PHYTOCHEMICAL STUDIES AND BIOLOGICAL ACTIVITIES OF THE

DICHLOROMETHANE EXTRACT OF THE PEPEROMIA PELLUCIDA.

NURHAYATI BINTI BIDIN

Bachelor of Science with Honours (Resource Chemistry)

Faculty of Resource Science and Technology

2008

Faculty of Resource Science and Technology

PHYTOCHEMICAL STUDIES AND BIOLOGICAL ACTIVITIES OF THE

DICHLOROMETHANE EXTRACT OF THE PEPEROMIA PELLUCIDA.

NURHAYATI BINTI BIDIN

This project is submitted in partial fulfilment of the requirements for the Degree of

Bachelor of Science with Honours

(Resource Chemistry)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2008

i

DECLARATION

No portion of the work referred in this dissertation has been submitted in support of an

application for another degree of qualification of this or any other university of institution of

higher learning.

_________________________

Nurhayati binti Bidin

Department of Chemistry

Faculty of Resource Science and Technology

University Malaysia Sarawak

ii

ACKNOWLEDGEMENT

Firstly, I would like to thank God because I’m able to finish up my report in time. Also

I would like to express my grateful appreciation to Mr. Chieng Tiong Chin for his guidance

and patience in guiding and helping me to accomplish my project.

I also would like to thank all the lecturers who willingly shared their expertise which

contributes some relevant informations throughout this project.

Not to forget, my thanks goes to all the lab assistants and my friends for their support

throughout the duration of this project. Finally, I would like to express my gratitude to my

lovely family for their support and encouragement.

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TABLE OF CONTENTS:

Pages

Declaration i

Acknowledgements ii

Table of Contents iii

List of Abbreviations vi

List of Tables vii

List of Figures viii

List of Appendices ix

Abstract x

CHAPTER 1

INTRODUCTION

1.1 Introduction 1

1.2 Objectives 3

CHAPTER 2

LITERATURE REVIEW

2.1 Description of Peperomia pellucida 4-5

iv

2.2 Chemical composition of Pepromia pellucida 5-6

2.3 Biological activities 6-7

CHAPTER 3

MATERIALS AND METHODS

3.1 General experimental procedures 8-9

3.2 Sample collection 9

3.3 Extraction 9

3.4 Isolation and purification of chemical constituents 10

3.4.1 Thin layer chromatography (TLC) 10

3.4.2 Iodine vapour test 11

3.4.3 Vanillin dipping (Vanillin Sulphuric Acid Reagent) 11

3.4.4 Column chromatography 11

3.5 Analysis of chemical constituents 12

3.5.1 Gas chromatography-Mass spectrometry (GC-MS) 12

3.5.2 Fourier Transform Infra Red Spectrometer (FTIR) 12

3.6 Determination of biological activities 12

3.6.1 Toxicity to Artemia salina 12-13

v

3.6.2 Termiticidal activity test 14

3.6.3 Antibacterial activity test 15

CHAPTER 4

RESULTS AND DISSCUSSION

4.1 Extraction 16

4.2 Analytical Thin Layer Chromatography (TLC) 16

4.2.1 Analytical TLC of dichloromethane crude extract 16-17

4.3 Column Chromatography of dichloromethane crude extract 19-21

4.4 Gas Chromatography - Mass Spectroscopy (GC- MS) 22-24

4.5 Fourier Transform Infra Red Spectrometer (FTIR) 24

4.6 Brine Shrimp Toxicity Test 25

4.7 Termiticidal Activity Test 26-27

4.8 Antibacterial Activity Test 28

CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS 29-30

REFERENCES 30-33

APPENDICES

vi

LIST OF ABBREVIATIONS

CC Column Chromatography

CHCl3 Chloroform

DCM Dichloromethane

FTIR Fourier Transform Infrared

GC Gas Chromatography

IR Infrared Spectroscopy

MS Mass Spectroscopy

NMR Nuclear Magnetic Resonance Spectroscopy

Rf Retention Factor

TLC Thin Layer Chromatography

UV Ultraviolet Spectrophotometer

vii

LIST OF TABLES

Page

Table 2.1 Antimicrobial activity of extractives from Hygrophilia 8

& Peperomia pellucida

Table 4.1 Analytical TLC using single solvent 18

Table 4.2 Combine fractions of the same Rf value 20

Table 4.3 TLC for scrapped bands 21

Table 4.4 Average death of Artemia salina as a function of concentration 25

for the DCM crude extract

Table 4.5 The average survivor of termites for DCM crude extract of 27

Peperomia pellucida

Table 4.6 Inhibition growth of bacteria 28

viii

LIST OF FIGURES

Page

Figure 2.1 Peperomia pellucida 4

Figure 2.2 Pellucidin A 6

Figure 2.3 Phenyl propanoid dil – apiol 6

Figure 3.1 Toxicity test for Artemia salina 13

Figure 3.2 The termiticidal test for Coptotermes sp. 14

Figure 3.3 Paper disc diffusion method 15

Figure 4.1 GC – MS Data for component 1 22

Figure 4.2 GC – MS Data for component 2 23

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LIST OF APPENDICES

Appendix 1: Pentadecane

Appendix 2: Ion Fragmentation of Pentadecane

Appendix 3: Eicosane

Appendix 4: Ion Fragmentation of Eicosane

Appendix 5: Ion Fragmentation of Eicosane

Appendix 6: FTIR Spectrum for Component 1

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Phytochemical studies and Bialogical activities of the Dichloromethane Extracts of

Peperomia Pellucida

Nurhayati binti Bidin

Resource Chemistry

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

A phytochemical and biological activity of Peperomia pellucida (P.pellucida) from the

Dichloromethane (DCM) extract is a study to determine the chemical components present in

P. pellucida. The percentage yield of the P. pellucida extract was 4.12%. The crude was

fractionated into several fractions using Column Chromatography. Then, the combined

fractions were purified by the 20 cm x 20 cm TLC plate. The two major chemical components

of the separated band from TLC were analyzed by Gas chromatography-Mass Spectrometry

(GC-MS). For component 1, one major peak shown at the retention time 6.409 where as the

component 2, three major peaks been observed at the retention time of 14.171, 26.828, and

28.292. Ion fragmentation data shown that compound at 14.171 min has the molecular weight

equal to 212.3 g/mol. Bioassay was carried out on Artemia salina, Coptotermes spp.,

Escherichia coli and Staphylococcus aureus. The results of bioassay indicate that the DCM

extract has low toxic properties.

Keyword: Peperomia pellucida; biological activity; structural determination.

ABSTRAK

Kajian fitokimia dan aktiviti biologi terhadap Peperomia pellucida (P. pellucida) daripada

ekstrak Diklorometana (DCM) merupakan kajian bagi menentukan komponen kimia yang

hadir dalam P. pellucida. Peratusan hasil ekstrak kasar P. pellucida ialah 4.12%. Hasil

ekstrak di fraksikan kepada beberapa fraksi menggunakan kromatografi turus graviti.

Pecahan yang terpilih ditulenkan dengan menggunakan Kromatografi Lapisan Nipis (KLN)

bersaiz 20 cm x 20 cm. dua jalur utama yang dipisahkan daripada KLN di analisis

menggunakan Kromatografi Gas- Spektroskopi Jisim (KG-SJ). Untuk komponen 1, satu

puncak utama ditunjukkan pada sela masa 6.409 minit, manakala untuk komponen 2, tiga

puncak utama dapat dikenalpasti pada sela masa 14.171 minit, 26.828 minit dan 28.292

minit. Data fragmentasi ion menyatakan sebatian pada minit 14.171 mempunyai berat

molekul 212.3 g/mol. Bioessei dilakukan ke atas Artemia salina, Coptotermes spp,

Escherichia coli dan Stapylococcus aureus. Keputusan bioessei menunjukkan bahawa hasil

ekstrak kasar mempunyai ketoksikan yang rendah.

Kata kunci: Peperomia pellucida; keactifan biologi; struktur.

1

CHAPTER 1

INTRODUCTION

1.1 Introduction

The piperaceae family consists of 5 genera and 1400 species. The majority of the species in

this family are Piper and Peperomia which are about 700 and 600 species respectively (Duke,

1985). The piperaceae family consists of none woody plant with simple leaves. Piperaceae is

naturally distributed throughout the tropical and subtropical area (Online a). This family has

been known by most taxonomists and also called as the “pepper family”.

Piper nigrum, from Koehler (1887)

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Scientific classification:

The family has five genera with about two or three thousand species, the best known genera

are Peperomia and Piper (Online b). Peperomia, also known as Kelampungan Air is a large

genus found in tropical and sub tropical region of the world. It is a herb known for its

medicinal properties. P. pellucida has shown its antibacterial activity against Staphylococcus

aureus , Bacillus subtilis , Pseudomonas aeruginosa , and Escherichia coli, it could have a

potential as a broad spectrum antibiotic (Bojo et al. 1994). The phytochemical and biological

studies of Peperomia pellucida (P. pellucida) have been carried out to characterize some

componenst in the plant. The phytochemical analysis will be focusing on the compound that

gives the positive results for the antibacterial screening. This is one of the successful methods

for the investigation of traditional medicines as sources of the new drugs (A.J. Vlietinck et al.,

1995). This study was done because the people especially in the villages using this plant as the

folk medicine. Therefore, this researched is carried out to know the active compounds that

exist in the plant. The sample for this research was collected around the UNIMAS compound

and the nearby villages. The sample was air-dried before it was extracted using DCM, then the

crude extracts was tested for the biological activity at different concentrations. At same time,

isolation and purification of compounds were also carried out by using chromatographic

techniques.

Antimicrobial compounds from plants represent a potentially novel source of antimicrobial

substances since they act against bacteria via mechanisms that are different from those of

currently used antibiotics and may thus have clinical values in treatment against resistant

microbial strains (Eloff, 1998). Therefore, the purposes of this study are to figure out the

compounds that give the biological activity against the bacteria, brine shrimps and termites.

The bacteria that involve in this study are the gram negative bacilli which is Escherchia coli

3

(E. coli) and the gram positive cocci, Staphylococcus aureus (S. aureus). E. coli can be found

in water, soil and vegetation. E. coli causes the wound infections, appendicitis, peritonitis, and

infection of the gall bladder (Monica, 1984). S. aureus is a yellow pigmented species. It may

cause boils, pimples, pneumonia, osteomyelitis, meningitis and athritis (Michael and John,

2006).

1.2 Objectives

The objectives of the study are to extract P. pellucida using the solvent extraction method.

The chemical constituents of the DCM extract are identified using Gas Chromatographic-

Mass Spectrometry (GC-MS). Bioassays are done for the purpose to determine the biological

activities of the extracts against bacteria, brine shrimp and termites.

4

CHAPTER 2

LITERATURE REVIEW

2.1 Description of Peperomia pellucida

Peperomia is one of the two large genera of Piperaceae family. Most of them are compact,

small perennial epiphytes growing on the rotten wood. More than 1500 species have been

recorded. P. pellucida is a herb plant with succulent, alternate, oval leaves and influorescences

in terminal spikes, auxiliary opposite to the leaves. The species grow well during rainy season

and in loose, humid soils under the shade of trees. The plant species has a history of

ethnomedicinal use. The crude extract of P. pellucida was found to have anti-inflammatory,

chemotherapeutic, and analgesic properties.

Figure 2.1 Peperomia pellucida (Online c)

5

The common peperomia is soft, fleshy-leaved and has thick stems, usually they would not

exceed 12” in height. It has a peppery flavor and can be eaten as vegetables. Most of them are

shade loving plants that scald easily if they receive direct sunlight, so they usually grown as a

house plant. They are easily propagated from stem or leaf cuttings in the warmer weather.

Many species are non-succulent such as p. coperata, which is the most popular varieties of

peperomia and is breed as a house plant. P. obtusifolia, is the species that occasionally seen in

commerce. This species is said to be originally from Tropical America and southern Florida.

It has thick dark green glossy leaves and can reach 6-8 inch in height (Online d).

2.2 Chemical composition of Peperomia pellucida

In folk medicine, P. pellucida has been used to treat abscesses, boils, skin wound and

conjunctivitis (Bojo et al., 1994). Other medicinal features of P. pellucida are to lower the

blood pressure (in Northeastern Brazil), and as a diuretic (in Guyana) (May, 1982). Since the

species give the positive results on folk treatment, therefore it must be containing some

compounds that contribute to these biological activities. Hence, some studies have been done

in determining the biologically active compounds in P. pellucida.

In the previous study in 2003 from Maria et al., stated that the aqueous extracts of the P.

pellucida have the effects on anti-inflammatory and analgesic activities. The study has been

done at Brazil, in May 1998. The results from their researches gave positive results on the

anti-inflammatory and analgesic activities.

Previous researches of the chemical analysis on the P. pellucida, the compound that can be

determined were apiol, 2,4,5-trimethoxystyrene, flavones, flavonols and phytosterols

(Manalo, 1983). A new dimeric ArC2 compound which was named pellucidin A (Figure 2.2)

6

along with the known phenylpropanoid dil-apiol (Figure 2.3) (Bernhard and Thiele, 1978),

were determined in the chemical studies on the methanol extracts of the aerial parts of the

species.

Figure 2.2 Figure 2.3

2.3 Biological activities

The better understanding of the study can be achieved by focusing on their biological

activities. The cytotoxicity of the chemical constituents of the plants can be determined using

the brine shrimp and termites test. Brine shrimp is the most common method and very easy to

work with. The method to conduct the brine shrimp is simple and cost saving. Rather than

other organisms, shrimp can tolerate many pollutants (McLaughin, 1991).

The potential for the plant to act as the anti-infective agents have been revealed by the

screening of the plant extracts and natural products on the antimicrobial agents. The study

from Khan and Omoloso (2002), proposed that in the butanol extracts, Peperomia pellucida

gave the best of the broad spectrum of antibacterial activity. The study took place in Papua

New Guinea. The results from their study reported that the fractions of P. pellucida that was

7

obtained from the petrol, dichloromethane, ethyl acetate, and butanol gave the good level of

antibacterial activity and the best fraction was the butanol fraction. The results are illustrated

in the Table 2.1.

TABLE 2.1 Antimicrobial activity of extractives from Hygrophila stricta and Peperomia

pellucida.

Microorganism Hygrophila stricta Peperomia pellucida Ref

Chl C P D E B C P D E B

Bacillus cereus G+ 8 18 16 8 14 8 12 10 12 18 16

B. coagulans G+ 10 18 12 16 14 8 14 8 14 20 18

B. megatarium G+ 8 20 18 18 16 10 16 14 14 18 16

B. subtilis G+ 8 18 16 12 10 8 14 14 10 18 16

Lactobacillus casei G+ 10 20 12 16 12 10 14 12 12 18 18

Agrobacterium tumefaciens G- 10 20 18 18 18 10 14 10 12 16 12

Citrobacter freundii G- 8 18 18 12 18 10 14 12 12 18 16

Enterobacter aerogenes G- 10 20 14 8 16 8 16 12 14 18 18

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CHAPTER 3

MATERIALS AND METHODS

3.1 General Experimental Procedure

There were several solvents used to carry out the experiments, which were cyclohexane

(HmBG Chemicals), dichloromethane (Analar® BDH Lab. Supplies), ethyl acetate (PC

Laboratory Reagent), methanol (HmBG Chemicals), ethanol (HmBG Chemicals), acetone

(Mallinckrodt Chemicals) and sulphuric acid (Merck KGaA).

Grinding of the samples was performed by using coffee blender (Blender Pensonic Dry

Blend). The sample of the Peperomia pellucida was soaked with dichloromethane (DCM).

The solvent was then removed using the rotary evaporator (Buchi Mode R-200). The sample

was freeze-dried for three hours using Freezone 18, 7755032-18L LABCONCO CORP. to

remove the excess water.

Analytical TLC was carried out using silica gel (Merck, 60 F254, 0.25 mm in thickness). Each

spot on the TLC was visualized under ultra violet (UV) light (UV-11). Iodine vapour

(Laboratory Reagents, 99% purity, Iodine resublimed, Ajax Chemicals) and vanillin dipping

(Vanillin for Synthesis, EWG-Nr: 2044652, Gehalt acidimetr., Merck) were used as

visualizing agent. Column Chromatography (CC) was packed with silica gel 60 (Kiesel gel

60, 0.063-0.2 mm, 70-230 mesh ASTM) and acid wash sand (Alfa Aesar, A19936) was used

to create one linear line at the bottom and top of the column chromatography.

GC-MS analysis (Hewlett Packard 6890 using column HP-5MS crosslinked, non-polar 5%

phenyl methyl siloxane with 30.0 m x 0.25 mm x 0.25 μm film thickness), FTIR (FT-IR

9

Spectrum GX, PerkinElmer) and NMR (JNM-ECA500, 500MHz) were used to identify the

component extracted from P. pellucida.

3.2 Sample Collection

The samples of Peperomia pellucida was collected from around the Unimas main campus and

the nearby villages. The samples were air-dried and the entire plants were used for the

extraction. The sample was ground to get the fine product to make it easier for further

processing steps.

3.3 Extraction of P. pellucida.

About 1.0 kg dried sample of Peperomia pellucida was extracted with DCM at the room

temperature for three days and filtered using filter funnel with the cotton wool and was re-

filtered using filter paper. The filtrate was dark green in colour. This step was repeated three

times and the filtrates were combined. Then, the solvent was evaporated to dryness under

reduced pressure by using rotary evaporator to give 16.481 g of DCM crude extract. The

crude extracts with different concentrations were used for bioassay and for further isolation

and purification purposes.

10

3.4 Isolation and Purification of Chemical Constituents

3.4.1 Thin Layer Chromatography (TLC)

The crude extract from the P. pellucida was subjected to TLC analysis using silica plates

(Aluminium sheets, 20 x 20 cm; silica gel 60 F254). The samples were spotted 1 cm from the

bottom line of the plate using capillary tube and were allowed to dry. The TLC plate was then

developed in a suitable solvents with appropriate ratio depend on the polarity of the samples.

The plate was then examined under UV light, iodine vapour and vanillin solution. All the

spots shown were then marked and the retention factor, Rf for each spot was determined using

the equation below:

Rf = Distance travelled by the component

Hexane, dichloromethane, chloroform, ethyl acetate, ethanol and methanol were used as a

mobile phase in the analytical TLC, while iodine vapour and vanillin dipping act as a

visualizing agents to observe any additional spots on the TLC. First, the analytical TLC was

done using single solvent to observe which one of the solvents can be combined to give the

best separation. From the observation, the crude extract of P. pellucida gave the best

separation using hexane and chloroform. Therefore, the crude was submitted to further

analysis in the column chromatography and hexane and chloroform with the ratio from pure

hexane, 4:1, 1:1, 1:4 and pure chloroform were chosen to monitor the fractionation in the

column chromatography.

Rf = Distance travelled by the component

Distance travelled by the solvent

11

3.4.2 Iodine Vapour Test

The iodine crystal was placed in the container and left for a few minutes until the container

was filled with iodine gas. The TLC plate was then put in the container until brownish yellow

spots were observed.

3.4.3 Vanillin Dipping (Vanillin- Sulphuric Acid Reagent)

The vanillin solution was prepared by dissolving 1 g of vanillin in 100 mL of ethanol and

added with 1 mL of concentrated sulphuric acid. The solution was stirred until it became

homogenous. The TLC plate was developed in the vanillin and dried using hair dryer until

spots were observed.

3.4.4 Column Chromatography (CC)

About 10.0 g of dichloromethane crude of the Peperomia pellucida was used for column

chromatography (CC) analysis. CC was carried out on silica gel 60 (Merck, 0.040-0.063) with

column sized 21 cm height x 4.4 cm diameter. The column was prepared by using 120 g of

silica gel and compacted with cotton to avoid the silica gel from being eluted out. Sand was

added in a linear line. Then, the column was filled with the slurry silica gel and left overnight

to make sure it was really compacted. Finally, the sample was added to the column and eluted

using the solvent with increasing polarity (solvent ratio). A series of 50 mL eluent was

collected and the fractions were subjected to TLC. The fractions with the same Rf value were

then combined.

12

3.5 Analysis of Chemical Contituents

3.5.1 Gas Chromatograph – Mass Spectroscopy (GC-MS)

GC-MS was performed by using Hewlett Packard 6890 with column HP-5MS crosslinked,

non-polar 5% phenyl methyl siloxane (30.0 m x 0.25 mm x 0.25 μm film thickness). Helium

was used as the carrier gas and the inlet temperature was 280oC while interface temperature

was 300oC. Exactly 1 μL of the fractions was diluted in 200 μL of dichloromethane and 1 µL

of the diluted sample was injected into the GC – MS.

3.5.2 Fourier Transform Infra Red Spectrometer (FTIR)

Functional group of the selected fraction was determined using FTIR procedure.

Approximately 1.0 mg of the samples was mixed with 100.0 mg of potassium bromide (KBr).

The mixture was compressed to the film about 1mm thick under pressure. The FTIR spectrum

was recorded in the range of 400 cm-1

– 4000 cm-1

using FTIR Spectrum GX, Perkin Elmer.

3.6 Determination of Biological Activities

3.6.1 Toxicity to Artemia salina

Biological activity against the larvae of Artemia salina was carried out using the protocol by

McLaughlin et al., (1991). Artemia salina eggs were hatched in the sea water. The eggs were

added into 1 L of sea water at the temperature of 27ºC and pH 7.5. The test samples were

prepared by dissolving exactly 2.0 mg of extract in 2.0 mL methanol. Then the solutions were

divided into 5 µL, 50 µL and 500 µL and transferred into different multi-dish holes in triplicate