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The Quantitative Determination of Rutin in Different Glycerinic Plant Extracts

by Solid-Phase Extraction and Thin-Layer Chromatography with
Densitometry

Article  in  JPC - Journal of Planar Chromatography - Modern TLC · January 1999

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 Determination
The Quantitative of Rutinin Different
GlycerinicPlantExtractsby Solid-PhaseExtraction
and Thin-LayerChromatography with Densitometry
Simona Cobzac, Gabriela Cimpan, Neli Olah, and Simion Gocan*

Key Words:

TLC
SPE
Densitometry
Plant extracts, glycerinic
Ribes nigrum
Sorbus domestica
Tilia tomentosa

Summary Thin-layer chromatography and densitometry are wide-

The quantitative determination of rutin from Ribesnigrum, Sorbus
domestica, and Tilia tomentosaglycerinic plant extracts has been
performed by TlC-densitometry. Solid-phase extraction (SPE)
was used to separatethe glycerinefrom the plant extracts studied.
The SPE recoveryof rutin was 100.4Vqi.e. loss of the substanceof
interest was negligible. Silica gel 60Frroplates were scannedat l =
260 nm (U[ fluorescencequenching), and at I -- 440 nm (visible
region) after spraying with 2-aminoethyldiphenylborinate.Similar
results obtained from the use of these two methods of detection.
1 Introduction
Although plant extractsare widely usedin homeopathicmed-
icine, for the treatment of different diseases,the control of
these drugs is often performed by qualitative analysis,
becausethe composition of a plant extract is very complex
and not always completely known. The quality control of
commercialplant extractsfollows the proceduredescribedin
the Homeopathic Pharmacopoeia[1], involving the so-called
'fingerprint'method which is, in fact, separationby thin-layer
chromatography.A photographof the obtained separationis
usuallyattachedto the analysiscertificate,and the separated
spots should have the same pattern as described in the
Homeopathic Pharmacopoeia[1]. The colors of the spots
and their position relative to standardsubstancesare impor-
tant characteristicsof plant extracts[2]. Quantitative analysis
is seldom applied in this field and only spectrophotometryis
includedin the pharmacopoeias [3-5].
S. Cobzac,G. Cimpan, N. Olah, and S. Gocan, 'Babeg-Bolyai'University,
Faculty of Chemistry and Chemical Engineering, Analytical Chemistry
Department, 11 Arany JanosStreet, 3400 Cluj-Napoca,Romania.
spreadmethodsfor the quantitativedeterminationof alka-
loids [6-8], glycosides[9], flavones110-12],and other com-
pounds from plant extracts 173,141.Alcoholic extracts or
alcoholic extracts containing glycerine are also used for
therapeutic purposes.Glycerinic plant extracts cannot be
analyzed directly by thin-layer chromatography because
glycerine hinders the separation.Glycerine should, there-
fore, be removedfrom the plant extractsamplebefore TLC
analysis[15].
This paper reportsthe quantitativeanalysisof rutin in three
glycerinic plant extracts - from Ribes nigntm,,Tilia tomen-
tosa, and Sorbusdomestica.It is known from the literature
that theseplants contain rutin [16]. Glycerinewas separat-
ed from the samplesby solid-phaseextraction (SPE). The
retained compoundswere eluted from the cartridge with
methanol, and the solution obtained was analyzed by
TLC-densitometry.
2 Experimental
The Ribes nigrum, Tilia tomentosa, and Sorbus domestica
plant extracts were obtained from Plantextrakt (Radaia,
Cluj, Romania) as solutionsin ethanol containing50Voglyc-
erine. For analysisof the samplesby TLC glycerinewas sep-
arated from the extractsby solid-phaseextraction.
2.1 Solid-PhaseExtraction
Glycerinic plant extract (1 mL) was diluted to 70 mL with
1% (vlv) methanol in water. SPE was performed on
Supelclean Envi-18 cartridges (Supelco, Bellefonte, PA,
USA). The cartridges were conditioned by passage of
methanol (5 mL) and IVo (u/u) methanol in water (5 mL).

26 vol. 12.JANUARY/FEBRUARY
leee Journalof PlanarChromatography
 QuantitativeTLC of Rutin in Plant Extracts

The sampleswere passedthrough the cartridge at 10 mL

OH
min*r, enablingretention of the componentson the adsor-
bent. The cartridge was then washed with l% (vlv)
methanol in water (5 mL), to remove nonselectively
retainedcompounds,and the cartridgewas dried in a gentle
air stream.The retained compoundswere eluted from the
cartridge with methanol acidified to pH 3.5 with HCI
(5 mL). The solventwas evaporatedfrom the eluate at 40"C
and the residue was dissolvedin methanol (1 mL). This
samplewasusedfor TLC separationand quantitativedeter-
mination of rutin bv densitometrv.

2.2 Determinationof SPE Recovery

Rutin was obtained from Fluka (Sigma-Aldrich Chemie Figure 1

GmbH, Steinheim,Germany).The SPE recoverywas deter- The structure of rutin.

mined by useof a standardsolutionof rutin (5.1mg rutin in

10 mL ethanol).This solution (1 mL) was mixed with glyc-
erine (1 mL) and the mixture was diluted to 70 mLwith lVo
(r.,/u)methanol in water. This solution was applied to the
SupelcleanEnvi-18 cartridge [previouslyconditioned with
methanol(5 mL) and77o(u/u)methanolin water (5 mL)] at
10 mL min-r. After compound retention the cartridge was
washedwith 1% (vlv) methanol in water (5 mL) and the
retained rutin was then eluted from the cartridge with
methanolacidifiedto pH 3.5 with HCI (5 mL).The solvent
was evaporatedfrom the eluate at 40'C and the residuewas
dissolvedin methanol (1 mL). This sample was used to o . tl,iL:)

determinethe recovervof rutin bv SPE.

-Cj . +!,,,.

2.3 Thin-LayerChromatography
B
TLC was performed on 10 cm X 10 cm silica gel 60F,ro II
plates (Merck, Darmstadt, Germany). The samples _._l
^ -F i-i r-r I1
I

processedby SPE were applied as spots,5 pL spot-r.1.5cm I

from the bottom edge of the plate, by use of a Desaga -, -t,-,,',
' ' 1i
I
AS-30 automated applicator.The mobile phase was ethyl {
L

'-r,::,-,,-,-]
acetate-formicacid-water,80 : 10 : 10 (vlv\, and the devel-
opment distancewas 8 cm. The plates were developedin
normal chamberspreviouslyequilibratedfor 30 min. After
developmentthe plates were dried in a gentle air stream
and then scannedin UV light at ), : 260 nm, by meansof a
ShimadzuCS-9000dual-wavelengthflying-spotscanner,in
reflectancemode. The plates were also scannedin visible
light at ), = 440 nm after sprayingwith a methanolic solu-
tion of 2-aminoethyldiphenylborinate (1%, vlv).

3 Resultsand Discussion
The structure of rutin is shown in Figure 1. The densito-
grams obtained from the extracts of the Ribes nigrum,
Sorbusdomestica,and Tilia tomentosasamples(prepared by
SPE before TLC), are shownin Figures2 and 3. The densi- 'r. !_r'-r j-r. ,_,,_' _au. i-rLJ , 15. i'r:r

tograms obtained from the plant extracts are overlapped Figure 2

with those obtained from a standard solution of rutin to The densitograms obtained by fluorescence quenching (,t = 260 nm) from the
enableidentificationof rutin by Ro value. extracts of (a) Ribes nigrum, (b) Sorbus domestica, and (c) Tilia tomentosa.

Journalof PlanarChromatography VOL. 12.JANUARY/FEBRUARY

1999 27
QuantitativeTLC of Rutin in Plant Extracts

pg/spot
Figure 4

The linear calibration plot for rutin, l = 260 nm (eq. (1).

Figure 3

The densitograms obtained at l = 44O nm, after spraying with 2-amino-

ethyfdiphenylborinate, from the extracts of (a) Ribes nigrum, (b) Sorbus domes-
tica, and (cl Tilia tornentosa.

The efficiency of recovery of rutin by SPE was very good,

100.4Vo.The experimentswere performedon rutin standard
solution,and the reported value is the mean of resultsfrom
three experiments.
The calibrationcurye for rutin includes 12 area valuescor-
responding to concentrationswithin the range 0.135-
2.55p,gspofr.Eachspotwasappliedin duplicateand scanned
pg/spot
three times; mean areaswere used to constructthe calibra- Figure 5
tion curve. Figure 4 showsthe linear calibration curvesfor The linear calibration plot for rutin, .tr= 440 nm (eq. (2)).
rutin, obtainedby densitometryat z\ : 260 nm. Eq. (1) is the
calibrationcurye from Figure 4.
dard error, and r is the correlation coefficient for 957o
A :2618.r(*t+21.0) + 18104.3(-r935.trr (1)
confidencelimits.
sa o: 640. 5, s ar
: 44 2 .1 ,s: 1 .2 2 0 ,r: 0 .9 9 7
Figure 5 showsthe calibration curve for rutin at i : 440 nm
where ,4 is the scanned area, c is the concentrationof after spraying the plate with methanolic 2-aminoethyl-
rutin (g spot-'), so,,and sal are the standarderrors of the diphenylborinate(IVo,vlv). Eq. (2) is the linear calibration
intercepta0 and the slopear, respectively,s is the fit stan- curve (the symbolshave the samesignificanceas for eq. (1):

28 voL. 12.JANUARY/FEBRUARY
lsss Journalof PlanarChromatography
 QuantitativeTLC of Rutin in Plant Extracts

A = 1188.6(629s1.6)
+ 291s7.6(62037.6)c (2) References

suo= 7324.8,sa1
: 974.5,s= 2524,r: 0.995
l1] Homoopathisches
Arzneibuch,Frankfurt,Vol. l, 1978;Vol. II,
The plates were scannedat two different wavelengths,at 1991.
t : 260 nm (fluorescencequenching),and at .,\ = 440 nm
after sprayingthe plate with 2-aminoethyldiphenylborinate, [2] H. Wagne4S. Bladt, and M. Zgainski,Drogen Analyse. Dr"inn-
schicht-Chromatographische Analyse von Arzneidrogen,
to enable comparison of the results obtained by the two
Springer,Berlin, 1983.
methods.The errors were slightlygreater for the rutin cali-
bration curve at u\ : 440 nm than for the experimentsper- t3] DAB 10,DeutschesArzneibuch10,Ausgabe1991,Deutscher
formed at .,\: 260 nm. This can be explainedby the higher ApothekerVerlag,Stuttgart,1991.
noiselevel obtainedafter densitometryof the sprayedplate.
The concentrationsof rutin in the plant extractsstudied,as [a] The United StatesPharmacopoeiaXXIII Revision,Natural
Formulas 18, United States PharmacopoeialConvention,
obtained from the linear calibration curves,are shown in Rockville,MD, 1995.
Thble 1.
t-5] Romanian Pharmacopoeia,Edilia X, Editura Medicala,
Table 1 Bucharest,1993.
The concentration of rutin (mg mLr) in Ribes nigrum, Sorbus dornes-
tica, and Tilia tomentosa, as determined by densitometry. 16l D.Antor'-PratsandJ.L. Harborne,Biomed. SystematicsEcol.
2l (lee3)45s.
), : 260 nm ), : 440 nm
l7l O. Paruuis,VB. Stric'ht,R. Vanhaelen-Fastre,
andM. Vanhaelen,
Tilia tomentosa 0.043-f 0.003 0.040-r 0.004 J. PlanarChromatogr.7 (1994)80.
Sorbusdomestica 0.045+ 0.003 0.050-1_0.004
Ribes nigrum 0.067-f 0.002 0.070| 0.003 [8] E. Soc'zewifiskl
and "I. Flieger,J. Planar Chromatogr. 9 (1996)
107.

tel P. Poukens-Renwart,M. Irls, and L. Angemont, J. Planar

4 Conclusion Chromatogr.6 (1993)43-5.

The quantitativedeterminationof rutin from complexmix- [ 1 0 ]P.C.SchmidtandK. Vogel,Dtsch.Apoth. Zrg.132 (1992)462.

tures, as plant extracts,can be performed rapidly and with
versatility by TlC-densitometry. If the alcoholic plant u 1 lK. Hoffmann-Bcthn,H. Lotter P Seligmanrz,and H. Wagner.
extract containsglycerine,this should be removed by SPE. PlantaMedica58 (1992)544.
Samplepreparationby SPE leadsto negligiblelossesof the
substanceof interest, becauseof the good recovery.The U2)A. Pieroni,D. Heimler,and Y Huung, J. Planar Chromatogr. 11
(1998)230.
TLC platescan be scannedin the UV (i = 260 nm) or can
be sprayedwith 2-aminoethyldiphenylborinate and exam- [13]J.P.lVikiema,R. Vanhaelen-Fastre,
and M. Vanhaelen,J. Planar
ined under visible light (n = 440nm), with similar results. Chromatogr.lf (1998)148.

Acknowledgments 114lG. MaQsik, J. Kowalslq,, H. Strzelec'ka,and E. Soc'zewifiski,

J. PlanarChromatogr.T(1994)129.
The authors are grateful to ProfessorNel Velthorst(Free
University,Amsterdam, The Netherlands)for donation of
[ 1 s ]S. Gocan and.S.Cctbzac,Revistade Chimie 47 (1996) 54.
the Shimadzu CS-9000 scanner, and to the Plantextrakt
Company (Radaia,Cluj, Romania) for kindly providing the lt 6l M ax Wi c'ht/, Teedrogen, Wissenschaftl iche Verlagsgese
Ilschaft
glycerinicplant extracts. mbH, Stuttgart,Germany,1989.

Ms received:November12, 1998
Acceptedby SN: November24,1998

Journalof PlanarChromatography V O L .1 2 .J A N U A R Y / F E B R U A1R9Y9 9 29

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