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

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29, 2014.