown in Table 2 using agar well diffusion method, significant activity of the leaves extract was observed against the tested bacterial strain. Among the various strains E. coli shows the maximum zone of inhibition and minimum zone of inhibition was noted against Staphyllococcus aureus. The increased concentration of plant extract may responsible for high zone of inhibition [19].

 

 


 

Table :2 Antibacterial activity of  Solanum nigrum aqueous leaves extract

S. No

Bacterial strain

Zone of Inhibition (mM)

30 µl

60 µl

90 µl

Standard drug

1

E. coli

12 ± 02

14 ± 03

17 ± 08

15 ± 55

2

B. subtilis

8 ± 15

10 ± 12

11 ± 12

13 ± 21

3

K. pneumoniae

7 ± 21

8 ± 18

9 ± 17

12 ± 45

4

B. cereus

8 ± 0.8

10 ± 11

11 ± 58

10 ± 11

5

S. aureus

6 ± 12

7 ± 25

8 ± 32

9 ± 09

 

 

 

Figure 4: Antibacterial activity of S. nigrum

 

 

 


Hepatoprotective activity of Solanum nigrum

Acetaminophen is the drug and most commonly used as a liver damaging agent in animal studies. The continues intake of acetaminophen will affect the metabolic functions of  liver may due to increased secretion of enzymes like Aspartame transaminase, alanine trasaminase and alkaline phosphatases [20] apart from it will increase the levels of Cholesterol, Tryglycerides and Total protein level during induction.  

 

 

 

 

 

 

Table 3 and figure-5 shows the effect of aqueous extract S. nigrum (300 mg/kg and 600 mg/kg) on serum biochemical markers in paracetamol induced liver toxoxcity. Hepatic damage causes elevated level of liver enzymes such as SGOT (serum glutamic oxaloacetic transaminase) and SGPT (serum glutamic pyruvic transaminase). Treatment with S. nigrum at 600 mg/kg revealed comparable activity with reference standard silymarin (25mg/kg). aqueous extract S. nigrum decreased the liver markers SGPT (102.33±4.23) and SGOT (45.83 ± 1.94)

 

 

 

 

 


 

Table 3.Changes in the activity of AST and ALT in Experimental Animals

Parameters

Group I

Group II

Group III

Group IV

Group V

Level of

Significance (p)

AST (IU/L)

100.67± 4.33

200.50±10.7

115.67±4.51

108.00±4.91

102.33±4.23

< 0.05

ALT (IU/ L)

44.83 ± 1.80

80.67 ± 4.82

56.33 ± 1.76

46.33 ± 2.21

45.83 ± 1.94

< 0.05

Group I – Control; Group II – Acetaminophen induced; Group III – Acetaminophen+ Silymarin; Group IV – Acetaminophen + Aqueous extract S. nigrum (300 mg/kg); Group V – Acetaminophen + Aqueous extract S. nigrum (600 mg/kg) the observations are given as Mean ± SD of 6 rats of each group. Groups IV and V were compared with Group I.

 

Figure 5: Changes in the activity of AST and ALT

 

 


Changes in the Level of Serum Total Bilirubin 

Table 5 and Fig. 6 present the changes in the level of serum total bilirubin. Here, paracetamol-induced liver damage was characterized by increased level of bilirubin and they return to normal level after 7 days of treatment with aqueous extracts from S. nigrum leaf.


 

 

 


Figure 6: Changes in the Activity of Bilirubin

 


Changes in the Level of Serum Total Protein

Table 5 and Fig. 7 present the changes in the level of serum total protein. Here, paracetamol-induced liver damage was characterized by decreased level of total protein and they return to normal level after 7 days of treatment with aqueous extracts from S. nigrum leaf.


 

 

Table 5.Changes in the levels of serum Bilirubin and Total Protein in Control and Experimental Rats

Parameters

Group I

Group II

Group III

Group IV

Group V

Level of

Significance (p)

Bilirubin (mg/dL)

0.19 ± 0.06

0.12 ± 0.15

0.15 ± 0.06

0.17 ± 0.02

0.18 ± 0.03

< 0.05

Total Protein (g/dL)

7.49 ± 0.29

7.19 ± 0.23

7.33 ± 0.28

7.31 ± 0.33

7.41 ± 0.38

< 0.05

 

 

Figure 7: Changes in the activity of Total Protein

 

 

 


Changes in the Level of Serum Total Cholesterol and Triglycerides

Table 6 and Fig. 8 present the changes in the level of serum cholesterol and serum triglycerides. Here, paracetamol-induced liver damage was characterized by increased level of total cholesterol and triglycerides and they return to normal level after 7 days of treatment with aqueous extracts from S. nigrum leaf.

 


 

Table 6: Changes in the level of total cholesterol and triglycerides in control and experimental rats.

Parameters

Group I

Group II

Group III

Group IV

Group V

Level of

Significance (p)

Cholesterol (mg/dL)

121.67 ± 5.62

349.50±15.6

115.00± 4.23

155.00±5.24

139.87±5.52

< 0.05

Triglycerides (mg/dL)

108.50 ± 5.14

219.83±10.20

112.33± 5.40

135.00±6.02

123.17±5.62

< 0.05

 

Figure 8: Changes in the Actvity TGL and cholesterol

 


Histopathology analysis

The changes in the hepatocytes during different conditions are shown in the figure 9. The normal cells and induced cells are having good difference are clearly shown in the figures. And the treatment with standard drug and Solanum nigrum low and high dose treatments are look like same structures explains the medicinal properties of the plnat extract.


 

 

 

(a)                                                                       (b)

                  

Figure9: Histaopathological changes of liver Normal, Induced, Treatment Standard and Treatment with Solanum  nigrum (a) 300 mg/kg body and (b) 600 mg/kg body

 


CONCLUSION:

On the basis of results obtained, it can be concluded that the aqueous extract of Solanum nigrum leaves seems to possess hepatoprotective activity in male albino rats. The observed protective activity of Aqueous S. nigrum may be due to the identified phytochemical compounds that are present in extracts. This finding justifies the use of this plant in traditional medicine in treatment of microbial infections, liver disease and supports the use of S. nigrum in the treatment of acetaminophen-induced hepatotoxicity.

 

REFERENCES:

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[2]     Acharya E, Pokhrel B. Ethno-medicinal plants used byBantar of Bhaudaha, Morang, Nepal. Our Nature. 4:2006;96-103.

[3]     Zakaria ZA, Gopalan HK, Zainal H, et al. Antinociceptive,anti-inflammatory and antipyretic effects of Solanum nigrum chloroform extract in animal models. Yakugaku Zasshi 126: 2006;1171-1178.

[4]     Rani P, Khullar N. Antimicrobial evaluation of somemedicinal plants for their anti-enteric potential againstmultidrug resistant Salmonella typhiPhytother Res. 18(8):2004; 670-673.

[5]     Ravi V, Saleem TSM, Maiti PP, Gauthaman K, Ramamurthy J. Phytochemical and pharmacological evaluation ofSolanum nigrum Linn. African Journal of Pharmacy and Pharmacology. 3(9): 2009; 454-457.

[6]     Akubugwo I. E, Obasi N.A, Chinyere G.C and Ugbogu A. Mineral and phytochemical contents in leaves of Amaranthus hybridus L and Solanum nigrum  L. subjected to different processing methods. African Journal of Biochemistry Research. 2 (2): 2008; 040-044.

[7]     Ali NS, Singh K, Khan MI, Rani S. Protective effect ofethanolic extracts ofSolanum nigrum on the blood sugar ofalbino rats. IJPSR. 1(9): 2010; 97-99.

[8]     Hanifa MA. Evaluation of Immunostimulant Potential of Solanum nigrum using fish, etroplussuratensis challenged with aphanomyces. International Journal of Pharma and Bio Sciences. 2(1): 2011; 429-443

[9]     Chinthana , Ananthi T. Protective effect of Solanum nigrum and Solanum trilobatum aqueous leaf extract on Lead induced neurotoxicity in Albino mice. Journal of Chemical and Pharmaceutical Research. 4(1): 2012;72-74.

[10]   Rawani A, Ghosh A Chandra G. Mosquito larvicidal activities of Solanum nigrum L. leaf extract against Culexquinque fasciatus. Parasitol Res. 107: 2010;1235–1240.

[11]   Balaji R, Prakash G ,Suganyadevi P , Aravinthan K M. Evaluation of cardio protective Activity of Methanolic Extract Of Solanum nigrum  Linn. in Rats. International Journal of Drug Development and Research. 3(3):2011; 139-147.

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[17]   Javed T, Usman AA, Sana R, Sidra R, Sheikh R. In-vitro antiviral activity of Solanum nigrum against Hepatitis C Virus. Virology Journal.8:2011; 26.

[18]   Elhag RAM, Badwi MAE, Bakhiet AO, Galal M. Hepatoprotective activity of Solanum nigrum extracts on chemically induced liver damage in rats. Journal on Veterinary Medicine and Animal Health. 3(4): 2011;45-50.

[19]   Rajeshkumar S, Antimicrobial effect of King of bitter Andrographis paniculata and traditional herb Aegle marmelos against clinical pathogens International Journal of PharmTech Research 2014-2015, 7(2), 325-329.

[20]   Rajeshkumar S, B. Tamilarasan, V. Sivakumar (2015) Phytochemical screening and hepatoprotective efficacy of leaves extracts of Annona squamosa against paracetamol induced liver toxicity in rats International Journal of Pharmacognosy  2(4): 178-185. DOI: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.2(4).178-8