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Study of the in vitro antimicrobial activity of harmine, harmaline and their derivatives
Journal of Ethnvpharmucology, Elsevier Scientific
289
35 (1992) 289-294
Publishers Ireland
Ltd.
Short Communication
Study of the in vitro antimicrobial activity of harmine, harmaline and their derivatives Aqeel Ahmad”, Khursheed Ali Khana, Sabiha Sultanaa, Bina S. Siddiquib, Sabira Begumb, Shaheen Faizib and Salimuzzaman Siddiquib ‘Department
of Microbiology
and ‘HEJ Research Institute of Chemistry, (Accepted
Peganum harmala L. (Zygophyllaceae), commonly known as harmal, grows wild in the semiarid regions of IndoPakistan subcontinent, Iran and Africa. Its different parts have been used in traditional systems of medicine for the treatment of a variety of human ailments (Dymock et al., 1890; Chopra et al, 1956). Peganum harmala is known to contain four main alkaloids, namely harmaline (Ci3Hi40N2) reported by Goebel, harmine (Ci3H120N2) isolated by Fritsche, harmalol (Ci2Hi20NZ) noted by Goebel and prepared by Fischer from harmaline, and peganine (C,,Hi20Nz) obtained by Merk and later found to be identical with vasicine isolated earlier by Hooper (Henry, 1949). Harmal alkaloids have been reported to possess hypotensive activity (Codding, 1983), while earlier the seeds have been shown to have a marked physiological activity (Elger, 1928) and have been used for curative purposes (El-Saad and EI-Rifaie, 1980). Preliminary work on the antimicrobial study of the crude ethanolic extract of seeds of Peganum harmala was perfomed by different groups of workers (Ross et al., 1980; Al-Sharma and co-
Correspondence
to: Aqeel
0378-8741/92/$05.00
Ahmad,
of Karachi,
0
Department
Karachi-75270,
of Microbi-
Pakistan.
1992 Elsevier Scientific
Printed and Published in Ireland
(Pakistan)
August 30 1991)
Introduction
ology, University
University of Karachi, Karachi-75270
Publishers Ireland
worker, 1979 and 198 1) who found that the extract possessed antimicrobial activity. The present study deals with the in vitro antimicrobial activity of harmine, harmaline and their derivatives tetrahydroharmine, harmol, harmalol and tetrahydroharmol against 12 Gramnegative and 11 Gram-positive bacteria and 16 species of fungi including 8 dermatophytes. Material and Methods Extraction
of P. harmala alkaloids
Harmine and harmaline (harmidine) were isolated from the seeds using the procedure described by Siddiqui (1962). Tetrahydroharmine was prepared by reduction of harmaline with zinc/HCl following the procedure reported earlier (Siddiqui et al., 1983). Preparation
of harm01 and harmalol
Harm01 and harmalol were prepared from harmine and harmaline, respectively, on refluxing the base with HI under the conditions reported previously (Siddiqui, 1962). Hydrochlorides
of harmol and harmalol
The hydrochlorides of harm01 and harmalol were prepared by treating the methanolic solution of the respective bases with freshly prepared ethereal solution of HCl. Ltd
290
Ham-dine
Harm01
Harmalol
Tetrahydroharmine
Preparation of tetrahydroharmol and its hydrochloride
To a solution of 1 g harmalol in 10% aqueous hydrochloric acid, zinc dust was gradually added with occasional shaking. The reaction mixture was heated on a water bath until the yellow colour of the solution disappeared. Unreacted zinc was filtered off and the filtrate ammoniated with prior addition of ammonium chloride, and the liberated base extracted with ethyl acetate. The ethyl acetate solution was treated with ethereal HCl and freed of the solvent. The colourless hydrochloride thus obtained formed flowers of needles on recrystallization from methanol.
Tetrahydroharrnol
pared in concentrations ranging from 50-500 &ml. Terbinafine in a concentration of 0.01-25 &ml was also used as a control antifungal antibiotic. Slants of each concentration were inoculated with standard amount of inoculum in triplicate and incubated at 29°C for l-3 weeks. The inhibitory effect was recorded at the end of the period. Results The results of the present study are presented in Tables 1 and 2. Discussion and Conclusions
Antibacterial activity
The antibacterial activity of harmine, harmaline and their derivatives was determined by the broth incorporation method using tryptone soya broth (Oxoid). CFU (107-10’) were inoculated in 5 ml tryptone soya broth tubes, in triplicate, containing varying concentrations (50-500 &ml) of the alkaloids and (0.01-100 pg/ml) of gentamicin (used as a control antibiotic), and incubated at 37°C for 24 h. The minimal inhibitory concentration (MIC) was recorded. Antifungal activity
Sabouraud dextrose agar (Oxoid) slants of harmine, harmaline and their derivatives were pre-
The present work represents an in vitro antimicrobial study of harmine, harmaline and their derivatives. These alkaloids were tested against 11 Gram-positive and 12 Gram-negative bacteria and 16 species of fungi, which included 8 dermatophytes, 6 filamentous fungi and 2 Candida species. Harmine was found to have antibacterial activity against both Gram-positive and Gram-negative bacteria. Apart from Vibrio cholerae, Salmonella schottmuelleri, Streptococcus pyogenes and S. faecalis, all the bacteria were inhibited at a concentration range of 100-500 &ml of harmine. The results are shown in Table 1. Harmaline and harm01 also showed antibac-
iNHIBITORY
Vibrio cholerae
Shigella dysenteriae Sh. flexneri Sh. sonnei
paratyphi A S. schottmuelleri Serratia marcesens
Proteus vulgaris Pseudomonas aeruginosa Salmonella
hydrophila Escherichia coii Klebsielia pneumonicre
G-&e Aeromonas
S. pyogenes
Staph. epi~rm~is Streptoco~~ faecalis S. h&is
B. subtilis Corynebacterium hofwnnii C. xerosis Sat&a &tea Staphylococ&~ aureus Staph. citreus
Grtm-psitive Baciiha pmilus
Cultures
MINIMAL
TABLE 1
250 >500
100
100 >500
250 250 250 250
500 500 500 500
200 250 >500
>500 500
250
>500 500
250
250
100
>500 200
2.50 >500
500 500
500 >500
500 500
250
250 250
200 250 >500
500 100 250
250 250 250
250 500
300 200
100 250
300 300 500
250 100 300
200 >500
250 100 500
100
250
Harmol
HARMALINE
>500 >SOO >500
>m >soo >soo
4 10 0.5
>SOO
>500 >500 >500
>500
>500
5 0.5
>500 >500 >500 >500
>500 >500 500 >500
>.500 >.500 >.500 500
1
0.5
1.5 2.5 >500 >500 >500 >500
>500
>soo
1 1.5
5
0.01 1
>soo
>500
>500 z-500
0.01 0.5 t0
0.1 0.05 0.5
0.05 0.01 0.05
Gen tamicin
BACTERIA
>500
>.500
>.500
>soo
500 >.500 >500
>SOO >500 >500
500 >!SOO >500
AGAINST
Tetrahydroharmol
500 500 >SOO
Tetrahydrohartnine
AND THEIR DERIVATIVES
>500 HO0
500
>500 >500
500 >sOO
>500
>500 >500
>sOO
>soo
500
>SOO
500 500
>500 >500
>soo
Harmalol
(MIC) OF HARMINE,
Harmaline
250 100
100
100
Harmine
MIC in &ml
CONCENTRATION
M
2
>500 >500
+++
+++
+++ +++
Fusarium moniliformi F. oxysporiwn
Ctidp Candiaia albicans C. tropicalis >500 >500
z-500
>500
>500
>500
>500
>500
> 500+ > 500+ >500 >500 >500
>500
>504l
>500
>500 z-500
>500
>500
>500
>500
>500
>500
> 500+ > 500+ >500++ >500 > 500+
500
500
aNot inhibited
at 45 &ml.
AND
>500 >500
>500
>500
>500
>500
>500
>500
>500 500 >500 500 > 500-t
500
500
250
Harmalol
HARMALlNE
> 500+
Harmol
OF HARMINE,
Harmaline
(MIC)
+, Slight growth; ++, moderate growth; +++, maximum growth. N.B: >500 with +/++ sign indicates slight/moderate growth but less than that of control.
>500 >500
>500
+++
>500
+++ >500
>500
+++
loo 100 100 5oa 100
100
+++
+++ +++ +++ +++ +++
100
75
+++
+++
Harmine
+++
flnw Curvellaria lunata Drechslera rostrata
Fiilltous Allescheria boydii Aspergillus
T. tonsurans T violaceum
longifusus T. mentagrophytes T. rubrum T. simii
jloccosum Microsporum canis Trichophyton
MIC in &ml
CONCENTRATION
Control
INHIBITORY
Epidermophyton
~topbytes
Cultures
MINIMAL
TABLE THEIR
>500 >500
>500
>500
>500
>500
>500
>500
>500 >500
> 500++ > 500+ >500
>500
>500
500
Tetrahydroharmine
DERIVATIVES
FUNGI
>500 >500
>500
>500
>500
>500
>500
>500
> 500+ > 500+
> 500+ > 500+ > 500++
>500
>500
> 500+
Tetrahydroharmol
AGAINST
5
10
20
10
IO
2
1
2.5
0.05 0.1 0.25 0.25 0.1
0.25
0.05
5
Terbinafine
293
terial activity to a lesser extent. The results showed that harm01 was slightly more effective than harmaline against Proteus vulgaris, Shigella dysent-
&ml.
eriae, Sh. flexneri, Sh. sonnei, Vibrio cholerae, Streptococcus pyogenes and Staphylococcus species
Tetrahydroharmol had no inhibitory effect up to 500 kg/ml. If we compare the amount of growth of dermatophytic fungi with control slants, almost all the alkaloids tested reduced the amount of growth of some fungi at higher concentration (Table 2). Gentamicin inhibited the growth of all the bacteria at 0.01-10 &ml. Most of the bacteria were inhibited at O.Ol- l~g/ml. Streptococcus faecalis and Pseudomonas aeruginosa were inhibited at 10 pg/rnl (Table 1). Terbinafine, used as a control antifungal antibiotic, inhibited all the dermatophytes at 0.050.25 &ml and the rest at l-20 pg/rnl (Table 2). All the tilamentous fungi and Candida species were found to be resistant to all the alkaloids and their derivatives tested.
(Table 1). The synthetic derivatives (harmalol, tetrahhydroharmine and tetrahydroharmol) were found to be almost ineffective at concentrations up to 500 &ml against the bacteria tested (Table 1). Al-Sharma and Mitscher (1979), while working on a broad spectrum activity of Iraqi P. harmala, found that ethanolic extract of the seeds possessed antimicrobial activity at concentrations of lOO1000 &ml; in another report (Al-Sharma et al., 1981) they mentioned that the antimicrobial activity was mainly due to harmine. Ross et al. (1980) who worked on the antibacterial activity of the ethanolic extract of the seeds of P. harmala, demonstrated that out of six fractions only two possessed antibacterial activity. From the active fraction they also isolated harmine, harmaline and harmalol by chromatography and showed that harmaline possessed comparatively more activity than harmalol but harmine appeared inactive. Alkaloids are poorly soluble or insoluble in water and form precipitate in the medium. Therefore the proper inhibitory concentration is difficult to determine by the medium incorporation method as used by Al-Sharma and Mitscher (1979). Secondly, Ross et al. (1980) used a disc diffusion method to study the antibacterial activity against 400-pg discs. However, the method they have described is not convincing. Ross et al. (1980) claimed that the antibacterial activity is due to harmaline and harmalol, not to harmine, while Al-Sharma et al. (1981) argued that the activity is mainly due to harmine. Our finding suggested that both hat-mine and harmaline possessed antibacterial activity (Table 1). Antifungal activity of these alkaloids showed that the harmine (at a concentration of 100 &ml) was effective against all the 8 dermatophytes used, while harmalol inhibited Microsporum canis, Trichophyton longtjiisus, T. rubrum and T. tonsurans at 500 &ml; and Epidermophytonjloccosum at 250
Harm01 inhibited only M. canis and T. longifusus, while tetrahydroharmine inhibited only E. floccosum at 500 &ml (Table 2).
References Al-Sharma, AS., Drake, D.L., Flynn, L., Mitscher, A., Park, Y. H., Rao, G.S.R., Simpson, A., Swayze, J.K., Veysoglu, T. and Wu, T.S. (1981) Antimicrobial agents from higher plants. Antimicrobial agents from Peganum harmala seeds. Journal of Natural Products 44, 145-747. Al-Sharma, A. and Mitscher, A. (1979) Comprehensive survey of indigenous Iraqi plants for potential economic value 1. Screening results of 327 species for alkaloids and antimicrobial agents. Journal of Natural Products 42, 633-642. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants. CSIR, New Delhi, p. 187. Codding, P.W. (1983) Structure-activity studies of B-carboiine. 1. Molecular structure and conformation of cis-3-carboxylic acid 1,2,3,4-tetrahydroharmine dihydrate. Canadian Journal of Chemistry 61, 529-532. Dymock, W., Warden, C.J.H. and Hooper, D. (1890) Pharmocographia Indicu. The Institute of Health and Tibbi Research. Republished under the auspices of Hamdard National Foundation of Pakistan, p. 71. Elger, F. (1928) Uber das Vorkommen von Harmin in einer sudamerikanischen Liane (Yage). Helvetica Chimica Acta II, 162-166. El-Saad and El-Rifaie. M. (1980) Peganum harmala, its use in certain dermatoses. International Journal of Dermatology 19, 221-222. Henry, T.A. (1949) The Plant Alkaloids. J. & A. Churchill Ltd., London,
pp. 488, 617.
294
Ross, .%A., Megalla, S.E., Bishay, D.W. and Awad, A.H. (1980) Studies for determining antibiotic substances in some Egyptian plants. Part II. Antimicrobial alkaloids from the seeds of Peganum harmala L. Fitoterapia 51, 309-312. Siddiqui, S. (1962) A reinvestigation of the alkaloidal constituents of Peganum harmala. Pakistan Journal of Scientific and Industrial Research 5, 207-211.
Siddiqui, S., Begum, S. and Siddiqui, B.S. (1983) Studies in harmine series of alkaloids. Part 11. Further new derivatives of tetrahydroharmine. Pakistan Journal of Scientific and Industrial Research 26, 53-58.
289
35 (1992) 289-294
Publishers Ireland
Ltd.
Short Communication
Study of the in vitro antimicrobial activity of harmine, harmaline and their derivatives Aqeel Ahmad”, Khursheed Ali Khana, Sabiha Sultanaa, Bina S. Siddiquib, Sabira Begumb, Shaheen Faizib and Salimuzzaman Siddiquib ‘Department
of Microbiology
and ‘HEJ Research Institute of Chemistry, (Accepted
Peganum harmala L. (Zygophyllaceae), commonly known as harmal, grows wild in the semiarid regions of IndoPakistan subcontinent, Iran and Africa. Its different parts have been used in traditional systems of medicine for the treatment of a variety of human ailments (Dymock et al., 1890; Chopra et al, 1956). Peganum harmala is known to contain four main alkaloids, namely harmaline (Ci3Hi40N2) reported by Goebel, harmine (Ci3H120N2) isolated by Fritsche, harmalol (Ci2Hi20NZ) noted by Goebel and prepared by Fischer from harmaline, and peganine (C,,Hi20Nz) obtained by Merk and later found to be identical with vasicine isolated earlier by Hooper (Henry, 1949). Harmal alkaloids have been reported to possess hypotensive activity (Codding, 1983), while earlier the seeds have been shown to have a marked physiological activity (Elger, 1928) and have been used for curative purposes (El-Saad and EI-Rifaie, 1980). Preliminary work on the antimicrobial study of the crude ethanolic extract of seeds of Peganum harmala was perfomed by different groups of workers (Ross et al., 1980; Al-Sharma and co-
Correspondence
to: Aqeel
0378-8741/92/$05.00
Ahmad,
of Karachi,
0
Department
Karachi-75270,
of Microbi-
Pakistan.
1992 Elsevier Scientific
Printed and Published in Ireland
(Pakistan)
August 30 1991)
Introduction
ology, University
University of Karachi, Karachi-75270
Publishers Ireland
worker, 1979 and 198 1) who found that the extract possessed antimicrobial activity. The present study deals with the in vitro antimicrobial activity of harmine, harmaline and their derivatives tetrahydroharmine, harmol, harmalol and tetrahydroharmol against 12 Gramnegative and 11 Gram-positive bacteria and 16 species of fungi including 8 dermatophytes. Material and Methods Extraction
of P. harmala alkaloids
Harmine and harmaline (harmidine) were isolated from the seeds using the procedure described by Siddiqui (1962). Tetrahydroharmine was prepared by reduction of harmaline with zinc/HCl following the procedure reported earlier (Siddiqui et al., 1983). Preparation
of harm01 and harmalol
Harm01 and harmalol were prepared from harmine and harmaline, respectively, on refluxing the base with HI under the conditions reported previously (Siddiqui, 1962). Hydrochlorides
of harmol and harmalol
The hydrochlorides of harm01 and harmalol were prepared by treating the methanolic solution of the respective bases with freshly prepared ethereal solution of HCl. Ltd
290
Ham-dine
Harm01
Harmalol
Tetrahydroharmine
Preparation of tetrahydroharmol and its hydrochloride
To a solution of 1 g harmalol in 10% aqueous hydrochloric acid, zinc dust was gradually added with occasional shaking. The reaction mixture was heated on a water bath until the yellow colour of the solution disappeared. Unreacted zinc was filtered off and the filtrate ammoniated with prior addition of ammonium chloride, and the liberated base extracted with ethyl acetate. The ethyl acetate solution was treated with ethereal HCl and freed of the solvent. The colourless hydrochloride thus obtained formed flowers of needles on recrystallization from methanol.
Tetrahydroharrnol
pared in concentrations ranging from 50-500 &ml. Terbinafine in a concentration of 0.01-25 &ml was also used as a control antifungal antibiotic. Slants of each concentration were inoculated with standard amount of inoculum in triplicate and incubated at 29°C for l-3 weeks. The inhibitory effect was recorded at the end of the period. Results The results of the present study are presented in Tables 1 and 2. Discussion and Conclusions
Antibacterial activity
The antibacterial activity of harmine, harmaline and their derivatives was determined by the broth incorporation method using tryptone soya broth (Oxoid). CFU (107-10’) were inoculated in 5 ml tryptone soya broth tubes, in triplicate, containing varying concentrations (50-500 &ml) of the alkaloids and (0.01-100 pg/ml) of gentamicin (used as a control antibiotic), and incubated at 37°C for 24 h. The minimal inhibitory concentration (MIC) was recorded. Antifungal activity
Sabouraud dextrose agar (Oxoid) slants of harmine, harmaline and their derivatives were pre-
The present work represents an in vitro antimicrobial study of harmine, harmaline and their derivatives. These alkaloids were tested against 11 Gram-positive and 12 Gram-negative bacteria and 16 species of fungi, which included 8 dermatophytes, 6 filamentous fungi and 2 Candida species. Harmine was found to have antibacterial activity against both Gram-positive and Gram-negative bacteria. Apart from Vibrio cholerae, Salmonella schottmuelleri, Streptococcus pyogenes and S. faecalis, all the bacteria were inhibited at a concentration range of 100-500 &ml of harmine. The results are shown in Table 1. Harmaline and harm01 also showed antibac-
iNHIBITORY
Vibrio cholerae
Shigella dysenteriae Sh. flexneri Sh. sonnei
paratyphi A S. schottmuelleri Serratia marcesens
Proteus vulgaris Pseudomonas aeruginosa Salmonella
hydrophila Escherichia coii Klebsielia pneumonicre
G-&e Aeromonas
S. pyogenes
Staph. epi~rm~is Streptoco~~ faecalis S. h&is
B. subtilis Corynebacterium hofwnnii C. xerosis Sat&a &tea Staphylococ&~ aureus Staph. citreus
Grtm-psitive Baciiha pmilus
Cultures
MINIMAL
TABLE 1
250 >500
100
100 >500
250 250 250 250
500 500 500 500
200 250 >500
>500 500
250
>500 500
250
250
100
>500 200
2.50 >500
500 500
500 >500
500 500
250
250 250
200 250 >500
500 100 250
250 250 250
250 500
300 200
100 250
300 300 500
250 100 300
200 >500
250 100 500
100
250
Harmol
HARMALINE
>500 >SOO >500
>m >soo >soo
4 10 0.5
>SOO
>500 >500 >500
>500
>500
5 0.5
>500 >500 >500 >500
>500 >500 500 >500
>.500 >.500 >.500 500
1
0.5
1.5 2.5 >500 >500 >500 >500
>500
>soo
1 1.5
5
0.01 1
>soo
>500
>500 z-500
0.01 0.5 t0
0.1 0.05 0.5
0.05 0.01 0.05
Gen tamicin
BACTERIA
>500
>.500
>.500
>soo
500 >.500 >500
>SOO >500 >500
500 >!SOO >500
AGAINST
Tetrahydroharmol
500 500 >SOO
Tetrahydrohartnine
AND THEIR DERIVATIVES
>500 HO0
500
>500 >500
500 >sOO
>500
>500 >500
>sOO
>soo
500
>SOO
500 500
>500 >500
>soo
Harmalol
(MIC) OF HARMINE,
Harmaline
250 100
100
100
Harmine
MIC in &ml
CONCENTRATION
M
2
>500 >500
+++
+++
+++ +++
Fusarium moniliformi F. oxysporiwn
Ctidp Candiaia albicans C. tropicalis >500 >500
z-500
>500
>500
>500
>500
>500
> 500+ > 500+ >500 >500 >500
>500
>504l
>500
>500 z-500
>500
>500
>500
>500
>500
>500
> 500+ > 500+ >500++ >500 > 500+
500
500
aNot inhibited
at 45 &ml.
AND
>500 >500
>500
>500
>500
>500
>500
>500
>500 500 >500 500 > 500-t
500
500
250
Harmalol
HARMALlNE
> 500+
Harmol
OF HARMINE,
Harmaline
(MIC)
+, Slight growth; ++, moderate growth; +++, maximum growth. N.B: >500 with +/++ sign indicates slight/moderate growth but less than that of control.
>500 >500
>500
+++
>500
+++ >500
>500
+++
loo 100 100 5oa 100
100
+++
+++ +++ +++ +++ +++
100
75
+++
+++
Harmine
+++
flnw Curvellaria lunata Drechslera rostrata
Fiilltous Allescheria boydii Aspergillus
T. tonsurans T violaceum
longifusus T. mentagrophytes T. rubrum T. simii
jloccosum Microsporum canis Trichophyton
MIC in &ml
CONCENTRATION
Control
INHIBITORY
Epidermophyton
~topbytes
Cultures
MINIMAL
TABLE THEIR
>500 >500
>500
>500
>500
>500
>500
>500
>500 >500
> 500++ > 500+ >500
>500
>500
500
Tetrahydroharmine
DERIVATIVES
FUNGI
>500 >500
>500
>500
>500
>500
>500
>500
> 500+ > 500+
> 500+ > 500+ > 500++
>500
>500
> 500+
Tetrahydroharmol
AGAINST
5
10
20
10
IO
2
1
2.5
0.05 0.1 0.25 0.25 0.1
0.25
0.05
5
Terbinafine
293
terial activity to a lesser extent. The results showed that harm01 was slightly more effective than harmaline against Proteus vulgaris, Shigella dysent-
&ml.
eriae, Sh. flexneri, Sh. sonnei, Vibrio cholerae, Streptococcus pyogenes and Staphylococcus species
Tetrahydroharmol had no inhibitory effect up to 500 kg/ml. If we compare the amount of growth of dermatophytic fungi with control slants, almost all the alkaloids tested reduced the amount of growth of some fungi at higher concentration (Table 2). Gentamicin inhibited the growth of all the bacteria at 0.01-10 &ml. Most of the bacteria were inhibited at O.Ol- l~g/ml. Streptococcus faecalis and Pseudomonas aeruginosa were inhibited at 10 pg/rnl (Table 1). Terbinafine, used as a control antifungal antibiotic, inhibited all the dermatophytes at 0.050.25 &ml and the rest at l-20 pg/rnl (Table 2). All the tilamentous fungi and Candida species were found to be resistant to all the alkaloids and their derivatives tested.
(Table 1). The synthetic derivatives (harmalol, tetrahhydroharmine and tetrahydroharmol) were found to be almost ineffective at concentrations up to 500 &ml against the bacteria tested (Table 1). Al-Sharma and Mitscher (1979), while working on a broad spectrum activity of Iraqi P. harmala, found that ethanolic extract of the seeds possessed antimicrobial activity at concentrations of lOO1000 &ml; in another report (Al-Sharma et al., 1981) they mentioned that the antimicrobial activity was mainly due to harmine. Ross et al. (1980) who worked on the antibacterial activity of the ethanolic extract of the seeds of P. harmala, demonstrated that out of six fractions only two possessed antibacterial activity. From the active fraction they also isolated harmine, harmaline and harmalol by chromatography and showed that harmaline possessed comparatively more activity than harmalol but harmine appeared inactive. Alkaloids are poorly soluble or insoluble in water and form precipitate in the medium. Therefore the proper inhibitory concentration is difficult to determine by the medium incorporation method as used by Al-Sharma and Mitscher (1979). Secondly, Ross et al. (1980) used a disc diffusion method to study the antibacterial activity against 400-pg discs. However, the method they have described is not convincing. Ross et al. (1980) claimed that the antibacterial activity is due to harmaline and harmalol, not to harmine, while Al-Sharma et al. (1981) argued that the activity is mainly due to harmine. Our finding suggested that both hat-mine and harmaline possessed antibacterial activity (Table 1). Antifungal activity of these alkaloids showed that the harmine (at a concentration of 100 &ml) was effective against all the 8 dermatophytes used, while harmalol inhibited Microsporum canis, Trichophyton longtjiisus, T. rubrum and T. tonsurans at 500 &ml; and Epidermophytonjloccosum at 250
Harm01 inhibited only M. canis and T. longifusus, while tetrahydroharmine inhibited only E. floccosum at 500 &ml (Table 2).
References Al-Sharma, AS., Drake, D.L., Flynn, L., Mitscher, A., Park, Y. H., Rao, G.S.R., Simpson, A., Swayze, J.K., Veysoglu, T. and Wu, T.S. (1981) Antimicrobial agents from higher plants. Antimicrobial agents from Peganum harmala seeds. Journal of Natural Products 44, 145-747. Al-Sharma, A. and Mitscher, A. (1979) Comprehensive survey of indigenous Iraqi plants for potential economic value 1. Screening results of 327 species for alkaloids and antimicrobial agents. Journal of Natural Products 42, 633-642. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants. CSIR, New Delhi, p. 187. Codding, P.W. (1983) Structure-activity studies of B-carboiine. 1. Molecular structure and conformation of cis-3-carboxylic acid 1,2,3,4-tetrahydroharmine dihydrate. Canadian Journal of Chemistry 61, 529-532. Dymock, W., Warden, C.J.H. and Hooper, D. (1890) Pharmocographia Indicu. The Institute of Health and Tibbi Research. Republished under the auspices of Hamdard National Foundation of Pakistan, p. 71. Elger, F. (1928) Uber das Vorkommen von Harmin in einer sudamerikanischen Liane (Yage). Helvetica Chimica Acta II, 162-166. El-Saad and El-Rifaie. M. (1980) Peganum harmala, its use in certain dermatoses. International Journal of Dermatology 19, 221-222. Henry, T.A. (1949) The Plant Alkaloids. J. & A. Churchill Ltd., London,
pp. 488, 617.
294
Ross, .%A., Megalla, S.E., Bishay, D.W. and Awad, A.H. (1980) Studies for determining antibiotic substances in some Egyptian plants. Part II. Antimicrobial alkaloids from the seeds of Peganum harmala L. Fitoterapia 51, 309-312. Siddiqui, S. (1962) A reinvestigation of the alkaloidal constituents of Peganum harmala. Pakistan Journal of Scientific and Industrial Research 5, 207-211.
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