Abstract:

Aim: To study the anti-mycotic activity of black cumin
seed (Nigella sativa) extract in vitro.

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Background: Nigella sativa Linneaus (N. sativa) (Family Ranunculaceae), a
herbal plant commonly referred to as black cumin seed and Karun-jeeragam (in
Tamil), is immune stimulating, hypotensive, anti-inflammatory, anti-cancerous,
antioxidant, hypoglycemic, spasmolytic and also has bronchodilating properties.
An antimycotic medication is a pharmaceutical fungicide or fungistatic used to
treat and prevent mycoses such as athlete’s foot, ringworm, candidiasis, etc.
Using herbal alternatives against chemical alternatives is thus explored in
this research.

Materials and Methods: Ethanolic extract of Nigella sativa was tested for
antifungal (antimycotic) activity against Candida albicans. Agar well diffusion
method was used and the zone of inhibition against the control sample,
chlorhexidine was measured and recorded.

Results: Nigella sativa extract displayed antimycotic property against Candida
albicans.

Conclusion: Fungal infections are prevalent throughout South India. Anti-mycotic
effect of herbs such as Nigella sativa can thus be purified and used as an
additional ingredient in mouthwashes and tooth paste.

Keywords: Nigella sativa, antimycotic, candidiasis, black cumin seed extract,
antifungal

 

Introduction:

Nigella sativa L. (Family: Ranunculaceae) is an indigenous herb of Southwest
Asia including regions of Iran, India, and Pakistan. Growing to a maximum
height of about 40– 70 cm, this plant has finely divided foliage and pale blue
and white flowers. From the fruit capsules, many small caraway-type black seeds
are produced (length: 2.5 to 3.5 mm and width: 1.5 to 2 mm).1 The plant is
known by various names in different languages; black cumin, black seed,
black-caraway (English), Karun-jeeragam (Tamil), Kalonji (Hindi), Habbah
Al-Sauda; seed of blessing (Arabic), Chernushka (Russian), çörek otu (Turkish),
and Cyah-daneh in Persian. For a long span of time, the seeds of this plant
have been used as a spice and additive in bread, cookies, and other dishes in
many Asian and Eastern countries.

Therapeutic benefits of
this miraculous spice and its active ingredients are being explored.2 N.
sativa has been extensively studied for its biological activities and
therapeutic potential and shown to possess a wide spectrum of activities viz.
as diuretic, antihistamic3, hypoglycemic4, antihypertensive, antidiabetic,
anticancer and immunomodulatory5, analgesic, antimicrobial, anthelmintic,
analgesics and antiinflammatory, spasmolytic, bronchodilator, gastroprotective,
hepatoprotective, renal protective and antioxidant properties. The seeds of N.
sativa are widely used in the treatment of various diseases like bronchitis,
asthma, diarrhea, rheumatism and skin disorders. It is also used as liver
tonic, digestive, anti-diarrheal, appetite stimulant, emmenagogue, to increase
milk production in nursing mothers to fight parasitic infections, and to
support immune system.

The composition of N.
sativa seed includes: fixed oil, proteins, alkaloid, saponin and essential oil.
The volatile oil (0.4-0.45 %) contains saturated fatty acids which includes:
nigellone that is the only component of the carbonyl fraction of the oil,
Thymoquinone (TQ), thymohydroquinone (THQ), dithymoquinone, thymol, carvacrol,
? and ?-pinene, d-limonene, d-citronellol, p-cymene, volatile oil of the seed
also contains: p-cymene, carvacrol, t-anethole, 4-terpineol and longifolene. Black
cumin seed have two different forms of alkaloids: isoquinoline alkaloid that
includes: nigellicimine, nigellicimine n-oxide and pyrazol alkaloid that
includes: nigellidine and nigellicine. N. sativa’s nutritional components are
vitamins, carbohydrates, mineral elements, fats and proteins that include eight
or nine essential amino acids. Black cumin seeds also have saponin and alpha
hederine and in trace amount has carvone, limonene and citronellol, as well as
provide relatively good amounts of different vitamins and minerals such as Fe,
Ca, K, Zn, P, Cu. The fixed oil (32-40 %) contains: unsaturated fatty acids
which includes: arachidonic, eicosadienoic, linoleic, linolenic, oleic,
almitoleic, palmitic, stearic and myristic acid as well as beta-sitosterol,
cycloeucalenol, cycloartenol, sterol esters and sterol glucosides.6

Most of the therapeutic
properties of this plant are due to the presence of thymoquinone (TQ) which is
a major active chemical component of the essential oil.7 The present study
was conducted to assess the antimycotic activity of ethanolic extract of
Nigella sativa against Candida albicans.

Candida albicans is a
commensal fungus commonly inhabiting human mucosal surfaces. C. albicans has adapted
to the human host and has evolved because of the specific demands of the human
host environment for a long time now. Peculiarly, colonizing C. albicans
strains can become opportunistic pathogens causing persistent mucosal
infections and life-threatening disseminated infections with high mortality
rates. The oral cavity because of its unique environment is a primary target
for opportunistic infections, particularly candidiasis.8

The resistance of infectious
disease causing micro organisms to various antibiotics has led to enormous
medical complications for the treatment 9 due to the frequent use of
commercial antimicrobial drugs to treat infections10 . Thus, leading to an
upsetting increase of fungal infections11, making it necessary for the
preparation of alternative drugs from medicinal plants12.

Hence, this study aims to
study the anti-mycotic activity of black cumin seed (Nigella sativa) extract in
vitro.

Materials and Methods:
Candida albicans was isolated from clinical samples and cultured and maintained
on subouraud’s dextrose agar medium at 30°C. Ethanolic extract of Nigella
sativa was prepared and used for the study.

 

Methodology:

The extracts were
prepared in the following concentrations in sterile water. 5mg/ml and 10mg/ml
and 20mg/ml. 100µl of extract of different concentrations were loaded on
sterile filter paper discs measuring 6mm in diameter, so that the concentration
of the extract on each disc was 500µg, 1000 µg and 2000µg respectively. The
discs were dried and kept aseptically. 13

 

Screening of Antifungal
Activity Disc Diffusion Technique

The ethanolic extract of Nigella
sativa was screened for antifungal activity by disc diffusion method. Activated
cultures of Candida albicans in Sabouraud’s broth was adjusted to 0.5 McFarland
standards 108 cfu/ml. 100 µl of the inoculum was introduced to molten
Sabourauds dextrose agar and poured in the sterile petri plates and allowed to set.
Sterile filter paper discs (6.0 mm diameter) impregnated with 2000µg/disc, 1000
µg /disc and 500 µg /disc of the plant extract dissolved in sterile water were
placed on fungal seeded plates and incubated at 28oC for 48 hrs. As a positive
control, Fluconazole (10 mcg /disc) and Amphotericin B (100 units /disc) were
used. Following an incubation period of 48 hrs, plates were removed from the
incubator and antifungal activity was evaluated by measuring zones of
inhibition of fungal growth. Clear zones within which fungal growth was absent
were measured and recorded as the diameter (mm) of complete growth inhibition.
The whole experiment was performed three times to minimize error. 14

Results :
The antimycotic activity of the extract at different concentrations was
screened by agar well diffusion technique and the zone of inhibition was
measured in mm diameter. The test solution inhibited the fungal strains with
varying degree of sensitivity for different concentrations. The antifungal
activity of the extract against C. albicans is shown in Table 1 and Figure 1. The
extract showed good antimycotic activity at different concentrations with
maximum zone of inhibition of 25mm at concentration 2000µg/ml.

Concentration
of the extract

Zone
of inhibition ( in mm diameter)

500µg/ml

16

1000µg/ml

20

2000µg/ml

25

Fluconazole
(10 mcg/disc)

24

Amphotericin
B (100 units/disc)

27

Table 1.

Figure 1- Graph showing antimycotic activity of ethanolic extract of Nigella
sativa.

Discussion:
In Hanafy MS et al. 1991’s study, Nigella sativa extract showed antibacterial
synergism with streptomycin and gentamicin and showed additive antibacterial
action with spectinomycin, erythromycin, tobramycin, doxycycline,
chloramphenicol, nalidixic acid, ampicillin, lincomycin and
sulphamethoxyzole-trimethoprim combination. It also exterminated a non-fatal
subcutaneous staphylococcal infection in mice when injected at the site of
infection.15

Randhawa MA et al. 2015
did a study where nano-particulated drugs-Amphotericin-B, Ketoconazole and
Thymoquinone (an active ingredient of Nigella sativa) was investigated in vitro
against Candida albicans yeasts and Candida biofilm and was found to be effective
in disinfecting both the Candida yeasts and Candida biofilm.16

According to the study
done by Shokri H et al. 2012 the mean zone of inhibition value for N. sativa
was found to be 40.8mm, showing strong anti-C. zeylanoides activities, which reinforces
the potential use of Nigella sativa for the treatment of candidiasis.17

N.H. Nadaf et al. 2015 performed
a study to analyze the anti-yeast activity of N.sativa extract by agar well
diffusion method against Candida albicans NCIM 3466. Supplementary exposition
of anti-yeast activity was done by scanning electron microscopy (SEM). The phytochemicals
were identified using GC–MS and the antioxidant properties checked by using
DPPH assay. The maximum anti-yeast activity was reported at pH 7 and 30 °C
temperature. The study confirmed the anti-yeast and antioxidant properties of
compounds extracted from N. sativa seeds which might be useful for further expertise
advancements in drug manufacturing.18

Khan MA et al. 2016 did an
in vitro study that checked the efficacy of Nigella sativa extract on the prevention
of Candida albicans growth on soft denture reliners. According to Khan MA 19,
N. sativa extract used in the study was significantly effective against candida
albicans.

Acoording to A.Naeini’s
study et al. 2008, Iranian medicinal herb oils including N.sativa and 13 other
oils out of the 16 oils testes confirmed a significant activity against C.
albicans tested with minimal inhibitory concentration (MIC) values ranging from
150 to 2300 ?g/ml using broth macrodilution method and the growth inhibition
zone ranging from 16 to 55 mm using disc diffusion method. 20

In the present study, Nigella
sativa was found to show good antifungal activity at different concentrations
with a maximum zone of inhibition of 25 mm at a concentration of 2000µg/ml.

Conclusion:
Despite all the significant
advancements in modern medicine, traditional herbs have given rise to many
important drugs. Medicinal plants are being studied for their potential
mechanism of action and therapeutic properties.21 Recent studies
suggest good correlation between medicinal use and the in-vitro anti-mycotic
activity of medicinal herbs. 22 The results of the present study evidently
indicate the antifungal activity of Nigella sativa extract and may be used as a
source for the isolation of active compounds that may serve as lead compounds
in antifungal drug development. Further studies on their cytotoxicity or
toxicity will be advantageous to know the possible harmful effects of this
extract for common used by the local communities.

Black cumin seed extract can thus undeniably be commercially modified to be
used as an antifungal drug to drastically reduce the incidence of candidiasis.
The anti-mycotic property can further be investigated to treat fungal
infections of other origins as well and in the synthesis of more advanced, strain
specific anti-fungal drugs.

Reconnoitering avenues in pharmacological benefits of herbs and spices can pave
way for safer, easier and cost effective developments in synthesis of drugs for
oral candidiasis and other fungal infections. Further research may however be
needed to explore the exact mechanism of action of Nigella sativa extract on C.
albicans. The anti-mycotic effect of herbs such as Nigella sativa can thus be
purified and used as an additional ingredient in mouthwashes and tooth pastes
to prevent fungal infections.

 

 

 

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