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Emilio Aguinaldo College College of Medicine Department of Pharmacology

Analgesic Activity of Peperomia pellucida (Ulasimang Bato) Aqueous Extract in Male Mice

A Research Paper Presented to:

Dr. Maria Stella T. Giron Dr. Nelia P. Cortes-Maramba Dr. Dan Villamangca

Submitted by: Group III Asuncion, Camille Carissa Claridad, Maru Hasan, Jomar Lee, Ming Ju Manesca, Ma. Rossini Racadio, Ma. Perpetua Ruedas, Jhoana-Lyn Tambut, 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 countrys 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, Sinawsinaw or Tangon-tangon in Bicol. It thrives in a tropical to subtropical climate. It is a shallowrooted 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, Ratear, Man-to-man or Clearweed throughout America; Konsaka Wiwiri in Guiana, Coraozinho or "Little Heart" in Brazil; and Lingua de sapo, Herva-de-vidro, Herva-de-jaboti or Herva-dejabuti 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 (Muoz 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 herbs 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 Reyes 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 hotplate 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. I. 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.

I. 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 hotplate method in one hour and two hours post-adminstration observation of reaction time to heat in seconds. II.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 55C and a temperature range of 52 -60 C 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 plants 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 A. 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). B. 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 Activity Ataxia Loss of Righting Reflex Analgesia Anaesthesia Respiratory Rate and Depth Corneal and Pinnal Reflex Paralysis of Forelegs, Hindlegs and head Loss of Screen Grip Startle Reaction Increase in Motor Activity Fine Body Tremors Coarse Body Tremors Fasciculations Convulsions Respiratory Rate Enolpthalmos Exopthalmos Palpebral Ptosis Pupil Size Nystagmus Lacrimation Bloody Tear Blanching Hyperemia Cyanosis Salivation Tail Erection Pilomotor Reaction Micturition Diarrhea Colpectasia Priapism Robichaud Test Circling Motion Tail Lashing Abdominal Gripping Head tap test Body Grasp Test Catalepsy Excess 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/kg Log dose interval=0.4 Log dose group Dose g/kg Log dose N # of animals died I II III IV V Negative Control (NSS) 0.75 1.88 4.69 11.77 29.56 1 ml -0.12 0.27 0.67 1.07 1.47 0 3 4 4 4 4 4 0/3 2/4 3/4 2/4 4/4 0/4

% mortality 0 50 75 50 100 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 A. 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)
B.

Research Instrument In 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.

C.

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 1 2 3 4 5 6 Biomarke r

Drug administered (ml)

Low Dose Peperomia pellucida aqueous extract NOAEL Peperomia pellucida aqueous extract High Dose Peperomia pellucida 1190 3.07 aqueous extract mg/kg Positive Control-Tramadol HCl 20 mg/kg Positive Control-Aspirin 480 mg/kg Negative Control (NSS) 1 Fatal Dose Peperomia pellucida 29.56 g/kg aqueous extract Biomarker 1 (male) death at 1st day post-administration Biomarker 2 (female) death at 2nd day post-administration Necropsy findings: both exhibited hemorrhage of peritoneum

log dose interval=0.2 Drug Dose Log dose 473.22 2.67 mg/kg 750 mg/kg 2.87

Preparation

50 mg/ml

1 mg/ml 20 mg/ml 1 1 g/ml

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.
A.

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 experiment pellucida no Heavy Metal Analysis yeAffect of Peperomia Use explored LD , AcuteExperiment Proper Data: Plant Toxicity Test:
Analgesic Activity of Aqueous Authentication Acceptable (Study o s outcome Level of Heavy observed toxidrome and Collation Metals? Exploratory test Authenticate? Peperomia Extract of limitation) NOAEL Statistical Analysis LD50 Validate established as the pellucida, NOAEL Interpretation dose Observe middle Toxidrome Finalization Observe NOAEL Presentation Record data LD50 comparable with established value?
5 0

Figure 1. Experiment Algorithm IX. Results and Discussion Table 3. Toxidrome at different dose levels Main Body System CNS Depression CNS Stimulation
General

Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia pellucida aqueous extract at 0.75 g/kg (Dose I) Parameter Animals Duration Responded (Time of Onset to Reversibility) Motor Activity A. 3/4 (F4, M3, M9) 1st 15 mins until 6th hr B. + (F10) 1st 15 mins until 6th hr Startle Reflex (+) 4/4 1st 15 mins until Day 6 Abdominal gripping (+) (M3, M9, F4) 1st 15 mins until 3rd hr

Observation

Subjective Observation

Catalepsy A. + B.

(M9) (M3, F4, F10)

1st 15 mins until Day2 1st 15 mins until Day 6

Main Body System

CNS Depressio n

CNS
Stimulation General Observation

Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia pellucida aqueous extract at 1.88 g/kg (Dose II) Parameter Animals Duration Responded (Time of Onset to Reversibility) Motor Activity A. 2/4 (F3, F7) 1st 15 mins until 3rd hr for F3 and until 4th hr for F7 2nd hour until Day 2 B. + (M2) Respiratory Rate () 4/4 1st 15 mins until 2nd hr Analgesia () 2/4 (M10, M2) 1st 15 mins until 4th hr st Loss of Pinnal Reflex (+) 4/4 1 30 mins until 2nd hr for F3, F7 and 3rd hr for M2, M10 Startle Reflex (+) 4/4 1st 15 mins until Death Circling Motion Head Tap A. Aggressive B. Passive Body Touch A. Aggressive B. Passive Excess Curiosity A. ++ B. + 2/4 (M10, M2) 4/4 4/4` 1st 15 mins and lasted for 30 mins 1st 15 min until 2nd hr 3rd hour until Day 6 (F3,F7) and until Death for M10, M2 1st 15 min until 2nd hr 3rd hour until Day 6 (F3,F7) and until Death for M10, M2 1st 15 mins until 1st hour 1st 30 mins until Day 6 for F3, F7 and until Death for M2, M10

Subjective Observation

4/4 4/4`

4/4 4/4

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 Duration Responded (Time of Onset to

CNS Depressio n CNS Stimulation Eyes Ears and Mouth General Observation

Motor Activity (+) Respiratory Depth () Analgesia () Loss of Pinnal Reflex () Startle Reflex (+) Enophthalmos (+) Blanching () Robichaud Test (+)

(F6, M4, M8) 2/4 (F6, M4) (M4, M8, M6) (M8) 4/4 (M8) 2/4 (F9, M8) 2/4 (M4, M8)

Reversibility) 1 15 mins until Death 1st 15 mins until Death 1st 15 mins and lasted for 45 mins 1st 15 mins until 2nd hr 1st 15 mins until Death
st

1st 30 mins until Death 1st 15 mins until Day 2 for F9 and until 4th hr for M8 1st 15 mins until Death for M4 1st 30 mins until Death for M8 F6: 1st 15 mins until 6th hr F9: 1st 15 mins until 5th hr st M4: 1 15 mins until death M8: 1hr after drug administration until Death F6: 6th hr until Death F9: 5th hr until Day 6 1st 15 mins until F6: 6th hr F9: 3rd hr M4: Death M8: Death F6: 2nd day until Death F9: 3rd hour until Death M4: 1st 15 min until 3rd hr M8: 1st 15 mins until Death F6: Last hr of Day 1 until Death M4: 5th hr until 6th hr

Head Tap A. Passive

4/4

B. Fearful Subjectiv e Observati on Body Touch A. Passive

2/4 (F6, F9) 4/4

B. Fearful Catalepsy () Excess Curiosity (+)

2/4 (F6, F9 ) 2/4 (M4, M8) 2/4 (F6, M4)

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 Duration

Responded CNS Depression Motor Activity (+2) Analgesia (+) Motor Activity (+) Fine Body Tremor (+) Coarse Body Tremor (+2) Fasciculation(+3) Convulsion (+) Startle Reflex (+) Enophthalmos (+) Palpebral Ptosis (+4) Abdominal gripping (+2) Head Tap A. Aggressive B. Passive Body Touch A. Aggressive B. Passive Excess Curiosity (M6) 2/4 (M6, F5) 2/4 (F8, M1) (F8, F5, M6) 2/4 (F5, F8) (F5) (F8) 4/4 (M6) (M6) 2/4 (F5, F8) (M1) (M6) (M1) (M6) (M1)

CNS Stimulation

Eyes
General Observation

(Time of Onset to Reversibility) 1st 15 mins until Death 1st 15 mins until 2nd hr 1st 15 mins and lasted for 90mins st 1 15 mins until Day2 1st 15 mins until 3rd hr 1st 15 mins lasted for 90 mins 1st 15 mins until 4th hr 1st 15 mins until Death 1st 15 mins until Death 1st 30 mins until Death 1st 15 min until Day 2 1st 15 min until Death 1st 15 mins until Death 1st 15 min until Death 1st 15 mins until Death 1st 15 min and lasted for 90 mins

Subjective Observation

Main Body System CNS Depression

Lethal and Non-lethal Toxidrome seen in mice administered with Peperomia pellucida aqueous extract at 29.56 g/kg (Dose V) Parameter Animals Duration Responded (Time of Onset to Reversibility) Motor Activity A. ++ (F2) 1st 15 mins until Death B. (M5, M7, FI) 1st 15 mins until Death Ataxia A. (F2) 1st 15 mins until Death B. ++ (M5, M7 and F1) 1st 15 mins until Death Loss of Righting Reflex A. (M5, M7 and F1) 1st 15 mins until Death B. ++ (F2) 1st 15 mins until Death Analgesia A. (M5, M7 and F1) 1st 15 mins until Death B. ++ (F2) 1st 15 mins until Death Respiratory Rate Mean: 50 breaths/33.97 sec (Increase in RR) Loss of Corneal Reflex (+) (F2) 1st 30 mins until Death Loss of Pinnal Reflex (+) (F2) 1st 30 mins until Death Paralysis: Forelegs (+) (F2) 1st 30 mins until Death Paralysis: Hindlegs

CNS Stimulation Eyes Ears and Mouth

A. + B. Paralysis: Head (+) Screen Grip Foreleg Loss (+) Screen Grip Hindleg Loss A. + B. ++++ Respiratory Rate Startle Reflex () Enophthalmos (+) Palpebral Ptosis (++) Lacrimation (+) Blanching (+) Hyperemia (+) Salivation (+) Robichaud Test A. + B. ++ Abdominal Gripping (+2)

2/4 (F2, M5) (M7) (F2) (F2)

1st 30 mins until Death 1st 30 mins until Death 1st hour until Death st 1 15 mins until Death

(M5) 1st hour until Death (F2) 1st hour until Death Mean: 50 breaths/33.97 sec (Increase in RR) 4/4 1st 15 mins until Death (F2) 1st 15 mins until Death (F2) 1st 30 mins until Death (F2) 1st 15 mins until Death 4/4 1st 30 mins until Death 2/4 (M7, F2) 45 mins until Death for M7 while until 5th hour for F2 st (F2) 1 15 min until Death 2/4 (F1, M7) (F2) 2/4 (F1, M5) 1st hour until Death 1st 15 min until Death F1 1st 15 mins until 3rd hour M5 1st 15 min until 6th hour st 1 15 min until Death 1st 15 min until Death 1st 30 min until Death 1st 30 min until Death

General Observation

Subjective Observation

Head Tap (Passive) Body Touch (Passive) Catalepsy A. +++ B. ++

4/4 4/4 (F2) 2/4 (M5, F1)

Main Body System CNS Depression CNS Stimulation Subjective Observation

Lethal and Non-lethal Toxidrome seen in mice administered with Normal Saline Solution 1ml (Negative Control) Parameter Animals Duration Responded (Time of Onset to Reversibility) Motor Activity () 4/4 1st 15 mins until Day 6 Startle Reflex (+) Excess Curiosity (+) 4/4 4/4 1st 15 mins until Day 6 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 4 th 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 4 th 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 3 rd 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 4 th and 5th dose groups which lasted until the 2nd and 3rd day respectively. It was also manifested by mice under the 1st dose group which lasted only for 3 hours. Based on the researchers 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 4 th 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 4th 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 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 controlsaspirin and tramadol showed increased analgesic activity while the middle dose decreased its effect. Decreased reaction time to heat of the negative control at twohour 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.

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 lowdose1 .141 1 lowdose2 Pair middose1 .941 2 middose2 Pair highdose1 .669 3 highdose2 Pair asprin1 - aspirin2 .029 4 Pair tramadol1 .027 5 tramadol2 Pair negctrl1 - negctrl2 .368 6 * 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-value One hour Between Groups Within Groups .001 Two hours .000

* 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 Low dose Group middle dose high dose Aspirin Tramadol negative control low dose high dose Aspirin Tramadol negative control low dose middle dose Aspirin Tramadol negative control low dose middle dose high dose Tramadol negative control low dose P-value One hour Two hours .779 1.000 .418 1.000 .438 .270 .000 .000 .989 .996 .779 1.000 .992 .999 .991 .257 .015 .000 .984 .999 .418 1.000 .992 .999 1.000 .449 .061 .000 .811 .972 .438 .270 .991 .257 1.000 .449 .087 .021 .813 .128 .000 .000

Middle dose

High dose

Aspirin

Tramadol

Negative control

middle dose high dose Aspirin negative control low dose

.015 .061 .087 .002 .989

.000 .000 .021 .000 .996

middle dose .984 .999 high dose .811 .972 Aspirin .813 .128 Tramadol .002 .000 * The mean difference is significant at the .05 level. IX. 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.

X. 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 subjects 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 , Mller 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 , Pea-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. Harrisons 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 mdicinales 1. [CD-Rom]. PROTA, Wageningen, Netherlands.

Muoz 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 Alteos Indians . J Ethnopharmacol . 2000;71:123-131. National Institute on Drug Abuse. 2009. 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. Tabers 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 (grams) DOSE TO BE ADMINISTERED

(mL) DOSE I M3 M9 F4 F10 DOSE II M2 M10 F3 F7 DOSE III M4 M8 F6 F9 DOSE IV M1 M6 F5 F8 DOSE V M5 M7 F1 F2 NEGATIVE CONTROL M11 M12 F11 F12 30.22 34.3 30.6 29.38 30.52 36.9 41.85 29.68 28.63 39.95 34.19 30.09 37.42 32.61 34.35 35.1 38.67 29.92 40.59 34.26 0.02 0.03 0.02 0.02 0.06 0.07 0.08 0.06 0.13 0.19 0.16 0.14 0.44 0.38 0.4 0.41 1.14 0.88 1.2 1.01

36.75 40.19 35.11 37.5

1 1 1 1

Appendix 2. Raw Data of the Analgesic Activity of Peperomia pellucida on Male Albino Mice DOSE GROUP WEIG HT DOSE TO BE OBSERVATION

(gra ms) LOW DOSE 14 24 31 44 2 13 25 39 MIDDLE DOSE 34 42 4 15 27 36 45 9 HIGH DOSE 6 17 23 33 41 3 11 22 NEGATIVE CONTROL 12 26 38 8 19 30 40 13.44 30.8 24.36 14.7 20.63 28.9 35.28 15.73 27.55 21.97 21.67 16.11 29.36 18.9 17.5 12.45

ADMINISTERED (mL) 0.26 0.21 0.21 0.15 0.28 0.18 0.17 0.12 MEAN

1ST HOUR 2.63 4.24 2.43 2.69 2.94 2.3 2.11 3.43 2.85 2.42 2.08 3.4 5.8 5.53 4.1 3.12

2ND HOUR 4.13 3.1 3.83 4.84 3.07 1.81 2.41 8 3.90 2.21 3.34 4.73 4.95 3.44 2.07 5.36 COD:ASPIRA TION 3.73 COD:ASPIRA TION 4.17 4.44 3.83 6.81 4.34 3.84 3.37 4.4 DEAD 3.39 3.32 3.18 3.18 2.36 2.65

18.4 15.25 31 28.4 15.22 16 18.64 28.75

0.28 0.23 0.47 0.43 0.23 0.24 0.28 0.43 MEAN 0.36 0.73 0.58 0.35 0.49 0.69 0.84 0.37 MEAN

DEAD 3.78 DEAD 4.86 3.12 3.66 4.86 4.76 5.23 2.93 4.20 DEAD 3.74 3.46 2.58 3.19 3.88 2.84

27.37 12.04 14.15 29.27 16.52 14.02 19.59

1 1 1 1 1 1 1

48 ASPIRIN 28 32 47 7 20 21 37 5 TRAMADOL 46 1 18 29 35 43 10 16

12.9

1 MEAN

3.3 3.28 4 3.4 DEAD 4.63 5.34 4.1 3.95 DEAD 4.51 4.33 12.25 4.48 DEAD 6.28 5.56 5.73 5.88 5.86

3.26 3.05 13.2 4.24 DEAD 7.88 7.76 7.02 6.79 DEAD 7.82 12.65 18.17 10.85 DEAD 9.18 14.67 27.4 5.27 14.03

16.49 21.44 12.74 12.06 38.85 18.47 14.47 25.6

0.4 0.51 0.31 0.29 0.93 0.44 0.35 0.61 MEAN

13.05 24.5 25.44 14.82 13.38 13.19 25.75 30

0.27 0.5 0.52 0.3 0.27 0.27 0.53 0.61 MEAN

Appendix 3 Mean reaction time of male mice one and two hours post administration of P. pellucida aqueous extract, negative and positive controls. Post-drug Administration Reaction Time in Seconds Drug Dose Group 1 hour 2 hour P. pellucida low dose P. pellucida middle dose P. pellucida high dose Positive Control (Aspirin) Positive Control (Tramadol) Negative control (NSS) 2.850.7 3.781.4 4.20.94 4.240.7 6.362.7 3.280.46 3.91.9 3.731.3 4.41.12 7.822.94 14.037.2 3.050.38

Appendix 4

Summary of animal mortality and corresponding necropsy findings Log dose group Dose g/kg 0.75 1.88 N # of animals died 2/4 % mortality 25 50 Necropsy Findings

I (M3, M9, F4, F10) II (M2, M10, F3, F7) III (M4, M8, F6, F9)

4 4

F10-Death by accident M2-death at day 3, no significant findings M10-death at day 4, no significant findings M4-death at day 2, enlarged intestines M8-death at day 1, enlarged intestines F6-death at day 4, no significant findings M6-death at day 1, enlarged intestines, (+) Intestinal Parasite F8-death at day 4, enlarged intestines M5-death at day 5, no significant findings M7-death at day 4, enlarged intestines F1-death at day 5, enlarged intestines F2-death at 5th hour, hepatomegaly with multifocal necrosis

4.69

75

IV (M1, M6, F5, F8) V (M5, M7, F1, F2)

11.77

2/4

50

29.56

4/4

100

Negative Control (NSS)

1 ml

0/4

Appendix 5 Proposed Time Frame DATE (2010) 04 February 4,5 February 14-19 February 21-26 February 3 March ACTIVITY Approval of Research Proposal Collection of P. pellucida Sample for authentication and heavy metal analysis. Collection of P. pellucida/Acute Toxicity Testing in Male Mice Purchasing and Preparation of other materials for experiment proper Collection of P. pellucida/Experiment Proper (Analgesic Test)

1-10 March 11 March

Collation, Statistical Analysis and Finalization of the Research Paper Research Presentation

Appendix 6 Budget Plan Sources of Expenses 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 Number of times (or items) 100 10 20 10 10 10 30 10 30 10 1 1 2 300php per person 150php per person 1000 50 3000 5php per tablet 33php per ampoule 106php per bottle 60php per galloon 150 330 106 60 1800 1000 50 3000 1449 Est. Php 15,945 Rate 80 php per mice Total 8000

Collection of Samples Transportation Meal Printing and Documentation Authentication of the Test Plant Heavy Metal Analysis Contingency

1 1 Grand Total