DEVELOPMENT OF NATURAL EYE DROP USING GREEN TEA

CHAPTER 1

INTRODUCTION

1.0 Background of study

Ophthalmic/ocular/eye drop preparations are topical preparations that
contain active pharmaceutical ingredients that is delivered through
either eyelids, conjunctive or lacrimal system. The administration to
the eye usually through container with dropper, or tube with nozzle
("Topical drug dosage forms for eye conditions", 2019) .It must be
sterile (solid, liquid, semi-solid).

Hydrating eye drops or also known as artificial tears is been used to

hydrate and moisture the eye which can relieve the eye comfort.
From the market, most of the eye drops commercialized formulations
contain propylene glycol, glycerin, hypermellose and etc. Glycerin is a
by-product manufacture of soaps and fatty acids and can be obtain
from fats and oils. Glycerin occurs naturally in animal and vegetable
fats and oils that are consumed as part of a normal diet (Sheskey,
Cook & Gable, 2009).

Form the far past, plants have been useful to the human kind. Mostly
for their medicinal values and the derivatives that have multipurpose
applications including in the pharmaceuticals formulation. Green tea
(Camellia sinesis) has a unique taste compared to regular tea and is
a worldwide beverage .Originally from southern and eastern Asia
(Van Wyk & Wink, 2004). It is mostly consumed mainly due to its
health value promoted by the ancient folk. It is the most widely
consumed beverage after water, due to its health, sensory, stimulant,
relaxing and cultural properties (Ahmed and Stepp, 2012). It is also
medicinally used to treat acute diarrhoea, weight loss, symptomatic
relief of skin disorders, and also taken as antioxidants.

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 Polyphenols is the active compound of the green tea. From the
polyphenols , flavonoids (catechins), are the common functional
components made up of 30% dry weight of the green tea leaves. The
catechins carry the potential properties of anti-oxidant. From various
research, green tea has been studied to act as photo-protection to the
skin and against ultraviolet damages thus increasing the skin
hydration and reducing the trans-epidermal water loss(TEWL).

1.0 Objective
1.0.1 General Objective
 To formulate the eye drop formulation with green tea.

1.0.2 Specific Objective

 To assess the formulation quality, pH, clarity, particulate
matter, particle size and sterility test.
 To observe the potential of green tea as active ingredient in
the eye drop.
1.1 Problem statement
 People nowadays tend to buy natural product based on plant
rather than animals. Hydrating eye drop that is available for marketed
mostly based on the active ingredient of glycerin.
 Glycerin occurs naturally in animal and vegetable fats and oils
that are consumed as part of a normal diet (Sheskey, Cook & Gable,
2009).
1.2 Hypotheses
1.2.1 Null hypothesis
 The leave extraction of the Camellia sinesis does not meet
the quality as active ingredient in the formulation of eye drop.
1.2.2 Alternative hypothesis
 The leave extraction of the Camellia sinesis does meet the
quality as active ingredient in the formulation of eye drop.

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 CHAPTER 2

LITERATURE REVIEW

2.0 Anatomy and physiology of the eye

Structure of the eye consists of layers and chambers as can be seen
on the figure 1 below.

Figure 1 : Structure of the eye (Willoughby et al., 2019)

The eye has 2 outer layers which is sclera and cornea. The white of
the eye is usually referred to the front of the sclera. Conjunctiva is the
part that will cover the sclera which extend to the eye lid and it is a
transparent membrane. Sclera consists of tough fibrous tissue which
will protect the eye from internal and external force thus the shape will
be maintained (Aulton & Taylor, 2018).

The sclera forms a connective tissue coat that protects the eye from
internal and external forces and maintains its shape (Willoughby et
al., 2019).

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 The cornea is the anterior part of the eye. It refracts and transmit
lights to lens and retina and also protect the eye against infection and
structural damage to deeper parts (Aulton & Taylor, 2018).

The cornea refracts and transmits the light to the lens and the retina
and protects the eye against infection and structural damage to the
deeper parts (Willoughby et al., 2019).

The surface of cornea and conjunctiva are covered by the film of

tears produced mainly by the lacrimal gland which will lubricate the
eye surface and protect from external particles parts (Aulton & Taylor,
2018).

2.1 Ophthalmic formulations (Topical: solutions)

Ophthalmic preparations are sterile products which intended for the
application to any of the ocular structure including any space adjacent
to ocular structure and its immediate surrounding spaces (The United
States Pharmacopeia, 2019).

Topical drug products are intended to administered to an ocular

surface component such as the eye lid, conjunctiva or cornea which
can produce systemic effects (The United States Pharmacopeia,
2019).

Main features of the eye for the ophthalmic drug delivery are
conjunctiva, cornea and lacrimal fluid ("Topical drug dosage forms for
eye conditions", 2019).

Absorption of the eye drops across the mucosa in the nasal cavity
that is near to the conjunctival sac helps eye drops with active
pharmaceutical ingredients reach the bloodstream (Winfield et al.,
2009).

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2.1.1 Eye drop formulation characteristics
2.1.1.1 pH adjustment

The control of the pH of the ocular formulation is very important as to

not damage or irritate the eye and the determinant of the stability of
the drug formulation. Ocular solutions ideally should be control at pH
7.4. Also the pH of the formulation should be maximize to the
therapeutic agent
(active ingredient) ("Topical drug dosage forms for eye conditions",
2019).

Normal tears have pH about 7.4 and the eye can tolerate the range
pH of about 3.0 to about 8.6 (The United States Pharmacopeia,
2019).

The prepared eye drop should be formulated at pH equivalent to tear

fluid value of 7.4 (Florence & Atwood, 2016).

2.1.1.2 Isotonicity
The solution is isotonic as the effective osmole concentration is the
same as another solution. Solutions that are isotonic with tears are
preferred. An amount equivalent to 0.9% sodium chloride (NaCl) is
ideal for comfort and should be used when possible (Uddin et al.,
2017).

2.1.1.3 Viscosity
More viscosity will increase the residence time in the eye and affect
the eye drop performance (The United States Pharmacopeia, 2019).
Viscosity enhancers are used in ophthalmic solutions to increase their
viscosity, enables the formulation to remain in the eye longer and
gives more time for the drug to exert its therapeutic activity or
undergo absorption for example Hydroxyethylcellulose,
Hydroxypropylmethylcellulose, Methylcellulose, Polyvinyl alcohol and
Polyvinylpyrrolidone (Uddin et al., 2017).

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2.1.1.4 Clarity
By filtration, clear eye drop solutions can be achieved as it will
eliminate the particulate matter. Sintered glass filters or membrane
filters 0.45-1.2 µm pore sizes are suitable (Florence & Atwood, 2016).

2.1.1.5 Drop size

Drop sizes may typically range from 20-70 µL. Drop size can be
controlled by weight or by volume , and it is typically evaluated during
product development (The United States Pharmacopeia, 2019).

2.1.2 Sterilization
Sterilization is a crucial requirement during the manufacture of
ophthalmic products and the common methods are moist heat under
pressure, dry heat, filtration, gas sterilization and ionizing radiation
(McDonnell, 2007).

2.2 Plant profile

2.2.1 Botanical Name
 Camellia sinensis (L.) Kuntze (Theaceae)
2.2.2 Synonyms
 Camellia thea Link
 Camellia theifera Griff.
 Thea bohea L.
 Thea sinensis L.
 Thea viridis L.

2.2.3 Plant Taxonomy

 Kingdom : Plantae
 Order : Ericales
 Family : Theaceae
 Genus : Camellia
 Species : C. sinensis
 Binomial name : Camellia sinensis (L.) Kuntze

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2.2.4 Plant Description

Figure 2: Green tea plant

 Habitat: Native from the South of China, it appears

cultivated as a bush of about 2,5m, in the high areas of Asia and
China with a warm and humid climate ("Green tea properties", 2019).
 Perennial tree of the tea family – Theaceae – up to 9 m..
Lanceolate or elliptic leaves, short petiolated, 5 -6 cm long; the
younger ones downy; the older ones glabrous. Lonely white or cream
flowers, sometimes paired in the axils of the leaves that hang from
short stems. Fruit in capsule with a single seed.
The variety Camellia sinensis assamica, from India, presents longer
leaves and a superior height to the Chinese variety Camellia sinensis
sinensis ("Green tea properties", 2019).

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2.3 Composition of Camellia sinensis (Chacko, Thambi, Kuttan &
Nishigaki, 2010)
Green tea contains polyphenols, which include flavanols, flavandiols,
flavonoids, and phenolic acids, these compounds may account for up
to 30% of the dry weight. Most of the green tea polyphenols (GTPs)
are flavonols, commonly known as catechins. Products derived from
green tea are mainly extracts of green tea in liquid or powder form
that vary in the proportion of polyphenols (45-90%) and caffeine
content (0.4-10%). The major flavonoids of green tea are various
catechins, which are found in greater amounts in green tea than in
black or Oolong tea (Vinson, 2000) .There are four kinds of catechins
mainly find in green tea: epicatechin, epigallocatechin, epicatechin-3-
gallate, and EGCG (Sano et al., 2001). The preparation methods
influence the catechins both quantitatively and qualitatively, the
amount of catechins also varies in the original tea leaves due to
differences in variety, origin, and growing conditions (Khokhar &
Magnusdottir, 2002).

Green tea is a popular neutraceutical as an antioxidant. Antioxidants

are compounds that protect cells against the damaging effects of
reactive oxygen species, such as singlet oxygen, superoxide, peroxyl
radicals, hydroxyl radicals, and peroxynitrite. An imbalance between
antioxidants and reactive oxygen species results in oxidative stress,
leading to cellular damage (Halliwell & Gutteridge, 1985). Catechins
are hypothesized to help protect against these diseases by
contributing, along with antioxidant vitamins (i.e., vitamins C and E)
and enzymes (i.e., superoxide dismutase and catalase), to the total
antioxidant defense system (Meki, Hamed & Ezam, 2009).

2.4 Ethanol as solvent to extract Camellia sinensis

Ethanol showed high extraction efficiency for fresh tea leaves. This
work has confirmed that green tea is a rich source of phenolics
compounds. Higher extraction temperature resulted in a significant
increase of the extraction yield (Setyopratomo, Puguh. 2014).

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2.5 Camellia sinensis in retaining the moisture of skin
 Anti-wrinkle effects of water extracts of teas in hairless
mouse
In this study, we found that (Camellia sinensis) water extract can
effectively reduce skin damage and promote anti-wrinkling processes
in UV-irradiated hairless mouse model, which is associated with
increased moisture capacity, decrease TEWL (Lee, Kim & Kim,
2014).

 Green tea Polyphenols provide Photoprotection, Increase

Microcirculation, and Modulates Skin Properties of Women.
We demonstrated that ingestion of green tea catechins improved skin
hydration, TEWL, density and elasticity (Heinrich, Moore, De Spirt,
Tronnier & Stahl, 2011).

 Applications of a stable green tea extract on human cheeks

TEWL was reduced to a significant level (p>0.005) with the active
formulation over the defined study period of 60 days (Mahmood,
Akhtar, Ali Khan & Ahmad, 2010).

 Improvement in skin barrier function following long-term

treatment with green tea and lotus in healthy adults, a step
towards future treatment of atopic dermatitis.
In green tea treated group, green tea treated side of the face has
shown gradual improvement in epidermal hydration and effect of
green tea mono treatment was superior compared to placebo as it
showed extremely significant improvements in skin epidermal
hydration through 60 days treatment course and TEWL -43.17%
reduction in TEWL at the end of study period (Mahmood & Naveed,
2013).

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2.6 Green tea to treat dry eye/protection to the eye
 Efficacy of Green Tea Extract for Treatment of Dry Eye and
Meibomian Gland Dysfunction; A Double-blind Randomized
Controlled Clinical Trial Study
Our study shows that, green tea extract improves ocular surface
inflammation, TBUT and meibum score in patients with evaporative
dry eyes and MGD, based on the OSDI symptoms questionnaire thus
this treatment can decrease the clinical signs and inflammatory
changes in evaporative dry eyes and MGD by suppressing the
inflammatory cytokine expression (Nejabat, Ali Reza, Zadmehr,
Yasemi & Sobhani, 2017).

 Green Tea Catechins and Their Oxidative Protection in the

Rat Eye
Analysis of eye tissues showed beyond a doubt that eye structures
absorbed significant amounts of individual catechins and the retina,
for example, absorbed the highest levels of gallocatechin, while the
aqueous humor tended to absorb epigallocatechin (Woods &
Bernstein, 2019) , (Chu et al., 2010).

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 CHAPTER 3

METHODOLOGY

3.0 Collection of the dried Camellia Sinesis leaves

 Forest Research Institute Malaysia or IHerb.com.

3.1 Authentication of the dried Camellia Sinesis leaves

 Research Management Centre UPM,Serdang

3.2 Materials and equipment

3.2.1 Materials
 Disodium edetate (0.1 % w/v)
 Benzalkonium chloride (0.01% w/v)
 Boric acid
 Hydroxypropylmethylcellulose (1.0%)
 Purified water USP
 Ethanol (20%)
 Cab-O-sil powder
 Filter paper
 Alcohol swab
 Alcohol spray

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3.2.2 Eye drop bottle

3.2.3 Equipment
 Beaker
 Test tube
 Conical flask
 Water bath
 Weighing balance
 pH meter
 Filter funnel
 Sintered glass filters or membrane filters
 Spatula
 Dropper
 Pipette
 Pi-pump
 Oven
 Glass rod
 Clarity test apparatus
 Syringe

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  Grade A laminar air flow
 UV-visible spectrometer

3.3 Preparation of the Extract Camellia Sinesis leaves

Crush the dried tea leaves in a blender and sieve
by 70/100 mesh sieve set.

For extraction, mix 16 g of the dried tea leaves

powder with 480 ml of the ethanol 20 % in a 500 ml
conical flask.

Put the mixture in a water bath with the temperature

of 60°C for 240 minutes.

Filtrate the extraction and put the liquid in an oven at

50 °C until it become concentrated.

Add Cab-O-Sil powder to the concentrate to produce

granules. Put the granules in oven, 50 °C overnight
to obtain dry granules.

Weight of extraction = (weight of dry granule – weight

of Cab-O-sil)

3.4 Determination of phenolics in the extract

 Thin layer chromatographic method
The TLC sheet acts as a stationary phase support. The solvent used
was petroleum ether: ethyl acetate in the ratio 7:3 respectively which
acted as mobile phase. 2µl of sample and control (solvent) were
taken and spot in TLC sheet and keep in a beaker containing mobile
phase. This set up was kept aside for 10-15 min. After that the TLC

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sheet was taken out and the bands visualized. According to the
number of component present bands were noted (Amico et al., 2008).
According to the number of components (polyphenols) in tea extract,
the bands were clearly visible. The results are as follows: in green tea
fresh leaves extract shows prominent seven bands and three bands,
green tea commercial leaves extract shows three bands and one
band in dust tea extract (Plate 1). From the results obtained it is
understood that dust tea undergoes oxidation process while
manufacturing and it loose the polyphenols (catechin) compounds
and so only one band is seen. This can be attributed to the lesser
antimicrobial activity of dust tea (S. Archana & Jayanthi, 2011).

Figure 3 : (S. Archana & Jayanthi, 2011)

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3.5 Phytochemical test of of the Extract Camellia Sinesis
leaves

3.5.1 Test for steroid

 Treat 4 mg of extract with 0.5 ml of acetic anhydride and 0.5
ml of chloroform. Then add slowly the concentrated solution of
sulphuric acid . Green bluish color is positive.
3.5.2 Test for tannins
 Gallic tannin test
Dissolve 0.5 ml of extract in 1 ml of water, mix uniformly and then
add 2 drops of ferric chloride solution. Blue color is positive.
 Catecholic tannin test
Dissolve 0.5 ml of extract in 1 ml of water, mix uniformly and then
add 2 drops of ferric chloride solution. Green black color is positive
3.5.3 Test for flavonoids
 Treat 2ml of extract solution with 1ml of lead acetate solution
and white colour is positive.
3.5.4 Test for glycosides
 Mix equal amounts of water extract and Fehling’s reagent in a
test tube, then boil in a water bath for 10 min. The formation of red
precipitate indicated the presence of glycosides.
3.5.5 Test for alkaloids
 Add A 10 g of the plant material to 50 ml of 4% HCl in a
steam bath, then treat 1 ml of the filtrate with Mayer’s reagent. The
appearance of white precipitate is an evidence for the presence of
alkaloids

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3.6 Preparation of eye drop
3.6.1 Amount and function of materials
MATERIALS FUNCTION AMOUNT
Green tea extract Active 0.6 g
ingredient
Disodium edetate (0.1 % w/v) Chelating agent 0.06g
Benzalkonium chloride Preservative 0.006g
(0.01% w/v)
Boric acid USP Buffer 1.14 g
Hydroxypropylmethylcellulose Viscosity agent 0.6 g
(1.0%)
Purified water USP Vehicle 60 ml

3.6.2 Preparation of solution

Weigh all the materials needed using weighing

balance,

Dissolve the active ingredient (green tea extract) with

some of the vehicle (purified water USP).

Disperse and mix all the excipients with glass rod

until dissolve.

Mix the mixture with the active ingredient solution

then add up the vehicle until desired amount.

3.6.3 Clarification
 Filter the eye drop solutions with sintered glass filters or
membrane filters 0.45-1.2 µm pore sizes.

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 3.6.4 Sterilization
 Filtration through membrane filter of 0.22 µm pore size into
the sterile eye drop container in the horizontal laminar air flow( with
effective HEPA filter).

3.7 Eye drop formulation quality control

3.7.1 pH
Evaluate the pH of the eye drop by using pH meter.

3.7.2 Clarity
i. By clarity test apparatus, observe the eye drop in front of both black
and white background with fluorescence lamp. When viewing at the
black background, white or light coloured particles might be seen
while when viewing at the white background, dark or black coloured
particles can be spotted easily.
If there is some floating particles been observed the result is said to
be positive, which means that the eyes drops solutions were not
properly filtered.
ii. By using UV-visible spectrometer to measure the clarity and compare
with other eye drop brand.

3.7.3 Drop size

Range from 20-70 µL. By weighing balance measure the average
weight of the drop and the density.

3.7.4 Determination of the catechins in the eye drop

By using UV-visible spectrometer to determine the catechin in the
green tea extract and eye drop. Spectral range of 200 to 500 nm,
spectral bandwith of 2nm and scan speed of 240nm per minute.

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 3.8 Data and statistical method
Using software Statistical Package for the Social Science or SPSS.
Data measured such as pH, drop size an concentration of catechins
will be analyze by sing SPSS v25.

REFERENCES

1 Attwood, D., & Florence, A. (2016). Physicochemical Principles of

Pharmacy. London: Macmillan Education, Limited.
2 Aulton, M., & Taylor, K. (2018). Aulton's pharmaceutics. Edinburgh
(Scotland): Elsevier.
3 Chacko, S., Thambi, P., Kuttan, R., & Nishigaki, I. (2010). Beneficial
effects of green tea: A literature review. Chinese Medicine, 5(1), 13.
doi: 10.1186/1749-8546-5-13
4 Chu, K., Chan, K., Wang, C., Chu, C., Li, W., & Choy, K. et al. (2010).
Green Tea Catechins and Their Oxidative Protection in the Rat
Eye. Journal Of Agricultural And Food Chemistry, 58(3), 1523-1534.
doi: 10.1021/jf9032602
5 Green tea properties. (2019). Retrieved from https://www.botanical-
online.com/en/medicinal-plants/green-tea-properties
6 Heinrich, U., Moore, C., De Spirt, S., Tronnier, H., & Stahl, W. (2011).
Green Tea Polyphenols Provide Photoprotection, Increase Sheskey,
P., Cook, W., & Gable, C. Handbook of pharmaceutical
excipients (8th ed., p. 283). Pharmaceutical press.
7 Kai On Chu, Kwok Ping Chan, Chi Chiu Wang, Ching Yan Chu, Wai
Ying Li, Kwong Wai Choy, Michael Scott Rogers, and Chi Pui Pang
Journal of Agricultural and Food Chemistry 2010 58 (3), 1523-1534
DOI: 10.1021/jf9032602.
8 Lee, K., Kim, S., & Kim, Y. (2014). Anti-wrinkle Effects of Water
Extracts of Teas in Hairless Mouse. Toxicological Research, 30(4),
283-289. doi: 10.5487/tr.2014.30.4.283
9 Mahmood, Tariq & Naveed, Akhtar & Khan, Barkat & Ahmad,
Mahmood & Khan, Haji M. shoaib & Zaman, Shahiq uz. (2010).
APPLICATIONS OF A STABLE GREEN TEA EXTRACT CREAM ON

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 HUMAN CHEEKS. INTERNATIONAL JOURNAL Of ACADEMIC
RESEARCH. 02. 121-126.
10 Mahmood, Tariq & Naveed, Akhtar. (2013). Improvement in skin
barrier function following long-term treatment with green tea and lotus
in healthy adults, a step towards future treatment of atopic dermatitis.
HealthMED. 7. 455-461.
11 Microcirculation, and Modulate Skin Properties of Women. The
Journal Of Nutrition, 141(6), 1202-1208. doi: 10.3945/jn.110.136465
12 Nejabat, M., Reza, S. A., Zadmehr, M., Yasemi, M., & Sobhani, Z.
(2017). Efficacy of Green Tea Extract for Treatment of Dry Eye and
Meibomian Gland Dysfunction; A Double-blind Randomized
Controlled Clinical Trial Study. Journal of clinical and diagnostic
research : JCDR, 11(2), NC05–NC08.
doi:10.7860/JCDR/2017/23336.9426
13 Sano, M., Tabata, M., Suzuki, M., Degawa, M., Miyase, T., & Maeda-
Yamamoto, M. (2001). Simultaneous determination of twelve tea
catechins by high-performance liquid chromatography with
electrochemical detection. The Analyst, 126(6), 816-820. doi:
10.1039/b102541b
14 S. Archana, S., & Jayanthi, J. (2011). Comparative analysis of
antimicrobial activity of leaf extracts from fresh green tea, commercial
green tea and black tea on pathogens. Journal Of Applied
Pharmaceutical Science, 08, 149-152.
15 Setyopratomo, Puguh. (2014). Extraction of phenolic compounds
from green tea using ethanol. ARPN Journal of Engineering and
Applied Sciences. 9. 1516-1521.
16 Uddin, M., Mamun, A., Kabir, M., Setu, J., Zaman, S., Begum, Y., &
Amran, M. (2017). Quality Control Tests for Ophthalmic
Pharmaceuticals: Pharmacopoeial Standards and
Specifications. Journal Of Advances In Medical And Pharmaceutical
Sciences, 14(2), 1-17. doi: 10.9734/jamps/2017/33924.
17 United States Pharmacopeial Convention. (2019). The United States
Pharmacopeia(pp. 6511-6516). Rockville, Md.

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18 Van Wyk, B., & Wink, M. (2004). Medicinal plants of the world (p. 75).
BRIZA PUBLICATIONS.
19 Vinson, J. (2000). Black and green tea and heart disease: A
review. Biofactors, 13(1-4), 127-132. doi: 10.1002/biof.5520130121
20 Willoughby, C., Ponzin, D., Ferrari, S., Lobo, A., Landau, K., & Omidi,
Y. (2019). Anatomy and physiology of the human eye: effects of
mucopolysaccharidoses disease on structure and function - a review.
21 Woods, M., & Bernstein, M. (2019). New evidence that green tea may
help fight glaucoma and other eye diseases - American Chemical
Society. Retrieved from
https://www.acs.org/content/acs/en/pressroom/presspacs/2010/acs-
presspac-april-21-2010/new-evidence-that-green-tea-may-help-fight-
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APPENDICES (Gantt chart)

MONTH (2019) March April May June July August September October November December January February

Research Area Planned

Identification
Achieved
Planned
Chapter 1 Achieved
Planned
Chapter 2 Achieved
Literature Review
Planned
Chapter 3 Achieved
Methodology
Planned
Check with Achieved
Supervisor
Planned
Oral Presentation Achieved
Planned
Draft Proposal 1 Achieved
Planned
Changes / Editing of Achieved
Proposal
Planned
Submission of Achieved
Proposal

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 Planned
Second draft
proposal Achieved

Planned
Final proposal Achieved
submission

Planned
Data colllection Achieved
Planned
SPSS Data entry Achieved
Planned
Data analyses Achieved
Planned
Thesis write up Achieved
Planned
Thesis presentation Achieved
Planned
Correction and Achieved
submission

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