PLEASE SCROLL DOWN FOR ARTICLE This article was downloaded by: [Das, Mukul][CSIR eJournals Consortium] On: 14 August 2008 Access details: Access Details: [subscription number 779749116] Publisher Informa Healthcare Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Critical Reviews in Toxicology Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713401167 Clinicoepidemiological, Toxicological, and Safety Evaluation Studies on Argemone Oil Mukul Das a ; Subhash K. Khanna a a Dyes and Food Adulterant Toxicology Laboratory, Industrial Toxicology Research Centre, Lucknow, India Online Publication Date: 01 January 1997 To cite this Article Das, Mukul and Khanna, Subhash K.(1997)'Clinicoepidemiological, Toxicological, and Safety Evaluation Studies on Argemone Oil',Critical Reviews in Toxicology,27:3,273 — 297 To link to this Article: DOI: 10.3109/10408449709089896 URL: http://dx.doi.org/10.3109/10408449709089896 Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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PLEASE SCROLL DOWN FOR ARTICLE
This article was downloaded by: [Das, Mukul][CSIR eJournals Consortium]On: 14 August 2008Access details: Access Details: [subscription number 779749116]Publisher Informa HealthcareInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
Critical Reviews in ToxicologyPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713401167
Clinicoepidemiological, Toxicological, and Safety Evaluation Studies onArgemone OilMukul Das a; Subhash K. Khanna a
a Dyes and Food Adulterant Toxicology Laboratory, Industrial Toxicology Research Centre, Lucknow, India
Online Publication Date: 01 January 1997
To cite this Article Das, Mukul and Khanna, Subhash K.(1997)'Clinicoepidemiological, Toxicological, and Safety Evaluation Studies onArgemone Oil',Critical Reviews in Toxicology,27:3,273 — 297
To link to this Article: DOI: 10.3109/10408449709089896
URL: http://dx.doi.org/10.3109/10408449709089896
Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf
This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.
Critical Reviews in Toxicology, 27(3):273-297 ( 1997)
Clinicoepidemiological, Toxicological, and Safety Evaluation Studies on Argemone Oil
Mukul Das* and Subhash K. Khanna Dyes and Food Adulterant Toxicology Laboratory, Industrial Toxicology Research Centre, Post Box 80, M.G. Marg, Lucknow-India, 226 001
*To whom all correspondence should be addressed.
ABSTRACT: Consumption of oil extracted from accidental or deliberate contamination of argemone seed to mustard seed is known to pose a clinical condition popularly referred to as Epidemic Dropsy. Several outbreaks of Epidemic Dropsy have occurred in the past in India as well as in Mauritius, Fiji Island, and South Africa. Clinico-epidemiological manifestations of argemone oil poisoning include vomiting, diarrhea, nausea, swelling of limbs, erythema, pitting edema, breathlessness, etc. In extreme cases, glaucoma and even death due to cardiac arrest have been encountered. The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Histopathological studies suggest that liver, lungs, kidney, and heart are the target sites for argemone oil intoxication. Studies have shown to elucidate the cocarcinogenic potential of argemone oil that can be correlated with the binding of sanguinarine with a DNA template. Pharmacological response in intestine revealed immediate stimulation of tone and peristaltic movements of the gut in the sanguinarine-treated animals. Argemone oiVSanguinarine caused a decrease in hepatic glycogen levels which may be due to the activation of glycogenolysis leading to an accumulation of pyruvate in the blood of Epidemic Dropsy cases. The increase in pyruvate levels causes uncoupling of oxidative phosphorylation leading to breathlessness, as observed in patients. Sanguinarine has been shown to inhibit Na+,K+-ATPase activity of different organs such as brain, heart, liver, intestine, and skeletal muscle, which may be due to the interaction with the glycoside receptor site on ATPase enzyme, thereby causing a decrease in the active transport of glucose. Argemone oil/alkaloid showed a Type I1 binding spectra with hepatic cytochrome P-450 (P-450) protein, thereby causing loss of P-450 content and an impairment of phase I and phase I1 enzymes. A green fluorescent metabolite of sanguinarine, benzacridine was detected in the milk of grazing animals. The delayed appearance of this metabolite in urine and feces of experimental animals suggests the slow elimination of the alkaloid. Argemone oil enhances hepatic microsomal and mitochondria1 lipid peroxidation, indicating that these two organelles are the sites of membrane damage. Furthermore, studies suggest that singlet oxygen and hydroxyl radical are involved in argemone oil toxicity. Several bioantioxidants show protective effect in argemone oil-induced toxicity in experi- mental animals. The line of treatment in argemone-intoxicated epidemics has so far been only symptomatic, and specific therapeutic measures are still lacking, although it has been suggested that diuretics, bioantioxidants, steroids, vitamins, calcium- and protein-rich diet had some beneficial effects on Epidemic Dropsy cases.
Edible oils and fats constitute an integral com- ponent of human diet. Besides providing energy, they serve as the only source of essential fatty acids, add special flavors and maintain cell mem- brane integrity.'-* The shortages of edible oils not only put economic pressure on imports but may also prompt the unscrupulous traders to indulge
in malpractices, thereby increasing the exent of adulteration.
Adulteration of edible oils with cheaper edi- ble or nonedible oils escapes visible detection due to complete miscibility and nonapparent changes in physical characteristics of the parent oil. A de- cennial survey study on edible oil quality revealed that the quality of mustard oil is occasionally be- ing tampered by mixing cheap oils and toxic adul-
terants. including Argemone oil and a nonpermitted fat-soluble synthetic dye, Butter yellow.’ In the early 1980s an epidemic desig- nated as Toxic Oil Syndrome was encountered in Spain, affecting more than 20,000 people through the ingestion of adulterated edible oil containing refined denatured rapeseed A rural market survey on mustard oil quality showed that 28% of examined samples from village markets of Uttar Pradesh were adulterated or substandard.’ In 1988, an outbreak, through the consumption of rape- seed oil adulterated with tricresyl phosphate, was encountered in Calcutta, India.* In 1990, every 4xth analyzed sample of mustard oil from
Lucknow City exhibited fluctuations from the minimum requisite standard specifications detailed in the Prevention of Food Adulteration (PFA) Act of India.y
Argemone oil can be extracted from the seeds of Argemone mexicana Linn (family Papaver- aceae), a native of the West Indies and naturalized in India. This plant grows widely (Figure 1) and is commonly known as Shailkanta in Bengal and Bharhhanda in Uttar Pradesh. It is also popularly known as Pivladhatura or Satyanashi, meaning devastating. The plant grows wild in mustard and other fields. Its seeds are black in color (Figure 2) and are similar to the dark-colored mustard seeds
FIGURE 1. Plant of Argemone mexicana showing flower, fruit, and leaves.
(Brassica nigra) in shape and size. Adulteration of argemone seeds in light yellow-colored mus- tard seeds (Brassica compestris) can easily be de- tected, but these seeds are rather difficult to visual- ize when mixed with black-colored mustard seeds. The rich oil-bearing argemone seeds yield approxi- mately 32 to 35% oil.loJ1 The physicochemical constants and the fatty acid composition of Arge- mone mexicana seed oil is given in Tables 1 and 2, respectively. Accidental or willful adulteration of edible oils with argemone oil, and comsumption of such contaminated oils, give rise to a number of clinical symptoms collectively referred to as Epidemic Dropsy. 12-19 Argemone seeds are used as a substitute because of easy availability, low
cost, and the fact that its oil has complete misci- bility with mustard
II. ALKALOIDS OF ARGEMONE OIL
The earliest report on the chemical investiga- tion of Argemone mexicana was that of Santos and Adkilen,21 who isolated protopine and ber- berine from its seeds. Mukherjee et a1.16 isolated a toxic substance, with an empirical formula, CZo H,, NO,, which was then identified as sangui- narine (Figure 3)? Later, observed that argemone oil contains at least two physiologi- cally active alkaloids. The major alkaloid, which
TABLE 1 Physiochemical Constants of Argemone mexicana Seed Oil
Constants Value Ref.
Acetyl value 22.0 24, 128 Acid value 3.5 Iodine value 49.8 Refractive Index 1.496 Saponification value 188.2
Specific gravity (30°C) 0.918 Unsaponified matter 1 . 1 %
Viscosity 8291 cal mol-I
is present in argemone oil to the extent of 87% of the total alkaloids, has a melting point of 190 to 19 l0C, and was identified as dihydrosanguinarine (Figure 3).” The other alkaloid that constitutes about 5% of the total alkaloids of argemone oil has been identified as sanguinarine. was able to extract 0.04% (w/v) sanguinarine/dihyd- rosanguinarine from Argemone mexicana seed oil, whereas Misra et al.24 could isolate the alka- loid to the extent of 0.13%. However, studies of Shenolikar et al.15 and our unpublished observa- tion indicates that the yield of alkaloids is 0.5 to 0.6% (w/v). Both the compounds, sanguinarine and dihydrosanguinarine are interconvertible by simple oxidation and reduction process. Sangui- narine alkaloid was previously isolated by Stermitz et a1.26 from Sanguinaria canadensis roots and is a member of the benzophenanthridine group of isoquinoline alkaloids. The chemical composi-
TABLE 2 Fatty Acid Composition of Argemone mexicana Seed Oil
tion of Argemone mexicana seeds and structures of the argemone alkaloids are given in Table 3 and Figure 3 , respectively. The other alkaloids, allocryptopine and coptisine have been isolated from some Argemone mexicana species.26 Bose et al.” observed that the main pharmacological ac- tivities of Argemone mexicana extracts are re- lated to the total alkaloid fraction.
H3C 0 OCH3
Berberine Protopine
Cheterythrne Coptisine
FIGURE 3. Chemical structures of alkaloids present in Argemone mexicana seed oil.
TABLE 3 Chemical Composition of Argemone mexicana Seeds: Alkaloid and Phenol Composition
Alkaloiddphenols Percentage Reference
Total alkaloids Dihydrosanguinarine Sanguinarine Berberine Protopine Cheletrythrine Coptisine
Total phenols Argemexitin
5,7-Dihydrochromone- 7-neohseperidose
111. OUTBREAKS OF EPIDEMIC DROPSY
The earliest reference to argemone oil poison- ing was made by Lyon,28 who reported four cases of poisoning in Bombay in 1877 from the use of this oil in food. Since then, Epidemic Dropsy has been reported from Bengal, Bihar, Orissa, Madhya Pradesh, Uttar Pradesh, Gujrat, Delhi, and Bombay mainly due to consumption of food cooked in argemone oil mixed mustard oil or occasionally by body massage with contaminated 0il.12,29-36 Thousands of people have been affected and many of them died during the Epidemic Dropsy. Recent- ly, there have been reports of several epidemics that occurred through consumption of argemone- adulterated edible oil, in the neighboring areas of Lucknow, India. More than 100 established poi- soning cases were encountered during 1988 to 1996 at Barabanki, Balamau (Hardoi), Bani Banthara (Unnao), Gonda, Sitapur, Sultanpur, Shahjahanpur, and Lakhimpur Kheri (unpublished observation).
Besides India, widespread epidemics have been reported from Mauritius, Fiji Islands, North- west Cape districts of South Africa, and possibly also M a d a g a ~ c a r . ~ ~ , ~ ~ Apart from South African study where the epidemic occurred through con- tamination in wheat flour, all the epidemics oc- curred through the contamination of mustard oil with argemone oil.
Several human feeding trials with argemone oil and argemone-adulterated mustard oil were
0.13 19, 134, 135 87.00 5.00 0.57 0.34 0.12 0.03
0.03 136, 137 -
~ n d e r t a k e n . ' ~ J ~ , ~ ~ - ~ ~ It was established that symp- toms similar to those of Epidemic Dropsy can be produced in human subjects by feeding argemone oil. This supported the presumption that arge- mone oil in mustard oil is responsible for Epi- demic Dropsy.
IV. CLINICAL SYMPTOMS OF EPIDEMIC DROPSY
The clinical manifestations of this disease have varied widely in different e p i d e r n i c ~ . ~ ' - ~ ~ . ~ ~ ~ On the basis of these epidemics, the following clini- cal picture has been drawn.
A. General Symptoms
The chief presenting symptom of the disease in every case was swelling of the feet and legs; both the legs were equally affected. On physical examination, the edema was found to be of the easily pitting type (known as bilateral pitting edema) (Figure 4). The swelling increased by the end of the day and after walking. Improvement was noticed after rest.35,46 The complaints and the general physical findings in Epidemic Dropsy cases are presented in Table 4 and 5, respectively. Fever, either intermittent or continuous, ranging
FIGURE 4. Patients showing characteristic pitting edema over lower limbs.
from 99 to 100.5"F was generally noted.3s Sig- nificant gastrointestinal disturbances included di- arrhea, with or without effortless vomiting, nau- sea, malena, anorexia and loss of taste, abdominal colic and vague discomfort in epigastrium were
TABLE 4 Complaints in Epidemic Dropsy Cases
1. Pain in abdomen 2. Loose stools 3. Vomitting 4. Pain in limbs 5. Burning sensation in
6. Redness in eyes 7. Swelling of feet 8. Redness on body 9. Breathlessness
10. Fever limbs and eyes 11. Weakness
o b ~ e r v e d . ~ ~ . ~ ~ Few patients had dry cough with slight expectoration, showing breathlessness on exertion. Some victims showed evidence of bron- chitis or pneumonia.42
TABLE 5 Physical Findings in Epidemic Dropsy Cases
Glaucoma has been reported to occur in about 5% of cases of Epidemic Dropsy.47 Hyperemia and a burning sensation of the eyes were observed in a few subjects. The intraocular tension was found to be raised in only a few cases. An asso- ciated high-tension glaucoma was observed in some of the victims of Epidemic Dropsy.36
C. Cutaneous Changes
Superficial flush and patchy erythema was a generalized feature. In a few cases, reddish-pur- plish blotches over lower limbs, not raised and that blanched on pressure, was an unusual feature (Figure 5).46 The face looked flushed because of marked capillary dilatation. Tender, dilated su-
perficial veins were seen in the lower limbs. Hy- perpigmentation was observed in some c a s e ~ . ~ ~ . ~ ~
D. Biopsy Findings
The essential histological changes induced by the disease in the various tissues and organs of man consist of the appearance of a large number of young and dilated capillaries. The young capillar- ies are filled with active endothelial cells contain- ing a considerable amount of protoplasm. Besides, there is a great deal of proliferation of endothelial cells around the dilated vessels.39 The pathologi- cal changes are readily seen in the skin. Skin biopsy studies revealed a capillary reaction consisting of marked proliferation of endothelial cells and dila- tion of capi l la r ie~ .~~ Reticulin staining showed that the cells were lying inside the capillary base- ment membrane. Hyperplasia of sweat glands was
FIGURE 5. An unusual feature in a victim of Epidemic Dropsy showing reddish-purple blotches over lower limbs.46
also observed in a few cases.48 According to regenerative changes in the parenchymal and kupf- Chopra and Chaudhuri, in addition to the changes already mentioned, there was indication of papil- lary and subpapillary edema resulting in flatten- ing out of the papillae. In the subcutaneous tissue, there was evidence of edema as shown by the looseness of the connective tissue fibers and an increase in the edematous collagenic fibers.
E. Cardiovascular Changes
Some of the victims of Epidemic Dropsy suf- fered from congestive cardiac failure and cardiac enlargement without decomposition. Persistent and relative tachycardia with a pulse rate usually vary- ing from 100 to 130 was observed. The most common cardiac abnormalities detected were high- voltage, short PR intervals. St-T changes (raised or depressed), T-inversions- or biphasic, and tall- peaked T waves. ’! I h
7’here was clinical and laboratory evidence of myocardial involvement and cardiac enlargement in some patients with pulmonary edema and other signs 01 cardiac failure. Arterial blood-gas studies done early during the illness showed arterial oxy- gen unsaturation and a respiratory alkalemia.49 Cardiac involvement was indicated by tachycar- dia and ballisto-cardiographic change^.^^,^*
F. Liver-Associated Manifestations
Apart from the liver being involved as a part of congestive cardiac failure, hepatomegaly as an isolated feature was observed in a few cases. The liver was tender, soft, and nonpulsatile. Jaundice or any other evidence of liver cell failure was not seen among such subjects with hepatomegaly.33,J6 Victims of Epidemic Dropsy showed no signifi- cant liver dysfunction but some showed nontender hepatornegaly.34 The pivotal feature was the dila- tation of sinusoids and central veins of the liver with the main changes occurring in the blood ves- sels which were dilated and surrounded by prolif- eration of endothelial cells.s1 Some evidence of liver cell injury was seen in the form of swelling of the cells with rarefied cytoplasm and occasion- al intracptoplasmic granular hyalin, stray focal ne- crosis, and lymphocytic infiltration and reactive
fer cells. Pregnant womenwho delivered at tern during the epidemic reportedly gave birth to still born fetuses.33
G. Hematological Changes
Serum protein estimation by paper electro- phoresis revealed normal values for total proteins in all the cases. There was a significant and con- sistent fall in serum albumin level and a rise in the amount of serum alpha, globulin.42
The plasma volume, blood volume, and avail- able thiocyanate space were estimated in patients of Epidemic Dropsy. The available thiocyanate space, the mean plasma volume, and interstitial fluid volume showed a rise. The total blood vol- ume was increased slightly, but there was a defi- nite contraction of RBC mass. The rise of plasma volume was mainly compensatory to replace the shrunken red cell volume and the total blood vol- ume therefore was not markedly altered.
A normocytic, hypochromic anaemia was a constant f e a t ~ r e . ’ ~ . ~ ~ In most of the cases. hemo- globin was below 10%. There was no leukocyto- sis. Eosinophilia (8 to 12%) was observed in 50% of the cases. Increased number of mononuclear cells were seen in 10% of the cases. Erythrocyte sedimentation rate was found to be raised. Pasricha et al.52 had earlier found that the sedimentation rate increased in every case and then observed that the increase was proportional to the severity of the disease. Blood urea nitrogen, blood uric acid, fasting blood sugar, and cholesterol were found to be well within the normal range.12
Among the electrolyte studies, a large num- ber of patients showed a reduction of serum so- dium. The majority of the readings were in the range of 123 to 132 mEq/l, normal values being 135 to 145 mEq/l. Serum potassium was normal in the majority of cases.32
V. MEDICINAL PROPERTIES OF ARGEMONE ALKALOID, SANGUINARINE
Sanguinarine is present in a large number of plants of Papaveracae and Fumaracae families.
Blood root plant (Sanguinaria canadensis L.), a monotype genus of the Papaveracae family, was used medicinally by native American Indians of many millennia. Alkaloid extract of S. canadensis have been used for over 300 years by practitio- ners of homeopathic medicine, principally as an expectorant and as an escharotic agent. Sangui- narine extract was a component in a paste used to treat accessible superficial neoplasm^.^^,^^ Studies have shown that sanguinarine possess antifungal, antibacteriophagal, antimicrobial, and antitumour a~tivity.,~-~O
VI. EXPERIMENTAL STUDIES ON ARGEMONE OWALKALOIDS
A. Short-Term Toxicity Studies
Animal feeding experiments in mice, rats, guinea pigs, rabbits, cats, monkeys, and pigs were carried out and it was observed that oil was toxic to rats and cat^.'^,^^ Pure argemone oil contains 0.5% sanguinarine alkaloid.25 Toxicity studies have been carried out using either argemone oil or the alkaloid sanguinarine. Only one study indicates that the toxicity of sanguinarine is two-and-a-half times higher than that of dihydr~sanguinarine.~~
1. Mice
An intraperitoneal injection of sanguinarine at a dose level of 40 mgkg body weight to mice was found to result in 100% mortality.61
2. Rats
The trial of different doses of argemone oil to albino rats by oral and subcutaneous routes showed that approximate LD,, value was 1.1 ml per kg and 1.3 ml per kg body weight, However, recent study has shown that the oral LD,, of sanguinarine was 1658 mgkg body weight to rats.63 Apparent toxicity signs were observed in rats fed upto 150 ppm pure sanguinrine in diet for 14 d and its treatment by gavage up to 0.6 mg per kg body weight for 30 d.63 Young albino rats fed
1 mg sanguinarine per day died within 1 week of admini~tration.~~ These results suggest that oral LD5o of argemone oil is much higher than that of 1.1 mUkg body weight as observed by Satyavati et a1.62
There was no mortality to rats treated intrap- eritoneally either with single (10 ml per kg body weight) or three doses (5 ml per kg body weight) of argemone oil. However, none of the animals receiving a second injection of argemone oil (10 ml per kg body weight) survived." Pure sangui- narine dissolved in peanut oil or ethylene glycol produced no signs of Epidemic Dropsy when in- jected to rats at a dose of 25 mg per kg body weight (equivalent to 5 ml argemone oilkg body ~ t ) . ~ , However, single intraperitoneal injection of sanguinarine (20 mg per kg body wt) was shown to cause 100% mortality to rats.23 Another study shows that LD50 of saguinarine by parenteral route was 50 mgkg body weight,64 which correlates well with the oil study."
Intramuscular injection of sanguinarine to rats at a dose of 50 mg per kg body weight did not cause any mortality.@ The intravenous LD,, value of sanguinarine was found to be 29 mg per kg body weight.63
3. Guinea Pigs
Guinea pigs did not appear to be a suitable test model for argemone oil toxicity, as they could not flourish well on mustard
4. Rabbits
Becci et al.63 showed that rabbits are also susceptible toward sanguinarine and reported an acute dermal LD,, value of greater than 200 mg per kg body weight.
5. Cats
La1 et al.39 observed growth retardation and weight loss in cats fed orally 5% argemone oil for 3 months.
Intramuscular administration and oral feed- ing of argemone oil to monkeys at a dose level of 1 .0 ml per pound of body weight for 4 weeks led to the development of edema and erythema along with distinct reddish angiomatous nodules over
Oral administration of pure sanguinarine at a dose of 1.6 mg per kg body weight to mon- keys produced no signs of Epidemic Dropsy."
7. Pigs
Oral feeding of 5% argemone oil in mustard oil for 3 months to pigs produced considerable gain in body weight.'9
B. Histopathological Observations
Animal feeding experiments to rats showed histological changes in the skin that were found to be similar to those in the skin of patients of Epi- demic Dropsy.39
'4s the chief effect of argemone oil is on the blood vessels, Chaudhuri and Chakrabarty"h showed vascular dilation in different internal or- gans in rats fed with argemone oil. They also administered varying percentages of argemone oil for 6 months to monkeys and observed that very low concentrations, up to 1 % or even 0.5% may be toxic when administered for such long periods. This clearly indicates the cumulative ef- fect of the toxicity of argemone oil.
The effect of argemone oil on the vascular system of rats was studied by Bhende6' using different oral doses over a period of time. The internal organs showed hyperemia of the capillar- ies and venules. A peculiar reaction was observed in the skin of feet ("the capillary reaction"). The capillaries looked like solid, distended cords filled with endothelial cells and almost without lumina. This lesion is considered specific for argemone oil. The capillary reaction produced by intracuta- neous injection is identical with the reaction in the skin produced by oral administration of the oil."8 Painting small quantities of oil on the skin of rats showed a similar capillary reaction. 6y
Reproduction of the symptoms of Epidemic Dropsy was. however, successful in monkeys due to the efforts of Chakravarty and Cha~dhuri .~" Monkeys showed signs of Epidemic Dropsy fol- lowing argemone oil administration either mixed with normal diet or by stomach tube. It was also reported that argemone oil, when applied to the skin, produced local erythema and cutaneous le- sions and changes in the internal organs showing that the oil is absorbed from intact healthy skin. This finding is extremely important in view of the fact that massage with mustard oil is quite popu- lar in India.
Argemone oil-feeding studies on monkeys, rats, and mice7' 72 revealed more or less similar clinical and histopathological changes. Liver from the treated rats and mice showed swollen hepato- cytes with hydropic changes. The nuclei of the cells also showed swelling and degenerative changes, all leading to necrosis. Marked prolif- eration of the veins and sinusoids separating liver cells and fatty infiltration was observed. The lungs revealed congestion and exudation of fluid and red cells into the alveoli in all species. The heart showed dilated vessels separating muscle fibers. The kidneys showed congestion of glomeruli. Dilated vessels and cellular infiltration in dermis was observed in the skin. Dilated vessels in the choroid layer of the eye were present in monkeys. Clinical symptoms like diarrhea, loss of weight, pitting edema, and cutaneous erythema were simi- lar to those observed in man.
An investigation was undertaken by Chakravarty et al.73 to study the toxicity of the argemone oil or alkaloids on the blood vessels. Intravenous or in- tramuscular injection of aqueous solution of total alkaloids (as hydrochloride) in rabbit ear showed large areas of vascularization persisting for a long time and the formation of rich intercommunicat- ing dilated plexus of vessels compared in fewer and more straight vessels in the controls. The latter showed no tendency to hemorrhage once well-formed vessels had developed, but this was consistently seen in the argemone-treated groups even up to a late stage.
Rats fed with a diet containing 5% argemone oil. calorically matched with control diet, for 60 d resulted in significant histopathological degen- erative changes in liver, lungs, kidneys, and heart
(Figure 6).74 Liver showed increase fibrosis, hy- perplasia of bile ducts, and congestion in a few portal tracts. Lungs of argemone oil-fed animals indicated congestion, thickening of interalveolar septa, and alveolar spaces were disorganized and irregular. Kidney showed vascular, glomerular congestion and patchy tubular lesions. Cardiac muscle fibers showed degenerative changes, which were more marked in auricular wall.74
Brink et al.49 claimed that dogs are easily intoxicated but appeared to develop a resistance to continuing doses of argemone oil.
C. Pharmacological Studies
Pharmacological experiments on dogs were conducted with a total alkaloid solution of hydro- chloride salt.27 While studying the blood pressure, it was found that low doses either had no effect or occasionally produced a slight rise, but a dose above 0.1 mgkg produced a definite fall in B.P. Atropine, adrenergic blocking agents, or antihistaminics did not modify the response. In the case of the heart, the total alkaloid fraction (1 .O mgkg) inhibited both auricles and ventricles in situ. It also antagonized acetylcholine and epi- nephrine response.
The alkaloid fraction, when given intrave- nously, stimulated respiration. It antagonized bar- biturate-induced respiratory depression at higher concentrations. The pharmacological response in intestine revealed immediate stimulation of tone and peristaltic movements of the gut in the treated animals. Atropine inhibited the increase in tone but did not affect the augmentory action of the alkaloid on peristalsis."
D. Carcinogenic and Mutagenic Studies
Argemone oil has been shown to be noncar- cinogenic as well as nontumorigenic over hybrid mice.75g76 However, cocarcinogenic activity of argemone oil cannot be ruled A metabolite of sanguinarine, benzacridine has been shown to produce skin tumor in mice and bladder cancer in rats.77 Because of the likely carcinogenic proper- ties and wide distribution of sanguinarine and its
metabolite, even at low concentration^,^^.^^ it is con- sidered responsible for some regional incidences of cancer, particularly esophageal and nasopharyngeal, in different parts of the world. Subramanyam et al.79 reported chromosomal aberrations such as chromo- somes erosions, breakages, fragmentation, and gaps in animals treated with argemone oil, indicating the possible mutagenic action of oil. However, sangui- narine was not shown to be mutagenic in Ames Salmonelldmicrosome assay.80 In different strains of Salmonella typhimurium, sanguinarine exhibited greater than 98% cytotoxicity at higher concentra- tion and graded decrease in cytotoxicity at each lower dose.80
E. Biochemical Aspects of Argemone- Induced Toxicity
1. Binding of Sanguinarine to DNA
Maiti et a1.81 showed that sanguinarine binds by monofunctional mode of intercalation. The binding of sanguinarine to DNA suggests the existence of more than one class of binding sites on the DNA template to which sanguinarine is bound by non-cooperative phenomenon.82 Bind- ing parameters determined from spectrophoto- metric measurements by Scatchard analysis ac- cording to an excluded site model indicate a very high specificity of sanguinarine binding of GC rich DNA.83 It may be pointed out at this stage that sanguinarine exhibits pH-dependent spectral changes. The typical spectral changes above pH 6.0 (decrease in absorption at 325 nm and in- crease in fluorescence emission intensity at 420 nm) can be explained by assuming formation of carbinolamine structure by hydroxylation of san- guinarine at C-6 in alkaline pH.
2. Effect on Enzymes
In assays carried out in vitro with rat liver post-mitochondria1 supernatant, sanguinarine was found to possess an inhibitory effect on the activ- ity of alanine and aspartate amino t ran~ferase.~~ A single i.p. dose (10 mg/kg) of sanguinarine de- creased the activities of the above enzymes in the
liver with corresponding rise in serum.85 Sangui- narine hydrochloride has also been found to inhibit the oxidation of pyruvate, lactate, and succinate from pigeon brain preparation^.^^ The observed in- hibition of pyruvate oxidation suggests the possi- bility of pyruvate accumulation in the blood of persons suffering from dropsy. A definite rise in blood pyruvate has been noticed in rats after a heavy dose of sanguinarine hydrochloride intrap- e r i t~nea l ly .~~ The inhibitory activity of argemone alkaloid depends on the reactivity of the iminium bond with nucleophilic sites, for example, the thiol groups, which may be present at the active sites of enzyme^.^^,^^
Sanguinarine has been shown to inhibit Na+, K+-ATPase from guinea pig brain and heart.86,89 Inhibition of ouabain binding by sanguinarine was time-dependent and resulted in a reduction of the equilibrium level of ouabain bound to enzyme preparation. The positive inotropic effect of san- guinarine on the heart may be due to interaction with the cardiac glycoside receptor site on Na+,K+- ATPase.88 Sanguinarine increases active Na+ efflux in intact cells from frog skeletal muscle but it inhibits the isolated Na+,K+-ATPase, presumably due to interaction with a site on the inner face of the membrane fragment. The stimulation of ac- tive Na+-efflux in the presence of ouabain may be due to interaction of sanguinarine with a site on the outer face of the membrane, perhaps the K+- activation site.YO
Studies of Cala et al.91 have shown that san- guinarine inhibits the ouabain sensitive K-Na pump and increases the downhill ouabain insen- sitive movements of K and Na in human red cells. These two effects have different temporal and con- centration dependencies and are mediated by two different chemical forms of sanguinarine. The oxi- dized charged form promptly inhibits the pump but does not affect leakage of K and Na. The re- duced, uncharged form of sanguinarine causes lysis of red cells but does not inhibit the pump. Sanguinarine also increases the conductance of bilayers formed from sheep red cell lipids. The effect is produced bythe uncharged but not by the charged form of ~anguinarine.~~ Recent studies have shown that sanguinarine (1 pmol) inhibit the transport of D-glucose (61 96) in an everted gut sac of the small intestine of rats while the transport of amino acids viz. aspartic acid, lysine and tyrosine
was unchanged.y2 Sanguinarine showed a dose dependent inhibition of intestinal and hepatic Na+,K+-ATPase in a non-competitive manner. The iminium compounds have been shown to possess varying inhibitory degree toward Na+,K+-AT- P a ~ e . ~ ~ It is suggested that sanguinarine interferes in the glucose uptake through blocking of sodium pump, via Na+,K+-ATPase thus inhibiting the ac- tive transport of glucose across intestinal bar- rier.92
Sanguinarine reduces the cholinesterase activi- ty.93 It stimulates the smooth muscles, antagonizes the action of histamine and 5-hydroxytryptamine, and has an oxytocic action like that of ~ i toc in .*~ Some features of Epidemic Dropsy (flushing, ery- thema, edema, redness, tachycardia, etc.) suggest- ed excessive histaminic activity. Sanguinarine was subsequently shown to inhibit pregnancy plasma diamine oxidase (histaminase) activity. This alka- loid may thus increase the histaminic activity by inhibiting its enzyme degrada t i~n .~~
Argemone oil feeding in rats for 3 months caused a decrease in the average albumidglobu- lin ratio and an increase in the glutamate oxaloac- etate transminase (GOT) activity in serum.61 Con- sumption of dietary intake of 5% argemone oil to rats for 60 d resulted in significant inhibition of hepatic alkaline phosphatase, GOT, and gluta- mate pyruvate transaminase activities with a con- comitant enhancement in Inhibition of RNA and DNA polymerase activities by sangui- narine has been shown by Belyaeva et aL9'
3. Role of Reactive Oxygen Species
Parenteral administration of argemone oil (5 ml per kg body weight) to rats for 3 d caused a significant increase in both nonenzymatic and NADPH-supported enzymatic hepatic lipid per- oxidation, a marker for membrane damage.'6 These studies suggest that hepatic microsomal as well as the mitochondria1 membranes are vulnerable to the peroxidative attack of argemone oil. The en- hancement of hepatic lipid peroxide formation has been shown to correlate with depletion of en- dogenous tripeptide, glutathione,' 1,74 which helps in maintaining the membrane integrity. Recent studies have shown that dimethylfuran, histidine, and beta-carotene, which are known scavengers
of singlet ~ x y g e n , ~ ’ substantially abrogated non- enzymatic NADPH and ferrous-dependent hepatic lipid pero~idat ion.’~,~~ Furthermore, scavengers of hydroxyl radical provide appreciable protection against argemone oil-induced hepatic microso- ma1 lipid peroxide formation. However, superox- ide dismutase and catalase, scavengers of super- oxide anion and hydrogen peroxide, respectively,* had no protective effect against argemone oil-medi- ated enhancement of lipid p e r o x i d a t i ~ n . ~ ~ . ~ ~ Thus, reactive oxygen species such as singlet oxygen and hydroxyl radical produced by argemone oil intoxication (Figure 7) may propagate an attack on lipid-rich membrane to cause enhanced lipid pero~idation.’~.~~ Antioxidants such as tannic acid, quercetin, butylated hydroxyanisole, butylated hydroxytoluene, alpha-tocopherol, riboflavin, or glutathione showed significant protection against argemone oil-induced hepatic microsomal lipid pero~idation.’~ Incidences of in vivo lipid peroxi- dation resulting in cellular, pathological, and physi- ological disturbances in various biomedical dis- orders such as beta-lipoproteinemia, erythropoetic protoporphyria, atherosclerosis, and inflammation are common occurrences.100 It can therefore be inferred that scavengers of reactive oxygen spe- cies as well as several anitoxidants could be use- ful against argemone oil-induced Epidemic Dropsy cases.
4. Metabolic Disposition Studies
In general, the acute or chronic exposure to argemone oil/alkaloids has been shown to cause toxic manifestations.1o1 Most of the exogenous compounds are metabolized in the body by the hepatic drug-metabolizing enzyme system. How- ever, it remains to be seen whether the parent compound or its biologically transformed me- tabolite is responsible for the major toxic effects of an alkal~id.’~’ Only during the last 2 decades, have investigators really begun to look into this aspect of toxicology of argemone oil/alkaloids.
It is now recognized that there are multiple isozymes of hepatic haem protein cytochrome P- 350 that have specificity for different sub- strates. 103~103 Single intraperitoneal administration of argemone oil (10 ml per kg body weight) to rats caused significant destruction of hepatic cyto-
chrome P-450 content, thereby inhibiting mono- oxygenases, including aminopyrine N-demethy- lase activity.” However, hepatic 0-deethylation of ethoxyresorufin, a specific substrate for cyto- chrome P-450 IA 1, IA2 isozymes were unaffected following a single parented administration of arge- mone oil. These results indicate that argemone oil has the specificity to alter different isozymic com- position of cytochrome P-45O.l1 Argemone oil caused significant depletion of glutathione content that may be due to the electrophilic character of the argemone alkaloid sanguinarine. Hepatic cyto- solic glutathione-S-transferase responsible for the catalysis of the conjugation reaction for biotrms- formation of toxic chemicals,1os was found to be inhibited as a result of reactive intermediate or lipid peroxides formed by argemone oil intoxication.”
Sanguinarine shows Type I1 binding with cyto- chrome P-45O.lo6 Type I1 binding spectra exhibit a peak at approximately 430 nm and a trough at 390 nm.Io7 A single i.p. dose (10 mg/kg) of san- guinarine has been shown to cause a significant loss of microsomal cytochrome P-450 and benzphetamine-N-demethylase activity.85 Sangui- narine, administered for 3 successive days, caused loss of microsomal cytochrome P-450,” inhibi- tion of aminopyrine N-demethylase and aryl hy- drocarbon hydroxylase activities and prolonga- tion of phenobarbital sleeping time in male rats.log
The argemone alkaloids sanguinarine and dihy- drosanguinarine have some structural similarity with methylene dioxyphenyl (MDP) compounds, which have the ability to destroy cytochrome P- 450 and inhibit monooxygenase activity through a reactive metabolic intermediate, possibly the free radical carbenes that form stable complex with cytochrome P-450 (Figure @.Io9 The inhibi- tory response of argemone oil might be the result of in-situ production of carbon monoxide through mixed function oxidase system, as did the MDP compounds, with the use of the carbon atom of the methylene dioxy group of the ring after cleav- ing the C-H bond,Io9 thus responding to the inhi- bition of the carbon monooxide-sensitive pigment cytochrome P-450. 11.8s
The overall speculative pathway of biometa- bolic clearance is shown in Figure 9.
The report on time clearance rate of argemone alkaloids in biological species are scanty. A green fluorescent metabolite of sanguinarine was de-
FIGURE 8. Mechanism of methylene dioxyphenyl compounds metabolism.10g
tected i n the inilk of goats fed on argemone leaves. This metabolite was tentatively identified as 3.4- henzacridine in the urine of treated rabbits by Hakiin et Recently, biometabolic elimination and organ retention profile of sanguinarine was qtudied in rats and guinea pigs."" This study re- 1 ealed that metabolic disposition of sanguinarine IS <low as the parent compound and/or its metabo-
co
lite did not appear in urine or feces of rats and guinea pigs after 6 h of single oral administration of sanguinarine (10 mgkg body wt.). Sanguinar- ine was found to be retained in the GI tract, liver, lung, kidney, heart, and serum even after 96 h, suggesting the overall target sites as observed by Upreti et al.74 The retention of sanguinarine and/ or its metabolite in serum even after 96 h may be
due to binding with plasma proteins,78 which in turn may lead to slow elimination. Sanguinarine and dihydrosanguinarine, having a molecular weight more than 300, are excreted through bile. However, no green fluorescent products were noticed in the bile.' l o Continued biliary passage of sanguinarine may expose the portal tracts con- taining bile ducts resulting in hyperplasia, fibro- sis, and cellular in f i l t ra t i~n .~~ The enterohepatic circulation of sanguinarine is quite prominent in rats and guinea pigs, as shown by appreciable elimination of sanguinarine and/or its metabolite(s) through feces.*1° From the overall metabolism, it can be proposed that sanguinarine may undergo N-demethylation like other alkaloidslo6 and then further opening the methylene dioxy ring as sug- gested by Wilkinson et al.lo9 These steps can be followed by rearrangement of carbon skeleton, a step not understood so far, to form a metabolite benzacridine (Figure 9).
5. Phototoxic Potential
Recent studies have shown the phototoxic property of sanguinarine to mosquito larvae (Aedes atropalpus) with an LD,, of 0.096 mg/ml, with near UV exposure when compared with 23.3 mg/ ml without UV light.'" The photochemical re- sults suggest that Type I1 (Singlet oxygen) and Type I (superoxide) mechanisms of photosensiti- zation are likely in competition. Studies by Tuveson et al. l2 with catalase deficient and pro- ficient strains of E. coli suggest that the Type I mechanism may be more important. Furthermore, the modest value of quantum yields singlet oxy- gen generation by sanguinarine, and the efficient electron donation of methyl viologen tend to sup- port the suggestion for the dominant Type I mecha- nism; nonetheless, the role of type I1 mechanisms may also be critical.lll Recent studies have shown the production of singlet oxygen following photo- sensitization of sanguinarine.' l3
+
DIHYOROSA NGUINARINE 1 . N - Demethylation 2 . Disappearance af oxygen functional groups 3. Rearrangement of the carbon skeleton
0 I 0
HzC' SANGUINARINE
3,4- BENZACRIDINE
FIGURE 9. Speculative pathway of metabolic clearance of Argemone alkaloid.
Sanguinarine inhibits both photosynthetic phosphorylation associated with femcyanide re- duction and cyclic photophosphorylation catalyzed phenazine methosulfate. 114 The alkaloid uncouples respiration and oxidative phosphorylation in rat liver mitochondria.’
Rukmini’I6 proposed that a solid “fatty acid derivative” is present in Argemone mexicana seed oil, which potentiates the toxicity of sanguinarine. The ‘fatty acid derivative’ proved to be the mix- ture of unusual C,, and C,, long-chain Keto acids. I L 7 Furthermore, these acids were also detected in Argemone ochroleuca seed oi1.’18 However, the significance of these keto fatty acids in the toxic- ity of argemone oil is not established.
7. Mechanism of Toxicity
Several lines of evidence are proposed to enumerate the mechanism of toxicity of argemone oilhlkaloid, as indicated in Figure 10. It has been suggested that the impairment of hepatic phase I and phase I1 enzymes by argemone oil” may decrease the rate of metabolism of the alkaloid, which in turn may be responsible for the slow elimination of the compoundmetabolite through urine and feces.110 The retention of sanguinarine in the GI tract, liver, lung, kidney, heart, and serum even after 96 h of exposurelIO indicates the chief target sites of toxicity.
The inhibition of Na+,K+-ATPase activity of heart by sanguinarine is due to interaction with the cardiac glycoside receptor site of the enzyme,s8 which may be responsible for producing degen- erative changes in cardiac muscle fibers in the auricular wall of rats fed argemone (Figure 6) and could be related to tachycardia and cardiac failure in Epidemic Dropsy patients.16.s0
The decrease in glycogen level following arge- mone oil in to~ica t ion~~ could be due to enhanced glycogenolysis leading to the formation of glu- cose- I -phosphate (Figure 1 1 ), which enters the glycolytic pathway resulting in an accumulation of pyruvate in the blood of experimental animals
and dropsy patients.23 The enhancement of glyco- genolysis can further be supported by the interfer- ence of sanguinarine in the uptake of glucose through the blocking of sodium pump via Na+,K+-ATPase thus inhibiting the active transport of glucose across intestinal barrier.92 It is well established that increased pyruvate concentration in blood un- couples oxidative phosphory1ation,’l9 which may be responsible for thickening of interalveolar septa and disorganized alveolar spaces in lungs of arge- mone oil-fed rats74 (Figure 6) and the breathless- ness observed in human victims.46
The decrease in hepatic glutathione levels with a concomitant increase in lipid peroxidation in argemone oil-intoxicated animals suggests the membrane-damaging potential’ of the alkaloid through the enhanced production of reactive oxy- gen spe~ies,’~,~* which may cause increased fibro- sis, hyperplasia of bile ducts, and congestion in a few portal tracts in liver of rats74 (Figure 6) and hepatomegaly with dilated blood vessels sur- rounded by proliferation of endothelial cells in argemone oil-intoxicated patient^.^^,^'
The binding of sanguinarine with GC-rich DNAs3 may result in the impairment of DNA polymerase acti~ity,9~ which could decrease the repair capacity leading to cellular metabolic/patho- logical changes. Because not much work has been carried out in this direction, the hypothesis re- quires more data to be generated.
Vll. THERAPEUTIC MEASURES IN DROPSY VICTIMS
No rational line of treatment based on the epidemiological observations and functional dis- turbances has so far been formulated. However, the following general treatment has been tried. Because edible oil adulterated with argemone oil is generally the primary source of exposure, it should be omitted from the diet. It has been shown by Sarkar23 that sanguinarine interferes with the oxidation of pyruvic acid and leads to its accumu- lation in the blood. It was inferred from Sarkar’s work that the mechanism of argemone intoxica- tion may be the same as that of vitamin B1 (thia-
FIGURE 10. Biochemical mechanism@) of toxicity of Argemone oil/alkaloid.
mine) deficiency that produces its symptoms by interfering with cell nutrition as a result of the blockage of carbohydrate metabolism at the pyru- vic acid stage, the exception being that sanguinar- ine blocks the sulfhydryl radical or an enzyme of the pyruvate dehydrogenase complex. Therapeu- tic administration of vitamin B 1 has, however, proven ineffective.I2O Vitamins C and K are advo- cated because of the capillary change and hemor- rhage noticed in the disease.
The limited trial of riboflavin and vitamin E in the outbreak of Barabanki District of Uttar Pradesh seems to have responded favorably.46 These results are in concurrence with the experi- mental studies where bioantioxidants provide a protective response in argemone oil-intoxicated rat^.^^,^* However, the lack of an adequate number of cases and the absence of controlled evaluation at this stage do not permit drawing any specific conclusion regarding the antioxidant mode of
G L Y GI ucose-6-phosphate c 0 L F ructose-6-p hos p hate Y S
J. I S J
J Pyruvate
Increased pyruvate uncouples oxidative phosphory- lation which may be responsible for breathlessness
FIGURE 11. Pathway of enhanced formation of pyruvate as a result of decreased glycogen level following argemone oil exposure.
therapy.i" It may be pointed out at this stage that the line of treatment in argemone-intoxicated vic- tims has so far been only symptomatic, and spe- cific therapeutic measures are still lacking.
It has been suggested that the diet should be rich in protein, with a moderate amount of fat and carbohydrate. Carbohydrate is restricted as its nor- mal metabolism is hampered. A high protein in- take is believed to restore the blood albumin and its colloid osmotic tension. A diet low in sodium or even without salt, in the severely edematous cases, has been suggested. A salt-poor diet helps i n the management of edema.120 Patients of Epi-
demic Dropsy in Bombay and suburbs responded favorably to such salt-free diets.32
Rest is generally advised to prevent cardiac strain and edema of legs due to the action of gravity. Diuretics, such as hydrochlorthiazide32 and laxatives intended to eliminate fluid and probably the toxin from the system, have been tried. The steroid therapy using pregniosolone was recommended i n the Bombay epidemicT2 probably due to the antiinflam- matory action of the steroids. Because a rise in blood histamine has been demonstrated in Epidemic Dropsy victims,27.94. I ? I antihistaminics, such as phenergan, have therefore been commonly prescribed.32J20
The studies of Ramasastri and B a b ~ ' ~ ~ have suggested that a level of 0.01% contamination of argemone oil in edible oil could be considered as permissible limits. The level of 0.01% contamina- tion with argemone oil is equivalent to an amount of 0.5 pg sanguinarine per milliliter of the sam- ple. 123 However, the contamination can increase in edible oil as high as 5.0 to 6.2 mg sanguinarine per milliliter where it can lead to the development of clinical manife~tati0n.l~~
IX. REMOVAL OF TOXIC COMPONENT(S) OF ARGEMONE OIL
Pasricha et al.Iz4found that argemone oil loses much of its toxicity when heated at 240°C for 15 min. This method of deactivation is, however, not satisfactory, as the oil still responds to the nitric acid test for argemone alkaloid. It may be quite possible that the oil can again become toxic after aerial oxidation. devised a method for re- moval of toxic alkaloids from argemone-adulter- ated mustard oil. This involved treatment with phosphoric acid followed by shaking with acid- activated Fuller's earth. Fuller's earth can finally be removed by filtration and the excess of phos- phoric acid neutralized with precipitated chalk. It was stated that purified oil in this way does not respond to the various tests for argemone oi1.Iz6 Sengupta and NairlZ7 found that similar deactiva- tion of the argemone oil can also be carried out by shaking the oil with Fuller's earth only at 140°C removing the toxic compounds Prakash et a1.Iz8 employed a 20% solution of ferric chloride as the deactivating agent. A slight modification in the normal refining of edible oils was suggested by Shen01ika1-l~~ for removing sanguinarine from contaminated oils. While the oil is treated with acid for degumming, steam is passed for 30 min. The condensed steam containing the sanguinarine salts is then tapped out. The oil is subjected to normal alkali refining and bleaching. This pro- cess removes up to 99.9% of sanguinarine. Con- taminated edible oils processed in this manner can be ingested. BoseI3O removed toxic alkaloids from oil by treating the oil (in hexane) with HCL,
removing the acid phase, and passing the hexane solution over bone-charcoal that absorbed the al- kaloids, giving detoxified oil.
It has been shown by Saboor13' that the aver- age specific gravities of the ordinary mustard seeds and argemone seeds are 1.133 and 1.088, respec- tively. Based on this observation, separation of the two seeds was devised using solution of com- mon salt in water having a specific gravity of 1.100. Being heavier, mustard seeds sink in the solution. Another method based on air elutriation has also been suggested for separating argemone and mustard seeds.132
ACKNOWLEDGEMENTS
The authors are grateful to the Director, Dr. R. C. Srimal, Industrial Toxicology Research Cen- tre, Lucknow, for his encouragement in the prepa- ration of this article. Thanks are due to Mr K. G. Thomas for his secretarial assistance.
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