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Spectrophotometric and Chromatographic Investigation of Bilberry Anthocyanins for Quantification Purposes
MICROCHEMICAL JOURNAL ARTICLE NO.
55, 12–23 (1997)
MJ961349
Spectrophotometric and Chromatographic Investigation of Bilberry Anthocyanins for Quantification Purposes,1 G. PETRI,*,2 U. KRAWCZYK,†
AND
´ . KE´RY* A
*Semmelweis University of Medicine, Institute of Pharmacognosy, Budapest, Hungary; and †Institute for Drug Research and Control, Warsaw, Poland
INTRODUCTION
Anthocyanins are intensively colored, water-soluble pigments known to be responsible for nearly all the pink and scarlet leaves and fruit of higher plants. They are important as coloring agents in the food and pharmaceutical industries. The anthocyanins of cherry, raspberry, blackberry, black currant, bilberry, vine grapes, elderberry, and some other plants are widely used as harmless pigments (2). Unlike the synthetic red pigments, anthocyanins are not stable and readily undergo changes during storage and processing of the raw material (3). These changes lead to loss of color, browning of products, and formation of precipitates in liquid extracts. Like flavonoids, anthocyanins are used in the treatment of disease states characterized by capillary bleeding associated with increased capillary fragility (4) (Fig. 1). They have a high free radical capturing effect in many diseases (Figs. 2, 3). Anthocyanin capsules are now being prepared for use in health regeneration and prophylaxis against cold viruses (8). However, the methods for standardization of these preparations are often of old types and not convenient for reproducing measurements. Thus, the aim of our investigation was to develop a good HPLC method for control. PLANT AND METHODS
Vaccinium myrtillus L. (Ericaceae): Bilberry, Huckleberry, Whortleberry (June– July: Leaves) This deciduous shrub is 20–60 cm high, with a creeping rhizome, numerous branched erect stems, and green angular twigs. The leaves are alternate, shortly petiolate, 0.8–3.5 cm long, ovate, acute, finely toothed, light green, with distinct crisscross venation. The flowers are 1–2, axillary. The corolla is pitcher-shaped, up to 6 mm in diameter, greenish-pink, with very short, turned-back lobes. The anthers are awned, opening by pores. The fruit is a berry, 6–8 mm in diameter, globose, black, and pruinose. Its habitat is heaths, moors, and woods, on acid soil. The active ingredients are tannin, myrtillin, pectin, arbutin, and vitamins B and C. It has effects as an astringent and a diuretic and reduces blood sugar level (leaves only). The parts used 1 2
From the special issue on the Seventh Italian-Hungarian Symposium on Spectrochemical Analysis. To whom correspondence should be addressed. 12
0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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QUANTIFICATION OF BILBERRY ANTHOCYANINS
FIG. 1.
13
Structures of the main Vaccinium myrtillus anthocyanins.
are mainly the dried or fresh fruits and, more rarely, the dried leaves; both are best dried in shade. Applications include use as a decoction against diarrhea, as a hot mouthwash for gingivitis, or as a gargle for inflammation of the throat. A decoction based on leaves is sometimes used but can have harmful side effects (hydroquinone poisoning) when used excessively or for long periods. The fresh fruits are sweet and very tasty. The freshly pressed juice is refreshing and a valuable source of vitamins B and C. The berries can be used in the home for soups, pies, preserves, jams, wine, etc. (1). In our investigation we used the isolated anthocyanidins of bilberry prepared in Italy, Inverni della Beffa factory, Milan (I); in Poland, Institute of Pharmaceutical
FIG. 2.
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Free radical induced membrane damage. From Feher et al. (5).
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FIG. 3.
Effects of vitamin E on arachidonic acid metabolism. From Feher et al. (5).
Industry, Warsaw (P); in Romania, Biofarm Bucharest (R); and in France, Laboratories Chibret Clermont-Ferrand (F) (6). TLC Method Anthocyanin extract was hydrolyzed and the aglycones were shaken out with isoamyl alcohol and identified by TLC in the following system: cellulose M.N. 300 F plate (layer thickness, 0.1 mm); developing system, formic acid:water:hydrochloric acid (6:5:1). Very polar, strongly acidified solvents are necessary for good preparation. Rf values of the main anthocyanidins Delphinidin Petunidin Cyanidin Malvidin
0.32 0.54 0.68 0.78
TABLE 1 Spectrophotometric Evaluation of Bilberry Extracts
Sample Malvin Extract Extract Extract Extract
standard I P R F
A11(535)*
Anthocyanin content†
A11(280)
A11(535) /A11(280)
558.92 84.66 380.21 19.20 7.80
100.00 15.15 68.03 3.44 1.40
268.09 69.85 240.60 51.90 28.70
2.08 1.21 1.58 0.37 0.28
* Specific absorbance (abbreviation of A1% 1cm). † In malvin chloride, % per dry weight.
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QUANTIFICATION OF BILBERRY ANTHOCYANINS TABLE 2 Chromatographic Data for Anthocyanin Separation by RP-HPLC Gradient Elution (System 10) Peak No.
Compound
tR
k*
1 2 3 4 5 6 7 8 9 10 11 12 13
Delphinidin-3-O-galactoside Delphinidin-3-O-glucoside Cyanidin-3-O-galactoside Delphinidin-3-O-arabinoside Cyanidin-3-O-glucoside Cyanidin-3-O-arabinoside Petunidin-3-O-glucoside Petunidin-3-O-arabinoside Malvidin-3-O-galactoside Malvidin-3-O-glucoside Malvidin-3-O-arabinoside Petunidin Malvidin-3,5-O-diglucoside
46.75 49.25 51.25 55.50 58.50 61.50 64.75 70.00 74.25 76.00 81.50 84.00 93.03
5.67 6.03 6.32 6.93 7.36 7.78 8.25 9.00 9.61 9.86 10.64 11.00 12.29
The anthocyanin extracts and malvin chloride standard solutions in 0.1 M HCl in methanol were applied to the plates. Anthocyanin spots were detected at 540 nm. We combined this TLC method with densitometry. The apparatus consisted of Shimadzu denzitometer, with a CS-930 high-speed, dual-wavelength TLC scanner and a DR-2 data recorder. Determination of Total Polyphenols and Tannins The official method of Ph.Hung., Ed.VII, with a modified concentration of solvent B and a new calculation was employed. For the determination of the crude drug, acetone:water (7:3, v/v) extracts of bilberry were taken. Sample preparation. Solution A containing about 0.050 mg of polyphenols in 1.00 ml was made by diluting the preparations and extracts with 40% ethanol. Then 10.00-ml aliquots of solution A were shaken for 1 h with 0.100 g of hide powder and filtered (solution B). To 1.00 ml of solvent A (/1.00 ml of methanol), 2.00 ml of solvent B reagents was added, and the absorbances were measured as described for the calibration curve. The content of compounds adsorbed on hide powder (tannins) was calculated as the difference between the total polyphenols and the amount of compounds not bound by hide powder. Determination of anthocyanins. Bilberry anthocyanin extracts were dissolved in 0.1 M HCl solution in methanol. UV–VIS spectra were recorded and absorbance was measured at 280 and 535 nm. Results were calculated in malvin chloride from the absorbance values at 535 nm. Standards Malvin chloride purum (691.04 g/mol) was obtained from Fluka AG. Authentic samples of delphinidin, cyanidin, malvidin, and petunidin glycosides were obtained from the Institute of Pharmaceutical Industry, Warsaw. Chlorides of delphinidin (338.70 g/mol), cyanidin (322.70 g/mol), malvidin (366.75 g/mol), and petunidin (352.71 g/mol) were obtained from Roth.
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FIG. 4.
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TLC densitometry fingerprints of bilberry anthocyanins.
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QUANTIFICATION OF BILBERRY ANTHOCYANINS
FIG. 5.
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TLC densitometry fingerprints of malvin chloride standard.
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FIG. 6. HPLC fingerprints: (a) extract P, (b) extract I, (c, d) malvin chloride standard. Partisil ODS, isocratic system B, detection at 535 nm, flow rate 1.0 ml/min (a–c), 1.5 ml/min (d).
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HPLC Techniques We worked with an internal standard method. The standard was malvidin–Cl (Fluka). HPLC apparatus used: 1. HP 1090 Hewlett–Packard liquid chromatograph with HP 10–40 diode array detector. 2. Pye Unicam 4100 liquid chromatograph with LC-XP gradient programmer CXPD pump, variable wavelength detector LC–UV–VIS, and PM 825 single pen recorder. A Rheodym 7125 recorder was used. The injection valve had a 20-ml 100 p. Columns: Li Chromosorb RP-18 10 mm Partisil 10-ODS C18 Nucleosil, equipped with RP-18 precolumn Isocratic systems: A. Tetrahydrofuran:formic acid:water (6:1:33) B. Tetrahydrofuran:formic acid:water (1:1:18) Gradient systems: 1. Solvent A: 5% formic acid; solvent B: MeCn; 0–30 min: 0.1–3.1% B, linear gradient; 31–45 min: 3.1–20.0% B, linear gradient; 46–80 min: 20.0% B, isocratic 9. Solvent A: 10% HCOOH; solvent B: MeOH:MeCN (1:1, v/v); 0–60 min: 0– 15% B, linear gradient; 61–90 min: 15% B, isocratic 10. Solvent A: 10% HCOOH; solvent B: MeCn; 0–60 min: 0–8% B, linear gradient; 61–90 min: 8% B, isocratic. Solutions were filtered through a Millipore sintered-glass filter prior to chromatography. Gradient procedure solvent mixtures were degassed with pure helium. Other operating conditions (flow rate, detection wavelength) are given in the figures. Retention was characterized by the following parameters: tR retention time of the component k * capacity factor: k * Å (tR 0 tM)/tM= where tM a
time taken for an unretained compound to transverse the column selectivity factor (relative retention characteristics of the two components A and B): a Å k *B/k *A .
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RESULTS
UV–VIS Spectrophotometry The results of the UV–VIS spectrophotometry of bilberry extracts are shown in Table 1. Anthocyanins are detected selectively in the visible region; our quantitative measurements were taken at 535 nm. Absorbance values in the UV depend also on the presence of other compounds (phenolic acids, tannins, etc.) in the extract. The extracts varied in anthocyanin content as well as in the ratio A11%cm (535)/A11%cm (280) . The anthocyanin content of extracts R and F were much lower than that of extracts P and I. Relatively high UV values of A11%cm (280) in extracts R and F suggest the presence of large amounts of other polyphenols in these samples. For the quantitative evaluation of anthocyanins the direct spectrophotometric method can be recommended, together with the estimation of the ratio A11%cm (535)/A11%cm (280) , which indicates the purity of the extract. Our results indicate that this ratio should be higher than 1.0 for extracts of good quality. Sometimes standardization of anthocyanin content after acidic hydrolysis and measurement of absorbance of aglycones is possible, but it is never exact, because if some proanthocyanidins are present in the extract, they form anthocyanins too after hydrolysis and give results higher than the real anthocyanin content. Therefore the visible/UV ratio is important for standardization. TLC Densitometry The characteristic ‘‘fingerprints’’ of bilberry extracts were obtained by TLC densitometry (Fig. 2). The standard of malvin chloride was separated into two main spots. Extracts I and P were separated into two or three main spots of aglycones and three main spots of glycosides. As the malvin standard was separated into two spots, the calculation was based on the total area of both peaks. The results of quantitative evaluation are presented in Table 2. The fingerprints of extracts P and I were similar, but extract P exhibited a higher total anthocyanin content. Extracts F and R could not be analyzed by TLC densitometry; because of the high content of impurities, the spots were tailing and good separation was not achieved (Figs. 4, 5). High-Performance Liquid Chromatography Isocratic elution—New RP-HPLC method. The extracts were analyzed by RPHPLC isocratic elution. The best results were obtained with tetrahydrofuran as organic modifier. Methanol or acetonitrile solutions in aqueous formic acid did not lead to sharp separation. HPLC analysis in an isocratic system consisting of THF:HCOOH: H2O, 1:1:18 (v/v), resulted in the characteristic fingerprints of bilberry anthocyanins. Under these conditions malvin standard was eluted as a single peak. Although isocratic elution allowed identification of bilberry extract and quantitative estimation of anthocyanin content, the separation of individual compounds was not achieved (Fig. 6.). Gradient elution—New RP-HPLC method. Full separation of bilberry anhtocyanins was achieved with RP-HPLC gradient elution. Methanol or tetrahydrofuran gradient in aqueous formic acid did not lead to sharp separation. Application of an acetonitrile:methanol (1:1, c/c) mixture as organic modifier improved the analysis, but better separation of delphinidin-3-O-glucoside and cyanidin-3-O-galactoside was obtained with acetonitrile gradient elution.
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FIG. 7. Chromatograms of bilberry extracts P and I. Partisil 10 ODS, gradient system 10, flow rate 0.5 ml/min, detection at 280 nm. Extract P: Polish; Extract I: Italian.
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TABLE 3 Composition of Bilberry Anthocyanin Extracts (Percentage Share of the Individual Compounds in the Total Amount of Anthocyanins) Peak No.
Compound
P
I
F
R
1 2 3 4 5 6 7 8 9 10 11 12
Delphinidin-3-O-gal. Delphinidin-3-O-gluc. Cyanidin-3-O-gal. Delphinidin-3-O-ara. Cyanidin-3-O-gluc. Cyanidin-3-O-ara. Petunidin-3-O-gluc. Petunidin-3-O-ara. Malvidin-3-O-gal. Malvidin-3-O-gluc. Malvidin-3-O-ara. Petunidin
12.47 14.46 6.34 14.76 — 11.44 12.03 7.89 4.28 14.32 1.99 —
13.52 15.47 10.92 12.87 5.72 9.49 8.19 7.67 2.86 9.49 2.60 1.17
— 9.35 — 19.42 12.95 5.75 12.95 20.86 — 18.70 — —
— — 33.01 33.01 — 19.18 — 14.78 — — — —
41.86 14.95 26.13 15.86
28.77 18.70 18.70 33.81
33.01 — 52.19 14.78
Summary Delphinidin glycosides Malvidin glycosides Cyanidin glycosides Petunidin glycosides
41.69 20.59 17.78 19.92
Because the eluted compounds were detected at 280 nm, it was possible to detect other polyphenols present in the extract. Phenolic acids, catechin, and some proanthocyanidins were eluted during 40 min of the procedure. The peaks of anthocyanins appeared within the next 50 min (Fig. 7). However, when the sample abounded in tannins, most of them were eluted together with anthocyanins and the baseline drifted. Chromatographic data on anthocyanin separation are specified in Table 2. Strong acidic conditions are required for the best sensitivity of the method, whereas in the qualitative analysis the same pattern of eluted compounds can be obtained with a less concentrated acidic component, 5% formic acid. The compositions of the extracts, based on RP-HPLC determination, are presented in Table 3. A preponderance of delphinidin glycosides was observed in all the extracts. Extracts I and P had similar compositions, except that we did not find cyanidin-3-Oglucoside in extract P. The content of malvidin glycosides was higher in extract P than extract I. In extract I, cyanidin glycosides prevailed over malvidin glycosides. Extracts F and R differed in their composition from each other and from extracts P and I. SUMMARY
We have compared different chemical methods for analysis of bilberry anthocyanin extracts. These can be performed by direct spectrophotometry or after chromatographic separation (TLC densitometry or HPLC). For quantitative evaluation of anthocyanins, we recommend the widely used direct spectrophotometric method, together with the estimation of the ratio A11%cm (535)/A11%cm (280) , which indicates the purity of the extract.
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TLC densitometry can be applied only to extracts of high purity. TLC separation of bilberry anthocyanins cannot be achieved with tannin-rich extracts. We have described a new RP-HPLC gradient procedure that seems the best method for standardization of bilberry extracts. It allows evaluation of the individual anthocyanins and detection of impurities such as phenolic acids and tannins. The same method can be used for other anthocyanin extracts, such as extracts of Althaea rosea, Vitis vinifera, and Sambucus nigra. Flow rate is the only HPLC parameter that should be changed as needed. REFERENCES 1. Laurent, E. Country Life Guides: Edible and Medicinal Plants of Britain and Northern Europe. Country Life Books, Hamlyn, London, 1981. 2. List, P. H.; Ho¨rhammer, L. In Vaccinium myrtillus L. Hagers Handbuch der pharmazeutischen Praxis, Vol. VI.C. Springer-Verlag, Berlin/Heidelberg/New York, 1979. 3. Iacobucci, G. A.; Sweeny, J. G. Tetrahedron, 1983, 30, 3005–3038. 4. Ga´bor, M. The Anti-inflammatory Action of Flavonoids. Akade´miai Kiado´, Budapest, 1972. 5. Fehe´r, J.; Csomo´s, G.; Vereczkei, A. Free-Radical Reactions in Medicine. Springer-Verlag, Berlin/ Heidelberg/New York, 1987. 6. Laboratories Chibret. Chem. Abstr., 1970, 72, 6238. 7. Krawczyk, U. Study and Research on Analytical Methods for Determination of Flavonoids and Anthocyanins in Some Plant Preparation. Dissertation (Supervisor: G. Petri), Med. Univ. Inst. of Pharmacognosy, Budapest, 1990. 8. Martindale, W. (Ed.) The Extra Pharmacopoeia. Pharmac. Press, London, 1982.
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55, 12–23 (1997)
MJ961349
Spectrophotometric and Chromatographic Investigation of Bilberry Anthocyanins for Quantification Purposes,1 G. PETRI,*,2 U. KRAWCZYK,†
AND
´ . KE´RY* A
*Semmelweis University of Medicine, Institute of Pharmacognosy, Budapest, Hungary; and †Institute for Drug Research and Control, Warsaw, Poland
INTRODUCTION
Anthocyanins are intensively colored, water-soluble pigments known to be responsible for nearly all the pink and scarlet leaves and fruit of higher plants. They are important as coloring agents in the food and pharmaceutical industries. The anthocyanins of cherry, raspberry, blackberry, black currant, bilberry, vine grapes, elderberry, and some other plants are widely used as harmless pigments (2). Unlike the synthetic red pigments, anthocyanins are not stable and readily undergo changes during storage and processing of the raw material (3). These changes lead to loss of color, browning of products, and formation of precipitates in liquid extracts. Like flavonoids, anthocyanins are used in the treatment of disease states characterized by capillary bleeding associated with increased capillary fragility (4) (Fig. 1). They have a high free radical capturing effect in many diseases (Figs. 2, 3). Anthocyanin capsules are now being prepared for use in health regeneration and prophylaxis against cold viruses (8). However, the methods for standardization of these preparations are often of old types and not convenient for reproducing measurements. Thus, the aim of our investigation was to develop a good HPLC method for control. PLANT AND METHODS
Vaccinium myrtillus L. (Ericaceae): Bilberry, Huckleberry, Whortleberry (June– July: Leaves) This deciduous shrub is 20–60 cm high, with a creeping rhizome, numerous branched erect stems, and green angular twigs. The leaves are alternate, shortly petiolate, 0.8–3.5 cm long, ovate, acute, finely toothed, light green, with distinct crisscross venation. The flowers are 1–2, axillary. The corolla is pitcher-shaped, up to 6 mm in diameter, greenish-pink, with very short, turned-back lobes. The anthers are awned, opening by pores. The fruit is a berry, 6–8 mm in diameter, globose, black, and pruinose. Its habitat is heaths, moors, and woods, on acid soil. The active ingredients are tannin, myrtillin, pectin, arbutin, and vitamins B and C. It has effects as an astringent and a diuretic and reduces blood sugar level (leaves only). The parts used 1 2
From the special issue on the Seventh Italian-Hungarian Symposium on Spectrochemical Analysis. To whom correspondence should be addressed. 12
0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1.
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Structures of the main Vaccinium myrtillus anthocyanins.
are mainly the dried or fresh fruits and, more rarely, the dried leaves; both are best dried in shade. Applications include use as a decoction against diarrhea, as a hot mouthwash for gingivitis, or as a gargle for inflammation of the throat. A decoction based on leaves is sometimes used but can have harmful side effects (hydroquinone poisoning) when used excessively or for long periods. The fresh fruits are sweet and very tasty. The freshly pressed juice is refreshing and a valuable source of vitamins B and C. The berries can be used in the home for soups, pies, preserves, jams, wine, etc. (1). In our investigation we used the isolated anthocyanidins of bilberry prepared in Italy, Inverni della Beffa factory, Milan (I); in Poland, Institute of Pharmaceutical
FIG. 2.
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Free radical induced membrane damage. From Feher et al. (5).
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FIG. 3.
Effects of vitamin E on arachidonic acid metabolism. From Feher et al. (5).
Industry, Warsaw (P); in Romania, Biofarm Bucharest (R); and in France, Laboratories Chibret Clermont-Ferrand (F) (6). TLC Method Anthocyanin extract was hydrolyzed and the aglycones were shaken out with isoamyl alcohol and identified by TLC in the following system: cellulose M.N. 300 F plate (layer thickness, 0.1 mm); developing system, formic acid:water:hydrochloric acid (6:5:1). Very polar, strongly acidified solvents are necessary for good preparation. Rf values of the main anthocyanidins Delphinidin Petunidin Cyanidin Malvidin
0.32 0.54 0.68 0.78
TABLE 1 Spectrophotometric Evaluation of Bilberry Extracts
Sample Malvin Extract Extract Extract Extract
standard I P R F
A11(535)*
Anthocyanin content†
A11(280)
A11(535) /A11(280)
558.92 84.66 380.21 19.20 7.80
100.00 15.15 68.03 3.44 1.40
268.09 69.85 240.60 51.90 28.70
2.08 1.21 1.58 0.37 0.28
* Specific absorbance (abbreviation of A1% 1cm). † In malvin chloride, % per dry weight.
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QUANTIFICATION OF BILBERRY ANTHOCYANINS TABLE 2 Chromatographic Data for Anthocyanin Separation by RP-HPLC Gradient Elution (System 10) Peak No.
Compound
tR
k*
1 2 3 4 5 6 7 8 9 10 11 12 13
Delphinidin-3-O-galactoside Delphinidin-3-O-glucoside Cyanidin-3-O-galactoside Delphinidin-3-O-arabinoside Cyanidin-3-O-glucoside Cyanidin-3-O-arabinoside Petunidin-3-O-glucoside Petunidin-3-O-arabinoside Malvidin-3-O-galactoside Malvidin-3-O-glucoside Malvidin-3-O-arabinoside Petunidin Malvidin-3,5-O-diglucoside
46.75 49.25 51.25 55.50 58.50 61.50 64.75 70.00 74.25 76.00 81.50 84.00 93.03
5.67 6.03 6.32 6.93 7.36 7.78 8.25 9.00 9.61 9.86 10.64 11.00 12.29
The anthocyanin extracts and malvin chloride standard solutions in 0.1 M HCl in methanol were applied to the plates. Anthocyanin spots were detected at 540 nm. We combined this TLC method with densitometry. The apparatus consisted of Shimadzu denzitometer, with a CS-930 high-speed, dual-wavelength TLC scanner and a DR-2 data recorder. Determination of Total Polyphenols and Tannins The official method of Ph.Hung., Ed.VII, with a modified concentration of solvent B and a new calculation was employed. For the determination of the crude drug, acetone:water (7:3, v/v) extracts of bilberry were taken. Sample preparation. Solution A containing about 0.050 mg of polyphenols in 1.00 ml was made by diluting the preparations and extracts with 40% ethanol. Then 10.00-ml aliquots of solution A were shaken for 1 h with 0.100 g of hide powder and filtered (solution B). To 1.00 ml of solvent A (/1.00 ml of methanol), 2.00 ml of solvent B reagents was added, and the absorbances were measured as described for the calibration curve. The content of compounds adsorbed on hide powder (tannins) was calculated as the difference between the total polyphenols and the amount of compounds not bound by hide powder. Determination of anthocyanins. Bilberry anthocyanin extracts were dissolved in 0.1 M HCl solution in methanol. UV–VIS spectra were recorded and absorbance was measured at 280 and 535 nm. Results were calculated in malvin chloride from the absorbance values at 535 nm. Standards Malvin chloride purum (691.04 g/mol) was obtained from Fluka AG. Authentic samples of delphinidin, cyanidin, malvidin, and petunidin glycosides were obtained from the Institute of Pharmaceutical Industry, Warsaw. Chlorides of delphinidin (338.70 g/mol), cyanidin (322.70 g/mol), malvidin (366.75 g/mol), and petunidin (352.71 g/mol) were obtained from Roth.
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FIG. 4.
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TLC densitometry fingerprints of bilberry anthocyanins.
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FIG. 5.
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TLC densitometry fingerprints of malvin chloride standard.
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FIG. 6. HPLC fingerprints: (a) extract P, (b) extract I, (c, d) malvin chloride standard. Partisil ODS, isocratic system B, detection at 535 nm, flow rate 1.0 ml/min (a–c), 1.5 ml/min (d).
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HPLC Techniques We worked with an internal standard method. The standard was malvidin–Cl (Fluka). HPLC apparatus used: 1. HP 1090 Hewlett–Packard liquid chromatograph with HP 10–40 diode array detector. 2. Pye Unicam 4100 liquid chromatograph with LC-XP gradient programmer CXPD pump, variable wavelength detector LC–UV–VIS, and PM 825 single pen recorder. A Rheodym 7125 recorder was used. The injection valve had a 20-ml 100 p. Columns: Li Chromosorb RP-18 10 mm Partisil 10-ODS C18 Nucleosil, equipped with RP-18 precolumn Isocratic systems: A. Tetrahydrofuran:formic acid:water (6:1:33) B. Tetrahydrofuran:formic acid:water (1:1:18) Gradient systems: 1. Solvent A: 5% formic acid; solvent B: MeCn; 0–30 min: 0.1–3.1% B, linear gradient; 31–45 min: 3.1–20.0% B, linear gradient; 46–80 min: 20.0% B, isocratic 9. Solvent A: 10% HCOOH; solvent B: MeOH:MeCN (1:1, v/v); 0–60 min: 0– 15% B, linear gradient; 61–90 min: 15% B, isocratic 10. Solvent A: 10% HCOOH; solvent B: MeCn; 0–60 min: 0–8% B, linear gradient; 61–90 min: 8% B, isocratic. Solutions were filtered through a Millipore sintered-glass filter prior to chromatography. Gradient procedure solvent mixtures were degassed with pure helium. Other operating conditions (flow rate, detection wavelength) are given in the figures. Retention was characterized by the following parameters: tR retention time of the component k * capacity factor: k * Å (tR 0 tM)/tM= where tM a
time taken for an unretained compound to transverse the column selectivity factor (relative retention characteristics of the two components A and B): a Å k *B/k *A .
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RESULTS
UV–VIS Spectrophotometry The results of the UV–VIS spectrophotometry of bilberry extracts are shown in Table 1. Anthocyanins are detected selectively in the visible region; our quantitative measurements were taken at 535 nm. Absorbance values in the UV depend also on the presence of other compounds (phenolic acids, tannins, etc.) in the extract. The extracts varied in anthocyanin content as well as in the ratio A11%cm (535)/A11%cm (280) . The anthocyanin content of extracts R and F were much lower than that of extracts P and I. Relatively high UV values of A11%cm (280) in extracts R and F suggest the presence of large amounts of other polyphenols in these samples. For the quantitative evaluation of anthocyanins the direct spectrophotometric method can be recommended, together with the estimation of the ratio A11%cm (535)/A11%cm (280) , which indicates the purity of the extract. Our results indicate that this ratio should be higher than 1.0 for extracts of good quality. Sometimes standardization of anthocyanin content after acidic hydrolysis and measurement of absorbance of aglycones is possible, but it is never exact, because if some proanthocyanidins are present in the extract, they form anthocyanins too after hydrolysis and give results higher than the real anthocyanin content. Therefore the visible/UV ratio is important for standardization. TLC Densitometry The characteristic ‘‘fingerprints’’ of bilberry extracts were obtained by TLC densitometry (Fig. 2). The standard of malvin chloride was separated into two main spots. Extracts I and P were separated into two or three main spots of aglycones and three main spots of glycosides. As the malvin standard was separated into two spots, the calculation was based on the total area of both peaks. The results of quantitative evaluation are presented in Table 2. The fingerprints of extracts P and I were similar, but extract P exhibited a higher total anthocyanin content. Extracts F and R could not be analyzed by TLC densitometry; because of the high content of impurities, the spots were tailing and good separation was not achieved (Figs. 4, 5). High-Performance Liquid Chromatography Isocratic elution—New RP-HPLC method. The extracts were analyzed by RPHPLC isocratic elution. The best results were obtained with tetrahydrofuran as organic modifier. Methanol or acetonitrile solutions in aqueous formic acid did not lead to sharp separation. HPLC analysis in an isocratic system consisting of THF:HCOOH: H2O, 1:1:18 (v/v), resulted in the characteristic fingerprints of bilberry anthocyanins. Under these conditions malvin standard was eluted as a single peak. Although isocratic elution allowed identification of bilberry extract and quantitative estimation of anthocyanin content, the separation of individual compounds was not achieved (Fig. 6.). Gradient elution—New RP-HPLC method. Full separation of bilberry anhtocyanins was achieved with RP-HPLC gradient elution. Methanol or tetrahydrofuran gradient in aqueous formic acid did not lead to sharp separation. Application of an acetonitrile:methanol (1:1, c/c) mixture as organic modifier improved the analysis, but better separation of delphinidin-3-O-glucoside and cyanidin-3-O-galactoside was obtained with acetonitrile gradient elution.
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QUANTIFICATION OF BILBERRY ANTHOCYANINS
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FIG. 7. Chromatograms of bilberry extracts P and I. Partisil 10 ODS, gradient system 10, flow rate 0.5 ml/min, detection at 280 nm. Extract P: Polish; Extract I: Italian.
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PETRI, KRAWCZYK, AND KE´RY
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TABLE 3 Composition of Bilberry Anthocyanin Extracts (Percentage Share of the Individual Compounds in the Total Amount of Anthocyanins) Peak No.
Compound
P
I
F
R
1 2 3 4 5 6 7 8 9 10 11 12
Delphinidin-3-O-gal. Delphinidin-3-O-gluc. Cyanidin-3-O-gal. Delphinidin-3-O-ara. Cyanidin-3-O-gluc. Cyanidin-3-O-ara. Petunidin-3-O-gluc. Petunidin-3-O-ara. Malvidin-3-O-gal. Malvidin-3-O-gluc. Malvidin-3-O-ara. Petunidin
12.47 14.46 6.34 14.76 — 11.44 12.03 7.89 4.28 14.32 1.99 —
13.52 15.47 10.92 12.87 5.72 9.49 8.19 7.67 2.86 9.49 2.60 1.17
— 9.35 — 19.42 12.95 5.75 12.95 20.86 — 18.70 — —
— — 33.01 33.01 — 19.18 — 14.78 — — — —
41.86 14.95 26.13 15.86
28.77 18.70 18.70 33.81
33.01 — 52.19 14.78
Summary Delphinidin glycosides Malvidin glycosides Cyanidin glycosides Petunidin glycosides
41.69 20.59 17.78 19.92
Because the eluted compounds were detected at 280 nm, it was possible to detect other polyphenols present in the extract. Phenolic acids, catechin, and some proanthocyanidins were eluted during 40 min of the procedure. The peaks of anthocyanins appeared within the next 50 min (Fig. 7). However, when the sample abounded in tannins, most of them were eluted together with anthocyanins and the baseline drifted. Chromatographic data on anthocyanin separation are specified in Table 2. Strong acidic conditions are required for the best sensitivity of the method, whereas in the qualitative analysis the same pattern of eluted compounds can be obtained with a less concentrated acidic component, 5% formic acid. The compositions of the extracts, based on RP-HPLC determination, are presented in Table 3. A preponderance of delphinidin glycosides was observed in all the extracts. Extracts I and P had similar compositions, except that we did not find cyanidin-3-Oglucoside in extract P. The content of malvidin glycosides was higher in extract P than extract I. In extract I, cyanidin glycosides prevailed over malvidin glycosides. Extracts F and R differed in their composition from each other and from extracts P and I. SUMMARY
We have compared different chemical methods for analysis of bilberry anthocyanin extracts. These can be performed by direct spectrophotometry or after chromatographic separation (TLC densitometry or HPLC). For quantitative evaluation of anthocyanins, we recommend the widely used direct spectrophotometric method, together with the estimation of the ratio A11%cm (535)/A11%cm (280) , which indicates the purity of the extract.
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TLC densitometry can be applied only to extracts of high purity. TLC separation of bilberry anthocyanins cannot be achieved with tannin-rich extracts. We have described a new RP-HPLC gradient procedure that seems the best method for standardization of bilberry extracts. It allows evaluation of the individual anthocyanins and detection of impurities such as phenolic acids and tannins. The same method can be used for other anthocyanin extracts, such as extracts of Althaea rosea, Vitis vinifera, and Sambucus nigra. Flow rate is the only HPLC parameter that should be changed as needed. REFERENCES 1. Laurent, E. Country Life Guides: Edible and Medicinal Plants of Britain and Northern Europe. Country Life Books, Hamlyn, London, 1981. 2. List, P. H.; Ho¨rhammer, L. In Vaccinium myrtillus L. Hagers Handbuch der pharmazeutischen Praxis, Vol. VI.C. Springer-Verlag, Berlin/Heidelberg/New York, 1979. 3. Iacobucci, G. A.; Sweeny, J. G. Tetrahedron, 1983, 30, 3005–3038. 4. Ga´bor, M. The Anti-inflammatory Action of Flavonoids. Akade´miai Kiado´, Budapest, 1972. 5. Fehe´r, J.; Csomo´s, G.; Vereczkei, A. Free-Radical Reactions in Medicine. Springer-Verlag, Berlin/ Heidelberg/New York, 1987. 6. Laboratories Chibret. Chem. Abstr., 1970, 72, 6238. 7. Krawczyk, U. Study and Research on Analytical Methods for Determination of Flavonoids and Anthocyanins in Some Plant Preparation. Dissertation (Supervisor: G. Petri), Med. Univ. Inst. of Pharmacognosy, Budapest, 1990. 8. Martindale, W. (Ed.) The Extra Pharmacopoeia. Pharmac. Press, London, 1982.
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