Emilio Aguinaldo College College of Medicine Department of Pharmacology Analgesic Activity of Peperomia pellucida (Ulasimang Bato) Aqueous Extract in Male Mice A Research Paper Presented to: Dr. Maria Stella T. Giron Dr. Nelia P. Cortes-Maramba Dr. Dan Villamangca Submitted by: Group III Asuncion, Camille Carissa Claridad, Maru Hasan, Jomar
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Emilio Aguinaldo CollegeCollege of Medicine
Department of Pharmacology
Analgesic Activity of Peperomia pellucida (Ulasimang Bato) Aqueous Extract in Male Mice
A Research PaperPresented to:
Dr. Maria Stella T. GironDr. Nelia P. Cortes-Maramba
Plant parts were thoroughly washed and cleaned to remove dirt and contaminants
Water was completely drained after washing
The plant’s young leaves and stalks were put in an icebox when transported from
the collection site and was processed immediately.
Plant parts were osterized
150 grams of the chopped plant parts were added with 150 ml distilled water and
boiled for 20 minutes in a glass vessel
The extract was cooled to room temperature and filtered
After filtration, the yield of the filtrate or the extract is 100%, this is equal to 1 g/ml
preparation
Aqueous extract was characterized based on pH = 6.0 and organoleptic qualities—
color: dark green, odor: grass-like, clarity: cloudy, consistency: watery.
*note that the extract have been prepared the same day as it was administered
B. Study Sample, Size and Randomization
Adult healthy male (25-30 grams) and female (20-25 grams) albino mice were used
after acclimatization for at least 1 day. The animals were housed in a cage under standard
laboratory conditions of a 12-h light/dark cycle and fixed temperature (25 ± 2 OC). Mice
were provided with water and pellets.
The animals were stratified according to gender (male and female) and were
randomly selected using the table of random numbers and were assigned (refer to table 1)
in one of the 5 log dose group and a negative control group (4 mice/group).
C. Procedure
1. Mice were fasted for 8-12 hours prior to experiment. Food and water were withheld
for the first 8 hours observation period of the first day. Thereafter, food and water
were given ad libitum.
2. Animals were weighed and separated by gender.
3. Animals were assigned to log dose group levels through randomization.
4. Dosage was calculated and prepared using the log dose interval.
5. Test drug and negative control were administered by gavage.
6. Using the multidimensional observation sheet, the following parameters were
observed as to onset of effect and reversibility:
Decreased Motor ActivityAtaxiaLoss of Righting ReflexAnalgesiaAnaesthesiaRespiratory Rate and DepthCorneal and Pinnal ReflexParalysis of Forelegs, Hindlegs and headLoss of Screen GripStartle ReactionIncrease in Motor Activity
Fine Body TremorsCoarse Body TremorsFasciculationsConvulsionsRespiratory RateEnolpthalmosExopthalmosPalpebral PtosisPupil SizeNystagmusLacrimationBloody Tear
Procedure mentioned above was followed and the preparation adjusted based on the
NOAEL, which is 750 mg/kg
Aqueous extract was characterized based on pH = 6.0 and organoleptic qualities—
color: dark green, odor: grass-like, clarity: cloudy, consistency: watery
To assure that the aqueous extract prepared for experiment proper is the same with
that used in exploratory toxicity test, a biologic test confirming the fatal dose, which
is 29.56 g/kg was performed using a pair (male and female) of albino mice (refer to
table 2). Fatal dose of the extracts prepared for exploratory toxicity test and
experiment proper was comparable.
*note that the extract should be prepared the same day as it would be administered
B. Study Sample, Size and Randomization
Adult healthy male albino mice weighing 13-30 grams were used after
acclimatization within 2 days. The animals were housed in a cage under standard
laboratory conditions of a 12-h light/dark cycle and fixed temperature (25 ± 2 OC). Mice
were provided with water and pellets.
The animals were randomly selected using the table of random numbers and were
assigned (refer to table 4) in one of the 6 groups (8 mice/group)
C. Research InstrumentIn order to test for analgesic activity of P. pellucida, the pain was induced thermally
to the subject using hot plate test or tail-flick test. This method used male adult mice in
groups of 8 per dose in comparison with the negative and positive control. The hot plate
with asbestos plate over was maintained at temperature of 52-60 degree celsius with a
constraining cylinder to prevent animal from jumping off, the response to or perception
of the subjects was modified to pain.
D. Procedure
1. Mice were acclimatized and fasted for 8 hours (except water) prior to test.
2. Each mouse was weighed and labeled.
3. Mice were randomly divided into six main groups as mentioned above.
4. Fasted mice were brought to the testing room. They were acclimatized in the testing
room for at least 10 minutes before the test began.
5. Hot plate with asbestos plate cover and beaker was prepared and maintained at a
constant temperature of 52 to 60oC inside the beaker.
6. Assigned dose for each male mouse was administered by gavage.
Table 2. Dose calculated and administered per group of Peperomia pellucida aqueous extract, biomarker (with corresponding result), positive and negative control groups. Drug dose of positive control groups based on equivalent surface area (dosage conversion factor) of man to mice=12.
Group Drug administered (ml) log dose interval=0.2 PreparationDrug Dose Log dose
Biomarker 1 (male) – death at 1st day post-administrationBiomarker 2 (female) – death at 2nd day post-administrationNecropsy findings: both exhibited hemorrhage of peritoneum
7. One hour after drug administration by gavage, each mouse was gently dropped into
the glass beaker on top of the hot plate and the time it takes for the mouse to do any
of the following: hindpaw lick, hindpaw flick or jumping off the beaker was recorded;
other types of behavior were ignored.
8. Mouse was removed immediately from the hot plate after a response is obtained. No
mouse has exceeded 30 seconds, which is the limit of exposure. Animals were tested
one a time and returned to the individual cage/plastic container.
9. Testing was repeated two hours after drug administration. It was ensured that the
reaction time is accurately observed and recorded with a timer in seconds.
10. Reaction time of each mouse was tabulated based on the negative control (NSS),
positive controls (Aspirin and Tramadol), low, middle and high dose for each drug
and appropriate statistical analysis was used to compare the treatment groups.
E. Data Processing Procedure
Data Analysis
Reaction time result was subjected for statistical analysis at 0.05 alpha level of
significance utilizing SPSS 13.0, a computer program for descriptive and inferential
statistical analysis. Data for multi-group comparisons was analyzed using One-way
ANOVA. Comparison between 1st hour and 2nd hour reaction time within groups was
analyzed using Paired T-test.
Collection of Peperomia pellucida
Plant AuthenticationAuthenticate?
Heavy Metal AnalysisAcceptable Level of Heavy
Metals?
Affect experiment outcome (Study limitation)
Acute Toxicity Test:
Exploratory testValidate established LD50Observe ToxidromeNOAELLD50 comparable with established value?
Use explored LD50, observed toxidrome and NOAEL
Experiment ProperAnalgesic Activity of Aqueous
Extract of Peperomia pellucida, NOAEL as the middle dose
Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia pellucida aqueous extract at 0.75 g/kg (Dose I)
Parameter Animals Responded
Duration(Time of Onset to
Reversibility)
CNS Depression
Motor ActivityA. ±B. +
3/4 (F4, M3, M9)¼ (F10)
1st 15 mins until 6th hr 1st 15 mins until 6th hr
CNS Stimulation
Startle Reflex (+) 4/4 1st 15 mins until Day 6
General Observation
Abdominal gripping (+) ¾ (M3, M9, F4) 1st 15 mins until 3rd hr
Subjective Observation
CatalepsyA. +B. ±
¼ (M9)¾ (M3, F4, F10)
1st 15 mins until Day21st 15 mins until Day 6
Main Body System
Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia pellucida aqueous extract at 1.88 g/kg (Dose II)
Parameter Animals Responded
Duration(Time of Onset to
Reversibility)
CN
S D
epre
ssio
n
Motor ActivityA. ±
B. +
2/4 (F3, F7)
¼ (M2)
1st 15 mins until 3rd hr for F3 and until 4th hr for F7
2nd hour until Day 2
Respiratory Rate (±) 4/4 1st 15 mins until 2nd hrAnalgesia (±) 2/4 (M10, M2) 1st 15 mins until 4th hrLoss of Pinnal Reflex (+) 4/4 1st 30 mins until 2nd hr for
F3, F7 and 3rd hr for M2, M10
CNS Stimulation
Startle Reflex (+) 4/4 1st 15 mins until Death
General Observation
Circling Motion 2/4 (M10, M2) 1st 15 mins and lasted for 30 mins
Subjective Observation
Head TapA. AggressiveB. Passive
4/44/4`
1st 15 min until 2nd hr3rd hour until Day 6
(F3,F7) and until Death for M10, M2
Body TouchA. AggressiveB. Passive
4/44/4`
1st 15 min until 2nd hr3rd hour until Day 6
(F3,F7) and until Death for M10, M2
Excess CuriosityA. ++B. +
4/44/4
1st 15 mins until 1st hour 1st 30 mins until Day 6
for F3, F7 and until Death for M2, M10
Main Body System
Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia pellucida aqueous extract at 4.69 g/kg (Dose III)
Parameter Animals Responded
Duration(Time of Onset to
Reversibility)
CN
S
Dep
ress
ion Motor Activity (+) ¾ (F6, M4, M8) 1st 15 mins until Death
Respiratory Depth (±) 2/4 (F6, M4) 1st 15 mins until DeathAnalgesia (±) ¾ (M4, M8, M6) 1st 15 mins and lasted for
45 minsLoss of Pinnal Reflex (±) ¼ (M8) 1st 15 mins until 2nd hr
CNS Stimulation
Startle Reflex (+) 4/4 1st 15 mins until Death
Eyes Enophthalmos (+) ¼ (M8) 1st 30 mins until Death
Ears and Mouth
Blanching (±) 2/4 (F9, M8) 1st 15 mins until Day 2 for F9 and until 4th hr for
M8
General Observation
Robichaud Test (+) 2/4 (M4, M8) 1st 15 mins until Death for M4
1st 30 mins until Death for M8
Sub
ject
ive
Obs
erva
tion
Head Tap A. Passive
B. Fearful
4/4
2/4 (F6, F9)
F6: 1st 15 mins until 6th
hrF9: 1st 15 mins until 5th
hrM4: 1st 15 mins until
deathM8: 1hr after drug administration until
DeathF6: 6th hr until DeathF9: 5th hr until Day 6
Body Touch
A. Passive
B. Fearful
4/4
2/4 (F6, F9 )
1st 15 mins untilF6: 6th hrF9: 3rd hr
M4: DeathM8: Death
F6: 2nd day until DeathF9: 3rd hour until Death
Catalepsy (±) 2/4 (M4, M8) M4: 1st 15 min until 3rd hrM8: 1st 15 mins until
DeathExcess Curiosity (+) 2/4 (F6, M4) F6: Last hr of Day 1 until
DeathM4: 5th hr until 6th hr
Main Body System
Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia pellucida aqueous extract at 11.77 g/kg (Dose IV)
Parameter Animals Responded
Duration(Time of Onset to
Reversibility)CNS
DepressionMotor Activity (+2) ¼ (M6) 1st 15 mins until DeathAnalgesia (+) 2/4 (M6, F5) 1st 15 mins until 2nd hr
CNS Stimulation
Motor Activity (+) 2/4 (F8, M1) 1st 15 mins and lasted for 90mins
Fine Body Tremor (+) ¾ (F8, F5, M6) 1st 15 mins until Day2Coarse Body Tremor (+2) 2/4 (F5, F8) 1st 15 mins until 3rd hrFasciculation(+3) ¼ (F5) 1st 15 mins lasted for 90
minsConvulsion (+) ¼ (F8) 1st 15 mins until 4th hrStartle Reflex (+) 4/4 1st 15 mins until Death
EyesEnophthalmos (+) ¼ (M6) 1st 15 mins until DeathPalpebral Ptosis (+4) ¼ (M6) 1st 30 mins until Death
General Observation
Abdominal gripping (+2) 2/4 (F5, F8) 1st 15 min until Day 2
Subjective Observation
Head Tap A. AggressiveB. Passive
¼ (M1)¼ (M6)
1st 15 min until Death1st 15 mins until Death
Body TouchA. AggressiveB. Passive
¼ (M1)¼ (M6)
1st 15 min until Death1st 15 mins until Death
Excess Curiosity ¼ (M1) 1st 15 min and lasted for 90 mins
Main Body Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia
System
pellucida aqueous extract at 29.56 g/kg (Dose V)Parameter Animals
RespondedDuration
(Time of Onset to Reversibility)
CNS Depression
Motor ActivityA. ++B. ±
¼ (F2)¾ (M5, M7, FI)
1st 15 mins until Death1st 15 mins until Death
AtaxiaA. ±B. ++
¼ (F2)¾ (M5, M7 and F1)
1st 15 mins until Death1st 15 mins until Death
Loss of Righting ReflexA. ±B. ++
¾ (M5, M7 and F1)¼ (F2)
1st 15 mins until Death1st 15 mins until Death
AnalgesiaA. ±B. ++
¾ (M5, M7 and F1)¼ (F2)
1st 15 mins until Death1st 15 mins until Death
Respiratory Rate Mean: 50 breaths/33.97 sec (Increase in RR)Loss of Corneal Reflex (+) ¼ (F2) 1st 30 mins until DeathLoss of Pinnal Reflex (+) ¼ (F2) 1st 30 mins until DeathParalysis: Forelegs (+) ¼ (F2) 1st 30 mins until DeathParalysis: Hindlegs
A. +B. ±
2/4 (F2, M5)¼ (M7)
1st 30 mins until Death1st 30 mins until Death
Paralysis: Head (+) ¼ (F2) 1st hour until DeathScreen Grip Foreleg Loss (+) ¼ (F2) 1st 15 mins until DeathScreen Grip Hindleg Loss
A. +B. ++++
¼ (M5)¼ (F2)
1st hour until Death1st hour until Death
CNS Stimulation
Respiratory Rate Mean: 50 breaths/33.97 sec (Increase in RR)Startle Reflex (±) 4/4 1st 15 mins until Death
EyesEnophthalmos (+) ¼ (F2) 1st 15 mins until DeathPalpebral Ptosis (++) ¼ (F2) 1st 30 mins until DeathLacrimation (+) ¼ (F2) 1st 15 mins until Death
Ears and Mouth
Blanching (+) 4/4 1st 30 mins until DeathHyperemia (+) 2/4 (M7, F2) 45 mins until Death for
M7 while until 5th hour for F2
General Observation
Salivation (+) ¼ (F2) 1st 15 min until DeathRobichaud Test
A. +B. ++
2/4 (F1, M7)¼ (F2)
1st hour until Death1st 15 min until Death
Abdominal Gripping (+2) 2/4 (F1, M5) F1 1st 15 mins until 3rd
hourM5 1st 15 min until 6th
hourSubjective Head Tap (Passive) 4/4 1st 15 min until Death
Observation Body Touch (Passive) 4/4 1st 15 min until DeathCatalepsy
A. +++B. ++
¼ (F2)2/4 (M5, F1)
1st 30 min until Death1st 30 min until Death
Main Body System
Lethal and Non-lethal Toxidrome seen in mice administered with Normal Saline Solution 1ml (Negative Control)
Parameter Animals Responded
Duration(Time of Onset to
Reversibility)CNS
DepressionMotor Activity (±) 4/4 1st 15 mins until Day 6
CNS Stimulation
Startle Reflex (+) 4/4 1st 15 mins until Day 6
Subjective Observation
Excess Curiosity (+) 4/4 1st 15 mins until Day 6
After inducing decoction of Peperomia pellucida by gavage, the male albino mice
belonging to five different dose groups exhibited central nervous system depression as
was shown in the tables. The mice exhibited decrease in motor activity with all five
doses, with 25% of the 5th dose mice moving very sluggishly even when handled and the
remaining 75% were quiet while exhibiting occasional and spontaneous movements.
Only 25% of the 4th dose mice and 75% of the 3rd dose mice exhibited depressed motor
activity. In the 2nd dose group 25% of the mice only moved when handled, and the
remaining 75% moved spontaneously however remained quiet. Analgesia was
manifested among mice from doses 2 to 5, 15 minutes post administration. All mice in
the 5th dose exhibited analgesia, 75% of the 3rd dose and 50% of both the 4th and 2nd doses.
Dose 5, which is the highest dose, scored positive for most of the CNS depression
parameters, in addition to the two mentioned above, dose 5 mice also showed respiratory
depression, ataxia, paralysis of both hind and forelegs, and loss of screen grip. Three dose
groups manifested loss of pinnal reflex, with doses 2 and 5 exhibiting it during the first
15 minutes post administration and dose 3 exhibiting it 15 minutes later. Among the
CNS stimulation parameters, fine and coarse body tremors were seen among 3 dose
groups, doses 1, 4, and 5, with dose 4 exhibiting the greatest amount of body tremors.
Under the 4th dose group, 25% of mice showed fasciculation during the first 90 minutes
post administration and in another 25%, convulsion was observed during the first 4 hours.
For the eye and ear parameters, enophthalmos and palpebral ptosis was seen in the
4th and 5th dose groups during the first 15 minutes post administration and was
intermittently evident until the end of the observation period. Blanching of the ears was
also observed in the 3rd and 5th dose groups. The 5th group started to show signs of
blanching 30 minutes post administration which lasted until the conclusion of the
observation period. In contrast to this, the 3rd group started to show signs of blanching 15
minutes post administration and it lasted only until the second day. Abdominal gripping
was manifested by 50% of mice under each of the 4th and 5th dose groups which lasted
until the 2nd and 3rd day respectively. It was also manifested by mice under the 1 st dose
group which lasted only for 3 hours.
Based on the researcher’s subjective observation, all mice under the 5th dose
manifested passive reaction to head and body tap until death. Only 25% of the mice
under the 4th dose group were passive, the other 25% showed aggression 15 minutes after
administration. All mice under the 3rd dose group showed passivity immediately after
administration which lasted until the 6th hour and 50% of mice were fearful to head and
body tap afterwards. All mice in the 4 th group showed aggression during the first 3 hours
post administration, and were then passive until the conclusion of the observation period.
Three out of the five dose groups showed an increase in curiosity after administration of
the aqueous extract of Peperomia pellucida with 25% of mice under the 4th dose group
manifesting it during the first 90 minutes. Excess curiosity was also manifested by half of
the mice belonging in the 3rd dose group, with 25% of it manifesting it during the last
hour of the first day until the end of the observation period, and the other 25% manifested
this behavior during the 5th hour which lasted only for 1 hour. All mice under the 2nd dose
group showed an excess in curiosity which started 15 minutes after the administration
and lasted until the 6th day.
The mice under the negative control group only manifested slight motor depression
among the toxidromes mentioned earlier. This was evident immediately after the administration
of NSS and lasted until the conclusion of the observation period.
Fifty percent (12/24) of the total population of mice used in the exploratory toxicity
testing died within 6 days observation period. Fifty percent (6/12) of mice died showed intestinal
showed hepatomegaly with multi-focal necrosis and another eight percent (1/12) died by
accident.
Figure 2. Logarithmic graph of log dose and percent mortality
In the Exploratory Toxicity Testing, the investigators started with 11.77 g/kg, which is
the established LD50 of P. pellucida aqueous extract, as the fourth dose (see table 1). There are
five log dose groups with 0.4 as the log dose interval. Two pairs of mice (2 males, 2 females)
were assigned to each group. The results were plotted to determine the LD50 and the No
Adverse Effect Level. Since the mortality result of the third (0.67 g/kg) and fourth dose (11.77
g/kg) are outliers, both were eliminated in the graph. The LD50 is estimated as the x-intercept of
the point of intersection of the line and the 50% mark of the y-axis (log dose 0.27). This is in log
dose so we get its antilog to get the LD50 (1.88 g/kg). The determined LD50 was not comparable
with the established LD50 of P. pellucida which is 11.77 g/kg ±20% (9.42-14.12). NOAEL is ¼
of the determined LD50 (1.88 g/kg ÷ 4), but based on the toxidrome, the first dose (0.75 g/kg)
with non-life threatening adverse effects was used as the NOAEL. This level was used as the
middle dose for analgesic testing of P. pellucida aqueous extract and from which the low and
high dose was computed with the log dose interval of 0.2 to be in a safe range (refer to table 2).
Figure 3. Comparison of 1 hour and 2 hours mean reaction time to heat of low, middle, high,
negative and positive control groups.
One hour post-administration of P. pellucida aqueous extract middle and high dose
exhibited analgesic activity compared with the negative control (NSS) and high dose also
showed comparable effect with aspirin. At two hours post-administration, all three doses
exhibited analgesic activity compared with the negative control. Comparing the mean reaction
time to heat of one hour and two hours post-administration of the extract, low dose, high dose,
and both positive controls—aspirin and tramadol showed increased analgesic activity while the
middle dose decreased its effect. Decreased reaction time to heat of the negative control at two-
hour post-administration of the NSS exhibited decreased tolerance to heat. Adverse reactions
observed post-administration of P. pellucida aqueous extract include decreased in motor activity,
ptosis and abdominal gripping.
Figure 4. Linear graph presentation of the percent difference of the reaction time to heat at 1st hour of low, middle and high dose P. pellucida against the negative control.
Figure 5. Linear graph presentation of the percent difference of the reaction time to heat at 2nd hour of low, middle and high dose P. pellucida against the negative control.
low dose (473 mg/kg)
middle dose (750 mg/kg)
high dose (1190 mg/kg)
% diff (1st hr)
-0.1311 0.1524 0.2805-15.00%
-5.00%
5.00%
15.00%
25.00%
35.00%
% DIFFERENCE (1st hr)
MEA
N %
CHA
NGE
low dose (473 mg/kg)
middle dose (750 mg/kg)
high dose (1190 mg/kg)
% diff (2nd hr)
0.2787 0.223 0.4426
2.50%
12.50%
22.50%
32.50%
42.50%
% DIFFERENCE (2nd hr)
MEA
N %
CHA
NGE
Percent difference of the mean reaction time to heat of each of the three doses of
P. pellucida aqueous extract and the negative control at one hour post-drug
administration showed a dose-response relationship although at low dose, analgesic
activity was not observed (refer to figure 3). At two hours post-administration of the
extract, dose-response relationship was not observed because the middle dose exhibited
lower analgesic activity compared with the low and high doses. Although the three dose
levels of P. pellucida aqueous extract exhibited analgesic activity compared with the
negative control and the high dose with aspirin at one-hour post-drug administration, the
effective dose fifty (ED50) cannot be determined because not one of the doses of the
extract produced prolonged reaction time to heat twice as that of the negative control.
Table 4. Paired T-test of one hour and two hours post-drug administration reaction time to heat of each dose level of P. pellucida aqueous extract and the control groups.
p-value Pair 1
lowdose1 - lowdose2
.141
Pair 2
middose1 - middose2
.941
Pair 3
highdose1 - highdose2
.669
Pair 4
asprin1 - aspirin2.029
Pair 5
tramadol1 - tramadol2
.027
Pair 6
negctrl1 - negctrl2.368
* The mean difference is significant at the .05 level.
Comparing the reaction time to heat between one-hour and two-hour post-drug
administration in all doses of P. pellucida aqueous extract and the negative control, there is no
significant difference. While both aspirin and tramadol exhibited increase in reaction time to heat
from one-hour to two-hour observation.
Table 5. One-way ANOVA of reaction time to heat between and within groups.
P-valueOne hour Two hours
Between Groups .001 .000
Within Groups* The mean difference is significant at the .05 level.
Analysis of Variance (ANOVA) to compare the reaction time to heat at one-hour and
two-hour observation between groups resulted in a statistically significant difference, that is, all
three doses of P. pellucida aqueous extract has lesser analgesic activity as compared with the
positive control groups.
Table 6. Multiple comparisons at one-hour and two-hours post-drug administration reaction time to heat of each group
Group GroupP-value
One hour Two hoursLow dose middle dose .779 1.000 high dose .418 1.000 Aspirin .438 .270 Tramadol .000 .000 negative control .989 .996Middle dose low dose .779 1.000 high dose .992 .999 Aspirin .991 .257 Tramadol .015 .000 negative control .984 .999High dose low dose .418 1.000 middle dose .992 .999 Aspirin 1.000 .449 Tramadol .061 .000 negative control .811 .972Aspirin low dose .438 .270 middle dose .991 .257 high dose 1.000 .449 Tramadol .087 .021 negative control .813 .128Tramadol low dose .000 .000 middle dose .015 .000 high dose .061 .000 Aspirin .087 .021 negative control .002 .000Negative control
Tramadol .002 .000 * The mean difference is significant at the .05 level.
XII.Conclusion
Based on the results obtained in the experiment, Peperomia pellucida aqueous extract
exhibited analgesic activity in middle dose (750 mg/kg) and high dose (1190 mg/kg) compared
with the negative control. Only the high dose extract has comparable analgesic effect with
aspirin at one-hour post-administration. But based on statistical analysis, P. pellucida aqueous
extract has lesser analgesic activity compared with the positive control groups.
XIII.Recommendation
Due to unavailability of resources the experiment had to impose measures which greatly
sacrificed several criteria which are highly significant in the conduct of the study. The experiment
was ideally designed to utilize 60 male albino mice weighing 20-25 grams each; however the
mice delivered were mostly of inappropriate weight. The researchers had to disregard the
imposed weight requirements which surely had great impact on this experiment. This has lead to
some number of deaths among subjects during the induction period due to aspiration. The
researchers, therefore, suggests that established criteria for the experimental subjects must strictly
be followed so as to avoid delay and cause the least harm possible among the study subjects, in
addition to this, greater validity of the results can also be obtained. Maintaining a heated beaker
with a constant temperature was another difficulty in this experiment. The researchers suggest
that vigilant monitoring of the beaker temperature must be done to achieve more reliable results
and avoid inducement of pain among subjects due to overly heated beakers. Lastly, observer bias
and variation regarding the subject’s reaction time must be carefully monitored to avoid
inconsistencies to assure the validity of the study.
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Taber’s Cyclopedic Medical Dictionary. Copyright 2005. F.A. Davis Company.Xu S , Li N , Ning MM , Zhou CH , Yang QR , Wang MW . Bioactive compounds from
Mean reaction time of male mice one and two hours post administration of P. pellucida aqueous extract, negative and positive controls.
Drug Dose GroupPost-drug Administration Reaction Time in Seconds
1 hour 2 hour
P. pellucida low dose 2.85±0.7 3.9±1.9
P. pellucida middle dose 3.78±1.4 3.73±1.3
P. pellucida high dose 4.2±0.94 4.4±1.12
Positive Control (Aspirin) 4.24±0.7 7.82±2.94
Positive Control (Tramadol) 6.36±2.7 14.03±7.2
Negative control (NSS) 3.28±0.46 3.05±0.38
Appendix 4
Summary of animal mortality and corresponding necropsy findings
Log dose group Dose g/kg
N # of animals
died
% mortality
Necropsy Findings
I (M3, M9, F4, F10)
0.75 4 ¼ 25 F10-Death by accident
II (M2, M10, F3, F7)
1.88 4 2/4 50 M2-death at day 3, no significant findingsM10-death at day 4, no significant
findingsIII (M4, M8, F6,
F9)4.69 4 ¾ 75 M4-death at day 2, enlarged
intestinesM8-death at day 1, enlarged intestinesF6-death at day 4, no significant findings
IV (M1, M6, F5, F8)
11.77 4 2/4 50 M6-death at day 1, enlarged intestines, (+) Intestinal ParasiteF8-death at day 4, enlarged intestines
V (M5, M7, F1, F2)
29.56 4 4/4 100 M5-death at day 5, no significant findingsM7-death at day 4, enlarged intestinesF1-death at day 5, enlarged intestinesF2-death at 5th hour, hepatomegaly with multifocal necrosis
Negative Control (NSS)
1 ml 4 0/4 0
Appendix 5
Proposed Time Frame
DATE (2010) ACTIVITY
04 February Approval of Research Proposal
4,5 February Collection of P. pellucida Sample for authentication and heavy metal analysis.
14-19 February Collection of P. pellucida/Acute Toxicity Testing in Male Mice
21-26 February Purchasing and Preparation of other materials for experiment proper
3 March Collection of P. pellucida/Experiment Proper (Analgesic Test)
1-10 March Collation, Statistical Analysis and Finalization of the Research Paper
11 March Research Presentation
Appendix 6
Budget Plan
Sources of Expenses Number of times(or items)
Rate Total
Samples Male Albino Mice
(25-35 grams)- Negative Control- 2 Positive
Control- Low Dose- Middle Dose- High Dose- Acute Toxicity
Test- Additional
Aspirin (80mg) Tramadol HCl
(100mg) NSS Distilled water
100
10
20
10101030
10
3010
11
80 php per mice
5php per tablet33php per ampoule
106php per bottle60php per galloon
8000
150330
10660
Collection of Samples Transportation Meal
2300php per person150php per person
1800
Printing and Documentation 1000 1000Authentication of the Test Plant