1

CHAPTER 1

INTRODUCTION

1.1 Background of Study

Nowadays, synthetic drugs used to treat various pathogenic fungi diseases

were readily available in pharmacy but many infectious diseases were

proven to be treated with herbal remedies in mankind history, so natural

product were said to provide unlimited opportunities in production of new

drugs (Parekh, 2007). Besides that, those synthetic drugs develop negative

side effects to human when it taken for long period also high cost incurred

in the development of the patentable synthetic drugs (Oladumoye, 2006).

Therefore there is an urgent need to discover new antifungal compounds

within plant with best solution to inhibit pathogenic fungi.

In plants, phytochemical compounds or secondary metabolites synthesized

in specific part or from all parts of the plant have beneficial medicinal

effects (Parekh, 2007). The words phyto in phytochemical means plant in

Greek was a chemical compound with double bond in their structure, so it

means that phytochemical was plant naturally active compound. The

primary function of phytochemical was to protect plants from parasitic

disease that brings damage to the plant. Besides that, it also source of

2

color, aroma and flavor for specific parts of the plant. Moreover,

phytochemical also useful in plant protection over environmental hazard

such as pollution, UV exposure and stress drought (Mamta, 2013). Mamta

(2013) claim that, in their research, phytochemical was beneficial to

human health as it control pathogenic attack of microorganism, when it

taking up orally in correct amount, termed as dietary phytochemical. This

dietary phytochemicals were usually found in green vegetables as well as

berries fruits such as cabbage, broccoli, strawberry and raspberries.

Casuarina equisetifolia L. was one of 17 plant species in genus Casuarina.

This evergreen tree originates from Australia and extends to islands of the

Pacific to South-East Asia (Swamy, 2013). The family of this plant

Casuarinacea, has widespread distribution as seashore tree and was well

known as Ironwood by Australians native. Others common name for this

plant include Beefwood, Bull-oak, Whistling-pine and Pokok rhu

(Ogunwande, 2011). These plants were usually planted near seashore and

functions as wind break (Ogunwande, 2011).

It was deciduous dicot angiosperm tree that superficially resembles a

conifer and does not fall in pine tree category (Pinyopusarerk, 1993). Al-

Snafi, (2015) claim that, C. equisetifolia flowers were tiny, light brown in

colour and divides to two types, male and female. The location of male

flower was at the tips of the leaf twigs, while location for female flower

was on branchlets below the leaf twigs. This plant has fruit that were

pinecone-like, brown, hard and warty. Besides that, their leaves were tiny

3

and scale like in whorls of 6 to 8 around the green with pine needle-like

leaf twigs with branches was slender and drooping. The tree said to have

wispy appearance with its bark was rough, gray brown and flanking while

its roots was hard and beefy red brown colour (Al-Snafi, 2015).

1.2 Problem Statement

Malaysia was a well kown countries riches in flora and fauna with

miscellaneous medicinal potential. Previous study in C. equisetifolia plants

reveals the presence of catechin, gallic acid, ellagic acid and lupeol

isolated form the plant which were antioxidants and also display

antimicrobial properties (Ogunwande, 2011). Besides that, the bark of this

tree had a role as traditional medicine and was widely used to treat

astringent, diarrhea, cough, ulcers and toothache (Nehad, 2012).

Candida albicans is a combination of genus Candida from Latin words

toga candida which means white robe worn by Roman senate candidates.

While the words albicans also from Latin words albicare, meaning “to

whiten” (Lynch, 1994). C. albicans were fungal yeast, that can causes two

major types of infections in human, superficial infection susch as oral or

vaginal candidiasis and life-threathening systemic function (Mayer, 2013).

Candidiasis is pathogenic to human being, as it attacks human with low

immunity system and cause serious infection (Lynch, 1994). Meanwhile,

A. niger was a fungi which causes Invasive Aspergillosis (AI) that spread

4

in air also causes serius symptoms for person with low immunity system

(Bobbarala, 2009). So the primary health care was to find better cultural

acceptability, better compatibility with human body and fewer side effect

(Parekh, 2007).

The root of C. equisetifolia plant were rarely studies for their antifungal

activity against pathogenic fungi such as C. albicans and A. niger as well

as the information about phytochemical constituents present in the root

part was currently lacking. Besides that, substance that can inhibit the

growh of pathogens or kill them also have no or lest toxicity to host cell

were considered candidates for developmnet of new antimicrobial drugs

(Ahmad, 2001). In this study, phytochemical constituents of the root part

of C. equisetifolia were screening and the antifungal activity of the root

were evaluate to see which pathogenic fungi inhibited by C. equisetifolia

root extract.

1.3 Significance of the Study

Natural products were widely invented nowadays as it has various

functions and usefulness in biological activities. About 1,200 species of

higher plants were found in peninsular Malaysia and 2,000 species found

in Sabah and Sarawak were reported to have medicinal values and have

been used for generations as traditional remedies (Philip, 2009). To

developed new drugs researcher must have knowledge about

5

phytochemical constituents in the plants. This experiment conducted to

study the phytochemical constituents in C. equisetifolia plants for the

presence of flavonoid, tannin, saponin, alkaloid and terpenoid to inhibit the

growth of pathogenic yeast fungal which were C. albicans that causes

Candidiasis and A. niger which caused Invasive Aspergillosis. Besides

that, this experiment also carried out to measure the retention factor value

for the presence of secondary metabolites using Thin Layer

Chromatography (TLC). The higher the properties of secondary

metabolites, the further separation of its retention factor, Rf value.

1.4 Objectives of the Study

The objectives of the study are:

1. To identify the presence of secondary metabolites in C. equisetifolia

root extract by using phytochemical screening.

2. To determine Rf value for each spot of secondary metabolites present

in C. equisetifolia root extract using Thin Layer Chromatography

(TLC).

3. To detect and evaluate inhibition of C. equisetifolia crude extract

towards cultured fungi, C. albicans and A. niger using disc diffusion

method and well diffusion method.

6

CHAPTER 2

LITERATURE REVIEW

2.1 Casuarina equisetifolia

C. equisetifolia was known by locals in Malaysia country as Pokok Rhu.

The valuable compound of this plant were its active primary and secondary

metabolites includes carbohydrates, alkaloids, protein, glycosides,

saponins, phenolic, flavonoid, tannins, steroids, gum, reducing sugar and

triterpenoids that were importance in pharmacological activities including

antimicrobial, antidiabetic and antioxidant (Al-Snafi, 2015). Other

function of the metabolites includes helping in giving flavor to food, as a

fragrance and food additives (Al-Snafi, 2015). Example of secondary

metaolites or phytochemical was bioactive flavonoid, efficient to trap or

scavenge free radicals and was an indigenous origin (Iqbal, 2015).

Phytochemicals present in different parts of the plants, such as in the roots,

stems, leaves, flowers, fruits and seeds. Function of phytochemicals were

to protect themselves as well as human against disease (Mamta, 2013).

Moreover, phenolic compounds presence in branchlets and bark of the

tree showned significant value in biological properties such as antioxidant

activity, antimicrobial effect, and modulation of detoxification enzymes

7

(Zhang, 2010). According to Hosses (2014), this plant phloem contains

many active metabolites rather than other parts of the plants and was

widely used in pharmaceutical as natural products. Thus this experiments

was conducted to determine the inhibition of fungi with C. equisetifolia

root extracts as a result of secondary metabolites present in roots of the

plants, and to determine its antifungal activities, whether it as effective as

the leaf and bark extracts.

Research by Hosses, (2014) indicated that the phenolic compound found in

leaves of C. equisetifolia have an antioxidant activity. Polyphenolic

compounds, like flavonoids, tannins and phenolic acids have the

antioxidant activity, so this plant can be made into antioxidant drugs. An

antioxidant in when present at low concentrations significantly prevents

oxidation of cell content like protein, lipid, carbohydrates and DNA.

Complex diseases like atherosclerosis, stroke, Alzheimer’s disease and

cancer can be prevent with the use of antioxidant-based drugs as treatment.

Moreover, C. equisetifolia was a well known as folk medicine to treat

diarrhea, cough, ulcers, toothache and diabetes. The parts of the plant such

as its bark, contains astringent and antioxidant properties importance as

remedy for diarrhea, dysentry, headache and fever cough (Swamy, 2013).

8

Figure 2.1 Casuarina equisetifolia species

2.2 Extraction of roots

The solvent used to extract secondary metabolites was an organic solvent

which were light petroleum ether (less polar), chloroform (medium polar)

and methanol (polar). Phenolic compound such as tannin and flavonoid

becomes importance as they have value in medicinal field. Technique used

to extract phenolic compounds such as secondary metabolite is solvent

extraction. It isolates plants antioxidant compounds from mixture of plant

crude extract with identified solvent (Sultana, 2009).

9

2.3 Phytochemical screening

The secondary metabolites, alkaloid, flavonoid, tannin, terpenois and

saponin were found and isolated from plants that have been studied

showing the compounds have value in treatment of anticancer,

antibacterial, analgesic, anti-inflammatory, antitumor, and antiviral. The

secondary metabolites can be tested with a crude methanol extract of bark

and leaves. Extraction of roots can also be used. The indicator to determine

qualitative result is expressed as (+) for phytochemical presence and (-) for

the absence (Iqbal, 2015).

2.3.1 Alkaloids

Alkaloid build of heterocyclic nitrogen atoms and its name derived

from “alkaline” meaning substance of nitrogen-contain base. It can

be synthesized naturally by large numbers of organism whether

animals, plants, bacteria and fungi. It also used as local anesthetic

and stimulant as cocaine. Its acts as inhibitors for microorganism

and reduce the risk of fungal infection in antibacterial and

antifungal activities (Mamta, 2013).

10

2.3.2 Flavonoid

It was a polyphenolic compounds mainly found in nature. The main

source of flavonoid were in vegetables and fruits. It have been use

during ancient times as treatment to certain disease. Its function

include oxygen free radical quenching and lipid peroxidation

inhibitor. It also valueable to used as antioxidant, antimicrobial and

anti-inflammatory as it inhibit tumor and development of lung

cancer. Besides that, it often used as anti-metastatic activity in

anticancer activity (Mamta, 2013).

2.3.3 Terpenoid

Terpenoid was natural product derived from five-carbon isoprene

units. They were multi cyclic structure that differ in functional

groups and basic of carbon skeletons between each other. It was

natural lipids that can be found in every class of organism and

largest group of natural product. It also acts as inhibitors for

microorganisms and reduce the risk of fungal infection. In

additions it act as neuropharmacological agents, anti-oxidants, and

cancer chemoprevention (Mamta,, 2013).

11

2.3.4 Tannin

Tannins were categorizing under heterogeneous group of high

molecular weight polyphenolic compounds. It has the ability to

form reversible and irreversible complexes with proteins and

polysaccharides. It was mostly found in fruits such as grapes,

persimmon and blueberry, and sometimes in chocolate. Research of

tannins was increased as biological action of tannin-containing

plant extract could inhibit deadly illness such as AIDS and various

cancers (Mamta, 2013).

2.3.5 Saponin

Saponin usually forms stable aqueous solution form such as soap.

This group includes glycosylated steroids, triterpenoids and steroid

alkaloids compound. Saponins function as protection from insect

attack in plant. Meanwhile, anti-carcinogenic properties of saponin

affect growth, food intake and reproduction in animals. It also

contains antioxidant properties (Mamta, 2013).

12

2.4 Candida albicans

Genus Candida were characteristically differentiated to have white

asporogenous yeast that capable of forming pseudohyphae and it was

member of class deuteromycetes known as “taxonomic pit” which

classified as yeast without known sexual stage or remarkable phenotypic

character (McCullough, 1996). Further, C. albicans was dimorphic yeast

that can switches from unicellular budding yeast to mycelial true hyphae

and believe to be an obligate associate of warm-blooded animal having no

sexual cycle (Candiracci, 2011). The virulence factors of C. albicans

which have been studied were cell wall of fungi and its adhesion ability.

The cell wall consists of polysaccharide, mannan and chitin. The outer

fibrillary layer of the cell wall consists of mannan and mannoprotein was a

mucous coat or capsule that will shed during infection (McCullough,

1996). Besides that, successful infection of host tissue by pathogenic

Candida depends upon its ability to adhere mucosal surface of host cell

(Gupta, 2008).

Diseases called Candidiasis was a fungal infection caused by yeasts that

belong to the genus Candida and commensal in human oral cavity as well

as vaginal mucosa. Because of pathogenic characteristics of the fungi, it

was capable to eliminate less adapted microorganism. Example was when

bacteria and fungi compete for limited nutrient in mucosal surface

(McCullough, 1996). Besides that, the host contributes at specific site or

tissue allows fungi to growth and adapts range of physiological extremes

13

like pH. This dimorphic yeast can invade host by immune defects, and use

of ill-fitting denture (Calderone, 2001).There were three types of

Candidiasis that infect humans, Esophageal Candidiasis, Vulvovaginal

Candidiasis and Invasive Candidiasis. Candidiasis symptoms develop due

to overgrowth of the fungi as well as its virulence factor (Gupta, 1970).

The main symptom that causes Candidiasis was weakened immunity

system. Symptoms such as white patches or plaques on tongue often

showed in people with Esophageal Candidiasis and this fungus usually

present along gastrointestinal tract (GI) and often affect tongue

(McCullough, 1996). Meanwhile diabetes, long-term use of broad

spectrum antibiotics and use of corticosteroid were symptoms showed by

people with Vulvovaginal Candidiasis. Besides that, people with very low-

birth weight infants, surgical patients, and patients with a central venous

catheter are exposed to Invasive Candidiasis (McCullough, 1996).

Treatment for Candidiasis was the used of amphotericin B or fluconazole

drugs. But this drug have negative side effect when it is taken for a long

duration (Candiracci, 2012) However, the problems can be solved when

natural product were invented to treat the disease as well as to avoid the

negative side effect. According to Al-Snafi (2015) fruit extraction obtain

from C. equisetifolia resulted in good antifungal activity against

pathogenic fungi such as C. albicans. While Ahmad (2001) said that plant

C. equisetifolia have broad spectrum of antibacterial activity as it inhibit

drug-resistant strain. According to Candiracci (2012), the flavonoid

14

present in honey flavonoid extract has the ability to inhibit pathogenicity

of C. albicans.

2.5 Aspergillus niger

A. niger was filamentous fungi identified to be one causative agent to

Invasive Aspergillosis (IA) (Sumathy, 2014). This fungal colony consist of

a compact white or yellow basal felt covered by a dense layer of dark

brown to black conidial heads. It was conidiophore which has smooth

walled, hyaline or turning dark towards the vesicle (Bobbarala, 2009). The

spore from Aspergillus species was responsible agents for the development

of Aspergillosis disease where it leads to reducing survival rate of human

population. It was because A. niger produce small hydrophobic conidia

that disperse easily into the air and survive broad range of environmental

conditions (Bansod, 2008).

Patients with prolong neutropenia, hematological malignancies, receiving

high dose corticosteroids therapy, underwent stem cell and solid organ

transplantation or with advanced HIV are becoming common victim of IA

as spores from the atmosphere will enter the lung and forms tangled mass

of fungus fibers. The fungal mass enlarge gradually and destroys the lung

tissue in the process, but usually does not spread to other areas unless

under certain circumstances (Bobbarala, 2009). The used of synthetic

drugs such as Amphotericin B to inhibit the fungal growth leads to

negative consumer reaction due to different ecological and medical

problems (Sumathy, 2014).

15

CHAPTER 3

METHODOLOGY

3.1 Materials

3.1.1 Raw materials

The raw material used was C. equisetifolia roots.

3.1.2 Chemicals

The list of chemicals used were nutrient broth media, distilled

water, light petroleum ether, chloroform, methanol,

dichloromethane, concentrated sodium hydroxide (NaOH), iron

(III) chloride (FeCl3), concentrated hydrochloric acid (HCL), potato

dextrose agar, and Wagner’s reagent.

16

3.1.3 Apparatus

The list of apparatus used were autoclave sterilizer, beaker,

capillary tube, centrifuge, conical and round bottom flask, cuvette,

electronic balance, filter funnel, filter paper, graduated cylinder,

incubator shaker, measuring cylinder, micropipette, micropipette

tips, petri dish, rotary evaporator, spectrophotometer, thin layer

silica gel paper (TLC plate), test tube, and UV light detector.

3.2 Methods

As shown in Figure 3.1 in appendix B methods used in this study were

collections of plant material, crude extraction, phytochemical screening, to

detect presence of alkaloid, flavonoid, tannins, terpenoids and saponin and

phytochemical analysis using TLC. Besides that, other methods were disc

diffusion method and well diffusion method used to determine antifungal

activity.

3.2.1 Collection of plant material

The fresh parts of C. equisetifolia roots were collected around

UiTM Pahang Campus, Jengka branch and the experiment was

conducted in Microbiology Laboratory 4. The fresh roots were dust

off from dirt and cut into fine pieces. The roots were let too dry on

17

room temperature until constant mass was obtained. The dried

roots were ground well into fine powder using mechanical grinder.

3.2.2. Extraction of plant material

500 g of C. equisetifolia root powder was then soaked in 1500 ml

light petroleum ether solvent for 1 day at room temperature. The

solvent-containing extracts then decanted and filtered using

Whatman No.1 filter paper. The extractions of the powder were

proceeding with chloroform and methanol orderly (Philip, 2009).

Each filtrate was concentrated under reduced pressure on rotary

evaporator till dried.

3.2.3 Phytochemical screening

The extract was analyses for the presence of phenolic compounds,

flavonoid, tannin, saponin, and alkaloid.

3.2.3.1 Test for flavonoid

Presence of flavonoid was determine using alkaline reagent test.

The extract was added with few drops of sodium hydroxide to give

intense yellow color. The presence of flavonoid detected by the

disappearance of yellow color after addition of dilute hydrochloric

acid (Khanam, 2014).

18

3.2.3.2 Test for tannin

Presence of tannin was determined using ferric chloride test. The

0.5 g extract was stirred with 10 ml distilled water and then filtered.

About 2 or 3 drops of 5 % of ferric chloride was added in test tube.

Presence of tannin was detected by the formation of bluish black

color or precipitate and taken as positive result (Iqbal, 2015).

3.2.3.3 Test for saponin

Presence of saponins was determined using Froth test. About 50

mg of extract was diluted with distilled water and made up to 20 ml

solution. Graduated cylinder was used for the suspension to be

shaken in 15 minutes. Presence of saponins detected by the

development of 2 cm layer of foam (Khanam, 2014).

3.2.3.4 Test for alkaloid

Presence of alkaloid was determined using Wagner’s test. A 6 ml

of dilute hydrochloric acid (HCl) was stirred with 15 mg of extracts

on a water bath for 5 min and filtered (Iqbal, 2015). About 1 or 2

drops of Wagner’s reagent was added. Presence of alkaloid was

detected by the formation of reddish-brown colour precipitate

(Khanam, 2014).

19

3.2.3.5 Test for terpenoid

Presence of terpenoids was determined using Salkowski’s test. The

100 mg crude extract was pour to 2 ml chloroform and shake,

followed by addition of 2 ml concentrated sulphuric acid (H2SO4)

along the side of the test tube to form a layer (Iqbal, 2015).

Presence of terpenoids was detected by the formation of reddish

brown colour at the interface (Khanam, 2014).

3.2.4 Thin Layer Chromatography (TLC) analysis

The TLC analysis was conducted to detect the presence of

secondary metabolites such as flavonoid, tannin, saponin, alkaloid

and terpenoid on 3.0 x 10 cm aluminum plates coated over silica

gel. The plant extract about 10 µl was applied on TLC sheet at

equal distance using capillary tube (Khan, 2010). The extracts

loaded sheets were dipped in watch glass to a depth of 1.0 cm from

bottom and allowed to cover the solvent over the top (Shafi, 2012).

The watch glass contains prepared solvent systems of different

polarities and TLC studies were carried out to select which solvent

system capable of showing better resolution (Sanjay, 2013). The

following solvent systems were tested to obtain the best separation

of crude extract plant, chloroform: methanol (9:1) ethyl acetate:

hexane: methanol (1:8:1) petroleum ether: chloroform: methanol:

20

distilled water (5:3:1:1) and petroleum ether: dichloromethane (2:8)

(Khan, 2010).

The develop TLC plates were air dried and observed under ultra

violet light (UV) at both 254 nm and 366 nm wavelength and

sprayed with various reagent later. The retention factor (Rf) value

determined by visualizing the movement of spots on plates and it is

calculated by using formula (Sanjay, 2013).

Rf = Distance travelled by compound

Distance travelled by solvent

(Rf - Retention factor)

3.2.5 Antifungal study

C. equisetifolia antifungal activities were investigated against two

(2) fungi species, C. albicans and A. niger. The pathogenic fungi

was obtained from Microbiology Laboratory, Universiti Teknologi

MARA, Campus Jengka, Pahang, Malaysia. The fungus was

maintained on potato dextrose agar (PDA) at 28 0C.

3.2.6 Inoculation of microorganism

C. albicans were pre-cultured onto nutrient broth in a rotary shaker

at 37 0C for 1 hour. Then, it was centrifuged at 60,000 rpm for 10

min and the pellet was suspended in saline water (Swamy, 2013).

21

Optical densities of incubated fungi were measured using a UV

spectrophotomer at 630 nm wavelength (Khanam, 2014).

Meanwhile A. niger was prepared from 3 to 5 days old culture

grown on PDA medium. A. niger was scrapped using sterile

spatula onto petri dishes contained PDA medium.

3.2.7 Sample preparation

Extracts for antifungal activities was tested at various

concentration. The roots extracts of C. equisetifolia from petroleum

ether, chloroform and methanol were weighed and dissolved in the

solvent itself to prepare stock solutions of 500.00 µg/µl

concentration. The same stock solution has been utilised to get

desired concentrations of 400.00 µg/µl, 200.00 µg/µl, 100.00

µg/µl and 50.00 µg/µl by the serial dilutions method using equation

(Koshy, 2009),

C1V1= C 2V2,

where C = concentration and V = volume.

3.2.8 Disc diffusion method for C. albicans

Antifungal activity of C. albicans with light petroleum ether,

chloroform, and methanol extracts of C. equisetifolia roots were

screened by using disc diffusion method using food-poisoned

technique (Khanam, 2014). The potato dextrose agar was

22

inoculated with C. albicans culture (1 day old) by point

inoculation. A filter paper disk which is 6 mm in diameter was

soaked with the extract and placed on an agar plate that has been

previously inoculated with a test organism (Tortora, 2014). Blank

disc was impregnated with petroleum ether, chloroform and

methanol followed by drying off was used as negative control. The

activity was determined after 24 hour of incubation at 28 0C. The

diameter of the inhibition zones were measured (Swamy, 2013).

Results of the disc diffusion were measure and expresses in terms

of zone of inhibition of the fungi growing around each disc in

millimeters as low activity (1–6 mm), moderate activity (7–10

mm), high activity (11–15 mm), very high activity (16–20 mm),

and no activity (-) ( Koshy, 2009).

3.2.9 Agar Well Diffusion method of A. niger

Antifungal activity of A. niger with light petroleum ether,

chloroform, and methanol extracts of C. equisetifolia roots were

screened by using agar well diffusion method with sterile cork

borer of size 0.8 mm. The fungi had been culture for 4 days old that

grown on potato dextrose agar (PDA) and were used for

inoculation of fungal strain on PDA plates. 100 µl of the petroleum

ether extract was introduced into the well, besides another 100 µl of

23

chloroform and methanol extracts of C. equisetifolia also

introduced serially in the agar plate (Bansod, 2008)

Incubation period of 24-48 hours at 28 0C was maintained for

observation of antifungal activity of plant extracts. The antifungal

activity was evaluated by measuring zones of inhibition of

founding the fungal growth surrounding the plant extracts. The

complete antifungal analysis was carried out under strict aseptic

conditions. The zones of inhibition were measured with antibiotic

zone scale in mm and the experiment was carried out in triplicates.

(Swamy, 2013).

3.3.0 Statistical analysis

Three samples of each plant extract were assayed. Each sample was

analyzed individually in triplicate and data were reported as means

(n = 3x3x3) ± SD (n = 3x3x3). Data was analyzed using a one-way

analysis of variance (ANOVA) using SPSS. A probability value of

p ≤ 0.05 was considered to denote a statistically difference (Sultana

2009).

24

CHAPTER 4

RESULTS AND DISCUSSIONS

4.1 Phytochemical screening

The phytochemical screening of petroleum ether, chloroform and methanol

crude extracts of C. equisetifolia roots revealed the presence of important

secondary metabolites such as alkaloid, flavonoid, tannin, terpenoid and

saponin as indicated in Table 4.1. Alkaloids were showed in all extract

indicated by formation of reddish brown colour while flavonoid shows in

chloroform and methanol extract and absence only in petroleum ether

extract. The positive observations of flavonoid indicate by the

disappearance of yellow colour when tested. Besides that, the presence of

tannins was observed by formation of bluish black colour or precipitate

that appears in the test tube and only methanol extract was positive. Lastly,

the presence of saponin was observed by development of 2 cm foam layer

and only chloroform extract was negative.

The phytochemical compounds detected, known to have medicinal

importance. According to Mamta (2013), alkaloid having wide

pharmacological activities including antihypertensive effects, antimalarial

activity and antiarrhythmic effect and mostly found in quinine. Flavonoid

25

according to research of Candiracci (2012), are one of the phenolic groups

that known as defense compounds. It exhibit several properties beneficial

to human and have antioxidant properties that function as protection agents

against free radical-mediated disease processes and reactive oxygen

species (ROS). It also acts as protection of biological system against the

harmful effects of oxidative processes on macromolecules such as lipid,

proteins, carbohydrates and DNA (Gupta, 2008). Besides that, tannin

compound present in the extract could be used as astringents or treatment

for diarrhea, as well as diuretics, against stomach and duodenal tumors. It

also functions as anti-inflammatory, antiseptic, and antioxidant (Koshy,

2009).

Similarly, terpenoids compound were known as anti-carcinogen,

antimalarial and anti-ulcers as well as antimicrobial agent. Its act as

inhibitor of micro-organism and reduces the risk of fungal infection

(Mamta, 2013). Other phytochemical, saponin, also known as

antimicrobial that can kill protozoan, as antioxidant that can impair the

digestion of protein and help in vitamins and minerals uptake in the gut. It

also acts as antifungal and antiviral (Mamta, 2013).

Comparison of the efficiency of the three extract indicate that all extract

inhibit C. albicans but methanol extracts show high efficiency of

inhibition. The secondary metabolites screening and identified in root

extract of C. equisetifolia may be responsible for the biological activities

26

shown by C. albicans and A. niger and the reason they could be

replacement of Ampotericin B, drugs that often used to inhibit fungi.

Table 4.1: Phytochemical screening of C. equisetifolia root extracts.

Phytochemicals

and Test

Positive

Observation

Petroleum

ether

Chloroform Methanol

Alkaloids

Wagner’s Test

Formation of

reddish brown

colour

+ + +

Flavonoids

Alkaline

Reagent Test

Disappearance of

yellow colour

_ + +

Tannins

Ferric Chloride

Test

Formation of

bluish black

colour or

precipitate

_ _ +

Terpenoides

Salkowski Test

Formation of

reddish brown

colour at interface

+ + _

Saponins

Froth Test

Development of 2

cm layer of foam

+ _ +

Key: + = present, _

= absent.

27

(a) (b) (c) (d) (e)

Figure 4.1: Phytochemical screening of secondary metabolites in C. equisetifolia root extract with petroleum ether.

(a) Alkaloid; (b) Flavonoid ; (c) Tannin; (d) Terpenoid and (e) Saponin.

28

(a) (b) (c) (d) (e)

Figure 4.2: Phytochemical screening of secondary metabolites in C. equisetifolia root extract with chloroform.

(a) Alkaloid; (b) Flavonoid; (c) Tannin; (d) Terpenoids and (e) Saponin

29

(a) (b) (c) (d) (e)

Figure 4.3: Phytochemical screening of secondary metabolites in C. equisetifolia root extract with methanol.

(a) Alkaloid; (b) Flavonoid; (c) Tannin; (d) Terpenoids and (e) Saponin.

30

4.2 Thin Layer Chromatography

Four (4) types of solvent systems has been tested to detect the best

separation of solvent extract, the solvent system of chloroform: methanol

(9:1) and petroleum ether: dichloromethane (1:4) exhibited higher spot

separation of extract in TLC compared to other. In Table 4.2 C.

equisetifolia produced 6 compounds in petroleum ether extract with

petroleum ether: dichloromethane (1:4) solvent system. Compound PE 6

showed highest Rf value of 1.00 and the PE 1 compound showed the least

Rf value of 0.13. Meanwhile in Table 4.3, chloroform extract with

chloroform: methanol (9:1) solvent system resulted, 5 compounds were

obtained with highest Rf value of 0.89 for CF 5. Chloroform extract show

three spots under visible light which has Rf value 0.63, 0.71 and 0.88,

while under UV254 all compounds spots separation showing black colour.

Besides that, 2 compounds present at UV 266 which Rf value were 0.53 and

0.78, and show fluorescent colour.

Lastly, methanol solvent extract reveals 6 compounds when solvent system

chloroform: methanol (9:1) was used as indicated in Table 4.4. The highest

Rf value for this solvent is 0.43 at compound ME 6. Besides that, when

tested under visible light, petroleum ether extracts does not show

separation, however, when tested under UV254, black spot separation is

detected, further experiments under UV266 reveal florescent colour at Rf

value 0.32 and 1.00.

31

Table 4.2 TLC analysis for petroleum ether extract

Solvent

system

Compound Rf value Visible light

colour

UV 254

colour

UV266

colour

PE:DCM

(1:4)

PE 1 0.13 - black -

PE 2 0.31 - black fluorescent

PE 3 0.41 - black -

PE 4 0.49 - black -

PE 5 0.56 - black -

PE 6 1.00 - black fluorescent

Note: PE- petroleum ether and DCM- dichloromethane.

32

Table 4.3 TLC analysis for chloroform extract

Solvent

system

Compound Rf value Visible light UV 254 colour UV266 colour

CF:ME

(9:1)

CF 1 0.55 - black fluorescent

CF 2 0.64 brown black -

CF3 0.71 brown black -

CF 4 0.79 - black fluorescent

CF 5 0.89 yellow black -

Note: CF - chloroform and ME - methanol

33

Table 4.4 TLC analysis for methanol extract

Solvent

system

Compound Rf value Visible light

colour

UV 254

colour

UV266

colour

CF:ME

(9:1)

ME 1 0.06 brown black -

ME 2 0.13 brown black -

ME 3 0.15 - black -

ME 4 0.20 - black -

ME 5 0.25 - black -

ME 6 0.43 - black -

Note: CF - chloroform and ME - methanol

34

FI (a) (b) (c)

Figure 4.4: TLC analysis of petroleum ether extract with solvent system petroleum ether: dichloromethane (1:4)

(a) Visible light; (b) UV254; and (c) UV266.

PE1

PE2

PE3

PE4

PE5

PE6

PE6

PE5

PE4

PE3

PE2

PE1

PE6

PE5

PE4

PE3

PE2

PE1

35

(a) (b) (c)

Figure 4.5: TLC analysis of chloroform extract with solvent system chloroform: methanol (9:1)

(a) visible light; (b) UV254; and (c) UV266.

CF1

CF2

CF3

CF5

CF4CF 2

CF 1

CF 5

CF 4

CF 3

CF 5 CF 4 CF 3 CF 2

CF 1

36

(a) (b) (c)

Figure 4.6: TLC analysis of methanol extract with solvent system chloroform: methanol (9:1)

(a) visible light; (b) UV254; and (c) UV266.

ME 6

ME 3ME 4

ME 5

ME 2

ME 1

ME 6

ME 6

ME 5

ME 5ME 4

ME 4ME 3

ME 3ME 1

ME 1

ME 2ME 2

37

4.3 Antifungal activity

Results obtained in the present study revealed that the tested material

extracts possess flavonoid, a compound that can inhibit C. albicans and

A.niger growth. Three replica of each plate were done. When tested by

disk diffusion method, the petroleum ether, chloroform and methanol root

extracts showed positive activity against C. albicans with zone of

inhibition present in all extracts. However, when tested with well diffusion

method for A. niger, there is no zone of inhibition present for all extracts,

although the preliminary phytochemical analysis revealed the presence of

saponin, flavonoid and tannin that can inhibit A. niger. Methanol extract

show slightly high antifungal activity compared to petroleum ether and

chloroform. The highest diameter of zone of inhibition (mm) was at 500

µg/µl of methanol extract with diameter 15.7 mm while the lowest was 5.7

mm against C.albicans while in A. niger it showed negative effect (0 mm).

It is quite possible that some root parts of the plant were ineffective in this

study because the concentration of the antifungal properties in the plant is

not sufficient so as to be effective. It is also possible that the active

chemical constituents were not soluble in petroleum ether, chloroform and

methanol.

38

Table 4.5: Antifungal activity of C. equisetifolia root extracts.

Microorganism Plant

parts

Petroleum ether

extracts

Chloroform

extract

Methanol

extract

C. albicans Roots + + +

A.niger Roots - - -

+ = present, - = absent.

39

Table 4.6: Antifungal activity of C. equisetifolia root extracts at various

concentration against C. albicans and A. niger .

Solvent extract Concentration

(µg /µl)

Zone of inhibition, ZI (mm)

C. albicans A. niger

PE extract 50

100

200

400

500

Negative control

5.0 ± 2.6

8.0 ± 1.0

9.0 ± 1.0

10.7 ± 0.5

12.7 ± 2.1

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

CF extract 50

100

200

400

500

Negative control

6.3 ± 5.7

10.0 ± 2.0

11.0 ± 2.6

12.3 ± 2.1

14.3 ± 0.6

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

Methanol extract 50

100

200

400

500

Negative control

5.7 ± 4.9

10.3 ± 3.2

13.3 ± 1.5

15.0 ± 1.7

15.7 ± 0.6

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

Inhibition zone are the mean including disc diameter (0.6 mm)

ZI were expressed as mean standard deviation of three replicates. Low activity (1-

6 mm), moderate activity (7-10 mm), high activity (11- 15 mm), very high activity

(16-20 mm), and no activity (-).The negative control for petroleum ether is

petroleum ether solution, negative control for chloroform is chloroform solution

and negative control for methanol is methanol solution.

40

(a) (b) (c)

Figure 4.7: Zone of inhibition against C. albicans of C. equisetifolia root extract indicated by arrow with,

(a) petroleum ether; (b) chloroform and (c) methanol

41

(a) (b) (c)

Figure 4.8: Zone of inhibition against A.niger of C. equisetifolia root extract with,

(a) petroleum ether; (b) chloroform and (c) methanol

42

Figure 4.9: The diameter of inhibition zone at different concentrations of

petroleum ether extract against C.albicans and A.niger.

Figure 5.0: The diameter of inhibition zone at different concentrations of

chloroform extract against C.albicans and A.niger.

2.6

1.0 1.0

0.5

2.1

0

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

50 100 200 400 500 Negative

control

Zo

ne

of

inh

ibit

ion

, m

m

Concentration of extract, µg/µl

C.albicans

A.niger

5.7

2

2.6 2.1

0.6

0

-2

-1

0

1

2

3

4

5

6

7

50 100 200 400 500 Negative

control

Zo

ne

of

inh

ibit

ion

, m

m

Concentration of extract, µg/µl

C.albicans

A.niger

43

Figure 5.1: The diameter of inhibition zone at different concentrations of

methanol extract against C.albicans and A.niger

In Figure 4.9, the mean standard deviation for three replicated zone of

inhibition against C. albicans for petroleum ether root extract, the highest

was 2.6mm at 50 µg/µl concentration, while lowest was 0.5 mm at 400

µg/µl concentration. Meanwhile, in Figure 5.0, the mean standard

deviation for three replicated zone of inhibition against C. albicans for

chloroform extract, 5.7 mm at 50 µg/µl concentration was the highest and

0.6 mm at 500 µg/µl concentration was the lowest. Besides that, the

highest standard deviation for methanol root extract was 4.9 mm at 50

µg/µl concentration and 0.6 mm at 500 µg/µl concentration was the lowest

that shown in Figure 5.1. All extract shows mean standard deviation for

zone of inhibition around range 1-6 mm which indicate low activity in the

4.9

3.2

1.5

15

1.7

0

-5

0

5

10

15

20

50 100 200 400 500 Negative

control

Zo

ne

of

inh

ibit

ion

, m

m

Concentration of extract, µg/µl

C. albicans

A.niger

44

inhibition of C. albicans. Further, the graph show that at minimum

concentration, C. albicans could be inhibited with the extract, so this

extract has strong antifungal activity. Besides that, to compared the

antifungal activity of C. albicans with A. niger, the A. niger indicated no

activity (0 mm).

Moreover, the drying process when doing the experiment may cause

conformational changes occurs in some of chemical constituents found in

these plants. Besides that, in this experiment, newly cultured A. niger is

used so the conidia, part of fungi that cause disease maybe not matured

enough to be inhibited by C. equisetifolia extracts. Further, according to

(Jefferey, 2011) members of same genus as A. niger, which was an A.

fumigatus capable in secreting secondary metabolites like alkaloid, cyclic

peptides and sesquiterpenoid, so the same characteristic could appear in A.

niger. Besides that, the secondary metabolites secreted by A. fumigatus

producing gliotoxin, a substance that can inhibit C. albicans activity.

45

CHAPTER 5

CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

In conclusion, root of C. equisetifolia showed the positive result of

bioactive compound such as alkaloids, flavonoids, terpenoids, tannin and

saponins. Flavonoid is known to have antimicrobial properties capable to

inhibit fungi. Further, A. niger could also secreting secondary metabolites

such as alkaloid, cyclic peptides and sesquiterpenoid. In this case, the

secondary metabolites properties in plant were not enough to inhibit the

fungi although the study showed inhibition with C. albicans. There were

medicinal value of this plant may as phytochemical presented in this study

are important in antifungal activity. This study also leads to the further

research in the way of isolation and identification of the Rf value as spot

separation from the root of C. equisetifolia using chromatographic and

spectroscopic techniques. Petroleum ether and methanol extract produced

6 compounds when treated with petroleum ether: dichloromethane (1:4)

and chloroform: methanol (9:1) respectively, while chloroform produced 5

compound in solvent system chloroform: methanol (9:1). Moreover,

petroleum extract, chloroform extract and methanol extract, all showed

positive result for zone of inhibition for C. albicans, while negative result

46

for A. niger. This showed that roots of C. equisetifolia also have potential

as antifungal agents as well as bark and leaves of this plant parts. This is

ongoing study as further research is needed to investigate the plants

biological activities.

5.2 Recommendation

In recommendation, C. equisetifolia root extract is a good substitute for

Ampotericin B which is synthetic drugs to treat Candidiasis. It is because;

this plant contains secondary metabolites that are important in medicinal

drugs such as flavonoid and alkaloid. Further research must be made to

detect if other secondary metabolites like glycosides, steroid and

triterpenoid in this plant also can inhibit C. albicans and A. niger. The

present of catechin, gallic acid, ellagic acid and lupeol isolated form the C.

equisetifolia plant which was antioxidants. So isolation of the compounds

from the plant can act as an alternative for antibiotic and can be

commercialized, so experiment to detect the antioxidant activity of C.

equisetifolia should be conducted.

47

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