CHAPTER 4 CHAPTER 4 CHAPTER 4 CHAPTER 4

Effect of Lactuca sativa on chemical

induced anxiety

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4. 1. Effect of Lactuca sativa extract on aluminium chloride induced

anxiety: behavioural changes in mice

4.1.1. Introduction

Aluminum (Al) has the potential to be neurotoxic in human and animals. It is present in many manufactured foods, medicines and is also added to drinking water for purification purposes (Newairy et al., 2009). Al is widely used in antacid drugs, as well as in food additives and tooth paste (Malekshah et al., 2005). Environmental pollution with different aluminium containing compounds, especially those in industrial waste expose people to higher than normal levels of Al (Kloppel et al., 1997). Particulate matters distributed by cement-producing factories contain high amount of Al and animals and population residing in the vicinity are exposed to the pollution (Fatima et al., 2001). Al is a possible contributing factor in Alzheimers disease (Campbell, 2002). However, epidemiological studies have indicated a link between Al in drinking water and Alzheimers disease and a variety of human and animal studies have implicated learning and memory deficits after Al exposure

(Schmidt et al., 2001; Exley, 2005; Buraimoh et al., 2012). Although aluminium has been implicated in Alzheimers disease, Parkinsonism, Dementia complex and causes extensive damage to the nervous system, to date the mechanism of Al neurotoxicity has not been fully elucidated (Niu et al., 2007). In recent research, aluminium has been reported to accelerate oxidative damage to bio molecules like lipid, protein and nucleic acids (Jyoti et al., 2007). The aim of this experiment was to evaluate the protective effects of Lactuca sativa against aluminium chloride on anxiety-related behaviour of albino mice

4.1.2. Methods

4.1.2.a. Experimental design

1% Tween 20 in distilled water was used for feeding the control group. The experimental group received Lactuca sativa extract suspended in 1% Tween 20 in

distilled water in varying concentrations. AlCl3 (10 mg/kg body weight, i.p.) was dissolved in distilled water, one group was fed with only AlCl3 and others along with various doses of hydro-alcohol extract of Lactuca sativa. Six mice were taken in each group. The doses of extracts were so adjusted as to administer 0.25 ml of the

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suspension of extracts for 15 days. The route of administration of the extract was by gavage.

Group I: Control

Group II: AlCl3 (10mg/kg bwt. Weight, i.p.) Group III: Lactuca sativa (100mg/kg bwt.)+ AlCl3 Group IV: Lactuca sativa (200mg/kg bwt.) + AlCl3 Group V: Lactuca sativa (400mg/kg bwt.) + AlCl3

4.1.2.b. Behavioral analysis

Open field test (Test was performed as described previously in the chapter no. 3) Elevated plus maze test (Test was performed as described previously in the

chapter no. 3) Light dark box test (Test was performed as described previously in the chapter

no. 3) Marble burying test (Test was performed as described previously in the

chapter no. 3)

4.1.2.c. Estimation of GABA in brain

Preparation of the mobile phase and the derivatizing agents were based on the methods described by Rowley et al. (1995). The mobile phase consisted of 0.1 M monosodium phosphate and 0.1 mM EDTA with 40% methanol (v/v) in water adjusted to pH 4.6 with 1 M phosphoric acid. Then, it was filtered through 0.45 m filters and degassed for 15 min. Stock solution (0.01 M) of GABA standard was prepared in double deionised water. To obtain agents for derivatization, OPA (22 mg, Sigma) was dissolved in 0.5 ml of absolute ethanol and 0.9 ml of sodium tetraborate buffer (0.1 M) adjusted to pH 10.4 with 5 M sodium hydroxide. The reaction of derivatization was performed at room temperature. Derivatizing agent (20 l) reacted with 1 ml of GABA standard and brain samples for 10 min in a polyethylene vial before injecting into the column. 25 microlitres of the resulting supernatant was chromatographed on a C 18 column in HPLC (Waters, India).

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4.1.3. Results and Discussion

4.1.3. a. Open field test

There was a total locomotor activity decrease in AlCl3-treated group of mice as evidenced by decline in the number of lines crossed and total mobility. The

locomotion activity increased significantly (p < 0.01) after the administration of hydro-alcoholic extract of Lactuca sativa at 200 and 400 mg/kg compared to only AlCl3-treated group. Treatment with the plant extract at 400 mg/kg body wt. showed anxiolytic activity in this paradigm, as there were significant differences between the

control as well as plant extract treated groups (Table 15). However, no significant effects were produced at 100 mg/kg body wt. of the plant extract.

4.1.3. b. Elevated plus maze test

There was decrease in time spent in the open arms and the open arm entries in AlCl3-treated groups (p < 0.01) compared to control group (Table 16). Hydro-alcoholic extract of Lactuca sativa at 200 mg/kg and 400 mg/kg significantly increased the time spent in the open arms. Entries in the open arms increased

significantly at the levels of 200 mg/kg (p < 0.05) and 400 mg/kg (p < 0.01). Plant extract at 100 mg/kg had no significant effects on any of the parameters that were measured on the EPM.

4.1.3. c. Light dark box test

The light/dark box is a characteristic tool used in the assessment of anxiety. The basic measure is the animals preference for dark, enclosed places over bright, exposed places. Time spent in the lit (light) half of the arena, and the related exploratory behaviours, are reliable parameters for assessing anxiolytic effects that may be useful in identifying and/or screening of anxiolytic and anxiogenic agents.

AlCl3-treated groups significantly decreased the time spent in light compartment (p < 0.01) compared to control group (Table 17). Significant increase in the time spent in the light compartment (p < 0.05) was seen with administration of 400 mg/kg of hydro-alcoholic extract of Lactuca sativa compared to control group (Table 28).

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4.1.3. d. Marble burying test

The number of marbles buried in the AlCl3-treated groups was significantly more than that of the control group of mice. The administration of 400 mg/kg of

hydro-alcoholic extract of Lactuca sativa decreased significantly the number of marbles buried (Table 18). However the effect was not significantly different in the other doses, 100 and 200mg/kg.

4.1.3. e. GABA estimation

Figure 18 shows the retention time of standard GABA and brain sample with retentation time of 6.063 and 6.087 min respectively. GABA levels were significantly decreased in the brain of AlCl3-treated groups in comparison to control group. On the other hand, it was found that GABA level was significantly increased in brain (p

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Note : LS-100, 200 and 400 are supplementation of Lactuca sativa at doses of 100, 200 and 400mg/kg body weight respectively, AlCl3 administration at a dose of 10 mg/kg body weight, i.p, to all animals except control group.

Open field test

Groups Time mobile (s/5min) Line crossing (No.) Control 180.9313.5 212.7513.2

AlCl3 98.50 9.3$ 122.7512.3$

LS-100+ AlCl3 132.1522.0* 161.0012.8*

LS-200+ AlCl3 164.3811.5* 186.5014.5*

LS-400+ AlCl3 187.9514.7** 224.6511.3**

Table 15. Effect of Lactuca sativa extract on the time mobile by mice behaviour in open field test against AlCl3. $ indicates p< 0.01compared with control, * indicates p

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Table 17. Effect of Lactuca sativa extract on the time spent in light zone by mice behaviour in light and dark test against AlCl3. $ indicates p < 0.01compared with control, * indicates p

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Figure 19. Correlation between behavioural parameters and brain GABA levels in anxiety animal models against AlCl3, (a) light dark box, (b) open field test, (c) elevated plus maze test, (d) marble burying test.

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4.2. Effect of Lactuca sativa extract on picrotoxin and flumazenil

induced anxiety; behavioural changes and biochemical changes in mice

4.2. 1. Introduction Picrotoxin (PTX) is the prototypic antagonist of GABAA receptors

(GABARs), the primary mediators of inhibitory neurotransmission (rapid and tonic) in the nervous system. PTX is clearly a non-competitive antagonist (NCA), acting not at the GABA recognition site but perhaps within the ion channel. Thus PTX appears

to be an excellent example of allosteric modulation, which is extremely important in protein function in general and especially for GABA receptors. PTX, is excitatory on the brain (analeptic) [not a depressant, like marijuana]. Such an agent may produce mood elevation and antidepressant, anxiety-generating, and alerting effects, as

opposed to the anxiolytic, sedative, and amnestic effects of GABA-enhancing drugs like benzodiazepines and ethanol (Olsen, 2006). Pixrotoxin was shown to antagonize inhibitory pathways in the nervous system activated by GABA, and GABA-enhancing drugs like barbiturates and benzodiazepines reverse its action (Liang et al., 2009).

Picrotoxin, a GABA A -receptor antagonist, produces seizures by blocking the

chloride-ion channels linked to GABA A receptors, thus preventing the entry of chloride ions into neurons. This leads to decreased GABA transmission and activity in the brain. Thus, convulsions arising from Picrotoxin are due to the decreased GABA A -receptor-mediated inhibition which tips the balance in favor of the

glutamate-mediated excitatory transmission (Stankevicius et al., 2008). The ability of SNE to attenuate seizures induced by Picrotoxin may possibly be due to an interaction with GABA A receptors and/or GABA neurotransmission.

Flumazenil is typically categorized as a benzodiazepine antagonist (Polc et al., 1982; Bonetti et al., 1988). Some studies have suggested that flumazenil also functions as a partial benzodiazepine agonist (Nutt et al., 1982; File and Pellow 1986). Flumazenil has been shown to exert some anxiogenic action on mice tested on an elevated plus maze, a situation involving relatively low levels of stress (Lee et al., 1991).

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Flumazenil selectively antagonises the central action of benzodiazepines by competitive interaction at the GABA-benzodiazepine receptor level. In addition animal studies have shown that flumazenil can influence dopamine metabolism in the

CNS (Joong et al., 2003). GABA mimetic activities have been used effectively in the treatment of acquired or non acquired chorea/ ballism (Becker et al., 1983). Administration of flumazenil induced ballistic movement. Ballistics movements that are performed with maximal velocity and acceleration can be considered ballistic

actions. Ballistic actions are characterised by high firing rates, brief contraction times and high rates of force developments. Flumazenil has rare CNS side effects such as seizure, agitation and anxiety (Spivey et al., 1993).

The aim of this experiment was to evaluate the protective effects of Lactuca

sativa against picrotoxin and flumazenil could have on anxiety-related behaviour of Albino mice

4.2.2. Materials and methods Picrotoxin and flumazenil was obtained from Sigma, USA. 5, 5'-dithiobis-(2-

nitrobenzoic acid), thiobarbituric acid, ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) were purchased from HiMedia, Mumbai, India. All other chemicals and reagents used were of analytical grade and obtained from local dealers in Mysore.

4.2.2.a. Experimental design The extracts of Lactuca sativa were suspended in a vehicle comprising 1% v/v

Tween 20 in distilled water. The suspending vehicle (0.25 ml) was used as control. Flumazenil and picrotoxin (1 mg/kg body weight) was dissolved in distilled water. Mice were grouped into chemical and plant treated groups (n=6) based on their body weight. The doses of extracts were so adjusted as to administer 0.25 ml of the suspension of extracts for 15 days. The route of administration of the extract was by gavage whereas flumazenil and picrotoxin was administered intraperitoneally.

GroupI: Control Group II: Flumazenil (1 mg/kg body weight) Group III: Lactuca sativa (100 mg/kg body weight) + Flumazenil Group IV: Lactuca sativa (200 mg/kg body weight) + Flumazenil Group V: Lactuca sativa (400 mg/kg body weight) + Flumazenil Group VI: Picrotoxin (1 mg/kg body weight)

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Group VII: Lactuca sativa (100 mg/kg body weight) + Picrotoxin Group VIII: Lactuca sativa (200 mg/kg body weight) + Picrotoxin Group IX: Lactuca sativa (400 mg/kg body weight) + Picrotoxin

4.2.2.b. Behavioural analysis

Elevated plus maze test (Test was performed as described previously in the chapter no. 3)

Vogles Test (Test was performed as described previously in the chapter no. 3) Light dark box test (Test was performed as described previously in the chapter

no. 3) Open field test (Test was performed as described previously in the chapter no.

3)

4.2.2.c. Chemical, biochemical and enzymatic assays (Tests were performed as described previously in the chapter No. 3)

4.2.2.d.Estimation of GABA in brain

(Analysis was performed as described previously in the chapter no. 4)

4.2.3. Results and discussion

The beneficial medicinal effects of plant materials typically result from the combinations of secondary metabolites present in the plants, through additive or synergistic action of several chemical compounds acting at single or multiple target sites associated with a physiological process (Briskin, 2000). This fact has a basis in the sense that medicinal actions of plants are unique to particular plant species or groups, consistent with the concept that combinations of secondary metabolites in a particular plant are often taxonomically distinct (Wink, 1999). According to Kaufman et al (2000), some plant products may exert their action by resembling endogenous metabolites, ligands, hormones, signal transduction molecules or neurotransmitters and thus have beneficial medicinal effects on humans due to similarities in their potential target sites (e.g. CNS, endocrine system etc.).

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4.2.3. a. Elevated plus maze test

The EPM test is based on the principle where exposure to an open arm of EPM evokes an approach-avoidance conflict that is considerably stronger than that evoked by the exposure to the enclosed arm (Hui et al., 2001; Powell et al., 2004).

The time spent in the open arms and the number of open arm entries decreased

in flumazenil and picrotoxin -treated groups (p < 0.01) compared to control group (Table 20). Hydro-alcoholic extract of Lactuca sativa at 200 mg/kg and 400 mg/kg significantly increased the time spent in the open arms. Entries to open arms increased significantly at the levels of 200 mg/kg (p < 0.05) and 400 mg/kg (p < 0.01). Plant extract at 100 mg extract had no significant effects on any of the parameters that were measured on the EPM.

4.2.3. b. Vogel Test in mice (Vogles Test)

Only few studies tried to apply the Vogles test to detect anxiolytic-like action of plant extracts and to be appropriate as a screening method for drugs that have apparent anti-anxiety actions. Licking in controls is suppressed, anxiolytics release this suppressed behaviour, while non-specific effects are assessed on non-punished

water drinking. Anxiolytics produced a significant anti-conflict effect, which means that these drugs increased the number of electric shocks mice received during the test session (Michel et al., 2007).

The number of licks and number of shocks in flumazenil and picrotoxin -treated groups (p < 0.01) decreased when compared to control group (Table 21). The number of licks and number of shocks increased significantly at the levels of 200

mg/kg (p < 0.05) and 400 mg/kg (p < 0.01). Plant extract at 100 mg/kg had no significant effects on any of the parameters that were measured on the Vogel Test.

4.2.3. c. Light dark box test

In light dark test, animals always try to spend more time in dark compartment compared to light box, out of fear of exposure to the new environment. Transitions have been reported to be an index of activity exploration because of habituation over time and the time spent in each compartment are reflection of aversion (Belzung et al., 1987).

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Flumazenil and picrotoxin -treated groups significantly decreased the time spent in light compartment (p < 0.01) compared to control group. Significant increase in the time spent in the light compartment (p < 0.05) was seen with administration of 400 mg/kg of hydro-alcoholic extract of Lactuca sativa compared to control group (Table 22).

4.2.3. d. Open field test

Open-field test shows the total distance travelled in the whole of the open-field arena by the mice. Though this parameter does not reflect changes in emotional behavior, it is important for evaluating the total locomotor activity of the animals

during the 5-minute trial. The two major behavioral variables evaluated in the open field test were the time spent in the zone of the field and the number of line crossings across the entire zone.

Locomotory activity decreased in flumazenil and picrotoxin-treated group of mice as evidenced by decline in the number of lines crossed and total mobility. Locomotion increased significantly (p < 0.01) after the administration of hydro-alcoholic extract of Lactuca sativa at 200 and 400 mg/kg compared to flumazenil and picrotoxin-treated group. Treatment with the plant extract at 400 mg/kg body wt. showed anxiolytic activity in this paradigm, as there were significant differences between the control as well as plant extract treated groups (Table 23). However, no significant effects were produced at 100 mg/kg body wt. of the plant extract.

4.2.3. e. Biochemical analysis

Recent data from several reports indicate that free radicals are involved in the

biochemical mechanisms underlying neuropsychiatric disorders in human. The results of several reports suggest that lower antioxidant defences against lipid peroxidation (Erkan et al., 2004).

A significant decrease in the serum total antioxidant level was observed in the group treated with flumazenil and picrotoxin as compared to control group. However treatment with the plant extract with flumazenil and picrotoxin increased the level of

total antioxidant in serum samples (Table 24).

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A significant increase in the brain nitrite, TBARS level was observed with the flumazenil and picrotoxin treatment. Administration of plant extract with flumazenil and picrotoxin treatment decreased the level of brain nitrite levels. Among the

treatments Lactuca sativa extract 400 mg/ kg body weight produced better response indicating protective effect against flumazenil and picrotoxin - induced damage (Table 24).

A significant decrease in the catalase activity and glutathione content was

observed in flumazenil and picrotoxin treated group of mice. The oral feeding of Lactuca sativa extract alone was unaltered when compared to control. The administration of Lactuca sativa extract (200 and 400 mg/kg body weight) reversed the flumazenil and picrotoxin induced reduction of catalase activity and glutathione

content (Table 24).

4.2.3. f. GABA estimation It is known that -aminobutyric acid (GABA) is one of the main inhibitory

neurotransmitters in the mammalian central nervous system and to some extent there

is a correlation between brain GABA content and its neuronal activity. Low levels of GABA, a neurotransmitter that reduces activity in the central nervous system, contribute to anxiety. A number of anxiolytics achieve their effect by modulating the GABA receptors (Lydiard, 2003; Nemeroff, 2003). GABA appears to play an important role in the pathogenesis of several neuropsychiatric disorders. Many of the

traditional agents used to treat psychiatric disorders are known to act, at least in part, by enhancing GABA activity, while some of the newer agents may exert their therapeutic effects exclusively via GABAergic actions.

Brain GABA levels significantly decreased in the flumazenil and picrotoxin -

treated groups in comparison to control group. Whereas, it was found that GABA levels significantly increased in (p

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GABA levels and dose dependant anxiolytic activity of plant extract as represented in figure 20.

Note : LS-100, 200 and 400 are supplementation of Lactuca sativa at doses of 100, 200 and 400mg/kg body weight respectively, flumazenil and picrotoxin

administration

at a dose of 1 mg/kg body weight, i.p, to all animals except control group.

Elevated plus maze test Groups Time spent in open arm (sec/5min) Control 69.614.36

Flumazenil (Flu) 38.34.89$ LS-100+Flu 61.312.4 LS-200+Flu 63.513.06* LS-400+Flu 82.416.78**

Picrotoxin (PTX) 53.115.75$ LS-100+PTX 89.318.9 LS-200+PTX 97.711.41* LS-400+PTX 113.711.6**

Table 20. Effect of Lactuca sativa extract on the time spent in open arm in mice behaviour in elevated plus maze against flumazenil and picrotoxin. $ indicates p < 0.01compared with control, * indicates p

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Light dark box test

Groups No. of entries to light zone Time in light zone (sec/5min)

Latency to first entry into light zone

Control 9.65.77 85.5120.1 48.918.2

Flumazenil (Flu) 6.02.45$ 94.3411.49$ 70.912.8$ LS-100+Flu 8.22.28 88.0610.9 47.910.3 LS-200+Flu

9.43.44* 103.815.0* 32.45.3* LS-400+Flu 12.21.78** 115.08.5** 15.74.6**

Picrotoxin (PTX) 6.82.28$ 61.1214.8$ 64.813.9$ LS-100+PTX 9.61.14 86.3616.7 42.35.8 LS-200+PTX

9.81.30* 96.8610.9* 25.64.2* LS-400+PTX

12.03.94** 126.4611.1** 14.66.4**

Table 22. Effect of Lactuca sativa extract on the time spent in light zone in mice

behaviour in light and dark test against flumazenil and picrotoxin. $ indicates p < 0.01compared with control, * indicates p

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Total antioxidant activity (%)

GSH (M/g tissue)

Nitrite (M/g tissue)

Catalase (U/mg protein)

TBARS (M/g tissue)

Control 70.52.6 72.11.6 40.24.1 2.650.26 98.52.8

Flumazenil (Flu) 32.63.5$ 29.30.9$ 108.36.1$ 0.730.08$ 180.34.9$ LS-100+Flu 44.62.9 34.51.1 91.65.9 0.840.01 159.15.2

LS-200+Flu 59.73.1* 49.60.8* 75.34.8* 1.030.06* 140.95.8*

LS-400+Flu 65.93.3** 61.61.8** 60.35.4** 1.840.10** 124.16.1**

Picrotoxin (PTX) 38.441.9$ 31.20.7$ 110.36.4$ 0.890.09$ 204.27.9$ LS-100+PTX

43.43.5 38.20.8 89.56.9 1.040.03 189.66.4

LS-200+PTX 54.62.7* 45.61.9* 71.35.4* 1.810.12* 160.69.4*

LS-400+PTX 67.22.9** 67.61.4** 61.66.4** 2.290.16** 138.36.7**

Table 24. Effect of Lactuca sativa extract on the total antioxidant, glutathione, nitrite,

TBARS and catalase in mice against flumazenil and picrotoxin. Data are presented as mean values (SD) from group of six mice. $ indicates p

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Figure 20. Correlation between behavioural parameters and brain GABA levels in anxiety animal models against Picrotoxin and flumazenil, (a) open field test, (b) elevated plus maze test, (c) Vogels Test, (d) light dark box test.

(b)

(c)

(d)

(a)

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4.3. SUMMARY AND CONCLUSIONS

The anti-anxiety activity of hydro alcohol extract of Lactuca sativa was treated to mice along with aluminium chloride which caused oxidative stress and anxiety by modulating behaviour in animal models like increased mobility in Open

field test, increased time spent in open arm of elevated plus maze, decreased number of marble buried in marble burring test and time spent in light zone and number of entries into light zone light dark box test.

Low levels of GABA, a neurotransmitter that reduces activity in the central nervous system, contribute to anxiety. A number of anxiolytics achieve their effect by

modulating the GABA receptors. Due to treatment of aluminium to animals GABA levels were decreased which was normalised by the treatment with Lactuca sativa.

These findings suggest that the hydro-alcoholic extract of Lactuca sativa possess significant anxiolytic activity that acts via modulation of behavior in light dark box test, elevated plus maze, antioxidant metabolite profile and GABA levels in brain, which were altered by the treatment of picrotoxin and flumazenil.

The result also showed that correlation does exist between the increase in

brain GABA levels and the delay in the onset of anxiety, suggesting that Lactuca sativa can be a major contender in the list of natural therapeutics for anxiety like disorders.