Research Articles

2017  |  Vol: 3(5)  |  Issue: 5(September-October)

Preliminary neuroprotective activity of aerial parts of Enicostemma littorale Blume extract in albino rats


Vinuth Chikkamath, Shanmukha I.*

PG Dept of Pharmacology S.C.S College of Pharmacy, Harapanahalli, Davanagere 583131

Karnataka, India.

*Corresponding author:

Shanmukha I.

PG Dept of Pharmacology, S.C.S College of Pharmacy, Harapanahalli, Davanagere 583131

Karnataka, India.


Abstract

Objective: The main aim of this research paper is to evaluate the neuroprotective activity of Enicostemma littorale Blume of 70% ethanolic extract against Monosodium Glutamate (MSG) and Aluminium fluoride (AlF3) treated albino rats. Materials and methods: The Enicostemma Littorale, extracted from Soxhlet apparatus unit, by using 70% ethanol and evaluated, in the course of free radical generation and excitotoxicity in Monosodium Glutamate (MSG) and Aluminium fluoride (AlF3) treated albino rats. Results and conclusion: The extract showed significant improvement in body weight, total protein, Catalase, super oxide dismutase, and lipid peroxide levels. The general behavior, locomotor activity, and region of the hippocampus were significantly protected by organ protective properties of ethanolic extract of Enicostemma Littorale Blume attribute to its antioxidant and behavioral properties.

Keywords: Neuroprotective, Enicostemma littorale Blume, Monosodium glutamate, Aluminium fluoride.


Introduction

Neurodegenerative disorders are a heterogeneous group of diseases of the nervous system, including the brain, spinal cord, and peripheral nerves that have many different etiologies (Ravindra et al., 2004). Acute and chronic neurodegenerative diseases are illnesses, associated with high morbidity and mortality and few or no effective options are available for their treatment. A characteristic of many neurodegenerative diseases which include stroke, brain trauma, spinal cord injury, amyotrophic lateral sclerosis, Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease is neuronal-cell death (Robert, 2003). Neuropathlogically, these are characterized by abnormalities of relatively specific regions of the brain and specific populations of neurons. Enicostemma littorale Blume a glorious perennial herb, belonging to the family Gentianceae. Upon literature survey, it was found that the hot extract it is being used for the treatment of diabetes, fever, stomach ache, dyspepsia, and malaria. It is also reported to possess antitumor (Dash, 2000), antiarthritic, hypoglycemic, and antimalarial activities (Katewa and Arora, 2001). There are reports that the plant possesses flavonoids, xanthenes, and so on in the aerial parts of this plant (Ghosal and Jaiswal, 1980). The flavonoids are known to have antioxidant, antiulcer, and anti-inflammatory properties. However, there are no reports on the Neuroprotective activity of the herb. As this herb is reported to contain alkaloids, catechins, saponins, sterols, triterpenoids, phenolic acids, flavonoids, and xanthones other related compounds. The hydro alcoholic extract of this herb was investigated for Neuroprotective activity by using various experimental models in albino rats

Materials and methods

Collection of plant Material and Preparation of extract

The herb of Enicostemma littorale Blume was collected from the Kanavihalli, near to Harapanahalli, Karnataka, India from June to August 2012, at the end of the flowering season. The herb was identified and authenticated by Prof .K. Prabhu, Department of Pharmacognosy, S.C.S. College of Pharmacy, Harapanahalli. A herbarium specimen No. SCSCOP.Ph.Col Herb.No.005/2011-2012.  The dried powder of aerial parts were defatted with Petroleum Ether, Chloroform, and then extracted with 70% ethanol using Soxhlet apparatus. The extracts were concentrated under reduced pressure, using rota flash evaporator and stored in airtight container in a refrigerator below at 10ºC.   

Experimental animals and housing conditions and ethical approval 

Albino Wistar rats weighing 150-250g were procured from Sri Venkateshwara Enterprises, Bengaluru. The animals were kept in quarantine section till monitoring of health status and subsequently transferred to the housing area. The animals were acclimatized for one week under laboratory conditions. They were housed in polypropylene cages with dust free rice husk as a bedding material and standard laboratory conditions of temperature (23 ± 2°C), humidity (40 ± 10%) and 12:12 h dark/light cycles. They were fed with standard rodent feed, (Gold Mohur Lipton India Ltd.) and water ad labium was provided. The husk in the cages was renewed thrice a week, to ensure hygienity and maximum comfort for animals. Ethical clearances for handling the animals was obtained from the Institutional animal ethics committee (IAEC), prior to the beginning of the project work and the registration no. SCSCP/583/5/2011-12 and dated was 26.11.2011 and care of laboratory animals was done as per CPCSEA guidelines, Ministry of Forests & Environment and Government of India.

Preliminary Phytochemical Screening

The preliminary phytochemical screening was carried out on Petroleum ether, Chloroform, 70% Ethanolic, and Aqueous extract for qualitative identification of type of phytoconstituents present (Kokate, 1999; Khandelwal, 2000).

Induction of Neurotoxicity

Neurotoxicity was induced in wistar rats by intraperitoneal injection of freshly prepared Monosodium glutamate (MSG) 2 g/kg and Aluminium fluoride (AlF3) 600 ppm in disease control, treatment and standard(Ascorbic acid) group animals as per experimental design.

Experimental design

Monosodium glutamate - induced Neurotoxicity (Ramanathan et al., 2007)

Overnight fasted rats were randomly divided into five groups of six rats (n=6) each as follows and treated for 7 days as follows;

Group 1: Received normal saline (i.p.) + vehicle (p.o.).

Group 2: Received MSG 2 g/kg (i.p.) + normal saline (p.o.).

Group 3: Received MSG 2 g/kg (i.p.) + 70% EEEL 250mg/kg (p.o)

Group 4: Received MSG 2 g/kg (i.p.) + 70% EEEL 500mg/kg (p.o)

Group 5: Received MSG 2g/kg (i.p.) + ascorbic acid 100 mg/kg, (i.p.) (reference Std.)

Aluminum fluoride - induced Neurotoxicity (Chandrashekhar et al., 2010)

The animals were divided into five groups of six rats (n=6) each as follows;

Group 1: Received normal saline (i.p.) + vehicle (p.o.).

Group 2: Received (AlF3) 600ppm + normal saline (p.o.).

Group 3: Received (AlF3) 600 ppm + 70% EEEL 250mg/kg (p.o).

Group 4: Received (AlF3) 600 ppm + 70% EEEL 500mg/kg (p.o).

Group 5: Received (AlF3) 600ppm + ascorbic acid 100 mg/kg, (i.p.) (reference Std.).

Results

Effect of 70% ethanolic extract on MSG-induced neurotoxicity

There was a marked change in the body weight in MSG-treated groups. Ethanolic extract showed a dose-dependent increase in the body weight of rats. Ethanolic extract at 500mg/kg dose increased the body weight by 66.11%.  Motor coordination and body balance were significantly affected in MSG administered rats when compared to normal control rats. Normal control rats showed the fall of time in 60.89 ±2.376s; whereas MSG alone treated rats showed the fall of time in 22.46 ± 1.771s. Pretreatment with 70% Ethanolic extract (250 and 500 mg/kg), significantly improved the motor coordination and body balance in MSG-treated rats (29.52±1.966 and 40.68 ± 2.446) respectively. It was further confirmed by histopathological observations.

Figure 1A. Shows the Histopathological Changes of different groups in MSG-induced rats

 

 

Table 1.  Effect of 70% Ethanolic Extract on Biochemical parameters, Body weight and Behavioral characters in MSG-treated rats

Treatment

GSH

LPO

SOD

CAT

TP

% Body weight change

Rota Rod Test

(s)

Locomotor Activity

(Count/5 min)

Mean

±

SEM

%

increase

Mean

±

SEM

%

inhibition

Mean

±

SEM

%

increase

Mean

±

SEM

%

increase

Mean

±

SEM

%

increase

Negative control

0.263± 0.002

----

0.111± 0.007

-----

0.042± 0.001

-----

0.411± 0.003

-----

0.666± 0.016

-----

------

60.89± 2.376

143.9 ± 2.300

Positive control

0.171±

0.001

-----

0.194±

0.001

-----

0.021±

0.008

-----

0.175±

0.007

-----

0.332±

0.023

-----

13.04

22.46 ± 1.771

41.37  ± 2.384

Standard

(Ascorbic

acid )

0.263±

0.001***

53.80

0.124±

0.001***

36.08

0.040±

0.001***

90.47

0.400±

0.009***

91.50

0.603±

0.031***

81.62

04.80

45.11±

2.683***

124.5  ±   1.743***

70% EEEL

(250mg/kg)

0.220±

0.007*

31.81

0.145±

0.004*

21.94

0.030±

0.002*

58.80

0.295±

0.019*

42.42

0.435±

0.033*

31.02

12.18

29.52±  1.966*

60.71±  8.880*

70% EEEL

(500mg/kg)

0.250±

0.010**

46.19

0.132±

0.003**

31.95

0.036±

0.003***

71.42

0.381±

0.013***

87.45

0.540±

0.021**

62.65

08.24

40.68 ± 2.446***

100.9  ±  3.653***

Each value is expressed as mean ± SEM (n = 6), where, NS represents non significant; ***P<0.001 – highly significant; **P<0.01- very significant; *P<0.05- significant, when compared to MSG alone treated rats. One-way ANOVA followed by Dunnett’s comparison test. *GSH- Glutathione, LPO- Lipid peroxides, SOD- Superoxide dismutase, CAT- Catalase, TP- Total Protein.

Figure 1B: Shows the Bar graphs of different groups in MSG-induced rats

 

 

 

 

Effect of 70% EEEL on Aluminium Fluoride (AlF3) – induced Neurotoxicity:

There was also a marked change in body weight in AlF3 treated groups. Ethanolic extract showed a dose-dependent increase in the body weight of animals. Ethanolic extract at 500 mg/kg dose, increased the body weight by 56.01%. Motor coordination and body balance were significantly affected in AlF3 administered rats when compared to normal control rats. Normal control animals showed the fall of time in 58.58±6.628s; whereas AlF3 alone treated rats showed the fall of time in 21.43±2.452s. Pretreatment with Vitamin-C and 70% (250 and 500 mg/kg) significantly improved the motor coordination and body balance in AlF3 treated rat 48.50±2.022, 29.34±2.239 and 42.78±1.997s) respectively. It was further confirmed by histopathological observations. There was a dose dependent inhibition of in-vivo LPO and increase in GSH, CAT and SOD in both the models. This was statically significant when compared to the positive control group. 

Figure 2A: Shows the Histopathological Changes of different groups in AlF3-induced rats.


Table2: Effect of 70% Ethanolic Extract on Biochemical parameters, Body weight change and Behavioral characters in AlF3 treated rats

Treatment

GSH

LPO

SOD

CAT

TP

% Body weight change

Rota Rod Test

(s)

Locomotor Activity

(Count/5     min)

Mean

±

SEM

%

increase

Mean

±

SEM

%

inhibition

Mean

±

SEM

%

increase

Mean

±

SEM

%

increase

Mean

±

SEM

% increase

Negative control

0.263± 0.002

----

0.111± 0.007

-----

0.042± 0.001

-----

0.411± 0.003***

-----

0.666± 0.016

-----

------

60.89± 2.376

143.9± 2.300

Positive control

0.171±

0.001

-----

0.194±

0.001

-----

0.021±

0.008

-----

0.175±

0.007

-----

0.332±

0.023

-----

13.04

22.46 ± 1.771

41.37  ± 2.384

Standard

(Ascorbic

acid )

0.263±

0.001***

 

53.80

0.124±

0.001***

36.08

0.040±

0.001***

90.47

0.400±

0.009***

91.50

0.603±

0.031***

81.62

04.80

45.11±

2.683***

124.5±   1.743***

70% EEEL

(250mg/kg)

0.222±

0.005*

29.81

0.153±

0.004*

22.94

0.029±

0.002

27.80

0.279±

0.019 *

36.42

0.435±

0.033*

31.02

12.18

29.52±  1.966*

60.71±  8.880*

70% EEEL

(500mg/kg)

0.250±

0.010**

46.19

0.132±

0.003**

31.95

0.036±

0.003***

71.42

0.381±

0.013***

87.45

0.540±

0.021**

62.65

08.24

40.68 ± 2.446***

100.9  ±  3.653***

Each value is expressed as mean ± SEM (n = 6), where, NS represents non significant; ***P<0.001 – highly significant; **P<0.01- very significant; *P<0.05- significant, when compared to MSG alone treated rats. One-way ANOVA followed by Dunnett’s comparison test. *GSH- Glutathione, LPO- Lipid peroxides, SOD- Superoxide dismutase, CAT- Catalase, TP- Total Protein.

Figure 2B: Show the Bar graphs of different groups in AlF3-induced rats.      

 

 

 

 

 

Discussion

Treatment with glutamate, as monosodium glutamate (MSG) - induced severe neurochemical damage and neurotoxic effects on any brain region (Johnston et al., 1998; Ortwo et al., 1997, Orti et al., 2006). Various investigators have previously demonstrated, some of the neurotoxicological signs induced by monosodium glutamate intake. These neurotoxicological signs are observed, after the administration of monosodium glutamate (2g/kg, i.p. for 7 days) in mouse and rats, including hypoxia-ischemia, hepatotoxicity, musculoskeletal pain and metabolic failures (Pavlovic et al., 2007). Excessive accumulating of glutamate in the synaptic cleft has been associated with excitotoxicity and glutamate is implicated in a number of neurological disorders (Mallick, 2007). However, there is accumulating evidence suggesting that glutamate-induced toxicity can be mediated through necrosis and apoptosis (Ankarcrona et al., 1995; Martin et al., 2012). One week administration of MSG in rats depleted the GSH, CAT and SOD levels and increased the LPO. The animals also exhibited aggressive behavior and in some animals fighting behavior was also observed.

The animals treated with AlF3(600ppm p.o for 7days)  in drinking water, revealed altered behavioral changes and causes oxidative damage to the brain was estimated by measuring LPO, SOD, CAT, GSH and total protein levels. The histopathological studies were performed in the striatal region of the brain (Thirunavukkarasu et al., 2012). A significant decline in the enzymatic activity like GSH, SOD, CAT and TP levels during monosodium glutamate and aluminum treatment, whereas LPO levels showed a significant increase, as compared to normal control. Synchronous administration of during monosodium glutamate and aluminum treatment reversed the enzyme levels to the normal. In the present study, it was observed that the plant possesses alkaloids, flavonoids and tannin and these constituents are reported to have organ protective properties. Hence, the organ protective properties may be attributed to the alkaloids, flavonoids and tannins constituents that are present in the test extract.

Conclusion

The 70% ethanolic extract of Enicostemma littorale Blume has a cerebroprotective activity against monosodium glutamate and aluminium fluoride induced cerebrotoxicity in Albino rats.

Acknowledgment

The authors would like to thank the Management, Principal, SCS College of Pharmacy, Harapanahalli for making available the infrastructure required for the study.

References

Ankarcrona M, Dypbukt J M,  Bonfoco E, Zhvotovsky V, Omenius S, Lipton SA et al. 1995. Glutamate induced neuronal death a succession of necrosis or apoptosis depending on mitochondrial function. Neuron, 15:961-973.

Chandra shekhar VM, Rani pariya VI, Ganapaty S, Parashal A, Muchandi AA. 2010. Neuroprotective activity of matricaria reticulates Linn against global model of ischemia in rats. Journal of Ethopharmacology, 127: 645-651.

Dash GK, Samanths A, Kannungo SK. 2000. Hepatoprotective activity of Indian medicinal plants. Indian Journal of Natural Products and Resources, 16:20.

Ghosal S, Jaiswal DK. 1980. Chemical constituents of genetianceae XXVIII; flavonoids of Enicostemma hysspifolicm (Wild) Verd. Journal of Pharmaceutical  Science, 69:53–56.

Johnston CA, Spined EJ, NegroVilar A. 1998. Effects of neonatal monosodium glutamate treatment on the hormonal and central monoaminergic dynamics associated with acute ether stress in the male rat. Brain Research Bulletin, 13:643-649.

Katewa SS, Arora A. 2001. Effect of certain ethnomedicinal plants. Indian Drugs, 38:6. Khandelwal KR. 2000. Practical Pharmacognosy techniques and experiments, 2nd (Eds), Nirali Prakashan Pune, pp. 149-56.

Kokate CK. 1999. Practical Pharmacognosy 4th (Eds), pp.149-56, New Delhi, Vallabha Prakashan.

Mallick HN. 2007. Understanding safety of glutamate in food and brain. Indian Journal of Physiology and Pharmacology, 51:216-234.

Martin LJ, Sieber FE, Traystman RJ. 2000. Apoptosis and necrosis occur in separate neuronal populations in hippocampus and cerebellum after ischemia and are associated with differential alterations in metabotropic glutamate receptor signaling pathways. Journal of cerebral blood flow and Metabolism, 20:153-167.

Orti GG, Bitzer Quintero OK, Beaszamte C, Rodriguez RS, Larios AF et al. 2006. Monosodium glutamate induced damage in liver and kidney: A morphological and biochemical approach. Biomedicine and Pharmacotherapy, 60: 86-91.

Ortwo SD, Beas ZC, Adame GG.  Feria Velsco AC. 1997. Effect of L-glutamate on cholinergic neurotransmission in various brain regions and during the development of rats, when administered prenatally. Neurochemistry International, 31:683-692.

Pavlovic V, Pavlovic D, Kocic G, Sokolovic D, Jevtovic Stoimenov T et al. 2007. Effect of monosodium glutamate on oxidative stress and apoptosis in rat thymus. Molecular and Cellular Biochemistry, 303:161-166.

Ramanathan M, Sivakumar S, Anandvijaykumar PR, Saravanababu C, Rathinavel Pandian P. 2007. Neuroprotective evaluation of standardized extract of Centella Asciatica in monosodium glutamate treated rats. Indian Journal of Experimental Biology, 45: 425-431.

Ravindra PS, Shashwat S, Suman K. 2004. Free radicals and oxidative stress in neurodegenerative diseases. Relevance of dietary antioxidants. Editor’s choice. Journal of Indian academy of clinical Medicine, 5(3): 218-25.

Robert MF. 2003. Apoptosis and caspases in neurodegenerative diseases. The New England Journal of Medicine, 348:1365-75.

Thirunavukkarasu SV, Lokesh U, Venkataraman S. 2012. Effect of Manasamitra   vatakam an Ayurvedic formulation on aluminium induced neurotoxicity in rats. Tropical Journal of Pharmaceutical Research, 11 (1):75-83.

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