Tubercle (1990) 71, 293-29.

5
0 Longman Group UK Ltd 1990

Tuberculostatic activity of henna (Lawsonia

inemis Linn.)

V. K. SHARMA*

S. M. S. Medical College, Hospital for Chest and Tuberculosis, Jaipur, India

Summary-The tuberculostatic activity of the herb henna Mawsonia irterrnis Linn.) was tested
in vitro and in viva. On Lowenstein Jensen medium, the growth of tubercle bacilli from sputum
and of Mycobacferium tuberculosis H3, Rv was inhibited by 6 yglml of the herb. In viva studies
on guinea pigs and mice showed that the herb at a dose of 5 mg/kg body weight led to
significant resolution of experimental tuberculosis following infection with M. tuberculosis
H3 ,Rv.

Introduction Materials and methods

The discovery of the tubercle bacillus by Robert
Koch in 1882 intensified the search for an effec-
tive and specific drug against tuberculosis. Today,
many bactericidal and bacteriostatic drugs are
used in combination all over the world with dra-
matic positive results and tuberculosis has
become a curable disease. Even with the advent
of such powerful SpCCifiC antimicrobials, a long
duration Of treatment with either daily Or inter-
mittent therapy is required. In the presently avail-
able regimens of 6-9 months duration, the
patients frequently stop taking drugs as soon as
the symptoms are ameliorated and the treatment
is therefore discontinued. We therefore wish to
find a specific drug which can cure tuberculosis in
a much shorter time. In this study we report the
tuberculostatic effect of a preparation derived
from henna (Lawsonia inermis Linn.), in vitro on
Lowenstein Jensen medium and in vivo in guinea
pigs and mice.
*Correspondence to: Professor V. K. Sharma, 4 Ja 6, Jawahar
Nagar, Jaipur - 302004, India
293
Preparation of the herbal drug
oh e d rug was prepared from henna (Lawsonia
inermis Linn.), a shrub indigenous to the Arabic
peninsula and Iran and widely cultivated in India.
A total of 250 g of powdered leaves was added to
1 1 of water, boiled for 15 min over low heat,
cooled and filtered. The filtrate was reduced to
l/3 of its original volume by heating on a water
bath. It was stored ready for use in airtight bottles
and its weight/ml was measured against water.
In vitro study
Five sputum samples, positive for tubercle bacilli,
were inoculated on to five drug-free control slo-
pes of Lowenstein Jensen (LJ) and on to another
five LJ slopes containing the preparation at a con-
centration of 6 ug/ml and incubated for 6 weeks.
In another study, Mycobacterium tuberculosis
strain Hs7Rv was likewise inoculated on to five
drug-free and five drug-containing LJ slopes and
incubated for 8 weeks.
294
In vivo study
A total of 20 guinea pigs with an average weight
of 500 g (range 490-606 g) were inoculated intra-
muscularly in the groin with 0.2 ml of a suspen-
sion containing 30000 M. tuberculosis HX7Rv by
means of a tuberculin syringe. The number of
bacilli was calculated by McFarlands nephelo-
meter standards for bacterial counts [l]. The ani-
mals were kept in four separate cages, five ani-
mals to a cage. Two cages served as controls. The
animals in the experimental group were given the
preparation of henna in a dose of 5 mg/kg body
weight intramuscularly daily for 15 days starting
from the day when the first of the control animals
died. All these animals were kept under observa-
tion for 15 weeks from the date of inoculation.
Animals, in either group, that survived for 15
weeks were killed and their viscera were sent for
histopathological examination. In another study,
40 mice were inoculated with the same dose of
H37R~ in the tail. Four animals were kept in each
cage. Mice surviving for 40 days were given the
drug, 5 mg/kg body weight daily by intramuscular
injection, for 1 month from the 40th day of inocu-
lation. The mice were kept for a period of 15
weeks.
Results
In vitro
After 6 weeks incubation all control media slopes
had more than 20 colonies with the typical
appearance of tubercle bacilli. On microscopic
examination, these colonies were acid-fast. None
of the slopes containing 6 pg/rnl of henna showed
any bacterial growth after 6 weeks and the water
of condensation was also negative for acid-fast
bacilli.
In vivo
Guinea pigs: Weight loss was observed in all the
guinea pigs: the weight dropped to an average of
300 g (range: 25@--350 g) over 2 weeks. All ani-
mals became sluggish in their activity from the
third day of inoculation and their appetite was
markedly reduced. Patchy loss of hair and erec-
tion of hair follicles all over the body was
observed from the 5th day of inoculation in
almost all animals. All 10 control animals died
within 5 weeks of inoculation: i.e. on days 23,24
(2), 26 (2), 27, 28, 31 (2) and 33. On macroscopic
SHARMA
Table 1 The numbers of untreated and treated mice
with tuberculous lesions and the numbers of animals
with various organs involved
Organs involved
Total Mice with Lymph
Group mice lesions Lung Kidney nodes Liver
Untreated 10 10 (100%) 9 10 10 9
Treated 30 9 (30%) 6 4 9 4
examination, tubercles were observed on the sur-
face of spleen, liver and lungs and the lymph
nodes showed necrotic areas. On microscopic
examination caseating necrosis with epithelioid
and giant cells were observed in all tissues.
In the experimental group, in which therapy
was started on day 23, all 10 animals started
improving by the fourth day of the initiation of
drug therapy. They regained their appetite and in
some cases it was increased so that it was neces-
sary to serve more food. They gained weight with
most regaining their normal weight of around
500 g within 3 weeks. The weight of three animals
exceeded their pre-experimental weight: one ani-
mal weighed 600 g and two animals weighed more
than 560 g. The remaining seven returned to their
normal weight of 500 g by the end of the 3 weeks.
Hair loss ceased and all animals became active
again within 4 weeks. All survived for 15 weeks,
after which they were killed and their viscera
were examined histopathologically. There was no
macroscopic or microscopic evidence of tubercu-
losis.
Mice: All the mice were sluggish in their activity
from the second week onward. Loss of hair and
appetite was observed in all animals and in four
cases there was an ulcer at the site of inoculation.
A total of 10 animals died during the first 35 days;
i.e. on days 21 (2), 22 (3), 33 (2), 34 (2) and 35.
The remaining 30 animals were treated from the
40th day for a period of 30 days. All these animals
survived for a period of 15 weeks, after which
they were killed. Nine of these animals had
macroscopical and microscopical evidence of
tuberculosis (Table 1).
Discussion
In 1945 Florey [2] reported some experiments on
the activity of certain antibiotics on experimental
TUBERCULOSTATIC ACTIVITY OF HENNA
tuberculosis.In none of these experimentswere Davey [9]. These
tuberculous lesions of animal arrested, though the
growth of tubercle bacilli was inhibited in culture.
The first drug which arrested tuberculous lesions
in guinea pigs was the compound diamino-
diphenyl sulphone (dapsone). Later, after discov-
ery of streptomycin by Waksman, quantitative
methods for determining the tuberculostatic
activity of antibiotics were developed [3].
Henna has been used to treat skin infections
such as Tinea and it is known to have antibacterial
properties which have been attributed to naph-
thoquinones, including lawsone [4]. Several other
agents with activity against the tubercle bacillus
have been isolated from plants. Japanese workers
isolated alkaloids from a vine named Stephonia
upharantha, and from a wistaria like plant re-
ferred to as ‘S. sasakii’, and the alkaloid capha-
ranthine has been used in the treatment and
prophylaxis of tuberculosis in Japan [5]. Chinese
workers investigated the activity of a series of
local plants against the tubercle bacillus and
Wang [6] reported prominent tuberculostatic
activity in an extract of coptis root (Coptis chi-
mensis). This activity lies in the alkaloid berberin
sulphate. In the Indian medical traditions of
Ayurveda and Unani, many plants are considered
to be useful in the treatment of tuberculosis [7,8].
In addition, Aerous calamus, Alpinia galanga,
Cucurbita maxima, Peposis, Eucalyptus citroidors
and Crewla populipholia are known to have some
activity against M. tuberculosis [S]. Some other
plants and plant-derived materials with such
activity have been described by Grange and
295
include Actuea spicatu, Galipea
officinalis, Piper cubeba, several resins and pro-
polis (bee glue).
In general, though, the use of herbs to treat
tuberculosis, although attempted in many coun-
tries, has not met with success. By contrast, this
study has revealed that an aqueous extract of
henna has strong in vitro and in vivo tuberculosta-
tic acitivity. Further evaluation of this herb is
therefore indicated.
References
1. Woffls G. McFarlands nephelometer standards for bacte-
rial counts. In Sonnenwirth AC, Jarett L, eds. Grud-
wohl’s, clinical laboratory methods and diagnosis. 8th ed.,
Vol. 2. St. Louis: CV Mosby Co. 1980.
2. Florey HW. Use of micro-organisms for therapeutic pur-
poses (Lister memorial lecture). Brit Med J 1945; 2: 635-
642.
3. Hinshaw HC, Garland LH. D&eases of the Chest. Phila-
delphia: Saunders Co.
4. Wren RC. Potter’s new cyclopaedia of botanical drugs
and preparations. Revised edition p 143. Saffron Walden:
CW Daniel Co. Ltd. 1988.
5. Nishikawa H. Screening tests for antibiotic action of plant
extracts. Jap J Exp Med 1949; 20: 337-349.
6. Wang VFL. In vitro antibacterial activity of some com-
mon Chinese herbs on Mycobacterium tuberculosis. Chi-
nese Med J 1950; 68: 169-172.
7. Chopra RN, Chopra IC, Handa KL, Kapur LD, eds.
Chopras’s Indigenous Drugs of India 2nd ed. Calcutta:
UN Dhur & Sons Pvt, Ltd 1958.
8. Indian Council of Medical Research. Medicinal Plants of
lndia Vol. 1 and 2. Delhi: ICMR 1976.
9. Grange JM, Davey RW. Detection of antituberculous
aetivity in plant extracts. J Appl Bact 1990; 68: 587-591.