p. pellucida

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

yes

noo

yes

yes

noo

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)



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


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)



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)



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


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)


Loss of Righting ReflexA. ±B. ++

¾ (M5, M7 and F1)¼ (F2)


AnalgesiaA. ±B. ++

¾ (M5, M7 and F1)¼ (F2)


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)


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)



Reversibility)CNS

DepressionMotor Activity (±) 4/4 1st 15 mins until Day 6

CNS Stimulation

Startle Reflex (+) 4/4 1st 15 mins until Day 6


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.

REFERENCES

Andrade MR , de Fatima Arrigoni-Blank M , Dmitrieva EG , Franzotti EM , Antoniolli AR , , Marchioro M . Anti-inflammatory and analgesic activity of Peperomia pellucida (L.) HBK (Piperaceae) . J Ethnopharmacol. 2004;91:215-218.

Arrigoni-Blank Mde F , Oliveira RL , Mendes SS , et al. Seed germination, phenology, and antiedematogenic activity of Peperomia pellucida (L.) H. B. K. BMC Pharmacol . 2002;2:12-19.

Aziba PI , Adedeji A , Ekor M , Adeyemi O . Analgesic activity of Peperomia pellucida aerial parts in mice . Fitoterapia . 2001;72:57-58.

Bayma JD , Arruda MS , Müller AH , Arruda AC , Canto WC . A dimeric ArC 2 compound from Peperomia pellucida . Phytochemistry . 2000;55:779-782.

Belardo LO. Some constituents of the volatile oil of Peperomia pellucida. Perfumery and Essential Oil Record 1967; 58:359

Bojo AC , Albano-Garcia E , Pocsidio GN . The antibacterial activity of Peperomia pellucida (L.) HBK (Piperaceae) . Asia Life Sci . 1994;3:35-44.

Chan-Bacab MJ , Peña-Rodriguez LM . Plant natural products with leishmanicidal activity . Nat Prod Rep . 2001;18:674-688.

Estrada, H, et al. Selection and Scientific Validation of Medicinal Plants for Primary Healthcare. Technical Report Series No.12.

Fauci, Bruniwald, et al. Harrison’s Principle of Internal Medicine. 17th Edition. McGraw Hill Incorported. 2007.

Freireich, EJ, et al. Quantitative comparison of toxicity of anti-cancer agents in mouse, rat, monkey and man. Cancer Chemotherapy Rep. 50, 219, 1966.

Galvez-Tan. Philippine Herbal Medicine 2009. Pansitpansitan.htm.

Katzung, B. Basic and Clinical Pharmacology 10th Edition. Copyright 2007. McGraw-Hill Company Inc.

Khan MR , Omoloso AD . Antibacterial activity of Hygrophila stricta and Peperomia pellucida . Fitoterapia . 2002;73:251-254.

Mosango, D.M., 2008. Peperomia pellucida (L.) Kunth. In: Schmelzer, G.H. & Gurib-Fakim, A. (Editors). Prota 11(1): Medicinal plants/Plantes médicinales 1. [CD-Rom]. PROTA, Wageningen, Netherlands.

Muñoz V , Sauvain M , Bourdy G , et al. A search for natural bioactive compounds in Bolivia through a multidisciplinary approach: Part III. Evaluation of the antimalarial activity of plants used by Alteños Indians . J Ethnopharmacol . 2000;71:123-131.

National Institute on Drug Abuse. 2009. www.drugabuse.gov.

http://www.drugabuse.gov/

Nodine, JH, Siegler, P. Animal and Clinical Pharmacologic Techniques in Drug Evaluation, 1964.

Noor, A, Roslida AH. Evaluation of Gastroprotective Effects of the Ethanolic Extract of Peperomia pellucida. 2009: 7678-683.

Quisumbing, Eduardo. Medicinal Plants of the Philippines. Katha Publishing Company Inc. 1978.

Ragasa CY , Dumato M , Rideout JA . Antifungal compounds from Peperomia pellucida . ACGC Chem Res Commun . 1998;7:54-61.

Sio, S. A Study on the Antihyperuricemic Effecet of the Aqueous Extract of Peperomia pellucida (L) HBK in Rats, 1993. University of the Philippines, Manila.

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

Peperomia pellucida . J Nat Prod. 2006 ; 69: 247-250.

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

p. pellucida

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