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Absorption of iron from ingested liver
482
Natural products
then killed. Two control groups, each of seven preg- sulphoxide(SMCSO)werepresentin both whole kale nant mice, were fed the basal diet or the basal diet and the haemolyticfraction, and support for the poscontaining 33% cellulose over the same period. Day sibility that this compound might be implicated in 0 was the day on which a vaginal plug was found. kale anaemiawas derived from the finding of high Blood samples were taken just before death and used concentrationsof volatile organicsulphurcompounds for the estimation of serum-protein levels. The in the rumen of an acutely poisoned goat. numbers of implantation sites and foetal deaths wece Administration of SMCSO to a hay-fed goat again recorded, and the live foetuses were weighed, exam- produced the type of haematologicalresponseseen ined for gross external and internal malformations with kale. The possibility that the active haemolysin and then cleared. stained and examined for skeletal wasproducedby rumenfermentationof SMCSO led anomalies. to in vitro studieswith rumencontents.Dimethyl diCompared with the other two groups, the bracken- sulphide was the main product of the early ferfed animals showed a significant reduction in body mentationstage,while later the proportions of methweight in spite of a higher food intake. Reproduction anethiol and dimethyl sulphide increased,although data parameters were similar for all the dams, how- the latter remaineda relatively minor component.Sever. The numbers of implants, foetal mortality and Methylcysteine, present to a smallerextent in kale, incidence of external malformations showed no sig- also gave rise mainly to methanethioland dimethyl nificant differences between the three groups, disulphidewith a minor amountof dimethyl sulphide. although the mean foetal weights in the bracken-fed While it thus appearsthat SMCSO is the primary group were significantly reduced. Examination of factor in the haemolytic activity of kale, it remains cleared foetuses showed an increase in rib anomalies to be established whether dimethyl disulphideand/or together with retarded ossification and incomplete methanethiolmay be the actual toxin. Haemolytic fusion of the sternebrae in the test group. The sugges- activity haspreviouslybeenassociatedwith di-n-protion that the bracken-fed animals might be protein- pyl and di-p-tolyl disulphidesas well as with thiols. deficient was rejected on the grounds that higher quantities of dietary protein were available to these 2890.Toxins isolatedfrom oriental mushrooms animals than to the controls, and it was therefore concluded that bracken itself was responsible for the Lin. J.-Y., Lin, Y.-J., Chen,C.-C.,Wu, H.-L., Shi, G.-Y. maternal and foetal toxicity observed. & Jeng, T.-W. (1974). Cardiotoxic protein from However bracken has been associated with anti- ediblemushrooms.Nature, Land. 252. 235. thiamine activity in some species (ibid 1973, 11. 163) and, sincethiaminedeficiencycan causegrowth retarA cardiotoxic protein, volvatoxin A, waspreviously dation in rat embryosand maternal toxicity in rats isolatedfrom the ediblemushroomVolvariella wlva(Brown & Snodgrass, J. Nun. 1965,87,353) it seems ceae by Lin et al. (Nature, Land, 1973, 246. 524). The that this factor may have contributed to the effects isolation of another cardiotoxic protein is now observed. reported,this time from Flammulina uelutipes, a mushmore conclusive information might have been room widely eatenin the Eastand cannedfor export. obtainedif larger numbersof animalshad beenused This toxin, named flammutoxin, was presentin the in eachgroupand morethan one doselevel had been mushroomsat a level of 320 ppm and had an LDSo studied.] of 2.45mg/kg. Unlike volvatoxin A, from which two components (namelyAI and AZ) were isolated,flammutoxincon2889. A haemolyticfactor in kale tained only one type of molecule,with a molecular Smith, R. H.. Earl, C. R. & Matheson, N. A. (1974). weight of 22,000.The amino acid compositionwas The probable role of S-methylcysteinesulphoxidein similar to that of volvatoxin A2. Flammutoxin, like kale poisoningin ruminants.Biochem. Sot. Eans. 2, volvatoxin A, haemolysedhumanblood cellsof group 101. 0, causeda ‘writhing reaction’ with a delay before onset and affected the electrocardiogram,depressing The anaemiacausedin cattle and goats by a high the ST segmentand inverting the T wave. In addition, intake of kale(Cited in F.C.7: 1971,9.759)alsooccurs flammutoxin induceda sharp fall in blood pressure, to a lessmarkedextent in sheep.The factor respon- caused swellingof Ehrlich ascitestumour cells and sible for the haematologicalchangesis not limited inhibited their respiration.These latter effects were to any one variety of kale but its chemicalidentity demonstratedalsowith volvatoxin A2, but this frachasso far remainedunknown. tion alonedid not elicit the writhing reaction.VolvaThe work cited above wasaimedat elucidatingthe toxin Al exhibited none of the biological activity nature of this haemolyticfactor, which was found to shownby volvatoxin A or A2, and it had no effect remainin the aqueousphaseafter exhaustivechloro- on the biologicalactivity of flammutoxin. form-extraction of largely deproteinizedand clarified kalejuice.This aqueousphase,which washighly toxic 2891. Absorptionof iron from ingestedliver whenadministeredto a goat, wasfractionatedby displacementchromatography.Fractions rich in acidic Naish,R., Kimber, C. L. & Deller. D. J. (1974).Liver amino acids were obtained and when these were iron: Changesinduced by cooking and acid-peptic pooled and admmisteredvia a rumen cannula to digestion.Br. J. Haemat. 26, 459. goats fed on hay, the haematological changes observedwere comparableto thosein goats fed exIn all attempts to curb the high rate of irondeficlusively on kale. ciency anaemiathroughout the world, attention has Relatively high concentrationsof S-methylcysteine to be paid not only to the total amount of iron in
Natural products the diet but also to the proportion of this total that can be absorbed from the human intestinal tract. Although the total iron content of staple foods has been well documented, some difficulty has been encountered in monitoring the changes induced in the physical and chemical state of dietary iron, and consequently in its absorbability, by the cooking of food and the process of digestion. The authors cited above have therefore studied the iron present in liver before and after cooking and/or enzymatic digestion and also its absorption after the different treatments. Guinea-pig liver was used for practical reasons and because its iron content and haem/non-haem iron distribution are comparable with those of the types of liver that constitute an important source of dietary iron. When guinea-pig liver was ‘cooked by heating to 100°C for 10 min, no alteration was observed in the distribution of iron between the haem and non-haem fractions, but compared with the findings in raw liver there was an increase in the high-molecular-weight fraction of liver iron as demonstrated by dialysis and molecular sieving on Sephadex G-200 and G-50 columns. Incubation of the liver homogenate with pepsin for 2 hr at pH 1.6 similarly failed to affect the haem/non-haem distribution pattern, but reduced the molecular weight of the iron complexes. When cooking was followed by this acid-peptic digestion, the overall increase in low-molecular-weight iron complexes was greater than that observed during acid-peptic digestion alone. Absorption studies were conducted in fasted female albino rats given intragastric doses of raw, cooked, digested, or cooked and digested homogenates containing 59Fe-labelled ferric chloride. Each dose contained the equivalent of 20 pg elemental iron. Cooking combined with acid-peptic digestion of the liver increased the liver-iron absorption by about 75%. a finding ascribed to the high percentage of low-molecular-weight iron components. No such increase was found, however, with liver subjected only to in uitro peptic digestion.
483
reduced by 18% and phosphorylation by 68%. Ascorbate oxidation was not affected by garlic oil, but phosphorylation was reduced by 64 and 79x, respectively, by 0.5 and I.0 fi garlic oil/3 ml. DAD (0.5 d/3 ml) reduced phosphorylation of glutamate by 53x, of succinate by 35% and of ascorbate by 59%, whereas DPD had relatively little effect. Thus, inhibition and uncoupling by garlic oil and DAD appear to affect phosphorylation sites I, 2 and 3. Respiration is more affected by these substances during oxidation of glutamate than during that of succinate or ascorbate. and phosphorylation is inhibited to a greater degree than is oxidation. The second paper concerns the lipid-lowering effect of DAD oxide (allicin) which occurs at a level of about 0.15% in garlic. In young rats given an oral dose of allicin of 100 mg/kg/day for 2 months. lipid levels in serum and liver were significantly reduced, the reduction being more marked in the cast of the liver lipids, but the proteins of serum and liver were not significantly affected. The change in lipids was due chiefly to a reduction in triglycerides and free cholesterol. The effect of allicin may be on lipid synthesis or catabolism, but interference with the biosynthesis of cholesterol and other lipid components is suggested by the capacity of allicin to combine with the sulphydryl group in cdenzyme A. which is essential for the biosynthesis of fatty acids. cholesterol, triglycerides and phospholipids, and by the finding that the cholesterol-lowering effect is most pronounced on free cholesterol. The reported therapeutic value of garlic in atherosclerosis receives support from these findings. 2893. More on tannin carcinogenicity Pradhan, S. N., Chung, E. B., Ghosh, B., Paul, B. D. & Kapadia, G. J. (1974). Potential carcinogens. I. Carcinogenicity of some plant extracts and their tannin-containing fractions in rats. J. natn. Cancer. Inst.
52.
1579.
Herbal remedies, beverages and food plants used by certain populations appear to have some connexion with a local prevalence of oesophageal cancer. It has been suggested that the tumorigenic agent in such commodities as sorghum and Krameria ixina may be the tannin (Cited in F.C.T 1971, 9. 898). This seems amply borne out by the evidence of the paper cited above, which describes the treatment of rats with injections of extracts of whole herbs or of tannin-containing or tannin-free fractions. For up to 75 wk, the rats were given weekly SC injections Garlic oil has been shown to be effective against of total aqueous extracts or fractionated extracts of root, Acacia insect pests, and both garlic oil and diallyl disulphide K. ixina (without root), K. triandra vulgare seeds. The total (DAD) have been found to inhibit protein synthesis villosa root and Sorghum extract and tannin-containing fraction of K. ixina, K. in mosquito larvae (George et al. Chemico-Biol. Interactions 1973, 6, 169). In an attempt to elucidate this triandra and A. villosa were all carcinogenic. The acaaction, the effect of garlic oil, DAD and dipropyl di- cia extracts were most potent and produced the first sulphide (DPD) on oxidative phosphorylation in the fibrous histiocytomas after 8 months. In contrast, a hepatic mitochondria of mice was investigated, using tannin-free extract of K. ixina produced tumours in only three of 60 rats. The total and tannin-containing three substrates. were less potent Glutamate oxidation was inhibited by 55 and 63%, extracts of K. ixina and K. triandra respectively, by concentrations of 0.5 and I.0 4 garlic than those of acacia, and the appearance of the histiocytomas was more delayed. A cold-water extract of oil in 3 ml reaction mixture, while phosphorylation was inhibited by 52 and 71% respectively. Succinate sorghum gave no indication of any carcinogenic oxidation in the presence of 1.0 fl garlic oil/3 ml was potential.
2892. Garlic alters enzyme systems George, K. C. & Eapen, J. (1974). Mode of action of garlic oil--Effect on oxidative phosphorylation in hepatic mitochondria of mice. Biochem. Pharmac. 23. 931. Augusti, K. T. & Mathew, P. T. (1974). Lipid lowering effect of allicin (diallyl disulphide-oxide) on long term feeding to normal rats. Experientia 30. 468.
Natural products
then killed. Two control groups, each of seven preg- sulphoxide(SMCSO)werepresentin both whole kale nant mice, were fed the basal diet or the basal diet and the haemolyticfraction, and support for the poscontaining 33% cellulose over the same period. Day sibility that this compound might be implicated in 0 was the day on which a vaginal plug was found. kale anaemiawas derived from the finding of high Blood samples were taken just before death and used concentrationsof volatile organicsulphurcompounds for the estimation of serum-protein levels. The in the rumen of an acutely poisoned goat. numbers of implantation sites and foetal deaths wece Administration of SMCSO to a hay-fed goat again recorded, and the live foetuses were weighed, exam- produced the type of haematologicalresponseseen ined for gross external and internal malformations with kale. The possibility that the active haemolysin and then cleared. stained and examined for skeletal wasproducedby rumenfermentationof SMCSO led anomalies. to in vitro studieswith rumencontents.Dimethyl diCompared with the other two groups, the bracken- sulphide was the main product of the early ferfed animals showed a significant reduction in body mentationstage,while later the proportions of methweight in spite of a higher food intake. Reproduction anethiol and dimethyl sulphide increased,although data parameters were similar for all the dams, how- the latter remaineda relatively minor component.Sever. The numbers of implants, foetal mortality and Methylcysteine, present to a smallerextent in kale, incidence of external malformations showed no sig- also gave rise mainly to methanethioland dimethyl nificant differences between the three groups, disulphidewith a minor amountof dimethyl sulphide. although the mean foetal weights in the bracken-fed While it thus appearsthat SMCSO is the primary group were significantly reduced. Examination of factor in the haemolytic activity of kale, it remains cleared foetuses showed an increase in rib anomalies to be established whether dimethyl disulphideand/or together with retarded ossification and incomplete methanethiolmay be the actual toxin. Haemolytic fusion of the sternebrae in the test group. The sugges- activity haspreviouslybeenassociatedwith di-n-protion that the bracken-fed animals might be protein- pyl and di-p-tolyl disulphidesas well as with thiols. deficient was rejected on the grounds that higher quantities of dietary protein were available to these 2890.Toxins isolatedfrom oriental mushrooms animals than to the controls, and it was therefore concluded that bracken itself was responsible for the Lin. J.-Y., Lin, Y.-J., Chen,C.-C.,Wu, H.-L., Shi, G.-Y. maternal and foetal toxicity observed. & Jeng, T.-W. (1974). Cardiotoxic protein from However bracken has been associated with anti- ediblemushrooms.Nature, Land. 252. 235. thiamine activity in some species (ibid 1973, 11. 163) and, sincethiaminedeficiencycan causegrowth retarA cardiotoxic protein, volvatoxin A, waspreviously dation in rat embryosand maternal toxicity in rats isolatedfrom the ediblemushroomVolvariella wlva(Brown & Snodgrass, J. Nun. 1965,87,353) it seems ceae by Lin et al. (Nature, Land, 1973, 246. 524). The that this factor may have contributed to the effects isolation of another cardiotoxic protein is now observed. reported,this time from Flammulina uelutipes, a mushmore conclusive information might have been room widely eatenin the Eastand cannedfor export. obtainedif larger numbersof animalshad beenused This toxin, named flammutoxin, was presentin the in eachgroupand morethan one doselevel had been mushroomsat a level of 320 ppm and had an LDSo studied.] of 2.45mg/kg. Unlike volvatoxin A, from which two components (namelyAI and AZ) were isolated,flammutoxincon2889. A haemolyticfactor in kale tained only one type of molecule,with a molecular Smith, R. H.. Earl, C. R. & Matheson, N. A. (1974). weight of 22,000.The amino acid compositionwas The probable role of S-methylcysteinesulphoxidein similar to that of volvatoxin A2. Flammutoxin, like kale poisoningin ruminants.Biochem. Sot. Eans. 2, volvatoxin A, haemolysedhumanblood cellsof group 101. 0, causeda ‘writhing reaction’ with a delay before onset and affected the electrocardiogram,depressing The anaemiacausedin cattle and goats by a high the ST segmentand inverting the T wave. In addition, intake of kale(Cited in F.C.7: 1971,9.759)alsooccurs flammutoxin induceda sharp fall in blood pressure, to a lessmarkedextent in sheep.The factor respon- caused swellingof Ehrlich ascitestumour cells and sible for the haematologicalchangesis not limited inhibited their respiration.These latter effects were to any one variety of kale but its chemicalidentity demonstratedalsowith volvatoxin A2, but this frachasso far remainedunknown. tion alonedid not elicit the writhing reaction.VolvaThe work cited above wasaimedat elucidatingthe toxin Al exhibited none of the biological activity nature of this haemolyticfactor, which was found to shownby volvatoxin A or A2, and it had no effect remainin the aqueousphaseafter exhaustivechloro- on the biologicalactivity of flammutoxin. form-extraction of largely deproteinizedand clarified kalejuice.This aqueousphase,which washighly toxic 2891. Absorptionof iron from ingestedliver whenadministeredto a goat, wasfractionatedby displacementchromatography.Fractions rich in acidic Naish,R., Kimber, C. L. & Deller. D. J. (1974).Liver amino acids were obtained and when these were iron: Changesinduced by cooking and acid-peptic pooled and admmisteredvia a rumen cannula to digestion.Br. J. Haemat. 26, 459. goats fed on hay, the haematological changes observedwere comparableto thosein goats fed exIn all attempts to curb the high rate of irondeficlusively on kale. ciency anaemiathroughout the world, attention has Relatively high concentrationsof S-methylcysteine to be paid not only to the total amount of iron in
Natural products the diet but also to the proportion of this total that can be absorbed from the human intestinal tract. Although the total iron content of staple foods has been well documented, some difficulty has been encountered in monitoring the changes induced in the physical and chemical state of dietary iron, and consequently in its absorbability, by the cooking of food and the process of digestion. The authors cited above have therefore studied the iron present in liver before and after cooking and/or enzymatic digestion and also its absorption after the different treatments. Guinea-pig liver was used for practical reasons and because its iron content and haem/non-haem iron distribution are comparable with those of the types of liver that constitute an important source of dietary iron. When guinea-pig liver was ‘cooked by heating to 100°C for 10 min, no alteration was observed in the distribution of iron between the haem and non-haem fractions, but compared with the findings in raw liver there was an increase in the high-molecular-weight fraction of liver iron as demonstrated by dialysis and molecular sieving on Sephadex G-200 and G-50 columns. Incubation of the liver homogenate with pepsin for 2 hr at pH 1.6 similarly failed to affect the haem/non-haem distribution pattern, but reduced the molecular weight of the iron complexes. When cooking was followed by this acid-peptic digestion, the overall increase in low-molecular-weight iron complexes was greater than that observed during acid-peptic digestion alone. Absorption studies were conducted in fasted female albino rats given intragastric doses of raw, cooked, digested, or cooked and digested homogenates containing 59Fe-labelled ferric chloride. Each dose contained the equivalent of 20 pg elemental iron. Cooking combined with acid-peptic digestion of the liver increased the liver-iron absorption by about 75%. a finding ascribed to the high percentage of low-molecular-weight iron components. No such increase was found, however, with liver subjected only to in uitro peptic digestion.
483
reduced by 18% and phosphorylation by 68%. Ascorbate oxidation was not affected by garlic oil, but phosphorylation was reduced by 64 and 79x, respectively, by 0.5 and I.0 fi garlic oil/3 ml. DAD (0.5 d/3 ml) reduced phosphorylation of glutamate by 53x, of succinate by 35% and of ascorbate by 59%, whereas DPD had relatively little effect. Thus, inhibition and uncoupling by garlic oil and DAD appear to affect phosphorylation sites I, 2 and 3. Respiration is more affected by these substances during oxidation of glutamate than during that of succinate or ascorbate. and phosphorylation is inhibited to a greater degree than is oxidation. The second paper concerns the lipid-lowering effect of DAD oxide (allicin) which occurs at a level of about 0.15% in garlic. In young rats given an oral dose of allicin of 100 mg/kg/day for 2 months. lipid levels in serum and liver were significantly reduced, the reduction being more marked in the cast of the liver lipids, but the proteins of serum and liver were not significantly affected. The change in lipids was due chiefly to a reduction in triglycerides and free cholesterol. The effect of allicin may be on lipid synthesis or catabolism, but interference with the biosynthesis of cholesterol and other lipid components is suggested by the capacity of allicin to combine with the sulphydryl group in cdenzyme A. which is essential for the biosynthesis of fatty acids. cholesterol, triglycerides and phospholipids, and by the finding that the cholesterol-lowering effect is most pronounced on free cholesterol. The reported therapeutic value of garlic in atherosclerosis receives support from these findings. 2893. More on tannin carcinogenicity Pradhan, S. N., Chung, E. B., Ghosh, B., Paul, B. D. & Kapadia, G. J. (1974). Potential carcinogens. I. Carcinogenicity of some plant extracts and their tannin-containing fractions in rats. J. natn. Cancer. Inst.
52.
1579.
Herbal remedies, beverages and food plants used by certain populations appear to have some connexion with a local prevalence of oesophageal cancer. It has been suggested that the tumorigenic agent in such commodities as sorghum and Krameria ixina may be the tannin (Cited in F.C.T 1971, 9. 898). This seems amply borne out by the evidence of the paper cited above, which describes the treatment of rats with injections of extracts of whole herbs or of tannin-containing or tannin-free fractions. For up to 75 wk, the rats were given weekly SC injections Garlic oil has been shown to be effective against of total aqueous extracts or fractionated extracts of root, Acacia insect pests, and both garlic oil and diallyl disulphide K. ixina (without root), K. triandra vulgare seeds. The total (DAD) have been found to inhibit protein synthesis villosa root and Sorghum extract and tannin-containing fraction of K. ixina, K. in mosquito larvae (George et al. Chemico-Biol. Interactions 1973, 6, 169). In an attempt to elucidate this triandra and A. villosa were all carcinogenic. The acaaction, the effect of garlic oil, DAD and dipropyl di- cia extracts were most potent and produced the first sulphide (DPD) on oxidative phosphorylation in the fibrous histiocytomas after 8 months. In contrast, a hepatic mitochondria of mice was investigated, using tannin-free extract of K. ixina produced tumours in only three of 60 rats. The total and tannin-containing three substrates. were less potent Glutamate oxidation was inhibited by 55 and 63%, extracts of K. ixina and K. triandra respectively, by concentrations of 0.5 and I.0 4 garlic than those of acacia, and the appearance of the histiocytomas was more delayed. A cold-water extract of oil in 3 ml reaction mixture, while phosphorylation was inhibited by 52 and 71% respectively. Succinate sorghum gave no indication of any carcinogenic oxidation in the presence of 1.0 fl garlic oil/3 ml was potential.
2892. Garlic alters enzyme systems George, K. C. & Eapen, J. (1974). Mode of action of garlic oil--Effect on oxidative phosphorylation in hepatic mitochondria of mice. Biochem. Pharmac. 23. 931. Augusti, K. T. & Mathew, P. T. (1974). Lipid lowering effect of allicin (diallyl disulphide-oxide) on long term feeding to normal rats. Experientia 30. 468.