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FINDING PHOTOTOXIC POTENTIAL OF CHLOROPHYLL AND

CHLOROPHYLLIN AS NATURAL LARVICIDES AND BACTERIOCIDES

Rudolf Surya Bonay


SMA Negeri 5 Jayapura
Jl. Angkasa Base-G Angkasapura, Jayapura, Papua

Abstract
Chlorophyll’s ability to make use of light energy and produce toxic
compounds through its chemical reactions shows that this compound is a potential
photosensitizer. This property has been used in cancer therapy. The application of
chlorophyll and its derivatives as phototoxins is a new breakthrough that has been
made as a manifestation of the writer’s concern over national problems of tropical
countries related to the control of malaria, dengue fever, and infectious diseases.
Research on the use of chlorophyll and chlorophyllin as phototoxins was
conducted, using larvicide tests on Anopheles aconitus and Aedes aegypti mosquitos
larvae and antimicrobe tests on Esceherichia coli and Baccilus subtilis. The results
show that chlorophyll extracted from suji leaves and chlorophyllin have antimicrobial
activity of 30.76-72.62%. Besides, chlorophyllin can also control the growth of
Anopheles aconitus and Aedes aegypti larvae. The results of the study indicate that
chlorophyll and chlorophyllin have phototoxin property. These findings on the
phototoxin potency of chlorophyll and chlorophyllin as natural larvicides and
antimicrobes need to be followed up to make use of the chlorophyll and its derivatives
that exist in abundance in nature.

Keywords : chlorophyll, chlorophyllin, phototoxin, larvicides, bacteriocides

1. BACKGROUND OF THE STUDY


Indonesia is a tropical country that has quite high rainfall and that gets the
sunlight all the year long. Such a climate is very suitable for the breeding of mosquito
species and other microorganisms that cause diseases like malaria, dengue fever, and
other infectious ones.
Various attempts have been made to prevent and overcome the problems, for
example, those using insecticides and antibiotics. Although the use of insecticides is
effective and economical, this technique has a detrimental effect if a high
concentration of the insecticides is accumulated in certain organisms and even in
human beings. The use of antibiotics to overcome infectious diseases proves to be
ineffective, with so many bacteria becoming resistant to antibiotics lately due to the
existence of new mutants. As a result, antibiotics cannot be used according to the
recommended dosages anymore. Therefore, efforts should be made to look for cheap
natural compounds that can be easily obtained, and that have little or even no side
effects.
Plants are biological natural resources rich in secondary metabolites that are
found abundantly in nature. Various natural compounds like terpenoids, flavonoid,
and alkaloid have often been studied in an attempt to look for natural larvicides and
antimicrobial compounds (Orjala, et al., 1994; Chariandy, et al., 1998; Mohanty and
Prakash, 2002; Li, et al., 2002; Ivanice, et al., 2004; Ponlawat, et al., 2005; and Nick,
et al., 1995). However, the compounds are of limited amounts so they are more
difficult to isolate. Chlorophyll, in contrast, exists in abundance so it is more easily
isolated. Therefore, one needs to make use of chlorophyll maximally.
Chlorophyll is important not only for plant growth. Its existence in foods can
also support the health of whoever consumes it. Some studies show that it is safe to
use chlorophyll in food industry (Sarkar, 1996; Negishi, 1989). In the medical field,
chlorophyll is recognized as a cure for one thousand and one diseases because it can
prevent and cure various kinds of diseases, like anemia, inflammation, constipation,
diabetes, etc (Limantara, 2004). Chlorophyll has also been used as a photosensitizer
in the photodynamic treatment for tumor and cancer /PDT (Dougherty, et al., 1998;
Moser, 1998; Rosenbach-Belkin, et al., 1998; Henderson, et al., 1991; Kessel, et al.,
1993). The photosensitizer in the PDT, in principle, applies photosynthesis
knowledge, especially the knowledge of the interaction between chlorophyll
molecules and light that produces “reactive” excitation compounds. This concept has
also been made use of in the application of chlorophyll as solar bio cells (Limantara,
2004).
Apart from the use of chlorophyll as a health supplement, a photosensitizer,
and a solar bio cell, the concept of the unique and universal interaction between
chlorophyll and light has also stimulated the writer to develop it as a phototoxin. The
idea of making use of chlorophyll as a phototoxin comes from the tetrapyrole ring
structure in chlorophyll that is a typical characteristic of phototoxin compounds.
Based on the above discussion, the objective of this research is to find out the
potential of chlorophyll’s phototoxin and its derivatives as natural larvicides and
antimicrobes. The desire to investigate this issue is a manifestation of the writer’s
concern over the problems that exist in Papua, the writer’s place of origin, due to the
outbreak of malaria, dengue fever, and other infectious diseases.

2. REVIEW OF RELATED LITERATURE


Chlorophyll and Chlorophyllin
Chlorophyll is the first living molecule that grows on the surface of the earth
in the form of moss (blue green algae) around 3.5 billion years ago. Chlorophyll is
green because, being ineffective of absorbing the green light wave, chlorophyll can
only reflect it. Chlorophyll’s basic structure is comprised of prophyrins (Figure 1),
and it contains tetrapyrole ring, one of which is reduced. The four pyrole rings are
bonded with a magnesium ion by nitrogen atoms. The fifth isocyclic ring (E) is found
near the third pyrole ring. At the fourth ring, the propionic acid substituent is
esterified with the diterpene alcohol phytol (long hydrocarbon tail) that is
hydrophobic in nature; while the other sides of the molecule in the chlorophyll’s
structure are hydrophilic in nature (Gross, 1991). The existence of the phytol enables
chlorophyll to be dissolved in fats but not in water.
A B

NH N
A B
N N
N HN Mg
N N
D C
H
E
H
H O
COOCH 3
O C
O

CH3 H CH3 H

Figure 1. Porphyrin Structure (A) and Chlorophyll Structure (B)


In plants, chlorophyll plays a role in photosynthesis process by converting
absorbed light energy into chemical energy and storing it as carbohydrate. Through
the photosynthesis process, plants provide various foods that we need daily, and even
the oxygen we breathe in.
Chlorophyll is found abundantly in green plants, among others is in suji
(Pleomele angustifolia) leaves which have been found to have higher contents of
chlorophyll than other plant species (Istichomah, 2004). This plant exists in
abundance in Indonesia and is usually planted as hedges (Rahayu, 1988). It is usually
used as a green food coloring, oil, and paper. The cooked young suji leaves can also
be consumed as a vegetable. Apart from that, in Ambon, people rub heated suji
leaves on the limbs of a person who suffers from beriberi, a disease caused by vitamin
B1 deficiency (Heyne, 1987).
Chlorophyllin is a semisynthetic derivative of natural chlorophyll which is
dissolved in water. The compound belongs to the phyllin group that is produced by
the reaction between chlorophyll and bases. Chlorophyllin can also be obtained by
adding sodium and copper to chlorophyll, so the pigment can be dissolved in water.
For its solubility in water, this compound is widely used in medical field.

Photochemistry of Phototoxin
Phototoxin is a compound whose chemical reaction has a toxic activity when
exposed to light. Phototoxin is known as an insecticide that has a low toxicity level
for mammals, but a very high one for insects. Besides, phototoxin can be used to cure
cancer, as an antibacterial agent, and to eradicate pathogenic fungi. Some of the
phototoxin compounds that have been studied and developed as phototoxins are,
among others, phorpyrin, synthetic coloring compounds used as food coloring such as
halogenated xanthene, thiophene alpha-terthienyl (alpha-T), hypericine and
coronatine. Meanwhile, no one has studied chlorophyll as a phototoxin.
The idea to use chlorophyll as a phototoxin, in principle, originates from the
use of chlorophyll as a photosensitizer in the PDT therapy that can produce singlet
oxygen toxic to cancer cells. The photochemical reaction that generates singlet
oxygen is represented by the Jablonski diagram (Figure 2) (Gilbert and Baggott.,
1991a).
Singlet
excited Triplet
state excited
C 1 F
state E O2 + S S(O)

A B G D G 3
O2
Ground state
Fig 2. A Jablonski diagram showing the various modes of excitation and relaxation in a
chromophore: (A) excitation; (B) fluorescence; (C) intersystem crossing (D)
phosphorescence; (E) non-radiative transfer of energy to singlet oxygen; (F) substrate
oxidation by singlet oxygen; (G) internal conversion

Illuminating the chromophore (the chemical group that gives colour to a molecule)
with light of appropriate wavelength excites it to excited singlet states (A). The
chromophore can relax back to the ground-state by emitting a fluorescent photon (B)
or to excited triplet states via intersystem crossing (ISC) (C). From the triplet excited
states, the chromophore can relax back to the ground state by emitting a
phosphorescent photon (D) or transferring energy to another molecule via a
radiationless transition (E). In the first-order approximation, radiative triplet to
singlet transition is quantum mechanically ‘forbidden’ since a change of electron
spins is required, however these transitions occur to a very small extent (Gilbert and
Baggott, 1991b). Additionally, the chromophore can also lose energy through internal
conversion or radiation-less transition during collision with other molecules (Gilbert
dan Baggott., 1991a) (G). In oxygenated environment, the chromophore readily
transfer its energy to ground state molecular oxygen (3O2) to produce singlet oxygen
(1O2) which can form adduct with organic substrates (S(O)) (F).
The photosensitizer and oxygen interact through the triplet state because
oxygen has a unique, triplet-ground state and low-lying excited states. The energy
required for the triplet to singlet transition in oxygen is 22,5 kcal mol-1, which
corresponds to a wavelength of 1274 nm (infrared light) (Turro, 1991). Thus,
relatively low energy is needed to produce singlet oxygen. Photochemical reactions
of this type are known as type-II photoreaction and are characterized by a dependence
on the oxygen concentration (O2) (fig. 3) (Foote, 1984). Although type II
photoreactions are commonly associated with singlet oxygen production, some other
compounds have triplet-ground states and can be involved in type II photoreaction;
among these are nitric oxide and vitamin A (Foote, 1984). In anoxic environment
excited photosensitizer (3P*) can react directly with organic substrate (S) by electron
exchange, producing an oxidized substrate (S+) and reduced photosensitizer (P-). In
hypoxic environments the reduced photosensitizer (P-) can react with oxygen to
produce superoxide anions (O2-) which can then form the highly reactive hidroxyl
.
radical (OH ) (Foote, 1984). ). The excited photosensitizer (3P*) can also react with
.
superoxide radical (O2 ) to produce superoxide anions (O2-) which can then create the
.
highly reactive hydroxyl radical (OH ) (Foote, 1984). Collectively, these reactions are
classified as type-I photoreaction and are characterized by a dependence on the target-
substrate concentration (fig. 3) (Foote, 1984). Although type II reaction are reported
to dominate during PDT, for example, (Henderson dan Dougherty, 1992), Foote has
suggested type I reaction may become more dominant under condition where the
photosensitizers are highly concentrated, and especially under hypoxic condition
(Foote, 1984).

Type-I photoreaction
3
P* + S P- + S+
P- + O2 P + O2-
hυ + 3
P+ + O2-
1
3
P* P* + O2*
P
Type-II photoreaction
3
P* + 3O2 1
P + 1O2
1
O2 + S S(O)

Fig. 3. Type-I and type-II photoreaction, where 1P is a photosensitizer in a singlet ground state,
3 *
P is a photosensitizer in a triplet excited state, S is a substrate molecule, P- is reduced
phosensitizer molecule, S+ is an oxidized substrate molecule, O2 is molecular oxygen
(triplet-ground state), O2- is the superoxide anion, O2* is the superoxide radical, P+ is the
oxidized photosensitizer, 3O2 is triplet ground state oxygen, 1O2 is oxygen in a singlet
excited state, and S(O) is an oxygen adduct of a substrate.

Oxygen in phototoxin
Singlet oxygen is so reactive that is has a lifetime that ranges from 10-100 µ s
in organic solvents. This restricts its activity to a spherical volume, 10 nm in
diameter, centered at its point of production. In an aqueous environment, singlet
oxygen´s lifetime is reduced to approximately 2µ s because the energy of oxygen-
hydrogen (O-H) stretching in water molecules nearly equals the excited-state energies
of singlet oxygen. The energy is dissipated as heat by the stretching and vibrational
motion of water molecules. Because singlet oxygen reacts so rapidly, phototoxin-
induced oxidative damage is highly localized to regions no larger in diameter than the
thickness of a cell membrane. Photodynamic damage is probably confined to targets
near to or within the hydrophobic region of the cell due to the hydrophobic character
of most photosensitizers.

Mosquitoes
Mosquitoes are relatively small and delicate insects, with slim bodies and long
legs. Male mosquitoes suck a plant liquid or nectar as their food; whereas, female
mosquitoes usually bite and suck blood. It is the female mosquitoes’ habit that has
caused mosquitoes to get the worst reputation in history compared to other blood-
sucking insects. The female mosquito’s bite can cause the spread of various kinds of
diseases. Mosquitoes may suck blood that contains disease agents. In the
mosquitoes’ bodies, the agent, then, develops, and finally it is spread to other hosts.
Various kinds of malaria-causing plasmodiums, dengue fever-causing DHF viruses,
chikungunya-causing chikungunya viruses, meningitis-causing JBE viruses, and
Wuchereria bancrofti as well as Brugia malayi that cause vilariasis (elephantiasis)
spread diseases this way.
Mosquitoes belong to dipteran order, culicid (culicidae) family, with three sub
families, namely, toxorhynchitinae, culicinae, and anophelinae. A mosquito’s pupa is
oval or like a coma with the abdomen tip resembling a tail, and it has a pair of air
tubes. Its larvae, with distinct heads, are aquatic. At the end of the abdomen
(anophelinae), the larva’s body is often covered with tuffs of bristles. The mosquito’s
eggs are usually placed in rows like rafts on the surface of water (culex), on water
plants (mansonia) or placed individually – one by one – on the walls of a water
(aedes)-containing vessel. The anopheles mosquito’s eggs are placed one by one on
the water surface, so they look like a boat with floats made of corrugated chorion.
Mosquitoes are insects that can successfully make use of their water
environment, including natural water and temporary as well as permanent man-made
water sources. Lakes, streams, ponds, marches, dams, irrigation ducts, stone water,
gutters, used cans, etc. can all act as breeding places for mosquitoes’ larvae.

Bacteria
Bacteria are the smallest organisms that can independently protect themselves.
Their sizes range from 0,1-50 µ m. To live, bacteria depend on the availability of
nutrients, pH, salinity, temperature, and water. Water is the medium vital for
microbes’ life, and it makes up 80% of the total mass of bacteria, so water plays a role
in spreading certain kinds of diseases.
The individual bacteria’s forms differ in terms of thickness, compositions, and
structures. Based on their reactions to a coloring substance, the bacteria’s cell walls
can be divided into two main forms. Bacteria that accept the coloring substance are
referred to as Gram-positive bacteria, and those that release the coloring substance
that has been applied are called Gram-negative bacteria. The cell walls of the Gram-
positive bacteria are relatively thicker (30-100 nm) and simpler than those of the
Gram-negative. Around 40%-80% of the cell wall is made up of a polymer complex,
namely, peptidoglycan (murein). The cell walls of the Gram-negative bacteria are 20-
30 nm thick. The outer layer is a polysaccharide; whereas, the inner layer close to the
cytoplasm membrane is made up of the periplasmic gel of the peptidoglycan. This
layer is 15 nm thick, and it represents 1%-10% of the cell’s dry weight.
One example of the Gram-positive bacteria is Baccilus subtilis; whereas, that
of the Gram-negative bacteria is Escherichia coli. B. subtilis, which is a non-
pathogenic bacterium that looks like a rod (baccilius) and usually lives in the soil. E.
coli, on the other hand, belongs to the enterobacteriaceae family (Henderson et al.,
2000). E. coli is pathogenic, and it looks like a rod (bacillus), with flagella all around
it.

3. MATERIALS AND METHODS


Materials and Instruments
The samples used to test the antimicrobes were chlorophyll that is found in
suji leaf extract and a chlorophyll derivative, namely, chlorophyllin; whereas, the
sample to test the larvicide was only chlorophyllin - a chlorophyll derivative that
dissolves in water. Other materials used were nutrient broth (NB), acetone, methanol,
Escherichia coli and Baccilus subtillis bacterial cultures, III and IV-stage Anopheles
aconitus and Aedes aegypti mosquito larvae. The Anopheles aconitus and Aedes
aegypti larvae were obtained from Research Development Vector and Deseases
Reservoar Institute, Salatiga. The instruments used were a five-watt tungsten lamp,
single beam UV-Vis Spectrometer, an incubator, laminar flow-cabinet, and other glass
equipment.
Methods
Extraction
Suji leaves were chopped up and finely ground (Figure 4). Then it was soaked
in a mixture of acetone and methanol solvents with a ratio of 3:7 (v/v) for 15 minutes
while occasionally stirred. After that, the mixture was strained using a paper filter,
and the obtained filtrate was evaporated.

Figure 4. Extraction of suji

Making the Chlorophyllin


The extracted chlorophyll was purified using a column chromotography with a
silica gel 60 (Merck) stationary phase, and solvent variation between hexane and
acetone : hexane with a ratio of 1:4 and 1:1 (v/v) as a mobile phase. The blue green
band which was pure chlorophyll a was then converted into chlorophyllin using the
Oster, et al.’s methods (1964a, b).

Antimicrobial Test
The bacterium culture was inoculated in NB medium, and then incubated for
24 hours. When the bacterium culture was 24-hours old, it was diluted with NB until
the optical density rate was 0.150.
3 ml bacterial solution in NB was put into a sterile reaction tube. Then the
sample was added to it such that the sample’s concentration in that solution was 1000
ppm. To assist the solubility of suji extract in the bacterial solution, 100 µ L of
acetone was added to the solution. As a control, tetracycline (an antibiotic that serves
as an antibacterium) and 100 µ L of acetone in the bacterial solution were used.
Larvicidal Test
The sample was dissolved in 25 ml of well water until the mixture had the
optical density (OD) of 0; 15; 25; 35; and 45 (equivalent to 261,6; 435,6; 610,8; and
784,8 ppm). Then 10 mosquito larvae together with their food were added into the
solution. The larvae used as animal test were Anopheles aconitus and Aedes aegypti
instar III and IV mosquito larvae.
The tests of phototoxin bioactivity on larvae’s mortality were conducted
through successive observations for 30 minutes in the dark (the zeroth hour), followed
by observations in the light for 24, 48, and 72 hours (Figure 5).

Figure 5. Testing Larvicidal

Irradiation
Due to the fact that the sunlight fluctuation cannot be controlled, the
experiment was done in a laboratory using a simple light source, namely, a 5-watt
tungsten lamp with the consideration that it could provide stable artificial light which
is polychromatic just like the sunlight. The light intensity was 900 µ mol photons⋅ m-
2
⋅ s-1, and the exposure periods were 24, 48 and 72 hours, unless otherwise specified.

4. DATA ANALYSIS
The data gathered were descriptively analyzed with three replications.

5. RESULTS AND DISCUSSION


Phototoxin compounds are organic compounds whose toxicity will increase or
emerge only when radiated with light. In principle, if a neutral molecule is exposed to
light, the light energy absorbed by the molecule is utilized to move more actively. In
this study, at the beginning of the antibacterium test as well as larvicide test, the
mixture was stored in a dark place for 30 minutes to minimize pigment degradation
before the observation was made, so that when exposed to light (tested in the light),
the pigment’s activity became optimal because of the interaction that happened
between the pigment and the light.
Antimicrobes
According to Proestos et al. (2004), the measurement of the antimicrobe
activity can be done using the change in optical density (OD) in a liquid growth
medium containing the antimicrobial compound. The effect of the antimicrobes on
the sample can be seen from the solution’s turbidity level after having been incubated
for 24 hours. In this study, this effect was shown by the difference in the optical
density rates of the bacterium solution with and without the addition of the sample.
As a comparison compound against suji extract and chlorophyllin, this study used
tetracycline antibiotics that have been proven to be able to hinder the growth of
bacteria, both Gram positive and Gram negative. The measured average optical
densities of the suji extract are presented in Table 1.

Table 1. Average optical density of bacterial solution + acetone with and


without suji extract and tetracycline
OPTICAL DENSITY ± SD
SAMPLE
Escherichia coli Baccilus subtillis
Bacterial solution + 100 µ L acetone 0,543 ± 0,0016 0,822 ± 0,0256

Bacterial solution + 100 µ L acetone + suji 0,185 ± 0,0422 0,069 ± 0,0042


extract
Bacterial solution + Tetracycline 0,128 ± 0,0042 0,135 ± 0,0333

The table shows that the optical density of the bacterial solution was higher than that
of the suji extract. This indicates that the suji extract had the ability to hinder the
growth of the bacteria inside it. Table 2 below presents the measured average optical
densities of the chlorophyllin.

Table 2. Average optical density of bacterial solution with and without


chlorophyllin and tetracycline
OPTICAL DENSITY ± SD
SAMPLE
Escherichia coli Baccilus subtillis
Bacterial solution 1,164 ± 0,0016 1,249 ± 0,0042

Bacterial solution + Chlorophyllin 0,723 ± 0,0651 0,342 ± 0,0030


Bacterial solution + Tetracycline 0,128 ± 0,0042 0,135 ± 0,0333

The high optical density level of the bacterial solution without chlorophyllin shows
that bacteria thrived well in the solution. On the other hand, the lower optical density
level of the chlorophyllin solution suggests that the growth of the bacteria in it was
hindered by the presence of the chlorophyllin.
Based on the optical density measurement results from two samples, it can be
inferred that the suji extract and chlorohyllin possess the antibacterial/antimicrobe
activity. A compound can be said to be an effective antibacterium if the compound
can hinder the growth of the bacteria or in some cases can even kill the bacteria.
Basically, there are two types of actions or ways in which antibacteria work, namely,
bactericidal action that can kill bacteria by damaging one of the organism’s
biochemistry function, and bacteriostatic action that only hinders the growth of the
bacteria (Lay, 1994).
The phototoxin effects of the suji extract and chlorophyllin as antibacteria are
shown in the following histogram.
Antimicrobial Activity

1.4
1.2
1
0.8
0.6
0.4
0.2
0
Suji leaf extract Chlorophylline Tetracycline
Sample

Figure 6. Average activity of suji extract,


chlorophyllin, and tetracycline
antibacteia on E. coli (□) and B. subtillis
(■)

Based on the antimicrobe tests conducted both on the Gram-positive bacteria (B.
subtilis) and Gram-negative bacteria (E. coli), it seems that the suji leaf extract,
chlorophyllin, and tetracycline – all three have stronger hindering strength against B.
subtillis than against E. coli. The difference in the sensitivity to Gram-positive
bacteria and that to Gram-negative bacteria may be attributable to the difference in the
cell wall morphologies. Pelczar (1986) states that the cell wall structure of the Gram-
positive bacteria (B. subtilis) is relatively simple because the cell wall only has one
layer, that is, the peptidoglycan layer, which makes it easy for the antimicrobe
compound to enter the cell and find its target to work on. In contrast, the cell wall
structure of the Gram-negative (E. coli) is relatively complex because the cell wall has
three layers, namely, lipoprotein, lipopolysaccharide, and peptidoglycan. It is difficult
for the antimicrobe compounds to enter the cell wall of the Gram-negative bacteria
because the bacteria have better protection against antimicrobe compounds compared
to Gram-positive bacteria. Besides, Gram-negative bacteria posses a selection system
for foreign substances found on the lipopolysaccharide layer.
Figure 6 also shows that chlorophyllin tends to have a larger percentage of
hindrance against E. coli and B. subtilis (37,8% and 72,62%) compared to suji leaf
extract (30,76% and 61,57%). This could be caused by the fact that chlorophyllin
used was a pure compound, as opposed to the suji leaf extract which was an impure
extract that still contained various mixed compounds, including other impure
substances. Therefore, with the same concentration level (1000 ppm), chlorophyllin
possesses higher specific activity than the suji leaf extract.

Larvisida
The mortality rates of the A. aconitus and A. aegypti instar III mosquito larvae
are presented in Table 3.

Tabel 3. Mortality rates of A. aconitus dan A. aegypti instar III mosquito


larvae at various chlorophyllin optical density
LARVAE MORTALITY± SD
Observation
A. aconitus A. aegypti
Time (hour)
0 15 25 35 45 0 15 25 35 45
0 0±0 0±0 0±0 0±0 1±1,73 0±0 0±0 0±0 0±0 1±1
24 0±0 2,7±3,79 5±1 8,7±2,31 10±0 0±0 1±1 2,7±2,08 6,3±2,08 8,7±2,31
48 0±0 4,7±3,79 5,7±0,58 9,3±1,15 10±0 0±0 2,7±1,53 4,3±1,15 8±2,65 8,7±2,31
72 0±0 5,3±3,21 5,7±0,58 9,3±1,15 10±0 0±0 4,7±1,15 5,7±1,53 8±2,65 9±1,73

According to Anonymous (2001), the toxic concentration can be said to be effective


when the percentage of the tested organisms’ mortality reaches 50%. At the 24 th –
hour observation, the sample at the optical density of 25-35 can be said to be quite
effective for A. aconitus and A. aegypti instar III larvae, because at that optical
density the percentage of the dead larvae reached 50%. At the 48th-hour observation,
the 50% mortality rate was reached at the optical density of 15-25, while at the 72nd –
hour observation, the 50% mortality rate was reached at a lower optical density level,
i.e., below 15 (A.aconitus) or 15-25 (A. aegyti).
The mortality rates of the A. aconitus and A. aegypti instar IV mosquito larvae
are presented in Table 4.

Tabel 4. Mortality rates of A. aconitus dan A. aegypti instar IV mosquito


larvae at various chlorophyllin optical density
Observation LARVAE MORTALITY± SD
Time (hour) A. aconitus A. aegypti
0 15 25 35 45 0 15 25 35 45
0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0,3±0,57
24 0±0 0,6±1,15 2,7±2,08 4±4,36 6±3,61 0±0 0,7±0,58 4±1 4,7±4,51 7,3±4,62
48 0±0 2±0 3,7±1,53 5,7±2,89 7,3±2,52 0±0 1,3±0,58 4,7±0,57 6±5,29 8±3,46
72 0±0 2,3±0,58 5,3±0,58 8±2,65 8,7±2,31 0±0 2,7±0,58 5,7±0,57 6,3±4,73 8±3,46

The table shows that at the 24th - hour observation, the optical density of 35-45 turned
out to be quite effective for A. aconitus and A. aegypti instar IV larvae, because at this
optical density, the number of the dead larvae reached 50%. At the 48th- hour
observation, the 50% mortality rate was reached at the optical density of 25-35, while
at the 72nd-hour observation, it was reached at the optical density of 15-25.
The results of chlorophyllin’s bioactivity test on A. aconitus and A. aegypti
instar III and IV mosquitos larvae showed that light was very influential in
chlorohyllin’s effectiveness. This was due to the absence of larvicides’ effects during
the dark storage (the 0th hour). However, when exposed to light, chlorophyllin’s
phototoxin activity on A. aconitus dan A. Aegypti mosquito larvae increased as
indicated by the increased number of the dead larvae. This finding indicates that
chlorophyllin had an activity as a toxin when radiated with light and became a
phototoxin compound. The increase in the mortality rate of the mosquito larvae until
72 hours observation seems to be also related to the testing duration of the toxic
material used. The longer the contact period between the toxic material
(chlorophyllin) and light and the target larvae, the more effective its larvicidal
activity.
The effectiveness of chlorophyllin’s phototoxin in killing A. aconitus dan A.
aegypti instar III dan IV mosquito larvae at various chlorophyllin densities is
presented in Figure 7.
Larvae Mortality

12
10 A
C
8 D
6 B
4
2
0
0 15 30 45
Optical Density of Chlorophylline

Figure 7. Increase in the mortality levels of A.


aconitus instar III (A), instar IV (B) and
A.aegypti instar III (C) and instar IV (D)
larvae in accordance with the increase in
the chlorophyllin’s optical density at the
24th-hour observation
The figure shows the higher the chlorophyllin’s optical density was, the larger the
number of the dead larvae was. This finding proves that chlorophyllin was indeed
effective as a phototoxin compound (larvicide).
Further analyses reveal that the III-stage larvae were more susceptible than the
IV-stage larvae. The susceptibility of each larva was related to its development
stadium. The younger larvae was more susceptible than the older larvae. The reason
for this could be that instar III larvae needed more nutrients to grow optimally. Along
with the nutrient absorption, more toxic compounds contained in the experiment
medium were also absorbed, resulting in a higher mortality rate of instar III larvae
than of instar IV larvae. On the other hand, instar IV larvae’s bodies are similar to
mosquitoes’ or called prepupa. Instar IV larvae have a typical characteristic, i.e.,
having rudimentary wings which are kept in special sacs as germinalis buds. The
buds are effective protective devices that make the larvae more resistant to toxic
substances.
In both the antibacterium and larvicide testing in which the samples were
exposed to light, the samples showed phototoxin activity, but when stored in the dark,
the samples did not display the effect of antibacteria and larvicides. This result
proved that light excited the chlorophyll in the suji leaf extract and the chlorophyllin,
causing detrimental effects on the bacteria and the larvae. This photosensitizer effect
is caused by the tetrapyrol ring that exists in the pigment (chlorophyll in the suji leaf
extract and chlorophyllin) which, when activated by light, interacts with oxygen
molecules and forms singlet oxygen that is toxic to cells. The tetrapyrol ring in the
porpyrin has photodynamic activity and its toxicity is not aroused in the dark.
The use of chlorophyll’s derivatives that are dissolved in water like
chlorophyllin is absolutely essential in the application of the compound to control
disease vectors whose habitats are in the water environment, for example, malaria or
dengue fever causing mosquito larvae and pathogenic bacteria. Some important
parameters such as high oxygen concentration in water (8.3195572 mg.L-1 at the
pressure of 1 atm, 25 0C) (Atkins, 1998; Anonim, 1965), the magnitude of quantum
yield of T1 formation of chlorophyll in the presence of oxygen (0.70-0.85) (Kraljic
dkk., 1979), triplet chlorophyll’s lifetime (ms order) and the low energy level needed
to produce singlet oxygen, i.e., 22.5 kcal/mol (Bellus, 1978) are important
determinant factors that highly support the use of chlorophyll as a phototoxin. The
presence of abundant dissolved oxygen allows the type II reaction mechanism to take
place (Macdonald and Dougherty, 2001). Coupled with the very high abundance of
chlorophyll, ease at isolating it, and little or pratically no molecular side effects that
have been supported by the findings of the study conducted by the writer, it is hoped
that the results of this study offer a new way of thinking in the use of chlorophyll and
its derivatives that are abundantly found in Indonesia in the efforts to overcome the
national problems in controlling disease vectors and infectious diseases.

6. CONCLUSIONS
Based on the results of the research on antimicrobes, it can be concluded that
the suji leaf extract has antibacterial activities of 30.76% (E. coli) and 61,57% (B.
subtilis), while those of chlorophyllin are 37,8% (E. coli) and 72,62% (B. subtilis).
To test the larvicides, at the 24th hour observation, the 50% mortality rate of instar III
larvae is reached at the chlorophillin’s optical density (OD) of 25-35, while that of
instar IV larvae is at the optical density (OD) of 35-45.

7. SUGGESTIONS
Because of the limited laboratory facilities in the place where the writer
worked, several important things could not be done. The writer regards that the
following points need to be further investigated to find out more about the phototoxin
mechanism of chlorophyll and its derivatives: (a) the measurement of the singlet
oxygen formation in water during light exposure, and (b) the formation of
chlorophyillin triplet state in water.

8. REFERENCES
Anonimous. (1965). Standard Methods for the Examination of water and wastewater,
12th ed., American public health association, New York
Anonimous. (2001). LC50 Lethal Concentration. ILPI, The MSDS Hyper Glossary.
http://www.ilpi.com/msds/ref/lc50.ht,l
Atkins, P.W. (1998). Physical Chemistry, 6th ed., Oxford University Press.
Bellus, D. (1978). Quenchers of singlet oxygen-A critical review in singlet oxygen
(Ratby, B and Rabek, J.F., eds) Wiley, Chichester. 61
Chariandy, C.M., Seaforth, C.E., Phelps, R.H., Pollard, G.V. dan Khambay, B.P.S.
(1998). Screening of medicinal plants from Trinidad and Tobago for antimicrobial
and insecticidal pproperties. Journal of Ethnopharmacology, 64 : 265-270
Dougherty, T.J., Gomer, C.J., Henderson, B.W., Jori, G., Kessel, M., Korbelik, J.,
Moan, J. and Peng, Q. (1998). Photodynamic therapy. J. Natl. Cancer Inst., 90 :
889-905.
Foote, C.S. (1984). Mechanism of photo-oxygenation. In Porphyrin Localization and
Treatment of Tumors. Doiron DR, Gomer CJ. (eds). Alam, R. Liss, New York. 3-
18
Gilbert, A., Baggott, J. (1991a). Molecular photochemistry. Essentials of Molecular
photochemistry. CRC press, Boca Raton. 1-10
Gilbert, A., Baggott, J. (1991b). Molecular photophysics. Essentials of Molecular
photochemistry. CRC press, Boca Raton. 91-144
Gross, J. (1991). Pigments in Vegetables. Van Nostrand Reinhold, New York
Henderson, B.W., Dougherty, T.J. (1992). How does photodynamic therapy work?
Photochem. Photobiol, 55 : 147-157
Henderson, B.W., Sumlin, A.B., Owczarczak, B.L. and Dougherty, T.J. (1991)
Bacteriochlorophyll a as photosensitizer for photodynamic treatment of
transplantable murine tumors. J. Photochem. Photobiol. B. Biol. 10, 303-313
Henderson, B.W., Wilson, M., MacNab, R. and Lax, A.J. (2000). Cellular
microbiology bacteria-host interaction in health and disease. Joh Wiley & Sons,
England
Heyne, K. (1987). Tumbuhan Berguna Indonesia vol I. Badan Litbang Kehutanan,
Jakarta, 526
Istichomah, K. (2004). Pengaruh lama penyimpanan daun terhadap komposisi pigmen
dan kandungan klorofil daun suji (Pleomele angustifolia). Skripsi FSM-UKSW,
Salatiga
Kessel, D., Smith, K.M., Pandey, R.K., Shiau, F.Y. and Henderson, B. (1993)
Photosensitization with Bacteriochlorins. Photochem. Photobiol. 58 : 200-203.
Kraljic, I., Barboy, N. and Leicknam, J.P. (1979). Photochem. Photobiol. 30(6) : 631-
633
Lay, B.W. (1994). Analisis Mikroba di Laboratorium. PT Grafindopersada, Jakarta.
Li, B., Yu, S., Hwang, J.Y. and Shi, S. (2002). Antibacterial Vermiculite Nano-
Material. Journal of Minerals and Materials Characterization and Engineering,
1(1) : 61-68
Limantara, L. (2004). Menambang Klorofil, Si Emas Hijau. Prosiding Seminar
Nasional Aplikasi Sains dan Matematika dalam Industri. Fakultas Sains dan
Matematika, Universitas Kristen Satya Wacana, Salatiga.
Macdonald, I.J. and Dougherty, T.J. (2001). Basic principle of photodynamic therapy.
Journal of Phorpyrins and Phthalocyanines, 5 : 105-129
Mohanty, S.S. and Prakash, S. (2002). Efficacy of Chrysosporium lobatum againts
larvae of malaria vector, Anopheles stephensi in the laboratory. Current Science,
83(12) : 1585-1588
Moser, J.G. (1998) Bacteriochlorophyllides, bacteriochlorins and
bacteriopheophorbides. In: Photodynamic Tumor Therapy (Edited by Moser,
J.G.), OPA, Amsterdam. 43-49
Negishi, T. (1989). Inhibitory effect of chlorophyll on the genotoxicity of 3-amino-1-
methyl-5H-pyrido[4,3-b]indole (Trp-P-2), Carcinogenesis, 10(1) : 145-9
Nick, A., Wright, A.D., Rali, T. and Sticher, O. (1995). Antibacterial triterpenoids
from Dillenia Papuana and their structure activity relationships. Phytochemistry,
40(6) : 1691-1695
Orjala, J., Wright, A.D., Behrends, H., Folkers, G. and Sticher, O. (1994). Cytotoxic
and Antibacterial dihydrochalcones from Piper aduncum. Journal of Natural
product, 57(1) : 18-26
Oster, G., Broyde, S.B., and Bellin, J.S. (1964a). Spectral Properties of Chlorophyllin
a. J. Am. Chem. Soc. 86: 1309-1313.
Oster, G., Broyde, S.B., and Bellin, J.S. (1964b). Photochemical Properties of
Chlorophyllin. J. Am. Chem. Soc. 86: 1313-1318
Pelczar, M.J. (1986). Dasar-dasar mikrobiologi 2. UI Press, Jakarta
Ponlawat, A., Scoot, J.G. and Harrington, L.C. (2005). Insecticide Suscetibility of
Aedes aegypti and Aedes albopictus across Thailand. J. Med. Entomol. 42(5) :
821-825
Proestos, C., Boziaris, I.S., Nychas, G.J.E. and Komaitis, M. (2004). Analysis of
flavonoids and phenolic acids in Greek aromatic plants : Investigation of their
antioxidant capacity and antimicrobial activity. Food Chemistry, 95 : 664-671
Rahayu, M. (1988). Ensiklopedi Nasional indonesia jilid 15. Cipta Abadi, Jakarta
Rosenbach-Belkin, V., Chen, L., Fiedor, L., Salomon, Y. and Scherz, A. (1998 )
Chlorophyll and bacteriochlorophyll derivatives as photodynamic agents. In:
Photodynamic Tumor Therapy (Edited by Moser, J.G.), OPA, Amsterdam. 117-
125
Sarkar, D. (1996). Clastogenic activity of pure chlorophyll and anticlastogenic effects
of equivalent amounts of crude extract of Indian spinach leaf and chlorophyllin
following dietary supplementation to mice, Environ Mol Mutagen, 28(2) : 121-6
Turro, N.J. (1991). Singlet oxygen and chemilluminescent organic reactions. Modern
Molecular Photochemistry. University Science Books, California. 583-593