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In vitro and in vivo bronchorelaxant effect in guinea pigs of “joshina” — A herbal polypharmaceutical
183
Journal
of Ethnopharmacology, 26 (1989) 183 - 187 Elsevier Scientific Publishers Ireland Ltd.
Short Communication
IN VITRO AND IN VIVO BRONCHORELAXANT GUINEA PIGS OF “JOSHINA” - A HERBAL POLYPHARMACEUTICAL
EFFECT
IN
K. KHETERPAL, T. KHANNA and R.B. ARORA Department of Pharmacology, Institute Nagar, New Delhi 110062 lIndiar
of History of Medicine
and Medical Research,
Hamdard
(Accepted November lo,19881
Introduction The common cold, catarrh, cough and associated respiratory complaints in response to seasonal variation are the commonest of human ailments. This is probably the major area wherein extensive self-medication is practised and wherein the greatest variety of drugs are indiscriminately used. Presently marketed syrups contain antihistaminic, antibiotic, expectorant, cough suppressant, analgesic and antipyretic constituents in an attempt to provide a multi-approach treatment. The dangers of such polypharmaceuticals cannot be overlooked as they frequently result in iatrogenesis. Therefore, there is a need to introduce effective remedies from nature which might be more in tune with the human body. In the Unani System of medicine, “joshina” is given to patients with cough, colds and catarrh and has been found to be effective without adverse effects. “Joshina” is a herbal preparation consisting of a concentrated aqueous extract prepared from seven plant ingredients (Table 11. Empirically some of the ingredients have been found to be effective in cough and cold. Malva rotundifolk is reported to have demulcent, antifungal and antiviral activity (Babber et al., 1982; Tripathi et al., 19821. In addition, it is prescribed in cases of bronchitis and cough (Nadkarni, 19761. Viola odorata and Onosma bracteatum are also claimed to have a demulcent action (Chopra et al., 19561. Glycyrrhiza glabra is reputed to be effective as an expectorant and demulcent in the inflammation of bronchial tubes (Anonymous, 19761. Experimentally glycyrrhizin, a glycoside obtained from Glycyrrhiza glubra, has been shown to produce anti-inflammatory effect on formaldehyde-induced rat paw edema in adrenalectomized rats (Gujral et al., 19611. A study using the clinically used form of “joshina” reported 03788741/89/$02.10 0 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
184
TABLE
1
COMPOSITION
OF “JOSHINA”
Plant name
Family
Part used (dried)
Composition
Viola odorata Linn. Onosma bracteatum Wall. Althaea officinalis Linn. Malva rotundifoliu Linn. Glycyrrhiza glabra Linn. Zizyphus vulgaris Lam. Cordia lutgolia Roxb.
Violaceae Boraginaceae Malvaceae Malvaceae Papilionaceae Rhamnaceae Boraginaceae
Flowers Seeds Seeds Seeds Rhizome Fruits Fruits
1.1 1.5 1.1 1.5 1.67 1.1 3.0
(g)
antitussive, expectorant and anti-inflammatory activity (Kheterpal et al., 1987). In the present paper, “joshina” has been further explored for bronchorelaxant activity in vivo and in vitro using guinea pigs. Materials and methods
Extract composition The sample of “joshina” obtained from Hamdard Trust Laboratories, Delhi, had the following composition: each dose of 10 ml represents the concentrated hot aqueous extract from the amount of ingredients specified in Table 1. Bronchoreluxant
activity
Guinea pigs (400 - 500 g) of either sex purchased from the All India Institute of Medical Sciences, New Delhi, were sacrificed after stunning. The trachea was removed and trimmed of excess tissue. A spirally cut 4-cm piece or a tracheal chain was suspended in an organ bath containing Krebs-Hensleit solution at 3’7 + l°C. The solution was bubbled with 95% oxygen + 5% CO, and allowed to equilibrate under a load of 1 g for 90 min with washing every 10 min. A cumulative concentration-response curve was then obtained with histamine (3.3 x 1O-7to 5.3 x lo+ M bath concentration) or acetylcholine (5.0 x lo* to 3.2 x lob4 M). The effect of each dose was recorded for 4 min. The same cumulative response to histamine and acetylcholine was repeated in presence of 40 and 80 d/ml (bath concentration) of “joshina” and EC,, values calculated and compared. In vivo bronchoconstriction was produced in guinea pigs (400-600 g) by exposing them to a lo/o histamine diphosphate aerosol using a standard nebulizer manufactured by Techno, Lucknow. Compressed air at a constant pressure of 180 mmHg was used to operate the nebulizer (Broadbent and Bain, 1964). Separate groups of guinea pigs were treated with “joshina” (4 ml/kg, p.o.1 or mepyramine (20 mglkg, i.p.1 1 h and 30 min prior to aerosol exposure, respectively. Preconvulsive time (the time taken by the animal from the time of exposure to the appearance of convulsion) was recorded and compared.
185
Results “Joshina” itself relaxed spirally cut bronchial muscle in a dose-dependent manner with 40, 80 and 160 &ml bath concentrations producing 0.18 +: 0.03, 0.48 + 0.06 and 0.86 -c 0.09 cm (mean + S.E.M., N = 10) of relaxation. Cumulative concentration-response curves to histamine and acetylcholine were established in tracheal chains by increasing the bath concentration in a logarithmic manner with maximal responses obtained at 5.3 x lO+ M and 8.0 x 10V5M, respectively (Figs. 1 and 21. “Joshina” (40 rl/mll shifted the logarithmic concentration-response curves in response to histamine and acetylcholine to the right (Figs. 1 and 21 with the EC, of histamine increased from 6.6 x 10T7M to 2.0 x 10q M and of acetylcholine from 1.8 x 10e5 M to 1.6 x 10F4M. The concentration-response curve of acetylcholine was shifted in a non-parallel fashion. In the in vivo experiments, the mean control preconvulsive time in guinea pigs exposed to 1% histamine aerosol was 75.8 s and this was increased to 117.0 and 191.3 s with l- and 2-h pre-treatments with “joshina” (4 ml/kg, p.o.1, respectively. Mepyramine (20 mglkg, i.p., 30 min prior to exposure) delayed convulsions 234.5 s after histamine aerosol (Table 2).
3.3
6.6 HISTAMINE
13.2
26.4
52.0
(x10-7M 1
Fig. 1. Effect of ‘joshina’ (40 d/ml) on the log concentration-response curve and EC,, of histamine on guinea pig tracheal chain: 0, histamine alone; A, ‘joshina’ + histamine.
186
100. 3
go-
=
60.
E w 5
70. 60 *
I
50--
$
40-
\ L
30-
g
20.
I ,
10.
I I
W
I
1
1
0.5
1
L,
I 2
ACETYLCHOLINE
0
4
16
32
blC%
Fig. 2. Effect of ‘joshina’ (40 &nll on the log concentration-response curve and EC,, of acetylcholine on guinea pig tracheal chain: 0, acetylcholine alone; A, ‘joshina’ + acetylcholine.
TABLE 2 EFFECT OF “JOSHINA” ON HISTAMINE AEROSOL-INDUCED A RESULT OF BRONCHOSPASM IN GUINEA PIGS
PRECONVULSIVE
P values indicate a comparison to control preconvulsive time using the Student’s values represent the mean f S.E.M. of six determinations per treatment. Pretreatment (dosage)
Pretreatment time(h)
Preconvulsive time (sl
TIME AS
t-test. Tabular
P
Saline control
_
75.8 f
8.1
_
Mepyramine 120 mglkg, i.p.1 Joshina (4 ml/kg, p.0.) Joshina (4 ml/kg, p.o.1
0.5
234.5 +
3.8
< 0.001
1
117.0 f
8.3
< 0.01
2
191.3 2 22.8
< 0.01
187
Discussion
“Joshina”, a herbal polypharmaceutical preparation has been reported to have high potential as an antitussive, anti-inflammatory and expectorant (Kheterpal et al., 19871. Since bronchoconstriction is a common characteristic of the chronic obstructive lung diseases, “joshina” was evaluated for its bronchorelaxant effect in vitro and in vivo in the present study. In vitro “joshina” alone produced a progressive relaxation of bronchial muscle using perfused spiral tracheal muscle. In addition, the EC, values of histamine and acetylcholine were increased when “joshina” was pre-incubated using the guinea pig tracheal chain technique. The results clearly indicated a non-specificity for acetylcholine receptors. In vivo, “joshina” significantly increased preconvulsive time in response to histamine aerosol in guinea pigs. These in vitro and in vivo studies suggest its use in chronic obstructive disorders. However, before making a definitive statement, a large number of experiments need to be done to delineate its mechanism of action. It seems worthwhile to subject the drug to more detailed studies due to worldwide interest in the development of clinically effective herbal drugs. Acknowledgements
The authors are grateful to Hakeem Abdul Hameed, History of Medicine and Medical Research, New Delhi, The technical assistance of T.A. Farooqui, A.R. Baghdadi ing assistance of Mr. Syed Masroor Hassan are gratefully
President, Institute of for facilities provided. and Sant Raj and typacknowledged.
References Anonymous (19761 MedicinaE Plunts of Indti Vol. 1, Indian Council of Medical Research, New Delhi, p. 438. Babber, O.P., Joshi, M.M. and Madan, R. (19821 Evaluation of plants for antiviral activity. Indian Journal of Medical Research ISuppL176,54 - 65. Broadbent, J.L. and Bain, W.A. (19641 Histamine antagonists. In: D.R. Laurence and A.L. Bacharach (Eds.1, Evaluation of Drug Activities: Pharmacometrics, Academic Press, New York, p. 491. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (19561 GZossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, p. 180 and 255. Gujral, M.L., Sareen, K., Phukan, D.P. and Amma, M.K.P. (19611 Antiarthritic activity of glycyrrhizin in adrenalectromized rats. Indian Journal of Medical Science 15,625 - 629. Kheterpal, K., Khanna, T., Arora, R.B. and Siddiqui, H.H. (19871 Study of potential of Unani polypharmaceutical preparation Joshina as antitussive and expectorant in experimental models. Indian Journal of Pharmacology 19, 200 - 204. Nadkarni, A.K. (19761 Indian Materiu Medica, Vol. 1, Popular Prakashna, Bombay, p. 763. Tripathi, R.N., Pandey, D.K., Tripathi, N.N. and Dixit, S.N. (19821 Antifungal activity in pollens of some higher plants. Zndiun Phytopathology 35(2), 345- 348.
Journal
of Ethnopharmacology, 26 (1989) 183 - 187 Elsevier Scientific Publishers Ireland Ltd.
Short Communication
IN VITRO AND IN VIVO BRONCHORELAXANT GUINEA PIGS OF “JOSHINA” - A HERBAL POLYPHARMACEUTICAL
EFFECT
IN
K. KHETERPAL, T. KHANNA and R.B. ARORA Department of Pharmacology, Institute Nagar, New Delhi 110062 lIndiar
of History of Medicine
and Medical Research,
Hamdard
(Accepted November lo,19881
Introduction The common cold, catarrh, cough and associated respiratory complaints in response to seasonal variation are the commonest of human ailments. This is probably the major area wherein extensive self-medication is practised and wherein the greatest variety of drugs are indiscriminately used. Presently marketed syrups contain antihistaminic, antibiotic, expectorant, cough suppressant, analgesic and antipyretic constituents in an attempt to provide a multi-approach treatment. The dangers of such polypharmaceuticals cannot be overlooked as they frequently result in iatrogenesis. Therefore, there is a need to introduce effective remedies from nature which might be more in tune with the human body. In the Unani System of medicine, “joshina” is given to patients with cough, colds and catarrh and has been found to be effective without adverse effects. “Joshina” is a herbal preparation consisting of a concentrated aqueous extract prepared from seven plant ingredients (Table 11. Empirically some of the ingredients have been found to be effective in cough and cold. Malva rotundifolk is reported to have demulcent, antifungal and antiviral activity (Babber et al., 1982; Tripathi et al., 19821. In addition, it is prescribed in cases of bronchitis and cough (Nadkarni, 19761. Viola odorata and Onosma bracteatum are also claimed to have a demulcent action (Chopra et al., 19561. Glycyrrhiza glabra is reputed to be effective as an expectorant and demulcent in the inflammation of bronchial tubes (Anonymous, 19761. Experimentally glycyrrhizin, a glycoside obtained from Glycyrrhiza glubra, has been shown to produce anti-inflammatory effect on formaldehyde-induced rat paw edema in adrenalectomized rats (Gujral et al., 19611. A study using the clinically used form of “joshina” reported 03788741/89/$02.10 0 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
184
TABLE
1
COMPOSITION
OF “JOSHINA”
Plant name
Family
Part used (dried)
Composition
Viola odorata Linn. Onosma bracteatum Wall. Althaea officinalis Linn. Malva rotundifoliu Linn. Glycyrrhiza glabra Linn. Zizyphus vulgaris Lam. Cordia lutgolia Roxb.
Violaceae Boraginaceae Malvaceae Malvaceae Papilionaceae Rhamnaceae Boraginaceae
Flowers Seeds Seeds Seeds Rhizome Fruits Fruits
1.1 1.5 1.1 1.5 1.67 1.1 3.0
(g)
antitussive, expectorant and anti-inflammatory activity (Kheterpal et al., 1987). In the present paper, “joshina” has been further explored for bronchorelaxant activity in vivo and in vitro using guinea pigs. Materials and methods
Extract composition The sample of “joshina” obtained from Hamdard Trust Laboratories, Delhi, had the following composition: each dose of 10 ml represents the concentrated hot aqueous extract from the amount of ingredients specified in Table 1. Bronchoreluxant
activity
Guinea pigs (400 - 500 g) of either sex purchased from the All India Institute of Medical Sciences, New Delhi, were sacrificed after stunning. The trachea was removed and trimmed of excess tissue. A spirally cut 4-cm piece or a tracheal chain was suspended in an organ bath containing Krebs-Hensleit solution at 3’7 + l°C. The solution was bubbled with 95% oxygen + 5% CO, and allowed to equilibrate under a load of 1 g for 90 min with washing every 10 min. A cumulative concentration-response curve was then obtained with histamine (3.3 x 1O-7to 5.3 x lo+ M bath concentration) or acetylcholine (5.0 x lo* to 3.2 x lob4 M). The effect of each dose was recorded for 4 min. The same cumulative response to histamine and acetylcholine was repeated in presence of 40 and 80 d/ml (bath concentration) of “joshina” and EC,, values calculated and compared. In vivo bronchoconstriction was produced in guinea pigs (400-600 g) by exposing them to a lo/o histamine diphosphate aerosol using a standard nebulizer manufactured by Techno, Lucknow. Compressed air at a constant pressure of 180 mmHg was used to operate the nebulizer (Broadbent and Bain, 1964). Separate groups of guinea pigs were treated with “joshina” (4 ml/kg, p.o.1 or mepyramine (20 mglkg, i.p.1 1 h and 30 min prior to aerosol exposure, respectively. Preconvulsive time (the time taken by the animal from the time of exposure to the appearance of convulsion) was recorded and compared.
185
Results “Joshina” itself relaxed spirally cut bronchial muscle in a dose-dependent manner with 40, 80 and 160 &ml bath concentrations producing 0.18 +: 0.03, 0.48 + 0.06 and 0.86 -c 0.09 cm (mean + S.E.M., N = 10) of relaxation. Cumulative concentration-response curves to histamine and acetylcholine were established in tracheal chains by increasing the bath concentration in a logarithmic manner with maximal responses obtained at 5.3 x lO+ M and 8.0 x 10V5M, respectively (Figs. 1 and 21. “Joshina” (40 rl/mll shifted the logarithmic concentration-response curves in response to histamine and acetylcholine to the right (Figs. 1 and 21 with the EC, of histamine increased from 6.6 x 10T7M to 2.0 x 10q M and of acetylcholine from 1.8 x 10e5 M to 1.6 x 10F4M. The concentration-response curve of acetylcholine was shifted in a non-parallel fashion. In the in vivo experiments, the mean control preconvulsive time in guinea pigs exposed to 1% histamine aerosol was 75.8 s and this was increased to 117.0 and 191.3 s with l- and 2-h pre-treatments with “joshina” (4 ml/kg, p.o.1, respectively. Mepyramine (20 mglkg, i.p., 30 min prior to exposure) delayed convulsions 234.5 s after histamine aerosol (Table 2).
3.3
6.6 HISTAMINE
13.2
26.4
52.0
(x10-7M 1
Fig. 1. Effect of ‘joshina’ (40 d/ml) on the log concentration-response curve and EC,, of histamine on guinea pig tracheal chain: 0, histamine alone; A, ‘joshina’ + histamine.
186
100. 3
go-
=
60.
E w 5
70. 60 *
I
50--
$
40-
\ L
30-
g
20.
I ,
10.
I I
W
I
1
1
0.5
1
L,
I 2
ACETYLCHOLINE
0
4
16
32
blC%
Fig. 2. Effect of ‘joshina’ (40 &nll on the log concentration-response curve and EC,, of acetylcholine on guinea pig tracheal chain: 0, acetylcholine alone; A, ‘joshina’ + acetylcholine.
TABLE 2 EFFECT OF “JOSHINA” ON HISTAMINE AEROSOL-INDUCED A RESULT OF BRONCHOSPASM IN GUINEA PIGS
PRECONVULSIVE
P values indicate a comparison to control preconvulsive time using the Student’s values represent the mean f S.E.M. of six determinations per treatment. Pretreatment (dosage)
Pretreatment time(h)
Preconvulsive time (sl
TIME AS
t-test. Tabular
P
Saline control
_
75.8 f
8.1
_
Mepyramine 120 mglkg, i.p.1 Joshina (4 ml/kg, p.0.) Joshina (4 ml/kg, p.o.1
0.5
234.5 +
3.8
< 0.001
1
117.0 f
8.3
< 0.01
2
191.3 2 22.8
< 0.01
187
Discussion
“Joshina”, a herbal polypharmaceutical preparation has been reported to have high potential as an antitussive, anti-inflammatory and expectorant (Kheterpal et al., 19871. Since bronchoconstriction is a common characteristic of the chronic obstructive lung diseases, “joshina” was evaluated for its bronchorelaxant effect in vitro and in vivo in the present study. In vitro “joshina” alone produced a progressive relaxation of bronchial muscle using perfused spiral tracheal muscle. In addition, the EC, values of histamine and acetylcholine were increased when “joshina” was pre-incubated using the guinea pig tracheal chain technique. The results clearly indicated a non-specificity for acetylcholine receptors. In vivo, “joshina” significantly increased preconvulsive time in response to histamine aerosol in guinea pigs. These in vitro and in vivo studies suggest its use in chronic obstructive disorders. However, before making a definitive statement, a large number of experiments need to be done to delineate its mechanism of action. It seems worthwhile to subject the drug to more detailed studies due to worldwide interest in the development of clinically effective herbal drugs. Acknowledgements
The authors are grateful to Hakeem Abdul Hameed, History of Medicine and Medical Research, New Delhi, The technical assistance of T.A. Farooqui, A.R. Baghdadi ing assistance of Mr. Syed Masroor Hassan are gratefully
President, Institute of for facilities provided. and Sant Raj and typacknowledged.
References Anonymous (19761 MedicinaE Plunts of Indti Vol. 1, Indian Council of Medical Research, New Delhi, p. 438. Babber, O.P., Joshi, M.M. and Madan, R. (19821 Evaluation of plants for antiviral activity. Indian Journal of Medical Research ISuppL176,54 - 65. Broadbent, J.L. and Bain, W.A. (19641 Histamine antagonists. In: D.R. Laurence and A.L. Bacharach (Eds.1, Evaluation of Drug Activities: Pharmacometrics, Academic Press, New York, p. 491. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (19561 GZossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, p. 180 and 255. Gujral, M.L., Sareen, K., Phukan, D.P. and Amma, M.K.P. (19611 Antiarthritic activity of glycyrrhizin in adrenalectromized rats. Indian Journal of Medical Science 15,625 - 629. Kheterpal, K., Khanna, T., Arora, R.B. and Siddiqui, H.H. (19871 Study of potential of Unani polypharmaceutical preparation Joshina as antitussive and expectorant in experimental models. Indian Journal of Pharmacology 19, 200 - 204. Nadkarni, A.K. (19761 Indian Materiu Medica, Vol. 1, Popular Prakashna, Bombay, p. 763. Tripathi, R.N., Pandey, D.K., Tripathi, N.N. and Dixit, S.N. (19821 Antifungal activity in pollens of some higher plants. Zndiun Phytopathology 35(2), 345- 348.