the potential of ornamental plant, sansevieria …
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THE POTENTIAL OF ORNAMENTAL PLANT,
SANSEVIERIA TRIFASCIATA TO INHIBIT THE
GROWTH OF ALEXANDRIUM TAMIYAVANICHII, A
TOXIC DINOFLAGELLATE
BY
IMA AMIRAH BINTI MOHD SUBERI
A thesis submitted in fulfilment of the requirement for the
degree of Master of Science (Biotechnology)
Kuliyyah of Science
International Islamic University Malaysia
JULY 2017
ii
ABSTRACT
Harmful algal bloom (HAB) is a phenomenon that can cause harm to human health
such as food poisoning and loss in aquaculture industries. However, in Malaysia,
research on mitigation of HAB is very limited. This study aims to determine the
potential of ornamental plant Sansevieria trifasciata in inhibiting the growth of HAB
species, Alexandrium tamiyavanichii. The crude extracts and active fractions of this
plant were tested on A. tamiyavanichii at different concentrations (0.001, 0.01, 0.1,
0.5, 1, 1.5, 2 and 2.5 mg/mL) for 24 hours. Toxicity test were conducted on artemia
using different concentrations of crude extracts (10, 50, 100 mg/mL) and active
fractions (10, 50, 100 and 500 mg/mL). Phytochemical screening was done to
determine the constituents by using standard procedures. Results obtained showed that
all concentration with different extracted method tested inhibited the growth of A.
tamiyavanichii. As the concentration increase, the pH of medium will decrease and
cause the removal efficiency to increase. The best removal efficiency was showed by
methanol fraction from ethanol fresh plant extract which was 93.86% at concentration
0.1 mg/mL and pH 7.46. Artemia showed 100% mortality at concentration 50 mg/mL
for ethanol fresh plant extract. For ethanol dried plant extract and distilled water fresh
plant extract, 100% mortality of artemia was at 50 mg/mL while distilled water dried
plant extract was at 100 mg/mL. All fractions showed 100% mortality of artemia at
concentration 500 mg/mL. In phytochemical screening tests, compounds detected in
ethanol fresh and dried plant extracts were alkaloid, tannins, glycoside, reducing sugar
and terpenoids whereas in distilled water fresh and dried plant extract detected
reducing sugar and terpenoids. For active fractions, all showed the presence of
alkaloid, tannins, glycoside, reducing sugar and terpenoids. The compounds detected
might have the function in inhibiting the targeted algae. Further study needed in order
to determine the bioactivity and its effect on HABs’ mitigation. This study showed
that S. trifasciata has the potential to be used for mitigation of HAB particularly A.
tamiyavanichii which help to minimize the impact of HAB in aquaculture field and
human or animal health.
iii
خلاصة البحث
إن الطحالب الدضرة الدنتشرة ىي أحد الظواىر الدؤذية للصحة البشرية لأثرىا في التسمم الغذائي والخسائر في صناعات الإستزراع الدائي. وعلى الرغم من ذلك فإن الأبحاث الدتركزة على التخفيف من انتشار
محدود جدا. تهدف ىذه الدراسة إلى التعرف على إمكانيات نبتة الزينة الطحالب الدضرة في ماليزيا ( في تثبيط نمو Sansevieria trifasciataالدسماة الذلب الثلاثية الأحزمة أو سانسيفييرا تريفاشياتا )
(. تم Alexandrium tamiyavanichiiالطحالب الدضرة من نوع أليكساندريوم تاميافانيتشي )ت والأجزاء النشطة منها ضد طحالب الأليكساندريوم تاميافانيتشي على تراكيز مختلفة اختبار الدستخلصا
ساعة. تم 24مغ/مل( لددة 2.5، و 2، و 1.5، و 1، و 0.5، و 0.1، و 0.01، و 0.001)، و 10القيام باختبار السمية ضد الأرتيميا )الروبيان الدلحي( للمستخلصات الخامة على تراكيز مختلفة )
مغ/مل(. تم 500، و 100، و 50، و 10مغ/مل( وللأجزاء النشطة على التراكيز ) 100 ، و50القيام بالدسح النباتي الكيميائي لتحديد الدكونات باتباع الطرق القياسية. أظهرت النتائج أن جميع التراكيز
فضت حموضة وبمختلف طرق الاستخلاص الدختَبرة قد أثبتت نمو الأليكساندريوم تاميافانيتشي. انخالوسط الاختباري بازدياد التركيز وأزالت أيضا احتمال ارتفاعها. تمت ملاحظة أن أفضل كفاءة للإزالة
% على 93.83كانت بالجزء الديثانولي من الاستخلاص الإيثانولي للنبات الطازج، حيث كانت بنسبة % على التركيز 100 . أظهرت الأرتيميا معدل موت بلغ7.46مغ/مل وحموضة معدلذا 0.1تركيز مغ/مل لدستخلصات الديثانول للنبات الطازج. أما بالنسبة لدستخلصات الإيثانول للنبات المجفف 50
مغ/مل، أما 50% على تركيز 100ومستخلصات الداء الدقطر للنبات النضر، كان معدل الدوت كل الأجزاء معدل موت مغ/مل. أظهرت 100مستخلصات الداء الدقطر للنبات المجفف كان على تركيز
مغ/مل. في الاختبارات النباتية الكيميائية، تم العثور على 500% على تركيز 100الأرتيميا بنسبة قلويدات، وتانينات، وقليكوزيدات، وسكر مختزل، وتيربينويدات. أما مستخلصات الداء الدكرر للنبات
ما بالنسبة للأجزاء النشطة فقد أظهرت كلها الطازج والمجفف فقد ظهر فيها سكر مختزل وتيربينويدات. أوجود قلويدات، وتانينات، وقليكوزيدات، وسكر مختزل، وتيربينويدات. القدرة التثبيطية لنمو الطحالب قد تمكن في ىذه الدركبات. ىناك حاجة إلى الدزيد من الدراسات لتحديد النشاط الحيوي وتأثيره التثبيطي
راسة أن لدى الذلب الثلاثي الأحزمة الإمكانية في استعمالو كمثبت لنمو على نمو الطحالب. أظهرت الدالطحالب الدنتشرة الدضرة، وبالتحديد نوع الأليكساندريوم تاميافانيتشي، مدا قد يساعد في التقليل من أثر
الطحالب الدنتشرة الدضرة في حقول الاستزراع الدائي وعلى الصحة البشرية والحيوانية.
iv
APPROVAL PAGE
I certify that I have supervised and read this study and that in my opinion, it conforms
to acceptable standards of scholarly presentation and fully adequate, in scope and
quality, as a thesis for the degree of Master of Science (Biotechnology).
.……………………………............
Normawaty Mohammad Noor
Supervisor
.……………………………............
Deny Susanty
Co-Supervisor
…………………………….............
Yukinori Mukai
Co-Supervisor
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and fully adequate, in scope and quality, as a thesis
for the degree of Master of Science (Biotechnology).
…………………………….............
Shahbudin bin Saad
Examiner
…………………………….............
Zainudin bin Bachok
External examiner
This thesis was submitted to the Department of Biotechnology and is accepted as a
fulfilment of the requirement for the degree of Master of Science (Biotechnology).
…………………………….............
Suhaila Mohd Omar
Head,Department of Biotechnology
This thesis was submitted to the Kuliyyah of Science and is accepted as a fulfillment
of the requirement for the degree of Master of Science (Biotechnology).
…………………………………….
Kamaruzzaman bin Yunus
Dean, Kuliyyah of Science
v
DECLARATION PAGE
I hereby declare that this thesis is the result of my own investigations, except where
otherwise stated. I also declare that it has not been previously or concurrently
submitted as a whole for any other degrees at IIUM or other institutions.
Ima Amirah binti Mohd Suberi
Signature………………………………… Date…………………………..
vi
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION OF
FAIR USE OF UNPUBLISHED RESEARCH
THE POTENTIAL OF ORNAMENTAL PLANT,
SANSEVIERIA TRIFASCIATA TO INHIBIT THE GROWTH
OF ALEXANDRIUM TAMIYAVANICHII, A TOXIC
DINOFLAGELLATE
I declare that the copyright holder of this thesis is jointly owned by the student
and IIUM.
Copyright © 2017 by Ima Amirah binti Mohd Suberi and International Islamic University
Malaysia. All rights reserved.
No part of this unpublished research may be reproduced, stored in a retrieval
system, or transmitted, in any form or by any means, electronics, mechanical,
photocopying, recording or otherwise without prior written permission of the
copyright holder except as provided below.
1. Any material contained in or derived from this unpublished research
may only be used by others in their writing with due
acknowledgement.
2. IIUM or its library will have the right to make and transmit copies
(print or electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieval
system and supply copies of this unpublished research if requested
by other universities and research libraries.
By signing this form, I acknowledged that I have read and understand the IIUM
Intellectual Property Right and Commercialization policy,
Affirmed by Ima Amirah binti Mohd Suberi
………………………… …………………………
Signature Date
vii
ACKNOWLEDGEMENTS
I would like to express my gratitude towards my main supervisor, Assoc. Prof. Dr.
Normawaty Mohammad Noor who always support, advised and give a hand
throughout my study. Also lots of thanks to my co-supervisors, Assoc. Prof. Dr. Deny
Susanti and Assoc. Prof. Dr. Yukinori Mukai for their useful advises and opinion.
Not to forget my friend, Sister Nor Salamah binti Mohamad Hidayat who also
help me during my difficulties. To my family especially my husband that always
understands and supports me until I manage to complete my study.
Lastly, I would like to thank Kuliyyah of Science, IIUM for the facilities
provided. This study also partially supported by FGRS grant from Ministry of Higher
Education, Malaysia.
viii
TABLE OF CONTENTS
Abstract……………………………………………………………………………... ii
Abstract in Arabic…………………………………………………………………... iii
Approval Page………………………………………………………………............. iv
Declaration Page…………………………………………………………………..... v
Copyright Page……………………………………………………………………… vi
Acknowledgements……………………………………………….………………… vii
List of tables………………………………………………………………………… x
List of figures…………………………………………………………..…………… xi
List of abbreviation………………………………………………………….……… xvii
List of symbols……………………………………………………………………… xviii
CHAPTER 1: INTRODUCTION…………………………………………………
1.1 Introduction…………………………………………………………….
1.2 Objectives………………………………………………………………
1.3 Hypothesis……………………………………………………………...
1.4 Significant of study…………………………………………………….
19
19
21
21
22
CHAPTER 2: LITERATURE REVIEW…………………………………………
2.1 Harmful algal bloom…………………………………………………...
2.2 HABs problem in Malaysia…………………………………………….
2.3 Alexandrium tamiyavanichii…………………………………………...
2.4 Mitigation of HAB …………………………………………………………...
2.4.1 Sansevieria trifasciata…………………………………………...
2.5 Phytochemical screening……………………………………………….
2.6 Toxicity test…………………………………………………………….
23
23
25
27
28
30
32
33
CHAPTER 3: METHODOLOGY………………………………………………...
3.1 Establishment of A. tamiyavanichii culture……………………………
3.2 Extraction of Sansevieria trifasciata……………………………………….
3.3 Fractionation process…………………………………………………..
3.4 Phytochemical screening……………………………………………….
3.5 Test of crude extracts and active fractions on A. tamiyavanichii……….
3.6 Toxicity test on brine shrimp (artemia)………………………………...
34
34
35
36
37
40
41
CHAPTER 4: RESULTS AND FINDINGS……………………………………...
4.1 Phytochemical screening……………………………………………….
4.2 Test of crude extracts and active fractions on A. tamiyavanichii……….
4.2.1 Ethanol fresh plant extract………………………………………
4.2.2 Ethanol dried plant extract………………………………………
4.2.3 Distilled water fresh plant extract……………………………….
4.2.4 Distilled water dried plant extract……………………………….
4.2.5 DCM fraction from ethanol fresh plant extract………………….
4.2.6 Methanol fraction from ethanol fresh plant extract……………...
43
43
44
44
49
54
59
64
69
ix
4.2.7 DCM fraction from ethanol dried plant extract………………….
4.2.8 Methanol fraction from ethanol dried plant extract……………..
4.3 Toxicity test on brine shrimp (artemia)………………………………...
4.4 Summary of results…………………………………………………….
4.5 Morphology observations……………………………………………...
74
79
84
87
88
CHAPTER 5: DISCUSSION……………………………………………………...
5.1 Phytochemical screening……………………………………………….
5.2 Test of crude extracts and active fractions on A. tamiyavanichii………
5.3 Toxicity test…………………………………………………………….
5.4 Conclusion……………………………………………………………..
89
89
91
96
97
REFERENCES…………………………………………………………………….. 99
x
LIST OF TABLES
Table No. Page No.
4.1 Phytochemical screening on crude extracts and active fractions (fresh
and dried plant sample).
43
4.2 Summary results of crude extracts. 87
4.3 Summary results of active fractions. 87
5.1 Comparisons of phytochemical compounds detected in S. trifasciata
in the study with other.
91
xi
LIST OF FIGURES
Figure No. Page No.
2.1 Alexandrium tamiyavanichii 27
2.2 Sansevieria trifasciata 31
3.1 Cultures of algae in the lab 34
3.2 (a) S. trifasciata plant, (c) plants were cut into small pieces, (d)
dried plant were blended and (e) extraction process rotary
evaporator
36
3.3 (a) VLC column and (b) extract with silica gel 37
3.4 Phytochemical screening process 40
4.1 Removal efficiency and cell density of 0.001 mg/mL ethanol
fresh plant extract on A. tamiyavanichii
46
4.2 Removal efficiency and cell density of 0.01 mg/mL ethanol
fresh plant extract on A. tamiyavanichii
46
4.3 Removal efficiency and cell density of 0.1 mg/mL ethanol fresh
plant extract on A. tamiyavanichii
46
4.4 Removal efficiency and cell density of 0.5 mg/mL ethanol fresh
plant extract on A. tamiyavanichii
47
4.5 Removal efficiency and cell density of 1 mg/mL ethanol fresh
plant extract on A. tamiyavanichii
47
4.6 Removal efficiency and cell density of 1.5 mg/mL ethanol fresh
plant extract on A. tamiyavanichii
47
4.7 Removal efficiency and cell density of 2 mg/mL ethanol fresh
plant extract on A. tamiyavanichi
48
4.8 Removal efficiency and cell density of 2.5 mg/mL ethanol fresh
plant extract on A. tamiyavanichii
48
4.9 Removal efficiency of ethanol fresh plant extract on A.
tamiyavanichii
48
xii
4.10 Removal efficiency and cell density of 0.001 mg/mL ethanol
dried plant extract on A. tamiyavanichii
51
4.11 Removal efficiency and cell density of 0.01 mg/mL ethanol
dried plant extract on A. tamiyavanichii
51
4.12 Removal efficiency and cell density of 0.1 mg/mL ethanol dried
plant extract on A. tamiyavanichii
51
4.13 Removal efficiency and cell density of 0.5 mg/mL ethanol dried
plant extract on A. tamiyavanichii
52
4.14 Removal efficiency and cell density of 1 mg/mL ethanol dried
plant extract on A. tamiyavanichii
52
4.15 Removal efficiency and cell density of 1.5 mg/mL ethanol dried
plant extract on A. tamiyavanichii
52
4.16 Removal efficiency and cell density of 2 mg/mL ethanol dried
plant extract on A. tamiyavanichii
53
4.17 Removal efficiency and cell density of 2.5 mg/mL ethanol dried
plant extract on A. tamiyavanichii
53
4.18 Removal efficiency and cell density of ethanol dried plant
extract on A. tamiyavanichii
53
4.19 Removal efficiency and cell density of 0.001 mg/mL distilled
water fresh plant extract on A. tamiyavanichii
56
4.20 Removal efficiency and cell density of 0.01 mg/mL distilled
water fresh plant extract on A. tamiyavanichii
56
4.21 Removal efficiency and cell density of 0.1 mg/mL distilled
water fresh plant extract on A. tamiyavanichii
56
4.22 Removal efficiency and cell density of 0.5 mg/mL distilled
water fresh plant extract on A. tamiyavanichii
57
4.23 Removal efficiency and cell density of 1 mg/mL distilled water
fresh plant extract on A. tamiyavanichii
57
4.24 Removal efficiency and cell densityof 1.5 mg/mL distilled
water fresh plant extract on A. tamiyavanichii
57
4.25 Removal efficiency and cell density of 2 mg/mL distilled water
fresh plant extract on A. tamiyavanichii
58
4.26 Removal efficiency and cell density of 2.5 mg/mL distilled 58
xiii
water fresh plant extract on A. tamiyavanichii
4.27 Removal efficiency and cell density of distilled water fresh
plant extract on A. tamiyavanichii
58
4.28 Removal efficiency and cell density of 0.001 mg/mL distilled
water dried plant extract on A.tamiyavanichii
61
4.29 Removal efficiency and cell density of 0.01 mg/mL distilled
water dried plant extract on A. tamiyavanichii
61
4.30 Removal efficiency and cell density of 0.1 mg/mL distilled
water dried plant extract on A. tamiyavanichii
61
4.31 Removal efficiency and cell density of 0.5 mg/mL distilled
water dried plant extract on A. tamiyavanichii
62
4.32 Removal efficiency and cell density of 1 mg/mL distilled water
dried plant extract on A. tamiyavanichii
62
4.33 Removal efficiency and cell density of 1.5 mg/mL distilled
water dried plant extract on A. tamiyavanichii
62
4.34 Removal efficiency and cell density of 2 mg/mL distilled water
dried plant extract on A. tamiyavanichii
63
4.35 Removal efficiency and cell density of 2.5 mg/mL distilled
water dried plant extract on A. tamiyavanichii
63
4.36 Removal efficiency and cell density of distilled water dried
plant extract on A. tamiyavanichii
63
4.37 Removal efficiency and cell density of 0.001 mg/mL DCM
fraction from ethanol fresh plant extract on A. tamiyavanichii
66
4.38 Removal efficiency and cell density of 0.01 mg/mL DCM
fraction from ethanol fresh plant extract on A. tamiyavanichii
66
4.39 Removal efficiency and cell density of 0.1 mg/mL DCM
fraction from ethanol fresh plant extract on A. tamiyavanichii
66
4.40 Removal efficiency and cell density of 0.5 mg/mL DCM
fraction from ethanol fresh plant extract on A. tamiyavanichii
67
4.41 Removal efficiency and cell density of 1 mg/mL DCM fraction
from ethanol fresh plant extract on A. tamiyavanichii
67
4.42 Removal efficiency and cell density of 1.5 mg/mL DCM
fraction from ethanol fresh plant extract on A. tamiyavanichii
67
xiv
4.43 Removal efficiency and cell density of 2 mg/mL DCM fraction
from ethanol fresh plant extract on A. tamiyavanichii
68
4.44 Removal efficiency and cell density of 2.5 mg/mL DCM
fraction from ethanol fresh plant extract on A. tamiyavanichii
68
4.45 Removal efficiency and cell density of DCM fraction from
ethanol fresh plant extract on A. tamiyavanichii
68
4.46 Removal efficiency of 0.001 mg/mL methanol fraction from
ethanol fresh plant extract on A. tamiyavanichii
71
4.47 Removal efficiency of 0.01 mg/mL methanol fraction from
ethanol fresh plant extract on A. tamiyavanichii
71
4.48 Removal efficiency of 0.1 mg/mL methanol fraction from
ethanol fresh plant extract on A. tamiyavanichii
71
4.49 Removal efficiency of 0.5 mg/mL methanol fraction from
ethanol fresh plant extract on A. tamiyavanichii
72
4.50 Removal efficiency of 1 mg/mL methanol fraction from ethanol
fresh plant extract on A. tamiyavanichii
72
4.51 Removal efficiency of 1.5 mg/mL methanol fraction from
ethanol fresh plant extract on A. tamiyavanichii
72
4.52 Removal efficiency of 2 mg/mL methanol fraction from ethanol
fresh plant extract on A. tamiyavanichii
73
4.53 Removal efficiency of 2.5 mg/mL methanol fraction from
ethanol fresh plant extract on A. tamiyavanichii
73
4.54 Removal efficiency of methanol fraction from ethanol fresh
plant extract on A. tamiyavanichii
73
4.55 Removal efficiency and cell density of 0.001 mg/mL DCM
fraction from ethanol dried plant extract on A. tamiyavanichii
76
4.56 Removal efficiency and cell density of 0.01 mg/mL DCM
fraction from ethanol dried plant extract on A. tamiyavanichii
76
4.57 Removal efficiency and cell density of 0.1 mg/mL DCM
fraction from ethanol dried plant extract on A. tamiyavanichii
76
4.58 Removal efficiency and cell density of 0.5 mg/mL DCM
fraction from ethanol dried plant extract on A. tamiyavanichii
77
4.59 Removal efficiency and cell density of 1 mg/mL DCM fraction 77
xv
from ethanol dried plant extract on A. tamiyavanichii
4.60 Removal efficiency and cell density of 1.5 mg/mL DCM
fraction from ethanol dried plant extract on A. tamiyavanichii
77
4.61 Removal efficiency and cell density of 2 mg/mL DCM fraction
from ethanol dried plant extract on A. tamiyavanichii
78
4.62 Removal efficiency and cell density of 2.5 mg/mL DCM
fraction from ethanol dried plant extract on A. tamiyavanichii
78
4.63 Removal efficiency and cell density of DCM fraction from
ethanol dried plant extract on A. tamiyavanichii
78
4.64 Removal efficiency of 0.001 mg/mL methanol fraction from
ethanol dried plant extract on A. tamiyavanichii
81
4.65 Removal efficiency of 0.01 mg/mL methanol fraction from
ethanol dried plant extract on A. tamiyavanichii
81
4.66 Removal efficiency of 0.1 mg/mL methanol fraction from
ethanol dried plant extract on A. tamiyavanichii
81
4.67 Removal efficiency of 0.5 mg/mL methanol fraction from
ethanol dried plant extract on A. tamiyavanichii
82
4.68 Removal efficiency of 1 mg/mL methanol fraction from ethanol
dried plant extract on A. tamiyavanichii
82
4.69 Removal efficiency of 1.5 mg/mL methanol fraction from
ethanol dried plant extract on A. tamiyavanichii
82
4.70 Removal efficiency of 2 mg/mL methanol fraction from ethanol
dried plant extract on A. tamiyavanichii
83
4.71 Removal efficiency of 2.5 mg/mL methanol fraction from
ethanol dried plant extract on A. tamiyavanichii
83
4.72 Removal efficiency of methanol fraction from ethanol dried
plant extract on A. tamiyavanichii
83
4.73 LC50 of ethanol fresh plant extract on artemia 85
4.74 LC50 of ethanol dried plant extract on artemia 85
4.75 LC50 of distilled water fresh plant extract on artemia 85
4.76 LC50 of distilled water dried plant extract on artemia 85
xvi
4.77 LC50 of DCM fraction from ethanol fresh plant extract on
artemia
86
4.78 LC50 of DCM fraction from ethanol dried plant extract on
artemia
86
4.79 LC50 of methanol fraction from ethanol fresh plant extract on
artemia
86
4.80 LC50 of methanol fraction from ethanol dried plant extract on
artemia
86
4.81 a: the ruptured cell, b and c: untreated cell
88
xvii
LIST OF ABBREVIATION
HAB Harmful algal bloom
PSP Paralytic shellfish poisoning
DSP Diarrheic shellfish poisoning
CFP Ciguatera fish poisoning
ASP Amnesic shellfish poisoning
sp. Species
L:D Light:dark
µm Micrometer
g Gram
VLC Vacuum liquid chromatography
mg Milligram
HCL Hydrochloric acid
mL Mililitre
mg/mL Milligram per mililitre
RE Removal efficiency
mm Milimetre
etOH Ethanol
MeOH Methanol
DCM Dichloromethane
dH2O Distilled water
Cells/mL Cells per mililitre
xviii
LIST OF SYMBOLS
% Percentage
ºC Degree celcius
19
CHAPTER 1
INTRODUCTION
1.1 INTRODUCTION
Harmful algal bloom (HAB) is a phenomenon occurred when there is single cell algae
of microscopic or macroscopic phytoplankton grow rapidly in huge number. It also
called as red tides as they composed of red pigments content dinoflagellates and cause
water change to red colour when bloom. However, the bloom colour is depending on
the algae type present and also their pigmentation. Over the last decade, this event had
been increasingly reported in various countries including Malaysia.
First HAB event in Malaysia was reported in year 1976 (Roy 1977). Bloom of
harmful algae caused big impact especially to our aquaculture field and economy. In
the year 2005 to 2006, there were bloom events in Penang that caused massive fish
kill and huge loss estimated not less than RM20 millions (Lim et al., 2012). The
causative organism responsible for this incident still remained unknown. Then, bloom
occurred in Sebatu, Melaka involved Alexandrium tamiyavanichii and caused three
people were hospitalised after consuming cultured green mussels that obtained from
newly established mussel’s farm (Lim et al., 2012). Recently, bloom of this species
was reported in Kuantan port and several people were hospitalised after consuming
contaminated shellfish. Others HAB reports that caused loss to economy were by
Cochlodinium polykrikoides at Kota Kinabalu, Sabah (Anton et al., 2008) and Perak
(Harun, Mohammad-Noor and Shahbudin, 2013). This indicates that HAB event in
20
Malaysia should be given great intention due to its high impact to aquaculture and
human health.
Several ways of prevention and mitigation have been reported to inhibit HABs.
One of the methods is by applying clay sprayed to affected water. This method has
been applied in Japan, Australia, South Korea and China (Pan et al., 2011). Harmful
algal cells in the affected water will be removed through flocculation and
sedimentation when clay is used (Pan et al., 2011). Other studies on HAB inhibition
used modified poly aluminium chloride-treated clay, acid treated clay (Pan et al.,
2011) and barley straw. Study conducted by Zhou et al., (2008) showed that man-
made diallyl trisulfide and garlic solution have similar algicidal affect towards algal
bloom. Both treatments introduced towards Alexandrium tamarense could inhibit the
growth at certain concentration.
In addition, research by Wei-dong et al., (2009) showed that extract of Chinese
fir has the potential to inhibit the growth of Alexandrium tamarense. Other studies
showed that Heterosigma akashiwo and Alexandrium tamarense effectively be
inhibited using seaweed such as Ulva pertusa, Corallina pilulifera and Sargassum
thunbergi (Renjun et al., 2007). All of these macroalgae released some
allelochemicals which able to inhibit the growth of both microalgae. Use of
allelopathy for HABs control became popular among researchers nowadays due to its
low cost, effectiveness and environmental friendly. However, in Malaysia, mitigation
of HABs is still in initial stage.
Therefore, in this study an attempt was made to use extract from ornamental
plant, Sansevieria trifasciata which commonly known as mother-in-law’s tongue or so
called snake plant to mitigate HAB particularly Alexandrium tamiyavanichii. There
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are approximately 70 distinct species of snake plant that have been identified (Alan
and Thomas, 1988). It is a succulent plant and originally found in tropical central
Africa and drier part of eastern and southern Africa. The leaves are flat and nearby
horizontal in shape (Carlquist and Schneider, 2007) with length about 70 to 100 cm
and 5 to 6 cm wide (Gautam et al., 2012). Previous studies showed that the species has
potential in medicinal, fodder, soil conservation and fibre (Khalumba et al., 2005).
Result from this study will be used in the management and mitigation of HAB event
in Malaysia.
1.2 OBJECTIVES
1. To isolate locally Alexandrium tamiyavanichii and establish into clonal culture.
2. To produce ethanol and water crude extract from fresh and dried plant sample of
Sansevieria trifasciata.
3. To produce active fractions from ethanol crude extract of fresh and dried plant
sample of Sansevieria trifasciata.
4. To determine the effect of crude extracts and active fractions on Alexandrium
tamiyavanichii and artemia.
5. To determine the constituents of phytochemical in Sansevieria trifasciata.
1.3 HYPOTHESIS
1. Clonal culture from locally Alexandrium tamiyavanichii will be successfully
isolated and cultured.
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2. Two fractions will be obtained with different polarities which are dichloromethane
and methanol fractions.
3. Crude extracts and active fractions will inhibit the growth of Alexandrium
tamiyavanichii but there will be no effects on artemia.
4. Several important phytochemical constituents will be detected in the plant.
1.4 SIGNIFICANT OF STUDY
Lots of cases involving harmful algal bloom have been globally reported including
Malaysia. HAB events were known to cause tremendous loss in economic, disturbing
to aquaculture field and harmful to human and animal health. Therefore, urgent
actions needed in order to minimize the effects. So, due to positive result obtained
from preliminary study using S. trifasciata on C. polykrikoides, extracts and active
fractions of this plant were used in this research for A. tamiyavanichii mitigation.
Plant extracts and fractions were used in this study due to environmental friendly
compared to method that use chemical.
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CHAPTER 2
LITERATURE REVIEW
2.1 HARMFUL ALGAL BLOOM
Harmful algal bloom (HAB) is a phenomenon that occurs throughout seawater and
freshwater cause by many factors including increasing of nutrient level which
contributed mostly from human activities such as aquaculture. The bloom of algae
normally is characterized by a high cell density of a single species. There are two
types of algal blooms i.e; toxic and non-toxic blooms. Toxic HAB is caused by
species of algae that has the capability to produce toxin and could harm human health
and aquaculture industry. Non-toxic HAB is caused by high cell density of a species
and cause problem to aquaculture industry.
Harmful algal bloom is caused by a group of phytoplankton (microalgae) such
as dinoflagellates, diatoms and blue green algae. Dinoflagellate is a free swimming
microalga. Examples of dinoflagellate that can cause harmful algal bloom are
Alexandrium spp., Pyrodinium bahamense, and C. polykrikoides. Then, diatom is a
dominant microalgae in the marine water and the most studied HAB species is
Pseudonitzschia spp.. For blue green algae, most of the species are freshwater. Among
species that have been identified as HAB species are Anabaena spp., Aphazinomenon
sp., Cylindrospermum sp., Microcystis, Oscillatoria, Nostoc, Hapalosiphon and
Nodularia (Gires, Lew and Asmat, 2002).
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There are several seafood toxicity that globally report which are paralytic
shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), ciguatera fish
poisoning (CFP), amnesic shellfish poisoning (ASP) and cyanobacteria toxins. For
PSP, toxin producers involved are P. bahamense var. compressum, A. tamiyavanichii
and A. minutum whereas bivalve mollusks are the primary vector. Main symptoms
showed by people that affected by PSP are paralyzed and death due to respiratory
failure (Gires, Lew and Asmat, 2002). Dinoflagellates Dinophysis, P. lima and P.
micans are toxin producers that responsible for DSP while mussel is the primary
vector. Diarrhea is the main symptom showed by people affected by this seafood
toxification (Gires, Lew and Asmat, 2002).
Benthic dinoflagellates involves in CFP as toxic producers are G. toxicus,
Ostreopsis spp. and Coolia spp. Marine fishes are the primary vectors responsible for
CFP and main symptoms showed by affected people also paralyzing and in extreme
cases death (Gires, Lew and Asmat, 2002). For ASP, it is known to be caused by
diatoms such as Pseudo-nitzschia. This algal produced domoic acid and contaminate
anchovies (Gires, Lew and Asmat, 2002). The final one is cyanobacteria toxins such
as anatoxin-a, microcystin and nodularin that caused by cyanobacteria or bluegreen
algae. All these toxins are harmful and can cause death. For anatoxins-a, species
involved are Anabaena spp., Aphazinomenon sp. and Cylindrospermum sp..
Microcystin toxin is produced by species of Microcystis, Oscillatoria, Anabaena,
Nostoc and Hapalosiphon while nodularins is produced by several species of
Nodularia (Gires, Leaw and Asmat, 2002).
According to Sunda et al., (2006), most of the red tide events documented
were caused by dinoflagellates which in bloom condition will discolour the effected