TOXICITY OF
DERRIS AND CUBE JOSEPH A. MATHEWS AND HOWARD D. LIGHTBODY Division of Pharmacology, Food and Drug Administration, U. S. Department of Agriculture, Washington, D. C.
CONOMIC considerations lead to the use of powdered derris or cube, or extracts made from them, as insecticides instead of the highly active principles, rotenone and its relatives, which have been isolated from these plants. Although in the earlier studies of the action of derris it was believed that rotenone was the essential constituent, recent work (3, 6) has strengthened the view that the roots of derris and cube possess more inseeticidal activity than can be accounted for by their rotenone content. Experiments reported in the preceding paper (4) indicate that rotenone may under certain circumstances be distinctly toxic to warm-blooded animals and must, therefore, be considered as potentially toxic to man. The present report is a similar study of the toxicity of derris and certain extracts made from it for warm-blooded animals, with an attempt a t correlation of the findings with the rotenone content as determined chemically, and with the toxicity for insects as determined on house flies.
given as a suspension in olive oil or as a solution in olive oil with the insoluble material removed by centrifugation, the amounts of powdered derris required to kill 50 per cent of a group of rats were approximately the same-that is, about 244 mg. per kg. Since the rotenone content of this sample of derris is 5.1 per cent, this amount of powdered derris would contain about 12.5 mg. of rotenone. In the previous paper it was reported that, given in solution in olive oil, about 25 mg. per kg. of rotenone were required to kill. The apparent rotenone value is, therefore, approximately twice the actual rotenone value or about 10 per cent. The equivalence in toxicity of the whole powder (the suspension) and the oilsoluble fraction would indicate that only those substances which are soluble in olive oil share in the lethal action, and that whatever remains in the marc is either innocuous or is in such a state that it does not.contribute to the toxicity. T h a t the latter supposition is correct, and that a considerable portion of the potentially toxic fraction of the whole root powder is notactive as a poisonousagent when ingested orally or when extracted by Oil, is indicated by the results of the administration of the acetone and ether extracts, respectively. Table I shows that the fatal doses from these fractions are, respec-
Experimental Procedure Three samples of Powdered derris root and one of cube were obtained through the courtesy of the Bureau of Entomology and plant Quarantine; that bureau also supplied information as to the rotenone content by determination, and the apparent rotenone or “rotenone based on toxicity.” This figure represents the amount of rotenone that would be present were all the toxicity for house flies due t o the rotenone. The materials were as follows: derris 2581, of which only that portion passing through a 100-mesh screen was used with an estimated rotenone content of 5.1 per cent (the original powder was rotenone 4.7 per cent, and rotenone based on toxicity not determined); derris 401, rotenone none, rotenone based on toxicity 5.5 er cent; derris 2217-M-1, rotenone 7.8 per cent, rotenone basecfon toxicity 15.0 per cent; cube 2218-M-1, rotenone 3.8 per cent, rotenone based on toxicity 8.5 per cent. For the determination of toxicity, young adult white rats were used. Prior to use, food was withheld for 16 hours. The materials studied were given by stomach tube, and the animals were subse uently kept under observation for 7 days, although rarely did ajatality occur after the third day. Since it was shown in the preceding paper (-4) that solution of rotenone in liquid fats affords a means of controlling particle size, and since particle size seems to be a factor in the toxicity, all materials used in the present study, unless otherwise stated, were administered in solution in olive oil. When or anic solvents were used, the solid extracts left on evaporation o? the solvent were powdered, triturated with oil, and warmed in a steam bath for several hours with frequent stirring, or were heated for a few minutes over a free flame to slightly over 100’ C. The mixtures were then cooled and made to volume with olive oil. Oil extracts of powdered roots were prepared in a similar manner, the residues being separated by centrifugation. The determination of toxicity consisted in finding the dose in milligrams of material per kilogram of rat weight necessary to kill 50 per cent of a oup of rats. Whenever the expression M. L. D. (minimum lethaydose) is used, it refers to this value.
TABLE1. R~~~~~~OF T~~~~~~~ D~~~~~~~~~~~~~ON D~~~~~ 2581 Dosage
Form
Mg. Guspenaionin olive oil
Olive oil extract
Aoetone extract in olive oil
Ether extractin olive oil
Effect of Solvent The symptoms of toxicity are essentially those described for rotenone in the preceding paper and need not be discussed here. Of the material obtained from the Bureau of Entomology and Plant Quarantine, derris 2581 was studied most extensively. The results of the toxicity determinations on this material are given in Table I. Table I shows that, whether
extract in olive oil
F
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No. No. Per of of Cent Apparent Rats Deaths Deaths Rotenone Mg. P e r cent 4 0 0 10 12.5 12 7 58 15 20 12 00 17.5 7 6 86 20 7 7 100 10 4 1 25 10 12.5 12 7 58 15 20 10 50 17.5 7 5 71 20 7 7 100 8 2 25 25 5 4 2 50 0 8 5 03 8 4 3 75 9 4 3 75 4 12 4 100 4 4 100 15 18 4 4 100 4. 25 20 4.6 4 1 25 5.1 08 3 38 6.1 12 0 50 7.8 6 5 83 10.2 4 4 100 15.4 4 3 75 20.5 4 4 100 25.0 4 4 100 8.3 12.’ l3 15.3 12 6 50 17.9 8 6 75 20.4 4 3 75 25.6 4 4 100
Dosage per Kg. yr Kg. Roteowder none
195 244 293 342 390 195 244 293 342 390 78 98 117 150 170 235 293 352 8o 90 100 120 150 200 300 400 500 250 300
360 400 500
d
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TABLE 11. COMPARISON OF CHEMICAL AND TOXICITY DATA Per Ceut Rotenone by Analysis Sample No. Derris 2581 5.1 Derris 401 None Derris 2217-M-1 7.8 Cube 2218-M-1 3.8 (I Rotenone based on toxicity.
Per Cent Toxicity to House FliesQ 6:5
15 8.5
--
-1M. L. D. of Root Powder, Mg./Kg.Per Cent Apparent Rotenone in PowderSuspension Olive Suspension Olive in oil Aaetone Ether CCln In oil Acetone Ether CCh olive oil extract extract extract extract olive oil extract extract extract extract 244 244 98 120 300 10 10 25 20 8.3 700 .. 960 3.6 2.6 ... 100 so 200 25 30: 1 .. 12.6 300 .. 370 ,. 8.9 .. .. 6.7
... ...
...
...
...
tively, 100 and 120 mg. per kg., equivalent to a n apparent rotenone value of 25 and 20 per cent. If the toxicity of the acetone extract were ascribed only to the rotenone present, the fatal dose would be of the order of 5 or 6 mg. per kg., far from the actual value of 25 mg. reported in the preceding paper. The carbon tetrachloride extract, however, did not prove as effective; the apparent rotenone value was 8.3 per cent, corresponding to an amount of powdered root of 300 mg. per kg.
Comparison of Toxicities As f u r t h e r check on the part played by oil in increasing availability as measured by lethal action, a solid acetone extract of derris 2581 was powdered and administered as a s u s p e n s i o n in starch paste. Although, as previously stated, such a n acetone extract in solution in olive oil has a fatal dose of about 100 mg. per kg., or a n apparent rotenone value of 25 per cent, that fed in starch had about half this activity, the fatal dose being 195 mg. p e r k g . , o r a n apparent rotenone value of 12.5 per cent. The increase in toxicity of the materials administered in olive oil as compared to that administered in starch suspension is less than would be expected from the results reported in the previous paper (4). There the toxicity of rotenone was increased as much as twenty times by administration in oil. If the toxicity of the starch suspension is largely due to nonrotenone materials, and this theory appears justifiable because of the low solubility of rotenone in water, then the enhancement of the rotenone fraction in the oil probably does not account for all of the f i v e f o l d i n c r e a s e shown by the figures. In order to test the completeness of extraction, the marc from the acetone extraction was fed in amounts u p to 10,000 mg.per kg. without any signs of toxicity. The carbon tetrachloride marc was reextracted with acetone, and the small amount of material was dissolved in oil and fed in amounts corresponding to 1000 mg. per kg. of the dried powder, without production of any symptoms. Either carbon tetrachloride changes the
.. ..
..
solubility, or the toxicity of the toxic material is reduced, since only approximately one-third as much activity can be obtained from the carbon tetrachloride as from the acetone extract. Results similar to those described were obtained in experiments with derris 401 which contains no rotenone (Table 11). The olive oil solution is here again more toxic than the carbon tetrachloride extract, the respective fatal doses being 700 and 960 mg. per kg., based on the original powder. The two extracts are as effective as though - they contained 3.6 and 2.6 per cent rotenone-i. e., apparent rotenone. Derris 2217-M-1, w h i c h c o n tained 7.8 per cent rotenone, was When extracts from derris or fed in the form of three extractscube root powders are fed to olive oil, acetone, and carbon tetrarats in olive oil solution, the chloride-with the results given in Table 11. Here again the same toxicity is greater than would order holds in the relative toxicities, be expected on the basis of the the acetone extract having a fatal rotenone content, indicating dose of about 80, olive oil about 100, that there may be present suband carbon t e t r a c h l o r i d e about stances, other than rotenone, 200 mg. per kg. Expressed as apparent rotenone, these values are which are physiologically ac31, 25, and 12.5 per cent, or aptive. proximately four, three, and two The relations between the times the actual rotenone content. toxicity of extracts and roteThese ratios are essentially the none content vary with the solsame as were found for derris 2581. Here again the total a p p a r e n t vent used and with the sample rotenone does not appear in the of derris from which they are olive oil extract, though the fracprepared. Acetone and ether tion is greater than that in sample extracts are about equally 2581. After the separation is made, toxic, and these solvents aphowever, olive oil does serve to enhance the availability, at least as pear to remove completely the shown in the toxicity figures. active s u b s t a n c e s . Carbon Cube 3218-M-1, which contains tetrachloride likewise appears 3.8 per cent rotenone, was fed t o to remove completely the toxic rats as the carbon tetrachloride agents but with partial loss of and olive oil extracts (Table 11). Here again the olive oil extract is physiological activity. Olive the more toxic of the two, and the oil fails to remove completely r e 1a t i o n s between rotenone and the principle from the powders apparent rotenone are similar to but does markedly accentuate those found for derris root powder. the toxic properties if the reTable I1 as a whole shows that there is no fixed relation between agents are extracted by acetone the rotenone content of any of the or ether and subsequently fed four root samples and the toxicities in oil solution. of the several extracts. In general, Rotenone content is not a the acetone extract is the most measure of toxicity of derris toxic and the carbon tetrachloride least toxic. The olive oil preparawhen administered orally to tion falls between the two, but it warm-blooded animals. The may approach one or the other of possible presence of still other the values. When the toxicities of substances that may be inthe various extracts are used for the jurious is discussed, calculation of the apparent rotenone ~~
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in the root, i t is seen that the chemical determination of rotenone will give no definite measure of how toxic the various preparations made from the root will be when fed.
the extractives is in optically active form, and Fink and Haller (1) have shown that the optically active derivative of deguelin, dihydrodeguelin, is more toxic to mosquito larvae than the corresponding inactive form. Whether or not the variations in apparent rotenone can be associated with changes in the optical rotation is not as yet apparent.
Toxic Constituents of Derris Extractives The toxic properties of the acetone extract of derris 2581, when attributed to rotenone, indicate a content of 25 per cent, five times that found by chemical examination. Since it is unlikely that a substance of toxic propertie? equal to rotenone could be present in such amounts and still remain unfound, i t seems more ressonabIe to suppose that there is present one or more substances of much greater pharmacological activity than rotenone, or else that studies based on the crystalline rotenone as isolated do not portray the real danger involved in human ingestion. In this connection Fink and Haller (1) believe that rotenone is probably to be regarded as the most important insecticidal constituent in derris extractives. The other well-characterieed crystalline compounds present (deguelii, tephrosin, and toxicarol) do not in the isolated form possess sufficient toxicity to account for the effectiveness of derris extractives. ?Key point out that, as isolated, only rotenone possesses optical activity. Haller and LaForge (2) have, however, shown indirectly that at least part of the deguelin present in 0
Vinegar from Dates BHAGWAN DAS AND J. L. SARIN Government Industrial Research Laboratory. P. 0. Shahdara Mills, Lahore, India HE raw material is the fleshy part of the date fruit (PRoeina sylvestria). The ripe fruit is bright red, and the fleshy edible part is 60 per cent of the weight of the fruit. The average percentage composition of the fleshy part is as follows: Moisture Raw fiber Sugars Mineral substances Not specified
20.20 38.53 35.80 2.97 2.50
To secure a rapid and uniform alcoholic fermentation, the fruit is worked into a juice by mixing it with an equal weight of water, boiling for an hour, and pressing. This procedure is repeated three times for maximum extraction. It is then filtered, made to known volume, and analyzed for sugars by standard methods. Although the amount of juice and its Balling degree vary considerably according to the ripeness of the fruit, its quality, extent of fermentation already developed in the fruit, and variety of the fruit, we may expect a yield of juice of about 15" Balling, equal to one and a half times the weight of the fruit. Juice containing 15 to 17 per cent sugar (specific gravity, 13" Balling) is inverted by boiling with hydrochloric acid (1 to 3 cc. per liter) and is then inoculated with a culture by the addition of 0.25 ounce of fresh yeast per gallon of juice to which previously have been added 70 cc. of a solution of nutrient salts (2 grams potassium phosphate, 0.2 calcium phosphate, 0.2 magnesium phosphate, and 10.0 ammonium phosphate dissolved in 860 cc. of water). A good fermentation sets in within 24 hours; the optimum temperature varies from 80" to 90" F. for the reaction which reaches its climax on the seventh day. The absence of reducing sugars indicates the end of fermentation. After the seventh day the Balling measurement is 1" to 2", and the acetic acid content is 0.25 gram per 100 CC. The fermented juice is then subjected t o acetous fermentation in an open vat by the inoculation of pure, young, and vigorous rnycodemza aceti, preferably by the addition of 10 per cent unpasteurieea "mother vinegar" and keeping the mixture still for 2 months. Li ht is excluded from the room as far as possible, since acetic fermentation is inhibited by the direct rays of the
VOL. 28, NO. 7
Acknowledgment The writers are indebted t o H. A. Jones, Division of Insecticide Investigation, and F. L. Campbell, Division of Control Investigations, both of the Bureau of Entomology and Plant Quarantine, for the selection of the samples of derris and cube and permission granted by that bureau to repeat in summary their data.
Literature Cited (1) Fink, D. E., and Haller, H. L., J . Econ. Entomol. (in press). ( 2 ) Haller, H. L., and LaForge, F. B., J. Am. Chem. Soc., 56, 241519 (1934). (3) Jones, H.A., Campbell, F. L., and Sullivan, W. N., J . Econ. Entomol., 28, 285-92 (1935). (4) Lightbody, H. D., and Mathews, J. A., IND. ENCI.CHBIM.,28,
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809 - (1936>.
(5)
Tatterafield,' F., and Martin, J. T., Ann. Applied Biol., 22, 578605 (1935).
RECEIVH~D March 26, 1936. .
sun. By the use of special cultures, the loss of acid during acetification can be materially reduced, since it is intimately associated with the life of the microorganism.1 Vinegar, when formed, is pasteurized and stored for some months in air-tight containers for aging. During this long storage period the vinegar deposits albuminous matter, bacterial cells, etc., and undergoes partial clarification.
Clarification Vinegar from inferior-quality dates often becomes cloudy or hazy on standing, which may be due to the presence of colloidal materials, pectin, protein, gums, and tannins. This objectionable property becomes apparent within 30 to 60 days or may be delayed for 12 to 15 months or more. This clouding may occur in both pasteurized and unpasteurized vinegar, in sealed bottles or in those exposed to the air through cotton lugs. This cloudiness is removed more easily by a process o f sedimentation and filtration than by simple filtration; the method employed is chiefly mechanical. Hyflo Filter-Cel (I to 2 per cent) is stirred with the vinegar; as it settles, it carries with it the albuminous particles causing the turbidity. By subsequent filtration under pressure, the sample filters brilliantly clear.
Sterilization After clarification, vinegar still contains acetic bacteria; on exposure to air, the bacteria grow on the surface and make the liquid turbid. Since all acetic bacteria perish at a relatively low temperature, it is sufficient to heat the vinegar to 150' F. by dipping the container in a water bath heated by steam. The vinegar is subsequently cooled by a current of cold water nearly to normal temperature. Sterilization also has the effect of maturing the vinegar and of giving it a softer taste and less acid aroma. This is probably due to its promoting the combination of the residual alcohol in the vinegar with acetic acid and thus accelerating the formation of the ester to which matured vinegar owes its flavor. The final vinegar is clear blackish red in color and smells like wine vinegar. It has a specific gravity of 1.18, a total acid content (as acetic) of 4.98 per cent, traces of ethyl alcohol, 2.17 grams of solids, and 0.17 gram of ash per 100 cc. Twelve to fifteen gallons of standard vinegar are obtained per 100 pounds of fresh fruit. The yield of vinegar from dried or cured fruit is greater because of the greater concentration of sugar. REICH~IVED May 11, 1936. 1 Mitchell, C. A., "Vinegar Manufacture and Examination," London. Charles Griffin t Co.
