Greater plantain leaf flavonoid contents, expressed as baicalin, were. 2.12 â 3.45%. The greater plantain (Plantago major L.), of the. Plantaginaceae family, is a ...
Pharmaceutical Chemistry Journal
Vol. 42, No. 4, 2008
A METHOD FOR QUANTITATIVE ESTIMATION OF TOTAL FLAVONOIDS IN GREATER PLANTAIN LEAVES D. N. Olennikov1 and L. M. Tankhaeva1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 4, pp. 40 – 42, April, 2008. Original article submitted February 2, 2006.
A method for the quantitative estimation of the total flavonoid fraction in greater plantain leaves was developed; the method had a relative error of no greater than 4%. Metrological analysis showed that the method was accurate and reproducible. Greater plantain leaf flavonoid contents, expressed as baicalin, were 2.12 – 3.45%.
perature 35°C, pressure 3.2 MPa, elution rate 150 ml/min, and UV detection at 244, 260, and 320 nm. Extraction of baicalin from the roots of the Baikal skullcap as follows. Ground material was extracted three times with 70% ethanol (1:10); extract was concentrated to dry residue, which was subjected to liquid-phase extraction with hexane. The aqueous residue was then acidified and extracted with a mixture of ethyl acetate and 95% ethanol (9:1). The interphase precipitate was collected by filtration, washed with cold 95% ethanol, and dried. The technical product was dissolved in a minimal volume of DMSO, and the solution was diluted with purified water (1:250 – 300) and kept at a temperature of 1 – 2°C. The precipitate was collected 12 h later, washed with purified water, and dried. Reprecipitation was repeated a further two times. Extraction stages were monitored by thin-layer chromatography (Silufol and Sorbfil plates; benzene:ethanol:methyl ethyl ketone:acetone (40:16:3:1); detection with 5% ammonium chloride solution in 95% ethanol) and HPLC. The physicochemical properties of the extracted product are shown in Table 1. Quantitative estimation of total flavonoid content in greater plantain leaves. Analytical samples of raw material were ground to a particle size passing through a mesh with openings 1.0 mm in diameter. About 1.0 g (accurately weighed) of ground material was placed in a 100-ml flask with a flange, 60 ml of 60% ethanol was added, a reflex condenser was fitted, and the flask was heated in a boiling water bath for 1 h. After cooling, the extract was filtered into a 200-ml measuring flask through a paper filter, which was washed with 10 ml of 60% ethanol. Extraction was repeated in the same conditions and the combined filtrate was made up to the mark with 60% ethanol (solution A). Solution A
The greater plantain (Plantago major L.), of the Plantaginaceae family, is a medicinal plant which is widely used in medical practice. Various investigators have extracted 12 flavonoid compounds from the leaves: baicalein, baicalin, scutellarein, hispidulin, hispidulin-7-glucuronide, plantaginin, homoplantaginin, 6-hydroxy-4¢-methoxyluteolin-7-galactoside, nepetin-7-glucoside, apigenin-7-glucoside, luteolin-7-glucoside, and luteolin-7-diglucoside [1 – 5]. The available literature includes reports on the quantitative contents of flavonoids in this species. The aim of the present work was to develop a method for the quantitative estimation of the total flavonoid content in greater plantain leaves. EXPERIMENTAL SECTION The plant material studied here consisted of leaves from the greater plantain procured via the pharmaceutical industry (OAO “ Krasnogorskleksredstva”). Absorption spectra were recorded using Cecil CE 2011 and Agilent 8453E UV-Vis instruments in quartz cuvettes with absorption path lengths of 1 cm. Optical rotation was measured using a Coers polarimeter, l = 10 cm, at 20°C. Melting temperatures were estimated using a IA-9100 melting point apparatus. HPLC was performed on a Millichrome A-02 microcolumn chromatograph. The chromatography conditions were: column 2 ´ 75 mm, sorbent ProntoSIL-120 – 5-C-18 AQ (No. 0322, 5 mm), tem-
1
Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, Ulan-Ude
208 0091-150X/08/4204-0208 © 2008 Springer Science+Business Media, Inc.
A Method for Quantitative Estimation of Total Flavonoids
TABLE 1. Comparative Physicochemical Characteristics of Baicalin Baicalin sample Aldrich (CAS ¹ Published data (series 120205) 21967-41-9) [7]
Parameter
M. p., °C (s, 1.0, DMSO) a 20 D UV, lmax, nm, EtOH UV, lmax, nm, + AlCl3 Main substance content (HPLC), %
219 – 221 –83
TABLE 2. Metrological Characteristics of the Method (Raw Material from OAO “Krasnogorskleksredstva”, Series 010104) n
x, %
S2
9 2.22 7.94 · 10
Sx –3
2.97 · 10
P –2
95
t ( p, f ) ± Dx, %
2.36
0.07
E, %
3.21
220 – 222 –85
245, 280, 312
245, 277, 313
290, 340
289, 340
94 – 96
209
95
1,2 1,1 1,0 0,9 0,8
(2 ml) was transferred to a 25-ml measuring flask and made to the mark with 60% ethanol (solution B). The optical density of solution B was measured using a spectrophotometer at a wavelength of 282 nm in a cuvette with an absorption path length of 10 mm. The reference solution was 60% ethanol. Parallel measurements were made of the optical density of a standard baicalin solution. The total flavonoid content (X, %), expressed as baicalin, per unit absolute dry weight of material was calculated as: X =
Dm st k V D st mk stV
´
100 ´ 100, 100 - W
where D is the optical density of the study solution; Dst is the optical density of the standard baicalin solution; kV is the coefficient of dilution of the study solution (2500); k stV is the coefficient of dilution of the standard baicalin solution (2500); m is the weight of the sample of raw material, g; mst is the weight of baicalin, g; W is the loss in weight on drying of the raw material, %. Preparation of standard baicalin solution. About 0.015 g (accurately weighed) of baicalin was placed in a 200-ml measuring flask, dissolved in 2 ml of DMSO, and made to the mark with 60% ethanol (solution A). Solution A (2 ml) was transferred to a 25-ml measuring flask and the solution volume was made up to the mark with 60% ethanol. The total flavonoid content could also be calculated using the specific coefficient of absorption of baicalin % 610 ) or using a calibration plot. Differences in values ( E11cm calculated by the three methods were no greater than 4 – 5%. Metrological analysis of the results was performed as specified in [8 – 11]. Instrument errors in the spectrophotometer (Kinstr) were calculated using potassium bichromate [12]. Regression analysis was performed on Advanced Grapher version 2.07 (Alentum Software Inc.). RESULTS AND DISCUSSION The flavonoid content of greater plantain leaves was complex. Studies of the shape of the absorption spectrum of ethanolic extracts of greater plantain leaves showed that the
1
0,7 0,6 0,5 0,4 0,3 0,2 0,1 0
2 260
280
300
320
340
Fig. 1. Absorption spectra of ethanolic extracts of greater plantain leaves (1) and baicalin solution (2) in 60% ethanol. The abscissa shows wavelength, nm; the ordinate shows optical density, U.
peak was at 282 nm, which was entirely explicable as nine of the 12 flavonoids extracted were 6-hydroxyflavones, which are characterized by peaks at 275 – 285 nm [13, 14]. Baicalin was selected as the standard substance, whose absorption peak (280 nm) is close to that of the greater plantain leaf extract. The analytical wavelength used in the present studies was 282 nm. The optical density curve was linear over the range 0.2 – 0.7 units for baicalin solutions of 3.5 – 11.5 mg/ml. The linear regression equation for the calibration plot was D = = 0.0630465x – 0.0205388 (standard deviation 1.01 ´ 10 – 2, coefficient of regression 0.9986, Kinstr = 0.98. Assessment of the optimal extraction parameters showed that the greatest level of flavonoid extraction occurred when 60% ethanol was used as the extractant. At a material:extractant ratio of 1:60, equilibrium occurred at 1 h of boiling. Double extraction ensured maximal extraction of flavonoids (up to 97% of the total content). The accuracy of the method was assessed using the standard experimental “added-found” method and analysis of the standard baicalin sample [15]. Analysis using the “added-found” method showed that the relative error was not greater than 3%. Analysis of the standard baicalin sample showed that the relative error of single measurements was within the range 0 – 3.13% and could be positive or negative. The mean error of three measurements for three samples was within the quite narrow range 0.85 – 1.98%. Thus, the component analyzed was esti-
210
mated with adequate accuracy such that the measurement results can be taken as correct. Deviations from the mean in three independent estimates were by no more than 2%, demonstrating satisfactory reproducibility. The metrological characteristics of the method are shown in Table 2. The relative error was no greater than 3.5%. This method was used to estimate flavonoid contents in nine material samples, values of 2.12 – 3.45% being obtained. Flavonoid contents in greater plantain leaves, expressed as baicalin, should be at least 2%. REFERENCES 1. N. P. Maksyutina, Khim. Prirod. Soedin., 7, No. 3, 374 – 375 (1971). 2. N. P. Maksyutina, Farmatsev. Zh., 27, No. 1, 59 – 63 (1972). 3. J. B. Harborne and C. A. Williams, Phytochemistry, 10 No. 2, 367 – 378 (1971). 4. S. A. Kawashty, Gamal-el-Din, M. F. Abdalla, N. A. M. Saleh, Biochem. Syst. Ecol., 22, No. 7, 729 – 733 (1994). 5. S. Nishibe, M. Murai, and Y. Tamayama, Nat. Med., 49, No. 3, 340 – 342 (1995).
D. N. Olennikov and L. M. Tankhaeva
6. D. N. Olennikov, T. M. Mikhailova, and L. M. Tankhaeva, Khim. Prirod. Soedin., 41, No. 2, 177 (2005). 7. V. M. Malikov and M. P. Yuldashev, Khim. Prirod. Soedin., 38, No. 4, 299 – 324 (2002). 8. Yu. I. Aleksandrov and V. I. Belyakov, Zh. Anal. Khim., 57, No. 2, 118 – 129 (2002). 9. M. I. Bulatov and M. P. Kalinkin, A Practical Handbook in Photocolorimetric and Spectrophotometric Analytical Methods [in Russian], Khimiya, Leningrad (1976). 10. A. N. Smagunova, Zh. Anal. Khim., 52, No. 10, 1022 – 1029 (1997). 11. K. Derfel, Statistics and Analytical Chemistry [in Russian], Mir, Moscow (1994). 12. Yu. Ya. Kharitonov, Analytical Chemistry (Analytics) [in Russian], Vysshaya Shkola, Moscow (2001). 13. O. I. Kostyuchenko, Rastit. Resursy, 13, No. 2, 403 – 417 (1977). 14. T. J. Mabry, K. R. Markham, and K. B. Thomas, The Systematic Identification of the Flavonoids, Springer-Verlag, Berlin, Heidelberg, New York (1970). 15. Basic Analytical Chemistry [in Russian], Yu. A. Zolotov (Ed.), Vysshaya Shkola, Moscow, Book 1.
