Inhibition of human pregnancy plasma diamine oxidase with alkaloids of Argemone mexicana--berberine and sanguinarine.
The adulteration of edible oil with Argemone oil is responsible for the clinical syndrome of epidemic dropsy. Some features of epidemic dropsy-flushing, erythema, tachycardia and cutaneous oedema suggest a hyperactivity of histamine. Antihistamincs-Hl receptor blockers-have been used with a variable degree of success in epidemic dropsy. But the mechanism of excessive histamine activity has not been well-understood in this disease of food adulteration.
The genus Argemone is composed of twenty-eight species, with a number of sub-species and varieties also being recognized. The species common in India is Argemone mexicana; the seeds of this plant resemble the seeds of the edible oil-mustard oil-used in the North and East India. The genera Argemone and Papaver are considered to be the most recent developments of the family Papaveracea. The evidence that papaverine potentiates histamine action and inhibits diamine oxidase suggested the possibility to one of the authors (A.B.V.), that the alkaloids of the related genus Argemone-berberine and sanguinarine may have a similar effect. The present paper reports positive findings of a significant inhibition in vitro of human plasma diamine , oxidase with berberine and sanguinarine-two principal alkaloids of Argemone mexicana.
Pregnant women in the second and third trimester were selected at random from the out-patient clinics at the Yale New Haven Hospital and at Dr. R. N. Cooper Municipal General Hospital, Bombay. At the time of selection, women with bad obstetric history or toxaemia of pregnancy were excluded from the study.
Collection and Preparation of Plasma
Five ml. of venous blood was obtained in a heparinized B-D Vacutainer or bulb, immediately centrifuged, and the supernatant plasma was frozen and stored at -10°C. At the time of the enzyme assay, plasma was thawed at room temperature. Five-tenths ml. of plasma was poured into a column (Kontes Glass Company No. F-1026-A) containing Permutit (Fisher Scientific Company); the permutit bed measured 60 x 7 mm. Five ml. of distilled water was added when the plasma meniscus entered the resin. For the enzyme assay, 1 ml. of the coloured effluent was collected.
Diamine Oxidase Assay15
The enzyme assay was carried out by incubation in glass-stoppered test tubes in a Dubnoff metabolic shaker at 37°C. The following mixture was pre-incubated for 10 minutes: prepared plasma, 0.25 ml; phosphate buffer, pH 6.9 and 0.1 M, 1.4 ml; and distilled water, 0.25 ml. The addition of substrate 0.1 ml. started the reaction; 0.1 ml of substrate consisted of putrescine hydrochloride (Calbiochem) and 14C-putrescine 1, 4-dihydrochloride (New England Nuclear) dissolved in distilled water to a final concentration of 11.9 µ moles putrescine base and 4.63 x 106 d.p.m. of 14C-putrescine per ml. The alkaloids-berberine hydrochloride (Unichem) and sanguinarine nitrate (Koch light)) were dissolved in water and 0.1 ml added to the pre-incubation mixture in serial dilutions with appropriate volume corrections. Protein content of the plasma was assayed before and after treatment.
After one-hour's incubation, the tubes were transferred to a boiling water bath for one minute, to stop the enzyme reaction. Control tubes, prepared identically, were heated to boiling at 0 rather than at 60 minutes. After cooling, Na2CO3, 200 mg was added to each tube. The mixtures were extracted twice with toulene-phosphor solution and the radioactivity was counted with a B-liquid scintillation spectrometer. Increasing concentration of the substrate-putrescine was used from 75 µgm, to study the effect of 1 x 10-6M concentration of berberine and sanguinarine. The effect of increasing concentration of pyridoxal phosphate (3.7 x 10-6M to 3.7 x 10-2 M) was also studied on berberine inhibition.
Calculations: The results were calculated as follows:
(c.p.m.60 - c.p.m.0) x P initial x 1.190 = µ moles of the products formed/ml of plasma
P final x (4.63 x 105) x 0.91 x 0.57
where c.p.m.60 and c.p.m.0 are the counts per minute observed after 60 and 0 minutes, respectively; P initial is the protein content of 1 ml. of untreated plasma and P final is the protein content of 0.25 ml. of Permutit-treated plasma added to the incubation mixture; and 0.91 and 0.57 represent correction factors for 91% extraction efficiency and 57 per cent counting efficiency, respectively. 1.19 represents the final concentration of putrescine in µ mols; 4.63 x 105 represents the d.p.m. per ml. of 14C-putrescine. The results were considered as microgram of product (? 1 pyrolline) formed per ml. of plasma. The inhibition by alkaloids is expressed as per cent inhibition, taking the basal D.A.O. activity as 100% active. No special statistical tests are applied as the results show a good dose-response curve and a dramatic inhibition of diamine oxidase by the alkaloids.
[Table - 1] shows the per cent inhibition of plasma diamine oxidase at different molar concentrations of berberine hydrochloride and sanguinarine nitrate. More than 50% of enzyme activity was inhibited by berberine and sanguinarine at 5 x 106 M concentration. The higher concentrations of both the alkaloids inhibited more than 90% of the diamine oxidase activity. The magnitude of inhibition is comparable with both alkaloids. There is a linear relationship between the concentration of the alkaloid and the degree of enzyme inhibition.
[Table - 2] shows the effect of increasing concentration of substrate on the inhibition of diamine oxidase by berberine (1 x 10-6 M) and sanguinarine (1 x 10-6 M). The presence of increasing concentration of substrate still permitted ~ 50% inhibition of diamine oxidase. However, with sanguinarine the increasing concentration of putrescine did partly neutralize the inhibitory effect.
Pyridoxal phosphate, a co-factor of diamine oxidase, was added to the pre incubation mixture at three concentrations-3.7 x 10-6 M, 3.7 x 10-5 M and 3.7 x 10-4 M. [Table - 3] shows that pyridoxal phosphate did not prevent the inhibition of diamine oxidase with berberine (5 x 10-6 M).
In a report covering 119 cases of epidemic dropsy, the symptom of redness over the oedematous part was observed in 75% of cases. Superficial flush and patchy oedema has been accompanied by local tenderness also. The histopathological study of the human skin also showed dilatation and proliferation of capillaries. It was theoretically proposed that over-production of histamine may be a factor responsible for some symptoms and signs.
However, no definitive studies of histamine metabolism have been reported in epidemic dropsy. Intense capillary congestion of ovaries in epidemic dropsies has been reported from India. The vascular changes produced by Argemone oil have been studied in rats.,  The clinical skin lesions were ascribed to excretion of alkaloids through the skin. Even clinical epidemic dropsy was induced in prisoners with contaminated edible oil. In those days of clinical investigative freedom, the emphasis was more on symptom-evolution and less on mechanisms. Water concentration and dilution tests, urea clearance and P.A.H. excretion test were carried out in three patients with normal results. The authors favoured the mechanism of oedema to be a fluid-losing angiopathy. They had not studied the histamine excretion in their patients. The pharmacological study of berberine has been of great interest to Indian scientists for many years; this is primarily because many berberine-containing plants are used in indigenous medicine. The hypotension induced by berberine was not ascribed to histamine liberation. Berberine blocks the stimulant action of histamine, acetylcholine, bradykinin etc. in the isolated guinea pig ileum. However, there was not much evidence suggesting the histaminase or diamine oxidase inhibiting action of berberine. The usefulness of berberine as an antibacterial and antidiarrhoeal agent has been proposed by several investigators.,  However, in epidemic dropsy diarrhoea is a common symptom. Berberine has also been reported to form a complex with DNA and may lead to diverse effects.
In the present study it was shown that berberine and sanguinarine at very low concentrations significantly inhibit diamine oxidase or histaminase. This would lead to a decreased catabolism of histamine and some of the clinical features of epidemic dropsy may be caused by this effect. However, it is desirable to show an increased excretion of histamine in patients with epidemic dropsy. Antihistaminics have been used in the treatment of epidemic dropsy with variable success. If an increased excretion of histamine is demonstrated in this condition, treatment with H2-receptor antagonist-cimetidine may prove to be beneficial in addition to Hl-receptor antagonists.
A competition of berberine with pyridoxal phosphate in the tryptophanase systems with E. coli has been demonstrated. However, in the present study pyridoxal phosphate did not significantly antagonize the 'in vitro' inhibition of plasma diamine oxidase. But it is difficult to extrapolate the results to the clinical situation. Studies with vitamin B6 in epidemic dropsy may provide interesting data.
Sanguinarine is quite a toxic alkaloid. The ubiquitousness of this alkaloid in many plants has raised problems of epidemiologic importances in relation to malignancy, glaucoma and other diseases. The inhibitory effect of sanguinarine on diamine oxidase adds another dimension to several pharmacological actions of this alkaloid. An important point for consideration is the wide-spread recommendation of berberine and sanguinarine-containing plants in indigenous system of medicine. Argemone mexicana has been used for many indications in India e.g. abortifaciant, purgative, antisyphilitic, antipruritic, etc. It is desirable that awareness is generated about the potential hazards of the plant.
In pregnancy, the plasma diamine oxidase-histaminase-is significantly raised. The enzyme is probably released from the placenta. The foetus produces tremendous amounts of histamine. Histaminase would protect- the mother from the effects of histamine. It has been claimed that in high-risk pregnancy, histaminase is low. It is interesting to note that if epidemic dropsy occurs in pregnant women it commonly results in still-birth or abortion.
We thank Dr. F. C. Redlich, Ex-Dean, Yale University School of Medicine and Dr. C. K. Deshpande, Dean, K.E.M. Hospital, Bombay, for the permission to use the clinical material. We thank Mrs. Giselle Chapo, Miss Doris Watts and Mrs. Uma Prakash for expert technical assistance. We thank Prof. R. S. Grewal, Director, CIBA-GEIGY Research Center for encouragement and guidance.