DEP AR'rMENT OP PHYSIOLOGY.

1940.

By

C. II. Wil�LHd\18, B.Sc., DIP. PnArrM.

and

H. J. G. HINmS, B.Sc.

lhpartmenl '�f l'hysiology, f!nivm·sity of Queensland.

I Rrpriutcd from ·The Australian. V olerinury J a'III'Hil7, Vol. 16, pp. 14-20.]

T!!O:UAS GILBRil'l' Ilovg� A<."ting GovPrnment Printer, Brisbanfl,

NuMBER 2.

QPl

V.l no.2

Reprinted from 'I'he Aust-ral·ian Veterina-ry Journnl, February, 1940, pages 14-20. -

-----�-----�------------ - - - �------ -- - ----�-------- --- --- ----

A vsT. VET. J ., J 940, Vol. 16.

'rHE 'rOXIC l'lWPERTIES OF SALVIA REFLEXA.

By C. H. YVrLI,JAMS, B.Se., Dip. Pharm., AND

H. J. G. HrNESJ B:Sc.

(From the Department of Physiology, University o£ Queensland.)

Introduction. 8aloia 1·ejlexa) commonly called wild mint Ot' miut weed, is well known

in Queensland and New South Wales as a plant poisonous to stock. Heavy mortalities have occurred in the field, and the responsibility of mint weed has been repeatedly confirmed by experimental feeding. An outstanding case occurred in the Pittsworth dis trict of (�ueensland on February 8th, 1932, when a:M bullocks died. The following description is taken from the report by Cory (1932):

"'l'he cattle were very hungry and thirsty. The drover camped the bullocks on the Saturday night iu a road in which practically nothing but \\'Hd mint existed, and owing to the stock being very hungry th ey naturally ate some. They were watered at dayl ight on the Sunday morning at the Springf'l, Pittsworth, and were on the mint for :five or six miles after watering. On Monday, 8th February, the bullocks commenced to die . Had the bullocks had other feed available they would not have eaten the mint to excess, which apparently happened. 'rhe mint is looked on by local stockowners as poisonous, but only when eaten in excess, which is a rare. occur re nce wHh local stock."

'I'he symptoms exhibited after ingestion of the plant are described as follows (Dept. Agric., N.S.W., 1935): "None shown immediately; later the animals stand apart, breathing rapidly. There is twitching of the muscles and 1he animal soon goes .down. If touched, violent twitching occurs, but the animal i� unable to get up. Death follows after a few hours."

The weed appears to be spreading rapidly in Queensland, and the C!neensland Poison Plants Committee decided to carry out further work on the toxic properties and constituents of the plant.

Samples of the !Jlant were collecled in the 'l'oowoomba district in NO\'emher, 19:37, and a further supply was obtained from the Springsure district in Deeember, 1938. All tests were carried out on dried ground material whieh lo:->t 11·5 per cent of its weight on dryi ng to constant weight :a.t 100° C. All analytical figures here reported are calculated on the material dried to <:onstant weight at 100° U.

Chemical Examination. 'rests for saponin, cyanogenetic and other toxie glycosides were negative,

but JH'PCipitates were obtained with all of the usual alkaloidal reagents. The substance resl)Qnsible for these precipitates can be extract�d by refinxing \dth acetone, <llld was shown to he choline. 'l'he substance so extracted cannot be shaken out of aqueous ammoniacal solution by chloroform. "\Vi th Florence's reagent <'haracteristic precipitates of the periodide were formed, and the platini<'hloride was prepared and identified microscopically. 'rhe percentage

Page 14

THE TOXIC PROPERTIIDS OF' SALVIA RliJF'LLGXJi.

of choline present in the plan t material as determined by precipitation as the mercurichloride and then weighing was found to be 0·05 per cent. In view of the comparatively low toxicity of choline and the smalJ amount present in the pl ant, it is improbable that this su bsta n c e is a fa ctor in poisoning by wild mint.

During the extraction of the plant· material, a considerable amount of inorganic material ·was deposited on cooling the acetone solution. 'l'h is material was mostly pota ssium nitrate together with a little ehloride.

Steam distillation of the dried material gave negligible amounts ol' essential oil.

At this stage, the s earch for the toxic principle seemed most unpromising, but on further reference to the literature on plant poisoniiJg we were impressed by the experiments on nitrite poisoning and its a;,;sociation with specie s of Tribulus as reported by Himington ( 1938). Accordingly, the nitrate and nitri te contents of the plant were next investigated.

(1) The Nitrate Content.--(a) Extraction: 'J'wo hundred granunes of the plant were boiled in water for thirty minutes . 'l'he filtered extract thus obtained was treated with lead sub-acetate and the precipitate removed by centrifuging. After removal of lead by hydrogen sulphide and passing carbon dioxide through the filtrate to remove hydrogen sulphide , the liqu id was

evaporated to about 50 ml., cooled in ice and the crude potassium nitrate was filtered off. This was re-crystallized after clarifying with norit and a

llnal yield of 6 grammes was obtained from the 200 grammes of n ir-dde d material.

(b) Estimation: For the estimation of nitrate, the following method was

used: Five grammes of the material are mixed with 50 ml. water in a beaker

and the weight of the beaker and contents determined. .After boiling gentiy for thirty minuteR, the water lo.�t by evapoeation i::l replaced. Cool. Filter; dilute 5 mi. of the iiltrate to 20 ml. with water. Mix 10 mi. of tllis dilution with :J ml. saturated solution of leud sub-acetate and iilter- bv vaeuum filter. Wash the eesidue several time" with a few mL of wate1· and add the washings to the filtrate. Shake the resulting liquid with a little norit and add 20 per cent . ammonium sulphate drop by drop to precipitate cxeess lead. :B�ilter with suction, wash with water and adjust the filtrate to 50 mi. Five mi. of this solution

. are evaporated to dryness on the water-oath aud the residue is

dried at 120° C. for five nd n utes . One ml. of pllenol disulphonic acid reagent i8 then added and the mixture heated on the watet··bath fm five minutes: Dilute, add excess of 1: 1 ammonia and make up to 50 ml. Compare in a colorimeter with a 8olution of potassium nitrate containing 0·2 gramme per litre, 5 ml. of which are 8imilarly treated. Per form a blank on the reagents. The amount of pot assium nitrate in the plant thus determined was fonnd to be 5 per cent.

(2) Nitr·ite Pro(luctio,n.·--'l'he plant mater·ial was free of nitrite. When macerated with water and a little chlmoform and left over n ight a strougly positive reaction for nitrate was obta ine d >vith tbe Gl'iess-Ilosnty 1•eagent. When measured quantitatively it was found that maximum nitrite production at room temperature required twenty hours maceration, the amount produced after eight hours being wry small. Some 40 per cent. of the nitra te present could be thus reduced. 'I'he reduction occurred at the expense of glucose, phosphate being necessary for the reaction.

Page 15

THE TOXIC PROPERTIES OF SALVIA RKFLEXA.

These observations confirm those of Rimington (1933). He showed that certain spec ies ·of 1'ribulus can have a high nitrate content which may be accompanied by an enzyme system that can reduce about 60 per cent. of this nitrate to nitrite. He also pointed out that the nitrate content of different species var ies greatly, as also does the activity of the enzyme system.

Nitrates and Nitrites as the Toxic Principle in Salvia Reflexa. It is well known that nitr ites have a powerful physiological action. In

addition to a vaso-dilator action, the nitrite radical has marked toxic properties through its power to convert hmmoglobin to methremoglobin. If sufficient conversion occurs, the respiratory function of the blood is so reduced that death ensues. .Accordingly, feeding experiments were carried out with sheep in order to test the hypothesis that the toxic properties of the plant were primarily due to nitrite poisoning.

In the first test , 800 grammes of the material were macerated in 5 litres of v,-ater for forty hours, and the liquid was expressed. .About three-quarters of a kilogram of the marc was force fed to a sheep without ill effects.

The expressed liquid measured 3 litres (containing about 11 grammes potassium nitrite) and was given as a drench to a 60 lb. sheep which had been staned and kept without water for some hours previously. .Apart from drowsiness and l aboured breathing, no ill effects were ohsPrverl until five and a half hours after drenching. The gait then became nnstearly and the animal staggered. Shortly afterwards it waR unable to stand and died in about ten minutes after going down, death being preceded by a few spaRms.

�tt rost-mortem examination was conducted immediately after death. The liver appeared congested and on section chocolate coloured blood flowed. The kidneys were congested and chocolate colonred, the lungs normal lmt Rlightly engorged·. The urine was clear and norma l in colour.

Blood samples taken at intervals after administration of the drench were Pxamined macroscopically for colour, and spectroscopically for changes in hwmoi!lohin pigments.

The first sample, one and a quarter hours after drenching, was red, but at two hom·s the colour had already changed to brownish red and the snh8eqnrnt samples became increasingly brown, at death being coffee coloured.

On speetroseopic examination they showed the absorption spectrum of metha•moglolJin "-itlt the difference that the bands in the yellow and in the

green were much more intense relativelv to that in the red than is the case ,�·ith ordinary methremoglobin. Halda�e and his co-workers (1897) have shown that this is due to the simultaneous presence in the blood of animals poiRoned hy nitrite of small amounts of nitric oxide-hmmoglohin, the abl-lorption hands of which coincide approximately with those of methmmo­globin in the yellow and green and so intensify them. The addition of ammonium s ulphide produced hmmoglobin and on agitation with air the absorption spectrum changed to that of oxyhremoglobin.

'l'he sample taken at death was centrifuged. The plasma was free of brown or red colour, showing that the methremoglobin is intracorpuscular and that its formation was unaccompanied hy hremolysis. Microscopic examination of the corpuscles showed them to be normal in size and intact.

The oxygen carrying capacity of the blood sample s was measured by the Haldane-Barcroft technique. For a short time after drenching this remained Page 16

THE TOXIC PROPERTIES OF SAL VIA REFLEXA.

constant at the �ormal value of 16 ml. 02 per 100 ml. blood, but then steadily fell to a value of 4·53 ml. 02 per 100 ml. blood at death. 'rhis may be interpreted as signifying the conversion of 72 per cent. of the hmmoglobin to methremoglobin before death.

There thus remained little doubt of the ability of the .plant enzyme to convert nitrate to nitrite and that under suitable conditions such conversion would occur. Bernheim and Dixon (1928) have shown, however, that liver tissue is capable of converting nitrate to nitrite, and Seekles and Sjollema(1932) showed that on administering a dose of nitrate to an animal, one-tenth of 'the nitrate was reduced to nitrite in the anim�l's body. This suggested that the reduction by plant enzyme thought necessary by Rimington ( 1933) might be accessory but not obligatory.

If we accept Seekles and Sjollema's (1932) conversion figures and take the toxic dose of sodiumnitrite for a sheep to be 0·1 to 0·2 grammes per kilo body weight, 3 to 6 grammes of nitrite or 30 to 60 grammes of nitrate would constitute a toxic dose for a 60 lb. sheep. We therefore determined the effects of a large dose of nitrate.

Eighty grammes potassium nitrate dissolved in 1i litres of wate1· were given as a drench to a sheep previously starved and kept without \,·ater for 48 hours. 'l'he animal showed little disturbance until just before death. which oecurred eight hours later. The blood picture closely resembled that of the previous experiment. The tota 1 oxygen capacity remained normal at 16 rnl. per 100 ml. blood for two hours and then steadily fell to a ntlne of 4·57 ml. per 100 ml. blood at death. representing a conversion of 71·5 per cen1. of the hmmoglobin to meth::emoglobin. 'rhe plasma of all the abn.ormal samples showed a strong reaetion for nitrite.

That the nitrate of the plant is capable of acting in a similar way and that the toxic dose can be much smaller was shown as follows:

Eight hundred grammes of plant material were boiled with 5· lih·es of water for 15 minutes, cooled, pressed, and the expressed liquid was given as

a drench to a 45 lb. sheep, the toxic dose of nitrate for which would be about 25 grammes. The volume of the expressed liquid was 3,100 rnl. ( = 25g. KN03).The dose was given at 11 a.m .. the animal was well at 5 p.m. the same day, hut was found dead at 8 a.m. the next morning. A post-mortem examination showed signs of metha'moglohin::emia.

Discussion. Under suitable conditions plants containing considerable amounts of

nitrate 'can prove toxic to stock. This is . brought about by the action of nitrites on h::emoglobin. The nitrite can lJe produced through the intermediary of both plant and animal enzymes. \Vith these facts established, we propose to discuss the occurrence of nitrates in plants. the mode of action of nitrites on the blood, the susceptibility of the grazing animal and to indicate lines of further work.

(A) The Accumulation of Nitra.tes in Plants.-All plants readily absorb nitrates and ammonia. which through interaetion with the products of photosynthesis are converted to organic nitrogen compounds, the final stage being the production of protein. The stoeage of uiteate in the aerial part of the plant will thus depend on the rate of supply. which is conditioned by the accunmlatiou of soil .nitrate, and on the rate of removaL which is accelerated by circumstances fayourable to photosynthesis.

Page 17

TI-m TOXIC PROPERTIES OF SADVIA REF'DEXA.

This' balance will be affected by man,y factors; climate, soil, age of plant and species being amongst the more important variables.

Thus Ohibmtll and Miller (19:n) have reported a nitrate content of 44 per cent. of the dry matter of rye grass grown on a sewage farm. Here nitrate supply appem·s to have been the dominating factor. A rapid survey of the literature �:�eerns to indicate that under similar conditions a variety of <lnDual and biennial plants can accumulate considerable amounts of nitrate.

Of the factors eoncerned, the conditions governing the supply of soil n.itrates are best nnderstood. Soil micro-orgaDisms can manufacture nitl·iite illOHt rapidly in neutral soils well supplied with organic matter maintained at Flnitable moisture contents. Drought retards microbial activity whilst pre�>erYing nitrate already formed; excessive rain leads to water: logging of the soil and to leaching of nitrates. Prescott and Piper · (1930)have shown the influence of soil moistm·c on nitrate accumulation in a South Austr·alian soil. There, nitrate formation is most rapid at 17 per cent. moisture and_ nitrate accumulation reaches a maximum in December. In Queensland, r;easons are more variable and most rain usually falls in the summer months. However, in districts falling within the thirty inch isohyet, a line which rougllly demarcates the "coastal" from the 11inland" country,

. and delimits the neutral and calcareous soils, falls of ra,in sufficient to cause heavy leaching occur only infrequently. Nitrate removal is brought about mainly by plant growth. Although systematic studies on soil nitrates have 110f. br.eu published for Queensland soils, these have be<�n made during the past J'e1Y ;years in co1medion with cotton growing. From conversations with Mr. W. (J. Wells* it is clear that nitrate accumulation is at times extraordinarilY high �{nd reaches a maximum in "normal" seasons in the month- of Decem he;. It is significant that most of the iield cases of'poisoning attributed to Salvia o<:cur in the period November to l!"ebruary. The spasmodic rains tend to provide sufficient soil moistur·e for nitrate formation without being sufficient for continuously high photosynthetic aetivity and so encourage storage of nitrate in the pl:ntt; of. Emmert and Ball (1933).

(B) 'Phe 1'omic Action of Nitmte8 and Nitrite8.-The reaction involved in the conversiou of hrernoglohin to methremoglohin is shown to be as follows (Heulmer and Meier, 1925): .

4-Hb02 + 4HN02 + 2Hz0 ---;. 4HbOH + 4HN03 + 02 At pH7 the eon version of oxyhmmoglobin to methremoglobin is complete

provided that 5 mols. of nitrite are present to 1 mol. of oxyhremoglobin, smaller amounts of nitrite causing less complete conversion. Haldane, Makgill and :M:avrogordato (1897) made a thorough investigation of the· anox::emia following nitrite poisoning. The dangerous symptoms depend, justas in carbon monoxide poii;oning, on the amount o:f' blood pigment rendered incapable of carrying oxygen to the tissues. Trans-itory methremoglobinmmia can occur, in which lfi to 40 per cent. of methmmoglobin can be formed with­out ill effects; if the amount rises to about 50 per cent. toxic symptoms appear, while 70 per cent. conversion causes death. HecO\'ery is brought about by the l'edueing enzymes present in the corpuscles, provided that the amount of nitrite present is not large enough to bring about a lethal conversion to methmmog-Iobin. Such recoveries followiug typical anoxa:-mia symptoms have been observed in the field following the poisoning of slwep and cattle by

• Director of Cotton Culture, lJi>pt. of Agrk. and Stock. Queensland. Page 18

THE TOXIC PROPEHTIES OF BALVIA REFLEXA.

Sa.lvia refiexa. If death is averted in this way the methremoglobin and nitric oxide hremoglobin soon disappear, leaving the blood normal. If the bodies of animals poisoned by nitrite are allowed to lie, the blood loses its brown colour and becomes purplish red. This is due to bacterial reduction of the methremoglobin to hremoglobin, which then combines with nitric oxide produced from excess nitrite present to produce nitric oxide hremoglobin which is red. It follows that post-mortem examination if made some time after death will fail to show methremoglobin.

(c) Nitr·ite Poisoning Under Field Con.ditions.-There are few recorded instances of nitrate poisoning of stock. Seekles and Sjollema (1932) showed (a) that 100 grammes potassium nitrate suddenly introduced into the rumen (by fi!'ltula) of a one year old ox caused toxic effects due to production of

meth:emoglobina>mia, (b) that a cow could eat in the course of a day herbage containiug 300 grammes KNO" without ill effects, and (c) that 62 grammes potassium nitrate per day to an ox fm· 116 days gave no ill effects. Another report states that four cows were killed by well water containing one ounce sodium nitratP per ga1lon (Fincher, 1936).

In the ordinary way, herbage containing high proportions of inorganic nitrogenous tompounds is unpalatable to stock, and the ma8sive dose of nitrate necessary for toxic effects will not l!e ingested. ·when, however. livestock have been travelling for some days on bare stock routes or are

untrucked after a long train journey, such considerations do not eount. An�­greeu or succulent herbage is greedily consumed. and if it happens to contain larg-e quimtities of nitrates, toxic effect!; follow.

In \'iew of this, the characteristic symptom� and signs of methremo glohinremia should be carefully looked for in the investigation of cases of plant poisoning occurring under such conditions and the nitrate content of all plants greedily eaten at the time should be determined. 'Ve feel that a possible explanation of many obscure eases of plant poisoning may be thus forthcoming.

Further inwstigation of the points raised in this discussion is being undertaken.

Summary. 11) Sttlt·"ia t·efiexa contains up to 5 per eent. of potassium nitrate on a

dry matter basis. The water extract of two pounds of the plant can cause death due to mcthremoglobiulPmia, the characteristics of which are discussed.

(2) Nitrate accumulation in plants in amounts sufficient to be toxic is most likely in the drier part!'! of the State following the spring and early summer rains.

·Acknowledgements.

Our thanks are due to field officers of the Department of Agriculture and Stock for providin� material; to Dr. J. Legg and Mr. Thorn who arranged and assh:ted with tlw feeding experiments. Mr. C. H. Finnemore kindly allowed one of us acerss to papers in his library which were unavailable in Brislmne.

References. Bemheim. F., and Dixon, M. ( 1928) .-Bio-ehem. J .. 22: 113. ChibnalL A. C., and Miller, E. J. (1931) .--J. bioi. Chem .. 90: 189. Cory, A. H. (1932).-Qd. agrie. J., 37: 233.

Page 19

THE TOXIC PROPERTlES OI<' SADVlA REl!'DEX,A.

Pamphl. Coun. sci. indu,str . .Res. Aust. ( 1935) .-No. 49.

Emmrrt, E. M., and Ball, F. K. (1933).--Soil Sci., 35: 295.

Fincher, M. G. (1936) .·--Cornell Vet., 26: 271. Abstr. in Ohern . Abstt. (1936). 30: 8389.

Haldane, J., Makgill, R H., and Mavrogordato, A. E. (1897).--,J. Physi.ol., 21: 160.

HPubner, W., and Meier, R. ( 1925) .-Nachr. Ges. Wiss. Gottingen, 73. Abstr. in Chern. A/Jstr. (1921). 21: 2757.

·

N.S.W. DcopL of Agriculture (1935).·--Agr-ir. Gaz. N.S.W., 46: 252.

Prescott, J. A., and Piper, C. IL (1930) .--J. agric. Sci., 20: 517. Rimington, C. (1933).--··S. Afr. J. Sci., 30: 472. Seekles, L., and :::ljollerna, B. (1932) .-A-rch. wiss. pm/{t. Tier-heillc., 65: 331. Abstr.

in Nutr-. Abstr. Ret'. (1933). 34: 329.

-�---·---------

.I.USTIV,LASIAN MEDICAL PURLIIIHINQ COM PAtH' LIMITILII