Quantitation Of Secondary Metabolites And Xanthine Oxidase Inhibitory Assay Of

Ethanolic Fraction Of Common Reed (Phragmites Australis) Ethanolic Extract

A Science Investigatory Project

Presented to the Faculty and Staff of

Jacinto P. Elpa National High School

Science Technology and Engineering

Tandag City

Life Science

Blesselle Ann A. Clar

Rhea Joy S. Caingcoy

Maria Jessa Spirit B. Naranjo

Researchers

Mrs. Ana Geran V. Millan

Research I Teacher

S.Y. 2017-2018
 Abstract

Inflammation has been the starting point to different fatal diseases, thought it may have

good effects, too much of it is clearly dangerous. The research on xanthine oxidase inhibitory

activity of Common Reed (Phragmites australis) as a xanthine oxidase inhibitor aims to

determine the phytochemical present in Common Reed to inhibit the formation of reactive

oxygen thouby protecting the cells from destruction and inflammation.

Common Reed leaf extract was prepared by cutting into small pieces and air-drying.

After which the researcher weighed 200 grams of it and ethyl alcohol so then it was carefully

placed inside the container macerated with 95% ethyl alcohol 48hr. to determine

phytochemicals present, the phytochemical analysis was then performed.

Phytochemical Analysis showed that tannins and flavonoids is present in Common Reed

that is a potential inhibitor of xanthine oxidase. Due to the protein-binding property of

tannins quantitative analysis was then calculated.

Results of the test showed that the quantitative determination of the total and water solute

phenolic and tannin content of plant material revealed a positive correlation with XO

inhibitory activity. Statistical analysis proved that positive control revealed the highest

inhibition and the plant extract was found to be concentration dependent.

Definition of Key Terms

The following terms are conceptually and operationally defined for better understanding

of the readers:

Inflammation. Is part of the complex biological response of body tissues to harmful

stimuli, such as pathogens, damaged cells, or irritants, and is a protective response

involving immune cells, blood vessels, and molecular mediators. The function of inflammation

is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from

the original insult and the inflammatory process, and initiate tissue repair.

Uv-vis. Refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet

visible spectral region. This means it uses light in the visible and adjacent ranges. The absorption

or reflectance in the visible range directly affects the perceived color of the chemicals involved.

In this region of the electromagnetic spectrum, atoms and molecules undergo electronic

transitions. Absorption spectroscopy is complementary to fluorescence spectroscopy, in

that fluorescence deals with transitions from the excited state to the ground state, while

absorption measures transitions from the ground state to the excited state.

Xanthine oxidase inhibitory assay. It is conceptually defined as the chemical compounds

that occur naturally in plants. the term generally used to refer to those chemicals that may have

biological significance, for example flavonoids, but are not established ass essential nutrients.
 TABLE OF CONTENTS

Title Page

Abstract

Definition of Key Terms

CHAPTER 1: INTRODUCTION

A. Rationale of the Study

B. Statement of the Problem
C. Research Objectives
D. Statement of the Null Hypotheses
E. Significance of the Study
F. Conceptual Framework
G. Scope and Limitation

CHAPTER 2: REVIEW OF RELATED LITERATURE

CHAPTER 3: MATERIALS AND METHODS

CHAPTER 4: RESULTS AND DISCUSSION

CHAPTER 5: SUMMARY, CONCLUSION AND RECOMMENDATION

REFERENCES CITED

ACKNOWLEDGEMENT

PICTORIALS

APPENDICES

RESEARCHERS’ LOGBOOK
 CHAPTER I

INTRODUCTION

A. Rationale of the study

Inflammation (from Latin: inflammatio) is part of the complex biological response of

body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective

response involving immune cells, blood vessels, and molecular mediators. The function of

inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues

damaged from the original insult and the inflammatory process, and initiate tissue repair.

The five classical signs of inflammation are heat, pain, redness, swelling, and loss of

function (Latin calor, dolor, rubor, tumor, and functio laesa). Inflammation is a generic

response, and therefore it is considered as a mechanism of innate immunity, as compared

to adaptive immunity, which is specific for each pathogen. Too little inflammation could lead to

progressive tissue destruction by the harmful stimulus (e.g. bacteria) and compromise the

survival of the organism. In contrast, chronic inflammation may lead to a host of diseases, such

as hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer

(e.g., gallbladder carcinoma). Inflammation is therefore normally closely regulated by the body.

Inflammation can be classified as either acute or chronic. Acute inflammation is the

initial response of the body to harmful stimuli and is achieved by the increased movement

of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A

series of biochemical events propagates and matures the inflammatory response, involving the

local vascular system, the immune system, and various cells within the injured tissue. Prolonged

inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells
present at the site of inflammation, such as mononuclear cells, and is characterized by

simultaneous destruction and healing of the tissue from the inflammatory process.

Common Reed (phragmites australis) is a genus of four species of large perennial grasses

found in wetlands throughout temperate and tropical regions of the world. Phragmites form

dense stands, which include both live stems and standing dead stems from previous years. The

plant spreads horizontally by sending out rhizome runners, which can grow 10 or more feet in a

single growing season, rapidly crowding out native grasses.

Common reed’s vigorous rhizomatous growth frequently results in dense and often

impenetrable stands. This alters the diversity of natural wetland communities. The thick litter

accumulation and dense vegetative growth prevents native plant species from growing, and can

alter the area’s hydrology. Wetland wildlife habitat is substantially degraded.

In this regard, the researchers of this study aims to determine the phytochemicals present

in Common Reed (Phragmites australis) and test its effectiveness as an alternative inhibitor of

xanthine oxidase activity.

B. Statement of the problem:

This study aims to determine the phytochemicals present in common reed extract and test

its effectivity as xanthine oxidase inhibitor.

Specifically, this study seeks to answer the following:

1. What phytochemical present in common reed that can inhibit the active of xanthine

oxidase?

2. What is the difference of the effectiveness of the allopurinol (positive control) with the

ethanolic extract from common reed?

C. Research Objectives:

To carry out this investigation, the following objectives will be evaluated. Specifically, the study

aims to:

1. Determine the phytochemical present in common reed that can inhibit the active of

xanthine oxidase.

2. Determine the difference of the effectiveness of the allopurinol (positive control) with the

ethanolic extract from common reed.

D. Statement of the Null Hypotheses:

The researchers formulated the following null hypothesis as response to the research

problems:

1. There is no significant phytochemicals present in common reed extract

2. There is no difference with the common reed ethanolic extract with the positive

control.

E. Significance of the Study

The importance of this study is to gain more knowledge about the ethanolic extract from

Common Reed (Phragmites australis) a bridge to minimize health issues, such as inflammation

Moreover, it serves as a reference for further studies about plants having xanthine oxidase

inhibitory assay. Specifically, this study will be beneficiary to:

Global The study will provide support for the search for new and useful source from plant used

in medicine throughout the world.

 Society This study will fill in for as instant solution to the increasing number of people suffering

side effects from the use of synthetic and dangerous chemicals which can cause threat to humans.

Department of Education When proven effective, this study might also be used for reference as

basin for further studies.

Poor people. This study will provide importance in determining the effective and cheap

alternative source of natural medicine out of commonly found natural resources in the locality.

F. Scope and Limitation

This study is limited on the determination of the phytochemicals present in common reed

Phragmites australis, and testing its effectivity as xanthine oxidase inhibitor, thus, we will test it

through in vitro which won’t deny any significant ethics. Furthermore, it focuses in alternative of

our positive control which is the alluporinol.

Ethanolic extract from common reed Phragmites Australis, is environmental friendly.

Also, this plant can be seen only in shallow water and wet lands, usually beside farmlands, lakes,

and river banks, etc.

G. Conceptual Framework

This conceptual framework shows the basic structure or the structural frame of our study.

Input Process Output

Independent Intervening Dependent Variable

Variable Variable
Phytochemicals
present and the rate of
Leaves and stem of UV-VIS analysis and
xanthine oxidase
common reed Xanthine Oxidase
inhibited
Inhibitory Assay
 CHAPTER II

REVIEW OF RELATED LITERATURE

This chapter gives information for the reader about the description and morphology of the

common reed also about its characteristics, efficacy as bioethanol, its medicinal values, related

literature, related studies and its other uses.

Figure 1. Common Reed

In the Philippines, common reed or commonly known as “tambo” is used as dust brooms are

made from the panicles, the hardy stems are used as firewood, Where bamboo is not available,

used for roofing ribs, culms used for pen handles, also it makes first-class paper, but difficult to

bleach, mouth pieces for musical instruments (clarinets, etc.)

Phragmites australis (common reed) is a perennial grass that grows in wetlands or near

inland waterways. Due to its fast-growing properties and low requirement in nutrients and

water, this arboreal variety is recognized as a promising source of renewable energy although

it is one of the least characterized energy crops.

This common reed forms large beds in shallow water; it has round, hollow stems, which

typically grow to 2m in height, but may reach 4m. These stems grow from a system of stout,
creeping rhizomes. The flat leaves taper into a point, and are attached to the stem by smooth

sheaths, which are loose so that the leaves all point in one direction in the wind. The flowers are

borne on highly branching purple inflorescences, which measure from 20 to 60cm in length. The

flowers are grouped into 'spikelets', which are 10-15 mm in length and support 1-6 flowers.

 Xanthine oxidase inhibitor is any substance that inhibits the activity of xanthine

oxidase, an enzyme involved in purine metabolism. In humans, inhibition of xanthine oxidase

reduces the production of uric acid, and several medications that inhibit xanthine oxidase

are indicated for treatment of hyperuricemia and related medical conditions including gout.

Xanthine oxidase inhibitors are being investigated for management of reperfusion injury.

(Nivorohzkin, 2006)

In experiments, numerous natural products have been found to inhibit xanthine oxidase in

vitro or in model animals (mice, rats). These include three flavonoids that occur in many

different fruits and vegetables: kaempferol, myricetin, and quercetin. More generally, planar

flavones and flavonols with a 7-hydroxyl group inhibit xanthine oxidase. An essential

oil extracted from Cinnamomum osmophloeum inhibits xanthine oxidase in mice. The natural

product propolis from selected sources inhibits xanthine oxidase in rats; the specific substance

responsible for this inhibition has not been identified, and the generality of these findings is

unknown. An extract of leaves of Pistacia integerrima also inhibits xanthine oxidase at a level

that appears to merit further research. (Wang, 2008)

Xanthine oxidase is a key enzyme responsible for hyperuricemia, a pre-disposing factor

for Gout and oxidative stress-related diseases. Only two clinically approved xanthine oxidase

inhibitors Allopurinol and Febuxostat are currently used for treatment of hyperuricemia.
However, owing to their side effects there is a need for new non-purine-based selective

inhibitors of xanthine oxidase. In the process of exploring novel xanthine oxidase inhibitors and

anti-oxidants, we screened the culture filtrate of 07 novel species of Muscodor, a sterile

endophytic fungi isolated from Cinnamomum and Aegle marmelos. Chloroform extract of M.

darjeelingensis exhibited the maximum xanthine oxidase inhibition in the qualitative and

quantitative assays. The IC50 of chloroform extract of M. darjeelingensis was 0.54 µg/ml which

was much lower to Allopurinol but higher when compared to Febuxostat. 88% reduction in uric

acid production was recorded by M. darjeelingensis chloroform extract which was similar to

allopurinol. The maximum anti-oxidant activity was exhibited by M. indica against the gallic

acid standard in the DPPH-free radical assay. Anti-oxidant activity index of M. indica was 7.7,

which was followed by M. kashayum with 5.4. M. darjeelingensis exhibited a moderate anti-

oxidant activity with anti-oxidant activity index of 1.63 in the DPPH assay. The present study is

the very first report of Muscodor species exhibiting xanthine oxidase inhibitory and anti-oxidant

activity together. Chloroform extract of M. darjeelingensis and M. indica stand out as potential

candidates for isolation and characterization of the xanthine oxidase inhibitor and anti-oxidant

compound, respectively. (Kapoor, 2016)

Allopurinol, the xanthine oxidase inhibitor, is the only drug available for the treatment of

gout. We examined the xanthine oxidase inhibitory activity of some commercially available

flavonoids such asepigallocatechin, acacatechin, myricetin, naringenin, daidzein and glycitein by

virtual screening and in-vitro studies. The interacting residues within the complex model and

their contact types were identified. The virtual screening analysis were carried out using

AutoDock 4.2 and in-vitro xanthine oxidase inhibitory activity was carried out using xanthine as

the substrate. In addition, enzyme kinetics was performed using LineweaverBurkplot analysis.
Allopurinol, a known xanthine oxidase inhibitor was used as the standard. The docking energy

ofglycitein was found to be -8.49 kcal/mol which was less than that of the standard (-4.47 kcal/

mol). All the selected flavonoids were found to exhibit lower binding energy (-8.08 to -6.03 kcal/

mol) than allopurinol. The docking results confirm that flavonoids showed greater inhibition of

xanthine oxidase due to their active binding sites and lesser binding energies compared to

allopurinol. This may be attributed to the presence of benzopyran ring in the flavonoids. In the

xanthine oxidase assay, IC50 value of glycitein was found to be 12±0.86 μg/mL, whereas that of

allopurinol was 24±0.28 μg/mL. All the remaining compounds exhibited IC50 values ranging

between 22±0.64 to 62±1.18 μg/mL. In the enzyme kinetic studies, flavonoids showed

competitive type of enzyme inhibition. It can be concluded that flavonoids could be a promising

remedy for the treatment of gout and related inflammatory disorders. Further in-vivo studies are

required to develop potential compounds with lesser side effects. (Iran, 2013)

The research of the isolation and xanthine oxidation inhibition activity of alkaloid

compound from Peperomia pellucida has been carried out. Alkaloid extract is isolated by column

chromatography and preparative TLC. Alkaloid isolate is identified spectroscopically by UVVis

spectrophotometer, FT-IR, and LC-MS/MS. Xanthine oxidase inhibition activity is carried out

by in vitro assay. The result showed that the alkaloid isolated probably has piperidine basic

structure. The alkaloid isolate has N-H, C-H, C = C, C = O, C-N, C-O-C groups and the aromatic

ring. The IC50 values of ethanol and alkaloid extract are 71.6658 ppm and 76.3318 ppm,

respectively. Alkaloid extract of Peperomia pellucida showed higher activity than ethanol

extract. (Fachriyah, 2017)

The search for novel xanthine oxidase (XO) inhibitors with a higher therapeutic activity

and fewer side effects are desired not only to treat gout but also to combat various other diseases
associated with the XO activity. At present, the potential of developing successful natural

products for the management of XO-related diseases is still largely unexplored. In the present

study, we have screened the methanolic extracts of various Jordanian medicinal plants for their

XO inhibitory activities using an optimized protocol. (Hudaib, 2011|)

Xanthine oxidase (XO), an enzyme widely distributed among mammalian tissues, is

associated with the oxidation of xanthine and hypoxanthine to form uric acid. Reactive oxygen

species are also released during this process, leading to oxidative damages and to the pathology

called gout. Available treatments mainly based on allopurinol cause serious side effects. Natural

products such as flavonoids may represent an alternative. Thus, a series of polymethoxyflavones

isolated and hemisynthesized from the bud exudates of Gardenia oudiepe has been evaluated for

in vitro XO inhibitory activity. Compounds 1, 2 and 3 were more active than the reference

inhibitor, Allopurinol (IC50 = 0.25 ± 0.004 μM) with IC50 values of (0.004 ± 0.001) μM,

(0.05 ± 0.01) μM and (0.09 ± 0.003) μM, respectively. Structure-activity relationships were

established. Additionally, a molecular docking study using MOE™ tool was carried out to

establish the binding mode of the most active flavones with the enzyme, showing important

interactions with its catalytic residues. These promising results, suggest the use of these

compounds as potential leads for the design and development of novel XO inhibitors. (Eur,

2018)

Activity-directed fractionation and purification processes were employed to identify

xanthine oxidase (XO) inhibitory compounds from the leaves of Perilla frutescens. The total

extract was evaluated in vitro on XO inhibitory activity and in vivo in an experimental model

with potassium oxonate-induced hyperuricemia in mice which was used to evaluate anti-

hyperuricemic activity. The crude extract showed expressive urate-lowering activity results.
Solvent partitioning of the total extract followed by macroporous resin column chromatography

of the n-butanol extract yielded four extracts and eluted parts. Among them, only the 70%

ethanol eluted part of the n-butanol extract showed strong activity and therefore was subjected to

separation and purification using various chromatographic techniques. Five compounds showing

potent activity were identified by comparing their spectral data with literature values to be

caffeic acid, vinyl caffeate, rosmarinic acid, methyl rosmarinate, and apigenin. These results

indicate that pending further study, these compounds could be used as novel natural product

agents for the treatment of hyperuricemia. (Wang, 2015)

The search for novel xanthine oxidase (XO) inhibitors with a higher therapeutic activity

and fewer side effects are desired not only to treat gout but also to combat various other diseases

associated with the XO activity. At present, the potential of developing successful natural

products for the management of XO-related diseases is still largely unexplored. In the present

study, we have screened the methanolic extracts of various Jordanian medicinal plants for their

XO inhibitory activities using an optimized protocol. Its results was six plants were found most

active (% inhibition more than 39%). These plants are Salvia spinosa L. (IC50= 53.7

μg/ml), Anthemis palestina Boiss. (168.0 μg/ml), Chrysanthemum coronarium L. (199.5

μg/ml), Achillea biebersteinii Afansiev (360.0 μg/ml), Rosmarinus officinalis L. (650.0 μg/ml),

and Ginkgo bilobaL. (595.8 μg/ml). Moreover, four more plants, namely Lavandula

angustifolia Mill. (28.7% inhibition), Helianthemum ledifolium (L.) Mill. (28.4%), Majorana

syriaca (L.) Kostel. (25.1%), and Mentha spicataL. (22.5%) showed a XO inhibitory activity in

the range of 22–30%. So then, the study showed that many of the tested plant species are

potential sources of natural XO inhibitors that can be developed, upon further investigation, into

successful herbal drugs for treatment of gout and other XO-related disorders.(Hudaib, 2011)
 In other experiments, with this objective, to screen Vietnamese medicinal plants for

xanthine oxidase (XO) inhibitory activity and to isolate XO inhibitor(s) from the most active

plant. The outcome was three hundreds and eleven methanol extracts (CME) belonging to 301

Vietnamese herbs were screened for XO inhibitory activity. Among these plants, 57 extracts

displayed XO inhibitory activity at 100 μg/mL with inhibition rates of over 50%. The extracts of

Archidendron clypearia (A. clypearia), Smilax poilanei, Linociera ramiflora and Passiflora

foetida exhibited the greatest potency with IC50 values below 30 μg/mL. Chemical study

performed on the extract of A. clypearia resulted in the isolation of six compounds, including 1-

octacosanol, docosenoic acid, daucosterol, methyl gallate, quercitrin and (-)-7-O-

galloyltricetiflavan. The compound (-)-7-O-galloyltricetiflavan showed the most potent XO

inhibitory activity with an IC50 value of 25.5 μmol/L. Overall, four Vietnamese medicinal plants

were identified to have XO inhibitory effects with IC 50 values of the methanol extracts below

30 μg/mL. Compound (-)-7-O- galloyltricetiflavan was identified as an XO inhibitor from A.

clypearia with IC50value of 25.5 μmol/L.( Tong, 2017)

The objective was to isolate and identify phytochemicals from roots of C. opaca and to

evaluate xanthine oxidase (XO) and alpha-amylase inhibitory activities of their methanolic

extract and its fractions.Results was Methanolic extract displayed significant activity against

both the enzymes with IC50 of 156.0 mg/mL and 5.6 mg/mL for XO and alpha-amylase,

respectively. Ethyl acetate fraction showed highest activity against both the enzymes with IC50

of 129 mg/mL and 4.9 mg/mL for XO and alpha-amylase, respectively. Chloroform fraction had

IC50 of 154.2 mg/mL and 5.5 mg/mL for XO and alpha-amylase, respectively. Aqueous fraction

exhibited significant efficacy against alpha-amylase (IC50 5.0 mg/mL). Hexane fraction showed

good activity against alpha-amylase in a dose-dependent manner but exhibited opposite trend
against XO. The compounds isolated from ethyl acetate fraction included limonene, vanillin,

lupeol, rutin, quercetin, b-sitosterol, Vitamin E, 2-hydroxyacetophenone, naphthalenone, 2,3,3-

trimethyl-2-(3-methylbuta-1,3-dienyl)-6-methylenecyclohexanone, and 2-benzenedicarboxylic

acid, mono(2-ethylhexyl) ester. They concluded that

Moderately polar phytochemicals of C. opaca roots possess exploitable inhibitory activity

against both the enzymes. (Saeed, 2014)

Tradescantia albiflora (TA) Kunth (Commelinaceae) has been used for treating gout and

hyperuricemia as folklore remedies in Taiwan. Therefore, it is worthwhile to study the effect of

TA extracts on lowering uric acid activity. The hypouricemic effects of TA extracts on

potassium oxonate (PO)-induced acute hyperuricemia were investigated for the first time. All

treatments at the same volume (1 ml) were orally administered to the abdominal cavity of PO-

induced hyperuricemic rats. One milliliter of TA extract in n-hexane (HE), ethyl acetate (EA), n-

butanol (BuOH), and water fractions has 0.28, 0.21, 0.28, and 1.03 mg TA, respectively; and the

plasma uric acid (PUA) level was measured for a consecutive 4 h after administration. Resulting,

All treatments at the same volume (1 ml) were orally administered to the abdominal cavity of

PO-induced hyperuricemic rats. One milliliter of TA extract in n-hexane (HE), ethyl acetate

(EA), n-butanol (BuOH), and water fractions has 0.28, 0.21, 0.28, and 1.03 mg TA, respectively;

and the plasma uric acid (PUA) level was measured for a consecutive 4 h after administration.

To sum it all up, Tradescantia albiflora extracts possess in vivo hypouricemic action in

hyperuricemic ratsT. albiflora extracts exhibited strong inhibitory activity against xanthine

oxidase (XO)Butenolide may play an important role in XO inhibitionThe extract bracteanolide A

was demonstrated potent XO inhibitory activity in vitro. Abbreviations used: TA: Tradescantia
albiflora, PO: potassium oxonate, HE: n-hexane, EA: ethyl acetate, BuOH: n-butanol, PUA:

plasma uric acid, XO: xanthine oxidase, MeOH: methanol, IP: intraperitoneal. (Sheu, 2016)

This study investigates the lowering of uric acid using longan extracts, including flowers,

pericarps, seeds, leaves, and twigs, on potassium-oxonate-induced hyperuricemia mice and its

inhibitory actions against xanthine oxidase (XO) activities. The findings revealed that ethyl

acetate fraction of longan extracts exhibited strong XO-inhibitory activity, and the flower

extracts (IC50 = 115.8 μg/mL) revealed more potent XO-inhibitory activity to those of pericarps

(118.9 μg/mL), twigs (125.3 μg/mL), seeds (262.5 μg/mL), and leaves (331.1 μg/mL) in vitro. In

addition, different dosages of longan extract (50-100 mg/kg) were administered to hyperuricemic

mice. The lowering effect of longan extracts on uric acid at 75 mg/kg markedly reduced plasma

uric acid levels in decreasing order: Flowers (80%) > seeds (72%) > pericarps (64%) > twigs

(59%) > leaves (41%), compared with allopurinol (89%). Finally, 10 isolated phytochemicals

from longan flowers were then examined in vitro. The results indicated that proanthocyanidin A2

and acetonylgeraniin A significantly inhibited XO activity in vitro. This is the first report

providing new insights into the urate-reducing effect of phenolic dimer and hydrolyzable tannin,

which can be developed to potential hypouricemic agents. (Fu, 2016)

 CHAPTER III

MATERIALS AND METHODS

RESEARCH DESIGN

This study will employ an experimental research design in the identification of the

xanthine oxidase inhibitory activity of Common Reed Extract. The xanthine oxidase inhibitory

activity will be done using xanthine oxidase assay kit and tested using the UV-Visible

spectroscopy.

RESEARCH LOCALE

The plant material was collected from Tandag City, Surigao Del Sur. The macerated

extract was taken to the University of the Immaculate Conception (UIC), Davao City where the

experimentation took place for quantitative phytochemical screening and xanthine oxidase assay.

Figure 1 Map of the University of the Immaculate Conception were

Experiment was done

SAMPLING DESIGN
 The non- probability purposive sampling design was employed in the collection of fresh

plant material. The collected sample selected limiting to the fresh samples only.

DATA GATHERING PROCEDURES

A. Plant Collection/ Identification

Plant samples was collected from Tandag City, Surigao Del Sur. The plant was weighed

to get the 200 grams of each sample. After which each 200 grams plant samples was carefully

placed inside the container macerated with 95% ethyl alcohol. After 48 hours, the macerated

plant material was filtered with Whatman No. 1 and was concentrated using rotary evaporator.

The crude extract was preserved at 5°C.

B. Estimation of Flavonoids in Common Reed (Phragmites Australis)

Total flavonoid content was measured by the aluminum chloride colorimetric assay

(Zhishen, et al., 1999). An aliquot (1 ml) of extracts and standard solution of catechin (100

mg/ml) was added to 10 ml volumetric flask containing 4 ml of distilled water. To this 0.3 ml 5

% NaNO2 were added. After 5 min, 0.3 ml 10 % AlCl3 was added. Then after 1 min, 2ml of 1 M

NaOH was added and the total volume was made up to 10 ml with distilled water. The solution

was mixed well and the absorbance was measured against prepared reagent blank at 510 nm.

Total flavonoid content of root extracts expressed as mg catechin equivalents (CE)/100 G fresh

weights. All samples were analyzed in triplicates.

C. Estimation of Total Phenolic Content

The determination of total phenolics based on Folin-Ciocalteu reagent assay (Singleton

and Rossi., 1965). An aliquot (1ml) of extracts and standard solution of Gallic acid (100 mg/ml)

was added to 25 ml volumetric flask, containing 9 ml distilled water. The distilled water itself

was used as blank. One ml of Folin-Ciocalteu reagent was added to the mixture and shaken.
After 5 min, 10 ml of 7% Na2CO3 solution was added to the mixture. The solution was diluted

to volume (25 ml) with distilled water and mixed. After incubation for 90 min at room

temperature, the absorbance against prepared reagent blank was determined at 750 nm with an

UV-Vis Spectrophotometer. The total phenolic content of root extracts expressed as mg Gallic

acid equivalents (GAE)/100 G fresh weights. All samples were analyzed in triplicates.

D. Test for Xanthine Oxidase Activity

The XO inhibitory activity was measured as previously reported. The substrate and the

enzyme solutions were prepared immediately before use. The reaction mixture contained an 80

mM sodium pyrophosphate buffer (pH = 8.5), 0.120 mM xanthine, and 0.1 unit of XO. The

absorption at 295 nm, indicating the formation of uric acid at 25°C, was monitored and the initial

rate was calculated. The methanolic dried extract, initially dissolved and diluted in the buffer,

was incorporated in the enzyme assay to assess its inhibitory activity at a final concentration of

200 μg/ml. Moreover, for the plants whose extracts showed enzymatic inhibition more than 35%,

the IC 50 evaluation was also performed. In this case, five different concentrations of the dried

extract (18.8, 37.5, 75, 150, and 300 μg/ml) were used to determine the concentration that

inhibits 50% of the XO enzyme activity (IC50 value). All assays were run in triplicate; thus,

inhibition percentages are the mean of three observations. A negative control (blank; 0% XO

inhibition activity) was prepared containing the assay mixture without the extract. Allopurinol

was used as a positive control in the assay mixture (Hudaib, M et al. 2011). The XO inhibitory

activity was expressed as the percentage inhibition of XO in the above-mentioned assay mixture

system, calculated as follows:

 where test inclination is the linear change in the absorbance per minute of the test

material, and blank inclination is the linear change in the absorbance per minute of the blank.

STATISTICAL ANALYSIS

This will utilize a One-Way Analysis of Variance to determine if there is significant

difference on the antioxidant property and xanthine oxidase activity of Common reed extract.

BIOSAFETY PROCEDURES

In performing the experiment the researcher shall observe the use of PPE (Personal

Protective Equipment) which includes eye protection, gloves, mask, laboratory gown and a

closed shoes. The hair shall be kept in a way that it does not dangle or cover the face. A thorough

review of the MSDS (Material Safety Data Sheet) of all chemicals to be used shall be done

before the experimentation process. Most importantly all chemical containers shall be labeled

properly before, during and after the experiment including the waste chemicals to avoid any

untoward incident.

ETHICAL CONSIDERATION

Proper handling of the chemical ethanol used was observed during the experimentation. The

used ethanol was disposed as a hazardous waste as defined by DENR ADMINISTRATIVE

ORDER No. 29 Series 1992 and Environmental and Protection Agency (EPA) due to its

flammability and ignitibility. The DENR Administrative Order Section 24.2 clearly encourages

proper management of hazardous wastes generated within the country by promoting; the

minimization of the generation of hazardous waste; the recycling and reuse of hazardous waste;

the treatment of hazardous waste to render it harmless; and the landfill of inert hazardous waste

residues. It also laid emphasis that hazardous waste shall be managed in such a manner as not to

cause or potentially cause –pollution; state of danger to public health, welfare and safety; harm
to animals, bird, wildlife, fish or other aquatic life; harm to plants and vegetation; or limitation in

the beneficial use of a segment of the environment. The waste generator shall be responsible for

the proper management and disposal of the hazardous waste.

 CHAPTER IV

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter presents the overall data of the study which include the phytochemical analysis and

xanthine oxidase inhibitory assay.

Phytochemical Analysis

Water Soluble Tannins and flavonoids as quercetin were determined

spectrophotometrically using the Milton Roy Spectrophotometer. Results of the test are shown

in table 1.

Table 1

Phytochemical Anlaysis of Common Reed (Phragmites Australis)

Phytochemicals Trial 1 Trial 2 Trial 3 Mean S.D.

Tannins, ppm 35.0 38.0 40.0 37.7 2.5166
Flavonoids, mg/ml 0.025 0.028 0.030 0.028 0.0025

Results of the test proved that flavonoids and tannins were substantially present in

Common Reed (Phragmites Australis). Accordingly, Flavonoids (Iio et al., 1985; Chang et al.,

1993), and certain other phenols (Hatano et al., 1989), polyphenols (Costantino et al., 1992) and

tannins (Hatano et al., 1990), as well as coumarins (Chang and Chiang, 1995), plant growth

regulators (Sheu and Chiang, 1996) and folic compounds (Lewis et al., 1984) have all been

reported to be potent XO inhibitors. The putative therapeutic effects of many traditional

medicines have been ascribed to flavonoids in particular due to their enzyme inhibitory and

antioxidant activity (Havsteen, 1983; Brandi, 1992). Therefore, the researchers expected positive

correlation between XO inhibitory activity and phenolic content of plant extracts. Moreover,

tannins, because of their protein-binding properties, may have interfered with our in vitro assays,
and so a quantitative analysis of the test plants was performed to distinguish the contribution of

non-tannin phenolics to XO inhibition from that attributable to tannins.

Figure 2 Calibration Curve of Tannins Determination

Figure 3 Calibration Curve of Flavonoids Determination

Inhibitory Assay of Plant Extract

In this study, Plants Common Reed (Phragmites Australis) was utilized for xanthine

oxidase inhibition which was associated for the treatment of gout, or diseases with associated

symptomologies such as rheumatism or arthritis. In this study, the presence of tannins and

flavonoids in the plant can be associated to its capacity to inhibit the xanthine oxidase enzyme.

Overall results are shown in table 2.

Table 2
 Xanthine Oxidase Inhibitory Assay of Common Reed (Phragmites Australis)

Treatments Trial Number Mean S.D.

1 2 3
18.8 ppm 9.8 7.5 8.8 8.7 1.1533
37.5 ppm 10.5 11.5 12.5 11.5 1.0000
75.0 ppm 18.8 18.2 20.0 19.0 0.9165
150.0 ppm 30.5 31.2 31.2 31.0 0.4041
300.0 ppm 39.0 40.5 40.1 39.9 0.7767
Positive Control 60.0 62.0 63.0
61.7 1.5275

Xanthine oxidase inhibitors (XOI) are typically used in the treatment of nephropathy and

renal stone diseases linked to hyperuricemia. XOI are agents that directly inhibit the synthesis of

uric acid in vivo. Certain active constituents present in crude plant extracts like flavonoids and

polyphenolic compounds have been reported to possess XOI. These findings have opened the

possibility of isolation of new natural compounds, which can be possible inhibitors of XO, and

led to the growing interest in the investigation of medicinal plants. The activity of flavonoids as

inhibitors of xanthine oxidase in vitro has been reported. Based from the results an increasing

concentration of Common Reed (Phragmites Australis) has resulted to increased XO inhibition

indicating the potentiality of the plant as xanthine oxidase inhibitor.

Inferential Statistics

To determine if there is an existing significant difference on the XO inhibitory activity of

Common Reed (Phragmites Australis) with the positive control allopurinol, One-Way Analysis

of Variance was utilized and results are shown in table 3.

Table 3

Xanthine Oxidase Inhibitory Assay of Common Reed (Phragmites Australis)

Treatments Mean S.D. F value P value Remarks

18.8 ppm 8.7 1.1533

37.5 ppm 11.5 1.0000
75.0 ppm 19.0 0.9165 1,153.88 0.0001 Significant
150.0 ppm 31.0 0.4041
300.0 ppm 39.9 0.7767
Positive Control 61.7 1.5275
Quantitative determination of the total and water-soluble phenolic and tannin content of

plants traditionally used for treating gout revealed a positive correlation with XO inhibitory

activity. Statistical analysis proved that positive control revealed the highest inhibition and plant

extract inhibitory activity of xanthine oxidase was found to be concentration dependent.

 CHAPTER V

SUMMARY, CONCLUSION AND RECOMMENDATION

In this point, you will know the summarization of the study, thought conclusion and

recommendation for future researcher.

Summary:

As shown in Chapter 4 (Result), it proves that the Common Reed (Phragmites asutralis)

has flavonoids and tannins through the Phytochemical analysis using the Milton Roy

Spectrophotomer. Thus, this lead the researchers for another test, the Xanthine Oxidase

Inhibitory Assay. Based from the results, an increasing concentration of Common Reed

(Phragmites australis) has resulted to increased XO inhibition indicating that the potentiality of

the plant as xanthine oxidase inhibitor. How even, before that process, a quantitative analysis

was performed due to the protein. Finding property of tannins, for it may have been interfered
with the in vitro assay. It is to show the contribution of non-tannin phenolics from the inhibition.

The results shows that Common Reed (Phragmites australis) can be an alternative to the positive

control, though it shows that there is a slow concentration compared to the positive control it is

still possible.

Conclusions:

According to the results of the results of this Investigaotry Project, it led the researchers

to the following conclusions.

1. The Phytochemicals present in the plant extract is tannins and flavonoids.

2. The concentration of the plant extract as a xanthine oxidase inhibitor is high, however the

positive control has the highest concentration.

3. The significance of the plant extract as a xanthine oxidase inhibitor is significantly high.

Recommendations:

Base from the summarize made and conclusions we hereby proposed this following

recommendations.

1. A further study about the other potentiality of Common Reed other than as a xanthine

oxidase inhibitor.

2. A further study of Common Reed as an anti-oxidant for this plant has the potential to be

one.

3. A further study of Common Reed besides medical purposes.

4. A further study that will identify the other uses of the phytochemicals present in this plant

extract.
 Any further studies is highly appreciated.

References Cited

Kapoor N, Saxena S. (2016, August). Xanthine oxidase inhibitory and antioxidant potential of

Indian Muscodor species. Retrieved from https://link.springer.com/article/10.1007/s13205-

016-0569-5

Asokkumar K, Madeswaran A, Umamaheswari M. (2012, February). Virtual Screening Analysis

and In-vitro  Xanthine Oxidase Inhibitory Activity of Some Commercially Available Flavonoids.

Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813288/

Fachriyah E, Ghifari M, Anam K. (2017). Isolation, Identification, and Xanthine Oxidase

Inhibition Activity of Alkaloid Compound from Peperomia pellucida. Retrieved from

http://iopscience.iop.org/article/10.1088/1757-899X/349/1/012017/pdf
Hudaib M, Tawaha K, Mohammad M, Assaf A, Issa A, Alali F, Aburjai T, Bustanji Y. (2011,

October). Xanthine oxidase inhibitory activity of the methanolic extracts of selected Jordanian

medicinal plants. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261066/

Grougnet R, Ortega M, Santi M, Paulino Z, Vera B, Bouzidi C, Dumontet V, Abin A. (2018,

Januray). Xanthine oxidase inhibitory activity of natural and hemisynthetic flavonoids from

Gardenia oudiepe (Rubiaceae) in vitro and molecular docking studies. Retrieved from

https://www.ncbi.nlm.nih.gov/pubmed/29207340

Wang W, Hou L, Zhang C, Shi H, Liu Y, Liu X, Guo B, Zhao D, Gao H. (2015, September 25).

Bioassay-Guided Isolation and Identification of Xanthine Oxidase Inhibitory Constituents from

the Leaves of Perilla frutescens. Retrieved from file:///C:/Users/Admin/Downloads/molecules-

20-17848%20(1).pdf
 Acknowledgement

We would like to express our deepest appreciation to the following that support and

helped us regarding our Science Investigatory Project. They are the people who contributed

much for the success of this endeavour.

To our beloved parents, for their unconditional love and support as we labored towards

this research.

To our research adviser, Mrs. Ana Geran Millan for giving us this opportunity and

giving us the additional information we need in doing this research and spending a time in

guiding us with our work.

To Mr. Venchie Badong, for helping and consulting us on our research study.

And most of all, to our Almighty God, for the strength, knowledge and for the gift of

life and wisdom, everything.

PICTORIALS
Collection of Common Reed (Phragmites australis) Cutting of Common Reed (Phragmites australis)

Washing of Common Reed (Phragmites australis) Drying of Common Reed (Phragmites australis)
Grinding of Common Reed (Phragmites australis) Macerated plant material Removal of ethanol to
with 95% Ethanol for 48hrs. the extract through
Rotary Evaporator

APPENDICES
 Curriculum Vitae

Personal Information

Name: Rhea Joy S. Caingcoy

Nickname: Rhea

Address: Prk. Quintos, Mabua, Tandag City

Birthday: January 2, 2002

Parents:

Mother: Irma S. Caingcoy

 Father: Rafael D. Caingcoy

Educataional Background:

Elementary: Special Science Elementary School

Tandag City

Secondary: Jacinto P. Elpa National High School

Tandag City

Curriculum Vitae

Personal Information

Name: Maria Jessa Spirit B. Naranjo

Nickname: Jessa

Address: Rivas St. Pob. Marihatag S.D.S

Birthday: March 6,2002

Parents:

Mother: Alice B. Simplicio

Father: Antonieto C. Simplicio

Educataional Background:

Elementary: Marihatag central Elementsry School

Marihatag S.D.S

Secondary: Jacinto P. Elpa National High School

Tandag City

Curriculum Vitae

Personal Information

Name: Blesselle Ann A. Clar

Nickname: Ann-ann

Address: Baex, Telaje, Tandag City

Birthday: December 9, 2002

Parents:

Mother: Anita A. Clar

Father: Virginilo L. Clar

Educataional Background:

Elementary: Tandag Pilot Elementary School

Tandag City

Secondary: Jacinto P. Elpa National High School

Tandag City

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