Kristina Bianca V.

Melendres BsBio2

INSTITUTE OF AGRICULTURE AND LIFE SCIENCES

CHEM 103
Final Requirement

Due: May 30, 2022, 5:00 PM

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In order to deepen and widen your knowledge on biochemistry, discuss exhaustively the major
components (i.e. introduction, methodology, results and discussions and conclusion) of the
following biochemistry related research articles. Furthermore, this activity of searching, reading,
and synthesizing research articles will somehow prepare you on how to make your Related Review
of Literature (RRL/ Chapter 2) and other components of your study proposal in the near future.
Search these articles using google or google scholar on your own so you will have the feel and the
experience of finding them.

1. Hayakari, R., Matsumiya, T., Xing, F. Tayone, J.C. Dempoya, J., Tatsuta,
T., Yashiro, T.A., Imaizumi, T., Yoshida, H., and Satoh, K. (2012). Effects
of Brazilian green propolis on double-stranded RNA-mediated induction of
interferon-inducible gene and inhibition of recruitment of
polymorphonuclear cells. Journal of Science and Food Agriculture.

Introduction
Propolis is a bee substance that has a vast array of biological activities,
namely antimicrobial activities, when taken orally. However, the cellular and
molecular mechanisms behind it are unknown.

Methodology
The A549 cells is kept at 37c in a 5% Co2 incubator in DMEM enriched with
10% fetal bovine serum. The culture medium is substituted with DMEM-0.5 % bovine
serum albumin for the chemostaxis experiment. Propolis powder has been provided
by Yamada Bee Farm. Propolis was dissolved with dimethyl sulfoxide and fed to the
culture medium for one hour before adding polyinosinic-polycytidylic acid. After then,
transfection takes place. Poly I:C was spontaneously transfected into A549 cells.
The cells were plated at a concentration of 1.5x105 cells per well in a 12-well culture
dish for 16-20 hours prior transfection. 1.5 microliters Lipofctamine 2000 was applied
to transfect poly I:C for the required time after 1 hour of propolis treatment, according
to the manufacturer's instructions. IFN-beta would then be measured using enzyme-
linked immunosorbent. To evaluate the amount of IFN-beta in the A549-conditioned
media, cells on a 24-well plate are exposed with propolis for 1 hour in advance to
ploy I:C transfection. After 24 hours, the medium is obtained, the cell suspension
was pelleted by configuration, and the precipitate was accumulated. The level of
IFN-beta in the control cells was determined using a human IFN-beta ELISAkit.
When it comes to the quantitative reverse transcriptase-polymerase chain reaction,
An illustra RNA spin Mini RNA Isolation Kit is utilized to collect total RNA from the
cells. Under the manufacturer's specifications, whole RNA was employed as a
substrate for single-strand cDNA synthesis with oligo(dT) primers and M-Mulv
reverse transcriptase. A CFX96 real-time PCR monitoring equipment was used to
identify the melanoma differentiation-associated gene 5, CCL2, CCL5, CXCL8/IL8,
myxovirus resistance 1, interferon-inducible gene 6-16, 2'-5'-oligoadenylate
synthase, 18S rRNA, and glyceraldehyde-3-phosphate dehydrogenase. Following
poly I:C transfection, the cells are cleaned twice in phosphate-buffered saline and
collected in hypotonic lysis buffer comprising a protease inhibitor cocktail for
immunoblot analysis. The lysate was cleaned by centrifugation at 12000 x g for 10
minutes at 4 degrees Celsius. On an 8.0 % sodium dodecylsulfate-polycrylamide gel
electrophoresis, a fraction of the cell lysate was separated by electrophoresis. The
proteins were then moved to a polyvinylidene fluoride membrane for further
encoding. After that, it was isolated for 1 hour at room temperature in TBST solution
containing 5% nonfat dried milk. The membrane is isolated with blocking buffer
containing one among 3 main antibodies: anti-MDAS, anti-RIG-I, or anti-beta-actin,
and incubated overnight at 4°C. The membrane was cultured for 1 hour at room
temperature with a 1:10 000 concentration in blocking buffer of a bovine anti-rabbit
antibody or an anti-mouse IgG antibody conjugated to horseradish peroxidase after
five washing with TBST. After cleaning in TBST, the immunoreactive bands were
detected with Luminata Crescendo Western HRP Substrate. Polymorphonuclear
leukocytes are prepared next. PMNs were extracted from arterial blood of healthy
volunteers utilizing a polymorphprep as indicated by the manufacturer. To break the
erythrocytes, the cell is treated in 0.45 % NaCl. After that, the remaining PMNs are
pelleted and resuspended in DMEM. For the chemotaxis experiment, the PMNs are
quantified and adjusted to a density of 1.0 x 105 cells mLi. In the medium A549 cells,
chemotactic activity is now assessed and combined. On a 24-well cell culture plate,
a 3-micrometer hole diameter chemotaxis chamber is established. In the lower
chamber, the cultured media from the cell treated with poly I:C in the exclusion of
propolis was applied. After adding PMN suspension to the upper chamber, the plates
were then incubated for 60 minutes at 37c. The filter membrane was then withdrawn
from the chamber and cell culture dish, and the bottom chamber was filled with
cultured media from cells treated with poly I:C in the inclusion or exclusion of
propolis. After adding the PMN suspension to the upper chamber, the plate was
incubated for 60 minutes at 37c. Following that, the filter membrane was removed
from the chamber and dyed with Glemsa. The transmigrated cells were counted
using a microscope.

Results
After removing the filter membrane from the chamber and cell culture dish, the
lower chamber was filled with culture media from cells treated with poly I:C in the
inclusion or exclusion of propolis. After adding the PMN suspension to the upper
chamber, the samples were incubated for 60 minutes at 37c. Following that, the filter
membrane was removed from the chamber and dyed with Glemsa. The
transmigrated cells were observed using a microscope. Propolis boosted the
production of myxovirus resistance 1 (MX1) but had no impact on interferon-
inducible gene 6-16 (G1P3) or 2'-5'-oligoadenylate synthetase (OAS). Because all of
these genes are IFN-inducible, propolis' antiviral signaling impact isn't always
controlled by IFN-ß autocrine regulation. By reducing dsRNA-induced interleukin-8
(IL8) and CCL5 expression, propolis pre-treatment inhibited polymorphonuclear
leukocyte (PMN) chemotactic activity in the cell-conditioned media.

Discussion
Since these genes is essential for host antiviral mechanisms including RNA
editing and translational silencing, elevation of type I IFN, 'fibroblast' IFN, and IFN-
beta is accountable for the antiviral immune reaction in the most of of adherent cells
during bacterial infection. Transfection of A549 cells with poly I:C, which imitated
dsRNA virus infection, stimulated IFN-beta expression, as initially disclosed. In
A5949 cells, we first showed that dsRNA did not induce additional type I IFN
members such as IFN-alpha, -epsilon, -kappa, or -omega. Despite the fact that
multiple lines of evidence imply that propolis has antiviral properties, no research has
been done to see how it affects IFN-beta expression. As a result, we looked into how
propolis affected IFN-beta production in response to dsRNA.

Conclusion
These data imply that propolis may decrease excessive inflammatory
responses during viral infection without altering innate immunity.
 2. Tayone, W.C., Tayone, J.C., Hashimoto, M. (2014). Isolation and Structure
Elucidation of Potential Anti-Dengue Metabolites from Tawa-tawa
(Euphorbia hirta Linn.). Walailak Journal for Science and Technology,
11(10): 825-832.

Introduction
Herbal medicines delivered by living beings that show organic exercises have
been made as strong drugs to combat diseases and spare millions of lives. Dengue
fever is caused by the dengue infection and is additionally known as break-bone
fever due to the extreme torment that an contaminated individual will encounter.
This illness is exceptionally genuine in tropical regions where Aedes
aegyptimosquitos live, and it is capable for more than 50 million cases of
dengue disease around the world each year. Euphorbia hirtaLinn., locally known as
tawa-tawa or gatas-gatas within the Philippines, is one such
potential characteristic item source. Individuals within the Philippines utilize a
wide assortment of therapeutic plants, especially in country regions, to treat
common infections. Since of the tall costs of over-the-counter drugs, home
grown plants have gotten to be a prevalent choice for both regular and year-
round infections. Various investigate considers have been conducted on Euphorbia
hirta Linn's organic exercises. Antibacterial and toxicological

Methodology
Euphorbia hirtaLinn is roughly 2.0 kg in weight. Herbs were harvested,
chopped, and sun-dried. For 48 hours, the dried plant was soaked in 6.0 L of a 50/50
EtOH/MeOH solution. A decoction was also used to make tea by boiling ca. Mix 20 g
of dried sample with 300 mL of water for 15 minutes. Before even being filtered,
concentrated in vacuum, and stored for the anti-dengue test, the tea was left to cool
down. Under the similar circumstances as compound 1, 2, and 3, the APCIMS
spectrum exhibited m/z 409.3800 [M+H-H2O]+. Compound 4 has an Rf value of 0.43
in TLC (20 percent EtOAc/hexane) with a protonated molecular ion proton peak at
413.3760, which relates to the molecular formula C29H48O. After some more
purification of Fr A2 with SiO2 gel chromatography, compounds 5 and 6 were
produced. Compound 6 was separated as a white powder with a TLC Rf value of
0.29 (20 percent EtOAc/hexane) and is slightly soluble in methanol. The APCIMS
spectrum shows m/z 397.3803 [M+H-H2O]+, which relates to C29H49: 39.3829 and
its chemical formula of c29H50O. The protonated molecular ion signal at 433.1103
indicates that compound 8 has the chemical formula C21H20O10. Euphorbia
hirtaLinn Metabolites are found in Compound 9.

Results and Discussion

Remarkably, the EtOAc fraction had the strongest anti-dengue effect. Dengue
virus serotype 1 plaque producing ability was lowered by 85% from 1400 to 200 PFU
using the EtOAc fraction. The EtOAc fraction continuously had a massive effect on
dengue virus serotype 2 infection (90 percent reduction). This suggests that the
tawa-tawa extract can eliminate the dengue virus, as well as the compound(s) that
do so can be located in the organic layer. The residue was then successively split
over silica gel column chromatography to give 1 (49.0 mg), 2 (9.0 mg), 3 (11.0 mg), 4
(2.0 mg), 5 (10.0 mg), 6 (30.0 mg), 7 (2.0 mg), 8 (4.0 mg), and 9 (4.0 mg) (27.0 mg).
The first identified metabolite was Yeild 1. The development of a double doublet
proton signal at 5.49 ppm (1H NMR) and carbon resonances (13C NMR) at 217.6,
157.6, and 117.2 ppm demonstrated the existence of a ketone and an olefinic
activity. In addition, the 1H NMR spectra of 1 revealed 8 singlet methyls. COSY,
DEPT, HMQC, and HMBC are additional 2D NMR investigations that complement
the prior explanation and confirm the planar structure of 1. As a result, taraxerone
was classified as number one. A combination of very up field doublets at 0.56 and
0.33 ppm (1H each, d both, J = 4.2 Hz) were discovered in the 1H NMR spectrum of
3 in CDCl3, correlating to a cyclopropyl methylene group of a cycloartane-type
triterpene. The moderately upfield olefinic proton signal at 5.10 ppm (1H, m) and two
singlet methyls at 1.68 (3H, s) and 1.61 (3H, s) ppm were identified by the ending
dimethylvinyl group in the side chain. The oxygenated methine signal at 3.28 ppm
(1H, m) and its carbon resonance at 78.9 ppm observed the formation of an alcohol
moeity of 3. Additional 2D NMR experiments (COSY, DEPT, HMQC, and HMBC)
back up the prior talks about its planar structure. Then, in the same way as 2 and 3,
a 28-nor oleanane derivative (4) was isolated. The presence of a triterpene molecule
was indicated by the 1H NMR spectra of 4. At 124.4 and 79.1 ppm carbon levels, it
found signals for the seven singlet methyl groups. Except for the presence of an
oxygenated proton at 3.18 ppm, the 1H and 13C NMR spectrum of 5 is substantially
identical to that of 1, showing that 5 is a reduced version of 1. This was substantiated
by the existence of a carbonyl ketone and an oxy-carbon signal. Compound 6 is a
triterpene because it has an alcoholic proton, an olefinic proton, and singlet methyl
groups. 6 was distinguished as -sitosterol since the 1H and 13C NMR results were
similar to the literature. Three flavonoid-like compounds have been isolated from
acetone-d6 extract, seven of which have been first recognized as 3-O-
arabinofuranoside of kaempferol. A downfield shift of C-4 at 89.8 ppm showed the
existence of a furanose sugar. The sugar unit was identified as a furan ring by NOE
correlations at H-2 (4.26 ppm) and H-6'. The existence of arabinofuranosyl as a
sugar moiety was further established by the fact that real D-arabinose produced
peracetate had similar GC profiles. The 3-O-rhamnopyranoside of kaempferol was
found to be compound 8. An AA'XX' system at 7.85 (H-2' and H-6') and an AX
system at 6.27 (H-6) were detected in the aromatic area of the 1H NMR spectra of
acetone-d6. The 3-O-rhamnopyranoside of quercetin was identified to be acetone-
d6. An ABX system at 7.50 (H-2') and a 2H AX system at 6.25 (H-6) were detected in
the aromatic area of the 1H NMR spectrum, indicating quercettin-type. Sugar protons
were assigned using COSY and coupling constant analysis. The axial orientations
among these molecules were identified because H-4 exhibited coupling constants of
9.5 and 9.3 Hz for H-5 and H-3, correspondingly.

Conclusion
Both the extract showed and infusion of Euphorbia hirtaLinn limited the
effectiveness of the dengue virus to produce plaques to some amount. One of the
nine identified chemicals could be the natural product(s) responsible for virus
neutralizing. Trivalent chromic increases glucose tolerance and promotes normal
glucose uptake by acting as a cofactor with insulin. Overexposure to chromate
compounds has been associated to respiratory illnesses. Low quantities of DNA-
bound chromium (III) ions may contribute to chromium mutagenesis and
carcinogenesis, according to these findings. Cr is among the four most commonly
used metals in tin can manufacturing. At pH 2.5 and 3.5, Cr is liberated at roughly 54
ppb and 16 ppb, respectively. Cr levels in fruit juices ranged from 31 to 50 parts per
billion. Especially fruit drinks in pouches, according to one research, include Cr.
Filipinos' usage of tinned fruit drinks has raised the number of fast food restaurants
has expanded and their desire for non-carbonated beverages has grown. The trace
metal Cr can be hazardous to the body if it is present in large quantities. The goal of
this research is to see if the current amount of Cr (VI) in canned juices offers a slight
health risk to humans.

3. Tayone, J.C. (2014). Spectrophotometric Determination of Chromium (VI)

in Canned Fruit Juices. International Journal of Sciences: Basic and
Applied Research, 19 (1): 426-432.

Introduction
Chromium is an important mineral for the preservation of normal physiological
functioning. Nutritional deficit in this mineral has been linked to a number of
illnesses. It has the ability to form a variety of ligand complexes with nucleic acids
and proteins, and it may play a role in glucose metabolism.

Methodology
Hexavalent chromium interacts with 1, 5-diphenylcarbazide in acidic solution
to give a reddish purple tint that may be measured by measuring its absorbance at
its highest absorption wavelength. A 4 mL volumetric flask with 4 mL of 0.01 percent
1,5-diphenylcarbazide was filled with 9.6 x 10-5 M standard chromium to determine
its maximum wavelength absorption (VI). The mixture was then diluted with 0.2N
sulfuric acid to the desired concentration. The absorbance was then measured from
200 to 800 nanometers (nm) using a 0.2N sulfuric acid solution as a reference.
Through applying beer’s law to 1,5-diphenylcarbazide, using a burette, 0.2, 4, 6, and
8 mL of 5.94 x 10-5M standard chromium (VI) solution were transferred to five 25mL
volumetric flasks containing 15 mL of diphenylcarbazide (DPC) solution. The
intensity of the solutions at maximum wavelength was measured using a Shimadzu
UV-VIS spectrophotometer160 after 30 minutes, and compared to a reference
solution produced by diluting 15 mL of DPC with 0.2N sulfuric acid. After that, the
samples are ready. 50g of each canned fruit juice sample was inserted in an empty
crucible that had already been weighted to a constant weight in three repetitions.
The crucible was then placed in a muffle furnace and heated for one hour at 550oC
until the ash turned white. After ashing, 1.5 mL of HCl was added and rotated to wet
all of the ash before adding 2.5 L of HNO3 to oxidate the chromium. The solution
was transferred to a 100 mL beaker and dried. The yellow brown color of free
bromine was produced by acidification, which was removed by adding 0.5 phenol.
Treatment following the standard addition method, a burette was used to deliver 4
mL of the test solution into two 10 mL volumetric flasks containing 4 L of DPC. At
543 nm, the absorbance was measured in comparison to a reference solution made
by diluting 4 L of distilled water to 10 L.

Results and Discussion

The absorbance of 1,5-diphenylcarbazide at various Cr (VI) concentrations
was plotted using Beer's Law to create a linear curve. A linear regression line or the
least square method were used to find the optimal straight line. Cr (vi)-DPC has a
maximum absorption wavelength of 543 nm. The quantity of inorganic mineral
material that remains after oxidation is indicated by the ash content. The ash content
of most fresh meals is less than 5%. Pure fats and oils have no or very little ash,
however processed foods like bacon can have as much as 11.6 percent. Fruits and
fruit juice can include 0.2 to 0.6 percent ash, but dairy products can have 0.5 to 5.1
percent ash. With 0.714 ppm, the pineapple orange taste sample had the highest Cr
(VI) concentration. Orange flavor sample number 4, 0.450 ppm, is followed by
samples 2 and 3, 0.426 ppm. All of these figures exceeded the legal limit in the
United States. EPA stands for Environmental Protection Agency.

Conclusion
At 543 nm, chromium (Cr) showed the maximum absorption. The ash
concentration was compared to the standard value for fruit juice and confirmed that
there was no adulterant. This concentration could threaten human health. More
stringent regulatory measures are required.
 4. Tayone, J.C. (2015). Biological and Chemical Characteristics of
Groundwater in a Rural Settlement Area of Davao Oriental American
Scientific Research Journal for Engineering, Technology, and Sciences,
14 (1): 94-99.

Introduction
Groundwater is the biggest unfrozen fresh water reservoir in the hydrologic
cycle. Rapid urbanisation had a significant impact on the level availability and quality.
Excessive extraction can result in surface subsidence, saltwater intrusion, and a
decrease in water supply, all of which are detrimental to ground water. The study's
goal was to assess the chemical and microbiological qualities of drinking water in
Badas, Mati City, Davao Oriental, Philippines. Its main purpose was to find out how
much MPN, pH, total dissolved solids, total hardness, chlorides, nitrates, sodium,
magnesium, and calcium were present.

Methodology
The study was conducted in the Philippines' Barangay Badas, Mati, Davao
Oriental. It was made up of eleven sitios with 26 puroks and 1,014 families in the
western section of Mati. The principal sources of water in this area were shallow
wells and springs. The faucet's edges were heated to gather water samples. Water
samples were taken after the pump had been running for ten to fifteen minutes. For
chemical analysis, a two-liter sample of water was taken. For the microbiological
characteristics, water samples were placed in a sterile container devoid of microbial
inhibitors. The collected samples were analyzed using various procedures suited for
each sample type.

Results and Discussion

Total coliform levels in water samples ranged from 2.2 to 16 MPN/100mL,
depending on the sampling site. These were above the maximum threshold set by
the Philippine National Standard for Drinking Water. Septic tanks were discovered to
be too close to the wells, potentially contaminating them. Anthropogenic activities
have a considerable impact on pH, which is one of the most important components
of evaluating water quality. Toxic metals may seep from water pipes if the pH is less
than 6.5, whereas pH greater than 8.5 has an unfavorable influence on the
disinfection process. The pH levels, on the other hand, were within the acceptable
drinking water guidelines, ranging from 7.0 to 7.15. Three sampling stations were
discovered to be over the limit. TDS levels in the study area ranged from 378 to 1088
ppm. The closely packed housing estate and rigorous irrigation activity at Station 2
resulted in the highest TDS concentration. Although high levels of TDS are not
generally harmful to humans, they can affect people with kidney and heart
conditions. The total hardness of the groundwater samples ranged between 271mg/L
and 659.5 mg/L. The levels in four sampling stations exceeded the allowable limit,
with station 2 near the coast having the highest level. This high level could also be
attributed to the large amount of agricultural waste and laundry waste disposed of
along this site. The chloride values ranged from 17.74 mg/L to 312.5 mg/L. Station
4's high chloride content was linked to its proximity to the coast. The chloride
concentration was found to be substantially within the permitted limits in general.
Nitrate concentrations ranged from 1.2 to 67.35 mg/L. The nitrate value for the
research region was found to be within the permitted range, with the exception of
station 2, which was located on an agricultural area. The sodium concentrations
varied from 35.63 mg/L to 227 mg/L. Near the coast, at station 4, the highest level of
sodium was observed. This station's sodium levels may rise due to saline intrusion.
Furthermore, the results show that nearby residents threw their laundry water near
the stations. Station 5 was found to have a low sodium level despite being located
far from the coast. People on a "salt-free" (low sodium) diet should avoid drinking
water containing sodium. The WHO's permitted values for magnesium and calcium
were 30 mg/L and 75 mg/L, respectively. With the exception of Station 5, all sample
stations exceeded the limit, with magnesium values ranging from 20.88 mg/L to
96.25 mg/L and calcium concentrations ranging from 74.25 mg/L to 117.5 mg/L. To
have clean drinking water, it is vital to conserve natural water sources.

Conclusion
The value of the most probable number (MPN) exceeded the Philippine
National Standard for Drinking Water's guideline. The studies suggested that
drinking untreated water was dangerous. Although the pH levels were within
permissible limits, the majority of the metrics differed from station to station.

5. Tayone, J.C. and Del Rosario, M. (2016). Molecular Modeling of

Debromolaurinterol Isolated from sea hare (Aplysia kurodai) using
Molecular Orbital Package Software. International Journal of Computer, 20
(1): 42-50.

Introduction
When molecules are seen in three dimensions, they are easier to perceive
and comprehend in chemistry. Between molecule atoms, bond lengths, bond angles,
and dihedral angles must all be addressed. This is where computers step in to assist
humans by performing the tedious tasks. The Molecular Orbital Package (MOPAC)
calculates, visualizes, rotates, manipulates, and optimizes the geometry of
molecules on a computer screen. Debromolaurinterol, produced from the Aplysia
kurodai sea hare, is a novel cytotoxic and antibacterial chemical. The structure and
biological activity were determined using Nuclear Magnetic Resonance (NMR) and
Mass Spectroscopy (MS). Bond lengths, angles, dihedral angles, heat of formation,
and other chemical constants that best define the molecule are still unknown. The
researchers wanted to make a molecular model of the chemical.

Methodology
The structure of the molecule debromolaurinterol was defined using a Z-
matrix, which is a means of characterizing the molecule atom by atom in terms of
bond lengths, bond angles, and dihedral angles. The study used MOPAC version
7.21 to do its calculations. MOPAC worked by describing the type of calculation and
controlling it with keywords. VECTORS, SYMMETRY, and BONDS were essential
words in the print final bond-order matrix. The program then read the initial molecule
geometry in z-matrix form. Iterative calculations were utilized to find a more realistic
and stable shape. The Z-matrix will next be produced by assigning a numerical value
to each atom in the molecule. After that, the origin or reference point was chosen, in
this case the fake atom XX1, which was placed at the coordinate system's origin.
Debromolaurinterol has the chemical formula C15H20O. The first atom (XX1) was
specified purely by its distance from atom 1 because the second atom was always
put on a preset axis. The bond lengths, bond angles, and dihedral angles were used
to specify the exact placements of the remaining atoms in the same way as before.
One line of keywords, two lines of user-defined text, and the z-matrix comprised
MOPAC or data sets. MOPAC input was subjected to iterative calculations to obtain
the most stable, realistic geometric molecular structure possible. Atoms were in the
first column, followed by bond length values, bond angles, and dihedral angles. The
zero in the last column indicated that the list had come to an end and there were no
more atoms to evaluate.

Results and Discussion

Organic compound formation heat is a crucial physical parameter that can be
used to predict molecule stability. Debromolaurinetrol was found to be more stable
than its constituent parts under ordinary circumstances, with a MOPAC value of -
6.75618 kcal/mol. The ionization potential of benzene (9.52 eV) falls as substituents
are added, and it is affected by the degree of electron migration. Debromolaurinterol
has an electronic energy of -17,174.16327 eV, which corresponds to the molecule's
minimal energy associated to its equilibrium bond length. It's worth noting that
molecules favor low potential energy since it causes atoms to repel one another,
increasing the distance between them and lowering the potential energy. Core-core
repulsion energy is the electrostatic interaction between positively charged particles.
The core-core repulsion MOPAC computation yielded a positive outcome. MOPAC
computed interatomic lengths can help synthetic chemists figure out if chemicals that
target certain reactive sites will produce the desired reaction products. This distance
should, in theory, be bigger than the distance between C30 and C25, which are on
opposite sides of the plane. Because of the aromatic OH, phenol can be easily
alkylated to form an ether molecule via the Williamson synthesis [13]. The
disubstituted phenol product debromolaurinterol may prevent the alkylation reaction
from proceeding. The bulky ortho substituents, notably the CH3's and cyclopropyl
moieties, may have a steric effect with –OH. Alkylation of –OH is difficult or
impossible, particularly when bigger alkyl groups are inserted into the phenol. One of
MOPAC's primary objectives is to generate a visual model of the chemical being
studied. A better grasp of structure can help organic chemistry and biochemistry in
particular. By rotating the molecule using several models, it may be seen from every
perspective.

Conclusion
Alkylation of –OH is difficult or impossible, especially when the phenol
contains larger alkyl groups. One of MOPAC's main goals is to create a visual model
of the chemical under investigation. Organic chemistry and biochemistry, in
particular, can benefit from a greater understanding of structure. The molecule may
be seen from any angle by rotating it using several models.

6. Tayone, J.C. and Del Rosario, M. (2017). Crude Extract Yield, Total
Phenolics, Total Flavonoids from the Ink of Sea Hare (Dolabella
auricularia). International Journal of Advanced and Applied Sciences,
4(11): 17-21.

Introduction
Dolabella auricularia is a soft-bodied mollusc with no protective shell that
moves slowly. It produces huge volumes of ink as a chemical defense against its
predators when agitated. The ink contains secondary metabolites acquired from their
algal diet, based to one study. Dolastatin and dolabellanin are two substances
identified from D. The purple ink of the sea hare has a wide range of biological
effects, including antioxidant and anti-allergic characteristics, and could be employed
chemically as anticancer medicines. anti-inflammatory. Anti-cancer activity and
cytotoxin Dolastatin 10 is now in phase II clinical trials and is expected to be
commercially viable in the coming years. Some of the compounds isolated from D
are dolastatin and dolabellanin. auricularia that have the potential to be used
chemically as anticancer agents The purple ink of the sea hare has a variety of
biological effects, including antioxidant and anti-allergic properties. anti-
inflammatory,. Anti-cancer activity and cytotoxin Dolastatin 10 is now in phase II
clinical trials and is expected to be commercially viable in the coming years. Davao
Oriental is home to a varied range of natural resources. The marine biodiversity of
Pujada Bay, which includes sea hare, is well-known. Local fisherman value the egg
strings, or "lukot," laid by sea hare because they give food and revenue.

Methodology
 Mati City, Davao Oriental, Philippines is home to Pujada Bay. Five sample
species were hand-picked at random along the coast. The purple ink was extracted
by gently pressing the organisms, which caused them to release it. All ink samples
were blended to make one composite sample. The mixture was centrifuged and
filtered through a Buchner funnel with mild suction. The crude extract was
maintained in a tightly sealed container at 0 to 5 degrees Celsius until it was
examined. The crude extract yield was obtained by multiplying the crude extract.
after 100 times concentration divided by volume of fresh ink samples analysis,
nextract solution. The ethanol and water portions were placed in ovens set to less
than 50oC and 100oC, respectively, after cooling and weighing for one hour. Folin-
ciocalteau methods are now used to determine total phenolics. After that, the
completed product was combined with water and allowed to sit for 90 minutes before
being diluted. Combining 25 to 150g/mL of Gallic acryl hydroxychloroethylene (GA)
with 80ml of ink samples in 80 percent methanol produced the calibration curve. The
aluminum chloride colorimetric method is used to determine total flavonoids.
Aluminum chloride forms stable acid complexes with the C-4 ketogroup and either
the C-3 or C-5 hydroxyl group offlavones and flavonols in the aluminum chloride
procedure. A 1 mL aliquot of the extractsolution was placed in a 10 mL volumetric
flask, filled to the mark (10 mL) with distilled water, and rapidly shaken for 5 minutes.
For the standard calibration curve, the absorbance was measured at 510 nm using
known doses of quercetin (50, 100, 150, 200, and 300 mg/L). All experimental
measurements were made in triplicate and the mean standard deviation was
calculated. A t-Test with a p-value of 0.05 was used to see if there was a significant
difference in extract yield, total phenolics, and total flavonoids when ethanol and
water were used as extracting solvents.

Results and Discussion

The crude extract yield of the ethanol fraction was p, which was much greater
than the water fraction's 0.05. The mean standard deviation of all experimental
measurements was computed.
When ethanol and water were used as extracting solvents, a t-Test with a p-
value of 0.05 was utilized to evaluate if there was a significant difference in extract
yield, total phenolics, and total flavonoids. As indicated by its higher value, ethanol is
more effective in extraction than water in this investigation. The increased yield of
ethanol extract may be due to the existence of polar and nonpolar components of the
chemicals found in sea hare ink. The efficiency of total phenolics extraction was
highly dependent on the type of sample and the type of extracting solvent.
Antioxidant activity is well known for phenols. It has redox properties that absorb and
neutralize free radicals, scavenge singlet or triplet oxygen, and decompose
peroxides. Fresh hare ink samples had total flavonoids ranging from 9.42 to 32.73
mg/mL. Hare ink's ethanol content was far more variable than the water fraction (p
0.05). The findings clearly suggest that seahorse ink may include beneficial
chemicals as well. Sea hare ink may include bioactive chemicals that have medicinal
promise. Algae includes chemicals that have cancer-fighting potential.

Conclusion
The sea hare's ink may include bioactive chemicals that could be used in
nutritional or medicinal products. Based on the properties of these chemicals, the
presence of phenolics and flavonoids in considerable amounts suggests that ink may
have antioxidant potential.

7. Tayone, J.C., Del Rosario, M., and Canencia, O.P. (2019). Extracts from
egg strings of sea hare (Dolabella auricularia): Yield, antioxidant activity,
zoochemical profile, and toxicity. International Journal of Advanced and
Applied Sciences, 6(1): 24-28.

Introduction
Marine environment has always been a source of marine natural products that
have a wide range of biological activities. The secondary metabolites from marine
organisms play a vital role in the development of a new drug to cure various human
diseases. About 116 genera of mollusks which include 36 species of sea hare have
contributed to the thousands of compounds already discovered since 1963. Sea hare
(Dolabella auricularia) is a shell-less mollusk that belongs to the class gastropoda
and family Aplysiidae. Sea hare can lay millions of eggs in an intertwined strand. The
egg strings of sea hare are found to contain primary metabolites that are needed by
the body. It is a good source of proteins and other minerals and found to be ideal for
human consumption (Pepito et al., 2015). Researches show that it also contains
secondary metabolites that are reported to have multiple biological effects including
antioxidant activity that neutralizes the toxic effects of free radicals (Simmons et al.,
2005). In the continuing quest for natural antioxidants and cytotoxic agents, this
study is perhaps the first attempt in Davao region, Philippines to establish such
biological activities of egg strings from sea hare using different extracting solvents
(ethanol, ethyl acetate, and hexane). The antioxidant activities of crude extracts were
evaluated by measuring their ability to scavenge the radical 2, 2-diphenyl-1-
picrylhydrazyl (DPPH). Zoochemical analyses were done for determining the
presence or absence of selected secondary metabolites and brine shrimp lethality
assay for its cytotoxic activity.
Methodology
About 6kg of egg strings of sea hare was collected in guang guang, pujada
bay in mati city. These eggs are cleaned with distilled water then dried. After it dried,
it was immersed in 95% ethanol and after 24-28 hours it is filtered. Its residue is
then discarded and the filtrated mixture is then concentrated in the vacuo. It is then
stored in a tightly stoppered vial until its analysis. After that, using a pipet, a 10 ml of
the extract is transferred into a empty dish and is placed in the oven for less than
500C for one hour. This process is repeated until constant weight is obtained.
A solution of DPPH is then prepared through dissolving 6 mg of DPPH in 50 ml
methanol. One ml of that DPPH solution is then immersed wth 1 ml of the extract .
the mixture is incubated at 370C for about 30 minutes. The absorbance of it has
decreased measuring at 517 nm. The radical scavenging activity is observed through
comparing the absorbance with the blank cotaining only DPPH solvent. As for the
zoochemical analysis, using selected established procedure the secondary
metabolites alkaloids, anthraquinones, coumarins, flavonoids, glycosides, phenols,
saponin, steroids, quinones, tannins, and terpenoids determination is done. Now to
test its toxicity of the extract, brine shrimp bioassay is carried out. 10 ml of the brine
shrimps is then transferred, using a pipette, into three different vial containing 4.5ml
of brine solution and 0.5 ml of the extract. A drop of yeast 3mg yeast mixed with 3ml
artificial seawater also added to serve as food. The vials is then illuminated and is
checked after 24 hours. The percentage of the lethality is determined by comparing
the mean surviving brine shrimp of the test and control tubes. Now for the statistical
analysis, it is carried out in triplicate and were expressed as the mean standard
deviation. The data are analyzed by analysis of variance following Fisher Pairwise
comparison test.

Results
The result shows that the yield of the extract of egg strings from the sea hare
for ethanol, ethyl acetate, and hexane using the dried samples is at p<0.05. It
showed a significant difference of the extract at different crude fractions. This
difference may be due to the fact that the efficiency of extraction and its yield are
affected not only by the extraction method, particle size of the sample, solvent used
and presence of interference but also the chemical composition of the sample. As for
the result of the antioxidant activity, using the change in absorbance produced by
reducing DPPH, the three extracts were measured. It showed the dose-response
curves of different extracts fractions. It had significant difference at p<005. For the
ethanol extract, it had a significant effects towards the percentage of inhibition. The
variable degrees of free radical scavenging property increased in dose-dependent
manner. DPPH radical’s percentage if inhibition ranged from 21.97% to 24.86% at
the highest tested dose of 1000 micrograms per milliliters and from 20.81% to
23.51% at the lowest dose with IC50>1000 micrograms per milliliters for all extracts.
As for the zoochemical analysis, it showed presence of secondary metabolites in
defferent test. Test for alkaloids showed positive for the ethanol extract only. Phenols
is also appeared in all extracts. As for saponins, steroids, and terpenoids, they
appeared to be in increasing concentration since the intensity of color change per
solvent was evident. The hexane fractions have high concentration compared to
ethyl acetate and ethanol. While tannins is present in moderate amount in ethyl
acetate, also small amount in hexane but absent in ethanol fraction. Anthraquinones,
coumarins, flavonoidds, glycosides, quinones are not present in the three extracts.
The antioxidant activity of the different extracts might have contributed to the
secondary metabolites presence in the egg strings. As for cytotoxic activity of the
extracts, the highest concentration was only 3.33%. this low mortality rate indicated
the absence of cytotoxic and antitumor components in the egg strings of sea hare.

Discussion
The results showed that the antioxidant activity of the extracts was dependent
on the extracting solvent used. The solvent used is ethanol and ethanol is said to be
the most frequent solvent being used in the extraction of antioxidants because of its
high polarity and hence can favorably extract polar compounds such as phenolic and
flavonoids that are effective antioxidants. Moreover, this study showed that the
extracts had proton-donating ability which served as free radical inhibitor or
scavenger and could act as primary antioxidant. With these, egg string of sea hare
can be a potential source of natural antioxidants and can be recommended to be
part of our diet to protect human health and promote general wellness.

Conclusion
Based on the antioxidant activity assay conducted on the different crude
extracts, compounds with nutritional and medicinal potential may be isolated from
egg strings. These potentials may be due to the secondary metabolites present like
alkaloids, phenols, steroids, tannin, saponins, and terpenoids which are known for
their biological activities. On the other hand, the low mortality of brine shrimp at high
concentration indicated the absence of cytotoxic compounds and confirms the edible
nature of egg strings. The result of this study increases the nutritional and medicinal
potential values and provide baseline information for another possible source of
future novel antioxidants that can be used in food and pharmaceutical industries.

8. Tayone, J.C., Ortiz, C.C. and Tayone, W.C. (2020) Selected mollusks from
Pujada Bay, Philippines: Heavy metal health risk assessment and
antibacterial activities. Asian Journal of Biological and Life Sciences, 9 (2):
177-184.

Introduction
The marine ecosystem is a home to various organism that is vital to the
community. However, its importance to humans is being threatened by various
pressures in the environment. Pollution in water bodies had become a significant
problem in many parts of the world due to both natural and anthropogenic
processes. Most of the chemicals used in industries, agricultural lands and domestic
wastes are disposed of in water channels that all eventually transported to ocean
waters. These various human activities may be the cause of heavy metal enrichment
in sediments and water that affects marine organisms’ health, diversity and
abundance. Heavy metal concentrations in an aquatic ecosystem can cause
carcinogenic, reproductive and developmental effects on marine organisms. On the
other hand, human exposure to heavy metals even in lower concentrations can pose
different health diseases and may lead to carcinogenic or non-carcinogenic health
risks. Marine invertebrates such as molluscs and crustaceans are known to
accumulate heavy metals in marine waters and sediments. Bivalves and gastropods
are the types of molluscs commonly used in bio monitoring of aquatic pollution.
These molluscs are bottom dwellers and filter-feeders. They primarily feed on
phytoplankton, inhale water with oxygen and absorb food particles through their gills.
Due to their feeding mechanisms, they are susceptible to different contaminants
present in their environment. However, marine organisms such as mollusc are found
to contain natural products and can become a potential source of novel
pharmaceutical compounds for anticancer, antioxidant, antibiotic and among others.
Several studies have been conducted on the different aquatic resources existing in
Pujada Bay, including environmental monitoring of contaminants in sediments, water
and marine organisms. However, no study has been reported on the human health
risk of heavy metals (cadmium and lead) and the antibacterial properties of four
selected mollusk species namely; Anadara maculosa, Antigona puerpera, Canarium
urceus and Lambis lambis in Guang-guang, Pujada Bay, City of Mati, Davao
Oriental. Hence, this study.

Methodology

Four (4) types of marine mollusk species were randomly handpicked and
collected in Guang-guang These were: A. maculosa, A. puerpera, C. urceus and L.
lambis. The collected bivalves and gastropods mollusk species were properly
cleaned with distilled water and blanched. Its soft tisues were removed from the
shells and air dried. One gram of dried, ground powdered sample was weighed
accurately in a porcelain crucible then placed in a muffle furnace at 500°C for 2 hour.
The ash is the moistened with 1 ml distilled water and immersed with 3-4 ml of
concentrated HNO3. The mixture was heated on a hot plate at 100 to 120°C to
evaporate excess nitric acid then transferred into a 50 mL volumetric flask and
diluted to mark with distilled water. The solution was used for the determination of
heavy metals, such as Cd and Pb, using the Atomic Absorption Spectrophotometer.
On the other hand, 4 L composite seawater samples were also collected to
determine the physicochemical parameters of water in two sampling stations in
Guang-guang, Pujada Bay. These include the pH, dissolved oxygen and total
dissolved solids of the water. The methods used followed the Standard Methods for
the Examination of Water and Wastewater. After the preparation of sample water,
about 20 g of each dried samples was treated with 95% ethanol for 24 to 48 hour
and then centrifuged and filtered. The residue was washed with fresh portions of
alcohol and the filtrate was then concentrated under vacuo at a temperature below
50°C using a rotary evaporator. The extract was stored in a tightly closed vial at 0 to
5°C until its analysis. The ethanolic extract of molluscs samples was subjected to
antimicrobial test against pathogenic gram-positive Staphylococcus aureus and
gram-negative Escherichia coli using agar well diffusion method. Ampicillin and
penicillin were used as standard drugs, respectively, for studying the activities of the
extract. After that, the melted agar was poured into dry sterile petri dishes and
solidified. A sterile cotton swab moistened with the test suspension that was
previously incubated in Muller Hinton Broth for 24 hr at 35°C was used to streak over
the entire surface of the agar. It was allowed to stand for 5 min. A cork borer was
sterilized by immersing it in an erlenmeyer flask containing ethanol and heated over
an alcohol flame. The agar was stabbed using the cooled cork borer to the bottom of
the dish to create a well. The well was filled with the previously prepared crude
ethanol extract of mollusc samples. The incubation period was 24 hour at 35°C. The
extract’s activity was determined by measuring the diameter of the zone of inhibition
and compared with the values produced from the standard drugs. All results were
expressed in terms of mean and standard deviation. Data were further processed to
estimate the risk analysis based on available standards.

Results
Bivalves, a. maculosa and A. puerpera showed 0.03 μg/g concentration of Cd
and Pb. As for the gastropods, C. urceus showed 0.06 μg/g of Cd concentration
where as L. lambis showed 0.08 μg/g of the same metal concentration. These values
are below the standard limit for Cd. However, Pb concentrations in both bivalves and
gastropods mollosks are all equal and below the standard level for Pb. In
assessment of the potential adverse impact of the heavy metal on the aquatic
ecosystem, its risk quotient is calculated and showed RQ value for Cd 0.06 μg/g in
A. maculosa and A. puerpera. As for C. urceus, it showed 0.12 μg/g and for L.
lambis, it showed 0.16 μg/g. For the RQs for Pb, all molluscs sample showed 0.2
μg/g. As for the assessment for human health risk indication, Hazard quotien is
applied to evaluate the non carcinogenic health risk of marine mollusc ingestion
using exposure values. It showed a value of 0.035 Cd in A. maculosa and A.
puerpera while in C. urceus it showed 0.069 and 0.092 in L. lambis. As for values for
Pb, all marine mollusk samples were 0.029.
Chronic daily intake obtained a value of 3.15 x10-5 Cd in both bivalve. While there is
7.03 x 10-5 C.urceus and 9.37 x 10-5 in L. lambis. As for the chronic daily intake of
Pb, all mollusk obtained 1.17 x 10-4. The carcinogenic risks of heavy metals through
marine mollusc consumption were evaluated and showed a value of 5.68 x 10-9 in
both bivavlves. As for C. urceus, it showed 1.14 x 10-8 and L. lambis have 1.52 x 10-
8. These values were less than the standard range of carcinogenic risk factor of 1.0
x 10-6 to 1.0 x 10-4. On the other hand, the of Pb in all the mollusk species was still
within the permissible range. As for the water sample, during the collection, have a
recorded temperature of 30 degree celcius in both stations 1 and 2. It has a pH
range of 6.5 to 9. Dissolved oxygen level in both sampling stations were 7.7 mg/L.
the total dissolved solids of 35,646 mg/L in station 1 was slightly higher than in
station 2. Now for the antibacterial activity, the mollusc extracts using ethanol solvent
showed different values of the zone of inhibition against E. coli and S. aureus. C.
urceus showed activity against S. aureus with a 10. 78 mm zone of inhibition but this
value is weaker compared to the 30.44 mm zone of inhibition showed by the
standard ampicillin drug.

Discussion
The observed differences between gastropods and bivalves in accumulating
Cd heavy metal in this study were probably due to the type of mollusc, their feeding
abilities, position in the water column, sedentary habits and other factors. The low
concentration of Cd and Pb in both bivalves and gastropod molluscs is an indication
of low levels of such heavy metals in the marine ecosystem of Guang-guang, Pujada
Bay. Human activities in the Bay, such as wastewater discharge may not contain
high amounts of heavy metals such as Cd and Pb. The risk quotients od Cd and Pb
were less than the permissible level of 1. RQ<1 indicates that there is a minimal
potential adverse effect to marine organisms because of pollutant exposure. As for
RQ>1, this indicates severe potential adverse effects to marine organims because of
pollutant exposure. Now for hazard quotient, values fpr Cd and Pb in bivalves and
molluscs are below 1. HQ<1 indicates no hazard for adverse health effects on
human health making the consumption of molluscs safe. Bivalves and molluscs’
chronic daily intake showed that CDI of Pb in molluscs is slightly higher compared to
CDI for Cd. This can disturb cellular activities. It will inflect oxidative stress causing
damage to DNA and interferes with the function of the organ system. As for
carcinogenicity of these metals, Pb affects human nervous system, hematopoietic
system and the renal. Exposure to Cd will also affect the renal, skeletal and
pulmonary organs. However, when consuming mollusc that have these metals, have
no potentials risk to humans since it did not surpassed the permissible range of
value fpr Cd and Pb.

Conclusion
The Cd and Pb concentrations in the four mollusks species, namely; A.
maculosa, A. puerpera, C. urceus and L. lambis collected from Guang-guang,
Pujada bay, Davao Oriental were below the standard limit of 0.5 μg/g set by FAO
No.210 (2001) and other international agencies. The health risk indices were all
below the acceptable level set by USEPA. Hence, Cd and Pb posed no potential
non-carcinogenic and carcinogenic risks to human health and are safe for
consumption. The seawater quality was good, based on the determined physico-
chemical characteristics of the water. Lastly, C. urceus showed an antibacterial
factor.

9. Tayone, W.C., Ishida, K., Goto, S., Tayone, J.C., Arakawa, M., Morita, E.,
and Hashimoto, M. (2020). Anti-Japanese Encephalitis Virus (JEV) Activity
of Triterpenes and Flavonoids from Euphorbia hirta. Philippine Journal of
Science, 149 (3): 603-613.
Introduction
Euphorbia hirta L. is known in the Philippines as “tawa- tawa.” It is a traditional
herb and abundant in open grasslands. Tawa-tawa is normally harvested by taking
the whole plant at its flowering stage and the decoction is prepared by boiling it for a
few minutes and then given to the patient as tea. The plant was reported by Patil’s
group to be effective for the treatment of gonorrhea, dysentery, boils, pulmonary
disorders, and jaundice. The plant is said to be effective as an anti-inflammatory,
antidiarrheic, anti-human immunodeficiency virus, antibacterial, anti-asthma,
antioxidant, and anti-proliferative. Moreover, it also has the potential to inhibit
angiotensin-bn converting enzyme and promote cartilage degeneration in arthritic
rats. In the course of our previous investigations exploring biologically active
metabolites from natural products, we also reported the isolation of nine metabolites
from E. hirta and their potential against dengue fever. JEV is a member of flavivirus
and is one of the leading causes of severe encephalitis in Asia and the Western
Pacific. Both JEV and dengue virus are transmitted by mosquitoes and share similar
viral structure, genome organization, and replication strategy in the host cell.
Therefore, it has been considered that anti dengue reagent targeting basic
propagation mechanism can also work as an inhibitory reagent for JEV, as well as
for other members of flaviviruses. In this paper, we report the anti-JEV activities of
the nine compounds isolated from the aforementioned plant and the isolation of the
additional four compounds, which were not included in our earlier study.

Methodology
The structure elucidation of isolated compounds was performed with NMR
spectroscopy. The 1H and 13C NMR spectra were recorded in CDCl3 and CD3OD
on a JEOL JNM-ECA500 spectrometer. The residual CHD2OD and 13CD3OD were
used as the internal standards. Splitting patterns are designated as s (singlet), d
(doublet), dd (doublet of doublet), t (triplet), q (quartet), m (multiplet), and br (broad).
Chemicals and solvents were purchased from Fujifilm Wako Pure Chemical
Industries and Sigma-Aldrich Co. LLC. Those were used without further purification.
Thin-layer chromatography (TLC) analyses were carried out using Merck silica gel
TLC silica gel 60 F254 plates. Silica gel column chromatographies were carried out
using Merck 707734. After acquiring the chemicals and solvents, plant sample was
collected from Mati City, Davao Oriental, Philippines in June 2018. The material was
first identified via indigenous knowledge and comparison with images from online
photos. After that preparation, extraction and isolation of secondary metabolites is
conducted. A whole plant of tawa-tawa for about 1kg was collected and washed
using tap water. It is then cut around 3 cm long and then air dried. After it fully dried,
it is soaked in 8L of 50% MeOH solution for 48 hours while frequently being stirred.
A 500 ml portion of the extract is then filtered and concentrated in vacuo until the
volume become approximately 20ml. this aqueous suspension is extracted with
100ml EtOAc and the aqueos layer is collected and concetrated in the vacuo. This
extraction obtained 1.2 grams. After the residue was diluted with methanol,
diatomaceous earth granule was added. The resulting suspension was concentrated
in the vacuo. As for the residue, it is loaded on a Φ 1.6 x 12 cm column and attached
to preparative ODS medium pressure column chromatography. The aqueous MeOH
was eluted under gradient condition to obtain quercetrin and myricitrin. The 10L of
the extract was filtered with cotton gauze and the filtrate was concentrated under
reduced pressure until all methanol was removed. It is then lyophilized. The obtained
paste was then suspended with EtOAc and the soluble portion was concentrated to
give the crude material of 23.2 g. It was then suspended with H2O and extracted
with EtOAc. The organic layer was concentrated to give the extract. After the residue
was diluted again with EtOAc, 20 g of silica gel was added and concentrated
carefully with a rotary evaporator. The residue was placed on a silica gel on a Φ 8 x
90 cm column and then developed with EtOAc/hexane solvent system and gave
fraction A (eluted with 100% n-hexane, 108 mg), fraction B (eluted with 30%
EtOAc/hexane, 7.90 g), fraction C (eluted with 60% EtOAc /hexane, 1.60 g), fraction
D (eluted with 100% EtOAc, 434 mg), and fraction E (eluted with 100% MeOH, 3.60
g). The fraction B was subjected to second silica gel column chromatography to give
fraction B-1 which is 363 mg, fraction B-2 that have 790 mg, fraction B-3 cotaining
400 mg, fraction B-4 500 mg, fraction B-5 4.6 g, and fraction B-6 1.3 g. Since fraction
B-2 formed solid particles, it was diluted with 10% EtOAc/hexane. Standing the
solution precipitated taraxerone. A portion of the fraction B-5 was subjected to
second silica gel column chromatography to provide fractions B-5-1 (1.2 g), B-5-2
(533 mg), B-5-3 (250 mg), and B-5-4 (25 mg). Fractions B-5-1 and B-5-2 were then
dissolved independently with hexane and, upon standing of the solutions,
precipitated taraxerol and β-sitosterol. The mother solution from fraction B-5-1 was
subjected to the third silica gel column chromatography to give 24-
hydroperoxycycloart-25-en-3β-ol and 25-hydroperoxycycloart-23-en-3β-ol along with
fractions B-5-1-1 (121 mg), B-5-1-2 (615 mg), B-5-1-5 (0.5 mg), and B-5-1-6 (43.6
mg). Further fractionation of fraction B-5-1-2 by preparative medium pressured ODS
column chromatography with ODS-SM 50 µm 120 A, Φ 3.0 × 17.0 cm under the
same conditions mentioned above gave lupeol. The other silica gel column
chromatography of fraction B-6 provided (23E)-cycloart-23-en-3β, 25-diol (9, 8.2
mg). The obtained samples were characterized by the 1H and 13C NMR spectra.
Compound 1 was further reacted with 200 µL acetic anhydride in pyridine (500 µL)
and stirred overnight at room temperature to give its expected heptaacetate
derivative in 30.1% yield. This result corroborates the presence of seven hydroxy
groups in 1. After that is the acetylation of quercetrin. A 10 mg of 1 was treated with
200 µL acetic anhydride in pyridine (500 µL) and stirred overnight at room
temperature. The reaction was checked and guided by TLC analyses until the
starting material has disappeared. The mixture was concentrated under reduced
pressure and co-evaporated several times with toluene to remove traces of pyridine.
The major product after preparative TLC gave 5 mg of the expected heptaacetate
compound. Now, for the cells and viruses,
HEK293A and Vero cells were cultured in Dulbecco's modified Eagle medium
supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% (v/v)
fetal bovine serum, in humidified air containing 5% CO2 at 37 °C. JEV-Chb was
amplified on HEK293A cells. JEV strain AT31 were grown in 293A cells. To prepare
these infected cells and cell culture supernatants, compounds 1 and 2 were
dissolved and diluted in sterilized water while 3, 4, 5, 6, 7, 8, and 9 were dissolved
and diluted in dimethyl sulfoxide. HEK293A cells were seeded with a density of 1 ×
104 cells/well into 96-well plates and incubated overnight. Subsequently, 1 µL of
diluted compounds were added to each well. Two hours after the addition of the
compounds or just solvent as a control, cells were inoculated with JEV-Chb or the
mock control with a multiplicity of infection (MOI) of 0.3. Culture supernatant was
taken 24, 48, or 72 hours after virus inoculation (hpi). The cell lysate was prepared
72 hpi. HiBiT-mediated luciferase activity of culture supernatants and cell lysates
were measured to evaluate virus proliferation. Wildtype JEV was inoculated into
sterile water (control) or 100 µM myricitrin-treated HEK293A cells with MOI of 0.3,
and the cell culture supernatants were taken 48 and 72 hpi. Focus forming units of
culture supernatants were determined to evaluate virus proliferation. After all of that,
HiBiT Luciferase Assay is conducted. Cells were lysed with lysis buffer, 150 mM
NaCl, 1% (v/v) Triton X-100, and complete protease inhibitor cocktail. HiBiT-
mediated luciferase activity of cell lysate or culture supernatant was measured using
Nano Glo HiBiT Lytic Detection System kit (Promega) following the manufacturer’s
protocol. Cell Viability Assay is also conducted. HEK293A cells with a density of 1 ×
104 cells/well in 96-well plates were treated with various concentrations of
compounds for 72 h. Cell viability was evaluated using Cell Titer Glo 2.0 reagent
(Promega) following the manufacturer’s protocol. As for virus titration, viral titers
were determined by a focus-forming assay. In brief, Vero cell monolayers were
inoculated with serially diluted viruses, and cultured in DMEM supplemented with 1%
methylcellulose for 36 hours. The cells were fixed with 4% paraformaldehyde in
phosphate-buffered saline and are permeabilized and blocked with 0.1% Triton X-
100 and 10% FBS in PBS. The cells were incubated with primary antibodies anti-
JEV NS3 antibody in PBS for 30 min at room temperature. Then, the cells were
incubated with secondary antibody in PBS for 30 min at room temperature. The
infectious foci were counted using a fluorescence microscope, and the viral
infectious titers were calculated as focus forming units per mL. As for analysis of the
statistics, values in graphs are shown as the mean standard deviation. P-values are
calculated by a two-tailed student’s t-test using the Microsoft Excel.

Results and discussion

In our pursuit for further investigation of E. hirta or tawa-tawa, four more
compounds were isolated in addition to compounds we isolated previously. The
crude sample after soaking for 48 hours was sequentially purified over silica gel
column chromatography to afford the nine compounds. To screen antiviral
compounds isolated from E. hirta, JEV-Chb is used, a recombinant JEV strain
carrying a sequence encoding a reporter peptide HiBiT fused to the capsid structural
protein. The effects of the nine isolated compounds on the growth of JEV-Chb were
examined. As a positive control of JEV propagation, inhibitor N2,N4-
dibenzylquinazoline-2,4-diamine is used. JEV-Chb was inoculated onto cell culture
supplemented with each compound and incubated for 24, 48, or 72 hours. The
amount of extracellular viral particles and intracellular pro-viral particles were
estimated by measuring the HiBiT luciferase activities from culture supernatant and
cell lysate, respectively. After the initial screening, we found that compounds 2, 6,
and 8 inhibited the accumulation of JEV-Chb in culture supernatant in a dose-
dependent manner. Compounds 6 and 8 also inhibited the accumulation of JEV- Chb
in cells while 2 did not suggesting that 2 affects viral assembly but not the protein
production. These two compounds also showed significant cytotoxic effects
indicating that the apparent inhibition of JEV-Chb propagation was a result of cell
death. In conclusion, out of the nine compounds we tested, only 2 was able to
successfully inhibit the propagation of JEV-Chb without detectable cytotoxicity. The
recombinant JEV-Chb strain was initially used in the anti-viral study for faster and
easier detection in real-time fluorescent microscopy. After 2 showed activity in the
preliminary screening against JEV-Chb, its anti-viral activity was then verified with
the nonrecombinant or wildtype strain. Results further confirmed a similar inhibitory
effect of 2 on the propagation of a wildtype JEV strain at 100 µM. However, 2 is
weaker compare to baicalein, a flavonoid with potent in vitro anti-JEV effects at all
different stages of JEV infection. 2 reduced the level of capsid in the culture
supernatant but not in the cells indicating that 2 affects assembly or secretion of the
viral particle rather than viral protein production. Compound 2, being the most polar
of the nine isolated metabolites, was the most active. Minor structural differences of
1 and 2 lead to significant differences in the ability to inhibit the production of the viral
particle. Independent studies have demonstrated that 2 has an antioxidative activity.
Other studies also showed that flavivirus infection induces oxidative stress. This
oxidative stress resulted in the intracellular accumulation of reactive oxygen species
(ROS), and a decrease in ROS levels through chemical or genetic inhibition
weakened the innate immune responses to flavivirus infection and facilitated
flavivirus replication. On the other hand, cells infected with flavivirus induce
oxidative-stress responses to control the antiviral, inflammatory, and anti-apoptotic
properties by inducing antioxidant gene expression.

Conclusion
Taken all together into account, myricitrin showed inhibition of JEV at 100 µM.
A more thorough study is needed on its cellular antiviral stress response
mechanisms and in vivo investigation to demonstrate myricitrin’s potential. Moreover,
further investigations are crucial in understanding and establishing at what stage
(early or late) of the cycle does 2 inhibits JEV replication.
 10. Tayone, J.C., Morales, J.T. and Jimenez, L.A (2015). Nutrient and
chemical composition of sea hare, Dolabella auricularia in Pujada Bay,
Davao Oriental. Davao Research Journal, 11:6-12.

Introduction
The Sea hare, Dolabella auricularia, locally known as donsol, is a small
marine gastropod. Like many other gastropods, it exhibits torsion, a phenomenon
that moves the mantle cavity from posterior to the front of the body twisting the
visceral organs through a 180-degree rotation. Sea hares are slow moving marine
invertebrates that usually lack morphological defense structures such as spines or a
protective shell. When threatened or placed in an unfavorable condition, they may
often start laying eggstrings. There are no records of predators eating sea hare’s
eggs strings which maybe due to distasteful chemicals that they obtain from their
algal diet, a kind of chemical defense to fight off potential predators or to force back
neighbors competing for space. Sea hare digestive gland extracts contain wide
variety of secondary metabolites like prepacifinol epoxide and its skin extracts
contained (-)-7-dehydrocholesterol. The species D. auricularia is also known to
contain hemocyanin. Moreover, sea hares are good source of pharmacologically
active compound, dolastatin 10, an anti-cancer agent which is now currently in phase
II clinical trials. Dolastatin peptides have been found to be the source of 20 potent
anticancer agents. Dolabellanin B2, a new isolate with antimicrobial factor, was also
found to contain 33 amino acid residues. This peptide was cytoxically effective
against some pathogenic microorganisms at 2.5-100 µg. mL-18. Further, sea hare is
also a good source of primary metabolites.
Methodology
The study site was conducted in Guang-guang, Pujada Bay, City of Mati,
Davao Oriental. Three sea hare individuals approximately weighing 1 kg each were
randomly collected. These were placed in a plastic container with sea water
maintained at 4ºC to retain the freshness of the samples and to prevent moisture
loss during transportation to DOSCST laboratory. These samples were then
eviscerated and cleaned by removing the internal parts and washed. These were cut
into pieces about half an inch and drained for 10 minutes in a strainer. About 2.0 g of
prepared samples with replicates (RS1, RS2 , RS3) were used for determining the
moisture content at DOSCST laboratory. The rest of the samples were air dried for
five days. Dried samples in three replicates (RS1, RS2 , RS3) were placed in a
properly labeled zip locked container and submitted to University of Immaculate
Conception - Science Resource Center laboratory for the chemical analysis. Egg
strings Samples. About a kilogram of egg strings samples were randomly collected.
This is a mixture of freshly laid greenish and matured brownish egg strings samples.
Samples were placed in a plastic container with seawater and prepared. About 2.0 g
of the fresh samples with three (3) replicates were used for the analysis of moisture
content which was done at DOSCST laboratory. The rest of the samples were air
dried. Dried samples were divided in three replicates (RE1, RE2 , RE3), placed in a
properly labeled zip lock container and submitted to University of Immaculate
Conception Science Resource Center for the chemical. After that chemical and
nutrient is analyzed. The different nutrients and chemical composition of the sea
hare and its egg strings were determined following the standard analysis suggested
by the standard method of Association of Official Analytical Chemists such as:
Gravimetric method for moisture and ash content, Soxhlet for the crude fat, Macro
Kjeldahl for the crude protein, UV-VIS for phosphorous and Atomic Absorption
Spectrophotometer for Na, Ca, Mg Fe and Zn. The concentration of each analysis
was reported as the mean of the three replicate samples.
Results and Discussion
The egg strings showed higher average moisture content (93.32%) than its
mother sea hare (82.78%). Water is often excluded in the list of nutrients but it is a
an essential dietary component and must be obtained either from drinking water or
from the products of the body’s metabolism. The results showed that egg strings
were rich in moisture and can contribute to the human's body water requirement. The
results showed that ash content of sea hare (39.78%) was higher than its egg strings
(28.96%). This difference can be attributed to their structural component. The egg
masses were elongated greenish spaghetti-like strings of gelatinous filaments while
sea hare had a large flattened, quick heavily calcified shell. The crude fat content of
egg strings (2.01%) was also higher compared to its mother sea hare (0.74%). In
any case, sea hare or egg strings, these marine species is a negligible source of fat.
Hence, this can be recommended for those who are in low fat diet. The protein
component of sea hare (33.25%) was higher compared to its egg strings (23.87%).
The result of this study shows that both sea hare and egg strings are rich in minerals
such as phosphorous (P), potassium (K), sodium (Na), calcium (Ca), magnesium
(Mg), iron (Fe) and zinc (Zn). These are needed by the body for normal growth and
development. The average phosphorus concentration in egg strings (226.6 grams
per 100 grams) was higher than the P in sea hare (28.84 grams per 100 grams). Egg
strings K’s content of 600 mg can supplement the needed amount as compared to
sea hare with only 380 mg per 100 gram sample. The same pattern was observed
for sodium. Egg strings had higher Na content of 8960 mg compared to sea hare
with 790 mg per 100 gram sample. Magnesium (Mg) content was higher in egg
strings (1010 mg) compared to sea hare with 480 mg per 100 gram sample. Egg
strings have iron (Fe) content of 17.5 mg and is higher compared to sea hare which
0.548 mg. Zinc (Zn) content of 28.2 mg in egg strings was higher than in sea hare,
10.326 mg per 100 gram samples.
Conclusion
The sea hare is a valuable source of food to coastal residents, especially in
Guang-Guang, City of Mati. The result of this study provided basic information on
sea hare’s nutritive value. It also showed that egg strings of sea hare were a better
source of the basic nutrients that a human body needs. Moreover, this study serves
as the baseline for further chemical studies that may somehow lead to the discovery
of pharmaceutical substances.
11. Tayone, J.C. (2019). Investigation of chemical composition and
antibacterial activity of ink from Sea hare (Dolabella auricularia). Walailak
Journal of Science and Technology, 17 (6): 600-607.
Introduction
The biological and chemical biodiversity of the marine environment is
immeasurable and is an extraordinary resource for the discovery of various drugs.
Marine organisms competing for survival in the challenging ocean environment
produce chemicals that provide ecological advantages. Dolabella auricularia or sea
hare is a marine organism that belongs to Phylum Mollusca. Dolabella auricularia is
normally found in sheltered bays or lagoons, in grass beds or on sandy-muddy
substrate. It feeds on a variety of brown, green and red macro algae. When
threatened, sea hares squirt a copious protective secretion from its gland in the
mantle cavity which contains chemicals that has biological activities. Dolabellanin
and dolastatin 10 are compounds isolated from D. auricularia that are already on its
phase II clinical trial as anticancer agent. Moreover, the ink also showed antibacterial
activity against pathogens P. aeroginosa and V. cholera. The chemical composition
of ink from sea hare depends on the algal diet, stage of life and its reproductive
cycle, environment, and geographical location. The richness of Pujada Bay’s
biodiversity is a great factor that will greatly influence the type of metabolites present
in the sea hare. In Davao Oriental, studies on mariculture, nutritive value, and
commercialization of sea hare and its egg strings were already conducted. However,
no current studies on ink has been made on its potential to be a source of
compounds which may eventually lead to the development of pharmacological
substances. The information obtained from this study will contribute to the growing
literature of Dolabella auricularia as a basis for further chemical studies of sea hare
coming from Davao Oriental that will hopefully lead to the discovery of another
bioactive and novel compounds.
Materials and methods
The study is conducted along the coast of guang-guang, pujada bay, mati city.
Samples are collected here. Sea hares are randomly collected and placed in a
laboratory pan. Ink is obtained through exerting a minimal pressure on the sea hare.
About 100 to 200 mL composite ink sample was collected and transferred into a
reagent bottle. About the same amount of volume of 95% ethanol is then added to
the sample, the ink. The sample is allowed to stand for 24 to 48 hours. After that it is
centrifuged, filtered, and subjected to rotary evaporator below 50 degree celsius to
concentrate it. This concentrate is used for both secondary metabolites screening
and antibacterial activity while fresh samples were used for the proximate
composition and mineral content determination. The proximate and mineral
composition of ink from sea hare were determined by following the standard official
methods of the Association of Official Analytical Chemists (AOAC). Gravimetric
method for moisture and ash content was used. Soxhlet for the crude fat, Macro
Kjeldahl for the crude protein, UV-VIS for phosphorous and Atomic Absorption
Spectrophotometer (AAS) for Na, P, Ca, Mg Fe and Zn. The concentration of each
analysis was reported as the mean of the three replicate fresh ink Samples. After
that secondary metabolites screening is then conducted. Test for saponin, steroids,
alkaloids, glycosides, flavanoids, tannins, and anthraquinone. After that the samples
antibacterial activity will be observed.
Results and discussion
On the average, collected sea hare samples measured 5 inches and weighed
300 grams. Its color was mottled green that camouflage with the sandy muddy
substrate as one of their defense mechanisms aside from their purple ink chemical
defense. The chemical composition of many marine organisms is said to be affected
and influenced directly by diet, stage of life and its reproductive cycle. The result for
proximate composition as wet basis showed that ink was composed mostly of water
(90.4 %), followed by crude fat (5.32 %), the ash content that speaks about the
mineral content of the ink for both macro and micro nutrients (2.04 %) and lastly
crude protein (0.75 %). Accordingly, among marine species, the concentrations of fat
vary more than protein and carbohydrate. This is due to their responses to
environmental conditions, physiological traits and feeding. The high crude fat content
(5.32 %) compared to protein and mineral content of the ink can be an indication of
greater ability of sea hare to store lipid. The concentration of protein maybe low
however, this should not be underestimated since it is a source of amino acids such
as phenylalanine and tyrosine that can be converted through metabolic process into
phenolic compound. As or the mineral content of ink from sea hare revealed that ink
can be a good source of minerals for both macro and micronutrients. The samples
contained a large quantity of sodium (0.662 %) which was followed by phosphorous
with a mean of 0.25 %. Magnesium and potassium were also present in moderate
quantity with the average amount of 664 µg/g of magnesium and 578.6 µg/g of
potassium while calcium concentration was only 310.8 µg/g. On the other hand, the
concentration of micronutrients iron and zinc were 2.146 µg/g and 1.898 µg/g
respectively. The phosphorous concentration in this study fell within the range for
most marine invertebrates (0.24 to 1.16 %). The result of this study is important in
consideration of the minerals (Ca, K, P, Na, Mg, Fe and Zn) usefulness in the body.
Calcium is needed for both therapeutic and prophylactic effect. Zinc is important for
sexual development, reproduction, and normal growth. Now the results of secondary
metabolites screening revealed that sea hare’s ink contained saponins, steroids and
flavonoids. The honeycomb formation of Froth test, blue color formation of
Libermann-Buchard test and development of red color solution with Bate-Smith and
Metacalf test indicated the presence saponins, steroids and flavonoids respectively.
Dragendorff’s and Mayer’s reagent, Keller-Kiliani, ferric chloride and Bortntrager
qualitative test showed negative results for alkaloids, glycosides, tannins and
anthraquinones respectively. The result of this study clearly showed that ink from
sea hare is a good source of secondary metabolites. Also, this result will now be a
basis for further studies to quantify the amount of such metabolites present. The ink
extract showed a weak antimicrobial activity compared to the standard drugs. The
6.0 mm zone of inhibition against E. coli and S. aureus was comparatively weak
compared to the standard ampicillin and penicillin which exhibited zone of inhibition
at 18 and 26 mm in diameter. Accordingly, sea hares may have different microbial
components with varying strength and concentration that greatly affects their
antimicrobial threshold. Hence, the low antimicrobial activity of D.auricularia from
Davao Oriental, Philippines may be attributed to the type of antimicrobial compound
present and its concentration. However, the presence of saponins, steroids and
flavonoids may also be responsible or had contributed to its seemingly weak
antimicrobial activity since these compounds generally have antibacterial factor.
Conclusions
This study had shown the proximate composition, mineral content, secondary
metabolites screening and antimicrobial activity of ink from sea hare of Pujada Bay,
City of Mati, Davao Oriental. Preliminary results of this study showed that ink of sea
hare from Davao Oriental was a good source of primary and secondary metabolites
and it had antimicrobial factor. These findings add knowledge to the potential
medicinal value of sea hare and can be a basis for further chemical studies that will
hopefully lead to the discovery of compounds with pharmacological importance.

12. Tayone, JC, Del Rosario, RM, Canencia, OP, and Tayone, WC. (2021).
Egg strings of Dolabella auricularia: Its In vitro Antioxidants and
Antibacterial Activities. Asian Journal of Biological and Life Sciences, 10
(2); 340-345.
Introduction
Egg strings of the sea hare, Dolabella auricularia are found to contain primary
metabolites that are needed by the body. It is a good source of proteins and other
minerals and found to be ideal for human consumption. Researches show that it also
contained secondary metabolites, which include phenols and flavonoids. These
compounds are reported to have multiple biological effects, including anti-bacteria
and antioxidant activity that neutralizes the toxic effects of free radicals. Phenolics
are one of the main secondary metabolites. On the other hand, flavonoids are known
to be potent anti-oxidants that are dependent on their molecular structures. The
antioxidant and free radical scavenging activities of phenols and flavonoids depend
on the hydroxyl groups attached to the large ring system. The amount of flavonoids,
phenols, the extent of antioxidant activity, and antibacterial properties are dependent
on the type of material extract and the solvent used for extracting such compounds.
Solvents with different polarities can include water, methanol, ethanol, acetone, ethyl
acetate, carbon tetrachloride, chloroform, or hexane. The different pattern of
solvents’ effect on the extraction of sample materials is also due to the different
geographical location of plants or marine organisms subjected to such analysis.
Further, the extraction process can also affect the results. This study validates the
medicinal and nutritional potential of egg strings of D. auricularia using the extracting
solvent of different polarities. It also provides benchmark information for the conduct
of further researches on sea hare egg strings leading to the possible development of
medicinal substances. This information can be a basis to strengthen the
implementation of policies to protect, conserve, and sustain such local resources.
This will lead to an increase in the productivity of sea hare, thereby also increase the
income of the local fisher folks and, eventually, contribute to the “no hunger”
sustainable development plan of the region.

Methodology
4 kg of egg strings is randomly picked along the coast of guang- guang,
pujada bay, mati city. It is washed with distilled water then dried through nitrogen
blanketing at 60 degree celsius. 20 g of the dried sample is added with 95% ethanol,
ethyl acetate, and hexane separately for 48 hours. The mixture is then filtered and
washed with fresh portions of each solvent. The filtrate was concentrated under
vacuo at a temperature below the boiling point of the extracting solvents using a
rotary evaporator. The concentration of the extract (mg/mL) was determined by
approximately weighing 10 mL of the extract in an empty dish. It was dried in an
oven with a temperature less than 50 degree Celcius for one hour, cooled, and
weighed until a constant weight was obtained. Flavanoids is assayed through
aluminum chloride colorimetric method. The aluminum chloride method is based on
the formation of stable acid complexes with the C-4 keto group and either C-3 or C-5
hydroxyl group of flavones and flavonols with aluminum chloride. About 1 mL aliquot
of the extract solution was added to a 10 mL volumetric flask with 4 mL distilled
water. Sequentially, 0.3 mL of 5% NaNO2 was added and allowed to stand for 5 min,
followed by the addition of 0.3 mL AlCl3 (10%). After 6 minutes, 2 mL of 1M NaOH
was added and diluted with distilled water up to the mark (10 mL). The solution was
vigorously shaken, and the absorbance was recorded at 510 nm wavelength. The
same wavelength was used for the generation of a standard calibration curve using
known concentrations of quercetin in ethanol (50, 100, 150, 200, and 300 mg/L). The
concentrations of flavonoids in the sample was calculated from the calibration plot
and expressed as mg quercetin per gram of sample. After all of that, phenolics is
then assayed through the method folin-ciocalteau. It is based on the formation of a
blue color developed due to the complex redox reaction of phenols with
phosphomolibdic acid present in folin-ciocalteau reagent 1ml of the extract is added
to the flask containing 9 ml distilled water followed by 1 ml folin-ciocalteau’s phenol
reagent. It is then thoroughly mixed. Then follows the addition of 10ml of 7% sodium
carbonate. The mixture will then be diluted to 25ml with distilled water and left at
room temperature to stand for 90 minutes. A blue color (molybdenum blue) complex
was developed. The absorbance was measured at 750 nm. A standard calibration
curve was prepared using 25 to 150µg/mL in 80% methanol of Gallic acid. The total
phenolics were expressed as µg Gallic acid equivalents (GAE)/g samples. Now, the
extracted samples are subjected to antimicrobial test against pathogenic gram-
positive Staphylococcus aureus and gram-negative Escherichia coliusing agar well
diffusion method. Ampicillin and penicillin were used as standard drugs, respectively,
for studying the activities of the extract. The melted agar was poured into dry sterile
petri dishes and solidified. A sterile cotton swab moistened with the test suspension
that was previously incubated in Muller-Hinton Broth for 24 hours at 37 degree
Celsius was used to steak over the entire surface of the agar. It was allowed to
stand for 5 min. A cork borer was sterilized by immersing it in an erlenmeyer flask
containing ethanol and heated over an alcohol flame. The agar was stabbed using
the cooled cork borer to the bottom of the dish to create a well. The well was filled
with the previously prepared crude extract samples. The incubation period was 24
hrs at 37 degree Celsius. The activity of the extract was determined by measuring
the diameter of the zone of inhibition and compared with the values produced from
the standard drugs. The measurement of the data is carried out in triplicate and is
expressed as the mean standard deviation. It is analyzed using analysis of variance
followed by Fisher Pairwise comparison test.

Results
The total flavonoids were expressed in terms of quercetin equivalent per
grams of egg strings with the linear equation based on the standard curve: y =
0.0005x + 0.0027, r2 = 0.9947. The concentration of flavonoids in the egg strings
sample had a significant difference at p<0.05. It ranged from 6.27 mg/g to 8.83 mg/g
in this order: ethyl acetate > ethanol > hexane. Using Fisher Pairwise Comparisons,
each extract differs significantly from each other. As for the total phenolics, it is
expressed in terms of Gallic acid equivalent and calculated with a linear equation
based on a standard curve: y = 0.0025x + 0.0291, r2= 0.9814. The highest
concentration of the total phenolics was measured in ethanol extract (0.17 mg GA/
g). This was followed by ethyl acetate extract with total phenolics content (TPC) of
0.13 mg GAE/ g and lastly hexane extract with 0.01 mg GAE/ g. A significant
difference was observed at p<0.05 with hexane showing significantly different from
the other two extracts. Now, the natimicrobial activity of the egg strings extract (1
mg/mL) using different solvents showed different values of the zone of inhibition
against E. coli (gram-negative) and S. aureus (gram-positive). It was the ethyl
acetate crude extract that gave promising results that were almost comparable with
the positive control.

Discussion
 This study quantifies the previous result which showed the presence of
different secondary metabolites that include phenols and flavonoids. It was the
phenols and flavonoids in the egg strings’ extract that is responsible for the
antioxidant activity that was measured using DPPH assay. Accordingly, the
secondary metabolites flavonoids, which include flavones, flavanols, and tannins,
have an antioxidant property that greatly depends on the hydroxyl group, specifically
the 3-OH. It can scavenge reactive oxygen species (ROS) or reactive nitrogen
species (RNS) because of the presence of phenolic hydroxyl groups in its structure.
The present study showed that the ethyl acetate extract had high total flavonoids at p
< 0.05 in the egg strings of sea hare. This is an indication that ethyl acetate
maximized the extraction of the total flavonoids in egg strings. These results only
prove that natural products like total flavonoid compounds can be obtained from
marine mollusks. It is believed that secondary metabolites of sea hare organisms are
obtained from their algal diet. Compounds with strong potential in treating cancer are
isolated from algae, which include dolastatin 10, which is also isolated from sea
hare. On the other hand, ethanol solvent was more efficient in extracting the total
phenolics from egg strings of sea hare. The efficiency of the extraction of the total
phenolic was greatly dependent on the type of sample and the kind of extracting
solvent used. Phenolics are generally known to have antioxidant activity. It has redox
properties that absorb and neutralize free radicals, scavenging singlet or triplet
oxygen, as well as decomposing peroxides facilitated by the presence of a hydroxyl
group. Hence total phenolic concentration rapidly estimates the antioxidant activity.
Moreover, the aqueous ink extract was effective in scavenging DPPH radicals and
exhibited reducing power activity with an IC50 at 0.94 mg/mL and 39.4 µg/mL,
respectively. Although these are lower than the IC50 of the standards used, it is still
said to be the strongest antioxidant activity of sea hare. The antibacterial activity
results of the ethyl acetate extract were very promising. The secondary metabolites
present in the egg strings could be responsible or had contributed to its antimicrobial
activity since these compounds generally have an antibacterial factor. The presence
of phenolics and flavonoids shows a broad spectrum of antimicrobial agents with
high antifungal activity against gram-positive and gram-negative bacteria. It is the
partial hydrophobic property of phenols that make it effective against microbes. It
either inactivates microbial adhesions or inhibits hydrolytic enzymes such as
proteases.

Conclusion
Based on the measured antioxidants and antibacterial activity assay
conducted on the different crude extracts, compounds with nutritional and medicinal
potential may be isolated from egg strings of D. auricularia. These potentials may be
due to the presence of phenols, flavonoids, and other secondary metabolites. The
findings of this work may add to the overall value of the nutritional and medicinal
potential of the egg strings from sea hare.