International Journal of Pharmacy and Biological Sciences

ISSN: 2321-3272 (Print), ISSN: 2230-7605 (Online)

IJPBS | Volume 8 | Issue 2 | APR-JUN | 2018 | 583-590
Research Article | Biological Sciences | Open Access | MCI Approved|
|UGC Approved Journal |

COLOCASIA ESCULENTA (L.) LEAF BIO-WAX AS

A HYDROPHOBIC SURFACE COATING SUBSTANCE FOR PAPER
FOR PREPARING HYDROPHOBIC PAPER BAGS
Akash Kalita and Nayan Talukdar*
*Department of Biotechnology, Assam down town University, Guwahati, Assam, India.

*Corresponding Author Email: nayan.new16@gmail.com

ABSTRACT
The surface of Colocasia esculenta (L.) (taro) leaf is covered with a layer of highly hydrophobic layer of bio-wax.
The main objective of the project was to isolate the bio wax layer of the leaves using organic solvent extraction
method using chloroform and coat it on to the surface of paper to obtain hydrophobic paper which might be later
used for making biodegradable hydrophobic paper bags. Also, the isolated bio-wax was subjected to various tests
like heat test, hydrophobicity test, anti-microbial test, quantitative analysis to check its viability for industrial uses.
The result of quantitative analysis showed that 1 gram of sample leaf contained about 0.116 gram of wax. It was
also observed that the paper coated with the bio wax attained hydrophobic property which was similar to the
Colocasia leaf. Heat sensitivity test showed that the bio-wax retains hydrophobicity even at high temperature. The
bio-wax also possesses anti-bacterial property.

KEY WORDS
Anti-bacterial, Bio-wax, Colocasia esculenta (L.), Hydrophobic, Heat sensitivity, Quantitative

INTRODUCTION the animals when swallowed by terrestrial or marine [3,

Plastic bags, without any doubt, have found usefulness 4] and the alarming concern is that the production of
due to the properties like high durability, non- plastic bags has increased several folds over few
corrosiveness, light weight, electrical and thermal decades. Around 5 billion plastic bags are used across
insulation, etc, that plastics possess [1]. But in spite of the globe [3]. So, attempts are made to create
having such varying range commercially useful alternatives like biodegradable plastic bags. But many
properties, plastic bags have become a global concern studies show that biodegradable plastic requires a
because of the fact that they possess serious threat to favorable environment [5] i.e. optimum temperature,
the environment because of their non-biodegradable moisture, presence of microbial population, etc for the
nature. Even process like incineration which is carried process of degradation and even if they degrade, it
out with the intention of destroying the plastic bags produces smaller particles of plastic called micro-
produce large amount of greenhouses gases and other plastics [5] which gives shows that even the use of
toxic gases like carbon dioxide, carbon monoxide, biodegradable plastic is not completely eco-friendly [6].
sulphur dioxide gases, etc [2] which does more harm Therefore, to avoid these problems, use of paper bags is
than good to the environment by causing air pollution. encouraged because it has been scientifically proven
Plastic bags are also the major cause of blockage of that paper bags do not cause as much harm as plastic
drainage systems, one of the reasons behind urban bags do [7]. Although the rate of biodegradation is slow
floods and water pollution. Not only plastic bags cause in landfills with low moisture, but recycling of paper
pollution to the environment, but also cause death of bags requires 91% less energy per pound as compared

International Journal of Pharmacy and Biological Sciences Akash Kalita and Nayan Talukdar* 583

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 ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.

to plastic bags and also reduce the production of toxic among the stapled vegetable crops of the world and
gases and use of water to a considerable amount [8]. global production is about 12 million tonnes from
But paper bags have their own limitations. Paper bags approximately 2 million hectares which gives an average
are not water resistant due to which the uses of paper yield of 6.5 tonne per hectare [16, 17]. The high
bags become very limited. On the other hand, if plastic abundance of the leaf gives it the potential to be a raw
films are used to laminate the paper bags to make it material for industrial uses.
water resistant then the product becomes non-
biodegradable or if they are coated with synthetic MATERIALS AND METHODS
compounds like slimicides or lacquer [9] to have Isolation of wax from surface of leaves
additional advantages over plain paper then their Fresh leaves of Colocasia esculenta were collected. One
degradation might cause water and soil pollution. leaf was cut into fragments. 20ml of Chloroform was
So, in an attempt to find suitable organic material which taken in a beaker. The leaf fragments were immersed in
might be able to solve the above-mentioned problems, the chloroform for 3 minutes. Glass rod was used to
answers were sought in the nature. Ariel surface of immerse the leaves completely. After 3 minutes the
plants contain hydrophobic water proofing wax (bio- chloroform was transfer into a new beaker. The
wax) on them which provides protection against chloroform now contains the epicuticular wax present
environmental stresses [10, 11]. In some plants this wax in the leaf fragments. A white cloudy layer of wax can be
is highly hydrophobic in nature [12]. One example of seen floating on the chloroform [18].
such plant which possesses leaves having such a layer of Wax confirmatory test
hydrophobic bio-wax is Colocasia esculenta, a plant Wax was extracted from the leaves by solvent
belonging to family Araceae [13] which is commonly extraction method. The solvent (chloroform) is
known as ‘taro’ is a perennial herb which can grow upto evaporated. The wax was then dissolved in ethanol and
1.5 meter (4 feet). It is found throughout the tropical transferred into a test tube. Few ml of distilled water
and subtropical regions and can grow in a wide range of was then added to the solution and shaken. The solution
dry and wet places. These leaves are highly hydrophobic turned milky white [19].
and when water falls on it, the water rolls off in the form Quantitative analysis bio-wax
of a droplet, a phenomenon known as ‘Lotus Effect’, Fresh leaves were collected and cut into fragments. 1.5g
which is self-cleaning, water- repellant property found of leaf fragment was measured. The leaf fragments
in some plants [14, 15]. So, the bio-wax of the Colocasia were then dipped into 10ml of chloroform for 3
esculenta leaves, if extracted, it could be used as a minutes. The chloroform was then discarded and the
coating substance on the surface of paper to attain a remaining chloroform on the leaf fragments were then
layer of hydrophobic bio-wax on the paper. The allowed to air dry. The weight of the leaf fragments
hydrophobic paper hence attained could be used as a were then measured again. The process was repeated
source of raw material for making paper bags. In for 2.5g and 3.5g with 15ml and 20ml of chloroform
addition to water resistance or hydrophobic nature, the respectively. By subtracting the weight of the leaves
most important advantage of this product will be that it after the chloroform treatment from the weight of the
will be completely biodegradable, and the time required fresh leaves the amount of wax can be calculated. The
for degradation will also very less due to its organic amount of bio wax present in 1gm of fresh leaves can be
nature. Moreover, Colocasia esculenta is highly calculated by the following formula:
abundant in nature. It is positioned at rank fourteenth

Average weight of wax per gram of leaf = Total weight of epicuticular wax (in gram)
Total weight of the leaf fragments (in gram)
Test for hydrophobicity coating on the Petri plate left was again dissolved in 3ml
Bio-wax was isolated from leaves by solvent extraction of chloroform to get high concentration of wax. The
method. The chloroform containing the wax was poured solvent containing wax was then poured on a
into a Petri dish and the solvent was allowed to rectangular piece of filter paper using a micropipette.
evaporate. After the solvent has evaporated the wax Test for hydrophobicity was then done by dropping

International Journal of Pharmacy and Biological Sciences Akash Kalita and Nayan Talukdar* 584

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 ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.

water on to it using pipette and was compared to a filter at room temperature. Different Petri plate was then
paper without the wax coating. The water resistance subjected to different temperature ranging from 30oC to
was evaluated by observing the time till which the paper 120oC for 2 minutes. The retention of hydrophobic
shows hydrophobicity. property and integrity of the wax was then observed.
Test for anti-bacterial property Removal of pigment from the wax
Nutrient agar media was prepared by dissolving 8.4gm The extracted wax contains greenish pigment in it which
of nutrient agar media in 300ml of distilled water and is due to the presence chlorophyll. To make the wax free
was then autoclave [20, 21]. It is then poured into 12 of any pigment the following method was used:
Petri plates and was allowed to solidify. E.coli and The sample leaf was exposed steam in a water bath for
Streptococcus species were then inoculated onto 6 Petri 30 minutes. The temperature was set at 100oC because
plates for each species one Petri plate of each are chlorophyll degrades at high temperature [22]. The
control. To the other five Petri plates of each species, colour of the leaf turns into brownish-green and
disc containing sample of varying concentration are becomes soft. The steam treated leaf was then treated
added. The plates were incubated in an incubator at with chloroform to extract the wax. The chloroform
35oC. The radiuses of zone of inhibition were then containing the wax was then poured into a Petri plate
measured for each species. and was left to evaporate. After the solvent gets
Heat sensitivity test evaporated a white layer of bio-wax can be seen. The
Wax was extracted in different Petri plates using Petri plate containing the wax was then kept in oven for
chloroform. The solvent was then allowed to evaporate then minutes at 60oC to remove water molecules.

RESULTS
Isolation of wax from surface of leaves

Fig 2: Solvent free layer of bio-wax on a Petri plate.

Fig 1: Cloudy layer of bio-wax on chloroform.

Wax confirmatory test

Fig 3: Wax confirmatory test

International Journal of Pharmacy and Biological Sciences Akash Kalita and Nayan Talukdar* 585

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Int J Pharm Biol Sci.

Quantitative analysis bio-wax

Sl. Weight of leaf fragments beforeWeight of leaf fragments after Amount of
No. chloroform treatment (in gram) chloroform treatment epicuticular wax
A (in gram) (in gram)
B A-B
1 1.5 1.28 0.22
2 2.5 2.20 0.30
3 3.5 3.15 0.35
Table 1: Comparative table of weight of the fresh leaves and chloroform treated leaves

Calculation:
Average weight of wax per gram of leaf = Total weight of epicuticular wax (in gram)
Total weight of the leaf fragments (in gram)

Average weight of wax per gram of leaf = 0.22 + 0.30 + 0.35 = 0.87 = 0.116grams
1.50 + 2.50 + 3.50 7.50
Test for hydrophobicity

SL. Time
Object for Test
NO. (in minutes)

1 Paper Coated with Bio-wax 30+

2 Paper without bio-wax coating 0

Table 2: Comparative table of duration of water resistance

Fig 4: Water on Petri plate without bio-wax Fig 5: Water on Petri plate with bio-wax layer

International Journal of Pharmacy and Biological Sciences Akash Kalita and Nayan Talukdar* 586

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Int J Pharm Biol Sci.

Fig 6: Comparison between bio-wax filter paper and normal filter paper when water droplets are dropped on
them.
Test for anti-bacterial property
Sl. No. CONCENTRATION Radius of zone of inhibition Radius of zone inhibition
OF WAX (in mg) for E. Coli (in mm) for Streptococcus (in mm)
1 Control None None
2 50 0 2
3 100 3 3
4 150 3.5 4
5 200 4 5
6 250 4.3 5.1
Table 3: Radius of zone of inhibition at 24 hrs

Fig 7: Graph for zone of inhibition

International Journal of Pharmacy and Biological Sciences Akash Kalita and Nayan Talukdar* 587

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Heat sensitivity test

TEMPERATURE
Sl. No. HYDROPHOBIC PROPERTY WAX INTEGRITY
(in oC)
1 30 Present Intact
2 40 Present Intact
3 50 Present Intact
4 60 Present Intact
5 70 Present Intact
6 80 Present Intact
7 90 Present Intact
8 100 Present Intact
9 110 Present Starts to degrade
Becomes semi-
10 120 Present
solid
Table 4: Hydrophobic property and integrity of wax at different temperatures

Removal of pigment from the wax

Fig 8: Normal solvent free bio-wax Fig 9: Solvent free bio-wax extracted from steam treated leaves

DISCUSSION be then used to make biodegradable hydrophobic paper

The present study shows that the plant Colocasia bags.
esculenta which is abundantly found not only in Assam It is observed that the bio-wax extracted by the process
and many parts of India but also all over the world of organic solvent extraction possesses a greenish
possesses a highly hydrophobic layer of bio-wax on its pigment which might be unsuitable for future industrial
leaves. The bio-wax can be extracted by from the leaves use. The pigment was successfully removed by treating
by organic solvent extraction method which in this case the leaves with steam (at 100oC for 30 minutes) prior to
is chloroform. During the course of study quantitative the process of extraction. The bio-wax extracted after
analysis shows that the amount of bio-wax present per steam treatment exhibited white colour rather than
gram of leaf is equal to 0.116 gram approximately. green which proved that the green pigment was
The extracted bio-wax shows hydrophobic property successfully eliminated by the process of steam
even when it is exposed to high temperatures which is treatment. It is due to degradation of chlorophyll at high
around 95-100 degree Celsius. These characteristics temperature provided by steam. The white coloured
make the bio-wax of Colocasia esculenta a suitable wax was subjected to a temperature of 60 oC for 15
substance for coating papers to make them minutes to remove any water molecule that may have
hydrophobic. The hydrophobic papers thus created may got trapped into the wax due to steam. The wax hence

International Journal of Pharmacy and Biological Sciences Akash Kalita and Nayan Talukdar* 588

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 ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.

extracted was free from water molecules and it even [5] Science for Environment Policy | In-depth Reports |
loosed its white colour and became almost transparent. Plastic Waste: Ecological and Human Health Impacts,
The heat did not denature the wax since it is stable even (2011).
[6] UNEP, Biodegradable Plastics and Marine
at 100 degrees Celsius.
Litter.Misconceptions, concerns and impacts on
The present study also shows that the bio-wax also
marine environments. United Nations Environment
possesses anti-bacterial property against E.coli and Programme (UNEP), Nairobi, chapter 3, p 21, (2015).
Streptococcus which can be an added advantage (All [7] Bags and Its Damaging Impact on Environment and
though the actual resistance against the bacteria of the Agriculture: An Alternative Proposal, Vol. 3, No. 4,
bio-wax might be more than the result shown is the (2013).
experiment.) [8] Bell K. and Cave S., Comparison of Environmental
Impact of Plastic, Paper and Cloth Bags, (2011).
[9] Marsh K. and Bugusu B., Food Packaging—Roles,
CONCLUSION
Materials,And Environmental Issues, (2007).
From the study it can be concluded that since the leaves [10] Post-Beittenmiller D., Biochemistry and Molecular
of the plant Colocasia esculent (L.) is abundantly Biology of Wax Production in Plants, (1996).
available and its bio-wax possesses high hydrophobic [11] Cunningham T., Characterizing the molecular
property, therefore the bio-wax could be used as a composition of epicuticular waxes of vegetation and in
surface coating substance for papers which might be surface sediments in Great Sippewissett marsh,
used for making biodegradable hydrophobic paper bags Falmouth MA, (2012).
which not only will reduce the use of plastic bags up to [12] Koch K., Hans-Jürgen Ensikat, The hydrophobic
coatings of plant surfaces: Epicuticular wax crystals
a great extent but also will help in reducing pollution in
and their morphologies, crystallinity and molecular
the environment.
self-assembly, (2008).
[13] Lakhanpaul S., Velayudhan K.C. and Bhat K.V., Analysis
ACKNOWLEDGEMENT of genetic diversity in Indian taro [Colocasia esculenta
We here by acknowledge the management of Assam (L.) Schott] using random amplified polymorphic DNA
down town University, Guwahati, Assam, India for their (RAPD) markers, p 603, (2003).
cooperation, encouragement, and for providing us the [14] Ensikat H. J., Ditsche-Kuru P., Neinhuis C.and Barthlott
W., Superhydrophibicity in perfection: the outstanding
opportunity to carry out the research. We would also
properties of the lotus leaf, 152–161, (2011).
like to acknowledge the Central Instrumentation
[15] Latthe S.S., Terashima C., Nakata K.and Fujishima A.,
Facility, Assam down town University for providing us Superhydrophobic Surfaces Developed by Mimicking
with all the requirements and space for the project. Last Hierarchical Surface Morphology of Lotus Leaf, 4256-
but not the list we would like to thank all the people who 4283, (2014).
helped us in carrying out the research and thereby [16] Rao V. R., Matthews P.J., Eyzaguirre P.B., Hunter D.,
helping us in achieving our goal. The Global Diversity of Taro: Ethno botany and
Conservation. Biodiversity International, Rome,
chapter 1, p 1, (2010).
REFERENCES
[17] Brooks F.E., Taro leaf blight. The Plant Health
[1] Thompson R.C., Moore C.J., Saal F.S., Swan S.H.,
Instructor. DOI:10.1094/PHI-I-2005-0531-01, (2005).
Plastics, the environment and human health: current
[18] Ganeva T., Stefanova M., Koleva D., Ruiz S.R., Isolation
consensus and future trends., (2009).
and recrystallization of epicuticular waxes from Sorbus
[2] Junod T.L., Gaseous Emissions and Toxic Hazards
and Cotoneaster leaves: 497–504, (2015).
Associated with Plastics in Fire Situations - A Literature
[19] Rothery M., Techniques: Biochemical Tests.
Review, (1976).
[20] HiMedia Laboratories, Nutrient Agar, M001.
[3] Jaconsen S., Plastic bag pollution, (2005).
[21] Aryal S., Nutrient Agar: Composition, Preparation, and
[4] Knoblauch J.A., Environmental Health News, Plastic
Uses, (2015).
Not-So-Fantastic: How the Versatile Material Harms
[22] Erge H.S., Karaden‹z F., Koca N., Soyer Y., Effect of Heat
the Environment and Human Health, (2009).
Treatment On Chlorophyll Degradation And Color Loss
In Green Peas, GIDA, 225-233, (2008).

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 ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.

*Corresponding Author:
Nayan Talukdar*
Email: nayan.new16@gmail.com

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