Southwestern University

Department of Medical Technology

Villa Aznar, Urgello St., Cebu City

Effect of Cassia alata Linn. on the growth of Candida

albicans in vitro

Submitted to:

Ms. Merafe O. Torregosa, RMT,MSMT

Submitted by:

Dela Serna, Bob Jansen

Dumdum, Ma. Jella Mae

Ermac, Rinest

Pelarca, Pearl Jade

Rama, Jenissa

Satera, Monalisa

BSMT 3E

RATIONALE

In the recent years, research on medicinal plants

has attracted a lot of attention globally. Large body

of evidence has accumulated to demonstrate the

promising potential of medicinal plants used in various

traditional, complementary and alternate systems of

treatment of human disease.

Cassia alata Linn., belonging to family

Caesalpiniaceae is a pantropical, ornamental shrub,

distributed from tropical America to India. It is

commonly known as Ringworm Senna, Seven Golden

Candlesticks, Candle Bush, Gelenggang (Malay) or

Akapulko (Filipino). The attractive shrub is named for

its flower buds which grow in a column and look like

fat yellow candles each complete with a flame. Its

leaves, which fold together at night, are bilateral and

symmetrically opposed. The leaf extracts of the plant

have been reported to possess medicinal properties and

used against ringworm, scabies, ulcers and other skin

diseases such as pruritis, eczema and itching. The

leaves contain chrysophanic acid, a fungicide that is

used to treat infections such as ringworm and athlete's

foot. Previous reports of antimicrobial activity

against human pathogens have been widely carried out

for Cassia alata Linn. Fuzellier et al. (1982) found that

aqueous extract from the leaves of Cassia alata and some

of its components, rhein, emodol, 4,5-dihydroxy-1-

hydroxy-methylanthrone and 4,5-dihydroxy-2-hydroxy-

methylanthraquinone, had antifungal activity against

some dermatophytes and yeast.

The aim of the present work is to evaluate the

antifungal activity of its aqueous flower and leaves

extract on human pathogenic fungi (Candida albicans).

There are over 20 species of Candida yeasts that

can cause infection in humans, the most common of which

is Candida albicans. Candida albicans is a diploid fungus

that grows both as a yeast and filamentous cells and a

constituent of the normal gut flora comprising

microorganisms that live in the human mouth and

gastrointestinal tract. Candida albicans lives in 80% of

the human population without causing harmful effects,

although overgrowth of the fungus results in

candidiasis.

Candidiasis is a fungal infection caused by

yeasts. It is often observed in immunocompromised

individuals such as HIV-infected patients. A weakened

or undeveloped immune system or metabolic illnesses

such as diabetes are significant predisposing factors

of candidiasis. A common form of candidiasis

restricted to the mucosal membranes in mouth or vagina

is thrush, which is usually easily cured in people who

are not immunocompromised. Very rarely, the infection

may become invasive spreading throughout the body,

resulting in fever along with other symptoms depending

on the parts of the body affected. In extreme cases,

these superficial infections of the skin or mucous

membranes may enter into the bloodstream and cause

systemic Candida infections. Candida infections can

affect the esophagus with the potential of becoming

systemic, causing a much more serious condition, a

fungemia called candidemia.

REVIEW OF RELATED LITERATURE

Cassia alata Linn., popularly known as “akapulko” is a

course, branched shrub found in a cool climate like

near rivers. It can grow up to 15 feet tall, and has

green alternate leaves, with even, pinnate leaflets.

The flowers are on a long pedicle with yellow color and

bloom from the bottom to the end. They have 4-5 petals.

It is used worldwide as a cure for, among others,

curvy, psoriasis and fungal infections. The

phytochemicals extract are rhein, emodol, 4,5-

dihydroxy-1-hydroxy-methylanthrone and 4,5-dihydroxy-2-

hydroxy-methylanthraquinone.

The juice from the leaves has been used as a cure

for poisonous bites and venereal eruptions. When mixed

with lime juice, it has been used for ringworm and

other skin diseases (Quisumbilg 1978). It is used as a

remedy for skin diseases, like herpes and ringworms

because of its chysophanic acid content ( Bunyiplana,

1963).

Its therapeutic efficacy against tinea versicolor

was tested for the first time involving humans

(Damodaran, 1994), a 10-year human study indicating

that the leaf extract can be reliably used as an herbal

medicine to treat pityriasis versicolor.

In a research done in Malaysia 1995, the ethanolic

extract of Cassia alata leaves was investigated for its

anti-microbial activities on several micro-organisms

including bacteria, yeast dermatophytic fungi. In

vitro, the extract exhibited a high activity against

various species of dermatophytoc fungi, but low

activity against non-dermatophytic fungi. However,

bacterial and yeast species showed resistance against

in vitro treatment with the extract.

Another similar study was done in Chiang Mai

University which showed that 35% ethanolic extract from

Cassia alata Linn leaves demonstrated antifungal

activity against T. mentagophyte, T. rubrum and M.

gypsum. The activity was determined by agar diffusion

method (Nanthachit).

In a study done by the Department of Pharmacology

in the University of the Philippnes, Manila, powder,

leaves, and lotion from akapulko and powder and

defatted extract from leaves of amplaya reduced the

genotoxic activity of dimethylnitrosamine,

methylmethanesulfate and tetracycline. This was shown

by the reduction of chromosome breaking effects of

these genotoxins as induced by the difference drugs

preparations from akapulko and amplaya (Balboa and

Sylianco, 1992).

In 1991, clinical trials revealed the

effectiveness of akapulko as an anti-fungal agent which

supported the Department of Health and Department of

Science and Technology in their launching of an

akapulko lotion in 1994.

Like any drugs, the Acapulco extract was also

tested for its side effects. Dr. Xenia Tigno and Dr.

Angelica Francisco found that excessive use of lagundi,

akapulko and damong maria can cause diverse effects.

The plant extracts consist of different chemical

components which may not be needed to treat a given

disorder. The haphazard use – just to facilitate

treatment of a given disorder – enhances other body

processes which could adversely promote other body

activities. Such scenario may lead to side effects. For

instance, frequent and excessive use of the akapulko to

treat a skin disorder can cause itchiness. This side

effect can be compared to a person who takes a dose of

paracetamol which eases body aches or pains but promote

dizziness. The National Research Council of the

Philippines (NRCP) explained that the side effects are

caused by other chemical components which are

superfluous to the cure of the skin disorders and

promote itchiness. The NRCP researchers recommend that

an extensive study be conducted to isolate or separate

the chemical components which are not needed to treat

skin disorders and avoid unwanted effects. Through

further investigation, a variety of medicine can be

developed for specific disorders without side effects.

Akapulko lotion produced from Cassia alata, Linn,

is clinically proven as an effective treatment for

tinea versicolor locally known as “an-an”. Clinical

trials have established its efficacy and safety in

comparison with commercially available anti-fungal

creams, particularly sodium thiosulfate. Akapulko

lotion is estimated to cost about 67% less than its

counterpart in the same therapeutic category. The year

1998 stamped another important milestone in herbal

medicine research and development. It saw the transfer

of akapulko lotion as anti-fungal to the private sector

for commercialization. Under a license agreement with

DOST-PCHRD, Pascual Laboratories Inc., will soon make

available akapulko lotion in the commercial market.

In eastern Nigeria, Cassia alata Linn. plants which

have frothing or foaming ability have been employed as

soap for bathing and for treatment of skin and wound

infections. Ethnomediacally, juice and extracts from

leaves of the plant are topically applied as anti-

inflammatory and antimicrobial agents, especially in

the treatment of skin diseases including eczemas, ring-

worms and pruritus (Benjamin, 1980; Benjamin and

Lamikanra, 1981; Oliver, 1986; Ayim, 1987; Akinde et

al., 1999). These plant materials are either used alone

or formulated into local soaps, ointments and creams

which are often commercially available. Due to lack of

evidence on the efficacy of herbal soap, and the poor

aesthetic presentation, these products are mostly

patronized by low income group in the local communities

in the past. But interestingly, the popularity of herb-

based soaps is increasing due to many years of

accumulated experience on their efficacy on topical

disorders. Currently, there are so many commercial

brands of herb-based soaps with good claims of efficacy

and are now enjoying increasing patronage. It is

therefore important to investigate these soaps to

validate the claims and also establish other useful

properties which will help in promoting public

acceptance and encourage wider usage.

Soaps act as emulsifiers or surfactants, softening

the horny-layer of the epidermis and acts as a

germicide by enhancing the permeability of microbial

envelope thereby disrupting the integrity of microbial

cells. Antimicrobial activity of soaps make them useful

agent for bathing, laundry, washing, and cleansing of

surfaces (Fuerst, 1978; Hugo and Russel, 1983).

The cleansing and germicidal properties of the

soapy-plants are comparable to those of the standard

soaps, which are salts of higher fatty acids. Crude

preparations of soapy plants are able to soften the

skin epidermis, enhance greater penetration and

cleansing of sores and acne and thereby promote rapid

healing and resolution of blemishes.

In the study they evaluated the antiseptic

potentials of Cassia alata-based herbal soap formulated

in our laboratory. Cassia alata is known to contain

some secondary metabolites like resin, saponin,

phenols, flavonoids, anthraquinone glycosides and

alkaloids (Akinde et al., 1999). These

phytoconstituents are also known to possess surface

activity and other soap related properties. In previous

studies, Cassia alata has been found to possess

excellent wound-healing properties (Benjamin and

Lamikanra, 1981; Palanichamy et al., 1991) and is also

useful in the treatment of eruptive and pustular skins

conditions by rubbing crushed fresh leaves on infected

area (Akinde et al., 1999).

The antimicrobial activity of Cassia alata

extracts has also been investigated against S.aureus,

S.aureus coagulase positive, B.subtilis,B. cereus

B.stearothermophillus, E.coli, V.cholerae, S.typhi,

S.dysenteriae and K.pneumoniae.

The acetone and ethanol extracts showed high

activity against nearly all test microorganisms. The

inhibitory effects are very close and identical in

magnitude and comparable with that of standard

antibiotics (Miconazole), when used.

The therapeutic efficacy of Cassia alata leaf

extract against Pityriasis versicolor (a skin fungus,

aka Tinea versicolor) has been reported. The leaf

extract can be reliably used as an herbal medicine to

treat this type of fungus. It has no side-effects. It

is also effective against infections caused by these

fungi: Candida albicans, Trichophyton mentagrophyte and

Aspergillus niger.

The phyto-chemical adenine, in the leaves has been

documented as an effective platelet aggregating

inhibitor (reduces sticky blood and arterial plaque).

Senna, like most Cassia species, contains a group of

phyto-chemicals named anthraquinones; they are known

for the laxative effect. Senna is more effective than

the antibiotic Erythromycin to increase the movement

of stool through the bowels. The U.S. Food and Drug

Administration (USDA) has approved Senna as a

nonprescription laxative. For cleaning of the bowel it

is excellent.

Asthma is a major cause of disability, health

resource utilization, and poor quality of life for

those who are affected. Prevalence data are lacking for

many countries in Africa, but recent estimates indicate

that nearly 50 million Africans currently have asthma.

The prevalence of the disease is the greatest (about 8%

of the population) in Southern Africa. It is

anticipated that with continued urbanization and

increasing westernization of lifestyles, the burden of

asthma in Africa will continue to increase considerably

in the coming decade.

The orthodox treatment for the management of acute

attack and day to day therapy of asthma may involve the

use of bronchodilators, expectorants, and

corticosteroids. Modern medicine is one of the largest

industries in the world. However, the use of herbal

remedies and traditional medicine are also rising

steadily. Moreover, much of the modern scientific

medicines have evolved from traditional medicine. With

the ability to extract such benefits from plants, our

traditional system deserves an objective and critical

examination. Large numbers of medicinal plants are used

ethnomedically in the treatment of asthma, but there is

a need to conduct pharmacological investigations to

ascertain their therapeutic values. Antiallergic

activity of cassia alata has been recently described by

Singh et al. The plant possesses laxative, anti-

inflammatory, antimutagenic, analgesic, and

antimicrobial properties. Chemical analyses of extracts

from Cassia alata yielded constituents as phenolics,

fatty acids, terpenoids, and anthraquinones. Infusion

of leaves and flowers is used to treat asthma in Congo.

The torrified seeds are used as a coffee substitute and

reportedly have antiasthmatic effect. Cassia alata is

used in India for the same properties.

The study of Pieme et al. has been designed to

evaluate the acute and subacute toxicities of aqueous-

ethanolic extract of leaves of Cassia alata and

provided evidence of the nontoxic effect of this

extract. In another study, rats fed dried ground leaves

of C. alata in their chow or ethanol extract added to

their daily drinking water developed hepatic lesions

accompanied by renal and intestinal damage. Growing

evidence has shown that some of the plant’s secondary

metabolites are toxic and/or carcinogenic, which can

induce adverse effects leading to mutation and/or

degenerative diseases. The risk from long-term use of

such remedies has not however been fully investigated,

especially in terms of their potential to cause

mutagenicity and carcinogenicity. Among short-term

toxicity assays, the comet assay is a very sensitive

test for the quantification of DNA damage. Although DNA

damage as revealed by the comet assay may not

necessarily result in permanent genetic damage, there

is consensus about a close association of unrepaired

DNA damage or error-prone repair processes, mutations,

and the induction of various types of cancer. In view

of the above considerations, and due to the lack of

information about Cassia alata genotoxicity, it is

necessary to perform investigation on the effects of

this herbal product which is often used as a

therapeutic modality on genetic alterations.

Evidence supports the potential role of

antioxidant agents in cancer prevention. We have

therefore undertaken to investigate the

antigenotoxicity of the aqueous-ethanolic extract from

Cassia alata, given that radical scavenging antioxidant

activity was reported from the aerial parts of this

species .

The aim of their study was to evaluate the

potential bronchodilator effect of extracts from Cassia

alata, and if any, to characterize their

pharmacodynamic profile and to investigate in vivo the

genotoxic and antigenotoxic effects of aqueous-

ethanolic extract of C. alata leaves.

Japanese researchers in 2003 reported that a leaf

extract evidenced anti-inflammatory activity. In

animal studies conducted in the Phillipines in 2002,

the leaves were reported to possess pain-relieving,

anti-inflammatory, antimutagenic, and hypoglycemic

actions. In 1994 a 10-

year human study was published in India which indicated

that a Guajava leaf extract can be reliably used as a

herbal medicine to treat Pityriasis versicolor (a type

of skinfungus) without side effects (20th of January

2006, Guardian newspapers). Another clinical trial on

human being was conducted in 1990 by Damodaran and

Venkataraman to study the therapeautic efficacy of

Cassia alata Leaf extract against Pityriasis

versicolor, Ethnopharmacol. 42:19-23, 1994 study. The

leaves are reported to be useful in treating

convulsion, gonorrhoea, heart failure, abdominal pains,

edema and is also used as a purgative.

RESEARCH METHODOLOGY

Research Design

This study will use aquasi-experimental design.

Research Subject

The study will utilize pure culture of Candida

albicans.

Research Environment

The study will be conducted at the Medical

Technology Laboratory in Southwestern University.

Research Instrument

The study will use vernier caliper to measure the

zone of inhibition.

Data Gathering Procedure

I. Acquisition of plant

The petals and leaves of Cassia alataLinn. will be

collected in Busay,Lahug, Cebu City.

II. Preparation of extract

The leaves will be shade dried and at room

temperature for 7 days to attained a constant weight.

It is not dried under sun so as not to lose some of the

biochemical constituents of the plant. It is then

pulverized with clean mortar and pestle to fine powder.

It is then stored in a sterilized glass container at

room temperature (25-30°C) until used. Ethanol soxhlet

extraction method as described by Akinyemiet al. (2000)

and Abdulrahmanet al. (2004) will be adopted for the

study. The stock extract will bestored in a sterile

bottle and kept in refrigerator at 7°C.

III. Preparation of Materials

A. Culture Media

The study will utilize Sabouraud Dextrose

Agar (SDA) plates obtained from the

Microbiology Department of Vicente Sotto

Memorial Hospital.

B. Antifungal Discs

Whatman filter paper no. 1 will be use to

prepare the discs approximately 6 mm in

diameter, which will be placed in a petri dish

and sterilized in a hot air oven. Dispense

0.005 ml (5 microliter) of the aqueous flower

and leaves extract of Cassia alata Linn.

usingsterile micropipette tips. Let it dry in

room temperature.

IV. Antimicobial Assay

A. Preparation

Using an inoculating loop, obtain an inoculum

from the pure culture of Candida albicans and

suspend it directly into a small volume of

sterile NSS. Repeat this procedure until the

turbidity matches that of the 0.5 McFarland

Standard.

B. Plate Inoculation

Dip in sterile nontoxic swab into the

standardized suspension. Remove the excess fluid

by pressing and rotating the swab against the

side of the tube above the fluid level. Streak

the swab evenly in three directions over the

entire surface of the agar plate, turning sixty

degrees each time to obtain a uniform inoculum.

Make a final sweep to the agar rim with the

cotton swab. Allow the plate to stand on the flat

surface for 3-5 minutes.

C. Application of the Antifungal Discs

Within 15 minutes after the plates are

inoculated, apply the antifungal impregnated discs

to the surface of the inoculated plates either

with mechanical dispenser or by hand with sterile

forceps. Gently press all discs onto the agar with

forceps or with an inoculating needle to insure

complete contact with agar surface. The special

arrangement of the discs should not be closer than

15 millimeter to the edge of the plate and far

enough to prevent overlapping zones of inhibition

at least 20 millimeter apart

Within 15 minutes after the discs are applied,

the plates will thenbe inverted and place in an

incubator at 37 degrees Celsius for 16-18 hours.

At the end of the 16-158 hours incubation,

examine the plates and measure to the nearest

millimeter the zone of complete inhibition.

C. Reading

After 18 hours, read the different zones of

inhibition using the vernier caliper.

REFERENCES

Naturia. “Seven Golden Candle Sticks.” Website:

http://www.naturia.per.sg/buloh/plants/candlesticks.htm

. Accessed on December 29, 2014.

Specialty Natural Products. “Cassia alata Linn”.

Website:

http://www.snpthai.com/en/herbal-extract/supplements/ca

ssia-alata-extract. Accessed on December 29, 2014.

Tropilab Inc. “Cassia alata.” Website:

http://www.tropilab.com/cassia-ala.html. Accessed on

December 29, 2014.

Wikipedia. “Candida albicans.” Website:

http://en.wikipedia.org/wiki/Senna_alata. Accessed on

December 29, 2014.

Wikipedia. “Senna alata.” Website:

http://en.wikipedia.org/wiki/Senna_alata. Accessed on

December 29, 2014.

Wikipilipinas. “Cassia alata.”“ Website:

http://en.wikipilipinas.org/index.php/Cassia_alata.

Accesed on December 29, 2014.