Antibacterial Activity of Aqueous Acetone Extracts from Acacia nilotica seeds

EXTRACTS FROM Acacia nilotica SEEDS

KHIDIR TAJELSEIR OTHMAN MUSTAFA

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

DECLARATION

I declare that this thesis titled “Antibacterial Activity of Aqueous Acetone

Extracts from

Acacia nilotica

Seeds” is my own work. Information and quotes that

were sources from journals and books have been acknowledged and mentioned where

in the thesis they appear. All complete references are given at the end of the paper.

Bogor, July 2009

Khidir Taj-Elseir Othman Mustafa

KHIDIR TAJELSEIR OTHMAN MUSTAFA. Antibacterial activity of aqueous

acetone extracts from

Acacia nilotica

seeds. Supervised by SUMINAR SETIATI

ACHMADI and NISA RACHMANIA MUBARIK.

The present study aimed at evaluating the

in vitro

antimicrobial activity of

aqueous acetone extracts of medicinal plant

Acacia nilotica

subsp.

nilotica

(Family:

Fabaceae

) seeds against Gram-negative bacteria

Escherichia coli

, Gram-positive

organisms

Staphylococcus aureus,

and Gram-negative marine bacteria

Vibrio

harveyi.

The crude extract of

A. nilotica

presented the highest anti-

S. aureus

activity

and was effective against all bacterial strains tested. The antibacterial activity of

aqueous acetone extract and isolated constituents of seeds and pods of

A. nilotica

were evaluated by the cup diffusion method against the three species of bacteria

.

The

crude extract was subsequently fractioned and monitored by bioassay leading to the

isolation of active fractions by further chemical analysis. These active fractions

showed highest antibacterial activity

in vitro

at 1000 ppm concentrations which

capable of inhibiting the growth of bacteria. The inhibition diameters were varied

from 0.1-0.6 cm. Sephadex LH-20, a liquid chromatography media, was used to

purify and to separate the active compounds, the result of the separations was 3

fractions, two were ethanol fractions, and one was acetone fraction. Two–dimension

thin layer chromatography showed these two fractions were not pure. Acute toxicity

test towards

Artemia salina

Lethal Dose 50 (LD

50

) result showed that the crude

extract of the seed has sufficient toxicity in low concentration, indicating that the

crude extract should be effective towards microorganism as well. Elucidation by

Fourier transform infrared and ultraviolet spectroscopy for these two fractions

presented good result which indicated they were part of tannins.

Key words: Acacia

nilotica

seeds, medicinal plants, antibacterial activity,

Escherichia

coli,

Staphylococcus

aureus, Vibrio harveyi

Summary

KHIDIR TAJELSEIR OTHMAN MUSTAFA. Antibacterial activity of aqueous

acetone extracts from

Acacia nilotica

seeds. Supervised by SUMINAR SETIATI

ACHMADI and NISA RACHMANIA MUBARIK.

Many efforts have been made to discover new antimicrobial compounds from

various kinds of plants. One of such resources is folk medicines. The increasing

prevalence of multidrug resistant strains of bacteria and the recent appearance of

strains with reduced susceptibility to antibiotics raises the specter of untreatable

bacterial infections and adds urgency to the search for new infection-fighting

strategies.

Acacia nilotica

is a tree 5-20 m high with a dense spherical crown, stems and

branches usually dark to black colored, fissured bark, grey-pinkish slash, exuding a

reddish low quality gum. Pods are strongly constricted, hairy, white-grey, thick and

softly tomentose. Its seeds number approximately 8000/kg.

 

A. nilotica

and other

acacia species are used in folk medicine by peoples in rural areas for illnesses caused

by bacteria, but this has not been confirmed by experiments.

Condensed tannins are natural preservatives as anti-fungal agents. This is the

reason for their presence in the outer tissues of plants – to help protect against attack

by a variety of pathogens including fungi. Condensed tannins are larger tannins and

toxic to many microbes. When condensed tannins are broken down their subunits

they are believed to be able to bind proteins but low molecular weight tannins are

toxic to fewer microbes because once the molecule is broken down the subunits loses

their protein binding abilities.

The present study aimed at evaluating the

in vitro

antimicrobial activity of

aqueous acetone extracts of medicinal plant

Acacia nilotica

subsp.

nilotica

(Family:

Fabaceae

) seeds against Gram-negative bacteria

Escherichia coli

, Gram-positive

organisms

Staphylococcus aureus,

and Gram-negative marine bacteria

Vibrio

harveyi.

From 400 g of the

A. nilotica

seeds which were extracted with aqueous

acetone

70% 

_{yielded 6% crude extract of the weight and its moisture content was}

12.58%. The addition of the FeCl

3

10% to the filtrate of the sample showed blackish

blue color, indicated the presence of tannins in the sample. Tannin content was

determined using butanol-HCl assay method (as % catechin) using U-2010

Spectrophotometer, wavelength 530 nm, and found the catechin content was 46.61%.

The crude extract of

A. nilotica

presented the highest anti-

S. aureus

activity

and was effective against all bacterial strains tested. The antibacterial activity of

aqueous acetone extract and isolated constituents of seeds and pods of

A. nilotica

were evaluated by the cup diffusion method against the three species of bacteria

.

The

crude extract was subsequently fractioned and monitored by bioassay leading to the

isolation of active fractions by further chemical analysis. These active fractions

showed highest antibacterial activity

in vitro

at 1000 ppm concentrations which

capable of inhibiting the growth of bacteria. The inhibition diameters were varied

from 0.1-0.6cm. Sephadex LH-20, a liquid chromatography media, was used to purify

and to separate the active compounds, the result of the separations was 3 fractions,

two were ethanol fractions, and one was acetone fraction. Two–dimension thin layer

chromatography showed these two fractions were not pure. Acute toxicity test

towards

Artemia salina

Lethal Dose 50 (LD

50

) result showed that the crude extract of

the seed has sufficient toxicity in low concentration, indicating that the crude extract

should be effective towards microorganism as well. Elucidation by Fourier transform

infrared and ultraviolet spectroscopy for these two fractions presented good result

which indicated they were part of tannins.

Acacia nilotica

originated from Gadarif area in the east of Sudan, was an

effective antibacterial plant as was confirmed by many other studies carried out

previously. It was found that the higher the concentration, the more effective the

antibacterial activity.

This research and the previous research confirmed the

usefulness of this plant

A. nilotica

as

a characteristic feature of medical plant and

pharmaceutical.

 

It is recommended that further work is required to learn a more specific area

in identifying the exact effective substances and their toxicity levels so as to find the

correct or safe levels of consumption.

© Copyrights property of Khidir Taj-Elseir Othman Mustafa, the year 2009

Copyrights is protected

Prohibited multiplies without written permission from Bogor Agricultural University,

partly or entirely in any form or by any means, electronic, photocopying,

 

ANTIBACTERIAL ACTIVITY OF AQUEOUS ACETONE

EXTRACTS FROM Acacia nilotica SEEDS

KHIDIR TAJELSEIR OTHMAN MUSTAFA

Thesis

As one of requirements to obtain degree of

Master of Science at

The Department of Chemistry

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

2009

Name : Khidir TajElseir Othman Mustafa

Registration Number : G451078131

Major : Chemistry

Approved by

Advisory Committee

………. ……...……….

Prof. Dr. Ir. Suminar S. Achmadi

Dr. Nisa Rachmania Mubarik, MSi

(Chairman) (Member)

Agreed

Coordinator of Major Dean of Graduate School

Programmer

………. ……..….……….

Prof. Dr. Ir. Latifah K. Darusman Prof. Dr. Ir. Khairil A. Notodiputro, M.S.

EXTRACTS FROM Acacia nilotica SEEDS

KHIDIR TAJELSEIR OTHMAN MUSTAFA

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

DECLARATION

I declare that this thesis titled “Antibacterial Activity of Aqueous Acetone

Extracts from

Acacia nilotica

Seeds” is my own work. Information and quotes that

were sources from journals and books have been acknowledged and mentioned where

in the thesis they appear. All complete references are given at the end of the paper.

Bogor, July 2009

Khidir Taj-Elseir Othman Mustafa

KHIDIR TAJELSEIR OTHMAN MUSTAFA. Antibacterial activity of aqueous

acetone extracts from

Acacia nilotica

seeds. Supervised by SUMINAR SETIATI

ACHMADI and NISA RACHMANIA MUBARIK.

The present study aimed at evaluating the

in vitro

antimicrobial activity of

aqueous acetone extracts of medicinal plant

Acacia nilotica

subsp.

nilotica

(Family:

Fabaceae

) seeds against Gram-negative bacteria

Escherichia coli

, Gram-positive

organisms

Staphylococcus aureus,

and Gram-negative marine bacteria

Vibrio

harveyi.

The crude extract of

A. nilotica

presented the highest anti-

S. aureus

activity

and was effective against all bacterial strains tested. The antibacterial activity of

aqueous acetone extract and isolated constituents of seeds and pods of

A. nilotica

were evaluated by the cup diffusion method against the three species of bacteria

.

The

crude extract was subsequently fractioned and monitored by bioassay leading to the

isolation of active fractions by further chemical analysis. These active fractions

showed highest antibacterial activity

in vitro

at 1000 ppm concentrations which

capable of inhibiting the growth of bacteria. The inhibition diameters were varied

from 0.1-0.6 cm. Sephadex LH-20, a liquid chromatography media, was used to

purify and to separate the active compounds, the result of the separations was 3

fractions, two were ethanol fractions, and one was acetone fraction. Two–dimension

thin layer chromatography showed these two fractions were not pure. Acute toxicity

test towards

Artemia salina

Lethal Dose 50 (LD

50

) result showed that the crude

extract of the seed has sufficient toxicity in low concentration, indicating that the

crude extract should be effective towards microorganism as well. Elucidation by

Fourier transform infrared and ultraviolet spectroscopy for these two fractions

presented good result which indicated they were part of tannins.

Key words: Acacia

nilotica

seeds, medicinal plants, antibacterial activity,

Escherichia

coli,

Staphylococcus

aureus, Vibrio harveyi

Summary

KHIDIR TAJELSEIR OTHMAN MUSTAFA. Antibacterial activity of aqueous

acetone extracts from

Acacia nilotica

seeds. Supervised by SUMINAR SETIATI

ACHMADI and NISA RACHMANIA MUBARIK.

Many efforts have been made to discover new antimicrobial compounds from

various kinds of plants. One of such resources is folk medicines. The increasing

prevalence of multidrug resistant strains of bacteria and the recent appearance of

strains with reduced susceptibility to antibiotics raises the specter of untreatable

bacterial infections and adds urgency to the search for new infection-fighting

strategies.

Acacia nilotica

is a tree 5-20 m high with a dense spherical crown, stems and

branches usually dark to black colored, fissured bark, grey-pinkish slash, exuding a

reddish low quality gum. Pods are strongly constricted, hairy, white-grey, thick and

softly tomentose. Its seeds number approximately 8000/kg.

 

A. nilotica

and other

acacia species are used in folk medicine by peoples in rural areas for illnesses caused

by bacteria, but this has not been confirmed by experiments.

Condensed tannins are natural preservatives as anti-fungal agents. This is the

reason for their presence in the outer tissues of plants – to help protect against attack

by a variety of pathogens including fungi. Condensed tannins are larger tannins and

toxic to many microbes. When condensed tannins are broken down their subunits

they are believed to be able to bind proteins but low molecular weight tannins are

toxic to fewer microbes because once the molecule is broken down the subunits loses

their protein binding abilities.

The present study aimed at evaluating the

in vitro

antimicrobial activity of

aqueous acetone extracts of medicinal plant

Acacia nilotica

subsp.

nilotica

(Family:

Fabaceae

) seeds against Gram-negative bacteria

Escherichia coli

, Gram-positive

organisms

Staphylococcus aureus,

and Gram-negative marine bacteria

Vibrio

harveyi.

From 400 g of the

A. nilotica

seeds which were extracted with aqueous

acetone

70% 

_{yielded 6% crude extract of the weight and its moisture content was}

12.58%. The addition of the FeCl

3

10% to the filtrate of the sample showed blackish

blue color, indicated the presence of tannins in the sample. Tannin content was

determined using butanol-HCl assay method (as % catechin) using U-2010

Spectrophotometer, wavelength 530 nm, and found the catechin content was 46.61%.

The crude extract of

A. nilotica

presented the highest anti-

S. aureus

activity

and was effective against all bacterial strains tested. The antibacterial activity of

aqueous acetone extract and isolated constituents of seeds and pods of

A. nilotica

were evaluated by the cup diffusion method against the three species of bacteria

.

The

crude extract was subsequently fractioned and monitored by bioassay leading to the

isolation of active fractions by further chemical analysis. These active fractions

showed highest antibacterial activity

in vitro

at 1000 ppm concentrations which

capable of inhibiting the growth of bacteria. The inhibition diameters were varied

from 0.1-0.6cm. Sephadex LH-20, a liquid chromatography media, was used to purify

and to separate the active compounds, the result of the separations was 3 fractions,

two were ethanol fractions, and one was acetone fraction. Two–dimension thin layer

chromatography showed these two fractions were not pure. Acute toxicity test

towards

Artemia salina

Lethal Dose 50 (LD

50

) result showed that the crude extract of

the seed has sufficient toxicity in low concentration, indicating that the crude extract

should be effective towards microorganism as well. Elucidation by Fourier transform

infrared and ultraviolet spectroscopy for these two fractions presented good result

which indicated they were part of tannins.

Acacia nilotica

originated from Gadarif area in the east of Sudan, was an

effective antibacterial plant as was confirmed by many other studies carried out

previously. It was found that the higher the concentration, the more effective the

antibacterial activity.

This research and the previous research confirmed the

usefulness of this plant

A. nilotica

as

a characteristic feature of medical plant and

pharmaceutical.

 

It is recommended that further work is required to learn a more specific area

in identifying the exact effective substances and their toxicity levels so as to find the

correct or safe levels of consumption.

© Copyrights property of Khidir Taj-Elseir Othman Mustafa, the year 2009

Copyrights is protected

Prohibited multiplies without written permission from Bogor Agricultural University,

partly or entirely in any form or by any means, electronic, photocopying,

 

ANTIBACTERIAL ACTIVITY OF AQUEOUS ACETONE

EXTRACTS FROM Acacia nilotica SEEDS

KHIDIR TAJELSEIR OTHMAN MUSTAFA

Thesis

As one of requirements to obtain degree of

Master of Science at

The Department of Chemistry

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

2009

Name : Khidir TajElseir Othman Mustafa

Registration Number : G451078131

Major : Chemistry

Approved by

Advisory Committee

………. ……...……….

Prof. Dr. Ir. Suminar S. Achmadi

Dr. Nisa Rachmania Mubarik, MSi

(Chairman) (Member)

Agreed

Coordinator of Major Dean of Graduate School

Programmer

………. ……..….……….

Prof. Dr. Ir. Latifah K. Darusman Prof. Dr. Ir. Khairil A. Notodiputro, M.S.

Thanks be to God Almighty for guiding, strengthening and for His endless

blessings that have seen this research work completed, in titled “Antibacterial

Activity of Aqueous Acetone Extracts from

Acacia nilotica

Seeds”. I would like to

express my gratitude to all those who gave me the possibility to complete this thesis. I

want to thank the Government of Indonesia for giving me scholarship and the

Government of Sudan for giving me permission to study in Indonesia and helping me

with support in Indonesia. Also I thank Bogor Agricultural University, IPB; I gave

my special thanks to the Department of Chemistry and Department of Biology to

their unlimited helping which made this work succeed. I have furthermore to thank

the Dean of the Graduate school, Mr. Khairil Anwar, and Miss Anissa Anwar, and

Mr. Drajat Martianto for their continuous helping and looking after us. I am deeply

indebted to my supervisors Prof. Dr. Ir. Suminar Setiati Achmadi, and Dr. Nisa

Rachmania Mubarik, M.Si whose guidance, stimulating suggestions, and

encouragement helped me in all the time of research for and writing of this thesis. I

will not forget thanks Dr. Irma Suparto as the examiner and Dr. Iman Rusmana for

usage of

Vibrio harveyi

. My former colleagues from the Department of Chemistry

supported me in my research work. I wont to thank them for all their help, support,

interest and valuable hints. Especially I am obliged to Miss Wulan Tri Wahyuni, Mrs.

Hariyanti, Miss Hasu Marty Linda, and Mr. Paulyasi Tokasaya Fiji. I also want to

thank Mr. Sabur, Mr. Iman, and Mrs. Heny for all their assistance on the laboratories

work. My Father was of great help in difficult times. My Daughter Naziha, as well as

my brother Mutaz for their stimulating support to do this thesis. Especially, I would

like to give my special thanks to my wife, Sewasin, who patient love enabled me to

complete this work. God bless you all.

Bogor, August 2009

Khidir Taj-Elsir Othman Mustafa

BIOGRAPHY

The writer Khidir Taj-Elsir Othman was born on 27

th

May, 1976 in Wad

Madani as a fourth child to Mr. Taj-Elsir Othman Mustafa and Mrs. Buthaina Abdul

Rahaman Mustafa from Sudan. Under Graduate was gone through the Chemistry

Study Program, Faculty of Science, Sudan University of Science and Technology,

from 1996 to 2000, higher diploma graduate school of chemistry, from 2003 to 2004.

The writer was accepted under the development country partnership program

scholarship offered by Indonesia Government to do Masters Degree in Bogor

Agricultural University in the Chemistry Study Program. The writer worked as

chemist in Hemaidan Soap Factory (February 2002 – February 2003) at the same

time he was teaching assistant in Sudan University of Science and Technology

(January 2002 – May 2005). On January 2003 – June 2004 he joined Medco

Company Ltd – Branch of Water Analysis as chemist and manager. Then he follow

Al-Mohandis Paint Factory (October 2004 – July 2005). Since August 2005 he settled

as Teaching Assistant in Gadarif University, Faculty of Education, Department of

Chemistry.

CONTENT

Page

CONTENT ………...…. xi

TABLES ………..…. xii

FIGURES ………..………. xiv

APPENDICES ……….…..………... xv

INTRODUCTION

Background ……….………….….. 1

Objectives, Purpose, and

Scope of Research ……….……….… 2

Place and Time of Study ...……….……….…. 2

LITERATURE REVIEW

Acacia nilotica

subsp.

nilotica

…………...……….………... 3

Tannins

……….……….… 5

Staphylococcus aureus ………...………..

7

Escherichia coli

………...

8

Vibrio harveyi

………... 9

Artemia salina

………..……….. 10

Lethal Dose 50 (LD

50

) ……….. 10

MATERIAL AND METHODS

Apparatus and Materials ...…... 12

Chemical Analysis

……… 12

Tannin Fractionation ……….. 13

Toxicity Test (LD

50

) ………..…... 14

Antimicrobial tests …..………... 14

RESULTS

Moisture content of the seeds ……….………. 15

Tannins content ………... 15

Ethanol and acetone extracts …….………... 15

Acute toxicity ………... 16

Antibacterial properties ………. 16

Chemical structure ………... 19

 

DISCUSSION

Phytochemical analysis ……… 20

Tannin content ……….. 21

Separation ………... 21

Acute toxicity test toward

A. salina

(LD

50

)

……..………...

22

Antibacterial Test ………. 23

Elucidation ………... 26

Comparing with Previous Reference about Antimicrobial from

A. nilotica

………..…... 27

CONCLUSION ………..……… 29

RECOMMENDATION ………..……….. 29

REFERENCE ……….………... 30

APPENDICES ……….……….………... 34

TABLES

Page

1 Acute toxicity of Karber’s Method ……….……… 16

2 Phytochemical analysis of

Acacia nilotica

leaves ……….. 20

3

Tannin content of various

Acacia nilotica

subspecies ……….………... 21

4 Toxicity Classes ………. 23

5 Antibacterial activity of plant extracts against

V. harveyi

………. 24

 

FIGURES

Page

1

Acacia nilotica

(a) Pods and (b) Seeds …………..……… 3

2 Chemical structure of flavan-3-ol; monomer units and condensed tannins ... 6

3 The presence of tannin using butanol-HCl assay ……….………... 15

4 TLC (a) one and (b) two dimensions of ethanol fraction, acetone fraction,

and crude extract ……….……… 16

5 Inhibition zones of (a)

V. harveyi and (b) S. aureus

………...

17

6

Inhibition zone of

V. harveyi

………..………. 18

7 Inhibition zone of

S. aureus

………...……. 18

8 Inhibition zone of

E. coli

………..………. 18

9 The UV spectrum of (a) ethanol fraction (b) acetone fraction ……….. 19

10 The FTIR spectrum of (a) ethanol fraction (b) acetone fraction ………. 19

APPENDICES

Page

1 Flow diagram of

A. nilotic

seeds testing procedures …………..……..……... 34

2 Calculation of moisture content ……….. 35

3 Result of butanol-HCl assay ………..……….……….. 36

4 Data of butanol-HCl assay ………..………...………... 37

5 Result of butanol-HCl analysis ……… 38

6 Row data of antibacterial tests ………..………... 39

7 Inhibition zone of bacteria and its index ………..……… 40

8 FT-IR spectrum catechin as standard ……….……....……….. 41

INTRODUCTION

Background

Many efforts have been made to discover new antimicrobial compounds from various kinds of plants. One of such resources is folk medicines. Systematic screening of them may result in the discovery of novel effective compounds. The increasing prevalence of multidrug-resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections and adds urgency to the search for new infection-fighting strategies.

Sudan throughout its long history has accumulated a rich body of empirical knowledge of the use of medicinal plants for the treatment of various diseases. Chemical studies of Sudanese medicinal plants provide a valuable material base for the discovery and development of new drugs of natural origin (Cowan 1999). Acacia nilotica and other acacia species are used in folk medicine by people in rural areas as a remedy for tuberculosis, leprosy, small pox, dysentery, cough, ophthalmic, toothache, skin ulcers, cancer, as astringents, antispasmodics, and aphrodisiac. A. nilotica species have been reported to have antihyper-glycaemic, antimicrobial, antiplasmodial, anti-inflammatory, analgesic, and antipyretic properties.

The bark and seeds are used as a source of tannins. The species is also used for medicinal purposes. The gum of A. nilotica is sometimes used as a substitute for gum Arabic (obtained from A. senegal) although the quality is inferior. The species is suitable for the production of paper and has similar pulping properties to a range of other tropical timbers (Nasroun 1979).

Tannins are large, phenolic polymers that bind proteins and inhibit enzymes. Many plants contain tannins for their anti-microbial and anti-herbivore effects. There are two main types of tannins. Condensed tannins are larger tannins and toxic to many microbes. When condensed tannins are broken down, their subunits are still able to bind proteins. Hydrolysable tannins are toxic to fewer microbes because once the molecule is broken down; the subunits lose their protein binding abilities (Speilman 2005). Condensed tannins are natural preservatives as

anti-fungal agents. This is the reason for their presence in the outer tissues of plants – to help protect against attack by a variety of pathogens including fungi (Peter 2004). Aqueous acetone is more effective extractive than that of alcoholic solvents and acetone extract inhibits tannin-protein interaction (Reed et al. 1985). The methanol extract of the pods of A. nilotica shows antihypertensive activities.

Objectives, Purpose, and Scope of Research

The objectives of this research were (1) to determine the antimicrobial activity of A. nilotica subsp. nilotica seeds extracts; and (2) to define the effective fraction of the extracts towards three species of bacteria(Staphylococcus aureus,

Escherichia coli, and Vibrio harveyi).

This research would like to confirm the usefulness of this plant as a medicinal plant. By carrying out this research, safety of people from illnesses

caused by bacteria may be enhanced and also medicinal effects of A. nilotica

seeds can be established. Trials were undertaken to examine the anti-bacterial activity of the seeds aqueous acetone extract against both Gram–negative and Gram–positive bacteria (three species, i.e. E. coli, S. aureus, and V. harveyi).

Furthermore, the effective fractions against these three bacteriawere determined.

Place and Time of Study

The research was carried out in Dramaga Bogor from December 2008 to June 2009, in the Analytical Chemistry Laboratory, the Organic Chemistry Laboratory, Department of Chemistry, and the Microbiology Laboratory, Department of Biology, in the Faculty of Mathematics and Natural Sciences (FMIPA), and in the Biopharmaca Study Center, LPPM, Bogor Agricultural University, Indonesia (IPB).

3   

LITERATURE REVIEW

Acacia nilotica subsp. Nilotica

Acacia nilotica subsp. nilotica (Family: Fabaceae) is a tree 5-20 m high with a dense spherical crown, stems and branches usually dark to black colored, fissured bark, grey-pinkish slash, exuding a reddish low quality gum. The tree has thin, straight, light, grey spines in axillaries pairs, usually in 3 to 12 pairs, 5 to 7.5 cm long in young trees, mature trees commonly without thorns. The leaves are bipinnate, with 3-6 pairs of pinnulae and 10-30 pairs of leaflets each, tomentose, rachis with a gland at the bottom of the last pair of pinnulae. Flowers in globulous heads 1.2-1.5 cm in diameter of a bright golden-yellow color set up either axillaries or whorl on peduncles 2-3 cm long located at the end of the branches. Pods are strongly constricted, hairy, white-grey, thick and softly tomentose. Its seeds number approximately 8000/kg (Fagg 2001).

A. nilotica (thorn mimosa) is a species of Acacia (wattle) native to Africa and the Indian subcontinent. It is also currently an invasive species of significant concern in Australia (Fagg 2001). A. nilotica which was introduced to Indonesia is subsp. indica (Djufri 2004).

(a) (b)

Figure 1 Acacia nilotica (a) pods and (b) seeds (Attenborough 1995).

Seeds are extremely hard-coated. They are oblong, 6½-11 mm long, 12-14 mm wide and 3½-4 cm thick, dark brown to blackish brown Fig (1-b). Pleurogram distinct oblong creamy white Fig (1-a), seed weight variable 1000 seed weight 100-250 g, equivalent to 4000 to 10000 seeds per kg (Kongevej 2008). A. nilotica belongs to family Fabaceae, subfamily Mimosoideae, tribe Acacieae, genus Acacia, species A. nilotica, and subspecies nilotica. Nine subspecies/varieties are

recognized, the most important are subsp. adstringens; subsp. kraussiana; subsp. nilotica; and subsp. tomentosa (Kongevej 2008).

Uses of A. nilotica. A. nilotica is one of the most important multipurpose

tree species of dry land Africa and South Asia. The wood is hard and used for general construction purposes and implements. It is fairly termite resistant. The wood yields an excellent fuel wood. Flowers are attracting bees and make a good base for apiculture. Pods are nutritious and make a high quality fodder for livestock especially during the dry season. The plant has alleged medical properties as the bark is used for treatment of cough, cancers, tumours of the ear, intestine pains and the roots to treat impotence (Kongevej 2008).

The pods and leaves are very popular with cattle. Pods are also used as a supplement to poultry rations in India (Fagg 2001). A. nilotica makes a good protective hedge because of its thorns. In Italian Africa, the wood is used to treat small pox. In Ethiopia certain parts of the tree are used as alactogogue. The wood is "very durable if water-seasoned" and its uses include tool handles and lumber for boats (Fagg 2001).

Also, A. nilotica be used as medicine. African Zulu take the (bark) for

cough. Furthermore, it acts as an astringent and it is used to treat diarrhea, dysentery, and leprosy. In West Africa, the bark or gum is used to treat cancers and/or tumors (of ear, eye, or testicles) and indurations of liver and spleen, and condylomas. Sap or bark, leaves, and young pods are strongly astringent due to

tannin, and are chewed in Senegal as an antiscorbutic. The bruised leaves are

poulticed and used to treat ulcers. In Lebanon, the resin is mixed with orange-flower infusion for typhoid convalescence. The Chipi use the root for tuberculosis. In Tonga, the root is used to treat tuberculosis. Egyptian Nubians believe that diabetics may eat unlimited carbohydrates as long as they also consume powdered pods (Fagg 2001).

Chemical Composition of Seeds and Pods. Annual availability of A. nilotica subsp. indica pods in India is about 600,000 metric tons. The tree is a browse tree with pods and leaves that make excellent fodder and are available

5   

when there is no grass. The digestible crude protein and total digestible nutrient values of acacia leaves are 10.21% and 66.46%, respectively (Barman & Rai 2003).

The total tannin content of acacia pods is 18.71% and that of the seeds. The mature seeds contain 234 g/kg crude protein, 126 g/kg crude fiber, 66.6 g/kg crude fat, and 234 g/kg carbohydrates. Potassium, phosphorus, magnesium, iron, and manganese occurred in high concentrations (Siddhuraju et al. 1995).

Storage and Viability. The dry seeds can be stored for several years even

at ambient temperature. Bruchid infested seed can be damaged during storage but the insects rarely re-infest new seed in storage (Kongevej 2008).

Tannins

Tannins are water-soluble polyphenols that are commonly foundin higher herbaceous and woody plants. They can be classified into two categories: hydrolysable and non-hydrolysable (condensed). Tannic acid is an important gallotannin belonging to the hydrolysableclass, while catechinbelongs to the non-hydrolysable class (Fig 2).Hydrolysabletannins are esters of phenolic acids and a polyol,usually glucose. The phenolic acids are either gallic acidin gallotannins, or hexahydroxydiphenic acid in ellagitannins. The hexahydroxydiphenic acid of ellagitannins undergoes lactonization to produce ellagic acid (Hagerman et al. 2006).

Proanthocyanidins (condensed tannins) are polymeric flavanoids. The flavanoids are adverse group of metabolites based on a heterocyclic ring system derived from phenylalanine (B) and polyketide biosynthesis (A). The most widely studied condensed tannins are based on the flavan-3-ols (-)-epicatechin and (+)-catechin (Fig 2). The best characterized condensed tannins are linked via carbon-carbon bond between C8 of the terminal unit and C4 of the extender. In addition to this linkage, related dimmers linked by C6 of the terminal unit and C4 of the extender have been isolated (Hagerman et al. 2006).

Figure 2 Chemical structure of flavan-3-ol; monomer units and condensed tannins (Hagerman et al. 2006).

Types of Tannins Effective against Bacteria. Tannins have been reported

to be bacteriostaticor bactericidal against S. aureus. Catechins fromtea leaf are composed mainly of (–)-epicatechin (EC), epigallocatechin (EGC), (–)-epicatechin gallate (ECG), and (–)-epigallocatechin gallate (EGCG). Oxacillin, ampicillin, and cefalexin showed increased antibacterial activity against methicillin-resistant S. aureus (MRSA) in the presence of catechin below microbiologically influenced corrosion (MIC). ECG markedly lowered the MICof oxacillin and other ß-lactams, but not of otherantimicrobial agents, for strains of MRSA. In the presence of plasma S. aureus forms a fibrin-rich biofilm that is highlyresistant to the immune system and conventional chemotherapy.Vermerris & Nicholson (2006) reported that 5% zinc oxide,0.12% calcium oxide, 0.25% magnesium oxide, 70% sucrose, and low pH inhibit plasma coagulation by S. aureus cells, and that the formationof biofilms would probably also be inhibited under these conditions. They further hypothesized that the inhibition of fibrin formationproduced by S. aureus cells could be an important strategy againstS. aureus infection in human skin. The purpose of theirstudy was to examine the antibacterial activity of several tanninson the coagulation of plasma by S. aureus

7   

and the effect ofincubating S. aureus for 24 h in the combination of conventional chemotherapy and tannic acid below the MIC.

Staphylococcus aureus

Staphylococcus aureus is a facultatively anaerobic, Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and has large, round, golden-yellow colonies, often with hemolysis, when grown on blood agar plates. The golden appearance is the etymological root of the bacteria's name: aureus means "golden" in Latin.

Staphylococcus aureus is catalase positive (meaning that it can produce the enzyme "catalase") and able to convert hydrogen peroxide (H2O2) to water and oxygen, which makes the catalase test useful to distinguish staphylococci from enterococci and streptococci. A large percentage of S. aureus can be differentiated from most other staphylococci by the coagulase test: S. aureus is primarily coagulase-positive (meaning that it can produce "coagulase", a protein product, not an enzyme) that causes clot formation while most other Staphylococcus species are coagulase-negative. However, while the majority of S. aureus are coagulase-positive, some may be atypical in that they do not produce coagulase. Incorrect identification of an isolate can impact implementation of effective treatment and/or control measures.

Staphylococcus aureus may occur as a commensal on human skin; it also occurs in the nose frequently (in about a third of the population) and throat less commonly. The occurrence of S. aureus under these circumstances does not always indicate infection and therefore does not always require treatment (indeed, treatment may be ineffective and re-colonization may occur). It can survive on domesticated animals such as dogs, cats and horses, and can cause bumble foot in chickens. S. aureus can infect other tissues when normal barriers have been breached (e.g., skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a collection of furuncles). In infants S. aureus infection can cause a severe disease Staphylococcal scalded skin syndrome (SSSS).

Staphylococcus aureus infections can be spread through contact with pus from an infected wound, skin-to-skin contact with an infected person by

producing hyaluronidase that destroy tissues, and contact with objects such as towels, sheets, clothing, or athletic equipment used by an infected person.

Deeply situated S. aureus infections can be very severe. Prosthetic joints put a person at particular risk for septic arthritis, and Staphylococcal endocarditic (infection of the heart valves) and pneumonia, which may be rapidly spread (Kluytmans et al. 1997).

Escherichia coli

Escherichia coli is Gram-negative, facultative anaerobic and non-sporulating. The cells are about 2 m long and 0.5 m in diameter, with a cell volume of 0.6-0.7 m3. It can live on a wide variety of substrates. E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide. Since many pathways in mixed-acid fermentation produce hydrogen gas, these pathways require the levels of hydrogen to be low, as is the case when E. coli lives together with hydrogen-consuming organisms such as methanogens or sulfate-reducing bacteria.

Optimal growth of E. coli occurs at 37 °C, but some laboratory strains can multiply at temperatures of up to 49 °C. Growth can be driven by aerobic or anaerobic respiration, using a large variety of redox pairs, including the oxidation of pyruvic acid, formic acid, hydrogen and amino acids, and the reduction of substrates such as oxygen, nitrate, and dimethyl sulfoxide.

Strains that possess flagella can swim and are motile, but other strains lack flagellum. The flagella of E. coli have a peritrichous arrangement. E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population. This process led to the spread of the gene encoding shiga toxin from Shigella to E. coli O157:H7, carried by a bacteriophage. E. coli normally colonize an infant's gastrointestinal tract within 40 hours of birth, arriving with food or water or with the individuals handling the child. In the bowel, it adheres to the mucus of the large intestine. It is the primary facultative organism of the human gastrointestinal tract. As long as these bacteria

9   

do not acquire genetic elements encoding for virulence factors, they remain benign commensalism.

Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis. In rare cases, virulent strains are also responsible for hemolytic-uremic syndrome (HUS), peritonitis, mastitis, septicemia and Gram-negative pneumonia. Recently it is thought that E. coli and certain other food borne illnesses can sometimes trigger serious health problems months or years after patients survived that initial bout (Vogt &Dippold 2005).

Vibrio harveyi

It’s a species of Gram negative, bioluminescent, marine bacteria in the genus Vibrio. V. harveyi are rod-shaped, motile (via polar flagella), facultatively anaerobic, halophilic, and competent for both fermentative and respiratory metabolism. They do not grow at 4°C or above 35°C. V. harveyi can be found free-swimming in tropical marine waters, commensally in the gut micro flora of marine animals, and as both a primary and opportunistic pathogen of marine animals, including Gorgonian corals, oysters, prawns, lobsters, the common snook, barramundi, turbot, milkfish, and seahorses. V. harveyi is responsible for luminous vibriosis, a disease that affects commercially-farmed penaeid prawns. Additionally, based on samples taken by ocean-going ships, V. harveyi is thought to be the cause of the milky seas effect, in which, during the night, a uniform blue glow is emitted from the seawater. Some glows can cover nearly 6,000 square miles (Austin & Zhang 2006).

Vibrio harveyi, which now includes Vibrio carchariae as a junior synonym, is a serious pathogen of marine fish and invertebrates, particularly penaeid shrimp. In fish, the diseases include vasculitis, gastro-enteritis and eye lesions. With shrimp, the pathogen is associated with luminous vibriosis and Bolitas negricans. Yet, the pathogenicity mechanisms are imprecisely understood, with likely mechanisms involving the ability to attach and form biofilms, quorum sensing, various extracellular products including proteases and haemolysins, lipopolysaccharide, and interaction with bacteriophage and bacteriocin-like substances (Austin & Zhang 2006).

Artemia Salina

Artemia salina is a species of brine shrimp, primitive, aquatic crustacean that are more closely related to Triops and cladocerans than to true shrimps. They can tolerate fluctuations in the salinity of water — they can live in water having much more or much less salt content than normal seawater. The resilience of these creatures makes them ideal test samples in experiments. The females can produce fertilized eggs in parthenogenesis. These eggs can remain in a dormant state, in cysts, for a number of years until they are placed in water. Some animals are capable of living in very odd and extreme environments. Excellent examples of extreme environments are found in highly saline lakes and pools. These vary considerably in their total osmotic concentration, and in the nature of the solutes present.

The ability of A. salina to live in such brines has long been known. Artemia can survive in several most unlikely media. For example, it has been known that Artemia is capable of living for days in solutions of potassium permanganate, potassium bichromate, and silver nitrate. Such statements as these might be taken as evidence that Artemia is highly impermeable. A study of the survival of Artemia in various media may thus give information concerning the permeability of the animal and possibly also about the regulatory mechanisms (Croghan 1957). Brine shrimp eggs were supplied by Ocean Star International Inc. Larvae were used within 1 d of hatching (Lee et al. 1999)

Lethal Dose 50 (LD50)

LD50 test involves the administration of a substance to a group of animals at increasing doses in order to determine the dose that kills 50 percent of the test subjects within a set time frame. Typically, administration of the test substance is via a tube inserted down the esophagus into the stomach. Other routes of administration include inhalation and applying the substance to the animals’ skin. Several dose levels of a chemical are given until the dose lethal to half of the test population is attained. The test is typically allowed to proceed for 14 days, at which time all the animals who have not died from the test substance are killed. Animals that have not died within the test period may be sick or near death. The

11   

LD50 provides no information on what system failure led to the death of the animals. Some deaths may be due to the quantity of the test substance causing gastric rupture or other morbidity unrelated to the toxicity of the test substance.

The designers of the LD50 test in 1927 acknowledged its serious inadequacies, intending it only for certain narrow medical purposes. Nevertheless, use of the LD50 test has become widespread as a general measurement of chemical toxicity. The LD50 has been challenged for decades as both unreliable and uninformative (Kaufmann & Cohen 1986).

MATERIALS AND METHODS

Apparatus and Materials

The apparatuses used were extraction apparatus, rotary evaporator, freeze dryer, TLC silica gel (thin layer chromatography), Sephadex LH 20, instruments for bacterial test, Fourier Transform Infrared Shimadzu 2010A Spectro-photometer, UV U- 2800 Spectrophotometer.

Acacia nilotica subsp. nilotica seeds collected from Gadarif area in east of Sudan were the plant parts examined for the antimicrobial test. These seeds were saved in dark plastic storage box set at room temperature. Three species of bacteria (S. aureus, E. coli, V. harveyi) were collected in Microbiology Laboratory, Department of Biology, Bogor Agricultural University.

Chemical Analysis

Moisture Content. About 1 g of the finely ground material was

transferred into a tarred wide-mouth weighing bottle and weighed accurately. The sample was dried at 105 °C in an oven for 4 hours, cooled in a desiccator for about 20 minutes and weigh again accurately. The procedure was repeated until three weightings at an interval of one hour until a constant weight. The moisture content (%) was calculated from the sample weight before and after drying. The equation used was the following:

MC % = {(W2 −W3)/ (W3 – W1)} × 100 ……… (1)

where W1 = weight of weighing bottle (g), W2 = weight of moist sample + weighing bottle (g), W3 = weight of dried sample + weighing bottle (g), MC % = moisture content (%).

Tannin Test. About 1 g of the extract was dissolved in 20 ml of hot

distilled water and filtered. Two to three drops of FeCl310% was added to 2 ml of the filtrate. The production of blackish-blue or blackish-green coloration was indicative of tannins (Adegboye et al. 2008).

13   

The Butanol-HCl Assay (Hagerman et al. 2006). A colorimetric assay

involves the oxidative cleavage of proanthocyanidins with ferrous sulfate. About 1g of the aqueous acetone extract was dissolved in 100 ml of the aqueous acetone. To (0.5 and 0.1) ml of aqueous acetone plant extract solution and to (0.0, 0.1, 0.2 and 0.5) ml of standard (catechin which was solved in aqueous acetone 70%), was added a 5-mL portion of an acidic solution of ferrous sulfate (77 mg of FeSO4⋅7H2O dissolved in 500 mL of 2:3 HCl/n-butanol). The tubes was loosely covered and placed in a water bath at 95 °C for 15 min. The absorbance was read at 530 nm. Tannin content in aqueous acetone extracts was determined (as % catechin) using U-2010 Spectrophotometer, wavelength 530 nm (used for the standard curve). The molecular extinction coefficient ε mol that can be used to convert the absorbance values to a concentration is equal to 34700 L mol-1cm-1.

% Tannin (mg/100 g) = (As/Ast) × Fd × 100% ……… (2)

where As = absorbance of the sample; Ast = absorbance of the standard; Fd = dilution factor.

Tannin Fractionation

About 400 g of seeds of A. nilotica were cleaned and dried in open air and powdered, submerged in 70% aqueous acetone, and 1% ascorbic acid, then the aqueous phase was cleaned up by several washing with n-hexane (25 ml, 6 times). The water layer was washed with ethyl acetate (10 ml, 10 times). Then the solvents were removed by rotary evaporation and freeze dried (Lorenz 2002). The crude extract was applied to the liquid column chromatography using Sephadex LH 20 (Van Soest et al. 1999) as stationary phase and washed with 95% ethanol to elute the tannin with low molecular weight. The substance which was adsorbed at Sephadex LH 20 was treated with aqueous acetone of 50%.

The fractions were monitored with one-dimension TLC using TBA (t -butanol: acetic acid:water, 3:1:1) as a solvent system (Saeidnia et al. 2008), and two-dimension TLC using TBA 14:1:5, followed with acetic acid 6%, and they were detected with short UV-ray (Harbone 2006). The separated fractions were tested for antimicrobial activity against three species E. coli, V. harveyi, and S. aureus. Inhibition zones of the bacteria testing were calculated. Subsequently, the

fractions that had microbial activity were elucidated using FT-IR and UV spectrophotometer (Appendix 1).

Toxicity Test (LD50)

Crude extract was dissolved in sea-water to obtain concentration series of

10, 15, 20, 25, 50 ppm; Tween-80 was added to the homogenized solvents. To each mixture ten A. salina larvae were introduced. After 24 hours, observation was done on the living and the dead of the larvae (Sukardiman et al. 2004). The equation used is the following:

log LD50 = Xk – d/2 ∑ (Pi + Pi+1) ………. (3)

where i = group number, 1, 2, 3, 4, 5, and 6; Xk = the max of logarithmic dosage; d

= the difference of two border upon logarithmic dosage, d = lgD_i+1 - lgD_i; and Pi =

the death rate of each group (Jun et al. 2006).

Antimicrobial Tests

Kirby Bauer Method (Gary et al. 1980) was done in a sterile petri dish for each strain of bacteria, each agar surface was completely covered with one of the bacterial strains with concentration of 106 CFU/ml (colony forming units per ml of bacteria), then addition of the various concentrations of different fractions individually and crude extract; 0, 50, 100, 200, 300, 400, 500, 600 and 1000 ppm using papers disk. The sample Petri dishes were incubated at room temperature for 24 hours, and diffusible antibacterial agents showed zone of inhibition. This procedure was followed for all microbes used except for V. harveyi that had a slight difference to the other two by using 50% SWT (sea water complete) instead of distilled water. This is because of the bacteria is a halophile (salt-loving). The index was calculated (means of the difference between diameter of inhibition zone and the diameter of paper disk over the diameter of paper disk).

15   

RESULTS

Moisture Content of The Seeds

From 400 g of the A. nilotica seeds which were extracted with aqueous acetone yielded 21g and its moisture content was 12.58% (Appendix 2).

The result of moisture content led to the dry weight of the sample was 349.68 g and the crude extract was 21 g then the percentage of the crude extract was 6% of the weight.

Tannin Content

The addition of the FeCl310% to the filtrate of the sample showed blackish blue color (Figure 3) indicated the presence of tannins in the samples. In aqueous acetone extracts as determined using butanol-HCl assay method using U-2010 Spectrophotometer at wavelength 530 nm, the tannins was 40.75% (Appendix 3) as % catechin. The data in Appendix 4 and the calculation is shown in Appendix 5.

Figure 3 The presence of tannin using butanol-HCl assay.

Ethanol and Acetone Extracts

Column chromatography separation for the crude extract gave three fractions, two ethanol fractions (F1 and F2) and acetone fraction (F3). These fractions were based on thin layer chromatogram one- and two-dimension. The

ethanol fraction F1 and acetone fraction F3 were more pure than F2 fraction (Figure 4).

(a) (b)

Figure 4 TLC (a) one and (b) two dimensions of ethanol fraction, acetone fraction, and crude extract.

Acute Toxicity

The result of acute toxicity as tested toward LD50 of A. salina showed that the crude extract of A. nilotica has enough toxicity in low concentration, indicating that the crude extract may be effective towards microorganism as well (Table 1).

Table 1 Acute toxicity of Karber’s method Group

number

Dose mg/kg

Dose differences

mg/kg = a Dead

Mean b

Product a*b

1 50 - 7 -

2 25 25 6 6.5 162.5

3 20 5 4 5 25

4 15 5 3 3.5 17.5

5 10 5 0 1.5 7.5

LD50 = 50 – (212/7) = 19.7 mg/kg T = 212.5

Antibacterial Properties

The extract sample and its fractions from the A. nilotica seeds obtained from Sudan Gadarif inhibited the growth of the three bacterial targets with strong

17   

inhibition zone (Figure 5). The antibacterial activity of aqueous extract were evaluated by the cup diffusion method against three species of bacteria: S. aureus, (Gram-positive organisms), E. coli (Gram-negative), and V. harveyi (Gram negative). One marine bacteria in the genus Vibrio and two human pathogenic bacteria.

The raw data of antibacterial test were presented in Appendix 6.

(a) (b)

Figure 5 Inhibition zones of (a) V. harveyi and (b) S. aureus.

Aqueous, methanol, and ethanol extracts showed significant antibacterial activity against all the bioluminescent, marine bacteria in the genus Vibrio and the two human pathogenic bacteria tested. Crude extract was subsequently fractioned and monitored by bioassay leading to the isolation of active fraction by further chemical analysis. This active fraction recorded highly significant antibacterial activity in vitro MIC 400-1000 ppm for E.coli, 100-1000 ppm for S.aureus and 100-1000 ppm for V. harveyi bacteria (figures 6-8). Appendix 6 also revealed that further separation of active fraction resulted in the loss of antibacterial activity, indicating synergistic effect of the isolated active fraction. With regards to purity, the fractions which were applied in testing for antibacterial activity were ethanol fraction F1 and acetone fraction F3 in addition to the crude extract. The zone of inhibition (Appendix 7) in case of crude extract varied from 0.1 to 0.4 cm. In case of ethanol fraction F1, it varied from 0.1 to 0.4 cm. For acetone fraction, it was from 0.1 to 0.6 cm. The highest degree of inhibition zone was observed against S. aureus and the lowest degree of inhibition zone was against E. coli by the crude extract and the ethanol and acetone fractions.

Figure 6 Inhibition zone of V. harveyi.

Figure 7 Inhibition zone of S. aureus.

19   

Chemical Stucture

Ethanol fraction and acetone fraction were characterized by FT-IR and UV spectrophotometer. UV spectrum of ethanol fraction (Figure 9-a) with solvent ethanol read in max = 248nm, 305 nm and 418 nm, and FT-IR absorbance of ethanol fraction (Figure 10-a), and FT-IR spectra of ethanol fraction (Figure 10-a) resemble FT-IR spectra of catechin (Appendix 8), but the UV showed only one max = 320 nm (Figure 9-b) and FT-IR spectrum of acetone fraction (Figure 10-b).

(a)

(b)

Figure 9 The UV spectrum of (a) ethanol fraction (b) acetone fraction.

(a)

(b)

Figure 10 The FTIR spectrum of (a) ethanol fraction (b) acetone fraction.

DISCUSSION

Phytochemical Analysis

Addition of 1% FeCl3 to the solution of crude extract in hot water gave blue color; meaning that A. nilotica seeds contained tannins. According to Table 5, Satish et al. (2006) phenolic compounds and tannins were found in both ethanol and methanol extracts, indicating that these compounds are favorably extracted in polar solvent. In addition to that, Reed et al. (1985) mentioned that aqueous acetone is a more effective extracting agent than alcoholic solvents. Aqueous acetone was used because acetone inhibits tannin-protein interaction (Hagerman et al. 1986). Phytochemical analysis of the aerial parts of the A. nilotica demonstrated the presence of tannin as shown by the blue color in the FeCl3 test evidence flavonoids and polyphenolic compounds in the flowers, tannin glycosides, volatile oil organic acids, coumarins, and carbohydrates in the seeds (Haj-Ali & Yagoub 2007). Phytochemical analysis of all the solvent extracts revealed the presence of carbohydrates and glycosides, phytosterols, phenolic compounds, saponins, flavonoids, proteins and amino acids, gums and mucilages in both methanol and ethanol extract in the leaves (Table 2).

Table 2 Phytochemical analysis of Acacia nilotica leaves(Satish et al. 2006)

Test for Petroleum

ether

C6H6 CHCl3 CH3OH C2H5OH

Carbohydrates and glycosides

-- -- -- ++ ++

Proteins and amino acids -- -- -- ++ ++

Saponins -- -- -- ++ ++

Phenolic compound/ tannins

-- -- -- ++ ++

Flavonoids -- -- ++ ++ ++

Further phytochemical analysis revealed that the antibacterial activity of methanol and ethanol extract is due to the presence of acidic compounds. Antibacterial activity was not observed in isolated compounds indicating the loss of antibacterial activity on further separation of the active fraction (Satish et al.

21   

2006). The stem ethanolic bark extract of the plant possessed the active compounds e.g. tannins, terpenoids, saponins, and glycosides (Banso 2009).

Table 3 Tannin content of various Acacia nilotica subspecies (Barnes 2006)

A. nilotica

subspecies

Native Tannins %

Pods and seed Bark

Adstringens Africa, Asia and middle east Source of tannins Source of gum and tannin,

Cupressiformis India and Pakistan ? ?

Hemispherica native to Pakistan ? ?

Indica Africa, Asia and middle east 18-27% 12-20%

kraussiana Africa and middle east 38% 38%

Leiocarpa Ethiopia, Kenya, Mozambique, Oman, Somalia, Tanzania

Source of tannins Source of gum

and tannin,

Nilotica Africa middle east Indian ocean

50% +++

Subalata Africa, India & Pakistan Source of tannin Source of tannin

Tomentosa Africa, India &Asia Deseeded 50% +++

Tannin content

According to the result of moisture content which was 12.58%, tannin content was determined using butanol-HCl assay method (as % catechin) using U-2010 Spectrophotometer, wavelength 530 nm, and found the catechin content was 46.61% (Appendix 5). A. nilotica subsp. nilotica conformed to Table (3) Barnes (2006) content tannins in pod seeds around 50% compared with 46.6% in this research. In many plants, there is a large fraction (some times >50%) of the tannins that can not be extracted (insoluble tannins) (Reed et al. 1985).

Separation

Column chromatography separation for the crude extract gave three fractions, two ethanol fractions F1 and F2 and acetone fraction F3. Based on thin layer chromatogram (Figure 4) ethanol fraction F1 and acetone fraction F3 were more pure than F2 fraction. With regards to purity, the fractions which were applied in testing for antibacterial activity were ethanol fraction F1 and acetone

fraction F3 in addition to the crude extract. Ethanol fraction and acetone fraction were elucidated by FT-IR, UV spectrophotometer.

The remaining water-soluble residue of the crude acetone extract of Bruguiera gymnorrhiza bark was applied to an LH 20 Sephadex column that was eluted with methanol-water (1:1) to remove carbohydrates but retain oligomers containing a high proportion of proanthocyanidins polymers, but that were elutable with acetone-water (1:1). The acetone-water eluted polymer was reacted with phloroglucinol as a capture nucleophile in the presence of catalytic amounts of acetic acid, and the reaction product was separated on LH-20 Sephadex using ethanol as the solvent to isolate a series of oligomeric proanthocyanidin-4-phloroglucinol adducts. Epicatechin-(4β-2)-phloroglucinol and epicatechin-(4β -8)-epicatechin-(4β-2)-phloroglucinol were major products of this reaction, but other (as yet unidentified) products are also present. In the course of study of these products, a compound appearing at Rf 0.45 and 0.67 on cellulose TLC plates

developed with t-butanol-acetic acid-water (3:1:1) and 6% acetic acid, respectively, gave a red coloration after spraying with vanillin-HCI. This compound was isolated in chromatographically pure form by subsequent chromatography on LH-20 Sephadex using ethanol-water (7:3) as an eluting solvent (Achmadi et al. 1993).

Acute Toxicity Test Toward A. salina (LD50)

From Karber’s method of calculation, LD50 values gave good result (Table 1). The result shows that the crude extract of A. nilotica has enough toxicity (LD50 19.7 mg/kg) in low concentration, indicating that the crude extract may be effective towards microorganism as well.

The result of LD50 =19.7 mg/kg of this research (Table 7)was denoted as extremely toxic group according Toxicity Classes of Gosselin et al. (1984). As a measure of toxicity, LD50 is somewhat unreliable and results may vary greatly between testing facilities due to factors such as the genetic characteristics of the sample population, animal species tested, environmental factors, and mode of administration. Another weakness is that it measures acute toxicity only (as opposed to chronic toxicity at lower doses), and does not take into account toxic

23   

effects that do not result in death but are nonetheless serious. There can be wide variability between species as well; what is relatively safe for rats may very well be extremely toxic for humans, and vice versa. In other words, a relatively high LD50 does not necessarily mean a substance is harmless, but a very low one is always a cause for concern (Hodgson 2004).

Table 4 Toxicity Classes (Gosselin et al. 1984)

Probable Oral Lethal Dose (Human)

Toxicity Rating or Class Dose For 70-kg Person (150 lbs)

6 Super Toxic Less than 5

mg/kg

1 grain (a taste - less than 7 drops)

5 Extremely Toxic 5-50 mg/kg 4 ml (between 7 drops and 1 tsp)

4 Very Toxic 50-500 mg/kg 30 ml (between 1 tsp and 1 fl ounce)

3 Moderately Toxic 0.5-5 g/kg 30-600 ml (between 1 fl oz and 1 pint)

2 Slightly Toxic 5-15 g/kg 600-1200 ml (between 1 pint to 1 quart)

1 Practically Non-Toxic Above 15 g/kg More than 1200 ml (more than 1 quart)

Antibacterial Test

The active principles identified in this study exhibited antimicrobial activity against all the test organisms (Figures 6-8). Several plants, which are rich in alkaloids, tannins and glycosides, have been shown to possess antimicrobial activity against a number of microorganisms. For example, Adebajo et al. (1983) investigated the antimicrobial activity of leaf extract of Eugenia uniflora and reported that tannins, glycosides and alkaloids were detected and that the ethyl acetate and methanolic leaf extract of the plant were active against E. coli, P. vulgaris, K. pneumonia, and Aspergillus niger. Tannins have been reported to prevent the development of microorganisms by precipitating microbial protein and making nutritional protein unavailable for them (Banso 2009).

The crude extract sample from the A.nilotica seeds obtained from Sudan Gadarif area inhibited the growth of the three bacterial targets with clear inhibition zone. The zone of inhibition in case of crude extract varied from 0.1 to 0.4 cm. In case of ethanol fraction F1, it varied from 0.1 to 0.4 cm. For acetone fraction, it was from 0.1 to 0.6 cm. The highest degree of inhibition zone was

observed against S. aureus and the lowest degree of inhibition zone was against E. coli by the crude extract and the ethanol and acetone fractions.

The large size of the zones of inhibition indicated the potency of the active principles of the plant (Appendix 7), it was recorded that an increase in the concentration of the extract yielded higher activity as shown by the diameter of zone of inhibition. The fact that organisms may need higher concentrations of extracts to inhibit or kill them may be due to their cell wall components (Banso 2009).

Vibrio harveyi. Lawsonia inermis and A. indica showed high antibacterial activity against V. harveyi (Table 5). Due to its less toxic effect to shrimp and potent antibacterial activity the acetone extract of L. inermis could form one of the best options for developing novel antimicrobial compounds for eco-friendly management of disease caused by V. harveyi (Asha et al. 2008).

Table 5 Antibacterial activity of plant extracts against V. harveyi (Asha et al. 2008)

Concentration (µ1) Zone of inhibition (mm)

L. inermis A. indica

5 7.0 ±0 6.0 ± 0

10 7.33 ± 0.47 6.67 ± 0.47

20 7.66 ± 0.47 7.33 ± 0.47

40 8.33 ± 0.47 10.33 ± 1.88

80 10.33 ± 0.47 12.33 ± 2.05

In Figure 6, out of the three fractions of aqueous extract of A. nilotica seeds, the best result was obtained by acetone fraction and the crude extract showed the lowest inhibition activity. The highest concentration (1000 ppm) having the highest inhibition zone of 0.6 cm for the best fraction (acetone). Both acetone and ethanol fractions are more pure compared to the crude extract, therefore, giving a better inhibition effect than the crude extract. This means that antibacterial substances in acetone fraction and ethanol fraction are more effective than the crude extract.

25   

Explore application of green tea to water treatment for shrimp aquaculture, especially for rearing young penaeid shrimp, the anti-microbial activity of green tea powder (GT) and green tea polyphenols (GTPP) against pathogenic bacteria V. harveyi were examined using nutrient rich medium and sterilized sea water. The results of the experiments showed that both GT and GTPP have a high antibacterial activity against the pathogen. In the nutrient rich medium (325 agar mixed with GT and GTPP), 1000 ppm of GT or 400 ppm of GTPP exhibited antibacterial activity against Vibrio harveyi. In sterilized sea water, the antibacterial activity was demonstrated at 800 ppm of GT or 300 ppm of GTPP (Sornsanit et al. 2002).

Asha et al. (2008) and Sornsanit et al. (2002) report about A. indica antibacterial activity against V. harveyi and anti-microbial activity of green tea powder (GT) and green tea polyphenols (GTPP) against pathogenic bacteria V. harveyi. Antibacterial activity of polyphenols had being researched many a time by previous and current researchers like the polyphenols from A. nilotica in this research.

Staphylococcus aureus. According to Figure 7, the bacterial inhibition by crude extract and acetone fraction gave the best effect at the highest concentration (1000 ppm). At lower concentrations both ethanol and acetone fractions gave higher inhibition effect, whilst at higher concentrations crude extract and acetone fraction gave higher inhibition effects. Generally, ethanol extract had an average antibacterial effect around the 0.25-0.4 cm inhibition zones. Unlike V. harveyi, where acetone fraction was more effective, for S. aureus crude extract can be noted as highly effective at higher concentrations (1000 ppm), therefore its use without further fractionation can be exercised. This would be important in reducing economic burden as in more processes carried out to separate effective compounds.

Escherichia coli. In E.coli inhibition (Figure 8), acetone fraction gave the best overall inhibition effect followed by crude extract and ethanol fraction respectively. It can be observed that acetone fraction and crude extract give less

difference with antibacterial effects as compared to ethanol fraction in higher concentrations.

The concentration used for antibacterial activity assay of A. nilotica leaves by Satish et al (2008) was 50µl of the aqueous extract and 106 CFU/ml of bacteria. Highly significant degree of activity was observed against S. aurous and E. coli. Antibacterial effect by 3.5 & 1.5 cm zone of inhibition was recorded in A. nilotica leaves against S. aurous and E. coli respectively (Satish et al. 2008).

According to Figures 6, 7, and 8, acetone fractions in higher concentrations from 500–1000 ppm gave the best inhibition effect for all three bacterial species which were use in concentration of 106 CFU/ml of bacteria. The average antibacterial activity against the three bacteria using all the fractions revealed that V. harveyi was the most inhibited organism. V. harveyi and E. coli they are gram negative bacteria, and the amino acid sequence of Vibrio harveyi acyl carrier protein (ACP) is 86% identical to that of Escherichia coli ACP, although five non conservative amino acid differences are concentrated in the loop region between helices I and II (Marc et al. 1997). These bacteria naturally resided from E. coli and S. aureus in humans, and animals, etc. whilst V. harveyi being in salt water.

Elucidation

Ethanol fraction showed clearer spectrum of UV and FT-IR. UV spectrum of ethanol fraction (Figure 9-a) with solvent EtOH giving a reading of max = 248nm, 305 nm and 418 nm. Maximum synthesis of catechin occurred at 190-320 nm that represented UV light, followed by blue light at 380-420 nm. MeOH max of 248nm and 305 nm (John 2003), FT-IR spectra of ethanol fraction (Figure 10-a) resemble FT-IR spectra of catechin (Appendix 8) which was indicated by the compound in ethanol fraction content structure of flavan. However, the UV spectrum of acetone fraction showed only one max = 320 nm (Figure 9-b) and FT-IR spectrum of acetone fraction (Figure 10-b). These results could be explained by where the ethanol fraction was more pure the acetone fraction was less pure.

Various functional groups have characteristic absorptions and their presence can be used for diagnostic purposes. FTIR spectra can be acquired in transmission

27   

mode with an FTIR microscope after drying a cell wall suspension on a BaF2 microscope slide (Sené et al. 1994), or by pressing a thin potassium bromide pellet containing finely ground plant tissue and measuring the absorbance of infrared light going through the pellet with the use of an FTIR spectrometer (Vermerris et al. 2002).

Lorenz et al. (2002) reported, the extraction with 70%(v/v) aqueous acetone was standard method to purified condensed tannins, this result was confirmed with chemical analysis, spectroscopy analysis, column chromatography Sephadex LH 20 which indicated that ethanol fraction F1 was tannin, with low molecular weight from 287 to 500 unit, and the acetone fraction was condensed tannins with high molecular weight from 500 to over 9000 unit. The result of the elucidation confirmed the qualitative test which showed that these compounds were part of the polyphenols; more specifically was the condensed tannins.

Comparing with Previous Reference about Antimicrobial from A. nilotica

Previous studies revealed that Acacia species are effective antibacterial plants. Haj-Ali & Yagoub (2007) reported that A. nilotica fruits were effective against S. aureus, E. coli and V. harveyi using ethanol and chloroform extracts. Satish et al. (2006) tested A. nilotica leaves amongst 46 other plants against S.aureus and found a high zone of inhibition (0.9–3.55 cm). This zone of inhibition range is higher than this research work (highest degree of inhibition zone of 0.6 cm). These results could be from Satish et al. (2008) using A. nilotica leave extract as crude extract and in case of this research, using A. nilotica seeds as fractions and crude extract

Tannins compounds showed relevant antibacterial activity against Gram positive and Gram negative bacteria content S. aureus and E. coli they found in aqueous acetone in addition to both ethanol and methanol extracts, meaning that these compounds are favorably extracted in polar solvent.

Manríquez-Torres et al. (2007) wrote in his article, the dry aerial parts of Acacia cochliacantha were successively extracted with hexane, ethyl acetate, and methanol by maceration. These extracts showed relevant antibacterial activity against Gram positive and Gram negative bacteria content S. aureus and E. coli.

The ethyl acetate and hexane residues of the flowers and leaves presented the best activity, which mean A. cochliacantha had a few content of tannins.

The antimicrobial activity of hot water and ethanolic extracts of six plant extracts, utilized in Pakistan for the cure of liver damage, were studied. The extracts of A. arabica, Nymphaea lotus,Sphareranthus hirtus, Emblica officinalis, Cinchorium intybus, and Cardus marianum were tested in vitro against seven bacterial species and two fungal species by well-diffusion method and microdilution methods. The patterns of inhibition varied with the plant extracts, the solvent used for extraction and the organisms tested. E. coli, Salmonella typhi, and Pseudomonas aeroginosa were the most inhibited microorganisms. The extract of Sphareranthu hirtus was the most active against multi-drug resistant P. aeruginosa and enterohemorrhagic E. coli 0157 EHEC. The ethanolic extract of S. hirtus exhibited a higher effect than the hot water extracts (Hassan et al. 2009).

Tannin toxicity tested to rumen microorganisms has been described for several bacteria species such as Streptococcus bovis, Butyvibrio fibrosolvens, Fibrobacter succinogenes, Prevotella ruminicola, and Ruminobacter amylophilis.

Enervosanone was found to have the strongest activity against four tested bacteria. Cambogin was found to be inactive against S. aureus. (-)-epicatechin was found to be inactive against all tested bacteria (Taher et al. 2005).

The previous researches used mostly the crude extract to test, in contrary to this in this work which used fractions of the crude extracts.

29   

CONCLUSION

Acacia nilotica originated from Gadarif area in the east of Sudan, was an effective antibacterial plant as was confirmed by many other studies carried out previously. It was found that the higher the concentration, the more effective the antibacterial activity. For fraction effectiveness, out of the three fractions, acetone fraction gave the best results against all three bacteria (V. harveyi, S. aureus, E. coli) at higher concentrations (as high as 1000 ppm). However, at lower concentrations (lower than 400 ppm) ethanol fraction was more effective toward S. aureus and E. coli. The acetone fraction was elucidated as condensed tannins which were larger tannins and toxic to many microbes. When condensed tannins are broken down to their subunits they are still able to bind proteins. In the case of ethanol fraction was tannins with low molecular weight, are toxic to fewer microbes because once the molecule will break down and its subunits lose their protein binding abilities. This research and the previous research confirmed the usefulness of this plant A. nilotica as a characteristic feature of medical plant and pharmaceutical.

RECOMMENDATION

It is recommended that further work is required to learn a more specific area in identifying the exact effective substances and their toxicity levels so as to find the correct or safe levels of consumption.

Lampiran 1 Rata-rata masa inkubasi

C. michiganensis

subsp.

michiganensis

pada

berbagai varietas tomat hasil inokulasi dengan pengguntingan petiol

daun

Varietas Tomat (hari setelah inokulasi) Ulangan

Sy It Eg Sv Lc Pd Sm Ms Vc Mg 1 7.00 4.67 6.33 6.00 6.67 4.67 6.33 4.67 4.33 5.33 2 6.33 4.33 4.67 6.33 4.67 4.33 6.33 6.67 5.67 6.00 3 6.67 6.67 4.67 4.67 4.33 6.00 6.67 5.67 6.00 6.33 4 6.33 5.33 6.33 6.33 5.00 5.33 6.67 5.33 4.33 6.67 5 6.00 4.67 4.67 5.67 6.67 5.33 6.33 4.67 5.00 6.00 Rata-rata 6.47a 5.13b 5.33b 5.80ab 5.47ab 5.13b 6.47a 5.40ab 5.07b 6.07ab Keterangan : Sy : Synta; It : Intrend; Eg : Eggy; Sv : Saviro; Lc : Locus; Pd : Perdana; Sm:

San Morino; Ms : Maestro; Vc : Viccario; Mg : Monggal; semua tanaman kontrol tidak menunjukkan gejala; a,b: huruf baku hasil pengujian beda nyata dengan DMRT pada taraf uji 5%, ,huruf yang sama menunjukkan tidak berbeda nyata

Lampiran 2

Berbagai tipe gejala pada beberapa varietas tomat hasil inokulasi

C.

michiganensis

subsp.

michiganensis

dengan pengguntingan petiol

daun

Varietas Tomat Ul

Sy It Eg Sv Lc Pd Sm Ms Vc Mg

1 Kerdil, daun keriting, daun kering layu, klorotik, kanker batang, tanaman kering, mati Klorotik , layu, kanker batang, mati Klorotik ,daun kering, kanker batang layu, klorotik, kanker batang layu, klorotik, kering, kanker batang kerdil, daun keriting klorotik, daun keriting, kanker batang layu, kanker, kerdil klorotik, layu, kanker batang 2 Klorotik , daun kering, layu,kan ker batang, bunga kering Kanker batang, daun kering kerdil, mati Klorotik , layu, kanker batang, daun kering, mati klorotik, daun kering, kanker batang, mati klorotik, kulit batang mengelu pas, kanker batang layu, tanaman mati kerdil, daun keriting klorotik, kanker batang Layu, klorotik, daun kering, kanker batang klorotik, kanker batang 3 Klorotik , daun kering, kanker batang Layu, klorotik, kanker batang, daun kering, mati Klorotik , kanker batang, daun keriting, daun kering, mati klorotik, layu, kanker batang, mati layu, kanker batang klorotik, kering, kanker batang kerdil, keriting, kanker batang klorotik, kerdil, keriting layu, kanker batang layu, klorotik, daun kering, kanker batang 4 layu, daun kering, Kanker batang, mati layu, klorotik, kanker batang, daun kering Layu, kanker batang Klorotik , layu, kanker batang, mati klorotik, layu, kanker batang, mati layu kerdil keriting, kanker batang, mati kerdil, tanaman mati klorotik, kerdil, keritiing klorotik, daun kering, kanker batang layu, klorotik, kanker batang 5 Daun kering, kanker batang, mati Layu, klorotik kanker batang, tanaman kering Layu, kanker batang, daun kering, mati Klorotik , daun kering, kanker batang, mati layu, kanker batang, mati layu, petiol cokat, kanker batang daun keriting, kanker batang klorotik, kanker batang, mati layu, kanker batang layu, klorotik, daun kering, kanker batang Keterangan : Sy : Synta; It : Intrend; Eg : Eggy; Sv : Saviro; Lc : Locus; Pd : Perdana; Sm: San Morino;

Ms : Maestro; Vc : Viccario; Mg : Monggal; semua tanaman kontrol tidak menunjukkan gejala

Lampiran 3 Nilai skoring dan jumlah tanaman yang mati pada berbagai varietas

tomat hasil inokulasi

C. michiganensis

subsp.

michiganensis

dengan

pengguntingan petiol daun

Nilai skoring

Ulangan

Sub

unit

Sy It Eg

Sv Lc Pd Sm Ms

Vc Mg

a 3 5 5

4 3 3 3 3

4

3

b 5 4 5 4 4 3 4 4

2

4

1

c 3 5 4

3 4 4 3 3

2

4

a 3 3 5

5 3 5 4 4

2

3

b 4 4 4 3 3 5 4 3

2

2

2

c 5 4 5

4 4 4 3 3

3

4

a 4 5 5

5 4 3 3 3

2

4

b 3 4 5 4 3 3 3 3

2

2

3

c 4 4 5

3 4 4 3 3

3

4

a 4 5 4

5 5 5 4 3

2

3

b 5 3 4 3 3 4 3 4

2

4

4

c 4 4 3

4 3 4 3 4

3

3

a 5 2 5

5 5 3 4 5

2

4

b 4 3 4 3 3 3 3 3

2

3

5

c 4 4 4

4 4 3 3 3

3

3

Keterangan : Sy : Synta; It : Intrend; Eg : Eggy; Sv : Saviro; Lc : Locus; Pd : Perdana; Sm: San Morino; Ms : Maestro; Vc : Viccario; Mg : Monggal; Jumlah tanaman yang mati pada varietas Sy: 4 (26.67%), It: 4 (26.67%), Eg: 8 (53.33%), Sv: 4 (26.67%), Lc: 2 (13.33%), Pd: 3 (20.00%), Sm: 0 (0.00%), Ms: 1 (6.67%), Vc: 0 (0.00%), Mg: 0 (0.00%); semua tanaman kontrol tidak menunjukkan gejala

Lampiran 4

Keparahan penyakit pada berbagai varietas tomat hasil inokulasi

C.

michiganensis

subsp.

michiganensis

dengan pengguntingan petiol

daun

Varietas Tomat (%) Ulangan

Sy It Eg Sv Lc Pd Sm Ms Vc Mg

1 73.33 93.33 93.33 73.33 73.33 66.67 66.67 66.67 53.33 73.33 2 80.00 73.33 93.33 80.00 66.67 93.33 73.33 66.67 46.67 60.00 3 73.33 86.67 100.00 80.00 66.67 66.67 60.00 60.00 46.67 66.67 4 86.67 80.00 73.33 80.00 73.33 86.67 66.67 73.33 46.67 66.67 5 86.67 60.00 86.67 80.00 80.00 60.00 66.67 73.33 46.67 66.67

Rata-Rata 80.00ab 78.67abc 89.33a 78.67abc 72.00bcd 74.67bcd 66.67d 68.00dc 48.00e 66.67d Keterangan : Sy : Synta; It : Intrend; Eg : Eggy; Sv : Saviro; Lc : Locus; Pd : Perdana; Sm:

San Morino; Ms : Maestro; Vc : Viccario; Mg : Monggal; semua tanaman kontrol tidak menunjukkan gejala; a,b,c: huruf baku hasil pengujian beda nyata dengan DMRT pada taraf uji 5% ,huruf yang sama menunjukkan tidak berbeda nyata

Lampiran 5 Waktu pembungaan pertama

pada berbagai varietas tomat hasil

inokulasi

C. michiganensis

subsp.

michiganensis

dengan

pengguntingan petiol daun

Varietas tomat (hari setelah inokulasi)

Sy It Eg Sv Lc Pd Sm

Ms Vc

Mg

Waktu

pembungaan

Pertama

31*) 37 30*) 39 33 32 33

39 40 39

Keterangan : Sy : Synta; It : Intrend; Eg : Eggy; Sv : Saviro; Lc : Locus; Pd : Perdana; Sm: San Morino; Ms : Maestro; Vc : Viccario; Mg : Monggal; semua tanaman kontrol tidak menunjukkan gejala; *) tidak berbunga lagi setelah pembungaan pertama

Lampiran 6 Media perbanyakan bakteri

Media NBY (Nutrient Broth Yeast Extract)

per l

Nutrient broth

8,0 g

Yeast ekstrak

2,0 g

K

2

HPO

4

2,0 g

KH

2

PO

4

0,5 g

Glukosa

2,5 g

Semua bahan yang digunakan dilarutkan dalam 1 l aquadest kemudian disterilisasi

dengana autoklaf pada suhu 121

0

C selama 15 menit. Setelah itu ditambah dengan

1 ml larutan MgSO

4

.7H

2

O 1M steril.