Spectrophotometric determination of hydroxycynnamic acid and related compounds in echinacea preparations


O. A. Zaporozhets,1 E. A. Krushinsksya,1 V. N. Barvinchenko,2 N. A. Lipkovskaya,2
and V. K. Pogorelyi2

Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 37, No. 12, pp. 11 – 14, December, 2003.
Original article submitted November 12, 2002. In recent years, preparations based on biologically active the total content of carboxylic acids rather than the content of substances from various species of Echinacea Moench genus HCA derivatives accounting for the immunostimulant action.
have occupy leading positions on the market of drugs pre- A more selective method for determining HCA derivatives pared from medicinal plants. Echinacea extracts possess (calculated for chicoric acid) is based on the optical absorp- immunostimulant, antiinflammatory, wound-healing, and tion in the UV spectral range [7], which is included into a antimicrobial, and antiviral properties [1, 2]. Nevertheless, pharmacopoeial article for Echinacea purpurea herbs [8] and no simple and reliable analytical methods for monitoring the into a temporal pharmacopoeial article for the echinacea rhi- quality of echinacea preparations have been developed so far.
zome and roots [9]. Unfortunately, this method is still insuffi- In most cases, there is no need for identification and ciently selective and reliable, which is related to the fact that quantitative determination of each component in a given the analytical signal of optical absorption at 330 nm reflects phytopreparation, since the biological effect is frequently de- the presence of both HCA derivatives and their oxidation termined to a considerable extent by the synergistic action ofseveral substances, including unidentified compounds pres- products (quinones) absorbing in the same region [10]. For ent in trace amounts [3, 4]. For product quality monitoring and standardization, it is expedient to determine one or sev- pharmacopoeial method [7] show virtually the same content eral compounds from each group of biologically active sub- of HCA derivatives in both fresh preparations and those with stances contained in a given plant, which account for the expired storage duration, which can hardly be correct. It pharmacological effect of a preparation based on this plant.
should also be noted that chicoric acid cannot be used as a One of the the main groups of biologically active sub- reference compound because it does not meet all the require- stances in echinacea includes hydroxycinnamic (caffeic) acid ments of the existing normative documents [11].
(HCA) and related compounds representing HCA conjugates Since the immunomodulant action of echinacea is due to with sugars, quinic acid, and tartaric acid [1, 3, 4]. HCA pos- the HCA derivatives, it is important to develop a method for sesses antibacterial, antifungal, and antioxidant properties their selective determination in the presence of other stained [5]. A key role in the immunostimulant action of HCA prepa- substances contained in a given phytopreparation. In order to rations is played by chicoric acid [1, 3, 4]. For this reason, increase the selectivity of analysis, it is necessary to find a methods capable of determining the total content of HCA reagent capable of selectively interacting with HCA deriva- and its derivatives are commonly used for quality monitoring tives. As is known, HCA and related compounds exhibit the and standardization of echinacea preparations.
properties of both carboxylic acids and polyphenols. Readily According to a temporal pharmacopoeial article for the hydrolyzable metal salts form complexes with such com- echinacea rhizome and root tincture [6], the preparation qual- pounds but do not react with with their oxidation products ity is checked by titration. However, this method determines [12]. The known method of spectrophotometric determina-tion [13] of a HCA complex with zirconium chloride requires 1 Kiev National University, Kiev, Ukraine.
prolonged time and shows unsatisfactory reproducibility of Institute of Surface Chemistry, National Academy of Sciences of Ukraine,Kiev, Ukraine.
0091-150X/03/3712-0632$25.00 2003 Plenum Publishing Corporation Spectrophotometric Determination of Hydroxycynnamic Acid
We have chosen HCA to be the reference compound and studied the interaction of Al(III) with HCA for developing a new spectrophotometric method for the quality control of EXPERIMENTAL PART
Materials and instruments. Analytical-grade HCA
(Reakhim) was additionally purified by recrystallizationfrom hot (T = 70°C) distilled water, followed by drying to constant weight at T = 130°C. A HCA solution with a con-centration of 0.2 g/liter was obtained by dissolving an accu- rately weighed amount of the purified compound in distilled water; AlCl (0.50 M) and NH Cl (10%) solutions were pre- pared by dissolving analytical-grade compounds (Reakhim) Fig. 1. Normalized optical absorption spectra of the aqueous solu-
in 0.01 M HCl and water, respectively.
tions of (1, 4 ) HCA, (2 ) chicoric acid [10], and (3, 5 ) echinaceatincture: (1 3 ) without Al(III); (4, 5 ) in the presence of Al(III) The optical absorption measurements were performed on a Specord M-40 (Carl Zeiss Jena, Germany) and KFK-3 (LOMO, Russia) spectrophotometers in 1-cm optical cells.
Acidity (pH) of the sample solutions was monitored with anEV-74 ionometer equipped with a glass electrode.
the optical absorption of echinacea preparations in this spec- Analytical procedure. The optical absorption of the
tral region is mostly due to chicoric acid and other HCA de- complex of aluminum(III) and HCA was studied as a func- tion of pH. The sample solutions were prepared by mixing In the presence of Al(III), the spectra of an HCA solution 1.0 ml of a 0.2 g/liter HCA solution (or 0.3 ml of echinacea and an aqueous echinacea extract (Fig. 1, curves 4 and 5, re- tincture), 2.3 ml of a 0.5 M AlCl solution, and the necessary amount of a 10% NH Cl solution (to obtain the desired pH) bathochromic shift of the absorption peak observed upon in- in a 25-ml measuring flask. The optical density of each solu- troduction of the metal ions is related to the formation of tion was measured at a wavelength of 335 nm (A complexes between HCA derivatives and Al(III) in solution.
Then, the optical density was studied as a function of the Based on the analogous properties and close absorption spec- Al(III) concentration in solution. The samples were prepared tra of HCA and chicoric acid in solutions with and without Al(III), and taking into account that HCA is readily available 0.08 – 6.0 ml of a 0.5 M AlCl solution, and a 10% NH Cl and can be purified by recrystallization from aqueous solu- solution (to adjust pH 4.8). The optical density of each solu- tions, we have chosen HCA as a reference compound for the tion was measured at a wavelength of 335 nm (A spectrophotometric determination of the total content of The calibration curve was constructed using solutions HCA and its derivatives in echinacea preparations. The anal- prepared in 25-ml measuring flasks from 0.1 – 10.0 ml of a yses were performed at a wavelength of 355 nm correspond- 0.02 g/liter HCA solution, 2.3 ml of a 0.5 M AlCl solution, ing to the maximum absorption of the HCA – Al(III) com- and 10% NH Cl solution (to adjust to pH 4.8). The optical density of each solution was measured in a 1-cm cell at a In order to determine the optimum conditions for deter- mining HCA derivatives, we have studied the optical absorp- tion of HCA solutions in the presence of Al(III) as a function RESULTS AND DISCUSSION
of pH, aluminum chloride content, and HCA concentration in The standard substance has to be chosen so as to meet two requirements: it should be readily available and possessproperties specific of the object of analysis. In the case under TABLE 1. Determining Hydroxycinnamic Acid in Standard Solu-
consideration, such properties are the values of characteristic optical absorption in the UV and visible spectral regions. The spectra of cinnamic and chlorogenic acids (HCA derivatives more readily available than chicoric acid) differ significantly from those of echinacea extracts [4]. As can be seen from Fig. 1, the absorption spectrum of an HCA solution (curve 1 ) is generally analogous to the spectra of chicoric acid (curve 2 ) [10] and echinacea extract (curve 3 ). This is evidence that O. A. Zaporozhets et al.
Fig. 2. Plots of the optical density (l = 1 cm) at l = 355 nm versus
Fig. 3. The optical density (l = 3 cm) of the aqueous solutions of
pH for the aqueous solutions of (1, 2 ) HCA and (3, 4 ) echinacea HCA in the presence of various concentrations of Al(III) tincture (ET): (1, 3 ) without Al(III); (2, 4 ) in the presence of Al(III) The analytical signal intensity is a linear function of the HCA solution. It was established that the maximum optical density concentration in the interval from 0.1 to 8.0 mg/liter, in of HCA solutions in the presence of Al(III) is observed at which the calibration plot is described by the equation pH 3.7 – 5.5 (Fig. 2, curve 2 ). The optimum pH interval forthe determination of HCA and its derivatives is from 4.5 to5.5: under these conditions, the absorption is virtually inde- = (0.002 ± 0.001) + (0.0841 ± 0.0005)C pendent of pH, which must improve the reproducibility ofanalyses. This pH interval also features a plateau in the ab- for r = 0.9998. The detection threshold for HCA is sorption of echinacea tincture in the presence of AlCl (Fig. 2, curve 4 ). Subsequent experiments were performed Metrological characteristics of the proposed method with solutions adjusted at pH 4.8 ± 0.1 by adding NH Cl so- were determined by analyzing a series of standard HCA solu- tions. The results summarized in Table 1 show that the spec- Investigation of the dependence of the analytical re- trophotometric procedure provides for satisfactory reliability sponse on the Al(III) content in solution showed that the op- timum concentration of triply charged aluminum ions is An analysis of echinacea preparations should be per- 0.045 mole/liter (Fig. 3). The best Al(III) compounds are formed with allowance for the absorption of components chloride and sulfate, whereas the use of Al(NO ) is undesir- (e.g., the products of oxidation of HCA and its derivatives) able because nitrate ions exhibit strong oxidative properties.
not forming complexes with Al(III). This “background” can Under the optimum conditions, the interaction of Al(III) be subtracted by measuring the spectra with reference to a with HCA and its derivatives in a mixed solution immedi- solution containing all the same components except for ately produces coloration that is stable for at least ~1.5 h.
Table 2 presents the results of statistical processing of the experimental data on the content of HCA derivatives (re-calculated for HCA) in echinacea tinctures with various ex-piry dates. As can be seen, oxidation and decomposition of TABLE 2. Determining the Total Content of Hydroxycinnamic
the complex of biologically active substances (including Acid and Its Derivatives (Calculated for HCA, mg/liter) inEchinacea Purpurea Rhizome and Root Tincture (Batch 14.05.99) HCA derivatives) in the plant extract in the course of storage Using Intrinsic Absorption Method [7] and the Proposed Procedure leads to a general decrease in the optical absorption signal in- tensity. The presence of other colored compounds is respon- HCA found, mg/liter (x ± Dx ) sible for the absence of a direct proportionality between method [7] based on the measurement of the intrinsic optical absorption of echinacea tinctures. For example, the results of analyses using the proposed method with allowance for the * Maximum storage time according to manufacturer’s instruction, intrinsic absorption showed that the content of HCA and its derivatives (and, hence, the immunomodulant activity) Spectrophotometric Determination of Hydroxycynnamic Acid
TABLE 3. Determining the Total Content of Hydroxycinnamic
Acid and Its Derivatives (Calculated for HCA, mg/liter) in Echinacea Purpurea Rhizome and Root Tincture (Samples 1 – 6),Echinacea Purpurea Extract (Sample 7), and Immunal Preparation (Samples 8 and 9) by the Proposed Method (n = 3, P = 0.95) Fig. 4. The optical absorption spectra of (1 5 ) echinacea prepara-
tions and (6, 7 ) model HCA – quercetin 2 : 1 and 10 : 1 solutions,respectively, in the presence of Al(III). Solution concentrations (vol.%): (1 ) Echinacea purpurea extract (Lubnyfarm, batch 21100), 0.48; (2, 5 ) Echinacea Purpurea rhizome and root tincture (Kiev,batches 03.02.01 and 05.03.96, respectively), 1.2; (3, 4 ) immunal(Lek, batches 1102810B and 43045009A, respectively), 1.2;C = 1.75 ´ 10 – 5 (6 ), 3.5 ´ 10 – 6 M with the proposed spectrophotometric analysis for HCA and = 0.045 M; pH 4.8 ± 0.1; l = 1 cm.
its derivatives in water – alcohol based phytopreparations.
Determining HCA and its derivatives in echinacea
preparations. To 2.5 ml of a water – alcohol based echina-
cea preparation in 25-ml measuring flask was added distilled
dropped by a factor of almost 5, while the existing method water to the mark and the mixture was thoroughly stirred and [7] showed the decrease to be only by a factor of about 1.7.
filtered through a paper filter. To 1-ml aliquot of the filtrate As is known [1], echinacea ethanol extract contains, in in a 25-ml measuring flask was added 2.3 ml of 0.5 M AlCl3 addition to HCA and its derivatives, comparable amounts of flavonoids (glycosides of apigenin, luteolin, kaempferol, pH 4.8 ± 0.1 with the aid of a 10% NH Cl solution, after quercetin, etc.) representing another class of natural which the flask was filled with distilled water to the mark.
polyphenols and exhibiting significant optical absorption in The solution was thoroughly stirred and characterized with the wavelength range of l = 320 – 380 nm [10]. We have respect to the optical density at l = 355 nm in a spectropho- used quercetin to study the effect of flavonoids on the results tometer (or photoelectrocolorimeter) in a 1-cm optical cell of determination of HCA and its derivatives in echinacea relative to reference solutions containing the same compo- preparations. It was found that quercetin, as well as HCA, nents except for Al(III) salt. The content of HCA and its de- forms a complex with Al(III), but the peak of absorption of rivatives is determined using the A this complex in the UV – VIS spectral range occurs at 433 nm [14]. Figure 4 shows the absorption spectra of model Table 3 presents the results of analyses for a series of wa- solutions containing HCA and quercetin in different ratios ter – alcohol based echinacea preparations and the complex (curves 6 and 7 ) in comparison to the spectra of echinacea preparation immunal from various manufacturers. The cor- preparations (curves 1 5 ) in the presence of Al(III). As can rectness of these results was confirmed by the method of be seen from this figure, even the spectrum of a mixed solu- standard additives. As can be seen, the content of HCA andits derivatives (and, hence, the immunomodulant activity) tion with an HCA/quercetin ratio of 10 : 1 (curve 7 ) clearly varies within broad limits even in products from the same reveals a peak related to the Al(III) – quercetin complex for- manufacturer. This scatter confirms the need for standardiza- mation. At the same time, the spectra of echinacea prepara- tions in the presence of Al(III) exhibit no absorption peak inthis region, which is evidence of the absence of quercetin andother flavonoids in solution. This can be related to a signifi- REFERENCES
cant (80-fold) dilution of the initial echinacea extract in the 1. A. V. Sereda and G. F. Moiseeva, Farmakom, No. 3, 13 – 23 course of the sample preparation. As a result, the concentra- tion of ethanol decreases from 40 to 0.5% and the flavonoids 2. V. K. Pogorelyi, V. V. Turov, V. N. Barvinchenko, et al., Chem. (poorly soluble in water) exhibit precipitation. Therefore, the Phys. Technol. Surf., No. 4 – 6, 301 – 320 (2001).
flavonoids present in echinacea preparations do not interfere 3. R. Rawls, Chem. Eng. News, Sept. 23, 53 – 60 (1996).
O. A. Zaporozhets et al.
4. L. M. Lysochenko, A. G. Kotov, Yu. V. Podpruzhnikov, et al., 10. V. A. Baraboi, Biological Action of Phenolic Compounds of Provizor, No. 6, 37 – 38 (1999).
Plant Origin [in Russian], Naukova Dumka, Kiev (1976), 5. A. V. Simonyan, Khim.-Farm. Zh., 27(2), 21 – 27 (1993).
6. Temporal Pharmacopoeial Article VFS 42U-100 / 38-194-96.
11. V. P. Georgievskii and A. I. Grizodub, in: Drug Technology and Standardization [in Russian], RIREG, Kharkov (1996).
Echinacea purpurea Rhizome and Root Tincture.
12. I. M. Korenman, Photometric Analysis: Methods for Deter- 7. V. A. Kurkin, O. I. Avdeeva, E. V. Avdeeva, et al., Rast. Res., mining Organic Substances [in Russian], Khimiya, Moscow 8. Pharmacopoeial Article FS 42-2371-94. Echinacea purpurea 13. V. V. Belikov and M. S. Shraiber, Farmatsiya, No. 1, 66 – 72 9. Pharmacopoeial Article FS 42U-44 / 4-663-00. Echinacea 14. V. V. Belikov and T. V. Tochkova, Farmats. Zh., No. 5, 40 – 44

Source: http://www.ecotest.univ.kiev.ua/nauka/krush_PCJ.pdf

Med. questionnaire

PLEASE COMPLETE IN BLACK INK AS THIS DOCUMENT MAY BE CONVERTED TO AN ELECTRONIC IMAGE Full Name of Proposer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Address . . . . .

A homoeopathic proving

ambulance got to me in eight minutes and that was from Tully 28 kilometers away. The guys put me on oxygen set to 28L a minute to force my airways open. Normally people will go on 14L a minute but I needed more because I was blacking out and my breathing was packing up……I woke This paper is adapted from articles placed in the up in intensive care two days later….it was a

Copyright © 2010-2014 Metabolize Drugs Pdf