ANTIANXIETY PROPERTY OF TECOMA STANS LINN LEAVES EXTRACT IN EXPERIMENTAL ANIMALS - RESULTS

 

5. RESULTS

 

5.1 Successive Solvent Extraction

            The leaves of Tecoma stans are collected and shade dried. Coarse powder is made from these dried leaves and subjected to extraction in increasing polarities. Various extracts are prepared by using suitable solvents like petroleum ether, chloroform, alcohol and aqueous solvent with Soxhlet apparatus. The various percentage yield, colour, consistency and solubility in water of different extracts was noted in Table No.01.

Table No. 01: Percentage yield, colour, consistency and solubility in water of different extracts

Plant part used	Extracts	Percentage Yield	Color	Consistency	Solubility in Water
Tecoma stans leaves	Petroleum	2.08	Light Yellowish green	Sticky	Insoluble
	Chloroform	1.95	Dark green	Sticky	Soluble
	Ethanol	1.96	Dark green	Sticky	Soluble
	Aqueous	2.13	Dark brown	Dry powder	Soluble

 

5.2 Preliminary Phytochemical Screening

            The preliminary phytochemical evaluation of different extracts of Tecoma stans leaves revealed the presence of flavonoids, carbohydrates, starch, gum, proteins, tannins, saponins and phenolic compounds. The results of phytochemical analysis were significant in ethanolic and aqueous extracts when compared with petroleum ether and chloroform extracts.

            This observation clearly indicates that most of the bioactive compounds of Tecoma stans are soluble in ethanol and water, hence we have selected ethanolic and aqueous extracts for our studies. The results of phytochemical analysis were shown in Table No.02.

 

Table No. 02. Preliminary phytochemical screening of Tecoma stans.

Phytochemical constituents	Pet ether	Chloroform	Ethanol	Aqueous
Alkaloids	-	+	+	+
Glycosides	-	++	++	++
Carbohydrates	-	+	+	+
Flavonoids	-	+	+++	+++
Saponins	-	+	+++	+++
Tannins	-	+	+++	+++
Steroids	-	-	++	++
Proteins	+	+	+	+
Fats and oils	++	-	-	-
Starch	-	-	++	++
Gums	-	-	+++	+++
Phenolic compounds	-	-	+++	+++

 

- absent  ++more clarity

+ indicates  +++ better response

5.3. Acute Toxicity Study:

     Acute toxicity studies for ethanolic, chloroform, aqueous extracts of Tecoma stan linn (Bignoniaceae) were conducted as per OECD guidelines 420 using female albino swiss mice. Each animal was administered aqueous and ethanolic extracts by oral route. The animals were observed for any changes continuously for the first 2 h and up to 24 h for mortality, there were no mortality and noticeable behavioural changes in all the groups tested. The extracts were found to be safe up to 2000 mg/kg body weight.

 

5.4 Elevated Plus Maze Test:

5.4.1 Ethanolic and aqueous extracts of TS leaves in EPM:

            The behavioural effects of diazepam, ethanolics extract and aqueous extracts EE1, EE2 and AE1, AE2 and  on the behaviour of mice in the elevated plus maze test were summarized in table 3 and figure 5.1 to5.4.

 

            Diazepam has increased the percentage of arm  entries  in  open  arms more significantly  (***p  <  0.001,  Figure  5.1) and moderately significant increase in  time spent in open arms  (**p<  0.01,  Figure  5.2),  whereas decreased the percentage of  arm  entries  in  closed  arms more  significantly  (***p  <  0.001,  Figure 5.3 ), and moderately significant decrease in the  percentage  of  time  spent  in  closed  arms   (**p  <0.01,  Figure 5.2) as compare to control group.

 

            It was seen that in  the aqueous extracts,  AE1 has no significant results.  But aqueous extract AE2 showed moderately significant increase in percentage  of arm entries (**p< 0.01,  Figure 5.1)  and time  spent in  open  arm  (**p<0.01,  Figure 5.2 ),  whereas  moderately significant  decrease in the  percentage of entries in closed  arm   (**p<0.01,  Figure  5.3 ),  and less significant decrease in time  spent  in  closed  arm(* p<0.05, Figure  5.4)  as  compare  to  control.

 

            It was seen that in  the ethanolic extracts,  EE1 has no significant results.  But ethanolic extract EE2 showed moderately significant increase in percentage  of arm entries (**p< 0.01,  Figure  5.1)  and time  spent in  open  arm  (**p<0.01,  Figure  5.2 ),  whereas  less significant  decrease in the  percentage of entries in closed arm   (*p<0.05,  Figure  5.3 ),  and time  spent  in  closed  arm(* p<0.05, Figure  5.4)  as  compare  to  control.

Table 3. Effects of ethanolic and aqueous extracts of TSL leaves from elevated plus maze test in mice.

Treatment	% open arm entry	% open arm time	% close arm entry	% close arm time
Control	5.883±0.703	73.50±10.87	12.33±1.256	211.8±11.17
Diazepam (2 mg/kg)	16.83±2.358***	152.2±20.15**	16.16±0.792***	131.7±19.82**
EE1 (400 g/kg)	9.83±1.302	79.83±10.61	14.00±1.238	204.5±9.664
EE2 (800 mg/kg)	15.67±2.376**	149.8±17.30**	8.16±0.477*	150.00±23.42*
AE1 (400 mg/kg)	10.00±0.966	83.83±10.37	10.50±0.670	202.3±10.25
AE2  (800 mg/kg)	15.00±1.966**	148±19.05**	7.83±0.542**	144.5±17.79*

 

All values are mean ± SEM, (n =6), one way ANOVA, followed by Dunnet’s test.

* p< 0.05, ** p < 0.01, *** p  < 0.001, when compared to vehicle treated group.

 

5.5 Hole Board Test:

5.5.1. Ethanolic and aqueous extracts of TSL leaves in hole board test:

            The behavioural effects of diazepam, aqueous extracts AE1, AE2 and ethanolics extract EE1, EE2  on the behaviour of mice in the Hole board test were summarized in table 4 and figure 5.5 to 5.8.

 

            Diazepam treated group shown highly significant increase in head dip latency (***p < 0.001, Figure 5.6), whereas moderately significant increase in head dip count, no. of assisted rearing and locomotion  (**p< 0.01, Figure 5.5, 5.7 & 5.8)  Further analysis showed that aqueous extract AE1 was insignificant in above paradigms. whereas AE2 showed moderately significant in head dip latency (**p < 0.01, Figure 5.6), head dip count (**p < 0.01, Figure 5.5), locomotion (**p < 0.01, Figure 5.7) and no of assisted rearing less significantly (*p < 0.05, Figure 5.8)  as compare to control group. 

 

            The ethanolic extracts, EE1 was insignificant in above paradigms.   Whereas EE2 showed moderately significant increase in  locomotion  (**p < 0.01, Figure 5.7) and less significant increase in head dip latency (*p < 0.05, Figure 5.6)  , head dip count (*p < 0.05, Figure 5.5)  , no of assisted rearing  (*p < 0.05, Figure 5.8). 

Table  4.  Effect of ethanolic and aqueous extracts of TSL leaves from hole board test in rat.

Treatment	Head dip count	Head dip latency	Locomotion	No. Of Assisted Rearing
Control	11.67±1.783	18.00±2.017	29.67±2.390	16.50±2.617
Diazepam 2 mg/kg	27.00±4.099**	33.67±2.390***	63.00±9.459**	30.83±3.842**
EE1 (400 mg/kg)	15.50±1.708	20.67±1.333	47.67±3.169	18.83±2.750
EE2 (800 mg/kg)	22.17±1.740*	27.83±2.960*	66.67±9.426**	28.17±3.198*
AE1 (400 mg/kg)	17.33±1.085	23.83±2.136	52.67±6.031	17.33±2.201
AE2  (800 mg/kg)	25.00±3.715**	32.83±1.833**	69.67±8.032**	29.83±3.177*

 

All Values are mean ± SEM, (n =6), one way ANOVA, followed by Dunnet’s test.

* p < 0.05, ** p < 0.01, *** p  < 0.001, when compared to vehicle treated group.

 

5.6 Light/Dark Transition Test:

5.6.1. Ethanolic and aqueous extracts of TSL leaves in Light/Dark transition test:

            The behavioural effects of diazepam, aqueous extracts AE1, AE2 and ethanolics extract EE1, EE2  on the behaviour of mice in the Light and Dark transition test were summarized in table 5 and figure 5.9 to 5.10.

 

            Diazepam treated group shown highly significant decrease in time spent in dark area (***p < 0.001, Figure 5.9) and highly significant increase in time spent in light area (***p < 0.01, Figure 5.10).

 

            It was seen that, in groups treated with the aqueous extracts (AE1, AE2) and ethanolic extracts ( EE2), showed  moderately significant decrease in time spent in dark area (**p < 0.01, Figure 5.9) and moderately significant increase in time spent in light area (**p < 0.01, Figure 5.10). Whereas EE1 showed  less significant decrease in time spent in dark area (*p < 0.05, Figure 5.9)and  less significant increase in time spent in light area (*p < 0.05, Figure 5.10)

Table 5. Effect of ethanolic and aqueous extracts of TSL leaves from light/dark transition test in mice.

Treatment	Time in dark area	Time in light area
Control	187.2±8.534	103.7±10.12
Diazepam 2mg/kg	93.83±4.963***	204.00±6.36***
EE1	153.3±8.663*	138.3±7.787*
EE2	130.2±7.534**	166.00±5.164**
AE1	145.2±6.560**	148.7±6.984**
AE2	125.3±3.765**	169.8±3.135**

 

All values are mean ± SEM, (n =6), one way ANOVA, followed by Dunnet’s test.

* p < 0.05, ** p < 0.01, *** p  < 0.001 when compared to vehicle treated group.

5.7 Open Field Test:

5.7.1. Ethanolic and aqueous extracts of TSL leaves in Open field test:

            The behavioural effects of diazepam, aqueous extracts AE1, AE2 and ethanolics extract EE1, EE2  on the behaviour of mice in the Open field test were summarized in table 6 and figure 5.11 to 5.14. Diazepam treated group shown highly significant increase in locomotion, no. of assisted rearing,  entries in the central square and time spent in central square   (***p < 0.001, Figure 5.11 to 5.14).            

 

            It was seen that in  the aqueous extracts,  AE1 has no significant results.  But aqueous extract AE2 showed moderately significant increase in locomotion, no. of assisted rearing and time spent in central square    (**p<0.01,  Figure 5.11, 5.13, 5.14  ),   less significant increase in entries in the central square (* p<0.05, Figure 5.12)  as  compare  to  control.

 

            It was seen that in  the ethanolic extracts,  EE1 has no significant results.  But ethanolic extract EE2 showed no significant results in entries in the central square and time spent in central square and  moderately significant increase in locomotion (**p< 0.01,  Figure 5.11)  whereas  less significant  increase in the  no. of assisted rearing (* p<0.05, Figure  5.14)  as compare  to  control.

 

 

Table 6. Effects of ethanolic and aqueous extracts of TSL leaves from Open field test in mice

Treatment	Locomotion	No. Of assisted rearing	Entry in central square	Time spent in central square
Control	59.50±7.093	13.67±1.647	3.83±0.600	19.33±2.552
Diazepam (2mg/kg)	142.3±8.999***	29.83±3.381***	7.66±0.714***	42.00±5.495***
EE1 (400 mg/kg)	69.33±7.172	17.17±2.023	5.16±0.600	22.00±2.295
EE2 (800 mg/kg)	156.7±11.28**	25.00±3.276*	5.16±0.307	24.50±2.487
AE1 (400 mg/kg)	83.00±7.45	16.83±1.167	5.00±0.365	21.83±1.939
AE2 (800 mg/kg)	138.0±7.844**	27.00±3.000**	6.33±0.557*	40.50±5.227**

 

All values are mean ± SEM, (n = 3), one way ANOVA, followed by Dunnet’s test.

* p < 0.05, ** p < 0.01, When compared to vehicle treated group.