Effect of Nigella sativa Extract on Bacteriology Parameter of

Multibacillar leprosy Patients Administering MDT-WHO: Three

Months Observation

Dian Kusuma Dewi Ramadhani, Renni Yuniati, Subakir, Asih Budi Astuti, Falah Faniyah

and Prasetyowati Subchan

Faculty of Medicine, Diponegoro University, Semarang, Indonesia

Keywords: multibacillar leprosy, Nigella sativa, bacterial index, morphology index, MDT-WHO

Abstract: MDT-WHO implementation has decreased leprosy global prevalence. However, number of new cases

detected is still high. Therefore, new appropriate strategy, besides early detection and WHO-MDT treatment

which are the main strategies in leprosy eradication, is needed. The aim of this study was to investigate the

effects of Nigella sativa extract on bacteriology parameters of multibacillar leprosy patients administering

MDT-WHO. It was Double Blind - Randomized Controlled Trial study which was conducted in Jepara,

Central Java Province, Indonesia during February to June 2016. This study was performed on 60 eligible

multibacillar leprosy patients administering MDT-WHO. Bacterial index (BI) was measured using Ridley

logarithmic scale, whereas morphology index (MI) was measured as percentage of solid bacteria to the all

forms of bacteria. There was no subject who dropped out. The characteristics of subjects were similar between

the two groups, except for the MDT duration at baseline, where MDT duration in control group was longer.

There was no significant difference in BI proportion and MI proportion in the two groups after treatment.

Average BI in month 2 and 3 tend to be lower than control although not statistically significant. There were

significant differences of average BI decline between control and treatment groups in month 2 (p=0.000) and

month 3(p=0.005). There were statistically significant differences of average MI and average MI decline

between control and treatment groups in month 1 (p=0,000, p= 0.001, respectively). This study demonstrated

that Nigella sativa extract affected average MI and BI decline differences. Its efficacy as adjuvant therapy

might be confounded by duration of MDT-WHO.

1 INTRODUCTION

Leprosy is still world burden. Leprosy is infectious

disease that does not only affect medical condition,

but also social, economy, culture, and national

defense aspects. In most of the cases, leprosy does not

cause disability when it first manifests. Conditions

contributing to disability and deformity are

preventable when early detection can be performed

(Kemenkes RI, 2012).

Multi Drug Therapy recommended by World

Health Organization (MDT-WHO) has decreased

leprosy global prevalence from 5.2 million cases in

1980 to 200,000 cases in 2014. However, new cases

detected in 2014 were still high that is about 220,000-

250,000 new cases are diagnosed every year (Smith

& Aerts, 2014). Indonesia is a country with the third

highest leprosy prevalence in the world after India

and Brazil. Central Java is the province which has the

second highest new cases of leprosy in Indonesia

(Kemenkes RI, 2012). Therefore, new appropriate

strategy, besides early detection and WHO-MDT

treatment which are the main strategies in leprosy

eradication, is needed. To date, there are some studies

that have investigated micronutrients as adjuvant

therapy in leprosy treatments as well as to prevent

contact from getting infection (Smith & Aerts, 2014;

Vazquez et al., 2014; Rahfiludin, 2011).

Lepromatous or multibacillar (MB) leprosy has

lower cellular immunological reaction against M.

leprae. Multibacillar leprosy was characterized by

high bacillary load and high number of skin lesions.

Thus, untreated MB patients are the main source of

leprosy transmission (Walker & Lockwood, 2006).

The presence of acid-fast bacilli on skin tissue

scrapings is considered as one of the main signs of

Ramadhani, D., Yuniati, R., Subakir, ., Astuti, A., Faniyah, F. and Subchan, P.

Effect of Nigella Sativa Extract on Bacteriology Parameter of Multibacillar Leprosy Patients Administering MDT-WHO: Three Months Observation.

DOI: 10.5220/0008149600090013

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 9-13

ISBN: 978-989-758-494-7

leprosy and sufficient to establish a diagnosis of

leprosy. In 1982, WHO recommended the use of

MDT as a standard treatment for leprosy and

introduced an operational classification of leprosy

which differentiated leprosy into PB and MB based

on Reitz serum smear. Bacterial Index (BI) and

Morphological Index (MI) are used for the serum

Reitz smear assessments. Bacterial Index is a semi-

quantitative measure of leprosy bacillus density in the

smear. BI helps in determining the type of leprosy and

assessing the outcomes of the treatment. Morphology

Index is a percentage of the solid form to all forms of

leprosy bacillus. The Morphology Index is useful for

assessing the transmission and the outcomes of the

treatment and determining drug resistance

(Kemenkes RI, 2012).

Black cumin/ black seed (Nigella sativa) belongs

to Ramunculaceae family and is popular as herbal

medicine.(6) The efficacies of Nigella sativa that

have been widely known by the community included

as anti-parasite, antimicrobial, anti-inflammatory,

improving liver and kidney function, treatment for

respiratory and digestive disorders, and immune-

modulator. Some studies showed that continue

Nigella sativa administration can improve immune

responses. Nigella sativa oil has potential

potentiation effect to cellular immune response

through Th1 modulation and Th2 suppression,

meanwhile the other contents can suppress cell-

mediated immune system (humoral) (Salem, 2005;

Boskabady et al., 2011).

To our knowledge, there has not been any

research article evaluating the effect of Nigella sativa

extract on bacterial parameter in MB leprosy patients.

Therefore, this study sought to study the effects of

Nigella sativa on bacteriological parameters of MB

leprosy patients.

2 METHODS

The double blind-randomized control trial was

conducted from February to June 2016 in Donorejo

Hospital, Pati Regency, Central Java Province,

Indonesia. Donorejo Hospital was referral hospital for

leprosy.

Eligible subjects for this study were those who

gave their consent after thorough explanation and met

inclusion and exclusion criteria. Subjects were men

and women aged 20-60 years who had not received

MDT-WHO or had been on MDT-WHO for at least 1

month, did not have leprosy reaction, did not pregnant

or breastfeed, and did not consume any drugs with

immune-suppressant or immune-modulator effects in

the past 1 month. All subjects who did not follow

research protocols or experienced drug eruption due

to MDT and Nigella sativa are excluded.

Multibacillary leprosy was diagnosed according

to WHO classification. Ritz serum from both ear

lobules and active lesion were stained with acid-fast

stain and observed under microscope to obtain MI

and BI indexes. BI was calibrated using Ridley

algorithm and scaled from 0 to +6 points. MI was

counted from the percentage of solid mycobacterium

to all mycobacterium observed in the smear. MI was

expressed as percentage.

Subjects who were enrolled to this study were

randomized into two groups, i.e. treatment group and

controlled group. Subjects in controlled group were

asked to take two capsules of 500 mg Nigella sativa

oil, three times daily for 3 months. Subjects in control

group were asked to take placebo capsules in the same

frequency and duration. Each placebo capsules

contain 1000 mg lactose and had the same appearance

as Nigella sativa oil capsules. All of subjects received

WHO-MDT. Evaluation of BI and MI through Reitz

serum smear was performed in month 1, 2, and 3.

This study was approved by Health Ethics

Committee of Medical Faculty, Diponegoro

University and Kariadi Central Hospital, Semarang,

Indonesia.

3 RESULTS

During study period there were 65 patients who

clinically diagnosed having MB leprosy and only 60

subjects had positive Reitz serum. Therefore, only 60

patients were enrolled to this study. Through

randomization, the subjects were divided equally into

treatment and control groups. There was no dropped

out subject. All of subjects’ characteristics, except

MDT duration, were similar in the two groups. The

MDT duration in the treatment group (Mean ± SD,

1.67 ± 1.3) was significantly shorter than in the

control group (Mean ± SD, 2.53 ± 1.1), p = 0.007.

RCD 2018 - The 23rd Regional Conference of Dermatology 2018

Table 1. Average BI and MI in month 0, 1, 2, and 3

Treatment Control P

N Mean ± SD

Median

(Min – Max.)

N Mean ± SD

Median

(Min – Max.)

BI Index

Month 0 30 4.53 ± 0.97 4 (3 – 6) 30 3.87± 0.70 4 (3 – 5) 0.007

Month 1 30 2.43 ± 0.94 3 (1 – 4) 30 2.13± 0.86 2 (1 – 3) 0.188

Month 2 30 0.23± 0.63 0 (0 – 2) 30 0.47± 0.73 0 (0 – 2) 0.097

Month 3 30 0 0 (0 – 0) 30 0.03 ± 0.18 0 (0 – 1) 0.317

MI Index

Month 0 30 32.00± 27.72 15 (10 – 80) 30 12.00 ± 9.61 10 (0 – 60) 0,000

Month 1 30 6.33 ± 5.56 10 (0 – 20) 30 4.67± 5.07 0 (0 – 10) 0,000

Month 2 30 0 0 (0 – 0) 30 0 0 (0 – 0)

Month 3 30 0 0 (0 – 0) 30 0 0 (0 – 0)

Table 2. Average of BI and MI decline difference

Treatment Control P

Mean ± SD

Median

(Min – Max.)

Mean ± SD

Median

(Min – Max.)

∆ IB month 1 2.10± 0.88 2 (0 – 3) 1.73± 0.69 2 (1 – 3) 0.064

∆ IB month 2 4.30± 0.95 4 (2 – 6) 3.40± 0.86 3 (2 – 5) 0.000

∆ IB month 3 4.53 ± 0.97 4 (3 – 6) 3.83 ± 0.70 4 (3 – 5) 0.005

∆ IM month 1 25.67± 25.28 10 (0 – 70) 7.33± 9.80 10 (0 – 50) 0.001

∆ IM month 2 0 0 (0 – 0) 0 0 (0 – 0)

∆ IM month 3 0 0 (0 – 0) 0 0 (0 – 0)

There was no significant difference of bacterial

index proportion at baseline (month 0) between

treatment group and control group. There were BI

proportion changes in both groups in month 1, 2, and

3. However, the changes were not statistically

significant (data was not shown). The average BI of

treatment group in month 0 is significantly higher

from the control group (p= 0.007). However, in the

following months the difference became

insignificant, and even average BIs in treatment

group tended to be lower in month 2 and 3 as shown

in Table 1.

There was no significant difference of average BI

decline (∆ BI) in month 1 between two groups.

However, the comparison was statistically significant

in month 2 (p= 0,000) and month 3 (p= 0,005) as

shown in the Table 2.

At baseline there was statistically significant

difference of MI proportion between the two groups

(p= 0.037). After 1 month observation, significant

difference of MI proportion was not observed (p=

0.394). Statistics analysis could not be obtained in

month 2 and 3 due to all subjects in group treatment

were in MI 0% (data was not shown).

Average MI at baseline and month 1 were

significantly different between the two groups as

shown in the Table 1.

Average of MI decline

(∆ IM) of treatment group in month 1 was

significantly higher than control group (p= 0.001) as

shown in the Table 2. Bivariate analysis could not be

obtained in month 2 and 3 due to average MI in both

groups were 0.

4 DISCUSSION

MDT duration between the two groups was

significantly different at baseline suggested that MDT

duration could confound interpretation of Nigella

sativa administration effect in this study. One or two

doses of rifampisin killed 10

organisms, decreased

organism numbers into 10

in MB leprosy, and

eliminated most of them. Rifampisin was the most

effective anti-leprosy drug and could decrease MI of

lepromatous leprosy into 0% in 5 weeks (Bryceson et

al., 1990). Therefore, significant MI and BI decline in

this study might be due to MDT or Nigella sativa

administration.

Administration of Nigella sativa capsules 1000

mg three times daily could significantly decrease the

average of BI decline difference on month 1, 2, and 3

compared to baseline. The average BI decline

difference in treatment group (4.53 ± 0.97) was

higher than the control group (3.83 ± 0.69). However,

Effect of Nigella Sativa Extract on Bacteriology Parameter of Multibacillar Leprosy Patients Administering MDT-WHO: Three Months

Observation

BI proportion difference between the two groups in

month 1, 2, and 3 were not statistically significant.

Those suggested that Nigella sativa did not result in

different BI outcomes.

At baseline, there was significant difference of MI

proportion in both groups. However, after 1 month

therapy, the proportion comparison became not

statistically significant. This suggested that Nigella

sativa did not significantly decreased MI decline in

treatment group compared to MI decline in control

group.

Immunity mechanisms relied on Accessory

Immune System (AIS) reactivation which was

responsible for leprosy and stimulation of T or B

lymphocytes. T cells regulated T cytotoxic cells

induction, B lymphocyte function and macrophage

function by releasing cytokines. Th1 cells release

IFN- which would activate macrophages to kill

bacteria or to inhibit microbe growth and would

trigger T cytotoxic cell response resulting in self-

healing disease (Williams & Kupper, 2012). In

contrast, Th2 cells facilitated humoral immune

response and inhibited cellular immune response

which caused progressive infection. There were cross

regulatory cytokines. Th1 released IFN- which down

regulated Th2. Th2 released IL-4, IL-5, and IL-10

which inhibited and down regulated Th1 and

macrophages. (Williams et al., 2012; Abulafia &

Vignale, 2001).

Studies had demonstrated that Nigella sativa

extract had immune-modulator effect. Nigella sativa

had strong potentiation effect on cellular immunity

(mediated by T cell), as well as suppression effect on

humoral immunity which was mediated by B

lymphocytes. A study which was conducted more

than a decade ago, showed that Nigella sativa could

increase human immunity when was used regularly.

Most of subjects who administered Nigella sativa for

4 weeks showed increasing T CD4 to CD8 ratio by

55% and increasing natural killer cell function by

30% (Salem, 2005).

Boskabady et al. studied about Nigella sativa

immune-modulator effects in albumin desensitized

guinea pigs and observed the increase of IFN- and

the decrease of IL-10, suggested that Nigella sativa

had inhibition effect on Th2 cell and its cytokines and

stimulation effect on Th1 and its cytokines

(Boskabady, 2011). Study on cytomegalo-virus

infected murine showed that serum IFN- level was

associated with undetected virus on the 10

day

(Salem, 2000). Study on allergic rhinitis patients

showed that supplementation with Nigella sativa 2

gram daily for 30 days would increase PMN

fagocytosis activity and intracellular killing (Isik et

al., 2010).

In lepromatous leprosy, immune response against

M. leprae was dominated by T-suppressor cells

(CD8

, CD28

), with only small amount of CD4

Naive T cells commonly could not be activated

immediately. In this type of leprosy, high T2 activity

also observed, which should increase IL-4, IL-5, and

IL-10 productions. These cytokines would stimulate

B lymphocytes transformation into antibody-

secreting cells which produced antibody.(Bryceson et

al., 1990; Williams & Kupper, 2012; Abulafia &

Vignale, 2001) Therefore, Nigella sativa

supplementation to MB leprosy patients

administering MDT-WHO would improve patients’

prognosis.

It was possible to increase Nigella sativa clinical

efficacy and effect on lipid metabolism by means of

increasing the dose. However, it would increase the

gastrointestinal side effect. Daily 40 mg/kg Nigella

sativa administration was well tolerated by adults and

children. There was no side effect observed, except

for children who received 80 mg/kg dose (Kalus et

al., 2003) Other toxicity study demonstrated that 1

g/kg Nigella sativa supplementation for 28 days did

not elevate liver enzyme and did not cause toxicity

effect on the liver function (Dollah et al., 2013).

There were some limitations in this study. There

was inhomogeneity of MDT duration at baseline due

to the difficulty of finding new untreated cases as the

study subjects. In addition, in Donorojo Hospital,

Reitz serum examination were only performed on

serum from right and left ear lobules, whereas this

study only included patients from Donorejo Hospital.

The other limitation of this study was the study only

gave one supplementation dose to subjects.

5 CONCLUSIONS

This study had not proved Nigella sativa effect on

decreasing BI and MI of MB leprosy patients.

However, there was significant difference of average

MI and BI decline between treatment and control

group. Further studies should consider the subjects

homogeneity especially for MDT duration, enroll

new untreated cases only, use various Nigella sativa

doses, and be performed in larger field.

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Effect of Nigella Sativa Extract on Bacteriology Parameter of Multibacillar Leprosy Patients Administering MDT-WHO: Three Months

Observation