ENZYME INHIBITION SREENING OF SELECTED INDIGENOUS Formatted: Space After: 0 pt, Line spacing: single

PLANTS IN MINDANAO Formatted: Different first page header

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A TThesis Proposal to the Faculty of Formatted: Space After: 0 pt, Line spacing: single

Pharmacy and Chemistry Program of

University of the Immaculate Conception
Davao City
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In Proposal Partial fulfillment of the Requirements Formatted: Space After: 0 pt, Line spacing: single

for the Degree of Bachelor of Science in Pharmacy

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Apalisok, Arris Gene

Gani, Sittie Sumaiyah

Laluna, Ivy Glad

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February 2019

Table of Contents:

Title Page

Table of Contents

Chapter 1

……

Chapter 2

…..

Chapter 3

…..
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Chapter 1

THE PROBLEM AND ITS SETTING

Background of the Study

P The Philippines is home to a diverse selection of plants;

however, a lot of these plants does not haveare not yet scientifically

recognition recognized hence they and therefore fall with those that are

understudied. A variety of indigenous plants in Mindanao are highly

favored and used by native communities in their traditional medicines. Commented [FB1]: Site source, then give 1 example with
source too….

These plants however, have not yet been confirmed to elicit

pharmacological effects nor contain active constituents because of the lack

of research.
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There are various ways to discover and determine promising

pharmacological actions of these plants. With the biological make up of

these plants, their phytochemical constituents and secondary metabolites as

the backbone for pharmacological actions. The biochemical components of

medicines, either it can naturally or synthetically derived, determines the

pharmacokinetic and pharmacodynamic behaviors (Stepensky, 2013).

Hence, different laboratory methods can be useful as qualitative or

quantitative tool to analyze plants’ phytochemical constituents that can

have pharmacological application that can later become a candidate as new

medicine for specific disease.

Laboratory methods utilized in drug discovery starts with qualitative

screening thereafter if turns out positive for specific pharmacologic action,

quantitative testing is employed before bioassays to be performed. Among

several qualitative methods useful. The chemical structure of any drug

determines its pharmacokinetics and pharmacodynamics (Stepensky, 2013).

Using Thin-Layer Chromatography (TLC) method, is highly regarded as

efficient tool to screen enormous samples for a specific pharmacologic action

or presence of active secondary metabolites. Indigenous plants are to be

profiled using TLC to generate basic information about their phytochemical

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properties and evaluate if it can be an effective sample for drug

development.the chemical structures contained in the extracts of the plants

in question can be identified and separated into their respective drug

classes. In addition to the phytochemical screening of the selected plants,

their physical structures would also be observed, this would be done with

the use of a Scanning Electron Microscope (SEM).

The Hence this study intends to analyze establish both the

phytochemical properties of different indigenous plants in Mindanao and

discover their activities against specific enzymes that are responsible for

specific pathologic conditions in the community.and physical structures of

the selected plants in search of lead compounds using TLC and microscopy

respectively. With the elucidation of the physical and chemical structures of

the plant samples, inferences can be made as to what possible therapeutic

effects these plants can exert and what respective structure is the cause of

such effect.

Statement of the Problem

The study intends to evaluate the active metabolitesphytochemical

constituents of the different plants extracts from selected indigenous plants

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in Mindanao, Philippines. Using Thin Layer Chromatography Method with

different solvents, it aims to answer the following questions:

1. What is are the Retardation Factor (RF) of the major spots?

1.2. What are the phytochemical constituents present?

3. In what solvent does the phytochemical constituent soluble?

2.4. Under Ultraviolet Radiation, are there any spots that are active?

3. Are there any metabolites present in the extracted plants?

4. What are the physical structures of the dried plant extracts under

Scanning Electron Microscope?

5. Are there any enzyme inhibition capabilities present in the plant

samples?

Theoretical Framework

Pharmacologically active constituents in plants are responsible for the

therapeutic activity of the drug. Depending on the particular activity of the

constituent and on the other constituents or ingredients with which it is

associated, certain principles may be placed on one or the other category

(Tyler, et.al). With the advent of chemistry, of modern pharmacotherapy has

depended more on the synthetic drugs, however due to raised safety

concerns and lower drug efficacy there is a growing interest to show direct
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evidence of the crucial role of natural secondary metabolites (Altemini,

et.al., 2015).

Chromatography is a perfect tool for analyzing plants constituents.

High performance liquid chromatography (HPCL) gives the biggest

separation possibilities and additionally can provide on the structure of

separated compounds (Choma, et. al.,2015). With the Scanning Electron

Microscope can derived a meaningful information at high resolution from

the samples that are subjected to treatment (Gileadi, et.al.,2003)

Conceptual Framework

INDEPENDENT VARIABLE DEPENDENT VARIABLE

Plant Samples 1. Retardation factor of

the spots present.
- Costus speciosus
2. Determination of
- Drynaria quercifolia
active spots.
- Eleusine indica
3. Shape and particle
- Euphorbia hirta size of the active
metabolites.
- Ervatamia pandacaqui
4.2. Structure of the
- Ipomea aquatica
functional group.
Formatted: No bullets or numbering
- Mimosa pudica
3. Enzyme inhibiting
capacity

5.
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Type of extracting
mobile phasesolvents:

Polar Solvent

Mid Polar Solvent

Non Polar Solvent

n Fig1. The Conceptual Paradigm of the Study

Figure 1 portrays the relationship of the independent variable to the

type of extracting solvent used, with the dependent variable: retardation of

the spots present, determination of active spots, shape and particle size of

the active metabolites, and structure of the functional group. Commented [FB2]: Revise this part…. Introduce the different
solvents

Significance of the Study

The researchers conducted the study to provide baseline research and

data to the characteristic structures of a select few indigenous plants found

in Davao.

To the Students: Those students who are inclined to science may have a

better outlook of those plants that are regarded as useless in the society.
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They can also help in propagating and preservation of this indigenous plant

from extinction.

To the Society: People that are unaware of those plants as they considered

them as ineffective can have a better understanding to its natural use. They

can also help in preservation of this indigenous plant for future benefit.

To the Researcher: Researchers could provide information about the

indigenous plants with having secondary metabolites that heighten the

components of these plants. This study may be one of the bases for a new

learning theory to arise.

To the Pharmaceutical Field: Since the selected indigenous plants used in

the study are commonly found, results from this study may greatly

contribute for further investigations.

Scope and Delimitation

This study will be limited only to the screening of extracts from seven

indigenous plants that will be collected in different areas in Davao City. The

sample that will be used in the study will be limited to the following

indigenous plants and their parts: leaves of the Spiral Ginger (Costus

barbatus/speciosus), leaves of Pakpak Lawin (Dynaria quercifolia), leaves of

Bila bila/ Paragis (Eleusine indica), Tawa-tawa (Euphorbia hirta) leaves, the
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leaves of Pandakaki (Ervatamia pandacaqui), leaves of Kangkong (Ipomea

aquatic), and roots of (Mismosa pudica).

The study will use Thin Layer Chromatography method to screen out

different constituents from the plant extracts and to identify their purity.

Structural analysis of the extracts of different indigenous plants will

be conducted by the use of Scanning Electron Microscope to identify the

physical structure of the active metabolites in the said plants.

Identification of the physical structure of the plant extracts will be

conducted at the UIC Science Research Center and all the other laboratory

procedures will be conducted at the analytical chemistry laboratory. The

duration of the study will be from second semester of third year college to

first semester of fourth year college.

Definition of Terms

The following terms are clearly defined in order to have a better

understanding of the study.

Thin Layer Chromatography (TLC) – is a chromatographic technique used

to separate the components of mixture using a thin stationary phase

supported by band inert backing.

Extraction – are ways to obtain desired constituents from plant material

with a suitable apparatus and solvent.

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Indigenous Plants – plants that are naturally occurring in Mindanao.

Xanthine oxidase - is an enzyme tthat catalyzes the oxidation of

hypoxanthine to xanthine, and of xanthine to uric acid.

Acetylcholinesterase - is the primary enzyme responsible for the hydrolytic

metabolism of the neurotransmitter acetylcholine into choline and acetate.

β – glucosidase - is an enzyme responsible for the hydrolysis of β-glucosidic

linkages in aryl-, amino-, or alkyl-β-D-glucosidase, cyanogenic glucosides,

and oligo- or disaccharides.

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Chapter 2

REVIEW OF THE RELATED LITERATURE

This chapter discusses the indigenous plants in Mindanao that will be

use in the study as well as their literature reviews for further information

including their histories, descriptions, phytochemistry and taxonomy. This

also discusses the Thin Layer Chromatography Method for detecting

secondary functional group and Scanning Electron Microscope for

analyzing the structure of the active metabolites.

Related Literature

Bioautography

Planar chromatographic analysis hyphenated with the biological

detection method is an effective, inexpensive technique for the

phytochemical analysis of plant extracts to identify bioactive lead/scaffolds.

(Muller, 2004)

Several spectroscopic and chromatographic analytical methods have

been developed for standardization of products from medicinal plants,

which include mass spectrometry (MS), gas chromatography-mass

spectrometry (GC-MS), liquid chromatography (LC), thin layer

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chromatography (TLC), high performance liquid chromatography (HPLC),

and high performance thin layer chromatography (HPTLC) to guarantee

their quality, efficacy, and safety (Valle, Puzon, Cabrera, Rivera, & Valle,

2016).

Enzyme inhibition

Enzymes are important molecular targets for lead discovery in

primary screening assays. The use of a TLC support to screen for potential

plant-derived enzyme inhibitors is a rapid method which is relatively free

of disturbances due to solvent. (Dewanjee S. Et al., 2015)

Diazotization

The basic principle of this method is that the enzyme converts α-

naphthyl acetate (substrate) into α-naphthol. α-Naphthol reacts with fast

blue B salt (chromogenic agent) to make a purple colored background on

the TLC plates, while AchE inhibitors produce white spots. Enzyme

inhibitors block the formation of α-naphthol and hence no purple coloration

is produced. (Dewanjee S. Et al., 2015)

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Paragis plant (Eleusine indica)

http://kbl.org.ph/wp-content/uploads/2017/10/paragis1-300x169.jpg

Figure 1. Paragis

Kingdom: Plantae

Subkingdom: Trachebionta

Division: Magnoliophyta

Class: Liliopsida

Subclass: Commelinidae

Order: Cyperales

Family: Poaceae

Genus: Eleusine

Species: indica

Paragis is an annual, erect, tufted, adventitious, glabrous grass, 10

centimeters to 1 meter in height. Leaves are 10 to 30 centimeters long,

sometimes involute when dry, 3 to 7 millimeters wide, distichous, rather

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flaccid, with flattened sheaths. Spikes are 3 to 6, all in a terminal whorl, or

one or two lower down, 2.5 to 10 centimeters long , 3 to 5 millimeters thick.

Spikelets are very numerous, crowded, 3- to 5-flowered, 3 to4millimeters

long, the first glume 1-nerved and small, the second, 3-nerved, and the third

and succeeding ones ovate, acute. For many non-model species, there is very

little genome information available for researchers to conduct

comprehensive investigations into the genetic mechanisms underlying their

unique features and functions (Jing, 2014).

Constituent’s vitexin and isovitexin has been isolated from Eleusine

indica L. Gaertn plant. The plant Eleusine indica L. Gaertn seems to be a

promising candidate with respect to its diuretic activity and anti-urolithiatic

activity and may be used as adjuvant to dietary therapy and drug treatment

for controlling kidney stones and as a diuretic (An J, 2014)

 16 Formatted: Right: 0.25"

Kang-kong (Ipomoea aquatica)

https://cdn.shopify.com/s/files/1/2231/8055/products/Kang_Kong_c394afb2-be7e-487d-88aa-

8a715f9514e1_1024x1024.png?v=1541096281

Figure 2. Kangkong

Kingdom: Plantae

Subkingdom: Viridiplantae

Division: Tracheophyta

Class: Magnoliopsida

Subclass: Asteranae

Order: Solanales

Family: Convolvulaceae

Genus: Ipomoea

Species: aquatic
 17 Formatted: Right: 0.25"

Swamp morning glory is a sprawling vine, annual or perennial,

creeping on mud or floating on water; stems terete, branched, hollow and

succulent when floating, otherwise solid and firm, up to 3 m long, to 1 cm

in diameter. Leaves emersed, glabrous, alternate; petioles succulent when

grown in water, 3-20 cm long; blades greenish-brown, triangular, ovate,

lanceolate, or linear, entire to dentate, 3-15 cm long, 1-12 cm across, bases

truncate, cordate, hastate, or sagittate, lobes rounded to acute, entire to

dentate. Inflorescences axillary cymes, with one to a few flowers; peduncles

0.5-18 cm long. Flowers perfect, hypogenous, large and showy; pedicels1-

6.5cm long, with minute bracts at base; sepals glabrous, unawned, ovate, the

inner slightly longer than outer, 7-10 mm long; corolla funnel shaped,

glabrous, pink, often with darker eye, sometimes white or cream, 2.5-5.5 cm

long, 2-4 cm wide; stamens included, shorter than corolla, adnate with

petals above the base, filaments hairy at the base, anthers dehiscing

longitudinally; carpels glabrous, locules mostly 2, style included, shorter

than corolla, ovules mostly 4. Fruit a capsule, glabrous, globose to ovoid, 8-

10 mm long; seeds 4 or fewer, brown or black, mostly pubescent, 3-ranked,

rounded on back, about 5mm long, about 4mm wide. The seeds have an

omega-shaped border that surrounds the hilum.

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Swamp morning glory is a very popular leaf vegetable, especially in

Asian markets. It is harvested from the wild and is also commonly

cultivated, especially in Asia. The plant also has traditional medicinal uses.

Various parts of this plant has been used for traditional treatment of

boils, fever, headache, insomia, laxative, piles, swelling, ulcer and wound

(Lakshmi, 2012)

The antibacterial efficacy of the leaf extract IA has been reported against

four pathogenic bacterial strains E coli, P. aeruginosa, S. aureus and M

luteus that indicated the extract has antibacterial property. (Khatun, 2012)

Makahiya (Mimosa pudica)

https://qph.fs.quoracdn.net/main-qimg-d96856bb1976d8c3c8e1c77181dcba9f.webp

Figure 3. Makahiya

Kingdom: Plantae

Subkingdom: Viridiplantae

Division: Tracheophyta

Class: Magnoliopsida
 19 Formatted: Right: 0.25"

Subclass: Rosanae

Order: Fabales

Family: Fabaceae

Genus: Mimosa

Species: pudica

Makahiya is an annual to perennial, more or less prostate creeping

plant. The plant can grow up to 1 meter tall, but is more likely to be 15-45

cm tall, the stems usually becoming woody. The plant is gathered from the

wild for local medicinal use. It is cultivated as a green manure and for soil

stabilization, and is sometimes also cultivated for its uses in folk medicine.

It is commonly grown as an ornamental, being valued especially for

the novelty of its leaves drooping downwards whenever touched. Mimosa

pudica is very widespread in Central America, South America and the

Caribbean. This taxon is known to occur within a number of protected areas

throughout the species range and seeds have been collected and stored by

the Millennium Seed Bank Project as a method of ex situ conservation. It is

common and not considered to be threatened or in decline. The plant is

classified as 'Least Concern' in the IUCN Red List of Threatened

Species(2013).
 20 Formatted: Right: 0.25"

Pandakaki (Ervatamia pandacaqui)

http://www.stuartxchange.org/PandakakiPuti2.jpg

Figure 4. Pandakaki

Kingdom: Plantae

Subkingdom: Tracheobionta

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Asteridae

Order: Gentianales

Family: Apocynaceae

Genus: Ervatamia

Species: pandacaqui
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Pandakaki is an erect, branched and smooth shrub, 1 to 4 meters high.

Leaves are elliptic-lanceolate to oblong-elliptic, 5 to 12 centimeters long,

narrowed at both ends, shining and short-stalked. Inflorescences are axillary

and terminal, peduncled, and have rather few flowers. Calyx is green,

ovoid, and short. Corolla is white, tinged with green, slender-tubed, 1.7

centimeters long and slightly enlarged upward; limb is 2 to 2.5 centimeters

in diameter, composed of five, spreading, falcate, lanceolate lobes. Follicles

are red or yellowish-red, oblong, 2 to 4 centimeters long, and longitudinally

ridged or keeled.

Other genus and species of pandakaki such as Tabernaemontana

catharinensis had several important biological activities such as

antimicrobial, anti-tumoral, antioxidant, anti-cholinesterasic, and anti-

inflammatory. Although several biological activities of T.

catharinensis extracts have been reported, few substances with

anticholinesterase activity able to minimize damage caused by oxidative

stress have been described in T. catharinensis. (I. J. C. Vieira et al., 2008). The

alkaloids present are 16-epi-affinine, coronaridine-hydroxyindolenine,

voachalotine, voacristine-hydroxyindolenine, and 12-methoxy-n-methyl-

 22 Formatted: Right: 0.25"

voachalotine which can be extracted from the aerial parts (leaves and

branches) and also in the root bark of T. catharinensis. (Pereira et al. (2008).

Tawa-tawa (Euphorbia hirta)

https://2.bp.blogspot.com/--ucB6Q_VRbA/WaJ2mWQUUcI/AAAAAAAAACY/hgsatoYg_gYpN-

7DqEAtrXHN25ZHhw2TgCLcBGAs/s1600/chamaesyce_hirta21.jpg

Figure 5. Tawa-tawa

Asthma weed is an annual, branched herb containing latex, prostrate

to ascending, with branches up to 50 cm long. An important medicinal herb,

it is often sold in local markets and through the internet.

Extraction (as the term is pharmaceutically used) is the separation of

medicinally active portions of plant (and animal) tissues using selective

solvents through standard procedures. The products so obtained from

plants are relatively complex mixtures of metabolites, in liquid or semisolid

state or (after removing the solvent) in dry powder form, and are intended

for oral or external use. Extraction methods used pharmaceutically involves

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the separation of medicinally active portions of plant tissues from the

inactive/inert components by using selective solvents. During extraction,

solvents diffuse into the solid plant material and solubilize compounds with

similar polarity (Afolayan N. S. & Ocoh A.L., 2008).

Successful determination of biologically active compounds from

plant material is largely dependent on the type of solvent used in the

extraction procedure. The choice of solvent is influenced by what is

intended with the extract. Since the end product will contain traces of

residual solvent, the solvent should be non-toxic and should not interfere

with the bioassay. The choice will also depend on the targeted compounds

to be extracted (Das, Tiwari & Shrivastava, 2010).

Pakpak-lawin (Dynaria quercifolia)

 24 Formatted: Right: 0.25"

https://encryptedtbn0.gstatic.com/images?q=tbn:ANd9GcRE6xvI18H1eYeCTpVrO_bMbg8Ln1Q1i2vXkJ4

YLwLjsero83mCCg

Figure 6. Pakpak Lawin

Kingdom: Plantae

Subkingdom: Tracheobionta

Division: Pteridophyta

Class: Pteridopsida

Subclass: Polypodiidae

Order: Polypodiales

Family: Polypodiaceae

Genus: Drynaria

Species: quercifolia

Drynaria quercifolia is a robust, epiphytic fern with a long, creeping

rhizome, growing up to 1 metre tall. It produces two types of annual frond

 25 Formatted: Right: 0.25"

- short, sterile ones up to 40cm tall that remain on the plant for several years

and have a main purpose of trapping organic material to provide nutrient

for the plant, and taller fertile shoots that produce spores. The root is

sometimes gathered from the wild for local medicinal use.

Phytochemical screening yielded phenols, tannins, alkaloids,

proteins, xanthoproteins, carboxylic acid, coumarins, and saponins.

Spiral Ginger (Costus barbatus/speciosus)

https://i.etsystatic.com/6728015/r/il/30a274/1109051091/il_570xN.1109051091_tetx.jpg

Figure 7. Spiral Ginger

Kingdom: Plantae

Subkingdom: Tracheobionta

Division: Magnoliophyta
 26 Formatted: Right: 0.25"

Class: Liliopsida

Subclass: Zingiberidae

Order: Zingiberales

Family: Zingiberaceae

Genus: Costus

Species: barbatus

Spiral ginger is a rhizomatous herbaceous plant. Stems are stout,

leafy, up to 2 meters or more in height, and 1.6 centimeters in diameter.

Leaves are spirally arranged, oblong, 15 to 30 centimeters long, with pointed

tip, short stalks and covered with soft hairs on the lower surface. Flowers

occur in very dense, solitary spikes. Spikes are very dense, solitary, terminal,

ovoid, 5 to 10 centimeters long; purple bracts ovate, 2.5 to 4.5 centimeters

long. Calyx is flattened, purple, 3 to 5 centimeters long, with 3 short, ovate

lobes. Corolla segments are white, oblong, 4 to 6 centimeters long. Lip is

white, suborbicular, 6 to 8 centimeters long, crinkled, irregularly and rather

finely toothed, the margins incurved and meeting. Stamen is flat, and

including the broad petaloid connective, is about 5 centimeters long and 12

to 15 centimeters wide. Fruits are capsules, ovoid to rounded, 1.5 to 2

centimeters long, red, crowned by the persistent calyx.

 27 Formatted: Right: 0.25"

Some folkloric claims said that Spiral ginger (Costus barbatus) in Visayas

were juice of the stems used for dysentery. In Ayurveda, rhizome has been

used for diabetes, fevers, asthma, bronchitis, intestinal worms, rashes. Roots

used for catarrhal fevers, coughs, dyspepsia, worms and skin diseases.

(StuartXchange, Apr. 2013)

An alkaloid ext. from Costus speciosus rhizomes had papaverine-like

smooth muscle relaxant, antispasmodic activities in lab. animals (S. K.

Bhattacharya, A. K. Parikh, P. K. Debnath, V. B. Pandey. N, C. Neogy,

Journal of Research in Indian Medicine, 1973, 8(1), 10-19.). Rhizomes are

given in pneumonia, rheumatism, dropsy, urinary diseases, jaundice and

leaves are given in mental disorders. Bruised leaves are applied in fever;

decoction of stem is used in fever and dysentery (The Wealth of India: First

Supplement Series (Raw Materials), National Institute of Science

Communication and Information Resources, CSIR, 2007, Vol 2, 211, 213.).

Related Studies Commented [FB3]: Add more literature, 3 more per plant….
Formatted: Space After: 0 pt

In a study conducted by Brieta, F. et al. (2015) wherein they use

Fourier Transform Infrared Spectroscopy (FTRI), presence of nitrogen,

carbonyl group, hydroxyl groups and aromatic rings are observed in Costus

speciosus, Euphorbia hirta, Ipomoea aquatica and Mimosa pudica. In addition, an

 28 Formatted: Right: 0.25"

orange precipitates which positively indicates the presence of alkaloids are

found in plants Bila-Bila, Kangkong, Makahiya, Pandakaki, Pakpak Lawin,

Spiral ginger, and Tawa-tawa.

In the research of Malalavidhane et al., (2001), Ipomoea aquatica’s

aqueous extract showed its effectiveness in reducing blood sugar level in

rats just like the oral hypoglycemic drug Tolbutamide. Also, the study of

Monvar, et. al. (2013) in Ipomea aquatica plant detected the presence of

carotenoids and flavonoids. It is said that the leaves of Ipomea aquatica

contains xanthophyll, taraxanthin, nicotinic acid and others. Seven aliphatic

pyrrolidine amides with branched and linear saturated C15-C19 acyl

moieties were detected in stem and leaves. Thus, the plant also exhibits

nootropic effect when tested to rat hippocampus. As a result of the

treatment of methanol leaf extract that significantly increases acetylcholine

content in the hippocampus for the rats improved learning.

Mimosa pudica according to (Apaya, K., 2010) exhibited 62.36%

xanthine inhibitory and showed the lowest IC50 of 32.8 μg/mL. The aqueous

extract also produced significant antinociceptive action when orally

administered to mice, using acetic acid induced writhing and thermal-

induced nociception test according to (Karthikeyan M., 2009). It showed that

the extract has high margin of safety because there was no death recorded
 29 Formatted: Right: 0.25"

even at 10 times of the effective dose therefore the LD50 of the extract was

high.

Reducing sugar, alkaloids, flavonoids, sterols, tannins and

triterpenoids was shown in the phytochemical analysis of Euphorbia hirta

(Shih, M., 2008). Quercetin, a type of flavonol, was isolated from the plant

which contains bioactive etities with potential atistress activity using TLC

purification of fractions from hydroalcoholic extract and the confirmation

was done with Spectroscpic results (FTIR, HMR and MASS) (Tiwar, N. Et.

al., 2015). It has been used as a treatment for asthma in folk medicine

(Watanabe et al, 2005). In the study of (Lanhers et al, 1991), it has been said

that E. hirta reduced inflammatory hyperalgia of rheumatoid arthritis while

in Freund’s adjuvant-induced rheumatoid arthritis mdel, it was ineffective.

In the study of El-Far, A. et. al. (2018), detected the presence of

alkaloids, glycosides, steroids, phenolic flavonoids, polyphenols, tannins β-

carotene in plant Costus speciosus. And in another study of Bhattacharya et

al., (1972), it has been reported that the total alkaloids isolated from the

rhizome of Costus specious potentiated the pharmacological actions of

acetylcholine both in-vitro and in-vivo.

The study of Dewanjee et al., 2014, as the basis if plant contains AchE

and will be inhibited by AchE inhibitor will produce white spots in TLC
 30 Formatted: Right: 0.25"

plates. If the plants extracts screened under α and β- glucosidase under TLC-

bioautography an alternative test is used which involves the cleavage of 2-

naphthyl-β-D-glucopyranose or 2-naphthyl-α-D-glucopyranoside by

respective α- or β-naphthol which formed reacts with fast B salt to give a

purple colored diazo dye. For the detection of active enzyme, the solution

of 2-naphthyl-α-D-glucopyranoside (for glucosidase) or 2-napthyl-β-D-

glucopyranoside (for β-glucoside) in ethanol and fast blue Bsalt in distilled

water are prepared. The mixture of naphthyl-glucopyranoside solution and

B salt solution sprayed onto the plate to give a purple coloration within 2-5

min.

In addition, for Xanthine oxidase, the enzymatic oxidation of xanthine

produces 𝑂2 which reduces the pale-yellow tetrazolium salt (NBT) to a

formazan and a purple background is obtained.

The moderate activity inhibition of enzymes should be between 15 to

50% and extracts having less than 15% inhibition will not show significant

inhibition of enzyme (Adersen et al., 2006). And plant extracts having

activities where enzymes percentage of inhibition is 60% or more are

considered to possess strong inhibitory activity (Khan et al., 2006).

 31 Formatted: Right: 0.25"

Chapter 3

METHODOLOGY

This chapters includes all the different procedures involved in

identifying and testing the enzyme inhibition activity of the selected

indigenous plants in Mindanao. This chapter contains research design,

research locale, research instruments and research procedures in identifying

the enzyme inhibition of the selected plants.

Research Design

The researchers will utilize an experimental research design. The Commented [FB4]: TLC?? A scientific laboratory method

researchers will be extracting the dried plant samples by following the

method used by Tiwari, P. et al (2011). The determination of

acetylcholinesterase will be based on detection by diazotization, as well as

β- glucosidase inhibition and xanthine oxidase method used by Dewanjee

et. al (2014).
 32 Formatted: Right: 0.25"

Research Locale Commented [FB5]: Plant source???

The diazotization test for acetylcholinesterase, test for β- glucosidase

inhibition and xanthine oxidase as mention method will be done at

Analytical Chemistry Laboratory as well as the reagents to be used in the

study are to be taken from Chemistry Laboratory. All the afore mentioned

method and solvents to be used are located at the University of the

Immaculate Conception. All the indigenous plants will be collected in any

areas of Davao City.

Research Material and Procedure

Collection and Authentication of Experimental Selected Plants

The selected indigenous plants will be collected within Davao City.

The plant collected with proper handling and will be placed in a black bag.

The plants samples will be preserved for authentication by and will be sent

to the Davao Museum.National Museum for authentication.

Preparation of Selected Indigenous Plants

The freshly collected leaves and parts of plants specified will be

subjected to air drying for 3 days. Two hundred grams (200 g) of the air-

dried plants material will be cut into small pieces and will be placed in an

Erlenmeyer flask and will be macerated in 80% ethanol for forty-eight hours

(48 hours). The plant material will be allowed to be submerged completely

 33 Formatted: Right: 0.25"

in the solvent. After 48 hours, it will be filtered through a Buchner funnel

and will be subjected to a Rotary Evaporation (RotaVap) machine at 40-60

ºC. the extract will be place in an amber bottle and stored in a cold place

until used. Commented [FB6]: Source?

Preparation for Thin Layer Chromatography Screening

TLC using different solvents( polar, mid polar, non polar)

Detection of Alkaloids Commented [FB7]: Use the book of GUEVARA….

Detection of Flavonoids

Detection of acetyl cholinesterase (AchE) by Diazotization Commented [FB8]: Source?

The samples were applied to silica gel TLC plates and migrated in a

suitable solvent. After the complete removal of solvent, the AchE solution Commented [FB9]: specify

is sprayed. Allow the incubation of enzyme for 20 min. at 37 ºC and them

spray the mixture of 1 naphthyl acetate and fast blue salt. After 1-2 min.

AchE inhibitors show white spots on a purple background.

Detection of β-glucosidase Inhibitor Commented [FB10]: source??

The samples were applied to silica gel TLC plates and migrated in a

suitable solvent. Using 2-napthyl-β-D-glucopyranose in ethanol and fast

 34 Formatted: Right: 0.25"

blue Bsalts in distilled water having ratio of 1:4 of the mixture. The mixture Commented [FB11]: ???

is sprayed onto the plate to give purple coloration within 2-5 min.

Detection of Xanthine Oxidase

The enzyme is suspended in agar and distributed on the TLC plate

(for direct measurement of enzyme activity on the TLC plate is not possible).

After solidification, the plate is immersed into a solution of xanthine at 38ºC

for 20 min in the dark. A purple background will be obtained on the plate

and hence the enzymatic oxidation of xanthine produces O2 which reduces

the pale-yellow tetrazolium salt (NBT) to a formazan.

References: