p. pellucida
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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
Dr. Dan Villamangca
Submitted by:
Group IIIAsuncion, Camille Carissa
Claridad, MaruHasan, JomarLee, Ming Ju
Manesca, Ma. RossiniRacadio, Ma. PerpetuaRuedas, Jhoana-LynTambut, Zhenilane
March 2010
I. Introduction
Hippocrates (460-377 B.C.), known as the Father of Medicine used several methods of
treating his patients and among these was the use of medicinal plants. Today, manufacturers of
herbal medicines took advantage of the scientific and folkloric uses of its extracts in order to
formulate preparations to make it available in a form which is safe, efficient, and convenient
for its target users. Still, many plants are claimed by our country’s medicinal folklore to have
therapeutic effects, however some still remain with scanty or without scientific basis.
A good example is the known use of extracts of Peperomia pellucida more commonly
known in the Philippines as Pansit-pansitan in Tagalog, Olasiman Ihalas in Bisaya, Sinaw-
sinaw or Tangon-tangon in Bicol. It thrives in a tropical to subtropical climate. It is a shallow-
rooted herb that is very succulent, erect, and branched, growing from 5 to 40 centimeters in
height. The leaves are small with pointed or blunt tip and heart-shaped base, pale green,
pellucid, and shiny. The stems are erect and very slender. It has a mustard-like odor when
crushed. When matured, the small fruits bear one seed which fall of the ground and propagate.
This plant has a very rich folkloric history of medicinal use. In the Philippines, whole plant
warm poultice is used for abscesses and boils (Quisumbing, 1978); crushed leaves for
headache and convulsions; infusion or decoction-against gout, kidney troubles and rheumatic
pain; leaf juice for colic and abdominal pains and externally as rinse for complexion problems.
Taken as a salad, it is believed that pansit-pansitan helps relieve rheumatic pains and gout
(Galvez-Tan, 2009). Peperomia pellucida as an herbal medicine is also widely used not only in
the Philippines but in different countries as well. It is known as Pepper Elder, Silverbush, Rat-
ear, Man-to-man or Clearweed throughout America; Konsaka Wiwiri in Guiana, Coraçãozinho
or "Little Heart" in Brazil; and Lingua de sapo, Herva-de-vidro, Herva-de-jaboti or Herva-de-
jabuti in South America. In the Amazon region, it has been used as a cough suppressant,
diuretic, emollient, and in the treatment of cardiac arrhythmia. According to Dalziel, it is used
as an ingredient in medicinal infusions for convulsion in West Tropical Africa.
Previous chemical investigation on this plant indicated the existence of flavonoids,
phytosterols, apiols, and substituted styrenes (Bayma, 2000). Carotol (13.41%) was the major
hydroxylated sesquiterpene in a chemical analysis of P. pellucida. Other compounds include
peperomins which have cytotoxic or anticancer activity in vitro. Isolated flavonoids include
acacetin, apigenin, isovitexin, and pellucidatin. Phytosterols such as campesterol and
stigmasterol have also been isolated. Xu et al (2006) isolated secolignans, lignans and highly
methoxylated dihydronaphthalenone from the whole plant.
The plant may have the potential as a broad spectrum antibiotic (Bojo, 1994), as an
antifungal (Ragasa, 1998), as an antimalarial (Muñoz et al, 2000). Other accounts report that
the crude extracts of P. pellucida also shows anti-inflammatory and analgesic properties. In
2002, study conducted by Arrogini-Blank and his colleagues showed that rats orally
administered P. pellucida aqueous extract 200 and 400 mg/kg exhibited anti-inflammatory
activity in the carrageenan test. They concluded that the mechanism of action is associated with
prostaglandin synthesis interference, as confirmed by results of an arachidonic acid-induced rat
paw edema study. This result was further supported by an anti-inflammatory and analgesic
activity of P. pellucida study conducted by Andrade et al in 2004.
Most studies assessed analgesic activity by the abdominal writhing test using acetic acid or
by the hot-plate test. In mice subjected to the acetic acid-induced writhing test, a P. pellucida
extract exhibited analgesic activity at 400 mg/kg, inhibiting pain by 50% compared with
controls. In another study conducted by Aziba and his colleagues (2001) using the same test,
they attained higher inhibition percentages (78%) when a methanolic extract of P. pellucida
210 mg/kg was used. They claimed that the difference in results may be associated with use of
different extracts, climatic conditions, and plant origin. An analgesic effect was observed in the
hot-plate test at lower concentrations of 100 and 200 mg/kg, which may indicate extract
activity against inflammatory and non-inflammatory pain.
Folkloric and experimental evidences cited revealed many potential medicinal value. The
researches were compelled to further investigate the herb’s analgesic activity because pain is
universally understood as a signal of disease and it is the most common symptom that brings a
patient to a physician's attention. Since different diseases produce characteristic patterns of
pain, it can provide important diagnostic clues and can be used to evaluate the response to
treatment. Once this information is obtained, it is the obligation of the physician to provide
rapid and effective pain relief (Fauci, et al. 2007). In light of this, it is imperative to pursue
studies on pain relief therapy and such involves searching and testing substances whether
synthetic or natural for analgesic properties.
Currently, groups of drugs (prescription and over-the-counter) used to relieve pain directly
and indirectly, include Opioid analgesics, Non-steroidal Anti-inflammatory Drugs (NSAIDs)
and steroids. Opioid agonists produce analgesia by binding to specific G-protein coupled
receptors that are located in the brain and spinal cord regions involved in the transmission and
modulation of pain. However, with frequent use, tolerance and physical dependence develops.
These drugs are one of the commonly abused prescription medications in 2009 as reported by
the National Institute of Drug Abuse of the United States National Institute of Health.
Concomitant adverse effects include sedation, respiratory depression, cough suppression,
miosis, truncal rigidity, nausea and vomiting, constipation, urinary retention, biliary tract colic
and prolonged labor in pregnant women.
Steroids are organic compounds containing in its chemical nucleus the
perhydrocyclopentanophenanthrene ring. Therapeutic synthetic and natural corticosteroids
belong to this group. Its usefulness is in its ability to suppress inflammatory and immune
responses and to alter leukocyte function. Thus, when inflammation is suppressed pain is
reduced. Common side effects of prolonged, high-dose steroid hormone therapy include
alterations in the sleep-wake cycle, fluid and sodium retention, muscle weakness, thinning of
the skin, cataract formation, diabetes mellitus, osteoporosis and immune suppression.
NSAIDs act as non-selective inhibitors of the enzyme cyclooxygenase, thus reduces
prostaglandins that promote inflammation, pain, and fever. The most prominent members of
this group of drugs are aspirin, ibuprofen, and naproxen. Acetylsalicylic acid (ASA) was the
first discovered member of the non-steroidal anti-inflammatory drugs (NSAIDs). It is often
used as an analgesic to relieve minor aches and pains. Although relatively safe, the use of ASA
is still prohibited in some conditions such as those individuals suffering from hypersensitivity
reactions. This drug and other salicylates are not to be used for any reason (treatment of any
viral disease) in children under age 15 because of its strong association with Reye’s syndrome.
Nowadays the use of pain medications is rampant and its adverse effects are continuously
overlooked. This condition calls for the discovery and development of more cost effective
medications which can be used in replacement for common pain relievers, which can be proven
to be safer and effective. Thus, this study on an herbal medicine, Peperomia pellucida is
pursued to determine its analgesic activity as this could be a potential herbal medicine if
proven effective.
II. Research Problem
Does the aqueous extract of Peperomia pellucida (Ulasimang bato) exhibit analgesic
activity among male mice using hot-plate method?
III.Objectives
General Objective
To determine the analgesic activity of the aqueous extract of Peperomia pellucida with
Aspirin and Tramadol among male mice using the hot-plate method.
Specific Objectives
1. To compare the analgesic activity of the aqueous extract of Peperomia pellucida in three
dose levels, the negative control (NSS), and the positive controls (Aspirin and Tramadol)
in male mice based on reaction time to heat using the hot-plate method in one hour and
two hours post-drug administration.
2. To compare with the negative control (NSS) and the positive controls (Aspirin and
Tramadol) the dose with the highest analgesic effect of the aqueous extract of Peperomia
pellucida in male mice based on reaction time to heat using the hot-plate method in one
hour and two hours post-administration.
3. To determine the ED50 (Graded Dose Response) of the analgesic activity of the aqueous
extract of Peperomia pellucida in male mice based on reaction time to heat using the hot-
plate method in one hour and two hours post-administration.
4. To observe for adverse effects from administration of the aqueous extract of Peperomia
pellucida in three dose levels among male mice.
IV. Hypothesis
A. Null Hypothesis
Aqueous extract of Peperomia pellucida has comparable analgesic activity with both the
positive controls (Aspirin and Tramadol) using the hot-plate method.
B. Alternative Hypothesis
Aqueous extract of Peperomia pellucida has greater analgesic activity compared with the
positive controls (Aspirin and Tramadol) using the hot-plate method.
V. Study Design
Randomized Controlled Trial is a study design in which one treatment is compared directly
with another/other treatment/s to determine which of the treatments would be of greatest
benefit. The study involved male albino mice as test animals to investigate the analgesic
activity of Peperomia pellucida. Three dose levels of the aqueous extract of Peperomia
pellucida were compared with two positive controls and a negative control using the hot-
plate method in one hour and two hours post-adminstration observation of reaction time to
heat in seconds.
VI. Significance of the Study
Patients do not have to suffer in pain, thus, it is of primary concern to alleviate it.
Through this study, the researchers would be able to scientifically validate the fokloric claim
that aqueous extract of Peperomia pellucida (Ulasimang Bato) has analgesic activity. And
since P. pellucida is an ubiquitous plant here in the Philippines, it can be a possible
alternative to commercially available analgesics (NSAIDs and Opioids) if proven to be
effective. Likewise, its use might be more economical and practical as prices of medicine is
quite expensive in the country. Pursuing this study can also promote the use of herbal
medicines alike as people nowadays are bombarded with the use of painkillers not knowing
the complications concomitant with its frequent use.
IX. Scope and Limitations of the Study
The study involved determining and comparing with controls the analgesic activity of
aqueous extract of the leaves and stems of Peperomia pellucida at three dose intervals in
male mice. Reaction time to pain using the hot-plate method was observed. The study was
conducted in one month time frame. Preliminary experiment for exploratory toxicity testing
to validate the established LD50 and to determine the No Adverse Effect Level (NOAEL) has
been undertaken. Due to unavailability of resources the sample size was sacrificed, from the
ideal ten (10) subjects it was reduced to eight(8) subjects to those groups. Some of the mice
dose were also of inappropriate weight. During the experiment proper, the researchers were
also unable to maintain a constant temperature to 55ºC and a temperature range of 52 -60 ºC
was used instead.
X. Methods
Before performing the Exploratory Toxicity Test and the experiment for Analgesic
Activity using Hot-plate Method of the aqueous extract of Peperomia pellucida, the
following were accomplished:
1. Proper Collection and Authentication
Peperomia pellucida young aerial parts (leaves and stalks) were collected from
Cavite. Sample of the plant was brought to the National Museum and was
authenticated (see appendix).
2. Heavy Metal Analysis
Sample of the plant parts from the area of collection were subjected for heavy
metal analysis to assure that the test plant is free of chemical contaminants that may
affect the outcome of the study. The Intertek Testing Services, a Bureau of Food and
Drugs (BFAD) recognized laboratory located in 2310 Pasong Tamo Extension,
Makati City performed the analysis using Atomic Absorption Spectrophotometry
(AAS). Heavy metals analyzed include Lead, Arsenic, Mercury, Cadmium and
Chromium. The result showed undetectable levels of heavy metals analyzed (see
appendix). Sufficient washing with water and along the process of extraction, other
possible contaminants such as microorganisms and dirt were eliminated.
Exploratory Toxicity Testing
The Median Lethal Dose (LD50) is the dose of a compound that causes fifty percent
mortality in a population, generally given as a single dose. All deaths occurring within 14
days are included. Estrada, et al. established that the LD50 of aqueous extract of Peperomia
pellucida is 11.77 g/kg in mice. Based from this established value, the investigators will
perform an exploratory test through observing and collating the changes that will be
produced after administering the extract using a multidimensional observation sheet.
Observations have been made every 15 minutes for the 1st hour post administration of the
extract, every 30 minutes for the 2nd hour and every hour (4 hours) for the first 6 hours and
then daily up to the 6th day. The No Adverse Effect Level (NOAEL) has been determined and
this served as the basis for computing the dose used for the experiment proper.
Objectives:
1. Validate the established LD50 of the aqueous extract of Peperomia pellucida and
observe the toxidrome that will be produced post-administration.
2. Determine the No Adverse Effect Level (NOAEL) of the aqueous extract of Peperomia
pellucida.
A. Materials
1. Negative control-Normal Saline Solution (NSS)
2. Plastic transparent observation cages, feeding bottles, syringes (1ml tuberculin and 3
ml), gavage tubes, pH paper for solvents, dissecting instruments for necropsy of
animals.
3. Peperomia pellucida aqueous extract 1 gm/ml preparation.
Preparation of the Aqueous Extract
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
BlanchingHyperemiaCyanosisSalivationTail ErectionPilomotor ReactionMicturitionDiarrheaColpectasia
PriapismRobichaud TestCircling MotionTail LashingAbdominal GrippingHead tap testBody Grasp TestCatalepsyExcess Curiosity
7. Dose range, NOAEL and the observed results were recorded.
8. Toxidrome was collated and LD50 validated.
Table 1. Computed log dose group and summary of animal mortality.
Established LD50 of Peperomia pellucida aqueous extract=11.77 g/kgLog dose interval=0.4Log dose group Dose g/kg Log dose N # of animals
died% mortality
I 0.75 -0.12 3 0/3 0
II 1.88 0.27 4 2/4 50
III 4.69 0.67 4 3/4 75
IV 11.77 1.07 4 2/4 50
V 29.56 1.47 4 4/4 100
Negative Control (NSS)
1 ml 0 4 0/4 0
Analgesic Activity Testing
A. Materials
1. Drugs: Aspirin 80 mg (positive control), Tramadol HCl 50 mg (positive control),
Normal Saline Solution (negative control)
2. Plastic transparent observation cages, Distilled water, pH paper for solvents,
Weighing scale, Oven, Hot plate, Tall beakers, Gavage tubes, Syringes, Needles,
Timer, Blender, Glass vessels
3. Test Material
Preparation of the Aqueous Extract
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
1 Low Dose Peperomia pellucida aqueous extract
473.22 mg/kg
2.67
50 mg/ml2 NOAEL Peperomia pellucida aqueous extract
750 mg/kg 2.87
3 High Dose Peperomia pellucida aqueous extract
1190 mg/kg
3.07
4 Positive Control-Tramadol HCl 20 mg/kg 1 mg/ml5 Positive Control-Aspirin 480 mg/kg 20 mg/ml6 Negative Control (NSS) 1 1
Biomarker
Fatal Dose Peperomia pellucida aqueous extract
29.56 g/kg 1 g/ml
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
ObserveRecord data
Data:CollationStatistical AnalysisInterpretationFinalizationPresentation
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Figure 1. Experiment Algorithm
XI. Results and Discussion
Table 3. Toxidrome at different dose levels
Main Body System
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
enlargement, thirty-three percent (4/12) showed no significant findings, Eight percent (1/12)
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
low dose.989 .996
middle dose .984 .999 high dose .811 .972 Aspirin .813 .128
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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Appendix 1. Table of the Administered Dose in each Mouse for Acute Toxicity testing
DOSE GROUP WEIGHT DOSE TO BE (grams) ADMINISTERED (mL)
DOSE I M3 30.22 0.02M9 34.3 0.03F4 30.6 0.02
F10 29.38 0.02DOSE II
M2 30.52 0.06M10 36.9 0.07
F3 41.85 0.08F7 29.68 0.06
DOSE III M4 28.63 0.13M8 39.95 0.19F6 34.19 0.16F9 30.09 0.14
DOSE IV M1 37.42 0.44M6 32.61 0.38F5 34.35 0.4F8 35.1 0.41
DOSE V M5 38.67 1.14M7 29.92 0.88F1 40.59 1.2F2 34.26 1.01
NEGATIVE CONTROL M11 36.75 1M12 40.19 1F11 35.11 1F12 37.5 1
Appendix 2. Raw Data of the Analgesic Activity of Peperomia pellucida on Male Albino Mice
DOSE GROUPWEIGH
T DOSE TO BE OBSERVATION (grams) ADMINISTERED (mL) 1ST HOUR 2ND HOUR
LOW DOSE 14 27.55 0.26 2.63 4.1324 21.97 0.21 4.24 3.131 21.67 0.21 2.43 3.8344 16.11 0.15 2.69 4.842 29.36 0.28 2.94 3.07
13 18.9 0.18 2.3 1.8125 17.5 0.17 2.11 2.4139 12.45 0.12 3.43 8
MEAN 2.85 3.90MIDDLE DOSE
34 18.4 0.28 2.42 2.2142 15.25 0.23 2.08 3.344 31 0.47 3.4 4.73
15 28.4 0.43 5.8 4.9527 15.22 0.23 5.53 3.4436 16 0.24 4.1 2.0745 18.64 0.28 3.12 5.36
9 28.75 0.43 DEADCOD:ASPIRATIO
NMEAN 3.78 3.73
HIGH DOSE
6 13.44 0.36 DEADCOD:ASPIRATIO
N17 30.8 0.73 4.86 4.1723 24.36 0.58 3.12 4.4433 14.7 0.35 3.66 3.8341 20.63 0.49 4.86 6.813 28.9 0.69 4.76 4.34
11 35.28 0.84 5.23 3.8422 15.73 0.37 2.93 3.37
MEAN 4.20 4.4NEGATIVE CONTROL
12 27.37 1 DEAD DEAD26 12.04 1 3.74 3.3938 14.15 1 3.46 3.328 29.27 1 2.58 3.18
19 16.52 1 3.19 3.1830 14.02 1 3.88 2.3640 19.59 1 2.84 2.6548 12.9 1 3.3 3.26
MEAN 3.28 3.05ASPIRIN
28 16.49 0.4 4 13.232 21.44 0.51 3.4 4.2447 12.74 0.31 DEAD DEAD7 12.06 0.29 4.63 7.88
20 38.85 0.93 5.34 7.7621 18.47 0.44 4.1 7.0237 14.47 0.35 3.95 6.795 25.6 0.61 DEAD DEAD
MEAN 4.51 7.82TRAMADOL
46 13.05 0.27 4.33 12.651 24.5 0.5 12.25 18.17
18 25.44 0.52 4.48 10.8529 14.82 0.3 DEAD DEAD35 13.38 0.27 6.28 9.1843 13.19 0.27 5.56 14.6710 25.75 0.53 5.73 27.416 30 0.61 5.88 5.27
MEAN 5.86 14.03
Appendix 3
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
1 50 50
Heavy Metal Analysis 1 3000 3000Contingency 1449
Grand Total Est. Php 15,945
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