Measurements have been made on the visible and UV absorption
spectra of jatrorrhizine chloride in pure aqueous, methanol,
ethanol a nd propa nol solutions and in the p resence of various
concentrations of e ither hydrochloric acid or potass ium h ydroxid e.
In all the solutions, potassium hydroxide conside rably modifies
the absorption curves. The modifications b ecome manifest in the
disappearance of the absorption maximum and in the appearance
of new bathochromic shifted maxima. In these changes isobestic
points were noticed in the absorption curves. At very high potassium
hydroxide concentrations in ethanol and propanol solutions, the
absorption maximum disappears from the vis ible region of the
spectrum.
It has been suggested that the addition of potassium hydroxide
causes the formation of the quinonoidic form of jatrorrhizine. The
carbinolic form probably appears only in ethanol and propanol
solutions

K. Weber

Z. Gašparec

English

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CR 0 AT IC A CHEMIC A ACT A 39 (1967) 175 CCA-468 547 .94 :545.82 Original Scientific Paper Studies on the Spectra of Protoberberine Alkaloids. II. Absorption Spectrum of Jatrorrhizine Z. Gasparec . and K. Weber Department of Forensic Medicine, Medical Faculty, University of Zagreb, Zagreb, Croatia, Yugoslavia Received March 20, 1967 Measurements have been made on the visible and UV absorp-tion spectra of jatrorrhizine chloride in pure aqueous, methanol, ethanol and propanol solutions and in the presence of various concentrations of either hydrochloric acid or potassium hydroxide. In all the solutions, potassium hydroxide considerably modifies the absorption curves. The modifications become manifest in the disappearance of the absorption maximum and in the appearance of new bathochromic shifted maxima. In these changes isobestic points were noticed in the absorption curves. At very high potassium hydroxide concentrations in ethanol and propanol solutions, the absorption maximum disappears from the visible region of the spectrum. It has been suggested that the addition of potassium hydroxide causes the formation of the quinonoidic form of jatrorrhizine. The carbinolic form probably appears only in ethanol and propanol solutions. Jatrorrhizine, the phenol alkaloid of the protoberberine structure, shows an absorption curve with three absorption maxima at 'A = 265, 280 and 334 mµ 1• As the quaternary ammonium base, jatrorrhizine should show the same kind of isomerism as berberine2 and consequently its absorption curves should show the same changes under the influence of potassium h ydroxide and hydrochloric acid as in the berber ine spectra3. We have not found any information on this in the literature and have therefore directed our investi-gations to the study of the jatrorrhizine spectra in various solvents in the presence of bases and acids. The results of our measurements have shown that the absorption curves of pure jatrorrhizine chloride solutions agree mainly with those of berberine chloride. This agreement leads to the conclusion that both compounds possess the same nature of molecular structure, i. e. a protoberberine structure with a quaternary nitrogen (I). The appearance of the absorption maximum at /, = = 240-3 mµ can be attributed to the phenol group contained in the jatror-rhizine molecule. The potassium hydroxide in the jatrorrhizine molecule causes changes probably different from those in the berberine molecule because the changes in the absorption curves also differ substantially. The phenol group, parff-cularly in solutions with low potassium hydroxide concentration, could be ~upposed to allow . the formation of a resonant structure so as to permit the possibility of a quinonoid form (II) . 176 Z. GASPAREC AND K. WEBER l 11 III The appearance of the quinonoid -structure can be explained with the disappearance of the absorption maxima at }, = 224-8, 340-50 and 432-40 ma (analogy: naphthol-naphthoquinone4•5). The quinonoid structure can also easily explain the deepening of. the colour (from orange to red). The appearance of isobestic points suggests that the quaternary and quinonoid forms are in dynamic equilibrium and that they change from one into the other under the influence of bases and acids. This supposition does not exclude the possibility of tautomerism bound to the quaternary nitrogen atom and the appearance of the carbinol form, but in weaker potassium hydroxide concentrations the quinonoid structure probably dominates. The increased concentration of potas-sium hydroxide in ethanol and propanol solutions of jatrorrhizine chloride leads to the disappearance of the solution colour and, associated with this, to an absorption maximum in the visible spectrum region and to a decreased value of the extinction coefficient maximum at /. = 372--404 mµ with a hypsochromic shift of about 15-40 mµ . This phenomenon could be explained with the fixation of the negative charge to th e oxygen because of the high potassium concentration which inhibits the formation of the quinonoid structure and allows the formation of the carbinol form (III). This assumption is supported by the fact that in the long wave ultraviolet region the absorption maximum is shifted hypsochromically while the absorption curves assume a form similar to that of berberine. Since at A = 270 mµ no new absorption was observed, probably no open aminoaldehydic form is created except in propanol solution of high potassium hydroxide concentration. Our examinations have further shown the kind of solvent which influences the position of the absorption maximum in solutions with a definite potassium hydroxide concentration. Since this effect was not observed in pure solutions or solutions containing hydrochloric acid, it may be supposed that a free electronic pair appears on the nitrogen, which agrees with our suppositions. EXPERIMENTAL The absorption curves of jatrorrhizine · chloride were measured in both pure aqueous, methanol, ethanol and propanol solutions, and in the presence of either hydrochloric acid or potassium hydroxide. The measurements were carried out in the visible and ultraviolet regions of the spectrum. For measurements in the visible region of the spectrum a Carl-Zeiss - Jena universal spectrophotometer was used and for measurements in the ultraviolet region a Unicam S. P. 500 spectrophotometer. The jatrorrhizine chloride used in these measurements was isolated from th£ rind of the root of Common barberry (Berberis vulgaris) 6, and purified by recry-stallisation from ethanol (960/o) until the chromatographic sample showed only SPECTRA OF PROTOBERBERINE ALKALOIDS 177 a redish stain, which, when exposed to the ultra-violet light of the quartz lamp, did not show any yellow fluorescence. The chromatography was carried out using the rising technique with 1°/o aqueous sodium carbonate solution. The stock solution of jatrorrhizine chloride were prepared by dissolving 0.0392 grams of jatrorrhizine chloride in 100 ml. of the respective solvent (water, methanol, ethanol and propanol). The solvents had previously been purified by distillation. The potassium hydroxide and hydrochloric acid were of analytical reagent grade. By dissolving them in the respective solvents the stock solutions were prepared (1 N). The Preparation of Samples for Measurements The samples to be measured were prepared in the same way as in the exami-nation of the berberine chloride spectra3• The concentration of jatrorrhizine chloride in the samples amounted to 5 X 10-5 M. The concentration of potassium hydroxide in the measured samples varied between 2.5 X 10-5 and 1 X 10-2 M, and that of the hydrochloric acid between 1 X 10-5 and 1 X 10- 2 M; both in equal intervals of 0.25 units· The solution samples were prepared in the same way as for measurements in the visible and ultravio.let regions of the spectrum. The measurements were made at room temperature. The spacings between the individual measured points varied between 3 and 5 mµ. RESULTS The experiments showed that the solutions of jatrorrhizine chloride in water, methanol and propanol are yellow coloured. The addition of hydrochloric acid does not change the colour of the solutions except in the propanol solution where its addition causes a colour change into lemon yellow. Potassium hydroxide changes the colour of the solutions into orange or pink, depending upon the potassium hydroxide concentration. A high concentration of potassium hydroxide reduces the colour intensity and makes the colour disappear in propanol solutions. Measurements in the Visible Region of the Spectrum The absorption curves obtained by measuring the pure solutions, (except the propanol solutions) show marked absorption maxima in water at 'A = -= 425 mµ, in methanol at 'A= 433 mµ, in ethanol at 'A= 436 mµ. The hydro-chloric acid in the propanol solution causes a marked absorption maximum at 'A = 440 mµ (Fig. 1). Hydrochloric acid does not influence the absorption curves of the other solutions. Potassium hydroxide changes the shapes of the absorption curves in all the solvents. As an example we refer to Figs. 1 and 2. The changes in methanol and ethanol are the same. The absorption curves of the solvent with a potassium hydroxide concentration 5 X 10-4 M exhibit one maximum and one minimum (Table I). Solution aqueous methanol ethanol propanol 6. water/propanol TABLE I Absorption minimum I maximum 425 mµ 455-8 mµ 446 mµ 487 mµ 4.55 mµ 500 mµ 460 mµ 515 mµ 60 mµ 178 Z. GASPAREC AND K. WEBER The above data show a bathochromic shift in the absportion maxima if solvents of lower dielectric constants are used. The bathochromic shift in solutions with potassium hydroxide is greater (60 m~l) than in solutions containing hydrochloric acid (15 mµ) . ~ t 8000 7000 6000 5000 3000 2000 .d''\. \2 ~ •rn 10 Concn.HCl=l0.10-• M 2. ----3 x Concn. KOH =5.0 .10-5M 4 e Concn. KOH=2.5.10-4M 600 . mM. Fig. 1. Visible absorption spectra of p.-opanolic solutions of jatrorrhizine chloride containing· various concentrations of hydrochloric acid and potassium hydroxide. t. I 6000 5000 4000 3 000 2000 1000 0 400 1 a Concn. KOH =00 . l 2 x Con en. KOH =5.0 .10-5M 3" Concn. KOH =1.0. l) -4M 4 • Concn. KOH= 5.0 . 10-• M 500 Fig. 2. Visible absorption spectra of aqueous solutions of jatrorrhizine chloride containing various concentrations of potassium hydroxide. SPECTRA OF PROTOBERBERINE ALKALOIDS 179 A further increase in the concentration of potassium hydroxide in aqueous solutions does not lead to substantial changes in the absorption curves. In ethanol solution, with an increase in the concentration of potassium hydro-xide above 5 X 10-3 M , the extinction coefficient values diminish changing the form of the curve substantially. The same was observed in propanol solutions (Fig. 3) with the difference that the changes already began at 1.5 X 10-4 M KOH and that solutions with potassium hydroxide concentrations 1000 7000 1000 • 000 4000 3000 2 000 1000 400 500 1 a Concn. KOH =2.5.10-4 M 2o Concn.KOH=2.5x 10·'M 3 x Concn. KOH =2.5 .10-'M 4"" Concn. KOH =2.5 .10·1 M 600 Fig. 3. Visible absorption spectra of p ropanolic solution of jatrorrhiziile ehloride containing various concentrations of potassium hydroxide. II -- x a methanol 7000 - - - - x o water 2000 1000 4 6 B 10·> ' 10· ' -M KOH Fig. 4. The influence of potassium h ydroxid e on the valu es of the extinction coefficient of the 1st and 2'nd maxima of a queous and methanolic solutions in the visible region of the spectra. 180 Z. GASPAREC AND K, WEBER of 2.5 X io-1 M and more were almost colourless. Figs. 4 and 5 show the values of the extinction coefficients of the 1st and 2nd absorption maxima with respect to the concentration of KOH. n -x o ethanol 7000 6000 5000 4000 3000 2000 1000 oL-~~~~~~~~~~~~~~~~~~~~~~~~__J 2 4 6 8 10·• .10"" 10-<- M KOH Fig. 5. The influence of potassium hydroxide on the values of the extinction coefficient of the 1st and 2nd maxima of ethanolic and propanolic solutions in the visible region of the spectra. In the visible region, all the absorption spectra show isobestic points: the spectrum of aqueous solutions at 'A = 440 mµ, the spectrum of methanol solutions at 'A = 457 mµ, the spectrum of ethanol solutions at 'A = 467 mµ and of the propanol solutions at 'A= 472 mµ. Measurements of the Ultraviolet Region of the Spectrum The absorption curves of pure solutions show four or five absorption maxima (Table II). TABLE II v Solution Absorption maxima II I III I IV I aqueous 224 mµ - 274 mµ I 344 mµ -methanol 226 m,t 240 m,t 265 mµ 350 mµ -ethanol 228 mµ 242 mµ 264 m,t I 348 mµ 404 mµ propanol - 243 mµ 266 mµ I 354 m>t -Potassium hydroxide causes two changes only, one of which is discernible in aqueous and methanol solutions (Fig. 6). Potassium hydroxide makes the maxima III and IV disappear. Maxima II becomes more marked and maximum V appeares. This is over when the potassium hydroxide concentration is 1 X 10-3 M in the aqueous solution, 7.5 X 10-4 M in the methanol solution, 5 X io-4 M in the ethanol solution and 2.5 X io- 4 M in the propanol solution. In these solutions the absorption curves show three maxima (Table III). 30 000 20 000 10 000 200 SPECTRA OF PROTOBERBERINE ALKALOIDS 300 1 eConcn.KOH·OO 2 o Concn. KOH •2.5.10"4 M 3 xConcn. KOH=7.5x10-4 M 4,, Concn. KOH •1.0 .10"' M 400 181 Fig. 6. UV absorption spectra of aqueous solutions of jatrorrhizine chloride conta ining various concentrations of potassium hydroxide. 30 000 20 000 10 000 200 1 o Concn. KOH• 0.0 2 x Concn. KOH• 2.5 .10-'M 3 ,, Concn. KOH• 7. 5 .10-'M 4• Concn. KOH·2.5.10-'M 4 _;. i -~-~--L-1 3 00 Fig. 7. UV absorption spectra of propanonc solutions of jatrorrhizine chloride containing various concentrations of potassium hydroxide. Besides the already mentioned changes (Fig. 7) an increase in the potassium hydroxide concentration in ethanol and propanol solutions caused the disap-pearance of maximum I, diminished the extinction coefficients of max~ma II and V and caused a hypsochromic shift in the latter. If this concentration 182 z: GASPAREC AND K. WEBER TABLE III Solution Absorption maxima I I II v aqueous I 228 mu 244 mµ 371 mµ methanol 230 mµ 248 mLL 388 m~L ethanol I 230 mµ 248 mµ 398 mft propanol - 248 mµ 404 mµ I in the propanol solutions is increased above 5 X 10-2 M , maximum II is: shifted bathochromically and the values of the extinction coefficient (Fig. 8). increase rapidly. ' l 30 000 20 ooo L 200 1 o Con en. KOH =5.o.10-4 M 2 x Con en. KOH =5.0.10-3 M 3 6 Concn . KOH= 1.0 .10-2 M 4e Concn. KOH=5.o.10-2 M 5 ~ Con en. KOH= 1.0 .10-1 M 300 Fig. 8. UV absorption spectra of propanolic solutions of jatrorrhizine chloride containing variou!t' concentrations of potassium hydroxide. In the ultraviolet region the spectra of jatrorrhizine chloride show two groups of isobestic points (Table IV). TABLE IV Solvent Groups of isobestic points First Second aqueous - - 352 mµ - -methanol 220 mµ 259 mµ 360 mµ - -ethanol 228 m~L 259 mµ 364 mµ 288 mµ 377 mu propanol 239 mµ 259 mµ 367 mµ 295 mµ 347-70 mµ SPECTRA OF PROTOBERBERINE ALKALOIDS 183 Acknowledgement. We wish to express our thanks to the management of the Pliva Drug Factory and particularly to Mr. V. Jakie, mr. ph., for having allowed us the use of the Unicam spectrophotometer. REFERENCES 1. K. Feist, W. Awe, and H. Et z rod t, Arch. Pharm. 272 (1934) 817. 2. W. Huck e 1, Theoretische Grundlagen der Organischen Chemie, I. Band, Akad. Verl. Leipzig, 1956, p. 257. 3. Z. Gaspar e c and K. Weber, Croat. Chem. Acta 38 (1966) 143. 4. W. R. Br ode, Chemical Spectroscopy, John Wiley and Sons, Inc., New York, 195.6. 5. R. A. Friedel and M. 0 r chin, Ultraviolet Spectra of Aromatic Compounds, John Wiley and Sons, Inc. , New York, 1951. 6. Z. G as p are c and D. B a r k o vi c, Farm. Glasnik 22 (1966) 383. IZVOD Studije spektara protoberberinskih alkaloida. II. Apsorpcioni spektri jatrorizina Z. Gasparec i K. Weber Izmjerene su apsorpcione krivulje jatrorizin-klorida u vidljivom i ultraljubi-castom spektralnom podrucju u vodenim, metanolnim, etanolnim i propanolnim otopinama bez i uz dodatak kalium-hidroksida u koncentracijama od 2,5 X 10-? do 5 X 10-1 M i solne kiseline u koncentracijama od 1 X 10- ;; do 1 X 10- 2 M. Na temelju mjerenja ustanovlj eno je da ciste otopine jatrorizin-klorida pokazuju apsorpcione krivulje s pet maksimuma apsorpcije i da solna kiselina nema znatnijeg utjecaja na oblik apsorpcionih krivulja. Kalium-hidroksid u svim otapalima znatno utjece na oblik apsorpcionih krivulja i na vrijednosti ekstinkcionih koeficijenata. Pri niskim koncentracijama kalium-hidroksida ustanovljeno je nestajanje maksi-muma apsorpcije i pojava novih batokromno pomaknutih maksimuma. Izmedu prve i druge skupine maksimuma zapazene su izobesticke tacke. Poveeanje koncentracije kalium-hidroksida u etanolnim i propanolnim otopi-nama uzrokuje smanjenje novo nastalog maksi.muma u dugovalnom ultraljubicastom spektralnom podrucju. Na temelju rezultata mjerenja moze se zakljuCiti da se jatrorizin u neutralnim otopinama i otopinama koje sadrie solnu kiselinu nalazi u obliku kvarterne amonium--soli koja je zuto obojena. Utjecajem kalium-hidroksida stvaraju se dva nova tautomerna oblika : kinoidni i karbinolni. Prvi utjecajem malih koncentracija kalium-hidroksida u svim otapalima, a drugi samo u etanolnim i propanolnim otopi-nama i u prisutnosti velike koncentracije kalium-hidroksida. Kinoidni i kvarterni oblici su invertibilni i stoje u dinamickoj ravnotezi. ZAVOD ZA SUDSKU MEDICINU I KRIMINALISTIKU MEDICINSKI FAKULTET ZAGREB PrimJjeno 20. ofojka 1967. 

Studies on the Spectra of Protoberberine Alkaloids. II. Absorption Spectrum of Jatrorrhizine

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