Metabolite Profiling of Ethyl Acetate Extract from Marsilea

crenata Presl. Using 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.

Ma’arif, B. and Agil, M.

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

DOI: 10.5220/0009841900002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

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.

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

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

No.

(min)

% Area

Measured

m/z

Molecular

Formula

Proposed Metabolite

Activity

1.272

0.3022

150.0280

Unknown

1.420

0.2059

119.0944

Unknown

2.118

1.0502

201.1728

11-Aminoundecanoic acid

2.598

1.7620

122.0842

2-Pyridylethylamine

Histamine agonist

(Kunkel and Dixon,

1984)

4.427

0.2680

301.1890

Megalanthonine

Antifungal (Reina

et al., 1998)

4.828

0.0245

378.1862

Tixocortol

Corticosteroid,

antiinflammatory

(Friedman and

Metcalfe, 1991),

decongestant

(Cuenant et al.,

1986)

4.930

0.0063

299.1944

Unknown

5.193

0.0799

315.1134

Unknown

5.342

0.1373

149.1203

Unknown

5.479

0.0713

431.2729

Unknown

5.662

0.0830

210.1255

Unknown

5.959

0.0335

519.3245

3,5-Isothiazoledicarboxamide, 4-

amino-N

-dicyclohexyl-N

[1-[[(3-methylbutyl) amino]

carbonyl]butyl]-

6.211

0.0193

545.3508

Unknown

6.623

0.0089

462.2615

Unknown

7.206

0.4010

196.1099

1-carboxy-3-hydroxyadamantane

7.972

0.1522

271.1930

Pyrrolidine, 1,1',1''-

(hydrazinylidenephosphoranylidy

ne)tris-

9.733

0.0992

256,1936

1H-Benzimidazole, 1-(2-

cyclohexylethyl)-5,6-dimethyl-

Antituberculosis,

antibacterial (Gobis

et al., 2015)

10.967

0.4997

191.1309

Unknown

11.448

1.0321

241.2772

Hexadecylamine

Antibacterial,

adjuvant for

diphtheria, tetanus

toxoid, and

antiinfluenza

(Attwood and

Florence, 2012)

11.630

0.5779

386.1728

Benzophenone, 2-(1-

ethylacetonyl)-3',4,4',5-

tetramethoxy-

11.882

0.0066

310.1203

Benzylbutylphthalate

Estrogenic activity

(Harris et al., 1997)

12.111

0.1000

310.1775

Portentol

Antioxidant,

anticancer

(Schröckeneder,

2012)

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

12.842

0.1933

3032925

Pregnan-3-amine

13.345

0.0078

228.1152

Bisphenol A

Estrogenic activity

(Hewitt and

Korach, 2010)

13.940

0.1502

567.4201

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

pin-6-amine, N,N-dibutyl-

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

dimethylethyl)-

14.077

0.1513

531.3416

Glycine, N-[[(E)-2-(4-

methoxyphenyl)diazenyl]carbony

l]leucyl-, compd. with N-

cyclohexylcyclohexanamine

(1:1)

15.038

3.7928

627.1884

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-phenyl-

16.970

36.4625

775.2261

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

Amino-20-chloro-17,25-

dihydroxy-5,8,10,24-

tetramethoxy-N-methyl-

15,29,31-trioxo-22-oxa-

14,30,32triazahexacyclo

[14.14.2.2

18,21

2,6

23,27

7,12

]hex

atriaconta-2(36),3,5

,7,9,11,18,20,23(33),24,26,34-

dodecaene-13-carboxamide

17.633

34.5167

592.2692

Unknown

17.885

10.8884

849.2460

SCl

Unknown

18.330

6.4550

701.2070

Unknown

21.658

0.0608

156.9950

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.

(min)

% Area

Measure

d m/z

Molecular

Formula

Proposed Metabolite

Activity

0.581

0.0068

124.9797

NCl

3,3-Dichloro-2-propen-1-

amine

1.500

1.0634

235.1421

Nitromethanetrispropanol

2.266

0.1459

122.0478

Nicotinamide

Activity of diphosphate

(ADP) -

ribosyltransferase

(Maurer et al., 2012),

anti-SIRT2 (Cui et al.,

2014).

4.016

0.0642

124.9789

Unknown

5.045

0.1590

149.1201

(S)-(+)-Methamphetamine

Increase activity of

neurotransmiter

norepinefrin and

dopamine, reduce

appetite (Ward et al.,

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

2016).

5.228

0.1070

431.2733

Unknown

5.445

0.0977

466.2989

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

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

terphenyl-2'-yl)-3-mesityl-1-

triazene

5.662

0.0169

519.3256

OCl

Unknown

7.206

4.6301

196.1102

1-carboxy-3-

hydroxyadamantane

8.006

0.2579

125.1882

12-Aminododecanoic acid

Hamper expression of

(Albertshofer et

al.,2005)

8.886

0.0908

119.0941

Unknown

10.53

1.4306

180.1148

2-tert-butyl-4-methoxyphenol

Antioxidant (Lee et al.,

2006)

11.01

0.6199

224.1886

Ethyl (4S)-5-cyclohexyl-2,2-

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

morpholinylsulfonyl)-L-

phenylalanyl]amino}-4-

pentenoyl]amino}-3-

oxopentanoate

11.37

0.2271

340.1314

Morachalcone A

Tyrosinase Inhibitors

(Nguyen et al., 2012),

inhibition of nitric oxide

(Joo et al., 2014),

pancreatic lipase

inhibitory (Jeong et

al.,2015)

11.56

3.0017

310.1200

Lintopride

Treatment of

gastrointestinal reflux,

nausea and vomiting

(Delvaux et al., 1995)

11.99

0.1281

332.1961

8-(4-Ethyl-1-piperazinyl)-3-

methyl-7-(2-methyl-2-propen-

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

2,6-dione

12.43

4.2855

503.3096

Pederin

Antioxidant, anticancer

(Ghoneim, 2013)

12.61

0.6065

693.3941

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)tetrahydro-

2H-pyran-2-yl]oxy}tetrahydro-

2H-pyran-2-yl]oxy}-9-

octadecenoyl]ami no}acetate

12.91

0.2985

363.3127

Unknown

13.20

0.7061

276.2087

Phenyl laurate

Antimicroba,

antihipertension (Edwin

and Edmund,1940)

13.46

0.3641

495.3566

Unknown

13.79

2.6389

531.3408

Unknown

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

14.30

0.8403

698.5889

Unknown

15.54

21.6948

698.5885

Unknown

16.71

11.5201

698.5896

Unknown

17.15

11.2271

592.2689

Pheophorbide A

Antiinflamation,

antioxidant (Vencl et

al., 2009), anti-HIV

(Zhang et al., 2003)

17.37

0.6928

592.2694

1,1'-(1,4-Butanediyl)bis{2,6-

dimethyl-4-[(3-methyl-1,3-

benzothiazol-2(3H)-

ylidene)methyl]pyridinium}

18.33

33.0776

698.5885

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.

REFERENCES

Adityara, R. A. 2017. Uji aktivitas antiosteoporosis fraksi

etil asetat daun Marsilea crenata Presl. dalam

meningkatkan kepadatan tulang trabekula femur

mencit betina. Skripsi : Universitas Airlangga.

Aemi, N. Y. 2012. Uji aktivitas antiosteoporosis fraksi n-

heksana daun Marsilea crenata Presl. dalam

meningkatkan kepadatan tulang trabekular vertebra

mencit betina. Skripsi : Universitas Airlangga.

Albertshofer, K., Siwkowski, A. M., Wancewicz, E. V,

Esau, C. C., Watanabe, T., Nishihara, K. C., Maier, M.

A. 2005. Structure - Activity Relationship Study on a

Simple Cationic Peptide Motif for Cellular Delivery of

Antisense Peptide Nucleic Acid. J Med Chem. Vol.

48(21): 6741–6749.

Attwood D., and Florence, A.T. 1983. Surfactant Systems:

Their chemistry, pharmacy and biology. Chapman and

Hall Ltd. London:.

Cos, P., Bruyne, T. D., Apers, S., Berghe, D. V., Pieters,

L., Vlietinck, A. J. 2003. Planta Med. Vol. 69: 589-

599.

Cuenant, G., Stipon, J. P., Plante-Longchamp, G.,

Baudoin, C., Guerrier, Y. 1986. Efficacy of Endonasal

Neomycin-Tixocortol Pivalate Irrigation in the

Treatment of Chronic Allergic and Bacterial Sinusitis.

J Otorhinolaryngol Relat Spec. 48(4):226-32.

Cui, H., Kamal, Z., Ai, T., Xu, Y., More, S. S., Wilson, D.

J., & Chen, L. 2014. Discovery of Potent and Selective

Sirtuin 2 (SIRT2) Inhibitors Using a Fragment-Based

Approach, 2. J Med Chem. Vol. 57(20):8340-8357.

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

Cui, J., Shen,Y., Li R. 2013. A Review: Estrogen

synthesis and signaling pathways during aging: from

periphery to brain. Trends in Molecular Medicine.

Vol. 19, No. 3.

Delvaux, M., Maisin, J. M., Arany, Y., Atlan, P., Prieto-

Cabanis, M. J., Canal, M., Frexinos, J. 1995. The

effects of lintopride, a 5HT-4 antagonist, on

oesophageal motility. Aliment Pharmacol Ther. Vol.

9(5):563-9.

Edwin, H. B., and Edmund, J. D. 1940. The Fries

rearrangement of phenyl laurate and phenyl stearate.

Journal of chemical society.

Friedman, B.S., and Metcalfe, D.D. 1991. Effects of

tixocortol pivalate on gastrointestinal disease in

systemic mastocytosis: a preliminary study. Clinical

and Experimental Allergy. Vol. 21:183-188.

Ghoneim, K.S. 2013. Human dermatosis caused by

vesicating beetle products (insecta), cantharidin and

pederin: an overview: world journal of medicine and

medical science. 1(1).

Gobis K., Foks H., Serocki M. 2014. Synthesis evaluation

of in vitro antimycobacterial activity of novel 1H-

benzo[d]imidazole derivatives and analogues.

European Journal of Medicinal Chemistry.

Harris A. C., Henttu P., Parker G. M., and Sumpter J. P.

1997. The Estrogenic Activity of Phtalate Esters In

Vitro. Environmental Health Perspectives. Vol. 105,

No. 8.

Hewitt C. S., and Korach S. K. 2011. Estrogenic Activity

of Bisphenol A and 2,2-bis(p-Hydroxyphenyl)-1,1,1-

trichloroethane(HPTE) Demonstrated in Mouse

Uterine Gene Profiles. Environmental Health

Perspectives. Vol 119, No. 1.

Jeong, J. Y., Jo, Y. H., Kim, S. B., Liu, Q., Lee, J. W., Jin,

E., Lee, M. K. 2015. Pancreatic lipase inhibitory

constituents from Morus alba leaves and optimization

for extraction conditions. Bioorganic & Medicinal

Chemistry Letters. Bioorg Med Chem Lett. Vol. 1;25

(11):2269-74

Joo, T., Sowndhararajan, K., Hong, S., Lee, J., Park, S. Y.,

Kim, S., Jhoo, J. W. 2014. Inhibition of nitric oxide

production in LPS-stimulated RAW 264.7 cells by

stem bark of Ulmus pumila L. Saudi J Biol Sci. Vol.

21(5):427-35.

Kunkel, H. G., and Dixon, F. J. 1984. Advances in

Immunology. Academic Press Inc. London. Vol. 35

Laswati, H. 2011. Green Clover Potentiates Delaying the

Increment of Imbalance Bone Remodeling Process in

Postmenopausal Women. Folia Medica Indonesiana.

Vol. 47. No. 2. Page 112-117.

Ma’arif B., Agil, M., Laswati H. 2016. Phytochemical

assessment on n-hexane extract and fractions of

Marsilea crenata Presl. leaves through GC-MS. Trad.

Med. J, 2016; 21(2):77-85.

Ma'arif, B., Agil, M., Laswati, H. 2018. Alkaline

Phosphatase Activity of Marsilea crenata Presl.

Extract and Fractions as Marker of MC3T3-E1

Osteoblast Cell Differentiation. Journal of Applied

Pharmaceutical Science Vol. 8(03), pp. 55-59.

Maurer, B., Sirt, D., Rumpf, T., Scharfe, M., Stolfa, D. A.,

Schmitt, M. L., Jung, M. 2012. Inhibitors of the NAD

+‑Dependent Protein Desuccinylase and

Demalonylase Sirt5. ACS Med Chem Lett. Vol. 3(12):

1050–1053.

Nguyen, N. T., Nguyen, M. H., Nguyen, H. X., Bui, N. K.,

Nguyen, M. T. 2012. Tyrosinase inhibitors from the

wood of Artocarpus heterophyllus. J Nat Prod. Vol.

75(11):1951-5.

Nurjanah, A. A., and Abdullah, A. 2012. Aktivitas

Antioksidan dan Komponen Bioaktif Semanggi Air

(Marsilea crenata). Jurnal Inovasi dan Kewirausahaan.

Vol. 1. No. 3. Page 152-158.

Ososki, A. L., Kennelly, E. J. 2003. Phytoestrogens: a

Review of the Present State of Research. Phytotherapy

Research. Vol. 17. Page 845-869.

Patil, J. S., Suresh, S., Sureshbabu, A. R., Rajesh, M. S.

2011. Development and Validation of Liquid

Chromatography-Mass Spectrometry Method for the

Estimation of Rifampicin in Plasma. Indian J Pharm

Sci. 2011 Sep-Oct; 73(5): 558–563.

Reina, M., Gonzalez-Coloma, A., Gutierrez, C., Cabrera,

R., Henriquez, J., Villarroel, L. 1998. Pyrrolizidine

Alkaloids from Heliotropium megalanthum. Journal of

Natural Products. Vol. 61, No. 11.

Schröckeneder, A. 2012. Towards the Total Synthesis of

Portentol A Formal Synthesis of Dimethylglutamine

The Crystal Structure of the Dess-Martin Periodinane

[Disertasi]. München: Ludwig Maximilians

Universität München.

Vencl, F. V, Gómez, N. E., Ploss, K., & Boland, W. 2009.

The Chlorophyll Catabolite , Pheophorbide a , Confers

Predation Resistance in a Larval Tortoise Beetle

Shield Defense. J Chem Ecol. Vol. 35(3), page 281–

288.

Villiers, T. J. 2009. Bone health and osteoporosis in

postmenopausal women. Elsevier : Best Practice &

Research Clinical Obstetrics and Gynaecology. Vol.

23. Page 73-85.

Ward, L. F., Enders, J. R., Bell, D. S., Cramer, H. M.,

Wallace, F. N., Mcintire, G. L., Supelco, S. 2016.

Improved Chiral Separation of Methamphetamine

Enantiomers Using CSP-LC – MS-MS, (2), 1–9.

Widiasari, F. A. 2017. Uji aktivitas antiosteoporosis fraksi

etil asetat daun Marsilea crenata Presl. dalam

meningkatkan kepadatan tulang trabekula vertebra

mencit betina. Skripsi : Universitas Airlangga.

Yang, T-S., Wang, S-Y., Yang, Y-C., Su, C-H., Lee, F-K.,

Chen, S-C., Tseng, C-Y., Jou, H-J., Huang, J-P.,

Huang, K-E. 2012. Effects of standardized

phytoestrogen on Taiwanese menopausal women.

Elsevier : Taiwanese Journal of Obstetrics &

Gynecology. Vol. 51. Page 229-235.

Zhang, H., Tan, G. T., Hoang, V. D., Hung, N. Van,

Cuong, N. M., Soejarto, D. D., Fong, H. H. S. 2003.

Natural Anti-HIV Agents. Part IV. Anti-HIV

Constituents from Vatica cinerea 1, 2(d), 263–268.

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