THE ANTISCORBUTIC FACTOR IN LEMON JUICE.

 XXII. THE ANTISCORBUTIC FACTOR IN LEMON JUICE.

 BY ARTHUR HARDEN AND SYLVESTER SOLOMON ZILVA.

 Biochemical Department, Lister Institute. 

(Received July 4th, 1918.) 

In a previous communication [1918] we have described an attempt to isolate the antiscorbutie principle by means of adsorption. The attempt proved unsuccessful but we were able to make a series of observationa in connection with the behaviour of the antiscorbutic factor in orange juice towards some adsorbents under certain conditions. We have since attempted to study the chemical nature of the factor in question adopting another method of investigation, namely that of fractionating an antiscorbutically active juice. We have chosen lemon juice for that purpose. As is well known the main bulk of the solids of lemón juice consists of citric acid. In the event of the antiscorbutic being associated with the citric acid, good yields of the acid could be obtained from the juice for further investigation. If on the other hand by removing the citric acid the solid residue remained active, the antiscorbutic would then be associated with a comparatively much smaller mass of extraneous matter offering a better opportunity of studying both its chemical and physiological properties. The latter has proved to be the case and we now describe in this communication a few experiments from an investigation in progress on the above lines.

 EXPERIMENTAL. 

The following procedure was adopted in order to remove the free citric acid from lemon juice. To freshly squeezed out lemon juice an excess of precipitated calcium carbonate was added with constant stirring. The resulting suspension was then treated with two volumes of absolute alcohol and filtered through a fluted filter. The residue remaining on the filter was then pressed out in a hand press and the liquid thus obtained filtered. Both filtrates, which should be clear, were combined and evaporated in vacuo at 35° C. until all the alcohol had been removed and were then made up with distilled water to the original volume of the lemon juice used. The solution thus obtained is very slightly acid to litmus, yellow in colour, sweetish to taste and retains the characteristic lemon flavour as well as the bitter taste of the lemon. On evaporating the solution to dryness and heating the residue at 110° for an hour the equivalent of 1 cc. of the original lemon juice yields a residue weighing about 15 mgms. The residue after heating becomes caramelised and syrupy and tastes very sweet. It reduces Fehling's solution freely. On incinerating the residue about 3 mgms, of inorganic constituent per cc. of original lemon juice is obtained. The inorganic residue resulting from 10 cc. lemon juice treated as above when dissolved in a known quantity of decinormal HĻ80, and titrated back required 2 cc. of N/10 H2S04 to neutralise the carbonates produced by the incineration of the small quantities of the salts of organic acids present and this when calculated on the basis of sodium citrate corresponds to 1-7 mgms. per cc. When the decitrated juice was treated with CaCl, and two volumes of absolute alcohol a scanty precipitate of a few mgms. was obtained from 10 cc. of the solution and this consisted of the calcium salt of some organic acid or acids, as it charred and amelled of burning sugar when heated. A sample of lemon juice evaporated to dryness and heated at 110° for one hour gave a total residue of 105 mgms. per co., 4 mgms. of which was inorganic. 

Experiments were instituted to test the therapeutic and other properties of the treated lemon juice. 

 _Expt. 1._ The purpose of this experiment was to test the antiscorbutic potency of the treated lemon juice. Quantities equivalent to 2 co., 3 cc., 5 cc. and 7 cc. were tried respectively on four guinea pigs. The animals received oats and bran _ad lib_ . and 50 cc. of autoclaved full milk per diem. The respective doses of the treated lemon juice were administered daily by hand. The animals were allowed to go on for about eighty days when they were all chloroformed. It will be seen from fig. 1 which represents the weight curves of the animals, that on 2 cc. guinea pig No. 71 just managed to maintain its weight. Although it was unable to grow on that dose it failed nevertheless to succumb to scurvy. The other animals in the experiment actually succeeded in putting on weight. At the post mortem examination guinea pig No. 72, which received a dose of 3 cc. showed slight haemorrhages in the femoral muscles and also slightly enlarged costochondral junctions. The other animals (Nos. 54, 55 and 71) showed no signs of scurvy at al. This experiment shows without any doubt that after removing the free citric acid the residual fraction of lemon juice retains some antiscorbutie activity.