Hydrocotyle verticillata REDUCES ETHANOL TOLERANCE AND NEUROTOXIC

EFFECTS ON COGNITIVE AND MOTOR FUNCTION OF Drosophila melanogaster

Arabelle L. Santos

Jesscel Mae J. Libiran

Giuliana B. Anupol

Researchers

Jerone S. Mejia
Research Adviser

Paul Mark B. Medina

Associate Professor II
University of the Philippines-College of Medicine-Manila
Research Consultant
Hydrocotyle verticillata REDUCES ETHANOL TOLERANCE AND NEUROTOXIC
EFFECTS ON COGNITIVE AND MOTOR FUNCTION OF Drosophila melanogaster

Arabelle L. Santos, Jesscel Mae J. Libiran, Giuliana B. Anupol

Juan R. Liwag Memorial High School

ABSTRACT

Alcohol consumption, which leads to complex disorders involving neurological

deficits, is one of the main contributors in chronic diseases’ overall global deaths, reaching 3
million, annually. Prescriptive drug Disulfiram is commonly used for the aversion of alcohol
cravings and dependency, however, it’s costly, commercially unavailable and of unsure long-
term effects. Thus, the researchers investigated the potential of Hydrocotyle verticillata Leaf
Crude Ethanolic Extract (HvLCEE) in reducing alcohol tolerance in Drosophila melanogaster
by means of conducting various assays. Via sedation assay, the effect of HvLCEE on the flies’
ethanol tolerance under 35% and 85% EtOH was evaluated. The percentage of active flies
shows that HvLCEE, at its highest concentration (1000 µg/mL), successfully heightened
ethanol sensitivity and reduced tolerance. Locomotion after chronic ethanol exposure was
assessed through the negative geotaxis assay. High concentrations of HvLCEE invoked motor
deficits and postural control loss, implying an increase in ethanol sensitivity and decrease in
tolerance. Lastly, the aversive phototactic suppression assay was conducted to determine the
flies’ learning ability after chronic ethanol exposure. The highest concentration of HvLCEE
exhibited the highest mean pass rate of flies on both genders in PC0 and PC6 assessments,
suggesting the extract’s potential to reduce cognitive impairment and improve memory and
learning ability. Moreover, HvLCEE exhibited 32.87% free radical scavenging activity in the
DPPH assay, indicating its antioxidative potential. These outcomes suggest that HvLCEE can
lower ethanol tolerance and improve cognition of D. melanogaster which could serve as
potential treatment in lowering alcohol consumption and preventing its neurotoxic effects.

Keywords: Alcohol tolerance, Motor dysfunction, Cognitive deficit, Drosophila melanogaster,

Hydrocotyle verticillata
 INTRODUCTION

Chronic diseases have been causing people to suffer all over the world, and have been

rapidly increasing in present times, accounting for 70% of all deaths as of recent. A number of

these diseases can be attributed to the harmful use of alcohol which leads to 3 million annual

deaths globally (World Health Organization, 2018). In 2016, the WHO stated that 68% of the

population ages 15 years and older are affected by alcohol consumption in the Philippines.

These drinkers were categorized into three being occasional drinkers with 38.9%, regular

drinkers with 11.1% and 4.8% are heavy drinkers (Vinluan, 2013).

Alcohol consumption induces grave consequences to the central nervous system such

as oxidative stress when metabolized by the body. The ethanol-induced oxidative stress in the

brain can invoke neuro-cognitive deficits and neurodegeneration (Haraoh et al., 2019)

impairing both cognitive and motor functioning thereby altering visuospatial perception and

inhibit behavioral responses (Fillmore MT, 2011). Further, long-term alcohol intake affects

many neurotransmitter systems that ultimately lead to the development of craving and alcohol-

seeking behavior (Valenzuela, 2014). On the other hand, alcohol tolerance, one of the

hallmarks of alcoholism, is defined as the consumption of larger quantities of alcohol to reach

the same level of response and can develop on cellular and neural levels (Farris et al., 2015).

On account of medical advancements, there has been a rise in the number of established

and commercially-available medications that alleviate the impairing effects of alcohol. One of

these medications is Disulfiram which maintain alcohol abstinence by altering neuronal

processes in the brain to verse alcohol cravings and dependency. However, several drawbacks

are to be expected with its use including costly expenses, and its effects on the long-term

outcome of alcoholism are yet to be proven (Moore and Wright, 1990). Thus, the researchers

set on providing a cost-effective and natural way to mitigate the neurotoxic effects of ethanol.
 In recent times, focus on plant research has increased all over the world and a large

body of evidence has been accumulated in highlighting the immense potential of medicinal

plants. Hydrocotyle verticillata, commonly known as water pennywort, is noted for its folkloric

medicinal properties, being an ingredient among Chinese herbal medications used for muscular

dystrophy. Some studies have also shown its effectiveness for reducing headache, and as an

antipyretic, antitumor, antioxidant, antiproliferative properties along with the presence of

compounds that benefit the liver (University of Connecticut, 2019). Moreover, H. verticillata

could possess similar characteristics to its relative Centella asiatica which is known to be

neuroprotective, and is used to repair neuromuscular disorders and increase general brain

function and memory (Sanchez and Watson, 2016).

Drosophila melanogaster is one of the most widely used model organisms focusing on

genetic and behavioral studies which include advantages in aging research including short

lifespan, low maintenance requirements, and similar responses to humans when it comes to

alcohol sensitivity (Heberlein, 2000).

To contribute to studies about alcoholism - ethanol tolerance in particular - this study

was conducted to evaluate the effect of Hydrocotyle verticillata leaf crude ethanolic extract

(HvLCEE) on the D. melanogaster by means of neurotoxicity in relation to its cognitive and

motor functions particularly on its learning, memory, and behavior. The results of this study

can contribute to the prevention of alcoholism as it serves as a testament to the relevance of D.

melanogaster and H. verticillata in the fields of neuroscience, medicine, and biology.

 METHODOLOGY

Water pennywort (Hydrocotyle verticillata)

Collection of plant sample

Ethanolic Extraction and Phytochemical Analysis

Preparation of treatments Preparation of the test organism

Weighing of HvLCEE Serial dilution of Preparation of culture Sedating flies using Separation of male
HvLCEE media CO2 tank and female flies

Separation of male
and female flies
In-vivo assays In-vitro assay

Toxicity Assay Sedation Assay Negative Geotaxis Aversive Phototactic DPPH Assay
Assay Suppression Assay

Percentage Number of Climbing Pass Pass Rate Percent Scavenging

Survival active flies Rate Activity

Statistical Analysis

Waste Disposal

Figure 1. Flow chart of activities

 MATERIALS AND METHODS

Experimental procedures for the in-vivo assays were performed at the Biological

Models Laboratory, Department of Biochemistry and Molecular Biology, University of the

Philippines-Manila.

I.) Collection and Identification of Plant Sample

Five (5) kilograms of H. verticillata were collected within the vicinity of Juan R. Liwag

Memorial High School, Gapan City in the province of Nueva Ecija and the voucher specimen

of the plant sample was submitted to the Institute of Biology, College of Science, University

of the Philippines – Diliman, Quezon City for the specific plant identification.

II.) Source of Test Organism

Wild type strain of Drosophila melanogaster was acquired and maintained at the

Biological Models Laboratory of the Department of Biochemistry and Molecular Biology

University of the Philippines - Manila.

III.) Extraction and Phytochemical Analysis of Plant Sample

The air-dried and homogenized H. verticillata leaves were brought to the Industrial

Technology Development Institute, Department of Science and Technology (DOST) National

Capital Region, Gen. Santos Avenue, Bicutan, Taguig City for the standardized ethanolic

extraction of the bioactive compounds. The obtained crude ethanolic extract was then subjected

to phytochemical analysis.

IV.) Preparation of the Drosophila melanogaster Growth Medium

Culture media preparation was based on the method of Velasco and Medina (2014).

The mass culture of Drosophila melanogaster used in this study was maintained on sweet

potato-yeast media consisting of 250g of sweet potato, 10g of sucrose, 17.5g of yeast and 7.7g
of agar in 500 mL distilled water. The mixture was allowed to boil and to cool down before the

addition of 6 mL of propionic acid in order to inhibit the growth of microorganisms. The food

media was transferred in food vials and stored in a laboratory chiller with set temperature

ranging from 23-250C.

V.) Preparation of Treatments

Serial dilution was performed in preparing varied concentrations (1, 10, 100, 500, and

1000, 3000, 5000, 10000 µg/mL) of Hydrocotyle verticillata Leaf Crude Ethanolic Extract

(HvLCEE) in which 1% dimethyl sulfoxide (DMSO) was used as diluent, following the

procedure of Gerber (2016). 1% DMSO was also used as the negative control.

Table 1. Experimental and Control Treatments

Treatments Solvent Solute

10000 µg/mL 15 mL of 1% DMSO 150 mg of pure extract

5000 µg/mL 7.5 mL of 1% DMSO 7.5 mL of 10000 µg/mL HvLCEE

3000 µg/mL 6 mL of 1% DMSO 9 mL of 5000 µg/mL HvLCEE

1000 µg/mL 10 mL of 1% DMSO 5 mL of 3000 µg/mL HvLCEE

500 µg/mL 7.5 mL of 1% DMSO 7.5 mL of 1000 µg/mL HvLCEE

100 µg/mL 12 mL of 1% DMSO 3 mL of 500 µg/mL HvLCEE

10 µg/mL 13.5 mL of 1% DMSO 1.5 mL of 100 µg/mL HvLCEE

1 µg/mL 13.5 mL of 1% DMSO 1.5 mL of 10 µg/mL HvLCEE

Negative Control
99 mL Distilled water 1mL Pure DMSO
(1% DMSO)
VI.) Preparation of Ethanol Concentrations

Ethanol vapor causes neurotoxicity and apoptotic degeneration of olfactory receptor

neurons in adult Drosophila melanogaster (French and Heberlein, 2009). Serial dilution was

performed in preparing varied concentrations such 35% and 85% using 95% of ethanol as stock

solution following the formula m1v1=m2v2. Different concentrations of ethanol were used on

in-vivo assays.

Table 2. Formulation of EtOH concentrations for in-vivo assays

Concentration Diluent Stock Solution

35% 63.16 mL of dH2O 36.84 mL of 95% ethanol

85% 10.53 mL of dH2O 89.47 mL of 95% ethanol

VII.) Toxicity Assay

In order to determine the toxic level of HvLCEE, twenty (20) flies of both genders were

exposed in the prepared concentrations (1, 10, 100, 500, 1000, 3000, 5000, 10000 µg/mL) of

the extract within 24-48 hours (acute exposure) to 72-96 hours (chronic exposure). 1000 µg/mL

of extract was dispersed in each of the fly vials and the number of dead and alive flies were

recorded every 24 hours post treatment application.

VIII.) Sedation Activity Assay

Alcohol tolerance resulting from short-term exposure to ethanol was measured and

analyzed in the sedation activity assay (Chan, 2013). D. melanogaster were placed into food

vials in 6 groups of 20 overnight (24 hours), in order to allow the flies to recover in a controlled

space, and preparation of ethanol solution was done a day before the assay. The flies were
transferred into empty testing vials and cellulose acetate plugs were inserted until it reached 2

cm below the top of the vial. Hereafter, each row of six vials was handled as a set at staggered

1 min interval. For the time 0 assessment, each vial was grasped individually with thumb and

forefinger then gently tapped on the table three times to knock flies to the bottom of the vial,

30 seconds were allowed to pass before counting the number of flies that are immobile or dead

and the number of immobile flies for each vial at time 0 min was recorded. Timer was started

continuously at time 0 and 1mL of ethanol was added to cellulose acetate plugs in a circular

motion for the first set of 6 vials at 5 sec intervals then a silicone plug was inserted to seal the

vial. The process was continued to the other sets of vials. At time 6 min, the first set of vials

was tested by grasping each vial, tapped on the table to knock flies, waited 30 sec then the total

number of flies that were sedated was recorded. The other sets of vials were tested as done for

the first set until all flies were sedated. The total number of flies in each vial was entered and

the percent active flies were then calculated at each assessment time-point and plot data.

Ethanol sedation was quantitated by the number of active flies on different time durations.

Sedation in this assay was operationally defined as flies standing in the absence of walking or

lying on their backs with or without flapping their wings as described by Sandhu et al. (2015).

IX.) Alcohol-induced Oxidative Stress and Neurotoxicity

The cellulose acetate plug was flooded with 1 mL of ethanol in concentrations of 35%
and 85%, and was then inserted into the empty vial such that the wetted side of the plug faced
towards the base of the vial. Flies were exposed to ethanol for five minutes daily. After each
ethanol exposure, the flies were returned to their food vial. This procedure was done for a total
of 15 days, allowing the flies to acclimatize for a day before subjecting them to the behavioral
assays (Medina et al., 2018).
X.) Negative Geotaxis Assay

Motor impairment resulting from ethanol-exposure was measured using the negative

geotaxis assay (Berger et al., 2011). Sorted groups of 20 male and female flies exposed to

varying concentrations of HvLCEE were placed into fly vials facing each other vertically joined

by tape. The flies were given at least one hour to recover from the CO2 anesthesia and 1 minute

to acclimatize in the setting. The flies were allowed to settle at the bottom of the vial by gently

tapping the vial. Afterwards, climbing activity was observed for a minute. The number of flies

per group passing the 8 cm minimum height marker for every 10-second interval for one minute

was measured and the pass rate or percentage of the total flies without observed motor deficits

was noted. In contrast, flies with motor deficits remained at the bottom or showed almost no

movements.

XI.) Aversive Phototactic Suppression Assay

To assess the learning ability and memory functions of the adult Drosophila

melanogaster, Aversive Phototactic Suppression Assay was utilized after the flies have

undergone chronic ethanol exposure (Berger et al., 2011). Since fruit flies are instinctively

phototactic (Hirsch and Boudreau, 1958), when given a choice between a light and a dark alley,

flies will choose the lighted alley more frequently (Le Bourg and Badia, 1995). To induce

avoidance behavior in the flies, 0.2% caffeine was used as a negative reinforcer as an

alternative for quinine. Flies were individually placed into a T-maze and allowed to choose

between a lighted and a darkened chamber. The T-maze consisted of a center column with a

trap door and two independent chambers. Filter paper was wetted with caffeine solution

(50 µL) and placed into the lighted chamber such that the caffeine/humidity provides an

aversive association. Afterwards, the training of the flies to suppress phototaxis with aversive

stimuli was done. The flies were individually transferred on to the chamber by screwing the
dark tube back to the maze. The flies were allowed to acclimate in the dark chamber then the

light source illuminating the lighted chamber was slowly turned on. Afterwards, the trap door

that separates the chambers was slowly opened. The flies that walked to the lighted chamber

within 10 seconds were considered as positively phototactic and ready to be trained for the

assay. However, flies that did not walk to the lighted chamber were excluded from the assay.

The flies that stimulated positive phototaxis were tapped back into the dark chamber then the

trapdoor was closed. The flies were allowed to acclimate for 30 seconds. The filter paper with

caffeine solution was then dispersed to the lighted chamber then the trapdoor was slowly

opened and the light was turned on. The individual fly was allowed to walk in to the caffeine-

coated light chamber then after one minute, it was tapped back to the dark chamber and the

procedure was repeated for 9 more times. Five test trials were then conducted to assess the

passing rate - when it failed to go into the lighted vial it was then recorded as learning. The

pass rate over five consecutive trials is recorded as PC0 (0 hr post conditioning). Six hours post

training, each fly was again subjected to 5 trials the same way as before then recorded as PC6

(6 hrs post-conditioning) or the average pass rate tested in both males and females which is an

indicator of short-term memory function (Le Bourg and Buecher, 2002).

XII.) DPPH Radical Scavenging Assay

The plant extract HvLCEE was submitted to Saint Mary’s University for the DPPH

Radical Scavenging Assay to determine the antioxidant property of the plant sample. Adapting

the methods of U. Kolak et al., (2006), the concentrated extract was used to make a stock

solution and aliquot was taken to make 1000 ppm dilution and 1000 ppm of catechin as control

(1mg/mL). One milliliter of prepared stock solution was mixed with four mL of 0.1 mm DPPH

solution in separate plastic cuvette. Reactions were done in triplicate. The prepared mixtures

were incubated in the dark at 37ºC for 30 minutes. The absorbance readings were monitored at

517 nm using a UV VIS spectrophotometer. A lower absorbance of the reaction mixture

indicated higher free radical scavenging activity. The radical scavenging activities were

compared to the activity of the control catechin.

The ability to scavenge the DPPH radical was calculated using the formula:

% Radical Scavenging Effect = [(A control ‐ A sample) /A control] × 100

Where A control was the absorbance of the control which is the DPPH without the test

sample and A sample was the absorbance of the test sample containing the mixture of the DPPH

and the sample. Catechin was used as the positive control.

 RESULTS AND DISCUSSION

I. Plant Authentication and Scientific Classification

Hydrocotyle verticillata was identified and authenticated as a member of Araliaceae

family consisting of around 52 genera and 700 species (R.J. Hodgkiss, 2016). Schefflera

stellata, one of the species of this family, contains high amount of phenolic compounds and is

associated with good antioxidant activity (Hebbar and Nalini, 2014). Another member of this

family, Panax ginseng relieves the symptoms of alcohol hangover (Lee MH, et al., 2014).

Because of its relation to the family Araliaceae, Hydrocotyle verticillata may have a potential

to possess similar properties and candidate for reducing alcohol tolerance.

II. Bioactive Compounds detected on HvLCEE

The phytochemical analysis was conducted by executing a qualitative observation on

the bioactive compounds present in (HvLCEE).

Table 2. Bioactive compound detected in the extract.

Sample Description Results (Hydrocotyle verticillata)

Sterols (++)

Triterpenes (-)

Flavonoids (++)
Hydrocotyle verticillata Leaf Crude
Ethanolic Extract Alkaloids (+++)

Saponins (+++)

Glycosides (+++)

Tannins (++)

Note: (+) Traces, (++) moderate, (+++) abundant, (-) Absence of constituents
 Bioactive compounds present in the plant sample contain health-promoting properties

that are associated with this study. Flavonoids, a compound moderately found in the

Hydrocotyle verticillata, exert neuroprotective mechanisms and provides defense against

oxidative stress and neurotoxicity (Journal of Agriculture and Food Chemistry, 2012).

Moreover, this compound constitutes a large group of polyphenolic compounds with numerous

effects on behavior and cognition. These effects vary from learning and memory enhancement

to an improvement of general cognition. Furthermore, flavonoids have been implicated in a)

neuronal proliferation and survival, by acting on a variety of cellular signaling cascades,

including the ERK/CREB/BDNF and PI3K/Akt pathway, b) oxidative stress reduction. From

an electrophysiological aspect, they promote long term potentiation in the hippocampus,

supporting the hypothesis of synaptic plasticity mediation. Together, these actions reveal a

neuroprotective effect of flavonoid compounds in the brain. Therefore, flavonoid intake could

be a potential clinical direction for prevention and/or attenuation of cognitive decline

deterioration which accompanies various brain disorders (Ioannis Bakoyiannis et al., 2019).

Saponins enhance antioxidant activities, reduce inflammation, and protect dopaminergic

neurons which are the main sources of dopamine or the neurotransmitters in the mammalian

central nervous system (Luo et al., 2010). Lastly, according to Fernandez et al., 2009,

glycosides induce anxiolytic effect, and with higher doses, a sedative affect which decreases

locomotor activity.
III. Toxicity Test of Hydrocotyle verticillata on Wild type Strain of D. Melanogaster

HvLCEE at 1, 10, 100, 500, 1000 µg/mL exhibits no toxic effect in both acute and
chronic exposure in both male and female flies indicating that these concentrations are safe
for the in-vivo assays.

Preliminary examination on the toxicity of H. verticillata was carried out by exposing

twenty (20) wild type D. melanogaster of both gender to the varied concentration of the extract

(1, 10, 100, 500, 1000, 3000, 5000 and 10000 µg/mL) at different time intervals such 24, 48,

72 and 96 hours to determine the safe concentrations to be used for the in-vivo assays.

Acute Toxic effect of HvLCEE on Female

Wild Type D. melanogaster
100

80
% Survival

60

40

20

0
24 48
Time Exposure (Hrs)
1% DMSO 1µg/mL 10µg/mL 100µg/mL 500µg/mL

1000µg/mL 3000µg/mL 5000µg/mL 10000µg/mL

Figure 2. HvLCEE exhibits no lethal effect in acute female fly lethality tests at 1 µg/mL to 1000 µg/mL
as it attains the 90% thresholds of the negative control (1% DMSO) imply that these concentrations
are safe for in-vivo assays.

The calculated percentage survival of the female flies exposed to lower concentrations

of the HvLCEE (1, 10, 100, 500, and 1000 µg/mL) were recorded as 100% in the first 24 to 48

hours post treatment application (hpta) indicating that the extract has no toxic effect on acute

exposure as it exhibits higher percentage survival compared with the 90% thresholds of the

negative control (1% DMSO). However, the higher concentrations (5000 and 10000 µg/mL)

of the extract established lethal effects on the female flies at early period of exposure with both

85% at 24 hpta and the 10000 µg/mL continuously decrease to 80% survival.
 Chronic Toxic effect of HvLCEE on Female
Wild Type D. melanogaster
100

80
% Survival

60

40

20

0
72 96
Time Exposure (Hrs)
1% DMSO 1µg/mL 10µg/mL 100µg/mL 500µg/mL

1000µg/mL 3000µg/mL 5000µg/mL 10000µg/mL

Figure 3. HvLCEE at high concentrations (3000, 5000 and 10000 µg/mL) exhibits toxic effect on female flies
after chronic exposure as it exibit lower than 90% thresholds of percentage survival of negative control.
Thus, these concentrations are not recommended for the preceeding in-vivo assays. However, Lower
concentrations are considered safe for in-vivo assay as it attain the 90% thresholds of the negative contol.

Moreover, HvLCEE was further assessed for the next 72 and 96 hours to consider the

effect of extract after chronic exposure on the flies. Interestingly, the extract at 1, 10, 100, 500,

and 1000 µg/mL concentrations still display no lethal effect. Although 1000 µg/mL recorded

95% survival at 72 hpta and 90% in 96 hpta, it is still considered to be safe as it obtained the

90% threshold of the negative control (1% DMSO). Thus, these concentrations are considered

as safe dosage for the female flies and recommended for the proceeding in-vivo assays.
 Acute Toxic effect of HvLCEE on Male Wild Type D.
melanogaster
100

80
% Survival

60

40

20

0
24 48
Time Exposure (Hrs)
1% DMSO 1µg/mL 10µg/mL 100µg/mL 500µg/mL

1000µg/mL 3000µg/mL 5000µg/mL 10000µg/mL

Figure 4. HvLCEE exhibits no lethal effect in acute male fly lethality tests at 1, 10, 100, 500, and 1 000
µg/mL as it obtains the 90% threshold of survival rate of the negative control (1% DMSO).

Chronic Toxic effect of HvLCEE on Male Wild Type

D. melanogaster
100

80
% Survival

60

40

20

0
72 96
Time Exposure (Hrs)
1% DMSO 1µg/mL 10µg/mL 100µg/mL 500µg/mL

1000µg/mL 3000µg/mL 5000µg/mL 10000µg/mL

Figure 5. HvLCEE at 1, 10, 100, 500, and 1 000 µg/mL was confirmed to be non-toxic to the male flies
after chronic exposure as the concentrations obtain the 90% threshold of the negative control.

The results of male fly toxicity revealed that 1, 10, 100, 500, and 1000 µg/mL

concentrations of the extract are non-toxic in both acute and chronic exposure. On the other

hand, HvLCEE at 3000, 5000 and 1000 µg/mL did not attain the 90% thresholds of the negative

control implying toxic effect on the male flies.

IV. Sedation Activity Assay

High concentrations of HvLCEE increases ethanol sensitivity of D. melanogaster

that were exposed to 35% EtOH and 85% EtOH.

The effect of HvLCEE on the ethanol sensitivity of the flies to ethanol was evaluated

by measuring the sedation activity after subjecting 20 flies of both genders to 35% and 85%

EtOH in 6-minute intervals for a period of 1 hour.

Sedation Activity of Male Flies Exposed in 35% EtOH

100
1%DMSO
80
% Active Flies

1µg/mL
10 µg/mL
60
100 µg/mL

40 500 µg/mL
1000µg/mL
20

0
0 6 12 18 24 30 36 42 48 54 60
Time intervals (Mins)

Figure 6. HvLCEE at 1000 µg/mL displayed the shortest sedation time thus increasing ethanol sensitivity. The highest
concentration of the extract performed the lowest percentage of active flies in all time point observation implying
that the extract reduce tolerance in ethanol compared with all treatments.
Different concentrations of HvLCEE exhibit varied effects on the ethanol sensitivity of

male flies exposed in 35% EtOH. It can be seen in the graph that 1000 µg/mL recorded the

shortest sedation time compared with the negative control and lower concentrations of extract.

Flies were rapidly sedated at 18 minutes wherein active flies reduced from 73.75% and

continuously decrease to 43% and 20% after 24 to 30 minutes and totally sedated at 48 minutes

time period of observation. On the other hand, 1% DMSO recorded higher percentage active

flies in all time period of observation in which there are 97.5 %, 63.75% and 41.25% at 18 to

30 minutes observation. Moreover, longer sedation time was observed in negative control

compare with the highest concentration. Thus, suggesting that HvLCEE at high concentration

increased ethanol sensitivity causing shorter sedation time.

 Sedation Activity of Female Flies Exposed in 35% EtOH
100

1%DMSO
% Active Flies

80
1µg/mL

60 10 µg/mL
100 µg/mL
40 500 µg/mL
1000µg/mL
20

0
0 6 12 18 24 30 36 42 48 54 60
Time Intervals (Mins)

Figure 7. Highest concentration (1000 µg/mL) exhibits increased ethanol sensitivity by its short sedation
time. It is also recorded a rapid decrease in percent active flies exposed to 35% EtOH in 18 minutes.

Similar observation was shown in the female active flies wherein the highest

concentration (1000 µg/mL) exhibited the shortest sedation time compared with all treatment

groups. Treatments such as 1µg/mL, 10µg/mL, 100µg/mL, and 500µg/mL and 1% DMSO

were observed with more active flies at 18 minutes to 30 minutes compared with 1000 µg/mL.

This result implies that the highest concentration of HvLCEE has a potential to enhance the

activity of GABAB receptors that was considered as one of the major regulators of neuronal

excitability and have been implicated in ethanol-induced behaviors in flies that promotes

sensitivity to ethanol sedation but reduces rapid ethanol tolerance (Dzitoyeva et al. 2003).
 Sedation Activity of Male Flies Exposed in 85% EtOH
100
1% DMSO
80 1 µg/mL
% Active Flies

10 µg/mL
60
100 µg/mL
40 500 µg/mL

1000 µg/mL
20

0
0 6 12 18 24 30 36 42 48 54 60
Time Intervals (Mins)
Figure 8. All treatments (1 µg/mL, 10 µg/mL, 100 µg/mL, 500µg/mL, and 1000 µg/mL) including negative
control (1% DMSO) displayed rapid decrease in the percentage active flies at 12 minutes period of observation
and total sedation for the next 12 minutes observation.

The active flies recorded for the male flies on the first period of observation is

noticeably lower at the highest concentration of the extract (1000 µg/mL) with 86.65%

compared with the negative control, 1 µg/mL, 10 µg/mL, 100 µg/mL, and 500 µg/mL with

98.35%, 96.65%, 90%, 91.67% and 90% respectively. Moreover, flies exposed in the high

concentrations (500µg/mL and 1000 µg/mL) recorded the earliest time of total sedation at 18

minutes compared with the other treatments.

This result was similar with the studies of Singh and Heberlein 2000 and Wolf et al.,

2002 which states that Drosophila melanogaster responds to acute ethanol exposure in which

low ethanol concentrations increase their locomotor activity and they lose postural control

leading to sedation when expose at higher concentrations. The implication in regulating

sensitivity to ethanol-induced impairment in flies are associated with molecular signaling

pathways of PI3K/Akt that enhances ethanol sedation (Eddison et al., 2011).

 Sedation Activity of Female Flies Exposed in 85% EtOH
100

80 1% DMSO
% Active Flies

1 µg/mL
60 10 µg/mL
100 µg/mL
40 500 µg/mL
1000 µg/mL
20

0
0 6 12 18 24 30 36 42 48 54 60
Time Intervals (Mins)

Figure 9. Flies exposed in HvLCEE at 1000 µg/mL exhibit the earliest sedation time and lowest active flies
indicating that the extract reduce alcohol tolerance even in high concentration.

Similar observation were revealed in the female flies exposed in 85% ethanol in which

the highest concentration of the extract exhibit the lowest percent active flies in the first period

of observation with 68.35% compared with 1% DMSO and 1 µg/mL, 10 µg/mL, 100 µg/mL,

and 500 µg/mL with 83.35%, 90%, 90%, 88.35% and 76.65% respectively.

Moreover, 1000 µg/mL maintain the lowest active flies in the next 12 minutes

observation and total sedation after 18 minutes. These result could be due to increased GABA

production in the brain results to a state of sedation and decreased anxiety, (Valenzuela, 1997)

and there is such activation in GABA receptors when flavonoids are present (Hanrahan et al.,

2011) since it provides more inhibiting effect towards the flies at higher concentrations hence

the shorter sedation time.

 V. Negative Geotaxis Assay

High concentrations of the HvLCEE established lower climbing pass rates in both

male and female flies in 35 and 85 percent ethanol resulting to a loss postural control and

motor deficits implying decrease in alcohol tolerance and increase in ethanol sensitivity.

Flies that have been exposed to chronic alcohol exposure were then subjected to the

negative geotaxis assay. Motor deficits resulting from ethanol exposure toxicity were measured

by counting the no. of flies that did not pass the 8 cm line within 10 seconds.

Climbing Pass Rate of Male Flies Climbing Pass Rate of Female Flies
100 (35% EtOH ) 100 (35% EtOH)
78a
Pass Rate (%)

80
Pass Rate (%)

80 64.5bc 66.5ab 66.5ab 59a

51.5d 53.5ab 51.5ab
60 52cd 60 45.5bc 47abc
36.5c
40 40

20 20

0 0

Treatments Treatments
Figure 10. Highest concentration of the extract decreases the tolerance of male flies as it displays a significant lower
climbing pass rate compared with negative control and confirmed by ONE-Way ANOVA (p<0.05). Bars with the same
letter are insignificant.

Varied concentrations of the extract exhibit different effect on the motor function of

male flies exposed in 35% EtOH. Results revealed that 10 µg/mL, 100 µg/mL and 500 µg/mL

recorded the highest pass rate with 66.5 %, 78% and 66.5 % which is significantly different

with 1000 µg/mL establishing 52% pass rate implying that HvLCEE at high concentration

increased the fly ethanol sensitivity and reduce ethanol tolerance resulting to higher motor

deficits.

On the other hand, the effect of HvLCEE on female flies was also assessed after chronic

exposure to alcohol at 35%. Likewise, with the result of the climbing activity of the male flies

exposed in the same concentration of ethanol, the negative control showed the highest pass rate
 with 59% followed by the lowest concentration of the extract (1 µg/mL) with 53.5% which is

statistically insignificant with each other. On the other hand 500µg/mL HvLCEE recorded the

lowest climbing pass rate with 36.5 % that is significantly lower with the negative control (1%

DMSO) indicating that the high concentration of the HvLCEE increase the sensitivity of the

flies in ethanol and reduce alcohol tolerance. Moreover, it can be describe that female flies

were observed to performed lower geotactic activity after long term exposure to alcohol and

tend to have a high motor deficits and loss postural control compared with male flies as they

tend to have slower metabolism of ethanol leading to an increased concentration in their body,

which then result to a greater sedation sensitivity (Devineni and Heberlein, 2013).

Further investigation on the effect of HvLCEE on alcohol tolerance were performed by

geotaxis assay in which flies were exposed in higher concentration of ethanol such 85%.

Climbing Pass Rate of Male Flies Climbing Pass Rate of Female Flies
(85% EtOH) (85% EtOH)
100 100
Pass Rate (%)

72.5a
Pass Rate (%)

80 71.5ab 80
61.5bc
56c 51.5a 51a
60 60
40b
36.5d 33d 30.5bc
40 40 25.5c 28.5c

20 20
0 0

Treatments Treatments
Figure 11. High concentrations of HvLCEE (500 µg/mL and 1000 µg/mL recorded the lowest climbing pass rate on both male and
female flies after chronic exposure at 85% ethanol indicating that the extract at high concentrations increase ethanol sensitivity.
HvLCEE (1000 µg/mL) was confirmed to be significantly lower compared with negative control in both sexes as revealed by
ANOVA Single Factor (p<0.05). Bars with the same letter are insignificant.

Interestingly, male flies treated with high concentrations of HvLCEE such as 500

µg/mL and 1000 µg/mL established the lowest climbing pass rate with 36.5% and 33.0 % and

confirm to be significantly lower with the negative control and among concentrations of the

extract such 1 µg/mL, 10 µg/mL and 100 µg/mL with 56 %, 61.5% and 71.5% respectively.
 VI. Aversive Phototactic Suppression Assay

HvLCEE prevents learning and memory deficits after chronic exposure to EtOH

implying that the extract has a potential to avert neuronal cell death and improved cognitive

functions.

A. Learning Assay

Learning ability of flies were assessed after chronic exposure to ethanol both 35% and

85% concentration by Aversive Phototactic Suppression assay in which sets of flies where

trained in a T-maze and determine the percentage pass rate after 10 trial of training and 5 time-

assessment as Post-Conditioning (PC0) (Berger et al., 2011).

Pass Rate of Male Flies-PC0 Pass Rate of Female Flies-PC0

(35%) (35%)
94a 100
100 86a 80a
Pass Rate (%)

62b
Pass Rate (%)

80
80
52b 60
60
40c 34c
40 28d 32cd 40
22c 26c
18c
20 20

0 0

Treatments Treatments

Figure 12. HvLCEE reduce cognitive impairment and improved learning of flies both male and female after
chronic expose to 35% ethanol as confirmed by ANOVA Single Factor (p<0.05). Bars with the same letter are
insignificant.

On the Post-conditioning 0 (PC0) assessment of flies learning, it can be seen in the

figure that the flies exposed in the high concentration of the extract 500 µg/mL and 1000 µg/mL

recognize the highest percentage of pass rate with 86% and 94% on male and 62% and 80% on

female implying a high learning function compared with the negative control (1% DMSO) that

established 28% in male and 18% in female which is significantly lower among treatments.
 Constantly, male and female flies that were exposed in high concentration of the extract

such 500 µg/mL and 1000 µg/mL also performed a significantly high percentage of learning

functions with 66% and 78% for male and 56% and 70% on female even at high concentration

of ethanol (85%). These results showed significantly higher compared with negative control

(1%DMSO) establishing 20% and 22% in male and female flies.

Pass Rate of Male Flies (85%) - PC0 Pass Rate of Female Flies (85%) -
100 100 PC0

Pass Rate (%)

78a
70a
Pass Rate (%)

80 66ab 80
58bc 56ab
60 50cd 60 46b
42d 42bc
40 40 30cd
20e 22d
20 20

0 0

Treatments Treatments

Figure 13. HvLCEE reduce cognitive impairment and improved learning of flies both male and female after
chronic expose to 85% ethanol. Highest concentration of the extract recorded significantly higher compared
with negative control and lower concentrations as confirmed by ANOVA Single Factor (p<0.05). Bars with the
same letter are insignificant.

Learning deficits in the negative control are observed as it recorded the lowest

percentage of pass rate on both male and female could be due to chronic exposure to the ethanol

that can unregulated the sensitivity of NMDA receptors in the brain which results in a

glutamate-induced cytotoxic response in the brain. The calcium influx through the NMDA

receptors that were taken up in the mitochondria causing the production of reactive oxygen

species that disrupt the function of mitochondria and plasma membrane and lead to impairment

of neurological functions such as abstract problem solving, visuospacial and memory function,

perceptual motor skills and even motor function (Harper and Matsumoto, 2005). On the other

hand, the increase in learning functions of flies in both genders could be due to the bioactive
 compounds present in HvLCEE in which it possesses Flavonoids which constitute a large group

of polyphenolic compounds with numerous effects on behavior and cognition. These effects

vary from learning and memory enhancement to an improvement of general cognition.

Furthermore, flavonoids have been implicated in neuronal proliferation and survival, by acting

on a variety of cellular signaling cascades and oxidative stress reduction. These actions reveal

a neuroprotective effect of flavonoids in the brain that could lead to prevention and attenuation

of cognitive decline deterioration which accompanies various brain disorders (Bakoyiannis et

al., 2019).

B. Short-term Memory Assay

Short-term memory of flies after chronic exposure in alcohol of flies was then

conducted after 6 hours of post-conditioning 0 to determine the effect of extract in maintaining

a short term-memory.

Pass Rate of Male Flies (35%) - Pass Rate of Female Flies (35%) -

PC6 PC6
100 100
76a
74a
Pass Rate (%)

80 68a 80
Pass Rate (%)

58b
60 60 46bc
38c 42c
34b 34c
40 28b 26b 40
22b
20 20
0 0

Treatments Treatments

Figure 14. HvLCEE prevents cognitive impairment and protect neuronal cell death at it exhibit short term
memory retention after chronic expose to 35% ethanol. Highest concentration of the extract recorded as
significantly high pass rate among treatments and negative control as confirmed by ANOVA Single Factor
(p<0.05). Bars with the same letter are insignificant.

After chronic exposure of flies on EtOH, it was expected to exhibit cognitive deficits

in which memories were expected to disremember caused by neurotoxicity. However, the

results revealed that HvLCEE at high concentrations recorded the highest short-term memory
 retention on both male and female flies compared with negative control. This result was

attributed to the potential of the bioactive compounds present on the plant extract as it inhibits

neuronal deaths caused by neurotoxicants and promote synaptic plasticity (Nehlig, 2012).

Pass Rate of Male Flies (85%) - Pass Rate of Female Flies (85%) -

100 PC6 100 PC6

76a

Pass Rate (%)

70ab
Pass Rate (%)

80 66a 80
60ab 62ab
54bc
60 46cd 48bc 60 44cd
34de 36d
40 26e 40
20 20
0 0

Treatments Treatments

Figure 15. HvLCEE prevents cognitive impairment and protect neuronal cell death at it exhibit short term
memory retention after chronic expose to 35% ethanol. Highest concentration of the extract recorded as
significantly high pass rate among treatments and negative control as confirmed by ANOVA Single Factor
(p<0.05). Bars with the same letter are insignificant.

A remarkable result was established on the post conditioning 6 (PC6) at highest

concentration of the extract 500 µg/mL and 1000 µg/mL as it recorded the highest percentage

of pass rate of 60% to 66% in male and 70% to 76% in short-term learning assay which were

highly significant with the negative control with 26% and 36% in male and female. The results

imply that the neurotoxic effect caused by chronic exposure of flies in the ethanol is reduced

by the bioactive compounds that is present on the HvLCEE. Moreover, the plant extract

contains Flavonoids that can induce brain perfusion, stimulate angiogenesis, neurogenesis and

changes in the morphology of neurons that are involved in learning and memory (Nehlig,

2012).
VII. Radical Scavenging Assay

The scavenging activity of HvLCEE against free radicals was evaluated by subjecting

the plant extract to DPPH Radical Scavenging Assay wherein the radical scavenging activities

were compared to the activity of the positive control, Catechin.

Table 4. Percent Radical Scavenging Activity of the HvLCEE and positive control
Sample Description Absorbance Reading Mean % Radical Scavenging
Absorbance Activity (RSA)
1 2 3
Water pennywort 0.097 0.096 0.094 0.096 32.87%
Catechin (control) 0.046 0.043 0.042 0.044 69.23%
* 1000 µg/mL are the concentration of the 2 samples tested.

The results of DPPH Radical Scavenging Assay revealed that HvLCEE exhibited

high percentage of Radical Scavenging Activity (RSA) with 32.87% which is statistically

insignificant with the positive control, Catechin with 69.23%. This implies that the extract has

the ability to scavenge DPPH free radicals or act as hydrogen donors (Prakash, 2001).

Moreover, the extract was noted with higher Radical Scavenging Activity as

compared with the previous works of Derilo et al., 2016 where in the plant extracts such as;

Tabernaemontana pandacaqui with 17.62%, Lantana camara with 27.05%, Piper betle with

12.66%, Ocimum basilicum with 24.32% and Artemisia clemensiana with 20.84% . This

suggests that HvLCEE is a potential antioxidant that can scavenge free radicals compared with

other medicinal plants.

The presence of phytochemicals and secondary metabolites can be correlated with

the radical scavenging activity of the plant. Alkaloids are proven to be one of the major
antioxidants found in natural products with different studies highlighting its antioxidant

properties (Yin et al., 2010). Furthermore, it is stated that flavonoids can prevent free radical-

induced injuries by stabilizing the reactive oxygen species through reacting with the reactive

compound of the radical (Korkina & Afanasev, 1997). A study by Hanasaki et al. in 1994 also

revealed that flavonoids are powerful free radical scavengers. These antioxidant properties can

be reflected in the H. verticillata extract as the phytochemical test showed an abundance of

these compounds.
 CONCLUSION

The toxicity test revealed that 1µg/mL to 1000µg/mL concentration of the Hydrocotyle

verticillata Leaf Crude Ethanolic Extract (HvLCEE) has no toxic effects on the flies in both

acute and chronic toxicity as it attained the 90% thresholds of the negative control.

HvLCEE increased ethanol sensitivity of the flies as tested in the sedation activity

assay. The highest concentration of the extract exhibited the shortest mean sedation time

steadily decreasing at 18 min, and is comparatively lower than all other concentrations,

including 1% DMSO, in both male and female flies under 35% ethanol. Interestingly, male and

female flies under 85% ethanol have no observable effect due to the high concentration of

ethanol sedating flies at all concentrations of HvLCEE very early on. This could be attributed

to the increased GABA production in the brain due to the presence of flavonoids known to

inhibit neurotransmission and ultimately lead to sedation (Hanrahan et al., 2011). Moreover,

HvLCEE at high concentrations inhibit motor functions of the flies. Flies at high concentrations

of the extract chronically exposed to both 35% and 85% EtOH exhibited lower climbing pass

rate than the negative control and the rest of the lower concentrations. Both suggest that high

concentrations of the extract increase ethanol sensitivity of the flies resulting to loss of postural

control and motor impairment, implying a decrease in ethanol tolerance. On the assessment of

flies’ learning ability flies exposed in high concentrations of the extract 500µg/mL and

1000µg/mL recognize the highest percentage of pass rate with 86% and 94% on male and 62%

and 80% on female indicating a high learning function compared with the negative control that

established 28% in male and 18% in female which is significantly lower among treatments.

Lastly, HvLCEE established 32.87% scavenging activity which is insignificant with

the positive control with 69.23% of Cathechin. This result suggest that HvLCEE is a potential

antioxidant that could be associated with neuroprotective property and reduces oxidative stress.
 RECOMMENDATIONS

1. Isolation of bioactive compounds present in the Hydrocotyle verticillata Leaf Crude

Ethanolic Extract (HvLCEE) to determine which compounds are responsible in reducing

ethanol tolerance and neurotoxicity on cognitive and motor functioning.

2. Identification of the neurological pathways and genes that are targeted by ethanol.

3. Provide a visualization and more in-depth insight on the effects of HvLCEE in the prevention

of ethanol-induced neurodegeneration and neurotoxicity.

4. Conduct an assay to test the long-term effects of HvLCEE on the tolerance of the flies, and

in prevention of the formation of withdrawal.

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 APPENDICES

Figure 16. Collection of plant sample.

Figure 17. Weighing of Hydrocotyle verticillata Leaf Crude Ethanolic

Extract (HvLCEE).
 Figure 18. Dissolving HvLCEE

Figure 19. Preparing varied concentrations of HvLCEE.by serial dilution

Figure 20. Varying concentrations of HvLCEE.

Figure 21. Preparing 1% DMSO.

Figure 22. 1% DMSO is used as the negative control.

Figure 23. Prepared materials for Culture Media.

Figure 24. Weighing of sweet potato.

Figure 25. Weighing of sugar.

Figure 26. Weighing of yeast.

Figure 27. Weighing of agar.

 Figure 28. Boiling of 500mL dH2O.

Figure 29. Putting 250mg of Sweet Potato.

 Figure 30. Mashing of sweet potato.

Figure 31. Adding sugar, yeast and agar.

Figure 32. Pouring 6mL of propionic acid for the culture media.

Figure 33. Pouring the culture media into the bottles.

 Figure 34. Prepared culture media for propagation of flies.

Figure 35. Pipetting of varying concentrations into the prepared culture media.
 Figure 36. Propagation of flies.

Figure 37. Transferring Drosophila melanogaster from the food vial to an

empty bottle for separation.
Figure 38. Opening the CO2 tank to anesthetize the D. melanogaster.

Figure 39. Anesthetizing D. melanogaster using CO2.

 Figure 40. Separation of D. melanogaster by sex.

Figure 41. Varying concentrations of HvLCEE that were used for the
Toxicity Assay.
 Figure 42. Checking survival rate.

Figure 43. Data collection of Toxicity Assay

Figure 44. Materials for the preparation of diluting EtOH

Figure 45. Dilution of 95% EtOH in dH2O

Figure 46. Exposing the D. melanogaster on ethanol for sedation activity
assay.

Figure 47. Tapping the vials three times every 6 mins.

 Figure 48. Data collection of Sedation Activity Assay

Figure 49. Pipetting ethanol into the cotton plugs for exposure.
Figure 50. Chronically exposing the D. melanogaster on ethanol for 5
mins.

Figure 51 Marking the 8cm minimun height marker.

 Figure 52. Preparation of setup for Negative Geotaxis Assay.

Figure 53. Subjecting the D. melanogaster to Negative Geotaxis Assay to

test its motor function.
Figure 54. Tapping the vials every 10-second interval for one minute.

Figure 55. Data collection of Negative Geotaxis Assay.

Figure 56. Materials for the preparation of Aversive Phototaxis Suppression Assay.

Figure 57. Dissolving caffeine.

 Figure 58. Wetting the filter paper with caffeine solution.

Figure 59. Transferring flies for Aversive Phototaxis Suppression Assay.

Figure 60. Acclimatization of flies for Aversive Phototaxis Suppression Assay.

Figure 61. Subjecting the D. melanogaster to Aversive Phototactic

Suppression Assay to test its memory function.
Figure 62. Certificate of Ethanolic Extraction of the Plant Sample (Hydrocotyle verticillata).
Figure 63. Certificate of Authentication of the Plant Sample (Hydrocotyle verticillata)
Figure 64. Certificate of the Results of Phytochemical Analysis.
Figure 65. Mean Percentage Data and Statistical Analysis of Negative Geotaxis Assay
of Male flies (35%)
Figure 66. Mean Percentage Data and Statistical Analysis of Negative Geotaxis Assay
of Male flies (85%)
Figure 67. Mean Percentage Data and Statistical Analysis of Negative Geotaxis Assay
of Female flies (35%)
Figure 68. Mean Percentage Data and Statistical Analysis of Negative Geotaxis Assay
of Female flies (85%)
Figure 69. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Female flies (35% PC0)
Figure 70. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Male flies (35% PC0)
Figure 71. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Female flies (35% PC6)
Figure 72. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Male flies (35% PC6)
Figure 73. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Female flies (85% PC0)
Figure 74. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Male flies (85% PC0)
Figure 75. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Female flies (85% PC6)
Figure 76. Mean Percentage Data and Statistical Analysis of Aversive phototaxis
suppression Assay of Male flies (85% PC6)
Figure 77. Statistical Analysis for the DPPH Scavenging Assay