Metabolite Profiling of Ethyl Acetate Extract from Marsilea crenata

Presl. Usin

UPLC-QToF-MS/MS

Burhan Ma’arif*

1, 2

, Mangestuti Agil

Doctoral Program of Pharmaceutical Sciences, Department of Pharmacognosy and Phytochemistry,

Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia.

Department of Pharmacy, Faculty of Medical and Health Science, Maulana Malik Ibrahim

State Islamic University, Malang, Indonesia

Department of Pharmacognocy and Phytochemistry, Faculty of Pharmacy,

Universitas Airlangga, Surabaya, Indonesia

Keywords: Marsilea crenata Presl., Metabolite Profiling, UPLC-QToF-MS/MS, Phytoestrogens

Abstract: Marsilea crenata Presl. is a plant that widely used as traditional food in Surabaya, Indonesia. Although in

some research it was known contain phytoestrogens which have activity in bone formation, the

phytochemical properties of M. crenata has not been completely confirmed yet. The aim of this research

was to determine the metabolite profile of ethyl acetate extract of M. crenata using UPLC-QToF-MS/MS,

which can be used as a reference for further research and utilization of M. crenata. Dried powder of M.

crenata was extracted with n-hexane followed by ethyl acetate. The 100 ppm of ethyl acetate extract in

DCM and methanol then injected 5 µl each into the UPLC-QToF-MS/MS. The results were analyzed by

Masslynx 4.1 software, and showed various types of compounds, either detected compounds (36

compounds), or unknown compounds.

1 INTRODUCTION

Marsilea. crenata Presl. is an aquatic plant that

widely used as an ingredient for traditional food in

Surabaya, Indonesia (Nurjanah and Abdullah, 2012;

Ma’arif et al., 2016).

Figure 1: Marsilea crenata Presl.

Some of the research that had been done showed

that 96% ethanol extract, n-hexane extract, and ethyl

acetate extract of M. crenata leaves can inhibit

osteoporosis in female mouse (mus musculus) with

mechanism of bone formation improvement

(Laswati, 2011; Aemi, 2012; Adityara, 2017;

Widiasari, 2017). Other studies were also showed

that n-hexane extract of M. crenata leaves can

increase the alkaline phosphatase production in

MC3T3-E1 preosteoblast cell differentiation

process, which indirectly also play a role in bone

formation improvement (Ma’arif et al., 2018).

This activity appears to be suspected because of

the phytoestrogens content in M. crenata, where

phytoestrogens can bind to estrogen receptors (ERs)

in osteoblasts to increase their activity (Cos et al.,

2003; Villiers, 2009). Phytoestrogens are a group of

compounds contained in plants which have estrogen-

like structures or can replace the function of

estrogen, both in association with estrogen receptors

(ER-dependent) and not (ER-independent) (Ososki

and Kennelly, 2003; Yang et al., 2012; Cui et al.,

2013).

Although it has great potential as a medicinal

plants, the phytochemical properties of M. crenata

has not been completely confirmed yet. This

research was done to identify the metabolite profile

of ethyl acetate extract of M. crenata using UPLC-

QToF-MS/MS, which can be used as a reference for

further research and utilization of M. crenata.

Maâ

Zarif, B. and Agil, M.

Metabolite Proﬁling of Ethyl Acetate Extract from Marsilea crenata Presl. Using UPLC-QToF-MS/MS.

DOI: 10.5220/0008357300500057

In Proceedings of BROMO Conference (BROMO 2018), pages 50-57

ISBN: 978-989-758-347-6

UPLC-QToF-MS/MS is a powerful technique used

for metabolite profiling which has improved in

performance of chromatographic resolution, speed

and sensitivity analysis, saves time, also reduces

solvent consumption (Patil et al., 2011),

The ethyl acetate extract was selected because in

the preliminary study using TLC visualizer, this

extract showed the best TLC profile (Figure 2).

Whereas metabolite profiling of n-hexane extract has

been done before (Ma’arif et al., 2016).

Figure 2: TLC profile of : a. 96% ethanol extract; b. n-

hexane extract; c. ethyl acetate extract; and d. metanol

extract; from M. crenata leaves at λ 366 nm.

2 MATERIAL AND METHODS

2.1 Material

2.1.1 Plant Material

M. crenata were collected in Benowo, Surabaya,

Indonesia at November 2017, and identified in UPT

Materia Medica, Batu, Indonesia at December 2017

with specimen number 1a-17b-18a-1. The leaves

were prepared to get dry powder of M. crenata.

2.1.2 Chemical

All chemicals were grade of analytical reagent and

used as received. N-hexane, and ethyl acetate as

solvent were purchased from Pharmacy Department,

Faculty of Medical and Health Science, Maulana

Malik Ibrahim State Islamic University.

Dichloromethane, metanol, acetonitrile, and formic

acid as solvent and mobile phase on UPLC-QToF-

MS/MS were purchased from Central Forensic

Laboratory Badan Reserse Kriminal Kepolisian

Negara Republik Indonesia.

2.2 Methods

2.2.1 Extraction

Dry powder of M. crenata leaves were extracted

with n-hexane first. Its residue then re-extracted with

ethyl acetate. In the preliminary study, the 96%

ethanol extract was obtained by directly extracting

dry powder of M. crenata, while methanol extract

was obtained from re-extracting residue of ethyl

acetate extract with methanol. All extraction process

was using ultrasonic assisted extraction method

(Sonica 5300EP S3). This process was repeated,

collecting all the supernatants, which were finally

evaporated in a rotary evaporator (Heidolph) to get

ethyl acetate extract.

2.2.2 Analysis with UPLC-QToF-MS/MS

A simple, rapid, reliable and precise reversed phase

UPLC-QToF-MS/MS method has been developed

and validated according to the regulator guidelines.

The ethyl acetate preparation was done using solid

phase extraction, 100 ppm of ethyl acetate extract in

DCM and methanol then injected 5 µl each into the

an ACQUITY UPLC

H-Class System (Waters,

USA) coupled to an MS detector Xevo G2-S QToF

(Waters, USA). Sample were separated on an

ACQUITY BEH C

(1.7 µm 2.1x50 mm) with

acetonitril + 0.05 % formic acid and water + 0.05 %

formic acid as mobile phase, with flowrate 0.2

ml/min. The results of UPLC-MS analysis was

processed using the Masslynx Version 4.1 software,

to obtain the data of peak and m / z spectra of each

detected peak. The compound content can then be

predicted using the chemspider website.

3 RESULTS AND DISCUSSION

A total of 300 g dry powder of M. crenata leaves

were extracted with n-hexane and then ethyl acetate

to produce 2.82 g extract. The dry powder need to be

extracted first with n-hexane to remove impurities

which may interfere with the identification process,

such as fatty acid compounds. Ethyl acetate extract

of M. crenata were analysed by UPLC-QToF-

MS/MS to better interpret the diversity of available

phytochemicals.

Metabolite Proﬁling of Ethyl Acetate Extract from Marsilea crenata Presl. Using UPLC-QToF-MS/MS

Table 1: Predicted compounds of ethyl acetate extract from M. crenata leaves in DCM solvent

No. RT (min) % Area Measured m/z Molecular Formula Proposed Metabolite Activity

1 1.272 0.3022 150.0280 Unknown Unknown -

2 1.420 0.2059 119.0944 Unknown Unknown -

3 2.118 1.0502 201.1728 C

11-Aminoundecanoic

aci

4 2.598 1.7620 122.0842 C

2-Pyridylethylamine

Histamine

agonist

(Kunkel and

Dixon, 1984)

5 4.427 0.2680 301.1890 C

Megalanthonine

Antifungal

(Reina et al.,

1998

)

6 4.828 0.0245 378.1862 C

S Tixocortol

Corticosteroid,

antiinflammato

ry (Friedman

and Metcalfe,

1991),

decongestant

(Cuenant et al.,

1986)

7 4.930 0.0063 299.1944 C

Unknown -

8 5.193 0.0799 315.1134 Unknown Unknown -

9 5.342 0.1373 149.1203 Unknown Unknown -

10 5.479 0.0713 431.2729 Unknown Unknown -

11 5.662 0.0830 210.1255 Unknown Unknown -

12 5.959 0.0335

519.3245

3,5-

Isothiazoledicarboxa

mide, 4-amino-

-dicyclohexyl-

-[1-[[(3-

methylbutyl) amino]

carbon

l]but

l]-

13 6.211 0.0193 545.3508 Unknown Unknown -

14 6.623 0.0089 462.2615 C

PS Unknown -

15 7.206 0.4010 196.1099 C

1-carboxy-3-

hydroxyadamantane

16 7.972 0.1522 271.1930 C

Pyrrolidine, 1,1',1''-

(hydrazinylidenephos

horanylidyne)tris-

17 9.733 0.0992 256,1936 C

1H-Benzimidazole,

1-(2-

cyclohexylethyl)-5,6-

dimethyl-

Antituberculos

is, antibacterial

(Gobis et al.,

2015)

18 10.967 0.4997 191.1309 Unknown Unknown -

19 11.448 1.0321 241.2772 C

N Hexadecylamine

Antibacterial,

adjuvant for

diphtheria,

tetanus toxoid,

and

antiinfluenza

(Attwood and

Florence,

2012)

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

20 11.630 0.5779 386.1728 C

Benzophenone, 2-(1-

ethylacetonyl)-

3',4,4',5-

tetramethox

21 11.882 0.0066 310.1203 C

Benzylbutylphthalate

Estrogenic

activity (Harris

et al., 1997

)

22 12.111 0.1000 310.1775 C

Portentol

Antioxidant,

anticancer

(Schröckenede

, 2012

)

23 12.842 0.1933 3032925 C

N Pregnan-3-amine -

24 13.345 0.0078 228.1152 C

Bisphenol A

Estrogenic

activity

(Hewitt and

Korach, 2010)

25 13.940 0.1502 567.4201 C

Dibenzo[d,f][1,3,2]di

oxaphosphepin-6-

amine, N,N-dibutyl-

2,4,8,10-tetrakis(1,1-

dimethylethyl)-

26 14.077 0.1513 531.3416 C

Glycine, N-[[(E)-2-

(4-

methoxyphenyl)diaze

nyl]carbonyl]leucyl-,

compd. with N-

cyclohexylcyclohexa

namine

(

1:1

)

27 15.038 3.7928 627.1884 C

1H,5H-Pyrrolo[3,4-

g][1,2,4]triazolo[1,2-

a]cinnoline-

1,3,8,10(2H,7H,9H)-

tetrone, 7-(3-chloro-

4-hydroxy-5-

methoxyphenyl)-

7a,10a,11,11a-

tetrahydro-2-methyl-

9-[(4-

methylphenyl)amino]

-7a-

hen

28 16.970 36.4625 775.2261 C

(1R,13S,16S,17R,28

R)-28-Amino-20-

chloro-17,25-

dihydroxy-5,8,10,24-

tetramethoxy-N-

methyl-15,29,31-

trioxo-22-oxa-

14,30,32triazahexacy

clo

[14.14.2.2

18,21

2,6

23,

7,12

]hexatriaconta-

2(36),3,5

,7,9,11,18,20,23(33),

24,26,34-dodecaene-

13-carboxamide

29 17.633 34.5167 592.2692 C

P Unknown -

30 17.885 10.8884 849.2460 C

OPSCl Unknown -

31 18.330 6.4550 701.2070 Unknown Unknown -

Metabolite Proﬁling of Ethyl Acetate Extract from Marsilea crenata Presl. Using UPLC-QToF-MS/MS

32 21.658 0.0608 156.9950 C

1H-Pyrazolo[3,4-

d]thiazole-

3,5(2H,6H)-dione

Table 2: Predicted compounds of ethyl acetate extract from M. crenata leaves in methanol solvent

No. RT (min) % Area Measured m/z Molecular Formula Proposed Metabolite Activity

1 0.581 0.0068 124.9797 C

NCl

3,3-Dichloro-2-

ropen-1-amine

2 1.500 1.0634 235.1421 C

Nitromethanetrispropa

nol

3 2.266 0.1459 122.0478 C

O Nicotinamide

Activity of

diphosphate

(ADP) -

ribosyltransfera

se (Maurer et

al., 2012), anti-

SIRT2 (Cui et

al., 2014

)

4 4.016 0.0642 124.9789 Unknown Unknown

5 5.045 0.1590 149.1201 C

(S)-(+)-

Methamphetamine

Increase activit

y of

neurotransmite

r norepinefrin

and dopamine,

reduce appetite

(Ward et al.,

2016).

6 5.228 0.1070 431.2733 C

Unknown

7 5.445 0.0977 466.2989 C

(1E)-1-(2,2'',4,4'',6,6''-

Hexamethyl1,1':3',1''-

terphenyl-2'-yl)-3-

mesit

l-1-triazene

8 5.662 0.0169 519.3256 H

OCl Unknown

9 7.206 4.6301 196.1102 C

1-carboxy-3-

hydroxyadamantane

10 8.006 0.2579 125.1882 C

12-Aminododecanoic

acid

Hamper

expression of

(Albertshofer

et al.,2005

)

11 8.886 0.0908 119.0941 Unknown Unknown

12 10.533 1.4306 180.1148 C

2-tert-butyl-4-

methoxyphenol

Antioxidant

(Lee et al.,

2006

)

13 11.013 0.6199 224.1886 C

Ethyl (4S)-5-

cyclohexyl-2,2-

difluoro-4-{[(2S)-2-

{[N-(4-

morpholinylsulfonyl)-

L-

phenylalanyl]amino}-

4-pentenoyl]amino}-3-

oxopentanoate

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

14 11.379 0.2271 340.1314 C

Morachalcone A

Tyrosinase

Inhibitors

(Nguyen et al.,

2012),

inhibition of

nitric oxide

(Joo et al.,

2014),

pancreatic

lipase

inhibitory

(Jeong et

al.,2015)

15 11.562 3.0017 310.1200 C

Cl Lintopride

Treatment of

gastrointestinal

reflux, nausea

and vomiting

(Delvaux et al.,

1995)

16 11.996 0.1281 332.1961 C

8-(4-Ethyl-1-

piperazinyl)-3-methyl-

7-(2-methyl-2-propen-

1-yl)-3,7-dihydro-1H-

urine-2,6-dione

17 12.431 4.2855 503.3096 C

Pederin

Antioxidant,

anticancer

(Ghoneim,

2013)

18 12.614 0.6065 693.3941 C

Methyl {[(9Z)-17-

{[(2R,3R,4S,5S,6R)-

4,5-dihydroxy-6-

(hydroxymethyl)-3-

{[(2S,3R,4S,5S,6R)-

3,4,5-trihydroxy-6-

(hydroxymethyl)tetrah

ydro-2H-pyran-2-

yl]oxy}tetrahydro-2H-

pyran-2-yl]oxy}-9-

octadecenoyl]ami

}

acetate

19 12.911 0.2985 363.3127 C

Cl Unknown

20 13.208 0.7061 276.2087 C

Phenyl laurate

Antimicroba,

antihipertensio

n (Edwin and

Edmun

,1940)

21 13.460 0.3641 495.3566 C

Cl Unknown

22 13.791 2.6389 531.3408 C

S Unknown

23 14.306 0.8403 698.5889 C

Cl Unknown

24 15.541 21.6948 698.5885 C

Unknown

25 16.718 11.5201 698.5896 C

Unknown

26 17.153 11.2271 592.2689 C

Pheophorbide A

Antiinflamatio

n, antioxidant

(Vencl et al.,

2009), anti-

HIV (Zhang et

al., 2003)

Metabolite Proﬁling of Ethyl Acetate Extract from Marsilea crenata Presl. Using UPLC-QToF-MS/MS

27 17.370 0.6928 592.2694 C

1,1'-(1,4-

Butanediyl)bis{2,6-

dimethyl-4-[(3-

methyl-1,3-

benzothiazol-2(3H)-

ylidene)methyl]pyridin

ium}

28 18.330 33.0776 698.5885 C

Unknown -

Table 1 and Table 2 summarise all the

compounds characterized in ethyl acetate extract of

M. crenata, including retention times, % area,

measured m/z, molecular formula, putative

compounds, and its activity based on references.

In total there were 32 peak of compounds

identified in the DCM solvent, and 28 peak in the

methanol solvent. The use of two types of solvent

aimed to elute the ethyl acetate extract optimally.

From all the peaks, only 36 peaks can be identified,

while the rest are unknown compounds.

Unknown compounds may be identified as

impure compounds which are still detected by the

instrument, or they may be a new compounds, which

is undetectable in chemspider database, especially

unknown compounds with high concentrations.

Based on the results of this study, it is not yet

known which compounds are likely to have activity

as phytoestrogens, but when viewed from the

activity data in Table 1 and Table 2, it is known

some compounds have activity as antioxidants.

Where antioxidants is one form of phytoestrogens

activity, the ER-independent pathway.

Phytoestrogens can work through two pathways,

both ER-dependent and ER-independent pathway.

Although most biological actions of phytoestrogens

are mediated through ERs in cells (ER-dependent),

its can exert antioxidant effects and suppress

oxidative stress through an ER-independent

pathway. Phytoestrogens effectively prevent pro-

oxidant stress by limiting ROS release from

damaged mitochondria, and provides antioxidant

activity in cells (Cui et al., 2013).

4 CONCLUSIONS

From UPLC-QToF-MS/MS analysis, it is concluded

that ethyl acetate extract of M. crenata leaves

contain various types of compounds, either detected

compounds (36 compounds), or unknown

compounds. The unknown compounds still need to

be investigated further, especially those with high

concentrations.

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Metabolite Proﬁling of Ethyl Acetate Extract from Marsilea crenata Presl. Using UPLC-QToF-MS/MS