Effect of Sample-Preparation Methods on the Quantification of Select Flavonoids

8/6/2019 Effect of Sample-Preparation Methods on the Quantification of Select Flavonoids

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M. Waksmundzka-Hajnos et al.476

The most important activity of these substances is sealing and augmen-tation of capillary blood vessels, anti-oxidative, anti-thrombotic, hypoten-sive, and antiphlogistic activity [2–4]. For this reason flavonoids are widelyused in therapy. Various medicinal plants contain flavonoids as the maincompounds determining their biological activity. For this reason there is a

need to develop adequate methods for analysis of flavonoids in plant mate-rial.The first, very important step in quantitative analysis of secondary me-

tabolites in plants is sample preparation, which includes such processes as apreparation of plant material (drying, grinding, sieving), extraction of com-pounds from the plant material, and purification of the crude extract.

The last of these is necessary because extraction of compounds fromplant material is usually performed by liquid–solid extraction (LSE). Thegoal of every extraction process is rapid and effective isolation of com-pounds from a matrix by use of a minimum amount of solvent. Traditionalmethods such as percolation, exhaustive Soxhlet extraction, or direct extrac-tion with a solvent at boiling temperature under reflux are most often ap-

plied in phytochemistry. Use of modern extraction methods, which requireshorter extraction times, use small amounts of solvents, enable simultane-ous parallel processing of several samples, and are automatic, is sometimesreported [5, 6]. The greater effectiveness of these methods results from useof solvents at higher temperature and pressure. Modern extraction meth-ods, which are usually more expensive [7], include: microwave assisted sol-vent extraction (MASE), accelerated solvent extraction (ASE), also calledpressurised solvent extraction (PLE), and supercritical fluid extraction(SFE). In MASE electromagnetic waves induce rapid movement of solventmolecules which generates thermal energy and causes rapid extraction ofanalyte at high pressure (closed system) or at normal pressure (open sys-

tem) [7, 8]. In ASE extraction proceeds at high pressure in a closed vesselheated in an electric thermostatted oven [9]. The extract is then rapidly re-moved from the extracting vessel with a fresh portion of solvent. Betweenconventional and modern techniques is ultrasound-assisted extraction(USAE) in which ultrasound loosens the matrix structures and makes con-tact with solvent molecules much easier [10]. This method has disadvan-tages, for example inability to renew solvent and need for filtration after theprocess [7, 10].

Crude extracts obtained by one of these methods must be purified be-fore analysis. Plant extracts contain many ballast substances both nonpolar(chlorophylls, waxes, lipids) and polar (tannins, sugars). To remove thesesubstances liquid–liquid extraction (LLE) is usually used. This takes advan-

tage of solubility differences of hydrophobic substances, with affinity fornonpolar solvents, and hydrophilic substances, with affinity for aqueous so-



Effect of Sample-Preparation Methods in HPLC of Flavonoids 477

lutions. Although the analytes can be easily obtained by evaporation of thesolvent, LLE has many disadvantages, for example emulsion formation andthe long time of treatment.

Purification can be also performed by solid-phase extraction (SPE). Themethod applies various adsorbents and ion-exchangers and is widely used

for different purposes [9].The objective of our work was comparison of different extraction tech-niques for isolation of flavonoid glycosides (rutin and isoquercetrin) fromtwo common plant materials used in therapy – the inflorescence of Sambu-cus nigra L. (Familia Caprifoliaceae) and herb of Polygonum aviculare L. (Fa-milia Polygonaceae). Determination of flavonoid levels is an important taskin investigations of plant material quality and in standardisation of plantdrugs. For this reason elaboration of adequate procedures for samplepreparation and analysis of this group of compounds is a serious problem.

Experimental

Plant Material and Standards

Inflorescence of Sambucus nigra L. and herb of Polygonum aviculare L., col-lected in spring 2004, were purchased from Herbapol herbal industrial plant(Lublin, Poland). Dry plant material was milled and sieved.

All flavonoid standards were purchased from Sigma–Aldrich (Stein-heim, Germany).

Extraction Procedures

Exhaustive extraction was performed in a Soxhlet apparatus. Plant material(2 g) was placed in filter paper in the thimble holder of the apparatus andextracted for 15 h with methanol.

Ultrasound assisted extraction was performed in an Intersonic IS-4 ul-trasonic bath equipped with a thermostat. An Erlenmeyer flask containingplant material (2 g) soaked with 50 mL methanol was placed in the bath for30 min. The extract was then filtered and the same plant material wassoaked with a fresh portion of methanol and again extracted in the ultra-sonic bath. This was repeated three times and the methanolic extracts were

combined. Extraction was performed at ambient temperature (20 ± 1°C) and

at 60°C.

MASE was performed with 80% methanol in water using a Plasmo-tronika (Wroclaw, Poland) UniClever BMZ bath. Dried and ground plant




material (2 g) was placed in a laboratory flask with 50 mL extractant in amicrowave bath and extracted using two-step extraction with 40% genera-tor power for 1 min and 60% generator power (600 W) for 30 min, in bothopen and closed systems.

ASE was performed with a Dionex (Sunnyvale, CA, USA) ASE 200 in-

strument and solvent controller. Plant material (1 g) was mixed with neutralglass and placed in the stainless steel extraction cell. Flowers of Sambucusnigra L. were soaked with 50 mL 80% methanol in water and herb of Poly- gonum aviculare L. was soaked with 50 mL of pure methanol. The extractions

were performed for 10 min at a pressure of 60 bar, temperature 100°C.All extraction procedures were repeated three times. The extracts ob-

tained were combined and purified by LLE and SPE.

Purification Procedures

LLE

All methanolic extracts (obtained by Soxhlet, USAE, MASE, and ASE) wereevaporated to dryness under reduced pressure. Dry residues, soaked with50 mL boiling water were cooled under refrigeration (4°C). After 12 h ex-tracts were filtered and extracted with diethyl ether (8 × 50 mL). Diethylether extracts were analysed quantitatively for the presence of phenolic ac-ids by RP-HPLC [11]. The aqueous solution was then extracted with ethylacetate (8 × 50 mL) and the ethyl acetate extracts were combined and evapo-rated to dryness. The dry residues were dissolved in methanol in graduatedflasks (5 mL) and flavonoids were analysed quantitatively by RP-HPLC.

SPE

C18 cartridges (Bakerbond) were conditioned successively with 10 mLmethanol, 10 mL distilled water, and 10 mL 80% aqueous methanol. The ex-tract dissolved in 80% aqueous methanol (5 mL) was applied to the column.The process was performed in an SPE chamber (Baker, Germany). Flavon-oids were eluted from the adsorbent bed by 10 mL 80% methanol and theeluates obtained were analysed by RP-HPLC.

SPE cartridges were packed with 0.3 g polyamide (Woelm, Germany)and the columns were conditioned with 50% methanol. The extract, dis-solved in 50% aqueous methanol (5 mL), was applied to the column and

eluted with 20 mL 50% aqueous methanol. The eluates obtained were ana-lysed by RP-HPLC.




To establish the accuracy of the SPE method recovery tests were per-formed for two types of SPE adsorbent and three selected methanolic ex-tracts of Polygonum aviculare herb and Sambucus nigra L. inflorescence (in80% aqueous methanol for C18 cartridges and in 50% aqueous methanol forpolyamide cartridges). Standard solutions of rutin and isoquercitrin

(C = 0.1 mg mL−1

in 80% aqueous methanol for C18 and in 50% aqueousmethanol for polyamide cartridges) were prepared. Volumes of 0.5, 1, and2 mL (I–III fortification levels) of that solution were combined separatelywith 2 mL samples of each of the examined extracts and submitted to SPEon C18 and polyamide cartridges, respectively.

HPLC Analysis

Analysis was performed with Dionex (USA) HPLC equipment, a UV–visible detector, and a Rheodyne 20-μL injector. Compounds were sepa-rated on a 250 mm × 4.6 mm, dp = 5 μm, Prodigy ODS2 column (Phenome-

nex, USA) with a Hypersil ODS precolumn (Altech Associates Applied Sci-ence, USA). Isocratic elution was performed with 21:79 MeCN–aqueous ace-tic acid, pH 3, for extracts of Sambucus nigra and 15:85 MeCN–aqueous ace-tic acid for extracts of Polygonum aviculare. Detection was by UV absorbanceat 350 nm. Qualitative determination was performed by comparison of thespectra of standards and extract components. The flavonoid content of theextracts was determined by use of calibration plots for every standard.Calibration data are listed in Table I .

Table I. Linear relationships between peak area and concentrations of rutin andisoquercetrin (n = 3)

Plant material Compound Regression equation R2 Linearrange

(μg mL−1)

LOD(μg mL−1)a

LOQ(μg mL−1)*

Rutin y = 695.3297x − 0.3424 0.9999 0.08-8.5 0.0234 0.07819Sambucusnigra inflorescence Isoquercetrin y = 987.9955x + 0.3149 0.9995 0.06-1.5 0.01376 0.06882

Rutin y = 827.4952x − 0.3699 0.9999 0.03-3.0 0.021 0.10500Polygonumaviculare herb Isoquercetrin y = 1170.7653x 0.9990 0.02-0.6 0.01498 0.07490

aLOD and LOQ calculated as the amounts for which the signal-to-noise ratio were10 and 3, respectively




Results and Discussion

In the first step of experiment the extraction yield of the two flavonoid gly-cosides from Sambucus nigra L. inflorescence obtained by use of the differentmethods was compared. Because of a previous report that methanol was thebest extractant for the flavonoids rutin and isoquercitrin [12] this solvent was

used in our experiments. It is clearly seen from the histograms presented inFigs 1a and 1b that most efficient method of extraction for this sample was ex-haustive extraction in a Soxhlet apparatus. It should, however, be noticed thatextraction yield of rutin by exhaustive pressurised liquid extraction was al-most identical (Fig. 1a). High yield of both flavonoids was obtained by mi-crowave-assisted extraction in an open system, and isoquercitrin was well ex-

tracted by ultrasound-assisted extraction at 60°C (Fig. 1b).

(a)

(b)Fig. 1. Dependence on extraction method of the extraction yield of rutin (a) and

isoquercitrin (b) from Sambucus nigra L. inflorescence

The next stage of the work was choice of the optimum method for puri-fication of crude Sambucus nigra inflorescence extracts. LLE, and SPE on two




adsorbents – C18 and polyamide – were investigated. All these samplepreparation procedures have previously been used for flavonoid fractionsfrom plant material [13]. As is apparent from the histograms presented inFigs 2a and 2b the most efficient method of purification was C18 SPE. The ru-tin and isoquercetrin content was highest for samples purified by SPE on

C18. For purification of extracts containing more polar flavonoids SPE onpolyamide can be also applied. Similar conclusions can be drawn for theother extracts obtained (Figs 3a and 3b). Exemplary typical chromatogramsare presented in Figs 4a and 4b.

(a)

(b)

Fig. 2. Dependence on the method used (crude extract, LLE, C18 SPE, polyamide SPE) of

purification yield of rutin (a) and isoquercitrin (b) from extracts (Soxhlet and ASE) ofSambucus nigra L. inflorescence




(a)

(b)

Fig. 3. Dependence on the method used (crude extract, LLE, C18 SPE) of the purification

yield of rutin (a) and isoquercitrin (b) from extracts (USAE and MASE) of Sambucusnigra L. inflorescence

From these histograms and chromatograms it is apparent that the puri-fication method used affects the composition of the extracts. The most effi-cient purification method is solid-phase extraction on octadecyl adsorbent,although the method changes to some extent the composition of extractscontaining highly polar compounds (Fig. 4a). The method is characterisedby high repeatability (RSD ≤ 1.12%). C18 SPE also enables efficient recoveryof the flavonoids (up to 93.4% for rutin and up to 90.1% for isoquercitrin).If liquid–liquid extraction is used, repeatability and recovery for these fla-vonoids are distinctly lower and LLE changes proportion of rutin to iso-

quercitrin, as is clearly apparent from the chromatogram presented inFig. 4b. It is possible that LLE results in partial hydrolysis of rutin (diglyco-




(a)

(b)

Fig. 4. Chromatograms obtained by HPLC (ODS with 21:79 MeCN–aqueous acetic acidas mobile phase) of the flavonoid fraction from Sambucus nigra L. inflorescence extract

purified by SPE on polyamide (a) and by LLE (b). R, rutin; I, isoquercitrin




side), (producing isoquercitrin (monoglycoside). In SPE on polyamide therepeatability is, again, relatively high (RSD ≤ 2.14%). Recovery of rutin ishigh – up to 93.27% – but recovery of isoquercitrin is very low – only ap-proximately 40%, because polyamide strongly retains hydrophobic com-pounds. For this reason polyamide SPE is not recommended for purification

of extracts containing isoquercitrin and related flavonoids.

(a)

(b)

Fig. 5. Dependence on the method of extraction of extraction yield of rutin (a) andisoquercitrin (b) from Polygonum aviculare L. herb

The same methods of extraction were used for Polygonum aviculare

herb. For this plant material the most effective extraction technique wasASE, as shown in Figs 5a and 5b. Other methods, for example ultrasonifica-




tion and microwave-assisted liquid extraction, resulted in lower but similaryields of rutin (Fig. 5a). Exhaustive Soxhlet extraction resulted in a low ex-traction yield. Although ASE was also the most effective technique for iso-quercitrin, high yields were also obtained by use of microwave-assisted ex-traction in open and closed systems. A relatively high yield of isoquercitrin

was also obtained by Soxhlet apparatus (Fig. 5b).These results are different from those obtained for extraction of thesame compounds from Sambucus nigra inflorescence. This can be caused bydifferent construction of the cell walls of flowers and herb. Phenolic com-pounds can be bonded to cell walls – to lignin – which means that tech-niques with forced flow of solvent result in higher yield from herb.

For Polygonum extracts, also, the most effective method of purificationwas C18 SPE, for both rutin and isoquercitrin (Fig. 6). A exemplary typicalchromatogram obtained from Polygonum aviculare extract purified by use ofC18 cartridges is presented in Fig. 7 .

Fig. 6. Dependence on the method used (LLE, C18 SPE, polyamide SPE) of purification

yield of rutin from extracts (USAE, MASE, ASE) of Polygonum aviculare herb

The results obtained for rutin and isoquercitrin extracted and isolatedfrom Sambucus nigra inflorescence and from Polygonum aviculare herbshould be compared with the results for phenolic acids reported in our re-cent paper [11]. For most acids (protocatechuic, p-hydroxybenzoic, vanillic,ferulic) isolated from Sambucus nigra inflorescence the highest yield wasachieved by extraction in a Soxhlet apparatus, as for the flavonoids investi-gated. Other extraction methods have distinctly lower similar yields bothfor flavonoids and phenolic acids, however. Liquid–liquid extraction seemsto be the most effective method for purification of phenolic acids, but simi-

lar high recoveries are also obtained by use of C18 SPE.




Fig. 7. Chromatogram obtained by HPLC (ODS with 15:85 MeCN–aqueous acetic acid asmobile phase) of the flavonoid fraction from Polygonum aviculare L. herb extract purified

by C18 SPE

Similarly to the flavonoids, results from extraction of phenolic acidsfrom the herb of Polygonum aviculare by the different methods are quite dif-ferent from those for Sambucus nigra inflorescence. For this plant materialexhaustive extraction in a Soxhlet apparatus results in low yields of pheno-lic acids and flavonoids whereas high extraction yield has been achieved by

use of microwave-assisted techniques, especially MASE in a closed systemfor phenolic acids. For flavonoids this technique was more effective thanSoxhlet extraction. It should be remembered that this technique was unsuit-able for extraction of non-polar furanocoumarins, possibly because of theeffect of isomerisation or decomposition of hydrophobic imperatorin andphellopterin with simultaneous high yield of the more polar xanthotoxinand bergapten from Archangelica officinalis [14] and Pastinaca sativa fruit ex-tract [15]. There are significant differences between the yield of pressurisedliquid extraction for compounds extracted from Polygonum aviculare – forflavonoids the yield was highest whereas for phenolic acids the yield waslow.

One can attempt to explain the different yields of methods used for ex-traction of phenolic compounds from different plant material. The differ-




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Effect of Sample-Preparation Methods on the Quantification of Select Flavonoids

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