OUR LADY OF FATIMA UNIVERSITY

COLLEGE OF MEDICINE

Dermoprotective capability of Coffea canephora robusta (Kapeng Barako) Silverskin

Extract on UV- B exposed Caenorhabditis elegans

First year Medicine, Section F2, Groups 6-10, AY 2017-2018

Department of Biochemistry and Nutrition
College of Medicine- Our Lady of Fatima University
Reylan David, MD; Sharmaine Paguirigan, MD; Remedios P. Santos, MD

Abstract
Philippines is one of the few countries that produces varieties of coffee. Coffea canephora
robusta, a variety found in Batangas is one of them. The silverskin of other coffee species were
found to inhibit UV-B induced cutaneous damage. C. c. robusta has been found capable of
inhibiting hyaluronidase (which degrades collagen) and NF-KB (a gene that up-regulates
proinflammatory cytokines). However, studies using the said coffee assessing
dermatoprotection are virtually non-existent. A study showed that UV (ultraviolet) exposure in
tropical countries is rated as extreme (based on UV index or UVI). This study therefore,
attempted to assess the dermoprotective capability of C. c. robusta's silverskin extract (CSE) on
UV-B exposed Caenorhabditis elegans using Qualitative analysis.
Methodology: Extracted and filtered coffee silverskin was first prepared. A series of tests which
included -2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay and Phytochemical Analysis was done.
C. elegans worms were obtained at the University of the Philippines-Manila. After exposure to
UV- B, the nematodes were placed in six mediums (negative control – distilled water, positive
control-ascorbic acid, and four concentrations of CSE- 0.01mg/ml, 0.1mg/ml, 0.5mg/ml and
1mg/ml). Eight daily microscopic observations were made by a reputed helminthologist.
Results: At day eight, following criteria in Table I, dermoprotective capability of CSE was noted
at 0.01mg/ml and 0.1mg/ml concentrations. DPPH assay performed showed no significant anti-
oxidant effect. CSE, based on phytochemical screen was positive for alkaloids, terpenoids,
terpenes, quinones, flavonoids and tannins.
Conclusion: Based on the results, Coffea canephora robusta may exhibit a potential significant
dermatoprotective effect. Further studies can be done to assess this effect and its potential
application in medical skin interventions.
Keywords: Coffee silverskin, anti-oxidant, dermoprotective capability, Coffea canephora
robusta, Caenorhabditis elegans

____________________________________________________________________________

1. INTRODUCTION
In the Philippines, coffee is one of
the most consumed products in the market,
driven by the growing Business Process
Outsourcing (BPO)1. Popular coffee shops
offered extensive range of organic coffee on
their menu; hence an increased in the
demand for fresh roasted and ground coffee
boosted its production2. This result in the

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production of a lot of coffee silverskin during Coffee silverskin which is the outer layer
the brewing process. of the green coffee beans or commonly
known as chaff in the industries that
Coffea canephora robusta also produced during roasting process 5. Study
considered a variety of C. canephora, has a had shown that it has a hyaluronidase
variety accounts for 95% of C. canephora inhibition effect and inhibits NF-kB, which
grown worldwide. This coffee species has upregulates the transcription of pro-
the highest caffeine content and an inferior inflammatory mediators and associated with
taste compared to Coffea canephora inflammation and cancer6.
arabica but is resistant to coffee diseases3.
Studies have shown that coffee
Coffee silverskin is also known as coffee silverskin have potential in prevention and
effluent which is discarded during treatment of diabetes mellitus and obesity7,
refinement process. It is considered a anti-inflammatory8, anti-fungal and anti-
9
pollutant due to presence of organic parasitic capabilities , as well as
materials which include tannins, dermoprotective properties10.
polyphenols, and caffeine. These
compounds may contaminate water sources Therefore, coffee silverskin extract
and thus contribute to significant morbidity. (CSE) may represent a readily available,
low-cost, and innovative product that can be
This kind of pollutant into the used in the pharmaceutical and cosmetic
atmosphere could cause ozone depletion industries.
and global warming. Ozone layer is our
protective shield that absorbs most of the C. elegans is a small, non-parasitic,
sun’s ultraviolet (UV) radiation; protecting us nematode worm, composed of a simple
from potentially harmful effects. Philippines epidermal epithelium and overlying cuticle
being a tropical country is characterized by which is homologous to mammalian skin11
relatively high temperature. Extremely high and it has the advantage of a relatively
UV levels (UVI of up to 19 for clear sky and predictable and short lifespan12.
up to 22 under broken cloud conditions)
were also measured at sea level in the C. elegans has calcineurin which
tropical Pacific4. This extremely high UV populates skin, including melanocytes and
levels could affect human health. keratinocytes, calcineurin has been shown
to increase the levels of matrix

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metalloproteinases (MMP) a degrading USA wherein some strains were provided

enzyme that is responsible for collagen by the CGC, which is funded by NIH Office
destruction, inhibition of calcineurin led to a of Research Infrastructure Programs (P40
significant increase in the lifespans of C. OD010440).
elegans13 also C. elegans encodes several
tyrosinases capable of generating melanin, 2.2 Preparation of Coffee
and melanin has been detected in the C. Silverskin and Extraction
elegans cuticle14.
C. c. robusta was roasted in a hot
Therefore, C. elegans is extremely plate at 75 degrees Celsius for 20 minutes
important as many experiments are and silverskin easily peeled off from the
impossible to human subjects and also it is coffee beans. When silverskin was
convenient and inexpensive. collected, it was cut into smaller pieces and
placed in a beaker with distilled water in a
2. MATERIALS AND METHODS ratio of 50 mg silverskin: 1 mL Distilled
water. Mixture was then boiled at 100
2.1 Acquisition and Identification of degrees Celsius for 5 minutes and
Coffea canephora robusta (kapeng continuously stirred. It was filtered through a
barako) and Caenorhabditis elegans funnel with filter paper; the product was a
hazy light brown filtrate. There were four
Coffea canephora robusta (kapeng concentrations of CSE, namely 0.01 mg/mL,
barako) was acquired at Lipa, Batangas 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL.
because the location was known as the first
Coffee Capital in the Philippines and it was 2.3 2,2-Diphenyl-1-picrylhydrazyl
easier to acquire especially in lowlands. A (DPPH) assay
sample of the coffee plant was submitted to
the Bureau of Plant Industry for For the DPPH radical scavenging
identification and verification. assay, 20 μL of CSE was diluted
appropriately in Dimethyl sulfoxide (DMSO)
Caenorhabditis elegans was was mixed with 180 μL of DPPH in
procured at the Biochemistry Department of methanol (40 μg/mL) in a 96-well plate15.
University of the Philippines – College of The plate was kept in the dark for 15
Medicine at Pedro Gil, Manila because they minutes, after which the absorbance of the
have ties with the University of Minnesota,

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solution was measured at 540 nm in a coloration indicated the presence the

Multiskan Ascent plate-reader. presence of hydrolysable tannins, while
brownish-green coloration indicated the
2.4 Phytochemical Analysis presence of condensed tannins.

2.5 C. elegans culture and

An amount of 50 mg/mL CSE was
Inoculation with CSE
used for Phytochemical Testing. CSE was
subjected to several tests that include
C. elegans N2 strain eggs were
Alkaloids, Terpenes, Terpenoids, Quinones,
used in the experiment. The researchers
Flavonoids and Tannins.
hatched the said species in Nematode
Growth (NG) medium and were cultured on
To test for Alkaloids, Wagner’s test
agar plates containing E. coli OP50 strain.
was used. Appearance of a reddish-brown
Upon reaching maturity on the 4th day, the
precipitate indicate a positive result.
worms were transferred to plates containing
NG medium, E. coli, and different
To test for Terpenes and
concentrations of CSE (0.01 mg/mL, 0.1
Terpenoids, Salkowski test was used.
mg/mL, 0.5 mg/mL, 1 mg/mL). A negative
Appearance of a reddish-brown coloration
control (Distilled water 150 μL) and also a
at interphase indicated a positive result for
positive control (Ascorbic Acid 150 μL) were
Terpenoids, while a golden yellow color
used (Figure 1). The worms (120 worms)
indicated a positive result for Triterpenes.
were incubated for 7 days at 15 degrees
Celsius. During this period, worms were
To test for Quinones, Sulfuric Acid
subjected daily to Ultraviolet-B radiation for
test was used. Appearance of a red color
45 seconds. Worms were scored as dead if
indicated a positive result.
they fail to respond to the platinum wire.

To test for Flavonoids, Alkaline

Reagent Test was used. Appearance of an
intense yellow color upon addition of base
and turns colorless on addition of acid.

To test for Tannins, Ferric Chloride

test was used. Appearance of blue-black

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Replicate Replicate
Set-up
1 2
Negative Control (Distilled Water) 10 10
Positive Control (Ascorbic Acid) 10 10
Coffee silverskin extract: 0.01 mg/mL 10 10
Coffee silverskin extract: 0.1 mg/mL 10 10
Coffee silverskin extract: 0.5 mg/mL 10 10
Coffee silverskin extract: 1 mg/mL 10 10
TOTAL= 120 worms 60 60

Table 1. Distribution of worms per Set-Up

After hatching and culturing C.

2.6 C. elegans Dermoprotective elegans for 3 days at 20 degrees Celsius,
the worms were transferred to plates that
Lifespan measurement is a direct contained NG (Nematode Growth) medium,
method that determines the consequence of E. coli, a corresponding CSE extract
aging and death. Researchers count live concentration, namely 0.01 mg/mL, 0.1
and dead worms in synchronized mg/mL, 0.5 mg/mL, and 1 mg/mL, and
populations at regular intervals (e.g., 1 or 2 distilled water as negative control. 10 worms
days) and discard dead worms to prevent per treatment were incubated for 7 days at
confusion while performing subsequent 20 degrees Celsius and transferred every 2
counting. Most organisms have defense days to fresh media plates for scoring of
mechanisms against UV radiation, such as viability. The worms were exposed to UV-B
nucleotide excision repair pathways, that radiation for 45 seconds during the time
repair damaged DNA Because UV radiation being. Worms were observed through
induces DNA lesions and produces free fluorescent microscope and counted as
radicals. Ultraviolet light has dose- dead if there were no response to a
dependent effects on the health and survival platinum wire. Worms were observed
of C. elegans, and in most cases, 10 to 30 through fluoroscopy for better magnification
2
J/m /min of radiation are used however, the of movement. The assays were done in
specific doses of UV radiation selected duplicate. Observation of the C. elegans’
depends on UV resistance properties of was by the researchers with the aid of an
worms strains used in assay. It is performed Helminthologist to provide criteria in scoring.
using different stages of worms to
determine its stage-specific effects15.

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2.7

Dermoprotective assessment of C. capabilities of C. c. robusta (kapeng barako)

elegans CSE

The following criteria (Figure 2) was

used by the Helminthologist to assess the
dermal quality and dermoprotective

Positive Negative
Dermoprotectability Dermoprotectability
Presence of Cuticle Absence Cuticle
Absence of Scarring Presence of Scarring
Absence of Molting Presence of Molting
Intact Epidermis Absence of
Epidermis
Alive Dead

Table 2. Criteria for Identifying Dermoprotectability of C. c. robusta silverskin extract on

C. elegans

3. RESEARCH DESIGN
Qualitative Analysis used as the 5. RESULTS and DISCUSSION
research design for this research.
5.1 DPPH Assay
4. DATA ANALYSIS The results of the assay (Figure 3)
This research used Qualitative indicate that CSE from C. c. robusta
analysis for the Criteria for exhibited properties of free radical
Dermoprotective capability of CSE. scavenging. As the concentration of the
Absolute Risk Reduction, Relative Risk extract increased, the free radical
Reduction, Control Event Rate and scavenging activity showed minimal
Experimental Event Rate were the
statical tools used in this experiment.

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changes; therefore it was proved that the

dermorpotective capability of the CSE does
not directly correlate to antioxidant activity.

Figure 1. Free radical scavenging of DPPH by CSE

5. 2 Phytochemical Analysis the presence of Alkaloids. Caffeine is

CSE has shown positive reaction to
tests for Alkaloids, Terpenes, Terpenoids,
Quinones, Flavonoids, and Tannins. These
mechanisms provided the dermoprotective
capability of the CSE on C. elegans as well
as its lifespan-extending effect.
composed of alkaloids and the presence of
these substances can have anti-oxidative
properties which have numerous health
benefits and pharmaceutical purposes.
However, it has showed that at dose-
dependent levels it has an inhibitory effect
on wound and cellular repair16.

A. Test for Alkaloids B. Test for Terpenes and

The formation of a Reddish-brown Terpenoids
precipitate when Wagner’s reagent was CSE exhibited a positive reaction to
added to CSE showed a positive result for Salkowski test for Terpenoids which showed

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a reddish-brown coloration while it also addition of a base and turns colorless on

exhibited a positive reaction to Salkowski addition of an acid. Flavonoids have shown
test for Triterpenes which showed a golden effects on cell excision capabilities. At high
yellow coloration. Terpenes have showed non-toxic concentrations, it provided CSE
anti-oxidative capabilities which provide the capacity to modulate DNA repair19.
protection against reactive oxygen
17
species . E. Test for Tannins
CSE also showed a positive reaction
C. Test for Quinones to Ferric Chloride test on the test for
CSE has shown positive results on Tannins. Appearance of blue-black
the Sulfuric Acid test by producing a red coloration will indicate the presence of
color when the reagent was added. hydrolysable tannins, while brownish-green
Quinones have showed medical and coloration will indicate the presence of
pharmaceutical benefits such as anti- condensed tannins. These compounds are
tumoral activity as well as anti-oxidative used for its numerous biological and
capabilities18. pharmacological effects, which possess
anti-inflammatory and antioxidant activity20.
D. Test for Flavonoids Tannins repair DNA through base excision
CSE showed a positive reaction to repair, Presence of tannins aid in DNA
the Alkaline Reagent test for Flavonoids repair mechanism through use of base
which showed an intense yellow color upon excision repair21.

The CSE concentration of

0.01mg/ml and 0.1mg/ml showed maximum
dermoprotective effect while 0.5mg/ml and
1mg/ml concentration showed toxicity in
higher concentration.

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Figure 2. Illustrates the dermoprotective effect of CSE on the Replicate 1

Figure 3. Illustrates the dermoprotective effect of CSE on the Replicate 2

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Dermoprotectability of CSE on C. decrease in the number of nematodes

elegans are illustrated in figures 1 and 2.
Upon initial observation (Day 0) all C.
elegans satisfied the criteria of having
positive dermoprotection (see table 2). On
the first day, a nematode in 0.50 mg/mL
concentration showed a negative
dermoprotection while the rest still have
positive dermoprotection. From the third day
up to the fifth day there are continual
having positive dermoprotection. On the
sixth day, 1 mg/mL concentration on
replicate 2 and negative control on replicate
1 both have no more nematodes that
satisfies the criteria for positive
dermoprotection. On the eight day, both
0.01 mg/mL and 0.1 mg/mL concentrations
on both replicates still have a few
nematodes that were able to satisfy the
criteria of having positive dermoprotection.

Setup PD ND
0.01 3 7 EER 30 ARR 10
(+) Control 2 8 CER 20 RRR 50
0.10 4 6 EER 40 ARR 20
(+) Control 2 8 CER 20 RRR 100
1
0.50 1 9 EER 10 ARR -10
(+) Control 2 8 CER 20 RRR -50
1.00 0 10 EER 0 ARR -20
(+) Control 2 8 CER 20 RRR -100

Setup PD ND
0.01 3 7 EER 30 ARR 10
(+) Control 2 8 CER 20 RRR 50
0.10 4 6 EER 40 ARR 20
(+) Control 2 8 CER 20 RRR 100
2
0.50 2 8 EER 20 ARR 0
(+) Control 2 8 CER 20 RRR 0
1.00 1 9 EER 10 ARR -10
(+) Control 2 8 CER 20 RRR -50

Table 3. Risk Reduction (Control Event Rate, Experimental Event Rate, Absolute Risk
Reduction and Relative Risk Reduction)

At 0.1 mg/mL concentration, there is a concetration there are 50% and 100%
100% decrease risk of injury to C. elegans, increase risk of injury respectively to C.
this is followed by 0.01 mg/mL which has 50 elegans.
% chance while at 0.50mg/mL and 1 mg/mL

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Figure 4: Scoring results for

identifying Dermoprotectability of C.
robusta silverskin extract on C.
elegans

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Figure 7. Dermal study of UV- B Irradiated C. elegans strain N2 exposed to various

concentration of CSE for days 0-8

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7. CONCLUSION  Comparative studies between other

The researchers therefore conclude types of coffee found in the
that coffee silverskin of Coffea. canephora Philippines.
robusta is protective of the epidermis of  Using genetically modified C.
Caenorhabditis elegans exposed under elegans that show Cadherin,
UV–B radiation. The concentration of Collagen and Adherin Junctions.
0.01mg/ml and 0.1 mg/mL showed  Experimental study of the effect of
maximum dermoprotective capability but coffee silver skin in human
has no correlation with antioxidant activity. epidermal cell.

At 0.1 mg/mL concentration, there is

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