Tubercle (1990) 71, 293-29.5 0 Longman Group UK Ltd 1990
Tuberculostatic inemis Linn.)
activity
of henna (Lawsonia
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.
Materials and methods
Introduction
The discovery of the tubercle bacillus by Robert Koch in 1882 intensified the search for an effective and specific drug against tuberculosis. Today, many bactericidal and bacteriostatic drugs are used in combination all over the world with dramatic 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 intermittent therapy is required. In the presently available 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
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 slopes of Lowenstein Jensen (LJ) and on to another five LJ slopes containing the preparation at a concentration 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.
293
294
SHARMA
In vivo study
Table 1 The numbers of untreated and treated mice
A total of 20 guinea pigs with an average weight of 500 g (range 490-606 g) were inoculated intramuscularly in the groin with 0.2 ml of a suspension containing 30000 M. tuberculosis HX7Rv by means of a tuberculin syringe. The number of bacilli was calculated by McFarlands nephelometer standards for bacterial counts [l]. The animals were kept in four separate cages, five animals 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 observation 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 inoculation. The mice were kept for a period of 15 weeks.
with tuberculous lesions and the numbers with various organs involved
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 animals became sluggish in their activity from the third day of inoculation and their appetite was markedly reduced. Patchy loss of hair and erection 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
Group
Untreated Treated
Total mice
Mice with lesions
Lung
10
10 (100%) 9 (30%)
9 6
30
of animals
Organs
involved
Kidney
Lymph nodes
10 4
10 9
Liver
9 4
examination, tubercles were observed on the surface 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 necessary 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 animal 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 tuberculosis. 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
295
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 diaminodiphenyl sulphone (dapsone). Later, after discovery 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 naphthoquinones, 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 referred to as ‘S. sasakii’, and the alkaloid capharanthine 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 chimensis). 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
include Actuea spicatu, Galipea several resins and pro-
officinalis, Piper cubeba,
polis (bee glue). In general, though, the use of herbs to treat tuberculosis, although attempted in many countries, has not met with success. By contrast, this study has revealed that an aqueous extract of henna has strong in vitro and in vivo tuberculostatic acitivity. Further evaluation of this herb is therefore indicated.
References 1. Woffls G. McFarlands nephelometer standards for bacterial counts. In Sonnenwirth AC, Jarett L, eds. Grudwohl’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 purposes (Lister memorial lecture). Brit Med J 1945; 2: 635642. 3. Hinshaw HC, Garland LH. D&eases of the Chest. Philadelphia: 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 common Chinese herbs on Mycobacterium tuberculosis. Chinese 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.
Tuberculostatic inemis Linn.)
activity
of henna (Lawsonia
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.
Materials and methods
Introduction
The discovery of the tubercle bacillus by Robert Koch in 1882 intensified the search for an effective and specific drug against tuberculosis. Today, many bactericidal and bacteriostatic drugs are used in combination all over the world with dramatic 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 intermittent therapy is required. In the presently available 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
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 slopes of Lowenstein Jensen (LJ) and on to another five LJ slopes containing the preparation at a concentration 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.
293
294
SHARMA
In vivo study
Table 1 The numbers of untreated and treated mice
A total of 20 guinea pigs with an average weight of 500 g (range 490-606 g) were inoculated intramuscularly in the groin with 0.2 ml of a suspension containing 30000 M. tuberculosis HX7Rv by means of a tuberculin syringe. The number of bacilli was calculated by McFarlands nephelometer standards for bacterial counts [l]. The animals were kept in four separate cages, five animals 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 observation 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 inoculation. The mice were kept for a period of 15 weeks.
with tuberculous lesions and the numbers with various organs involved
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 animals became sluggish in their activity from the third day of inoculation and their appetite was markedly reduced. Patchy loss of hair and erection 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
Group
Untreated Treated
Total mice
Mice with lesions
Lung
10
10 (100%) 9 (30%)
9 6
30
of animals
Organs
involved
Kidney
Lymph nodes
10 4
10 9
Liver
9 4
examination, tubercles were observed on the surface 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 necessary 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 animal 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 tuberculosis. 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
295
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 diaminodiphenyl sulphone (dapsone). Later, after discovery 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 naphthoquinones, 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 referred to as ‘S. sasakii’, and the alkaloid capharanthine 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 chimensis). 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
include Actuea spicatu, Galipea several resins and pro-
officinalis, Piper cubeba,
polis (bee glue). In general, though, the use of herbs to treat tuberculosis, although attempted in many countries, has not met with success. By contrast, this study has revealed that an aqueous extract of henna has strong in vitro and in vivo tuberculostatic acitivity. Further evaluation of this herb is therefore indicated.
References 1. Woffls G. McFarlands nephelometer standards for bacterial counts. In Sonnenwirth AC, Jarett L, eds. Grudwohl’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 purposes (Lister memorial lecture). Brit Med J 1945; 2: 635642. 3. Hinshaw HC, Garland LH. D&eases of the Chest. Philadelphia: 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 common Chinese herbs on Mycobacterium tuberculosis. Chinese 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.
