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Philippine Science High School

Main Campus

FINDING AN ALL-NATURAL UV-PROTECTIVE

COMPOUND FROM Sargassumsp. EXTRACT

Paul Gilbert L. Castro

Ernesto Paulo M. Garcia

Francis Andrew S. Forbes

March 2010

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Finding an All-Natural UV-Protective Compound from

Sargassumsp. Extract

by

Paul Gilbert L. Castro

Ernesto Paulo M. Garcia

Francis Andrew S. Forbes

Submitted to the Faculty of the

Philippine Science High School - Main Campus

in partial fulfillment of the requirements for

Science and Technology Research 2

March 2010

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ABSTRACT

Ultraviolet rays are rays from the sun which can cause harm in our skin form sunburn

to cancer. The need for protection is becoming more and more important. However,

commercial sun blocks are made of synthetic substances that may harm our skin.

A natural alternative that is both safe for our skin and the environment yet provides the

same amount of protection from the suns harmful rays is needed now more than ever. One

organism that can survive under the heat of the sun without fearing the dangers of UV-caused

mutations is the seaweed Sargassum. It is a member of Phaeophyceae known to have

Mycosporine- like Amino Acids that absorb UV rays.

Water-soluble extracts from Sargassum sp. were successfully integrated with a

carboxymethyl cellulose (CMC) gel base to form a UV-protective sun block.

Spectrophotometeric testing of the water extract and zinc oxide, a known UV-absorbing

compound, showed that zinc oxide is more absorbent than the water extract. Testing of the

Sargassum-CMC gel and Nivea commercial sun blockusing a UV transilluminator showed that

the Sargassum-CMC gel may be UV protective but it did not perform better than the commercial

sun block. Furthermore, the gel compound made from Sargassumsp. was effective only above

80% concentration, yet even at this concentration it was still less effective compared to the

commercial sun block.

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i

APPROVAL SHEET

This research work entitled, Finding an All-Natural UV-Protective Compound

from Sargassum sp. Extract by Ernesto Paulo M. Garcia, Paul Gilbert L. Castro, Francis

Andrew S. Forbes, presented to the Faculty of the Philippine Science High School Main

Campus in partial fulfillment of the requirements in Science & Technology Research 2, is hereby

accepted.

________________________________Kent D. Kawashima

Research Adviser

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ACKNOWLEDGMENT

The group would like to give thanks to the following individuals which without whose

contributions would have made the success of this research project a virtual impossibility.

Firstly, the group would like to thank Dr. Marco Nemesio E. Montano of the University

of the Philippines Marine Institute of Science Unit whose expert advice on the field helped

immensely in doing our preliminary research on the project. The group would also like to

acknowledge the contribution of Sir Kent Kawashima in supervising the groups progress in the

project and for his input in the design of the experimental methodology. Lastly, the group would

like to thank Aldon Galido for his technical assistance in the making of the groups tarpaulin

presentation. And of course, the greatest thanks is reserved to the name of God for with his

divine grace and mercy: all is possible.

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iii

TABLE OF CONTENTS

TITLE

Approval Sheet

Acknowledgment

Table of Contents

List of Figures iv

List of Tables v

Introduction 1

Review of Related Literature

I. Ultraviolet RadiationII. UV ProtectionIII. Sargassum sp.IV. Carboxymethyl celluloseV. Mycosporine-like amino acids

3

4

4

4

5

Materials and Methods 7

Results and Discussion

I. Testing the UV-absorbing capabilities of the solution usingspectrophotometer

II. Analyzing and comparing data with Zinc Oxide

9

10

Summary and Conclusions 13

Recommendations 14

Bibliography 15

Appendix

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iv

LIST OF FIGURES

FIGURE TITLE PAGE

1 Absorbance with respect to wavelength 11

2 UV-Absorbance tested on a UV lamp 12

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v

LIST OF TABLES

TABLE TITLE

1 Absorbance ofSargassum sp. extract 18

2 Absorbance of 3.5% Zinc Oxide solution 18

3 Absorbance of 7.0% Zinc Oxide Solution 19

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INTRODUCTION

Background of the Study

Sargassum sp. is a genus from the class of seaweed called Phaeophyceae or brown

seaweedone of the three main types of seaweed. Sargassumsp.can grow to a maximum of

16 meters in length and have an average lifespan of 3 to 4 years which is longer than normal

seaweed. They are found in most salt water bodies in Asia, including the Philippines, and

have many uses nutritionally and medicinally.

Research shows that brown seaweed, like most water-dwelling organisms contain

Mycosporine-like Amino Acids or MAA in their body structures (Encyclopedia Brittanica,

2009). These amino acids are presumably used to protect the seaweed from harmful UVA

and UVB radiation from the sun (Dunlap & Shick, 2002).

Statement of the Problem

Most presently available commercial sun blocks are made with artificial ingredients

and may have harmful side effects that users may not be aware. Examples of such side

effects are allergic reactions and skin irritation. These irritations are especially common on

children because they have more sensitive skin than adults do. Another drawback of using

sun blocks with artificially-made chemicals is environmental concerns. These artificial

chemicals may prove harmful to the environment if they are not disposed of correctly and

safely.

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A possible solution to such a problem is to find a suitable substitute for the artificial

ingredients used in commercial brand sun blocks. Such a compound must complete three

criteria to be deemed suitable to be a substitute for the artificial ingredients: the compound

must be naturally-occurring, the compound must have UV-protection with equal

performance to the artificially made products and the product must cause no negative side

effects on the user.

Significance of the Study

The results of this research can be used to develop a fully all-natural sun block with

extracts from Sargassumsp. as the base ingredient. It would be as effective in blocking UV

rays as currently available brands of sun block and would be safer to use and better for the

environment. This is because the seaweed sun block, unlike commercially sold sun block

will not be made with artificial ingredients. And because the sun block shall be naturally

made with seaweed which is commonly used as an ingredient in food; there will be less risk

of skin irritation appearing, development of rashes or other side effects even with sensitive

skin upon application. There will also be less risk in environmental safety because the

potentially harmful synthetic chemicals are replaced with naturally-occurring compounds.

Scope and Limitations

The study was limited to Sargassumsp. and to the water-soluble compounds that may

be found in it. The procedures used limited the ability to fractionate and purify the extracts

into more specific partitions.

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The lotion or cream base used to carry the test substance did not have UV protective

properties to ensure that the integrity of the data gathered is preserved. Any putative UV-absorbing

or reflecting capability was assumed to be caused by the extract alone and not from the base or any

other ingredients added.

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REVIEW OF RELATED LITERATURE

I. Ultraviolet radiationUV radiation or ultraviolet radiation is very common nowadays especially with the

ozone layer not yet recovering. According to the National Aeronautics and Space

Administration (2001), ultraviolet rays are a form of light that is invisible with the naked eye.

It is called ultraviolet because it is just beyond the violet end of the visible spectrum. It has a

wavelength of 40 nm up to 400nm. According to Zeman (2009), ultraviolet light is classified,

arranged in increasing wavelength, into five types: Vacuum UV, Far UV, UVC, UVB, and

UVA. The first three types are not yet studied thoroughly because it is absorbed by the

atmosphere. However, the UVC rays are used for germicidal purposes even though they

cause temporary blindness (Zeman, 2009). UVB rays are the most dangerous among the five

types. According to the National Aeronautics and Space Administration or NASA (2001),

UVB rays are absorbed by DNA causing the DNA bonds to break. Most of the broken

DNA bonds are repaired by certain proteins, however if few DNA bonds remain broken, it

can cause skin cancer or carcinoma (NASA, 2001). Nearly all of the UVB rays are

supposedly blocked by the ozone layer but since the ozone layer was diminished, the amount

of UVB rays that made it through the troposphere has increased. UVA rays are the most

common UV light we are exposed of because the ozone layer absorbed only a few of them.

People need this type of UV light for Vitamin D synthesis and it has a skin darkening effect

which some people want (Zeman, 2009). According to the NASA (2001), UV levels are

significantly higher at the region where the equator lies than in the places at the poles. That

is why people in tropical regions are more prone and exposed to UV rays.

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II. UV ProtectionThere are two main things that keep UV rays from getting onto people: sunscreens

and sunblocks. According to Brannon (2006), these two things are very different especially

on how they protect people from UV radiation. Sunblocks reflect and disperse the UVA and

UVB rays. The chemicals used in sunblocks are usually titanium oxide and zinc oxide.

However, Brannon (2006) cites that most of the sun blocks are greasy and irritating to

people who have sensitive skin. Sunscreens absorb and spread the UVA and UVB rays

instead of reflecting it. They are typically composed of benzophenones which are giving the

UVA protection and cinnamates and salicyates which are for UVB protection (Bragg, n.d.).

The disadvantage of sunscreens is that it degrades after a few hours of sunlight.

III. Sargassum sp.Sargassumsp. are members of the class Phaeophycae or commonly known as brown

algae. According to Encyclopedia Britannica (2009), brown algae are common at oceans,

even a place is named after the Sargassumsp.: Sargasso Sea. It is named Sargasso Sea because

there are manySargassumsp. there floating. It can live for three up to four years. Seaweeds

have a thallus body or a plant-like body without real roots, leaves and stems (Campbell,

1999). The thallus consists of three parts: holdfast, blades and stipe. The holdfast is the one

that looks like a root but it functions just like the roots of the plant. The stipe is the stem-

like part. It is also the one contributes the most in the length of the seaweed. The last part,

blades, is the one that looks like leaves of a plant. Some seaweeds have blades with air inside

making them float.

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IV. Carboxymethyl CelluloseAccording to Chaplin (2009), Carboxymethyl Cellulose or CMC is made through the

reaction of cellulose, alkali and chloroacetic acid. Its structure is shorter than of cellulose. It

has a shape of a rod in low temperatures and has a coiled shape in high temperatures

(Chaplin, 2009). It is used in food as a thickener and emulsion stabilizer due to its

controllable viscosity. Ice cream is one example of food that CMC is used at.

V. Mycosporine-like amino acidsMycosporine-like amino acids (or MAA) are water soluble, transparent, have low

molecular weight that absorbs UV radiation (Oren & Gunde-Cimerman, 2007). They are

seen in marine life. That is because marine animals are the ones who are most exposed in

UV radiation. In order, to cope up marine animals developed into making or acquiring MAA

(Shick & Dunlap, 2002). It is said that the number of MAA is inversely proportional to the

depths of the organisms which have acquired MAA. That is, if an organism lives in the deep

parts of the ocean it has less variety of MAA than of the one that lives in shallow parts but

according to Shick & Dunlap (2002), it is because of the response of the organism to the UV

exposure instead of being related to the values of depths.

MAA have many different ways to protect organisms from UV radiation (Shick &

Dunlap, 2002). In microalgae, MAA acts as a sunscreen by lying free in the cytoplasm. In

more complicated organisms, MAA acts also as a sunscreen but not inside the cells, they are

in the superficial parts of the tissues. For example, in sea anemones, instead in the endoderm

parts MAA are highly concentrated on the ectoderm part.

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According to Shick & Dunlap (2002), the requirements of being a good natural

sunscreen is that is should be an effective absorber of UV radiation and can disperse the

energy absorbed without making free radicals or transferring it to UV reactive cells and

MAA have all the requirements.

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Acquisition ofSargassum sp. samples

Water extraction of potential UV-protective agents from Sargassumsp.

Testing the UV-protective capabilities ofthe solution using spectrophotometer

Integration of the Sargassumextract withCarboxymethyl Cellulose as a base

Testing the UV-protective capabilities ofthe gel using UV transilluminator

MATERIALS AND METHODS

Acquisition ofSargassum sp. samples

Sargassumsp. was obtained from Dr. Nemesio Montano, a professor at U.P. Diliman

who specializes in marine biology and had the dried raw seaweed in stock.

Water extraction of potential UV- protective agent from Sargassumsp.

Simple water extraction was used to extract the UV-protective extract. The samples

were blended in an osterizer with water until the resulting mixture is almost homogenous.

The resulting product was filtered using filter paper. The filtrate was stored in a refrigerator

while the solids were discarded.

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Testing the UV-protective capabilities of the solution using spectrophotometer

The absorbance of the Sargassum filtrate, and a blank solution of water, were

measured from 400nm to 800nm using a spectrophotometer. The same was done with the

positive control zinc oxide. Data points gathered were plotted as an absorbance curve

showing maximum and minimum absorbance measurements for all three samples.

Integration of the Sargassumextract with Carboxymethyl Cellulose as a base

The Sargassum extract was mixed with carboxymethyl cellulose (CMC) in order to

create a gel compound. Miztures of 5%, 10%, 20%, 40% and 80% concentrations of extract

versus CMC were made.

Testing UV- protective capabilities using a UV transilluminator

The gel compound was tested with tonic water against Nivea commercial sun block. Using

a UV transilluminator, the glow of the mixtures was observed.

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RESULTS AND DISCUSSION

I. Testing the UV-protective capabilities of the solutionIn the preliminary experiments done on the raw Sargassum sp. extract, it was

confirmed that Sargasuumsp. does contain compounds in its body structure that is able to

block radiation from certain wavelengths of light. But the effects of these compounds are

limited compared with the ingredients found in commercially available sun blocks.

The radiation absorbance ofSargassum sp. extract was measured using

spectrophotometry from the light wavelengths ranging from 400 to 800 (which was the

maximum and minimum wavelengths in the available spectrophotometer) in 20nm intervals

(Fig.1). The finding were then compared with the radiation absorption of a 7.0% zinc oxide

solution (which is the typical concentration found in commercial sunblocks) and a 3.5%

solution of zinc oxide.

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Figure 1. Absorbance measurements of the Sargassumsp. extract versus zinc oxide from 400

nm to 800 nm.

One can see from the data gathered that the 7.0% Zinc Oxide solution is effectively

absorbent in a larger span of wavelengths than with the Sargassumsp. extract. It can also be

seen that at their top absorbance rating, the 7.0% Zinc Oxide solution has a higher peak

absorbance rating than the Sargassum extract but only by 0.26 units of absorbance and it

also occurs in a higher wavelength of light.

II. Testing UV- protective capabilities using a UV transilluminatorIn the second experiment setup done for this project, it was proven that the Sargassm

sp. does have UV-protective properties. But like in the preliminary experiment, the data

gathered also showed that the sun block with Sargassumsp. extract has a significantly weaker

effect compared with commercially available sun blocks when it comes to UV-protection.

The UV-protective potential of the Sargassumsp. based sun block was measured by

mixing the Saraguumsp. extract with carboxymethyl cellulose in 5 different concentrations of

5%, 10%, 20%, 40% and 80% (Fig.2B-F). Then, these solutions were mixed with tonic water

in 1:2 ratios and exposed to a UV lamp which causes plain tonic water to emit a blue glow

because of a chemical reaction in tonic water when exposed to UV radiation. The intensities

of the glow of the 5 batches of solution were then captured and compared with each other

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and also with a negative control of pure tonic water (Fig.2A) and a positive control of

Nivea commercial sun block (Fig.2G) mixed with tonic water in the same 1:2 ratio.

A B

C D

E F

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Figure 2. UV-absorbance testing using a UV transilluminator. (A) Pure tonic water; (B) 5%

extract, left, pure tonic water, right; (C) 10% extract, left, pure tonic water, right; (D) 20%

extract, left, pure tonic water, right; (E) 40% extract, left, pure tonic water, right; (F) 80%

extract, right, pure tonic water; and (G) 80% extract, left, Nivea commercial sun block,

right.

The results of the experiment showed a sudden decrease in the brightness of the

glow of the solutions with increasing Sargassum sp. extract concentration, only showing

evident difference in brightness compared with the control negative in the 80% solution.

Though this does prove that Sargassum sp. does have UV-protective potential, it was later

proven that this potential is insignificantly small compared to the UV-protective potential

observed in the positive control.

G

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SUMMARY AND CONCLUSION

The extracts which came from Sargassum sp. exceeded the performance of the 3.5%

Zinc Oxide but 7.0% Zinc Oxide performed better in the UV spectrophotometer. The gel,

which was created by adding carboxymethyl cellulose and the extract, worked in preventing UV

rays from making the tonic water to glow but with only the gel that has 80% extract while the

ones with 5%, 10%, 20%, and 40% did not succeeded. Also, the commercial sunblock executed

the task better than all of the gels. It means that the extracts have the capacity to block UV rays

but not enough to match with the execution of commercial sunblocks with the said task. Relying

alone with the extracts will not operate similar to those of commercial sunblocks.

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RECOMMENDATIONS

During the experiments, found several problems were found with the methodology

especially in the extraction process of the Sargassumsolution. To make any future attempts of

replicating this research, a few suggestions were made to make the extraction process easier.

Firstly, use filter paper with relatively large pores in extraction. Whatman Filter Paper

type 42 was used and this resulted in the extraction process taking significantly longer than

the expected time. Also, to further speed up the extraction process, it would be advisable to

have more than one filtration set-up so that making the solution would be done faster with

multiple filtration set-ups proceeding simultaneously.

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BIBLIOGRAPHY

Baker, C. (1982). Methylcellulose & sodium carboxymethylcellulose: uses in paper conservation. TheBook and Paper Group, 1. Retrieved December 7, 2009 from site: http://cool.conservation-us.org/coolaic/sg/bpg/annual/v01/bp01-04.html

Bragg, S. (n.d.). How do sunscreens work?. Retrieved October 9, 2009 from site:http://beauty.about.com/od/summertanning/f/hsunscreenswork.htm

Brannon, H. (2006). Sunblock. Retrieved October 9, 2009 from site:http://dermatology.about.com/od/glossarys/g/sunblock.htm

Brown algae. (2009) In Encyclopedia Britannica. Retrieved October 9, 2009 from site:http://www.britannica.com/EBchecked/topic/81647/brown-algae

Campbell, N., Reece, J. & Mitchell, L. (1999). Biology (5th ed.). Canada: Addison Wesley Longman,Inc.

Chaplin, M. (2009). Water Structure and Science. Retrieved December 07, 2009 from site:http://www1.lsbu.ac.uk/water/hycmc.html

Dunlap, W. & Shick, J. (2002). Mycosporine-like amino acids and related gadusols: biosynthesis,accumulation and UV-protective functions in aquatic organisms. Annual Reviews, 64, 223-262. Retrieved October 9, 2009 from site: http://www.umsms.siteturbine.com/faculty/faculty.../Shick&Dunlap:ARP02.pdf

Gorbushina, A., & Volkmann, M. (2006). A broadly applicable method for extraction andcharacterization of mycosporines and mycosporine-like amino acids of terrestrial, marineand freshwater origin. FEMS Microbiology Letters, 255, 2. Retrieved August 11, 2009 fromsite: http://www3.interscience.wiley.com/cgi-bin/fulltext/118603246/HTMLSTART

Gunde-Cimerman, N., & Oren, A. (2007). Mycosporines and mycosporine-like amino acids: UVprotectants or multipurpose secondary metabolites. FEMS Microbiology Letters, 269, 1.Retrieved October 9, 2009 from site: http://www3.interscience.wiley.com/cgi-bin/fulltext/118512218/HTMLSTART?CRETRY=1&SRETRY=0

Hader, D., & Klisch, M. (2008). Mycosporine-like amino acids and marine toxinsthe common andthe different. Marine Drugs, 6. Retrieved August 11, 2009 from site:http://www.mdpi.com/1660-3397/6/2/147/pdf

Lew, B. (2007). How does a spectrophotometer work?. Retrieved October 9, 2009 from site:http://www.cbst.ucdavis.edu/education/courses/spring-2007.../lewfinaldraft.doc

National Aeronautics and Space Administration. (2001). Ultraviolet radiation. Retrieved October 9,2009 from site: http://www.nas.nasa.gov/About/Education/Ozone/radiation.html

Zeman, G. (2009). Ultraviolet radiation. Retrieved October 9, 2009 from site:http://www.hps.org/hpspublications/articles/uv.html

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Table 1. Absorbance of

Sargassum sp. extract

Table 2. Absorbance of 3.5% Zinc

Oxide solution

Appendix

Wavelength Absorbance

400 nm 0.44

420 nm 0.46

440 nm 0.44

460 nm 0.44

480 nm 0.43

500 nm 0.42

520 nm 0.40

540 nm 0.40

560 nm 0.38

580 nm 0.36

600 nm 0.34

620 nm 0.34

640 nm 0.32

660 nm 0.31

680 nm 0.29

700 nm 0.28720 nm 0.27

740 nm 0.27

760 nm 0.25

780 nm 0.24

800 nm 0.24

Wavelength Absorbance

400 nm 0.82

420 nm 1.14

440 nm 1.18

460 nm 0.99

480 nm 0.82

500 nm 0.58

520 nm 0.46

540 nm 0.41

560 nm 0.31

580 nm 0.25

600 nm 0.19

620 nm 0.14

640 nm 0.11

660 nm 0.09

680 nm 0.09

700 nm 0.05

720 nm 0.05

740 nm 0.03

760 nm 0.02

780 nm 0.02

800 nm 0.01

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Table 3. Absorbance of 7.0% Zinc Oxide Solution

Wavelength Absorbance

400 nm 1.24

420 nm 1.35440 nm 1.44

460 nm 1.44

480 nm 1.43

500 nm 1.40

520 nm 1.34

540 nm 1.26

560 nm 1.23

580 nm 1.13600 nm 1.08

620 nm 1.00

640 nm 0.93

660 nm 0.88

680 nm 0.83

700 nm 0.78

720 nm 0.77

740 nm 0.74760 nm 0.70

780 nm 0.68

800 nm 0.66