The document summarizes a study that investigated the antidepressant effects of a standardized hydroethanolic extract of Asparagus adscendens in mice. Key findings include: 1) The extract showed antidepressant-like effects in behavioral tests like the forced swimming test and tail suspension test at doses of 50-100 mg/kg. 2) It normalized monoamine neurotransmitter levels and reduced oxidative stress markers in the brain. 3) The extract also reduced elevated corticosterone levels, suggesting it may help normalize hyperactivity of the hypothalamic-pituitary-adrenal axis, another pathogenic factor for depression.

1. Journal analysis
Dr Manish Mohan
PG Student
SGMCRF

2. JOURNAL CLUB
Dr Manish Mohan
PG Student
SGMCRF

3. IJP
Antidepressant‐like effect of a
standardized hydroethanolic extract of
Asparagus adscendens in mice

4. • Department of Pharmaceutical Sciences and Drug Research,
• Punjabi University, Patiala, Punjab, India

5. Contents
• Introduction
• Methodology
• Results
• Discussion

6. Introduction
• Depression is a chronic, recurrent psychiatric disorder (21%)
• WHO ----4th leading cause of disability by 2020.
• Monoaminergic theory - 5-hydroxytyrptamine (5-HT), norepinephrine (NE), and
dopamine (DA).
• Monotherapy – resistance

7. Cont…..
• TCA–SSRI combination.
• Necessity to develop the newer antidepressant drugs with more safety and
efficacy.
• Asparagus adscendens Roxb. (Liliaceae)
( safed musali or dholi musali)

8. Cont…..
• Aphrodisiac and brain tonic.
• Roots -ant filarial, antidiabetic, antistress, chemo modulatory, aphrodisiac, antifungal, and
antioxidant activities.
• Vitamin C, xanthophylls, Vitamin E, β-carotene, and Shatavarin IV
• Palmitic acid, stearic acid, asparanin C, asparanin D
• Geriforte-----neurological disorder

9. Cont…..
• current study -antidepressant effect of root extract of A. adscendens and
its possible mechanisms

10. Materials and Methods
• Swiss Albino mice of either sex weighing 20–30 g
(3–4 months of age)
• The animals were housed on a 12-h light/dark cycle
• Temperature (22°C ± 2°C)
• Humidity (50 ± 10%).

11. Plant collection and preparation of extract
• “Council of Scientific and Industrial Research -Institute of Himalayan
Bioresource Technology”
• Roots ---- washed with water, shade-dried, and ground to a moderately
coarse powder
extraction
• Percolation method with ethanol: water (50:50 v/v) at room temperature

12. Cont…..
• Extract was evaporated to dryness using a rotary evaporator
Lyophilization
Stored at 4ºC for further use
• The percentage yield of the extract was found to be 44.7% w/w.

13. Phytochemical analysis and standardization of the plant
extract using high-performance-thin-layer chromatography
• Alkaloids, carbohydrates, glycosides, saponins, steroids, triterpenoids,
flavonoids, tannins, proteins, and amino acids
• Shatavarin IV ----biomarker for the standardization of the hydroethanolic
extract
HPTLC

14. Cont…………
• Presence of shatavarin IV was confirmed by TLC using ethyl acetate:
methanol: water (7.5:1.5:0.5, v/v/v) as a mobile phase
• A stock solution of hydroethanolic extract (30 mg/mL) and Shatavarin IV
(1 mg/mL) was prepared in methanol chromatogram
426 nm

15. Methodology
• Six groups of animals (n = 6)
• Group I: naive
• Group II: saline (10 mL/kg/day, i.p.)
• Group III: imipramine (15 mg/kg/day, i.p.) 14 days
• Groups IV, V, and VI: AAE
(25, 50, and 100 mg/kg/day, i.p., respectively)

16. Cont…..
• Behavioral assessment - 14th day.
• Sacrificed by decapitation cortex and hippocampus

17. Acute toxicity test of the extract
• 25, 50, and 100 mg/kg; i.p
• Percentage mortality and gross behavioral changes were observed during 24
h after treatment

18. Behavioral assessments
1. Forced swimming test
25 cm in height, 14 cm in diameter
Filled with 20-cm high water (25°C ± 2°C)
Duration of immobility was observed for 6 min

19. Cont……
2. Tail suspension test
Mice were individually suspended by tail (6 min)
Immobile
( when they hung passively and completely motionless.)

20. Cont……
3. Open‐field test
locomotor and exploratory behaviors
40 cm × 40 cm × 30 cm
25 equal squares (8 cm × 8 cm)
- crossings, rearing……….5 Minutes..

21. Estimation of monoamines using high‐performance
liquid chromatography with electrochemical
detection
High-performance liquid chromatography with electrochemical detection
 Brain samples - homogenized in an ice-cold solution of 0.1 M
perchloric acid Centrifuged at 14,500 g for 30 min at 4°C.
 The clear supernatants were filtered through a 0.45-μm pore membrane.

22. Cont……
 The separation was done in an isocratic elution mode at a column
temperature of 35°C using mobile phase
( 0.15 M sodium dihydrogen phosphate, 2.28 mM heptane sulphonic acid,
and 1 mM ethylenediaminetetraacetic acid (pH 5.4), containing 10% methanol
at a flow rate of 0.9 mL/min.)
monoamine neurotransmitters - in ng/g wet weight tissue.

23. Cont…….
• The order of elution
• Norepineprine (6.481 min)
• DA (15.437 min)
• 5-HT (38.144 min)

24. Biochemical estimation
• Brain samples were homogenized in an ice-cold 10% w/v (0.05 M, pH 7.4)
phosphate buffer centrifuged at 6000 g for 20 min at 4°C.
• Clear supernatant
• Thiobarbituric acid-reactive substances (TBARS)
• Reduced glutathione (GSH)
• Catalase (CAT)
• Total nitrite levels

25. Estimation of thiobarbituric acid‐reactive
substance
• An index of lipid peroxidation
• Method described by Oakes and Van der Kraak
• The absorbance was measured at 532 nm
microplate reader(BIORAD)
• TBARS value was expressed as nanomoles/mg of protein

26. Estimation of reduced glutathione
• Reduced GSH Absorbance at 412 nm.
Microplate reader(BIORAD)
• Values were expressed as micromoles per gram of wet tissue.

27. Estimation of catalase
• CAT activity -----The modified method of Chance and Maehly
• Calculated by using the molar extinction coefficient of H2O2 (0.071
mM/cm)
• Expressed as micromoles of H2O2 oxidized per minute per milligram
protein.

28. Estimation of total nitrite level
• Absorbance –(at 540 nm)
• (BIORAD-Microplate Reader, Logotech, ISE Group, Germany)
• Standard curve was plotted using 10–100 mM of sodium nitrite.
• All the values were expressed as ng/g of wet tissue

29. Serum corticosterone measurement
• Spectrofluorometer
• The standard curve was plotted using 10–100 ng of corticosterone
• All the values were expressed as ng/mL

30. Statistical analysis
• One-way analysis of variance (ANOVA)
• Student–Newman–Keuls post hoc multiple comparison test

31. Results

32. Phytochemical analysis and standardization of the
plant extract using high‐performance‐thin‐layer
chromatography
• Alkaloids, carbohydrates, glycosides, saponins, steroids, triterpenoids, flavonoids,
tannins, proteins, and amino acids.
• HPTLC analysis----Shatavarin IV
• The spots of the entire chromatogram were visualized at 426 nm
• Shatavarin IV - 9.4 mg/kg of the hydroethanolic extract

34. Acute toxicity studies
• No mortality
• No neurotoxic effects with respect to disturbances in motor coordination up
to the highest dose (i.e., 100 mg/kg).

35. Effect of Asparagus adscendens extract on forced
swimming test
• One-way ANOVA---There were significant differences between the treatment
groups
• Student–Newman–Keuls test-----AAE (25, 50, and 100 mg/kg) significantly
decreased the duration of immobility time as compared to vehicle control group
• Increased the swimming time at 50 and 100 mg/kg as compared to vehicle control
group

39. Effect of Asparagus adscendens extract on tail
suspension test
• Student–Newman–Keuls test
• Significant decrease in the immobility time attenuated by the administration
of AAE at 50 and 100 mg/kg
• Imipramine also decreased the immobility time after administration of 15
mg/kg dose

41. Effect of Asparagus adscendens extract on
open-field test
• No significant effect -----number of crossing and rearing

42. Effect of Asparagus adscendens extract on
monoamine neurotransmitters levels in cortical
and hippocampal areas of mouse brain
• Changes in norepinephrine levels
• Student–Newman–Keuls test indicated a significant decrease in the cortical
and hippocampal NE levels of vehicle control as compared to naive group

45. Changes in dopamine levels
• significant increase cortical and hippocampal DA levels
( AAE-treated groups as compared to vehicle control)
• no significant change in DA level
(imipramine-treated group as compared to vehicle control group)

47. Changes in serotonin levels
• No significant change was observed in hippocampal serotonin level in
between the groups
• A significant increase
( cortical serotonin level of AAE-treated groups as compared to vehicle control
group)

49. Effect of Asparagus adscendens extract on
lipid peroxidation,
reduced glutathione, catalase, and total nitrite
levels in
cortical and hippocampal areas of mouse brain

51. Effect of Asparagus adscendens extract on
serum corticosterone level

52. Discussion
• Current study demonstrate the significant antidepressant-like effect of
AAE as observed in the behavioral tests on mice
• TST and FST---widely accepted behavioral tools
• AAE reduced the duration of immobility in the FST and TST.
• Peak effect observed at 100 mg/kg in both the models.

53. Cont…..
• None of the test doses affected the locomotor activity
• Major Neurochemical Process In Depression
• impairment of monoamine neurotransmitter levels
• hyperactivity of hypothalamic–pituitary–adrenal (HPA) axis
• oxidative damage by inducing an imbalance between the in vivo pro-oxidant
and antioxidant status

54. Cont…..
• human studies HPA dysfunction elevated corticosterone levels
• Studies free radical generation increased during depression
• Bacopa monniera Wettst, Withania somnifera
• AAE significantly increased the activity of CAT and reduced GSH levels in both
cortex and hippocampus

55. Cont…..
• It also showed significant reduction in the level of lipid peroxidation marker and
total nitrite levels in both cortex and hippocampus
• Hyperactivation of HPA axis is another additional pathogenic factor for depression
increase in corticosterone levels
• AAE downregulation of corticosterone normalizing the HPA
hyperactivity

56. THANK YOU