Optimization of the sonication extraction method of Hibiscus tiliaceus L. flowers

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Ultrasonics Sonochemistry 13 (2006) 242–250

Optimization of the sonication extraction method ofHibiscus tiliaceus L. flowers

Maria Ines Soares Melecchi a, Valeria Flores Peres a,b, Claudio Dariva c,Claudia Alcaraz Zini a, Fernanda Contieri Abad a, Migdalia Miranda Martinez b,

Elina Bastos Caramao a,*

a Chemistry Institute, UFRGS, Porto Alegre, RS, av. Bento Goncalves, 9500, CEP 91501-960, RS, Brazilb Universidad de la Habana, Cuba

c URI-Campus de Erechim, RS, Brazil

Received 20 August 2004; accepted 14 February 2005

Available online 1 July 2005

Abstract

The influence of several experimental parameters on the ultrasonic extraction of Hibiscus tiliaceus L. flowers were investigated:

extraction time, solvent polarity, sample amount, solvent volume and sample particle size. It was concluded that the most influential

variables were extraction time and solvent polarity. The optimized procedure employed 5 g of ground flowers, 150 mL of methanol

and 140 min of extraction. The extracts were fractionated using preparative silica columns and the resulting fractions were analyzed

by GC/MS. Some saturated hydrocarbons, fatty acids, fatty acid methyl esters, phytosterols, and vitamin E were identified in the

plant extracts.

� 2005 Elsevier B.V. All rights reserved.

Keywords: Sonication; Hibiscus tiliaceus L.; GC/MS

1. Introduction

Identification of organic compounds from plant ex-

tracts is of great importance, mainly because they can

be used as an excellent source of phytotherapics. The

usual extraction procedures for the isolation of organic

compounds of medicinal plants are maceration, percola-

tion, and Soxhlet extraction [1]. These techniques usu-

ally require long extraction times, providing lowefficiencies. Moreover, many natural products are ther-

mally unstable and may degrade during conventional

processes where the matrix is kept in a boiling solvent,

such as Soxhlet extraction [2,3].

1350-4177/$ - see front matter � 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.ultsonch.2005.02.003

* Corresponding author. Tel.: +55 51 3316 7213; fax: +55 51 3316

7304.

E-mail address: [email protected] (E.B. Caramao).

Many reports on the beneficial effects of medicinalplants ultrasound extractions have been published [4–

6], and the main reported improvements have been

found to be enhanced efficiency and shortening of

extraction time [1,2,7]. A most likely mechanism for

ultrasonic extraction is an enhanced mass transfer, the

improved penetration of the solvent into the vegetal

due to cell disruption, and capillary effects [8,9].

The effects of the collapse of cavitation bubbles pro-duced by ultrasound on the cell walls of plants can be

described by the following mechanisms [10,11]: some

plant cells have glands filled with essential oil and one

characteristic of external glands is that their skin is very

thin and can be easily destroyed by sonication, thus

facilitating release of essential oil contents into the

extraction solvent. On the other hand, ultrasound can

also facilitate the swelling and solvatation of plant

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M.I. Soares Melecchi et al. / Ultrasonics Sonochemistry 13 (2006) 242–250 243

materials causing enlargement of the pores of the cell

wall. Better swelling will improve the rate of mass trans-

fer and, occasionally, break the cell walls, thus resulting

in increased extraction efficiency and/or reduced extrac-

tion time.

Other advantages of this technique are its high repro-ducibility, the possibility of using a wide range of sample

sizes and the low cost of the whole process [12–14].

The optimization of ultrasound operational parame-

ters (solvent polarity, sample particle size, etc.) accord-

ing to a specific plant matrix (leaves, flowers, stems,

etc.) is also of main importance in order to achieve high

extraction efficiency [8]. A further step is the use of pre-

fractionation techniques, such as preparative liquidchromatography to allow a reasonable characterization

of the individual constituents of complex mixtures of

plant extracts using gas chromatography with mass

spectrometer detector (GC/MS) or other highly efficient

instrumental technique [15,16].

Hibiscus (H.) tiliaceus L. is a typical plant of tropical

climate found in mangrove areas. Its flowers are used in

traditional medicine in the Asian and African countries[15], but information concerning its chemical composi-

tion is scarce in the scientific literature.

The goal of this work is the optimization of the H. til-

iaceus L. flowers sonication extraction and the chemical

characterization of the extracts obtained. Optimization

of the extraction process was performed by testing dif-

ferent sonication times, polarity of the extractor sol-

vents, particle size, sample amounts, and mass offlowers/volume of solvent extractor ratio. The extracts

were fractionated using silica gel preparative liquid

chromatography and the resulting fractions were ana-

lyzed by GC/MS.

2. Experimental

2.1. Equipments

An ultrasonic cleaning bath model Thorton T-14,

with 90 W of potency, 40 KHz of frequency and

0.27 W cm�2 of radiation intensity was used. The inter-

nal dimensions of the water bath were of

24 cm · 14 cm · 10 cm. The bath was kept at constant

temperature (25 �C) during all the extraction processesby changing the water after each 30 min of extraction.

The extracts were filtered and concentrated under re-

duced pressure.

GC/MS analyses were made in a quadrupole analyzer

Shimadzu QP 5050A using a capillary column-OV-5

(30 m · 0.25 mm · 0.25 lm). The chromatographic con-

ditions were: split injection (1:40); helium (Air Products,

Rio de Janeiro, Brazil) at a flow rate of 1.5 mL min�1;ionization by electronic impact with 70 eV. The column

temperature program began at 50 �C and reached

280 �C with a heating rate of 5 �C/min and remained

at the final temperature for 50 min. Injector and inter-

face were kept at 280 �C. Several compounds were iden-

tified comparing their mass spectra and retention data

with the ones of standard compounds. Other com-

pounds were only tentatively identified comparing theirmass spectra with the Willey library mass spectra.

2.2. Materials

All reagents were p.a. grade, and were purchased

from Merck GmbH (Darmstadt, German). Solvents

(n-hexane, benzene, dichloromethane, ethyl acetate and

methanol) were distilled twice and stored in dark flasks

until use. Silica gel 60 (Merck GmbH) having a particle

size of 0.063–0.200 mm (70–230 mesh ASTM), was em-

ployed in the preparative liquid chromatographic frac-

tionation (PLC). It was activated at 140 �C for 4 h.

2.3. Sample preparation

H. tiliaceus L. flowers were collected in Florianopolis,Santa Catarina—Brazil in January 2003. Sampling was

performed only once, as H. tiliaceus L. is a seasonal

plant. The plant was catalogued in the Botanical Insti-

tute of the Federal University of Rio Grande do Sul

(UFRGS), Brazil, with the herbarium number ICN:

113936, and identified by Dr. B. Irgand (Bio-sciences

Institute/Department of Botanical UFRGS, RS, Brazil)

as H. tiliaceus L. Flowers were oven dried, with air cir-culation, at 40 �C.

2.4. Sonication extraction: study of some parameters

The extractor solvents for the first sonication proce-

dure were hexane, ethyl acetate, and methanol. In paral-

lel another study was accomplished by using only

methanol as solvent extractor (5 g of sample for

150 mL solvent). All fractions underwent a chromato-

graphic fractionation scheme using preparative silica

columns.

Optimization of sonication procedure was firstly per-formed using a fractional factorial drawing 25�1. The

proposed matrix provided 16 experiments [17,18] and

the evaluated parameters were:

X1 (extraction time): the effect was evaluated using 40

and 180 min;

X2 (solvent polarity): two solvents of opposite polar-

ities, n-hexane (non-polar) and methanol (polar);X3 (amount of sample): 5 and 15 g;

X4 (particle size): the whole intact flowers and ground

flowers;

X5 (mass of sample/volume of solvent ratio): 1:10 and

1:30.

Table 1

Variables and results of the fractional factorial drawing5�1

Run Variables and values Mass yield (%)

X1 X2 X3 X4 X5 Experimental Theoretical

1 (+1) 180 (�1) Hexane (+1) 15 (�1) Ground (+1) 1:30 4.53 3.96

2 (+1) 180 (�1) Hexane (�1) 5 (�1) Ground (�1) 1:10 2.00 2.03

3 (�1) 40 (+1) Methanol (�1) 5 (+1) Whole (+1) 1:30 9.00 7.98

4 (�1) 40 (�1) Hexane (+1) 15 (�1) Ground (�1) 1:10 2.00 1.16

5 (�1) 40 (�1) Hexane (+1) 15 (+1) Whole (+1) 1:30 2.26 3.29

6 (+1) 180 (�1) Hexane (�1) 5 (+1) Whole (+1) 1:30 4.40 4.16

7 (�1) 40 (�1) Hexane (�1) 5 (�1) Ground (+1) 1:30 2.20 3.09

8 (+1) 180 (+1) Methanol (�1) 5 (+1) Whole (�1) 1:10 5.00 3.98

9 (+1) 180 (+1) Methanol (+1) 15 (+1) Whole (+1) 1:30 9.40 8.85

10 (�1) 40 (+1) Methanol (�1) 5 (�1) Ground (�1) 1:10 5.80 5.85

11 (�1) 40 (�1) Hexane (�1) 5 (+1) Whole (�1) 1:10 1.20 0.96

12 (+1) 180 (+1) Methanol (+1) 15 (�1) Ground (�1) 1:10 5.20 6.71

13 (�1) 40 (+1) Methanol (+1) 15 (�1) Ground (+1) 1:30 5.73 5.24

14 (+1) 180 (�1) Hexane (+1) 15 (+1) Whole (�1) 1:10 1.93 1.83

15 (+1) 180 (+1) Methanol (�1) 5 (�1) Ground (+1) 1:30 5.20 6.11

16 (�1) 40 (+1) Methanol (+1) 15 (+1) Whole (�1) 1:10 2.53 3.11

(�1): lower level; (+1): higher level.

X1: extraction time in minutes, X2: solvent polarity, X3: mass of the sample in grams, X4: particle size, X5: mass of the sample/volume of solvent in

grams per milliliter.

Fig. 1. Comparison between the theoretical and experimental values obtained for mass yield using a fractional factorial drawing 25�1.

244 M.I. Soares Melecchi et al. / Ultrasonics Sonochemistry 13 (2006) 242–250

Solvent volumes employed were 50, 150 or 450 mL,

according to the sample amount and to the mass of

the sample/solvent volume ratio.

Mass yield (y %) and chromatographic profile (CG/

MS) were employed as parameters for extraction com-

parisons. Statistical evaluation of the results was per-formed using Statistics for Windows version 6.0

software. Once optimum experimental conditions were

established, duplicate experiments were performed

according to them (5 g of ground flowers and 150 mL

of methanol). Extraction time was further investigated

using 60–340 min, setting time intervals of 40 min, in or-

der to determine when solvent saturation occurs, and if

long extraction times can cause degradation of the ex-tract constituents.

2.5. Chromatographic fractionation of methanol extracts

Fractionation of methanol extracts obtained in

different extraction times were performed using a meth-

od developed by Wang et al. [19] modified by da Luz

[20]. In this methodology, silica gel was used as solid

phase and n-hexane, hexane/benzene (1:1), dichloro-

methane, ethyl acetate, and methanol as elution sol-vents. A glass column (40 cm · 11 mm), was packed

with 3.0 g of previously activated silica. A thin layer of

anhydrous sodium sulfate (approximately 1.0 g) was

added on the top of the column, and the column was

conditioned with 20 mL of hexane. The sample (approx-

imately 500 mg of extract) was firstly mixed with 1.0 g of

silica and 1.0 mL of dichloromethane, forming a fine

Table 2

The influence of the extraction time on the mass yield using methanol

as extractor solvent

Run Time (min) Mass yield (%)

1 60 9.82

2 100 10.25

3 140 12.82

4 180 12.84

5 220 12.96

6 260 15.57

7 300 17.10

8 340 14.60


slurry. After solvent evaporation, the dry mixture was

added on the top of the column. The elution was per-

formed using 20 mL of each one of the following sol-

vents: n-hexane, hexane/benzene (1:1),dichloromethane, ethyl acetate, and methanol. The five

fractions of each extract were evaporated under a gentle

flux of nitrogen. Fractions obtained with n-hexane, hex-

ane–benzene and dichloromethane were analyzed using

GC/MS without prior treatment. Those obtained with

ethyl acetate and methanol were derivatized using BF3

in methanol [21] before the chromatographic analysis.

3. Results and discussion

Investigation of sonication extraction parameters was

investigated using a fractional factorial drawing 25�1

Table 3

Compounds from fraction 1 of the PLC (eluted with n-hexane) of the metha

Compoundsa Extraction time in minutes

60 100 140 180

1. Pentadecane

2. Hexadecane · · ·3. Heptadecane · · ·4. Prysthane · · ·5. Octadecane · · · ·6. Phythane · · ·7. Nonadecane · · · ·8. Eicosane · · ·9. Uneicosane · · ·10. Docosane · · ·11. Tricosane · · · ·12. Tetracosane · · · ·13. Pentacosane · · · ·14. Hexacosane · · · ·15. Heptacosane · · · ·16. Octacosane · · ·17. Nonacosane · · · ·18. Triacontane · · · ·19. Heneitriacontane · · · ·20. Dotriacontane · · ·21. Tritriacontane · · ·22. Tetratriacontane · · ·a ‘‘·’’ means that the compound was detected.b tR = average value of duplicate retention times in minutes.

with two levels (+1 and �1) for each parameter. Data

treatment was performed with Statistics for Windows

version 6.0 software, having mass yield percent (y %)

as the dependent variable. The following equation was

obtained for mass yield:

yð%Þ ¼ 4.27þ 0.431X 1 þ 1.70X 2 þ 1.06X 5 þ 0.634X 1X 3

þ 0.734X 4X 5

Experimental values of mass yield and those obtained

with the above equation (theoretical value) are shown

in Table 1.

The analysis of the equation allowed to observe thatthe variable X1 (extraction time) showed a significant

positive effect (+0.431 X1), indicating a direct relation

between extraction time and mass yield. Such a relation

is also valid for other solid-liquid extraction techniques.

Variable X2 (solvent polarity) also showed a significant

positive effect (+1.70 X2), that is, higher polarity allows

higher mass yields. This behavior might be determined

by the nature of the secondary metabolites present inthe plant species under study. The parameter mass of

the sample/sample volume (X5) showed also a positive

effect (+1.060). The variables such as particle size (X4)

and amount of sample (X3) did not influence signifi-

cantly the extraction process. However, the interrelation

between these variables X1X3 (extraction time and

amount of sample) and X4X5 (particle size and ratio

mass/solvent) presented positive effects, indicating that

nol extract of H. tiliaceus L. flowers

tRb

220 260 300 340

· 30.63

· 32.02

34.49

34.60

· · · · 36.83

36.97

· · 39.02

· 40.77

· 42.97

· · 45.04

· · 46.72

· · · 48.48

· · · · 50.35

· · · · 52.02

· · · 53.52

· · · · 54.91

· · · · 56.89

· · · 58.53

· · · · 61.00

· 64.25

· · · 66.97

· · 71.45

Table 4

Compounds from fraction 2 of the PLC (eluted with hexane/benzene 1:1) of the methanol extract of H. tiliaceus L. flowers

Compoundsa Extraction time in minutes tRb

60 100 140 180 220 260 300 340

1. Tetradecanoic acid methyl ester · 33.65

2. Pentadecanoic acid methyl ester · 35.91

3. Neophytadiene · · · · · · · 37.47

4. Hexadecenoic acid methyl ester · 37.68

5. Hexadecanoic acid methyl ester · 38.14

6. Phytol · · · · · 38.15

7. Phytol isomer · · · · · 38.73

8. Heptadecanoic acid methyl ester · · · 39.89

9. Octadecadienoic acid methyl ester · 41.57

10. Octadecenoic acid methyl ester · 41.77

11. Octadecenoic acid · · 43.00

12. Octadecanoic acid methyl ester · · 42.77

13. Nonadecenoic acid methyl ester · 43.73

14. Eicosenoic acid methyl ester · 45.37

15. Eicosanoic acid methyl ester · 45.77

16. Tricosane · · · 47.27

17. Heneicosanoic acid methyl ester · · 47.29

18. Tetracosane · · · 48.63

19. Docosenoic acid methyl ester · 48.93

20. Pentacosane · · · · · · · 49.97

21. Docosanoic acid methyl ester · · 50.75

22. Hexacosane · · · · 51.85

23. Tetradecanal · · 52.22

24. Tetracosanoic acid methyl ester · · 52.83

25. Heptacosane · · · · · · · 53.16

26. Octacosane · · · · 54.24

27. Squalene · · · · · · 55.14

28. Docosanol · · · · · · 56.36

29. Nonacosane · · · · · · · 56.71

30. Triacontane · · · · · 58.91

31. Octacosanoic acid methyl ester · · · · · · 60.88

32. Heneitriacontane · · · · · 61.20

33. Nonacosanoic acid methyl ester · · 62.05

34. Tritriacontane · · 67.40

a ‘‘·’’ means that the compound was detected.b tR = average value of duplicate retention times in minutes.

Table 5

Compounds from fraction 3 of the PLC (eluted with dichloromethane) of the methanol extract of H. tiliaceus L. flowers


60 100 140 180 220 260 300 340

1. Hexadecenoic acid methyl ester · · · · · 39.00

2. Hexadecanoic acid methyl ester · · · · · · · 39.48

3. Heptadecanoic acid methyl ester · · 41.49

4. Linoleic acid methyl ester · · · · · · · 42.90

5. Octadecenoic acid methyl ester · · · · · · · 42.99

6. Octadecatrienoic acid methyl ester · · · · · · 43.05

7. Nonadecanoic acid methyl ester · · · · · · 43.44

8. Octadecinoic acid methyl ester · · · 43.69

9. Octadecadienoic acid methyl ester · · · 44.34

10. Eicosanoic acid methyl ester · · · · · · 46.61

11. Tetracosanoic acid methyl ester · 55.79

12. Vitamin E · 63.08

13. Stigmastenone · · 72.78

14. Ergostenone · · 75.69

15. Pregnenedione · 77.36



Table 6

Compounds from fraction 4 of the PLC (eluted with ethyl acetate) of the methanol extract of H. tiliaceus L. flowers


60 100 140 180 220 260 300 340

1. Nonanoic acid · · · · · · · 23.05

2. Noninoic acid · 23.88

3. Tetradecene · · · · · · · 26.00

4. Hexadecene · · · · · · · 31.25

5. Benzophenone · · · · · · · 32.99

6. Tetradecanoic acid · · · · · 35.85

7. Methyl-benzophenone · · · · · · · · 35.69

8. Octadecene · · · · · · · · 35.98

9. Trimethyl pentadecenone · · · 37.70

10. Pentadecanoic acid · · · · · 38.07

11. Hexadecenoic acid · · · · · 39.68

12. Hexadecanoic acid · · · · · · · 40.81

13. Octadecenoic acid methyl ester · 42.97

14. Octadecadienoic acid · · · 43.87

15. Octadecadienoic acid methyl ester · · 44.34

16. Octadecenoic acid · · · · 44.54

17. Eicosene · · 56.85

18. Stigmastenol (isomer) · · 60.79

19. Vitamin E · · · · · · 63.04

20. Ergostenol · · · · 64.75

21. Ergostatrienol · 66.18

22. Fucosterol (isomer) · · · · 66.79

23. Stigmastadienol (isomer) · · · 68.07

24. Fucosterol (isomer) · · · · 68.38

25. Acetyloxy-androstanone · · · · · 68.74

26. Stigmastenol (isomer) · · · · · · · · 70.38

27. Fucosterol (isomer) · · · · · 70.91

28. Stigmastadienol (isomer) · · · · · · · 71.11

29. Fucostenone (isomer) · · · · · 72.03

30. Stigmastenone (isomer) · · · · 72.42

31. Fucostenone (isomer) · · 73.09

32. Stigmastadienol-acetate · 73.36

33. Stigmastadienone · · · · · · · 73.62

34. Hydroxy ergostenone · · · · · 74.80

35. Dimethyl androstenol-acetate · 75.12

36. Stigmastenone (isomer) · · · · · · · 76.76

37. Ergostadienone · · · · · · · 77.40

38. Stigmastadienenol (isomer) · · · · 77.97

39. Methyl, ethyl-colestanone · · · 85.85



a large amount of sample results in longer extraction

times and a large particle size demands higher volumes

of solvent.

Based on the results obtained with this theoretical

model, longer extraction times and higher solvent polar-

ity might result in enhanced extraction yields.

Fig. 1 shows the correlation of the experimental data

with those obtained using the theoretical model. Exper-iments 2, 6, 10, 11, 14 show an appropriate agreement

between experimental and the theoretical data. The best

extraction yields were achieved under the experimental

conditions of trials 3 and 9.

In order to further understand the influence of extrac-

tion time in the extraction yield, different times were

tested from 60 to 340 min. The other parameters were

kept constant: a sample amount of 5 g (X3), methanol

as solvent (X2), 1:30 as mass of sample/solvent volume

ratio (X5), and the whole plant of H. tiliaceus L. (X4).

The results are shown in Table 2.

These results show that the longer the extraction time

the higher the mass yield obtained, when methanol is

used as solvent. At extraction times longer than

300 min, a decrease in the mass yield was observed,which may be explained considering the possibility of

organic compounds decomposition by the effect of the

sound waves. This is one of the limitations of this

technique.

The tentative identification of the chromato-

graphic peaks for each PLC fraction is shown in Tables

3–8.

Table 7

Compounds from fraction 4 of the PLC (eluted with ethyl acetate) of the methanol extract of H. tiliaceus L. flowers after the derivatization with BF3

in methanol


60 100 140 180 220 260 300 340

1. Nonanoic acid · · · · · · 21.52

2. Noninoic acid (isomer) · · · · · 22.20

3. Noninoic acid (isomer) · · · · · 25.20

4. Undecanoic acid · 28.63

5. Dodecanoic acid · · · · · 30.03

6. Nonanodioic acid · · · · · 30.65

7. Tetradecanoic acid · · · · · · · · 34.44

8. Pentadecanoic acid · · · · · · · 37.38

9. Hexadecenoic acid · · · · · · · 38.71


11. Heptadecenoic acid · 39.81

12. Octadecinoic acid · · 40.51

13. Heptadecanoic acid · · · 41.47

14. Hexadecadienoic acid · · · 41.85

15. Octadecinoic acid · 42.24

16. Octadecadienoic acid · · · · · · 42.90

17. Octadecenoic acid · · · · · 43.03

18. Octadecatrienoic acid · · 43.08

19. Octadecanoic acid · · · · · · 43.43

20. Octadecinoic acid (isomer) · · · · · · · 43.71

21. Nonadecanoic acid · · 45.47

22. Nonadecenoic acid · · · 46.72

23. Eicosanoic acid · · · · · 47.14

24. Docosanoic acid · · · · · · · 49.94

25. Tricosanoic acid · · 52.09

26. Tetracosanoic acid · · · · · 53.27

27. Pentacosanoic acid · · 55.09

28. Hexacosanoic acid · · · 56.94

29. Stigmastenol (isomer) · 59.68

30. Heptacosanoic acid · 58.91

31. Stigmastadienol · 61.19


33. Octacosanoic acid · · · 62.17


35. Nonacosanoic acid · 65.14

36. Triacontanoic acid · 68.91

37. Fucostenona (isomer) · 71.91

38. Sigmastenone · 74.64

39. Stigmastenol (isomer) · 76.33

40. Fucostenone (isomer) · · 77.15

41. Stigmastadienone · 77.91

a ‘‘·’’ means that the compound was detected. Fatty acids were identified as methyl esters.b tR = average value of duplicate retention times in minutes.


Table 3 shows the identified saturated linear hydro-

carbons ranging from 15 to 34 carbon atoms found in

the hexanic fraction of the H. tiliaceus L. methanolic ex-

tract. Extracts resulting from 60 to 140 min of extractionshowed the same composition, except for the intensity of

the chromatographic peaks and mass yields. Saturated

linear hydrocarbons in Hibiscus flowers have not yet

been reported in the literature.

The second fractions (Table 4) show the presence of

some hydrocarbons, some fatty acid methyl esters

(FAME), and some phytosterols. It was also observed

that starting from 180 min some components were not

detected and others (that were not detected in shorter

elution times) showed up. A different behavior was no-

ticed when extraction was extended to 340 min, as sev-

eral esters of fatty acids were also detected. Thesecompounds were absent when extraction times were

shorter.

The chromatographic fractions obtained with dichlo-

romethane (Table 5) show some fatty acid methyl esters

(FAME) and sterols. Saturated and unsaturated esters

were found, being the saturated more abundant than

the other ones. The presence of FAME and steroids in

the flowers and stem of other species of the Hibiscus

Table 8

Compounds from fraction 1 of the PLC (eluted with methanol) of the methanol extract of H. tiliaceus L. flowers after the derivatization with BF3 in

methanol


60 100 140 180 220 260 300 340

1. Octanoic acid · · · · 17.14

2. Nonanoic acid · · · · 20.22

3. Dodecanoic acid · · · 28.72

4. Nonanodioic acid · · · 29.34

5. Hexadecene · · · 30.47

6. Tetradecanoic acid · · · · · · 33.84

7. Pentadecanoic acid · · · · 36.23

8. Hexadecenoic acid (isomer) · · · · · · · · 37.77


10. Hexadecenoic acid (isomer) · · · · · 39.81

11. Heptadecanoic acid · · · · 40.30

12. Octadecinoic acid · · · 40.44

13. Octadecadienoic acid (isomer) · · · · · · · · 41.65

14. Octadecenoic acid · · · · · · · 41.78

15. Octadecatrienoic acid · · · · 41.72

16. Octadecenoic acid (isomer) · · · · · · · 42.06

17. Octadecanoic acid · · · · 42.49

18. Octadecinoic acid (isomer) · · · 42.43

19. Nonadecanoic acid · · · 43.78

20. Eicosanoic acid · · 45.75

21. Docosanoic acid · · 49.20

22. Tricosanoic acid · · · · 50.64

23. Androstanol methyl acetate · 51.19

24. Tetracosanoic acid · · · 52.82

25. Pentacosanoic acid · · · 54.50

26. Hexacosanoic acid · · · 56.35

27. Heptacosanoic acid · · · 58.36

28. Octacosanoic acid · · · 60.71

29. Nonacosanoic acid · · · 63.45

30. Fucosterol · · 64.58

31. Ergostenol · · 64.83

32. Stigmastadienol · · 65.76

33. Stigmastenol · · · · 64.31

34. Fucosterol · 68.40

35. Fucostenone · · 69.14

36. Fucostenone · 70.54

37. Stigmastenone · · 73.18

38. Stigmastadienone · · 73.83

a ‘‘·’’ means that the compound was detected. Fatty acids were identified as methyl esters.b tR = average value of duplicate retention times in minutes.


was already reported in the literature [14,15], although

there is no report of these compounds for the H. tiliac-

eus species.

The presence of FAME in the previous fraction of the

340 min extract (Table 4) is probably due to premature

elution of these compounds resulting in their absence

in fraction 3 (Table 5).

The chromatographic fractions obtained with ethylacetate, were analyzed without derivatization (Table 6)

and after derivatization (Table 7). This allowed identifi-

cation of some free fatty acids, while methylation facili-

tated the chromatographic separation and analysis.

Vitamin E (a-tocopherol) and also some phytosterols

were found in the ethyl acetate fraction. These com-

pounds were already reported in other Hibiscus species

[14,15]. Extracts obtained after 140 min of extraction

contained a total of 31 compounds that were absent

when shorter extraction times were employed: free fatty

acids, FAME, unsaturated hydrocarbons, ketones, phy-

tosterols and a-tocopherol. The last PLC fractions were

analyzed after derivatization and the peak identificationis showed in Table 8. As it can be seen, the chromato-

graphic profile of these fractions is very similar to those

obtained in the previous fractions (Table 7). This results

can be explained, as both solvents employed are polar

(ethyl acetate and methanol).


4. Conclusions

Optimization of the sonication extraction parameters

of H. tiliaceus L. flowers, using a fractional factorial

drawing 25�1 indicated that the most influential param-

eters are solvent polarity and extraction time. Extractiontime of 140 min resulted in better mass yields having

methanol as solvent, while methanol provided higher

yields than hexane. Even though theoretical data pre-

dicted by the fractional factorial drawing 25�1 showed

that longer extraction times would result in higher

yields, experimental data have shown that longer extrac-

tion times allow artifact formation.

Compounds identified in the H. tiliaceus L. flowersextracts were: saturated hydrocarbons (from 15 to 34

carbon atoms), methyl ester of fatty acids, a-tocopherol,and phytosterols. No one of these compounds was for-

merly reported in the literature for H. tiliaceus L. flow-

ers. Qualitative composition of ultrasonic extracts was

the same found in the extracts obtained using conven-

tional extraction techniques, such as Soxhlet extraction,

among others [22].The presence of bioactive compounds in the H. tiliac-

eus L. flowers extracts, such as phytosterols may be

linked to the phytotherapic properties of this plant.

Some phytosterols have shown to exhibit cancer preven-

tive, anti-neoplastic, anti-pyretic, and immuno-modulat-

ing activity [23].

Acknowledgment

The authors thank FAPERGS, CNPq and FINEP

for their financial support.

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Optimization of the sonication extraction method of Hibiscus tiliaceus L. flowers

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