For Peer Review

Gradient x Isocratic Elution CCC on the Isolation of

Verbascoside and Other Phenylethanoids: Influence of the

Complexity of the Matrix

Journal: Planta Medica

Manuscript ID: Draft

Manuscript Type: Original Papers

Date Submitted by the Author: n/a

Complete List of Authors: Leitao, Gilda; Universidade Federal do Rio de Janeiro, Instituto de Pesquisas de Produtos Naturais Pinto, Shaft; Universidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e Alimentos Oliveira, Danilo; Universidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e Alimentos Guimares, Michelle; Universidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e Alimentos Crdova, Wilmer; Universidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e Alimentos Leitao, Suzana; Universidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e Alimentos

Keywords: Extraction, Isolation, Liquid-Liquid Partition Chromatography < Extraction, Isolation, Preparative Chromatography < Extraction, Isolation

Georg Thieme Verlag KG, P.O. Box 30 11 20, D-70451 Stuttgart, Germany www.thieme.de/plantamedica

Manuscript submitted to editorial office

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UNIVERSIDADE FEDERAL DO RIO DE JANEIRO INSTITUTO DE PESQUISAS DE PRODUTOS NATURAIS

Centro de Cincias da Sade, Bloco H. Av. Carlos Chagas Filho, 373,Ilha do Fundo, Rio de Janeiro, 21.941-920

Tel:3938-6513 Fax: 3938-6512

Rio de Janeiro, October 3, 2014

To the Editor of Planta Medica

C/C Guest Editor of the Special Issue on Modern Separation Techniques for the

Isolation of Natural Products Prof. Dr. Jean-Hugues Renault

Dear Editor,

We are submitting the manuscript (mini-review) entitled Gradient x Isocratic

Elution CCC on the Isolation of Verbascoside and Other Phenylethanoids:

Influence of the Complexity of the Matrix by Gilda Guimares Leito, Shaft Correa

Pinto, Danilo Ribeiro de Oliveira, Michelle Guedes Guimares, Wilmer Hervet Perera

Cordova, Suzana Guimares Leito, for possible publication at the above mentioned

special issue, of Planta Medica as previously proposed to Prof. Jean-Hugue Renault.

This work has not been published elsewhere, completely, in part, or in any other

form, and the manuscript has not been submitted to another journal. All authors concur

with the submission of the manuscript.

Hoping to hear from you soon,

Kindest regards,

Gilda Guimares Leito

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Gradient x Isocratic Elution CCC on the Isolation of Verbascoside and Other

Phenylethanoids: Influence of the Complexity of the Matrix

Gilda Guimares Leito1, Shaft Correa Pinto

2,3, Danilo Ribeiro de Oliveira

3, Michelle

Guedes Guimares3, Wilmer Hervet Perera Cordova

3, Suzana Guimares Leito

3

Affiliation

1Universidade Federal do Rio de Janeiro, Instituto de Pesquisas de Produtos Naturais.

2Universidade Federal do Rio de Janeiro, Curso de Farmcia/Campus-Maca.

3Universidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e Alimentos,

Faculdade de Farmcia.

Correspondence

Prof. Dr. Gilda Guimares Leito

Universidade Federal do Rio de Janeiro, Instituto de Pesquisas de Produtos Naturais, Av.

Carlos Chagas Filho, 373, Centro de Cincias da Sade, Bloco H, Ilha do Fundo, 21.941-

902, Rio de Janeiro, RJ, Brazil. E-mail: ggleitao@nppn.ufrj.br . Phone: +5521 3938-6795

ext.213 Fax: +55213938-6512

Abstract

Verbascoside is a phenylethanoid glycoside widely distributed in Nature,

especially among the order Lamiales, occurring in numerous plants which are constituents

of folk medicine preparations. It is responsible for a vast array of biological activities

including antitumor, antimicrobial, anti-inflammatory, anti-thrombotic and wound healing

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properties. This natural compound was isolated from Lantana trifolia and Lippia alba f.

Intermedia (Verbenaceae) using either gradient or isocratic elution modes. The ethyl

acetate extracts of Lippia alba and Lantana trifolia, rich in phenylethanoids and

flavonoids, were fractionated using the step-gradient elution approach. The four-step

solvent system composed of hexane-ethyl acetate-n-butanol-water (0.4:1:X:1), where X =

0.1 (solvent system A); 0.3 (solvent system B); 0.5 (solvent system C) and 0.7 (solvent

system D) allowed the isolation of verbascoside, along with other phenylethanoids and

flavonoids from the two plants. The difference in the complexity of the two plant extracts

demanded different purification steps, which included a second HSCCC purification,

using isocratic elution mode. Verbascoside was further isolated from the butanol extract of

L. trifolia using the solvent system ethyl acetate-butanol-water 1:0.2:1 on an isocratic run.

Keywords

Lantana trifolia, Lippia alba f. Intermedia, Verbenaceae, step-gradient countercurrent

chromatography, phenylethanoids, verbascoside, 2-O-acetyl-verbascoside

Introduction

Verbascoside (or acteoside) is a phenylethanoid glycoside widely distributed in

Nature, especially among the order Lamiales, occurring in numerous plants which are

constituents of folk medicine preparations to treat inflammation and microbial infections

[1]. In a recent review on its occurrence and biological significance, Alipieva et al. [1]

report that, other than being found in many Verbascum species, verbascoside has also been

found in more than 200 plant species of 23 families. Verbascoside is responsible for a vast

array of biological activities including antitumor, antimicrobial, antioxidant, anti-

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inflammatory, anti-thrombotic, photo protective, besides possessing wound healing and

chelating properties [1-2]. It has been suggested as a prototype for the development of a

new antifungal drug for the treatment of cryptococcosis, especially for Cryptococcus

neoformans [3]. On a study on the inhibitory effects of verbascoside on prolyl

oligopeptidase, it was suggested that this compound has important neuroprotective activity

[4].

The family Verbenaceae comprises about 100 genera and approximately 2000

species distributed in the tropics and subtropics, with only a few native genera from

temperate regions [5]. Lantana and Lippia are two important genera, which contain

phenylethanoids, flavonoids, terpenoids and other compounds with relevant biological

activities [6,7]. Lantana trifolia L. and Lippia alba f. Intermedia (Moldenke) are small

shrubs occurring in all regions of Brazil and are extensively used in folk medicine in the

form of teas as a sedative, and in the treatment of gastric and respiratory disorders [8, 9].

The separation of phenylethanoids from crude plant extracts and other sources by

DCCC [10-12], CPC [13] and HSCCC [14-17] has been addressed before and some

examples can be found in the literature. The reported data include separations in one step

CCC, two steps CCC as well as the combination of CCC with other chromatography

techniques. The support-free all liquid nature of this technique makes it especially useful in

the isolation and purification of phenylethanoids since they are very polar compounds,

with many hydroxyl groups, which make them strongly adsorbed onto the solid support

during conventional separations [14]. In previous works from our group we used the

gradient elution approach for the separation of phenylethanoids and iridoids from extracts

of Stachytarpheta cayennensis [18] and a two dimensional approach combining gradient

and isocratic elution modes for the purification of phenylethanoids and flavones from the

ethyl acetate extract of Lantana trifolia, [9].

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The present work describes the isolation of phenylethanoids from the ethyl acetate

extract of L. alba and from the butanol extract of Lantana trifolia, and discusses different

strategies of gradient and isocratic elutions in CCC according to the complexity of plant

extracts, using verbascoside as target compound.

Results and Discussion

Verbascoside, an important bioactive compound, has been isolated from many plant

sources by the use of countercurrent chromatography [13, 15, 17, 19-26].

Samples of plant extracts with a broad polarity range are ideally fractionated by the

use of gradients, which is not very common when separations are performed by

countercurrent chromatography. The choice of an appropriate solvent system for a gradient

elution in CCC can be a difficult task. One of the reasons is because any change in the

composition of one liquid phase of a biphasic liquid system may induce changes in the

other liquid phase [27, 28]. According to Foucault [29], the system ethyl acetate-n-butanol-

water (EBuWat) can be used for gradient separations in the normal phase mode because all

aqueous phase compositions in this system contain more than 90% water. The organic

phases of this system are mixtures of ethyl acetate and n-butanol with a small amount of

water. The gradient elution approach with the solvent system ethyl acetate-n-butanol-water

10:x:10 was previously used by our group for the isolation of verbascoside from both ethyl

acetate extracts of Stachytarpheta cayennensis roots [18] and Lantana trifolia aerial parts

ethyl acetate extract [9]. For S. cayennensis the gradient elution in four steps afforded the

isolation of 3 phenylethanoids (isomartynoside, verbascoside and isoverbascoside) and two

iridoids. For the ethyl acetate extract of L. trifolia, however, a two dimensional approach,

using both gradient elution, followed by isocratic elution CCC had to be used instead.

Also, hexane had to be added to that solvent system.

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The same strategy of gradient elution was used here, due to the complexity of the

extracts from L. alba. Various ratios of the solvents in the solvent system EBuWat were

tested but the system proved to be too polar for the effective separation of the compounds

in the extracts. Therefore, hexane was introduced as a fourth solvent, in a similar way as

for L. trifolia ethyl acetate extract. The optimization of the number of steps of the gradient

composed of hexane-ethyl acetate-n-butanol-water 4:10:X:10 (v:v:v) was done with a

series of test tube experiments. The aim of these experiments is to set two extreme

situations: one in which we have all (or at least the major part of) compounds in the upper

phase and the other where we have the opposite situation (all compounds in the lower

phase). The majority of compounds in the extract were retained in the lower phase

(aqueous) when the proportion of 1 was used for n-butanol, whereas a gradual inversion of

the partition coefficients occurred when the n-butanol ratio was raised to 7.

In order to verify the effectiveness of the chosen gradient steps, the purification of

the ethyl acetate extract from Lippia alba f. Intermedia was fist performed at the 80 ml coil

on a semi-preparative scale with the above solvent system, in four steps where X = 1

(solvent system A); 3 (solvent system B); 5 (solvent system C) and 7 (solvent system D).

This fractionation afforded 2-O-acetylverbascoside, (Fr 43-45, 33.5 mg, step C, Fig. 4),

verbascoside, (Fr 66-67, 78.5 mg, step C, Fig. 3) and a third phenylethanoid glycoside, (Fr

68, 11.4 mg, step D, structure under investigation). This separation was scaled-up to 3g

in the 310ml coil and 182 mg of 2-O-acetylverbascoside (Fr 58-60, step C), 589 mg of

verbascoside (Fr 77-80, step C) and 31 mg of the third phenylethanoid glycoside, (Fr 81-

82, step D) were obtained. A good correspondence between the preparative and semi-

preparative separations was observed (based on TLC results).

Verbascoside (Fr 18-19; 29.5mg; 80.5%) was further isolated from the butanol

extract of L. trifolia (as a continuation of the previous work by our group), along with 2-

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O--apiosylverbascoside (Fr 30-31; 39.3mg; 81.8%), using the EBuWat solvent system

(10:2:10) on an isocratic run.

The isolation and purification of 2-O-acetylverbascoside (2-acetylacteoside) and

verbascoside (acteoside) from Cistanches salsa (C.A. Mey) G. Beck was made by Lei and

co-workers [17] in a two-step isocratic HSCCC purification with the solvent system ethyl

acetate-n-butanol-ethanol-water 4:0.6:0.6:5, v:v. The isolation and purification of

verbascoside and isoverbascoside from seeds of Plantago psyllium was also achieved by Li

and co-workers [14] using again a two-step isocratic HSCCC fractionation, with the

solvent system ethyl acetate-water 1:1. The same compounds were isolated from an extract

of Beagle dogs faeces by HSCCC with ethyl acetate-n-butanol-ethanol-water 35:6:6:50

followed by purification with the same solvent system in the ratios 30:10:6:50 [15].

Verbascoside and oraposide from Orobanche rapum-genistae were separated on a single

run HSCCC with the solvent system ethyl acetate-acetonitrile-water 2.6:2.6:4.8 [16]. A

variation on the EBuWat system was made by Xie and co-workers by the addition of

glacial acetic acid to the system when purifying echinacoside and verbascoside purified

from Cistanche tubulosa [20]

The gradient system employed here proved to be effective for the separation of

verbascoside from the ethyl acetate extract of Lippia alba, different from the previous

results obtained in the case of Lantana trifolia ethyl acetate extract where the same

approach wasnt effective in the purification of verbascoside probably due to the greater

complexity of this extract when compared to that of Lippia alba. The isolation of

verbascoside with the gradient system employed was dependent on the complexity of the

extracts as shown by the results obtained with L. alba and L. trifolia. This is an useful

strategy as most of previous isolation and purification of these compounds described in the

literature involve more than one HSCCC step.

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The butanolic extract from L. trifolia is less complex than the ethyl acetate extract.

Thus, isocratic elution was efficient to isolate verbascoside and 2-O--

apiosylverbascoside from this extract. The EBuWat system can be used in gradient mode

to improve the separation and, consequently, the purity of the isolated compounds.

All isolated compounds were identified by 1H and

13C NMR and compared with literature

[10, 17].

Materials and Methods

Plant material

Lippia alba f. Intermedia (Moldenke) was collected growing wild in the banks of

Trombetas River, were collected in the city of Oriximin, Par State, Brazil, S 1o

45`49.0``

W 55o

52`19.2`` and Lantana trifolia was collected in the wild, at Mendes (Rio de Janeiro

State, Brazil). Plants were identified by Prof. Dr. Fatima G. Salimena, from Federal

University of Juiz de Fora, where voucher specimens are deposited, with the respective

numbers: CESJ 39530 and CESJ 30801, respectively.

Extraction of Plant Material

Aerial parts of Lippia alba f. Intermedia and Lantana trifolia were exhaustively extracted

with ethanol 96oGL. The ethanol extracts were fractionated by liquid-liquid partition

between water and hexane, dichloromethane, ethyl acetate and n-butanol, in this order.

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Choice of the Solvent System for CCC fractionations

Small amounts of the ethyl acetate extract of Lippia alba were dissolved in separate test

tubes containing the solvent system ethyl acetate-n-butanol-water in the ratios 8:2:10;

7:3:10; 6:4:10; 5:5:10; 3:7:10 and 2:8:10. The test tubes were shaken and the compounds

allowed to partition between the two phases. Equal aliquots of each phase were spotted

beside each other, separately, on TLC plates developed with the solvent system ethyl

acetate-acetone-water 25:8:5. The results were visualized after spraying TLC plates with

NP-PEG followed by visualization under UV light (365 nm). Another set of test tube

experiments was performed by introducing hexane into the ethyl acetate-n-butanol-water

system. The resulting biphasic solvent mixture hexane-ethyl acetate-n-butanol-water, in the

ratios 4:10:X:10 v:v: (where x = 1, 3, 5 and 7), were tested again by the shake-flask test

with small amounts of the ethyl acetate extracts from both plants as described before. The

step gradient was defined as hexane-ethyl acetate-n-butanol-water (4:10:X:10), where X =

1 (solvent system A); 3 (solvent system B); 5 (solvent system C) and 7 (solvent system D).

The choice of solvent system for the butanol extract of L. trifolia was made by

testing mixtures of ethyl acetate/n-butanol/water (EBuWat, 10:x:10, v/v; x=0, 1, 2 and 4).

HSCCC Separation by Gradient Elution

Semi-preparative separation of L. alba f. Intermedia ethyl acetate extract

Fractionation of the extract of Lippia alba f. Intermedia was first performed on a 80 ml

coil of a triple polytetrafluoroethylene multi-layer coil from a P.C. Inc. apparatus (15 mL +

80 mL + 280 mL, 1.6 id). The pump was a Dynamax Model SD-200. A Dynamax FC-1

fraction collector was also used. The CCC column was equilibrated with solvent system A

(aqueous phase as stationary). One gram of the ethyl acetate extract was dissolved in 2.5

ml of each phase of solvent system A and injected into the coil with a Rheodyne valve. The

organic phases of solvent systems A (tubes 1-20), B (tubes 21-40), C (tubes 41-60) and D

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(tubes 61-80) were used as the mobile phases in the head-to-tail elution mode, at a flow

rate of 2.0 ml/min, 4 ml/fraction, 850 rpm. In these conditions, the aqueous stationary

phase (solvent system A), VS, initially retained in the CCC column was 81 ml (SF =

88.75%; VM = 9 ml). At the end of the final step of the gradient the column content was

extruded.

Preparative separation of L. alba f. Intermedia

Preparative fractionation was performed in the 310 ml coil (80 + 230 ml, medium + large

coils) of the P.C. Inc. apparatus. Three grams of the ethyl acetate extract were dissolved in

15 ml of both phases of solvent system A and injected in the 310 ml coil (previously

equilibrated with stationary and mobile phases of system A, VS = 277 ml, VM = 33 ml, SF =

89.35 %). The aqueous phase was used as the stationary phase, while the organic phase

was used as the mobile phase in the head-to-tail elution mode, at a flow rate of 3.0 ml/min

(solvent pump SD-200, Dynamax), and the revolution rate was 850 rpm. Fractions of 12

ml were collected (fraction collector FC-1, Dynamax), solvent system A: tubes 1-27;

solvent system B: tubes 28-55; solvent system C: tubes 56-83 and solvent system D: tubes

84-117. At the end of the final step of the gradient the column content was extruded.

Isocratic HSCCC Separation

Fractionation of the butanol extract of L. trifolia was performed in isocratic elution mode

with the solvent system ethyl acetate-n-butanol-water 10:2:10. The stationary lower phase

was pumped into the coil. Then, the mobile upper phase was pumped (2 ml/min.) with

rotation in the tail-to-head direction (normal phase). The sample (500mg) was dissolved in

5 ml of the biphasic solvent system and injected in the 80 ml coil of the P.C. Inc. apparatus

after establishment of hydrodynamic equilibrium (Vm=19 mL, Sf=76%)

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The same pump and fraction collector described above were used. Fractions of 4 ml were

collected, rotor rotation was stopped at tube 20 and the column content was extruded in a

total of 60 fractions.

HPLC analyses of extracts and pure isolated compounds

The HPLC profiles of the ethyl acetate extracts from leaves of L. trifolia and L. alba were

obtained by gradient-elution with the mobile phase composed of methanol- 1% aqueous

acetic acid (pH 3.0) at a flow rate of 1 ml/min. The linear gradient was from 20:80 to 33:67

over 50 min, on a Lachrom HPLC System (Merck, Darmstadt, Germany) equipped with an

interface D7000, pump L-7100, diode array detector (DAD) L-7450A and solvent degasser

L-7612. The injections were done manually with a Rheodyne injection valve. The analyses

were carried out on a Rexchrom S5-100-ODS column (Regis Technologies Inc., Illinois,

U.S.A., 5 m particle size, 250 x 4.6 mm i.d.). The UV detection was performed from 220

nm to 350 nm. The sample (2 mg/ml) injection volume was 20 l. Fractions obtained from

HSCCC were analyzed in the same conditions.

Supporting information

Acknowledgements

Thanks are due to CNPq, CAPES and FAPERJ for financial support and scholarships.

Conflict of Interest

The authors declare no conflict of interest.

References

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For Peer Review

18 Leito GG, de Souza PA, Moraes AA, Brown L. StepGradient CCC Separation of

Phenylpropanoid and Iridoid Glycosides from Roots of Stachytarpheta cayennensis (Rich.)

Vahl. J. Liq. Chrom. & Rel. Technol. 2005; 28: 2053-2060.

19 Han L, Ji L, Boakye-Yiadom M, Li W, Song X, Gao X. Preparative isolation and

purification of four compounds from Cistanche deserticola Y.C. Ma by high-speed

counter-current chromatography. Molecules 2012; 17: 8276-8284.

20 Xie C, Xu X, Liu Q, Xie Z, Yang M, Huang J, Yang D. Isolation and purification of

echinacoside and acteoside from Cistanche tubulosa (schrenk) wight by high-speed

counter-current chromatography. Journal of Liquid Chromatography & Related

Technologies 2012; 35(18): 2602-2609.

21 Duan WJ, Zheng ZJ, Geng YL, Liu JH, Wang X. Preparative isolation and purification of

C6-C2 natural alcohol and benzyl ethanol from Forsythia suspensa by high-speed

countercurrent chromatography. Acta Chromatographica 2011; 23(3): 499-507.

22 Li L, Rong T, Yang R, Liu C, Young JC, Zhu H. . Isolation and purification of

phenylethanoid glycosides from Cistanche deserticola by high-speed counter-current

chromatography. Food Chemistry 2008; 108(2): 702-710.

23 Li L, Tsao R, Liu Z, Liu S, Yang R, Young JC, Zhu H, Deng Z, Xie M, Fu Z. Isolation

and purification of acteoside and isoacteoside from Plantago psyllium L. by high-speed

counter-current chromatography. Journal of Chromatography A 2005; 1063(1-2): 161-169.

24 Mei LH, Kyu KJ, Man JJ, Bi CC, Sung LS. Analysis of the inhibitory activity of

Abeliophyllum distichum leaf constituents against aldose reductase by using high-speed

counter current chromatography. Archives of pharmacal research 2013; 36(9): 1104-1112.

25 Jun X, Feng T, Jing Z, Caibo Y, Qi L. Separation, purification and quantification of

verbascoside from Penstemon barbatus (Cav.) Roth. Food chemistry 2012; 135(4): 2536-

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Georg Thieme Verlag KG, P.O. Box 30 11 20, D-70451 Stuttgart, Germany www.thieme.de/plantamedica

Manuscript submitted to editorial office

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For Peer Review

26 Berthod A., Brown L., Leito GG, Leito SG, in D. Barcel, Alain Berthod (Editors)

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Comprehensive Analytical Chemistry; Elsevier, NewYork, 2002, Vol. XXXVIII, p.37.

27 Berthod A., Brown L., Leito GG, Leito SG, in D. Barcel, Alain Berthod (Editors)

Countercurrent Chromatography The Support Free Liquid Stationary Phase,;

Comprehensive Analytical Chemistry; Elsevier, NewYork, 2002, Vol. XXXVIII, p.37.

28 Wu S, Wu D, Liang J, Berthod A. Modelling gradient elution in countercurrent

chromatography: efficient separation of tanshinones from Salvia miltiorriza Bunge. J. Sep.

Scoences 2012; 35:964-976.

29 Foucault AP, in Foucault AP (Editor) Centrifugal Partition Chromatography.

Chromatographic Science Series. Marcel Dekker Inc., New York, 1995, Vol. 68, p.90.

Page 15 of 15

Georg Thieme Verlag KG, P.O. Box 30 11 20, D-70451 Stuttgart, Germany www.thieme.de/plantamedica

Manuscript submitted to editorial office

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