RESULTS

Phytochemical Analysis

The dried powder of DA was green, bitter, and had no specific odor. Phytochemical screening of the ethanolic extract of DA revealed the presence of glycosides, flavonoids, tannins, and steroids in the extract (Table 15.1).

Acute Toxicity Study

According to OECD guidelines no. 425, an oral dose of the ethanolic extract of up to 2000 mg/kg bodyweight did not cause any untoward effect in the mice. No changes were observed in behavior,

TABLE 15.1

Results of Phytochemical Screening of an Ethanolic Extract of Dysphania ambrosioides Chemical Test Results

A) Carbohydrates

1) Molisch’s Test

-ve

2) Benedict Test

-ve

B) Alkaloids

1) Mayer's Test

-ve

2) Wagner’s Test

-ve

C) Glycosides

1) Modified Borntrager’s Test

+ve

2) Legal’s Test:

+ve

D) Proteins and amino acids

1) Xanthoproteic Test

-ve

2) Ninhydrin Test

-ve

E) Flavonoids

1) ShinodaTest

+ve

2) Lead acetate Test

+ve

F) Phytosterols

1) Salkowski’s Test

+ve

2) Liebermann Burchard’s Test

+ve

G) Tannin

1) Gelatin Test

TABLE 15.2

Transfer Latency of Mice in Different Treatment Groups on the Probe Day

Group

Treatment

Dose (mg/kg)

Transfer latency (s)

1

Control (normal saline)

10 ml/kg, p.o.

6.500 ±0.6455*

II

Scopolamine control

0.4 (i.p.)

11.500 ±1.323#

III

Standard (tacrine)

3 (i.p.)

4.500 ±1.041**

IV

DyslOO

100 (p.o.)

6.000 ±1.581*

V

Dys200

200 (p.o.)

5.250±0.8539**

All the values represent the mean ± standard error of the mean (SEM) (n = 6). # denotes P<0.5, ## denotes P < 0.01 and ### denotes P<0.001 when compared with the control, and * denotes P<0.05, ** denotes P<0.01 and *** denotes P< 0.001 when compared with the scopolamine control, as a result of one-way analysis of variance (ANOVA), followed by Dunnett’s test.

motor, or neuronal functions. The extract did not cause any anatomical changes. Hence, 100 and 200 mg/kg oral doses of the extract were used in the study.

EPM

Effect on Transfer Latency (TL)

On the probe day, the effects of the vehicle (saline), tacrine, scopolamine, DyslOO and Dys200 on TL were recorded. The scopolamine-treated group showed a significant increase in TL (Pc 0.05) when compared witli the saline control group, indicating memory impairment in the mice, whereas tacrine (the standard drug) caused reduced TL in mice (PcO.Ol), indicating amelioration of scopolamine-induced amnesia in the animals. Treatment witli DyslOO or Dys200 before exposure to scopolamine also exhibited decreased TL in the mice, compared with the scopolamine-treated group (Table 15.2 and Figure 15.1).

Barnes Maze

Effect on Escape Latency and Poke Error

All the animals showed a decrease in escape latencies and poke errors across the trials. Mice in the scopolamine-treated group showed longer ELs and made more errors, compared with the untreated control group (PcO.Ol). Mice in the tacrine-treated group exhibited the shortest ELs and made fewer poke errors (P<0.01) in the trials. A significant decrease in ELs (PcO.Ol) and poke errors (P<0.05) were also observed in the case of animals treated with DyslOO and Dys200, prior to scopolamine treatment, when compared with the scopolamine-treated group (See , Figure 15.1, and Table 15.3).

MWM

Effect on Escape Latency and Path Length

Mice In the scopolamine-treated group showed a significant increase in EL, when compared with the untreated control group (PcO.Ol), whereas mice in the standard group showed a significant decrease (Pc0.001), in the EL than the scopolamine induced group. Consecutive training sessions of 14 d in the MWM revealed significant retention of memory in the test groups. DyslOO- and Dys200-treated mice rapidly learned the location of the hidden platform as reflected by a

Effect on escape latency shown by mice in different treatment groups in the Barnes maze model

FIGURE 15.1 Effect on escape latency shown by mice in different treatment groups in the Barnes maze model. All the values represent the mean ± standard error of the mean, SEM (n=6). # denotes P < 0.05, ## denotes P < 0.01 and ### denotes P < 0.001 when compared with the control and * denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001 when compared with the scopolamine control, as a result of one-way analysis of variance (ANOVA), followed by Dunnett’s test.

TABLE 15.3

Effect on Numbers of Poke Errors (Barnes Maze Model) Made by Different Treatment Groups on the Probe Day

Group

Treatment

Dose (mg/kg)

Number of poke errors

1

Control (Normal saline)

10 ml/kg, p.o.

1.400 ±0.5099**

II

Scopolamine

0.4 (i.p.)

7.000± 1.378##

III

Standard (tacrine)

3 (i.p.)

2.000 ±0.8944**

IV

DyslOO

100 (p.o.)

2.600± 1.249*

V

Dys200

200 (p.o.)

2.800±0.8602*

All the values represent the mean ± standard error of the mean (SEM) (n=6), # denotes /'<0.05. ## denotes P<0.01 and ### denotes P< 0.001 when compared with the control, and * denotes P< 0.05, ** denotes P< 0.01 and *** denotes P< 0.001 when compared with the scopolamine control, as a result of one-way analysis of variance (ANOVA), followed by Dunnett’s test.

decrease in their latencies from day 1 to day 15, indicating the normal acquisition behavior. On the probe day, mice treated with DyslOO or Dys200 prior to treatment (P< 0.001) with scopolamine revealed significant decreases in EL, indicating alleviation of scopolamine-induced amnesia (Figure 15.2).

Mice in the scopolamine-treated group showed a longer path length (P<0.05), compared with the control group, whereas mice in the standard group (P<0.01) showed a significant decrease in the path length, compared wuth those in the scopolamine-treated group. Similarly, the path length (distance travel to reach the platform) was significantly shorter in both DyslOO- and Dys200-treated groups (P<0.05) compared w'ith scopolamine treated group (Table 15.4 and Figure 15.5).

Effect on escape latency showed by different treatment groups of rats in the MWM model

FIGURE 15.2 Effect on escape latency showed by different treatment groups of rats in the MWM model. All the values represent the mean ± standard error of the mean (SEM) (n=6). #denotes P < 0.05, ##denotes P < 0.01 and ### denotes P < 0.001 when compared with the control, and ^denotes P < 0.05, **denotes P < 0.01 and ***denotes P < O.OOlwhen compared with the scopolamine control, as a result of one-way analysis of variance (ANOVA), followed by Dunnett’s test.

TABLE 15.4

Path Lengths of Different Treatment Groups on the Probe Day (MWM)

Group

Treatment

Dose (mg/kg)

Path length (m)

1

Control (normal saline)

10 ml/kg, p.o.

0.1945±0.0319*

II

Scopolamine

0.4 (i.p.)

0.3803±0.05008 #

III

Standard (tacrine)

3 (i.p.)

0.1485±0.04831**

IV

DyslOO

100 (p.o.)

0.1993±0.03424*

V

Dys200

200 (p.o.)

0.1643±0.05006*

All the values represent the mean ± standard error of the mean (SEM) (n=6), # denotes /'<0.05. ## denotes P<0.01 and ### denotes P< 0.001 when compared with the control, and * denotes P< 0.05, ** denotes P< 0.01 and *** denotes P< 0.001 when compared with the scopolamine control, as a result of one-way analysis of variance (ANOVA), followed by Dunnett’s test.

TABLE 15.5

Total Time Spent in the Target Quadrant by Different Treatment Groups on the Probe Day (MWM)

Group

Treatment

Dose (mg/kg)

Time spent in target quadrant (s)

1

Control (normal saline)

10 ml/kg, p.o.

29.48 ±5.795

II

Scopolamine

0.4 (i.p.)

24.68 ±5.773

III

Standard (tacrine)

3 (i.p.)

43.00 ±4.282

IV

DyslOO

100 (p.o.)

32.08±7.014

V

Dys200

200 (p.o.)

33.33 ±3.659

All the values represent the mean±standard error of the mean (SEM) (n=6). # denotes /'<0.05 when compared with the saline control, and * denotes P<0.05 when compared with the scopolamine control, as a result of one-way analysis of variance (ANOVA), followed by Dunnett’s test.

Effect on Total Time Spent in the Target Quadrant

The total time spent in the target quadrant was measured by removing the platform from the target quadrant (Q4) and then allowing the animals to explore the maze. Analysis of the swimming performance during the probe trial revealed that animals which had received scopolamine spent less time in the target quadrant, whereas mice which had received DyslOO or Dys200 prior to scopolamine treatment spent comparatively more time in the target zone (Table 15.5 and Figure 15.6).

DISCUSSION

AD is a debilitating neurodegenerative disorder, the incidence of which continues to rise worldwide (Kulkarni et al., 2011). AD involves progressive loss of memory and cognition, characterized by aphasia, apraxia, and agnosia (Saikia et ah, 2018). Despite the availability of a number of allopathic medicines for the treatment of AD, searches for herbal remedies with high potency and fewer side effects than the allopathic drugs is going on. The present study investigated the therapeutic efficacy of an ethanolic extract of D. ambrosioides for memory enhancement. Scopolamine was used to induce the impairment of memory in the experimental animals. In the acute toxicity study, no death was observed up to a dose of 2000 mg/kg. The mice were physically active, indicating that the median half-maximal lethal dose (LD50) could be higher than 2000 mg/kg in mice.

The elevated plus maze, Morris water maze, and Barnes maze models were employed to assess the memory-enhancing effects of the ethanolic extract of D. ambrosioides. These models are commonly used for assessment of anti-amnesic property of drugs.

The results of the EPM task revealed that mice, after oral treatment with DyslOO or Dys200, prior to scopolamine treatment, showed a significant decline in TL quite similar to the effects observed after administration of the standard nootropic drug, tacrine. This suggests the possible memoryenhancing property of both DyslOO and Dys200. Moreover, both the extracts were assessed for their cognitive enhancement effect on mice against scopolamine-induced amnesia, using MWM. Reversal of scopolamine-induced amnesic parameters in the MWM test reflected increased retention of memory in the animals. Both the DyslOO and Dys200 groups showed remarkable reductions in EL and path lengths, when compared with the scopolamine group. In addition to this, the animals also spent more time in the target quadrant searching for the platform removed from the target quadrant, which is an indicator of cognitive improvement. The Barnes maze test was performed on rats in order to assess the retention of memory in animals. EL and the number of poke errors were recorded as markers of memory enhancement. The two test groups, DyslOO and Dys200, each took comparatively less time to find the target hole and made fewer poke errors, in contrast with the scopolamine-treated group.

Hence, it was revealed from the study that administration of an ethanolic extract of D. ambrosioides (15 d oral administration) showed a pronounced effect on reversal of scopolamine-induced memory loss in rodents. Comparatively, the higher dose (200 mg/kg) of DA extract showed greater activity than the lower dose (100 mg/kg) of the extract. This is the first preclinical study investigating the memory-enhancing activity of the ethanolic extract of DA. In Peru, the plant is also reported to be used ethnomedicinally as a memory enhancer (Potawale et al., 2008). Oxidative stress plays an important role in aggravating the pathogenesis of AD. It facilitates abnormal accumulation of A|3- and tau protein-initiated neurotoxicity (Dhingra and Kumar, 2012; Kim et al., 2016; Uddin et al., 2016). Scopolamine causes memory impairment in experimental animals by blocking the cholinergic neurotransmission (Pushpalatha et al., 2013), whereas oxidative stress induced by scopolamine can also lead to a decline in memory (Fan et al., 2005). An antioxidant property of DA has also been reported (Barros et al., 2013; Nascimento et al., 2006). Thus, the memory-enhancing activity of the ethanolic extract of DA might be due to multiple factors in the extract, with properties such as antioxidant and elevated cholinergic neurotransmission.

CONCLUSION

The reversal of the amnesic behaviors in the behavioral models, following administration of scopolamine, suggest the increased memory retention capacity of the ethanolic extract of DA. The amelioration of scopolamine-induced amnesia by the extracts may be associated with the antioxidant properties of the plant. This study supports the traditional claim of the plant as a memory enhancer. However, biochemical analysis and insight into the probable underlying mechanism(s) will be required in order to understand the clinical efficacy of the plant extract.

ACKNOWLEDGMENTS

The authors express their deep gratitude to the Dean and the Director of Research (Vety), College of veterinary science, Assam Agricultural University, Khanapara, Assam, for providing us with the facilities with which to carry out this research work.

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