Khảo sát thành phần hóa học và hoạt tính sinh học của bần trắng (sonneratia alba) và bần ổi (sonneratia ovata), họ bần (sonneratiaceae), mọc ở rừng ngập mặn cần giờ tp hồ chí minh

UNIVERSITY OF SCIENCE  NGUYEN THI HOAI THU STUDY ON CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF SONNERATIA ALBA AND SONNERATIA OVATA SONNERATIACEAE GROWING IN CAN GIO MANGROVE FOR

UNIVERSITY OF SCIENCE



NGUYEN THI HOAI THU

STUDY ON CHEMICAL CONSTITUENTS AND

BIOACTIVITIES OF SONNERATIA ALBA AND

SONNERATIA OVATA (SONNERATIACEAE)

GROWING IN CAN GIO MANGROVE FOREST

– HO CHI MINH CITY

DOCTORAL THESIS IN CHEMISTRY

Ho Chi Minh City, 2015

UNIVERSITY OF SCIENCE



NGUYEN THI HOAI THU

STUDY ON CHEMICAL CONSTITUENTS AND

BIOACTIVITIES OF SONNERATIA ALBA AND SONNERATIA OVATA (SONNERATIACEAE)

GROWING IN CAN GIO MANGROVE FOREST

– HO CHI MINH CITY

Speciality: Organic chemistry

Examination board:

Assoc Prof Dr Tran Cong Luan (2nd Reviewer)

Assoc Prof Dr Pham Dinh Hung (3rd Reviewer)

Assoc Prof Dr Phan Minh Giang (1st Independent Reviewer)

SUPERVISORS: PROF NGUYEN KIM PHI PHUNG

PROF POUL ERIK HANSEN

Ho Chi Minh City, 2015

ORIGINALITY STATEMENT

The work presented in this thesis was completed in the period of September 2010 to September 2014 under the co-supervision of Professor Nguyen Kim Phi Phung, University of Science, Vietnam National University, Ho Chi Minh City, and Professor Poul Erik Hansen, Roskilde University, Denmark

In compliance with the university regulations, I declare that:

1 Except where due acknowledgement has been made, the work is that of the author alone

2 The work has not been submitted previously, in whole or in part, to qualify for any other academic award

3 The content of the thesis is the result of work which has been carried out since the official commencement date of the approved PhD study program

4 Ethic procedures and guidelines have been followed

Ho Chi Minh City, June 2015

PhD Candidate

NGUYEN THI HOAI THU

First and foremost, I offer my sincerest gratitude to my supervisor Prof Nguyen Kim Phi Phung, who has been supporting me throughout the course of my research with her patience, kindness, invaluable advice, guidance, encouragement and comments

I would also like to greatly acknowledge Prof Poul Erik Hansen, my Co-supervisor, for his kind scientific guidance, support, and suggestions to my study

In addition, I am indebted to Dr Vo Van Chi and the deceased Pharmacist Phan Duc Binh, who provided me with the scientific name of the

four species in the Sonneratia and Avicennia genera

I am thankful to Dr Le Duc Tuan, from the Management Board for HCMC Environmental Protection Forests, for his help, kindness and advice during my plant sample collection

I would like to acknowledge the encouragement and insightful comments from the rest of the Examination Board

Special thanks go to the Foreign Language Center of the University of Science for their editing of the English version of my work

I would like to acknowledge the Department of Science and Technology –

Ho Chi Minh City for supporting me

Additionally, I would like to thank all my friends at the Department of Organic Chemistry Laboratory, University of Science, for providing me with their constant encouragement

Finally, an honorable mention goes to my family and friends, without whose understanding and support this thesis would not have been completed successfully

ACKNOWLEDGEMENTS i

APPENDICES

HR–ESI–MS : High Resolution–Electrospray Ionization–Mass

NMR : Nuclear Magnetic Resonance

LIST OF TABLES

Page Table 3.1

lines of some isolated compounds Acetylcholinesterase inhibition acitivities of some extracts and isolated compounds

LIST OF FIGURES AND SCHEMES

and 80

of 98

Drugs of natural origin, either products derived semi–synthetically from natural products or synthetic products based on natural product models, play an invaluable role in the drug discovery process Research on natural products is making important contributions to chemistry as well as biology and pharmacy.

Mangrove plants are plants that grow chiefly on tidal coasts of tropic and subtropic regions of the world The unique ecology and traditional medicinal uses of mangrove plants have attracted the attention of researchers over the years, and as a result, reports on biological activities as well as chemistry of mangrove plants have increased significantly in recent years

Nevertheless, a number of mangrove species such as Sonneratia alba J.E Smith, Sonneratia ovata Backer, (Sonneraticeae) have not yet been much studied,

or have not even been studied in Vietnam That is the reason why this work wants to focus on these species

The thesis, entitled “Study on chemical constituents and bioactivities of

Sonneratia alba and Sonneratia ovata (Sonneratiaceae) growing in Can Gio

mangrove forest – Ho Chi Minh City” aimed to achieve the following objectives Objectives

 To understand the chemical constituents of the two Sonneratia alba and Sonneratia ovata species growing in Ho Chi Minh City’s Can Gio mangrove

forests, in order to contribute to the knowledge of the phyto-chemical properties

of the Sonneratia genus

 To perform cytotoxicity assay against three cancer cell lines (Hela, NCI–H460 and MCF–7) and acetylcholinesterase inhibitions of extracts and isolated compounds to look for the bioactive ones

CHAPTER 1 LITERATURE REVIEW

1.1 SONNERATIA GENUS – BOTANICAL DESCRIPTION

The Sonneratia genus belongs to Sonneratiaceae family, which is widely

distributed in mangrove forests along the coasts of the Indian Ocean and the Pacific Ocean, where can be found strong waves, salt water or brackish water and coral

reefs In Vietnam, there are 5 species from this genus [1], namely Sonneratia alba J.E Smith, Sonneratia ovata Back., Sonneratia caseolaris (L.) Engl., Sonneratia apeltala Buch.Ham and Sonneratia griffithii Kurz

1.1.1 SONNERATIA ALBA J E SMITH

Vietnamese name: Bần trắng

Tree up to 15 m tall; pneumatophores erect, tapering, stem up to 1 m big, bark like guava’s Leaves simple, opposite, obovate or oval, thick, brittle, apex broadly rounded or even slightly emarginated; 10–12 pairs of veins of the leaf, convex on

above side Big flower, petals absent, stamens numerous, white; 6–8 calyx lobes

Berries 4–5 cm broad on stared–shape calyx, bitterness, small and numerous seeds

Sonneratia is one of representative pioneer species on newly formed, sandy mud in estuarine areas It can tolerate higher salinities than other Sonnerata species

It is found from East Africa throughout the Indian subcontinent, Southeast Asia,

1.1.2 SONNERATIA OVATA BACKER

Vietnamese name: Bần ổi

Big tree 4–5 m tall, sometimes up to 10 m, tinged red young bark, round branch Leaves simple, opposite, broadly ovate to suborbicular, thick, brittle; 10–12 pairs of veins of the leaf, no convex on above side Inflorescence umbel terminal, petals absent; 6–8 calyx lobes finely warty on outer side; stamens numerous, white Berries with erect calyx lobes; numerous seeds Flowers March–April, fruits June–July [1]

Grows on inland river banks with brackish water flooded by average to high tide [2]

1.2 CHEMICAL STUDIES OF SOME SPECIES OF SONNERATIA GENUS 1.2.1 SONNERATIA ALBA J E SMITH

In 2013, Sharmin Asad et al [7] reported the presence of stigmasterol (1),

β-sitosterol (2), β-sitosterol-4-ene-3-one (4), oleanolic acid (12) and lupeol (18) in

Sonneratia alba leaves

In 2004, Kanchuree Chaiyadej et al [17] isolated a mixture of stigmasterol (1)

and β-sitosterol (2), oleanolic acid (12), lupeol (18), betulinic acid (21) and

2,6-dimethoxy-p-benzoquinone (45) from the stems of S alba

In 2003, Hirosuke Oku et al [68] reported that lipid compositions of S alba

leaves collected on Iriomote island, Okinawa, mainly consisted of 11 lipid classes including polar lipids, triterpenoic alcohols, sterols, triacyl glycerols, wax esters and sterol esters Of these lipid classes, stearyl oleate (wax ester) was the main component comprising 19.3% of total lipids, followed by sterol ester with 16.8%

In 2002, Hiroshi Azuma et al [8] investigated the chemical composition of the floral scents of S alba native to Iriomote island, Japan The floral scents of S alba consisted mainly of trans-β-ocimene (47), 2,4-dithiapentane (48), heptan-2-one (49)

and methyl 2-methylbutanoate (50)

In 2002, W.M Bandaranayake [10] reviewed that barks, leaves, stems and

roots of S alba contained cyclitol, polyol, sucrose, glucose, fructose, condensed and

hydrolysable tannins, minerals, nucleotides Fruits of this species were used as traditional poultice in swellings and sprains

In 1968, J.B Lowry [60] reported that the stems of S alba contained ellagic

acid and its derivative (31–34)

1.2.2 SONNERATIA OVATA BACKER

In 2009, Shi-Biao Wu et al [115] isolated seven compounds from the fruits of Sonneratia ovata: oleanolic acid (12), maslinic acid (14), (–)-(R)-nyasol (26), (–)-

(R)-4-O-methylnyasol (27), 3-hydroxy-6H-benzo[c]chromen-6-one (28), dihydroxy-6H-benzo[c]chromen-6-one (29) and hovetrichoside C (44)

3,8-In 2008, Zheng Zhe and Pei Yue–hu [127] isolated seven known compounds

from S ovata including β-sitosterol (2), stigmast-4-ene-3-one (4), ursolic acid (15),

ocotillol (17), lupeol (18), betulin (20) and 1β-hydroxylupeol (22)

1.2.3 SONNERATIA CASEOLARIS (L.) ENGL

In 2010, Ashok Kumar Tiwari et al [97] isolated oleanolic acid (12), daucosterol (6) and luteolin (40) from the fruits of Sonneratia caseolaris

β-In 2009, Shi–Biao Wu et al [115] reported the isolation of nine compounds from the fruits of S caseolaris including oleanolic acid (12), maslinic acid (14), (–)- (R)-nyasol (26), (–)-(R)-4-O-methylnyasol (27), 3-hydroxy-6H-benzo[c]chromen-6- one (28), 3,8-dihydroxy-6H-benzo[c]chromen-6-one (29), luteolin (40), luteolin 7-

In 2008, Tian Minqing et al [95] isolated 24 compounds from the stems and twigs of the medicinal mangrove plant S caseolaris with eight steroids: stigmasterol (1), β-sitosterol (2), 3-O-palmitoyl-β-sitosterol (5), daucosterol (6), 6'-

-glucopyranoside) (8), cholesterol (9) and cholest-5-en-3β,7α-diol (10), nine

triterpenoids: oleanolic acid (12), 3β-O-acetyloleanolic acid (13), ursolic acid (15),

3β,13β-dihydroxyurs-11-ene-28-oic-28(13)-lactone (16), lupeol (18), betulin (20), lup-20(29)-ene-3β,24-diol (23), 3β-hydroxylup-20(29)-ene-24-oic acid (24) and 3β-

O-(E)-coumaroylalphitolic acid (25), three flavonoids: luteolin (40), quercetin

benzenecarboxylic acid derivatives: methyl gallate (30), 3,3'-di-O-methylellagic acid (33), 3,3',4-tri-O-methylellagic acid (34) and di(2-ethylhexyl)benzene-1,2-

dicarboxylate (51)

In 2006, Samir Kumar Sadhu et al [80] reported the isolation and

(41) from the leaves of S caseolaris

In 1984, R W Hogg and F T Gillan [34] reported that in fresh leaves of S caseolaris the content of fatty acids, sterols and leaf-wax hydrocarbons were 2000,

150 and 138 µg per 1 g of fresh weight, respectively Among them, although lauric acid was present in the least quantity but it was a potential antimicrobial agent, suitable for external application It was inexpensive and useful to control the infection in hospitals

In 1968, J.B Lowry [60] reported that the stems of S caseolaris contained

derivative of ellagic acid (31)

1.2.4 SONNERATIA APETALA BUCH HAM

In 2005, Ji Qingfei et al [40] isolated 7 compounds from Sonneratia apetala,

includings stigmast-5-en-3β,7α-diol (3), β-amyrin palmitat (11), lupeol (18), lupeone (19), betulinic acid (21), physcoin (35) and (±)-syringaresinol (39)

1.2.5 SONNERATIA HAINANENSIS W.C.KO, E.Y.CHEN & W.Y.CHEN

alkyl aromatics, namely integracins A (36), integracins B (37) and

15’-dehydroxyintegracin B (38) from the stems and leaves of Sonneratia hainanensis

In 2010, Hai–Li Liu et al [58] isolated two diphenacylpiperidine alkaloid,

sonneratine A (53) and (±)1-(2-piperidyl)-4-(p-methoxyphenyl)-butan-2-one (54)

from the stems and leaves of the Hainan mangrove Sonneratia hainanensis

1.2.6 SONNERATIA PARACASEOLARIS W.C.KO, E.Y.CHEN & W.Y.CHEN

In 2011, Xue–Lian Chen et al [18] reported a R-alkylbutenolide dimer, paracaseolide A (52) from the mangrove plant Sonneratia paracaseolaris

COMPOUNDS ISOLATED FROM SOME SCPECIES OF

H H OH COOH CH 3 3β-Hydroxylup-20(29)-ene-24-oic acid (24)

H OH Coumaroyloxy H COOH 3β-O-(E)-Coumaroylalphitolic acid (25)

Phenolics

R

H Luteolin (40)

Glc Luteolin 7-O- -D -glucopyranoside (41) (+)-2,3-Dihydrokaempf erol (43)

Quercetin 3-O- -L -arabinoside (42)

O HO

OH O

OH OH

2 3 5 7

1' 3' 4'

O HO

OH O

OH

2 3 5 7

1' 3' 4'

OH

4 5 7

3 1' 4'

O OHHO

HO O

Other compounds

SONNERATIA GENUS

1.3.1 SONNERATIA ALBA J E SMITH

The fruits of Sonneratia alba are used as poultice on swellings and sprains

[10]

S alba ripe fruits are traditionally used to expel intestinal parasites while half–

ripe fruits are usually applied for cough treatment [79]

The antimicrobial property of the S alba extracts by different solvents, n–

hexane, ethyl acetate and methanol were tested At a dose of 1.5 mg/disc, the methanol and ethyl acetate extracts showed the most effective inhibition against

tested strains The Gram negative bacteria Escherichia coli with the inhibition zone

of 17.5 mm appeared to be the most sensitive strain, followed by the Gram positive

bacteria Staphylococcus aureus (12.5 mm) and Bacillus cereus (12.5 mm) [79]

A polysaccharide obtained from the S alba leaf extract possessed a very

effective anti–diabetic property which reduced the blood sugar level by 19.2% during the first 6 hours and further to 66.9% after 12 hours [67]

A study found that different extracts of S alba bark showed significant

antioxidant, cytotoxic and antimicrobial properties The chloroform partitionate and

the methanol crude extract of S alba bark showed significant antioxidant property,

value of 12 and 14 µg/mL, respectively, in comparison with the positive control

standard (vincristine sulphate), n–hexane, carbon tetrachloride and chloroform

soluble partitionate of the methanolic extract, respectively [6] At the concentration

of 400 µg/disc, the carbon tetrachloride extract showed moderate inhibitory activity

against various Gram positive bacteria such as Bacillus cereus (10 mm), Bacillus subtilis (11 mm), Sarcina lutea (12 mm) and Gram negative bacteria such as Pseudomonas aeruginosa (10 mm) and Shigella dysenteriae (12 mm) and showed

mild antifungal activity [6]

Sonneratia extracts were used as ingredients of skin cosmetic cream Samples were prepared from dried, pulverized plants of the Sonneratiaceae (such as S alba.,

S caseolaris, S ovata, S griffithii Kurz., S gulngai N.C.Duke, S lanceolata Bl.)

extracted with 1–100 times of water or ethanol, etc., or extracted with carbon

dioxide supercritical fluid to obtain the corresponding extract This extract was then

mixed with oily component, surfactant, additive, etc, to form the cosmetic cream for

the assay The obtained product possessed the ability to maintain skin moisture and

smoothness [5]

Patil Priya D [73] reported that leaves and stems of S alba contained

γ–linolenic acid with the yield of 36.2 and 11.0 %, respectively Knowing that

evening prime rose and borage oil contained this fatty acid with the yield of around

25%, therefore, they can be used as an alternative source of γ–linolenic acid which

belongs to the family of 3–omega and 6–omega needed for our body's proper

functioning

Oleanolic acid (12) and betulinic acid (21), isolated from the stems of S alba,

exhibited antimycobacterial activity against Mycobacterium tuberculosis H37Ra

with MIC values of 25 and 50 mg/mL, respectively In addition,

1.3.2 SONNERATIA OVATA BACKER

According to traditional uses, the juice of Sonneratia ovata is used to treat

hemorrhages [10]

From the fruits of S ovata, three compounds (–)-(R)-nyasol (26),

(–)-(R)-4’-O-methylnyasol (27) and maslinic acid (14) were isolated and found to show

moderate cytotoxic activity against the rat glioma C-6 cell line using the MTT assay

1.3.3 SONNERATIA CASEOLARIS L ENGL

The whole plant or part of the plant is widely used in the traditional medicinal

systems of some countries For instance, in Myanmar, the fruit is used for poultice

In Malaysia, the skin of old fruits is used for helminthic treatment, half–ripe fruit

for cough and pounded leaves for hematuria and small pox In Bangladesh, the plant

is traditionally used as an anti–diabetic, astringent, antiseptic and in arresting

hemorrhage [75]

The methanolic extract of fruits of Sonneratia caseolaris significantly reduced

19.3, 27.6, 28.6 and 41.4% of glucose level in mice’s serum, when they were orally

administered by the methanol extract of S caseolaris at doses of 50, 100, 200 and

400 mg per kg body weight, respectively [75]

A report showed that levels of serum glucose, total cholesterol, triglycerides and low density lipoprotein cholesterol levels in Wistar rats were reduced when they were administered with dried leaf powder [4]

According to Wetwitayaklung P [109], the methanol extract of S caseolaris

The fruits of S caseolaris showed toxic against mosquito larvae [10]

In 2008, Tian Minqing et al [95] isolated luteolin (40) from the stems and twigs of S caseolaris and proved that in the in vitro cytotoxic assay against SMMC–7721 human hepatoma cells, this compound exhibited significant activity

In 2012, A A Laith et al [51] reported that the methanol extract of S caseolaris was effective on all Gram negative bacteria including Klebsiella pneumonia, Shigella dysenteriae, Enterobacter cloacae, Enterobacter sakazakii,

maltophilia and Aeromonas hydrophila with the MIC from 1.65 to 6.25 mg/mL and

In 2003, Nuntavan Bunyapraphatsara et al [15] showed that the calyces of S alba exhibited strong antioxidant activity followed by the stamens of S caseolaris The fruits of S caseolaris showed strong lipid peroxide formation inhibition

reductase induction activity

Oleanolic acid (12) isolated from the fruits of S caseolaris showed potent

pretreatment of rats with 12 displayed significant antihyperglycemic activity in starch tolerance test [97]

CHAPTER 2 EXPERIMENTAL

2.1 GENERAL EXPERIMENTAL PROCEDURES

NMR spectra were recorded on a Bruker Avance III spectrometer, at 500

The HR–ESI–MS was recorded on a HR–ESI–MS MicroOTOF–Q mass spectrometer on a LC– Agilent 1100 LC–MSD Trap spectrometer

Melting point of compounds was measured by a Büchi melting point apparatus The specific rotation was measured by a polarimeter ‒ A Krüss Optronic, Germany

followed by heating Gravity column chromatography was performed with silica gel 60 (0.040 – 0.063 mm, Himedia) The HPLC system was performed on an Agilent 1200 (USA) equipped with a G1311A quad pump, G1322A degasser, UV–VIS detector MWD G1365D and auto–sampler G1329A The separation was carried out on an Eclipse XDB–C18 column (4.6 × 150 mm i.d., 5 μm) with

a C–18 guard column

Instruments used were in Vietnam, at the Central Analysis Laboratory of the University of Science, Vietnam National University–Ho Chi Minh City and the Laboratory of the Department of Organic Chemistry, University of Science, Vietnam National University – Ho Chi Minh City and overseas, at the Laboratory

of the Department of Science, Systems and Models, Roskilde University,

Denmark and at the Laboratory of the Department of Chemistry, Chulalongkorn University, Thailand

Solvents used such as n–hexane, petroleum ether 60–90, chloroform, ethyl

acetate, methanol, acetic acid with the purity of over 98% for TLC and CC were

over 99.8 % were purchased from Sigma Aldrich Acetic acid, water and acetonitrile used for HPLC analysis were purchased from Merck

Vietnamese name: Bần trắng (Figure 2.1)

The leaves of Sonneratia abla were collected in Can Gio mangrove forest

in Ho Chi Minh city, Vietnam in December 2009 The scientific name of the plant was determined by the deceased pharmacist Phan Duc Binh and by the

the herbarium of the Department of Organic Chemistry, University of Science, Vietnam National University–Ho Chi Minh City

Figure 2.1 Sonneratia alba J.E Smith Figure 2.2 Sonneratia ovata Backer

Vietnamese name: Bần ổi (Figure 2.2)

The leaves of Sonneratia ovata were collected in Can Gio mangrove forest

in Ho Chi Minh city, Vietnam in March 2010 The scientific name of the plant was determined by the deceased pharmacist Phan Duc Binh and by the botanist

herbarium of the Department of Organic Chemistry, University of Science, Vietnam National University–Ho Chi Minh City

2.3 EXTRACTION AND ISOLATION

The air–dried powder of leaves (13,035 g) was macerated with methanol (50 L x 3) at room temperature for 48 hours and after filtration the methanolic

solution was concentrated at reduced pressure to yield a residue of 2,011 g This

crude extract was suspended in water with 10% of methanol and was partitioned first with petroleum ether (60–80 ºC) and then with ethyl acetate Finally, the remaining aqueous solution was evaporated to dryness to result in the remaining aqueous residue After evaporating at reduced pressure, three types of extracts were obtained: petroleum ether (690.0 g), ethyl acetate (415.0 g) and the remaining aqueous residue (885.0 g)

The petroleum ether extract was subjected to silica gel column chromatography (CC) (column: 120 x 6 cm), eluted with a solvent system of petroleum ether–ethyl acetate (stepwise 9:1, 4:1, 1:1, 0:1) and then ethyl acetate–methanol (stepwise 9:1, 4:1, 0:1) to give four fractions (E1–E4) Fraction E2 (90.0 g) was subjected to the silica gel CC (column: 120 x 6 cm) and eluted with petroleum ether–ethyl acetate (stepwise 9:1, 4:1, 1:1, 0:1) to give five subfrations (E2.1–E.2.5) Subfraction E2.1 (12.9 g) was further separated by the silica gel

CC and eluted with petroleum ether–chloroform (stepwise 9:1, 4:1, 1:1, 0:1) to obtain 12 (1,208.0 mg), 20 (15.0 mg) and 21 (448.8 mg) The same procedure was applied on subfraction E2.2 (6.9 g), eluted with chloroform–methanol (stepwise 1:0, 9:1, 4:1, 1:1) to afford 14 (5.0 mg), 55 (63.7 mg) and 97 (30.4 mg) The silica gel CC, eluted with chloroform–methanol (stepwise 1:0, 9:1, 4:1,

1:1) was applied on subfraction E2.3 (11.4 g) to give 8 (8.8 mg), 25 (17.0 mg),

30 (24.6 mg) and 87 (3.0 mg) and on subfraction E2.4 (14.9 g) to yield 33 (10.0 mg), 62 (44.7 mg) and 70 (24.8 mg)

The ethyl acetate extract was chromatographed on the silica gel CC (column: 120 x 6 cm) eluted with petroleum ether–ethyl acetate (1:4, 0:1) and then ethyl acetate–methanol (stepwise 9:1, 4:1, 1:1, 0:1) to give five fractions (A1–A5) Fraction A1 (6.6 g) was subjected to silica gel CC, eluted with chloroform–methanol (9:1) to obtain 89 (5,012.0 mg) Fraction A2 (40.0 g) was chromatographed on silica gel (column: 100 x 4.5 cm), eluted with ethyl acetate–methanol (stepwise 1:0, 9:1, 4:1, 1:1) to give five subfractions (A2.1–A2.5) Subfraction A2.2 (4.5 g) was subjected to reversed–phase RP–18 silica gel CC, eluted with water–methanol (stepwise 9:1, 4:1, 1:1, 0:1) to afford 88 (3.0 mg) and 93 (134.8 mg) Fraction A4 (30.0 g) was subjected to a silica gel CC (column: 100 x 4.5 cm) eluted with with ethyl acetate–methanol (stepwise 1:0, 9:1, 4:1, 1:1, 0:1) to afford eight subfraction (A4.1–A4.8) Subfraction A4.2 (1.2 g) was chromatographed on silica gel and eluted with dichloromethane–ethyl acetate–methanol (5:5:1) to afford 46 (29.6 mg), 66 (14.8 mg), 61 (13.5 mg) and

90 (11.0 mg) Subfraction A4.3 (4.0 g) was subjected to silica gel column chromatography (CC), eluted with a solvent system of ethyl acetate–methanol–water to obtain 58 (7.0 mg), 71 (21.0 mg), 84 (16.5 mg) and 85 (30.8 mg)

The procedure of extraction and isolation of compounds from the leaves of

the Sonneratia alba was presented in Scheme 1

The chemical structure of isolated compounds was studied using the spectroscopic methods of MS, 1D and 2D–NMR as well as by comparison of their data with those in the literatures

2.3.2 EXTRACTION AND ISOLATION OF THE SONNERATIA OVATA

The air–dried powder of leaves (10,913 g) was macerated with methanol (3x50 L) at room temperature for 48 hours and after filtration the methanolic

solution was concentrated at reduced pressure to yield a residue of 1,512 g The same procedure used with Sonneratia alba was applied to this residue This crude

extract was suspended in water with 10% of methanol and was partitioned first with petroleum ether (60–80 ºC) and then with ethyl acetate After evaporating at reduced pressure, three types of extracts were obtained: petroleum ether (245.0 g), ethyl acetate (390.0 g) and remaining aqueous residue (865.0 g)

The petroleum ether extract was subjected to silica gel column chromatography (CC) (column: 120 x 6 cm), eluted with a solvent system of petroleum ether–ethyl acetate (stepwise 9:1, 4:1, 1:1, 0:1) and then ethyl acetate–methanol (9:1, 4:1, 1:1) to give nine fractions (E1–E9) Fraction E2 (17.4 g) was subjected to the silica gel CC (column: 100 x 4.5 cm) and eluted with petroleum ether–chloroform (stepwise 9:1, 4:1, 1:1, 1:0) to give three subfrations (E2.1–E.2.3) Subfraction E2.2 (3.4 g) was further separated by silica gel CC and eluted with petroleum ether–chloroform (9:1, 4:1, 1:1) to obtain 2 (260.4 mg), 5 (10.1 mg) and 57 (972.3 mg) Fraction E8 (18.7 g) was applied on a silica gel CC (column: 100 x 4.5 cm) eluted with chloroform–methanol–water (stepwise 20:1:0, 9:1:0, 20:6:1, 14:6:1) to give five subfractions (E8.1 to E8.5) Subfraction E8.2 (2.3 g) was further separated by reversed–phase silica gel CC and eluted with water–methanol (4:1, 1:1, 0:1) to obtain 98 (120.4 mg)

The ethyl acetate extract was chromatographed on the silica gel CC (column: 120 x 6 cm) eluted with petroleum ether–ethyl acetate (1:4, 0:1) and then ethyl acetate–methanol (stepwise 9:1, 4:1, 1:1, 0:1) to give fractions A1 to A9 Fraction A2 (1.2 g) was subjected to RP–18 silica gel CC, eluted with water–methanol (5:5) to obtain the mixture of 74a and 74b (23.0 mg) Fraction A3 (1.7 g) was chromatographed on silica gel eluted with dichloromethane–ethyl acetate (20:1) to afford 26 (7.2 mg), 77 (25.0 mg), 82 (5.0 mg) and 86 (5.3 mg) Fraction A4 (8.4 g) was chromatographed on silica gel eluted with dichloromethane–methanol (20:1; 10:1) to afford 56 (15.1 mg), 64 (8.9 mg), 65 (14.2 mg), 78 (8.6 mg), 80 (32.8 mg) and 95 (31.5 mg) The same procedure was applied to fraction A5 (13.0 g) and eluted with dichloromethane–methanol (9:1; 4:1) to afford 40 (8.2 mg), 67 (16.1 mg), 69 (8.0 mg), 72 (23.4 mg) and 88 (12.2 mg) Fraction A6 (3.7 g) was chromatographed on Sephadex LH 20 eluted with methanol and then

was chromatographed on silica gel eluted with dichloromethane–methanol (9:1; 4:1) to afford 70 (17.4 mg) and 93 (6.7 mg) Fraction A7 (24.0 g) was chromatographed on silica gel (column: 50 x 6 cm) eluted with chloroform–methanol–water (stepwise 20:1:0, 9:1:0, 20:6:1, 14:6:1) to afford ten subfractions (A7.1–A7.10) Subfraction A7.6 (1.8 g) was subjected to reversed–phase RP–18 silica gel CC, eluted with water–methanol (stepwise 20:1, 9:1, 4:1, 1:1) to afford

46 (98.5 mg), 81 (6.0 mg) and 96 (6.3 mg) The same procedure was applied to subfraction A7.7 (2.8 g) to obtain 41 (175.4 mg), 59 (3.0 mg) and 75 (340.0 mg) and to subfraction A7.8 (2.2 g) to yield 76 (16.0 mg) and 94 (95.6 mg) Fraction A8 (30 g) was chromatographed on silica gel (column: 50 x 6 cm) eluted with chloroform–methanol–water (stepwise 20:1:0, 9:1:0, 20:6:1, 14:6:1) to afford ten subfractions (A8.1–A8.10) Subfraction A8.4 (1.3 g) was subjected to RP–18 silica gel CC and eluted with water–methanol (stepwise 8:2, 7:3, 1:1, 0:1) to obtain 79 (25.0 mg) Subfraction A8.6 (1.0 g) was subjected to reversed–phase RP–18 silica gel CC, eluted with water–methanol (20:1, 9:1, 4:1, 1:1) to afford

71 (4.5 mg) The same procedure was applied to subfraction A8.7 (2.4 g), eluted with water–acetonitrile (20:1, 9:1, 4:2) to give 68 (10.8 mg) and 73 (5.1 mg) Subfraction A8.9 (4.4 g) was subjected to reversed–phase RP–18 silica gel CC, eluted with water–methanol (9:1, 4:1, 1:1) to obtain 60 (23.7 mg), 61 (21.0 mg),

62 (66.0 mg), 63 (15.0 mg), 91 (22.2 mg) and 92 (3.0 mg)

The procedure of extraction and isolation of compounds from the

Sonneratia ovata leaves was presented in Scheme 2

The chemical structure of isolated compounds was studied using spectroscopic methods of MS, 1D and 2D–NMR as well as by comparison of their data with those in the literatures

basis set using the GIAO method [24], [110] Correlation coefficients between

Fig 3.3

2.5 ACID HYDROLYSIS OF 94 AND 98

D

Acid hydrolysis of 98: Compound 98 (10 mg) in a solution of 2N HCl in

methanol (5 mL) was refluxed at 80 ºC for 20 hours to yield a mixture of a fatty acid methyl ester (98A), a free sphingosine long chain base (98B) and a methyl

with chloroform The hexane solution was evaporated to dryness and analyzed by

probability of 84.4% (98A) The chloroform solution was evaporated to dryness and then was purified by silica gel column chromatography to afford 98B (2.9

HRESIMS (Appendix 58.4) with the pseudomolecular ion peak at m/z

2.6 BIOLOGICAL ASSAYS

2.6.1 CYTOTOXIC ACTIVITIES AGAINST THREE CANCER CELL LINES

AND HUMAN PRIMARY FIBROBLAST (NORMAL CELL)

Determination of cytotoxic activities against the HeLa (human epithelial carcinoma), NCI–H460 (human lung cancer) and MCF–7 (human breast cancer) cell lines and cytotoxic activities against the human primary fibroblast (normal cell) of tested samples were performed at the concentration of 100 g/mL using the antiproferative Sulforhodamine B (SRB) assay with camptothecin as the positive control

The Sulforhodamine B (SRB) assay was done followed the two documents [44], [86]

Samples were sent to be in vitro tested at the Faculty of Biology, University

of Science, Vietnam National University–Ho Chi Minh City, 227 Nguyen Van

Cu Street, District 5, Ho Chi Minh City, Vietnam, under the management of Assoc Prof Ho Huynh Thuy Duong

All cells were cultured in E’MEM medium (Eagle's Minimal Essential Medium) supplemented with 10% foetal bovine serum (FBS), 1% of 2 mM L–glutamine, 50 IU/mL penicillin, 50 g/mL streptomycin and maintained at 37

o

cells were treated with pure compound while the control wells were added only

by 100 µL medium All experiments were in triplicate The plates were incubated

cultures were fixed by adding 50 µL of cold 50% (w/v) trichloroacetic acid per

distilled water and air dried for 12–24 h Then a solution of 50 µL of SRB (0.2 % w/v in 1% acetic acid) was added to each well and was allowed to stay at room temperature for 30 min The SRB solution was removed out of plates by rinsing 4 times with a 1% glacial acetic acid solution (200 µL/well) The plates were air–dried for 12–24 h The bound SRB was solubilised to each well by adding 200

µL of 10 mM Tris Base (pH 10.5) The plates were shaken gently for 20 min and the optical density of each well was read using a scanning multiwall spectrophotometer at a test wavelength of 492 nm and a reference wavelength of

620 nm The optical density (OD) of SRB in each well is directly proportional to the cell number The negative control contained all components except for the fact that the sample was replaced by medium containing 0.25% DMSO The blank contained all components except for the fact that cells were replaced by medium (The blank included blank of negative control and blank of tested

sample) The OD value in the following equation was OD492 which was

)B(S)B(Cactivity Inhibitive

%

N N

T T N

The result of the cytotoxic activity against three cancer cell lines was presented in Table 3.21 and Table 3.22

2.6.2 ACETYLCHOLINESTERASE (ACHE) INHIBITION BIOLOGICAL

ASSAY

 Chemicals used in the acetylcholinesterase biological activity

(Sigma)

(BSA)

Anti–AChE activity of some extracts and isolated compounds was determined by Ellman’s method [26] with galanthamine as the positive control

Samples were in vitro tested at the Faculty of Pharmacology, University of

Medicine and Pharmacy, 41 Dinh Tien Hoang Street, District 1, Ho Chi Minh

City, Vietnam under the management of Assoc Prof Vo Phung Nguyen, or were tested at the Faculty of Biology, University of Science, Vietnam National University– Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam, under the management of Dr Quach Ngo Diem Phuong The principle of the method is that the enzyme acetylcholinesterase (AchE) hydrolyzes the substrate [Acetylthiocholine iodide (ATCI) is used as the substrate] to give a compound This compound will further react with Ellman reagent, 5,5’-dithio-bis-2-nitrobenzoate ion (DTNB) to give 2-nitrobenzoate-5-mecaptothiocholine and 5-thio-2-nitrobenzoate The latter possesses a yellow color and therefore, the rate of color production is measured at 405 nm by a spectrophotometer All samples were tested in triplicate

Acetylcholinesterase inhibition assay was done in 96 well plate Briefly,

125 L of 3 mM DTNB, 25 L of 15 mM ATCI and 50 L of buffer, 25 L of sample (tested sample was dissolved at three concentrations: 1.0 mg/mL, 0.5 mg/mL and 0.25 mg/mL in buffer containing 10% dimethylsulfoxide DMSO) were added to the wells followed by 25 L of 0.22 U/mL AChE The microplate

by a ELISA microplate reader (Bio–Tek Instrument, USA)

The negative control contained all components except for the fact that the tested sample was replaced by buffer containing 10% DMSO The blank contained all components except for the fact that AChE was replaced by buffer (Blank included blank of negative control and blank of tested sample) Galantamine was used as positive control with three concentrations (0.1 mg/mL, 0.05 mg/mL, 0.025 mg/mL in buffer containing 10% DMSO) The percentage of AChE inhibitory activity was calculated using the following equation:

x100%

)B(C

)B(S)B(Cactivity Inhibitive

%

N N

T T N

BT : Optical density of blank of tested sample All experiments described in this study were performed in triplicate

The result of acetylcholinesterase inhibition was presented in Table 3.23

Scheme 1: Extraction and isolation procedure for Sonneratia alba

Dried ground material of leaves of

E3 (60.0 g)

E4 (220.0 g)

- Maceration with methanol at room temperature (3x50 L)

- Filtration, evaporation at reduced pressure

- Dissolution in solvent system of methanol : water (1:9)

- Partition with petroleum ether and then with ethyl acetate

- Evaporation at reduced pressure

E2.3 (11.4 g)

E2.4 (14.9 g)

E2.5 (25.3 g)

A2 (40.0 g)

A3 (107.0 g)

A4 (30.0 g)

A5 (47.0 g)

89 (5,012.0 mg)

88 ( 3.0 mg)

93 (134.8 mg)

A4.1 (1.8g)

A4.2 (1.2 g)

A4.3 (4.0 g)

A4.4-A4.8 (21.6g)

CC PE: EA (9:1 – 0:1)

A2.1 (3.2 g)

A2.2 (4.5 g) A2.3-A2.5 (24.7 g)

Scheme 2: Extraction and isolation procedure for Sonneratia ovata

Petroleum ether extract (245.0 g)

Ethyl acetate extract (390.0 g)

Remaining aqueous residue (865.0 g)

- Dissolution in solvent system of methanol : water (1:9)

- Partition with petroleum ether and then with ethyl acetate

- Evaporation at reduced pressure

E8.2 (2.3 g)

E8.3-E8.5 (8.5 g)

A4 (8.4 g)

A7 (24.0 g)

A8 (30.0 g)

74a & 74b (23.0 mg)

Dried ground material of leaves of

Soneratia ovata

(10,913 g)

- Maceration with methanol at room temperature (3x50 L)

- Filtration, evaporation at reduced pressure

Methanolic crude residue (1,512 g)

Silica gel column chromatography (CC) PE: EA (9:1 – 0:1)

EA: M (9:1 – 1:1)

A2 (1.2 g)

A9 (165.0 g)

A1 (2.9 g)

E1

(22.7 g)

E2 (17.4 g)

E3-E7 (95.1 g)

E8 (18.7 g)

E9 (25.8 g)

(13.0 g) (3.7 g)A6

CHAPTER 3 RESULTS AND DISCUSSION

3.1 CHEMICAL STRUCTURE ELUCIDATION

From two species of the genus Sonneratia, 58 compounds were isolated

including 7 new compounds and a mixture of two new ones along with 50 known ones The new compounds were identified by checking with Scifinder software at Roskide University, Denmark in June 2014 The chemical structure of all compounds was elucidated on the basis of NMR and MS spectroscopic experiments The isolated compounds were divided into seven groups as listed below:

mixture of two new ones and nine known ones)

known ones)

known ones)

3.1.1.1 Structure elucidation of β-sitosterol (2)

Compound 2 was obtained from fraction E2.2 of Scheme 2 of Sonneratia ovata with essential physical data as described below

Discussion of the chemical structure

(>CH–O, H–3) The 13C–NMR data of 2 displayed 29 carbon signals, including two

with the data in the literature [95]

-Sitosterol is the common sterol of plant and was previouly isolated from S ovata [127], from leaves, stems and twigs of S alba [7], [17] and from stems and twigs of S caseolaris [95] It possessed various pharmacological properties such as

hypercholesterolemic, gastroprotective, platelet aggregation inhibitor, cytotoxic

50 μg/mL) cell lines] [131]

3.1.1.2 Structure elucidation of 3-O-palmitoyl-β-sitosterol (5)

Compound 5 was obtained from fraction E2.2 of Scheme 2 of Sonneratia ovata with essential physical data as described below

Discussion of the chemical structure

Detailed analysis of NMR data of 5 indicated that it possessed a similar structure to that of β-sitosterol The difference was the presence of signals for an additional alcyl group in 5 This was evidenced by the observation of a signal of

Table 3.1 NMR spectroscopic data of compounds 2, 5 and 8

spectrum Besides, the 13C–NMR spectrum also displayed carbon signals at δC

terminal methyl group The HR–ESI–MS showed the pseudomolecular ion peak at

was determined as hexadecanoyl or palmitoyl The structure of 5 was thus identified

as 3-O-palmitoyl-β-sitosterol by comparing its NMR data with those in the

literature [95] This compound was also reported to be found in stems and twigs of

29 26 27

Compound 8 was obtained from fraction E2.3 of Scheme 1 of Sonneratia alba

with essential physical data as described below

3.4, 3.5 and 3.6): Table 3.1

Discussion of the chemical structure

The NMR spectra of 8 were very similar to those of 5, including the proton and carbon signals of a β-sitosterol skeleton and a palmitoyl moiety However, 8 had one more β-glucose unit This was evidenced by the presence of a doublet

methylene carbon at δC 64.6 and four oxymethine signals from 71.0 to 75.0 ppm of

a sugar unit

The COSY spectrum showed the connection of H–1’/H–2’/H–3’/H–4’/H–

moiety at C–6’ of the glucose unit

The HR–ESI–MS showed the pseudomolecular ion peak at m/z 837.6563

comparison of its NMR data to those reported in the literature [95], the structure of

was previously reported to be found in stems and twigs of S caseolaris [95]

3.1.2.1 Structure elucidation of betulin (20)

Compound 20 was obtained from fraction E2.1 of Scheme 1 of Sonneratia alba with essential physical data as described below

Discussion of the chemical structure

of a lupane type triterpene It corresponded to the presence of doublet proton signals

28b) confirming the presence of a second hydroxyl group at C–28 in the structure of

20

characteristic of an α–oriented hydrogen at C–3 of a 3β–hydroxytriterpene Comparison of spectroscopic data of 20 with those in the literature [96] suggested that 20 was lup-20(29)-ene-3,28-diol or a more commonly name betulin

Betulin occurred in more than 200 different types of plant species with the

highest amount ranging from 10 to 25% found in the birch barks (Betula species) [41] It was also found in Sonneratia ovata [127], stems and twigs of Sonneratia caseolaris [95] This compound has been reported to exhibit anti–HIV, anti–

carcinogenic, anti–flu, anti–inflammatory, anti–hypoxic, anti–allergen, anti–

hepatoprotector, aphidifuge, cytotoxic, hypolipemic, detoxicant and adaptogenic properties [96], [121]

3.1.2.2 Structure elucidation of betulinic acid (21)

Compound 21 was obtained from fraction E2.1 of Scheme 1 of Sonneratia alba with essential physical data as described below