Study on chemical constituents and biological activities of some plants of boerhaavia genus (nyctaginaceae)

diffusa 19 Figure 3.1 Comparison of CD and absorption spectra of compound 59 Scheme 1: Extraction and isolation procedure for Green Boerhaavia diffusa 27 Scheme 2: Extraction and isol

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY

OF BOERHAAVIA GENUS (NYCTAGINACEAE)

Subject: Organic chemistry

Code number: 62 44 27 01

The first reviewer: Assoc Prof Trần Hùng

The second reviewer: Assoc Prof Phạm Đình Hùng

The third reviewer: Assoc Prof Trần Công Luận

The first independent reviewer: Assoc Prof Nguyễn Thị Hoàng Anh

The second independent reviewer: Dr Nguyễn Lê Bảo Khánh

SUPERVISORS: PROF NGUYỄN KIM PHI PHỤNG

PROF DOMINIQUE GUILLAUME

Ho Chi Minh City, 2014

TABLE OF CONTENTS

2.4 Biological assays 25

Chapter 3: RESULTS & DISCUSSION

APPENDICES

HR-ESI-MS : High Resolution - Electrospray Ionization - Mass Spectrometry

LIST OF TABLES

Table 3.1: 1H–NMR spectroscopic data of the known compounds 11, 21 and

Table 3.2: 13C–NMR spectroscopic data of the known compounds 11, 21 and

Table 3.3: 1H–NMR spectroscopic data of the known compounds 9, 10, 15,

Table 3.4: 13C–NMR spectroscopic data of the known compounds 9, 10, 15,

Table 3.5: 1H–NMR spectroscopic data of the new compounds 59, 60, 61

Table 3.6: 13C–NMR spectroscopic data of compounds 59, 60, 61

Table 3.7: 1H–NMR spectroscopic data of the known compounds 62, 18 and

Table 3.8: 13C–NMR spectroscopic data of the known compounds 62, 18 and

Table 3.9: 1H–NMR spectroscopic data of the known compounds 65, 39, 36,

Table 3.14: 1H–NMR spectroscopic data of the new compound 72,

Table 3.15: 13C–NMR spectroscopic data of the new compound 72,

vii

Table 3.16: 1H–NMR spectroscopic data of the known compounds 80, 81, 47

Table 3.17: 13C–NMR spectroscopic data of the known compounds 80, 81, 47

Table 3.19: NMR spectroscopic data of the known compounds 84, 85

Table 3.21: 1H–NMR spectroscopic data of the known compound 91 and the

Table 3.22: 13C–NMR spectroscopic data of the known compound 91 and the

Table 3.24: Inhibitive activity of cytotoxic activity against three cancer cell

Table 3.25: Acetylcholinesterase inhibition of some extracts and isolated

viii

LIST OF FIGURES AND SCHEMES

Figure 2.4: Comparison of TLC the four methanolic extracts of green

stem-leave (GS), red-purple stem-leave (RS), green root (GR) and

Figure 2.5: HPLC chromatograms of (a) red-purple and (b) green B diffusa 19 Figure 3.1 Comparison of CD and absorption spectra of compound 59

Scheme 1: Extraction and isolation procedure for Green Boerhaavia diffusa 27

Scheme 2: Extraction and isolation procedure for Red-purple Boerhaavia

ix

CHAPTER 1: LITERATURE REVIEW

Boerhaavia chinensis[w1] Boerhaavia coccinea[w2] Boerhaavia coulteria[w3]

Boerhaavia diffusa[w4] Boerhaavia erecta[w5] Boerhaavia gracillima[w6]

Boerhaavia herbstii[w7] Boerhaavia intermedia[w8] Boerhaavia linearifolia[w9]

Boerhaavia pterocarpa[w10] Boerhaavia purpurascens[w11] Boerhaavia repens[w12]

2

Boerhaavia scandens[w13] Boerhaavia tetrendra[w14]

Boerhaavia triquetra[w15] Boerhaavia wrightii[w16]

Figure 1.1 Some species of Boerhaavia genus

1.1 BOERHAAVIA GENUS – GENERIC DESCRIPTION

Boerhaavia is a genus of 40 species, most of which are widely distributed

in tropical and subtropical areas of the world.[19][69] Some of the species are treated as complex groups They are usually coastal plants spreading in sandy areas, gravelly plains, or on rocky slopes Owing to the polymorphism and the possible existence of hybrids between members of the complexes, many authors

of regional floras have indicated that the distinctiveness at the species level,

especially in the pantropical Boerhaavia diffusa complex and Boerhaavia repens

complex, were rather difficult.[49][76][78]

The genus Boerhaavia is characterized by elongate stems with prominent

nodes, slender forking cymose or rarely racemose or spicate inflorescences with small, scale-like free bracts, small flowers with campanulate, cylindric, funnel-shaped perianth limb separated by a constriction from the sulcate, enlarged lower

portion of the perigone, and anthocarps that are clavate or oblong, often glandular.[19]

According to the literature, among 40 species of Boerhaavia, 6 species are found in India They are Boerhaavia diffusa, Boerhaavia erecta, Boerhaavia

rependa, Boerhaavia chinensis, Boerhaavia hirsute and Boerhaavia rubicunda.[41] This genus Boehaavia represented in Taiwan by five species:

Boerhaavia coccinea Mill, Boerhaavia erecta L., Boerhaavia repens L., Boerhaavia glabrata Bl and Boerhaavia hualienense Chen  Wu sp nov.[66]

In Vietnam, the generic description of this genus by the Vietnamese botanists is not settled The Vietnamese botanist Pham Hoang Ho[2] mentioned

that there were three species of Boerhaavia in Vietnam including B diffusa L (Vietnamese name: nam sâm or nam sâm bò), B chinensis (L.) Asch  Schw

(Vietnamese name: nam sâm trung quốc) and B erecta L (Vietnamese name:

nam sâm đứng) The pharmacist Do Tat Loi[3]

wrote that there was only one

species of Boerhaavia, that was B diffusa L., or B procumbens Wight, or B

repens L The botanist Vo Van Chi[1] declared that there were two species and

they were B diffusa L (or B repens L.) and B chinensis (L.) Asch  Schweinf

(or B repanda Willd)

Some pictures of Boerhaavia species have been found widely on the

internet (Figure 1.1)

1.1.1 BOERHAAVIA COCCINEA M.[20][66][78]

Perennial herbs, prostrate to ascending, up to 80 cm tall Stems terete, 2-5

mm across, green flushed with purplish red, glandular puberulent and sparsely pilose Leaves somewhat fleshy, opposite, in unequal pairs; blades ovate, broadly ovate to suborbicular, upper surface yellowish green, green to bluish green, obscurely glandular to glabrous, lower surface pale, pubescence on veins,

margins entire, cartilaginous, undulate to slightly lobed, distinctly long-ciliate ca

1 mm long, obtuse to rounded at the apex, truncate to subcordate at the base, the larger one 3-8 cm long and 2-7 cm wide, with a long petiole up to 4 cm long, the

sparsely puberulent, neither surface punctate Inflorescences terminal, forked ca

3-6 times equally, diffuse, occasionally with sticky internodal bands; branches divergent, terminating in compact subumbellate or capitate, 2-5-flowered clusters Flowers pedicel shorter than 0.5 mm; bract at base of perianth quickly deciduous, 1, lanceolate, 0.8-1 mm; perianth purplish red to reddish pink or nearly white, campanulate beyond constriction, 1-1.5 mm; stamens 2-3, included

or barely exserted Fruits 1-9 per cluster, gray-brown to brown, obpyramidal, 4.5 mm × 1-1.2 mm, apex broadly conic, with sparse or moderately dense stipitate-glandular hairs on ribs, less densely pubescent or glabrous in sulci (rarely entirely glabrous); ribs 5, round, smooth; sulci 2-2.5 times as wide as base

3-of ribs, not rugose, not papillate.[69]

B diffusa was botanically reported possessing the green color of stems and

leaves.[4][8][58]

1.1.3 BOERHAAVIA ERECTA L.[66]

Annual herbs, slightly woody at base; taproot tappered Stems usually erect, sometimes decumbent, profusely branched primarily distally, 20-120 cm, minutely puberulent with bent hair basally, usually glabrous, rarely sparsely

puberent distally Leaves mostly in basal half of plant; larger leaves with petiole 6-50 mm, blade broadly rhombic-ovate, triangular-ovate, ovate, oval, or lanceolate, 20-80 mm × 10-45 mm (distal leaves smaller, proportionately narrower), adiaxial surface usually glabrous, sometimes minutely puberulent, usually punctate with small patches of small brown cells, base obtuse to round, margins entire or sinuate, usually with sticky internodal bands; branches strongly ascending, terminating in irredular umbellate or subracemose clusters of flowers, not all pedicels attaching at same point (flowers occasionally borne singly) Flowers pedical 0-5 mm; bracts at base of perianth deciduous, usually 2, narrowly to broadly lanceolate, 0.5-1 mm, apex often acuminate; perianth whitish, usually tinged with pink or purple (bright pink) between lobes and in tube, campanulate beyond constriction, 1-1.5 mm; stamens 2-4, slightly exserted Fruits 1-11 per cluster, pale greenish to straw colored or tan, narrowly obconic 2.7-4 mm × 1.2-1.5 mm, apex truncate or broadly low conic, glabrous; ribs 5, acute, slightly rugose adjacent to sulci; sulci 0.5-1 times as wide as base of ribs, slightly to prominently coarsely tranverse rugose, not papillate.[69]

B erecta occasionally forms mixed populations with B intermedia

without apparent intergradation Rarely, some specimens seem to combine features of either species, particularly with regard to inflorescence structure The two species bloom simultaneously and are visited by small insects Given the presumed close relationship and weedy habitats of each, hybridization seems possible Usually, the two species can be distinguished by the differences in fruit

length, the appearance of a crownlike apex of the nearly mature fruits of B

erecta (apex of ridges slightly expanded, apex of fruit slightly conic), and the

more precisely constructed terminal umbels of B intermedia Both species, particularly B intermedia, may produce entire inflorescences with branches

terminating in single flowers.[69] Woodson et al (1961) suggested that B erecta hybridizes with the perennial B diffusa.[79]

6

1.1.4 BOERHAAVIA REPENS L.[66]

Perennial herbs, prostrate, ascending to erect, up to 50 cm tall Stems fleshy, 2-5 mm across, green flushed with purplish red, pubescent, becoming glabrous and woody towards the base Leaves somewhat fleshy, opposite, in unequal pairs; blades ovate, ovate-lanceolate to lanceolate or elliptic, upper surface green, flushed with purplish red, puberulent or subglabrous, lower surface pale, pubescent on veins, margins entire, cartilaginous undulate to slightly lobed, glabrous to distinctly short-ciliate, obtuse at the apex, usually minutely apiculate, truncate, rounded to subcordate at the base, the larger one 3-5.5 cm long and 2-4 cm wide, with a long petiole up to 2.5 cm long, the smaller 1.5-2.5 cm long and 1-3 cm wide, with petiole less than 1.5 cm long; petioles pale flushed with purple, pubescent, the leaves in the portion from stem to inflorescence gradually reduced Inflorescences of pedunculate cymes or pseudoumbels, auxillary, or a small terminal panicle, the peduncules 1-8 cm long, 2-4 branched, capillary at apex, green flushed with purplish red, pubescent and glanduar to glabrous, each branch of peduncle subtended by a bract, bracts linear lanceolate, puberulent, 1.5 mm long, caducous Flowers 3-6, clustered at the apex of peduncle, subsessile; bracteoles usually 2, subtending a flower, in an unequal pair, the smaller slightly above, linear-lanceolate, 1 mm long and 0.2

mm wide, the larger below, lanceolate, 1.2 mm long and 0.3 mm wide, all those pale yellow, flushed with purple, puberulent

1.2 BOERHAAVIA GENUS –PHARMACOLOGICAL STUDIES

1.2.1 BOERHAAVIA COCCINEA M

There has been no publication on the pharmacological activities of

Boerhaavia coccinea L to the best of our knowledge

1.2.2 BOERHAAVIA DIFFUSA L.[5]

Rawat et al (1997)[59] reported on hepatoprotective effect of B diffusa Their studies also revealed that the administration of aqueous extract of B diffusa

(2 ml/kg) exhibited more effectively hepatoprotective activity than the powder

form More importantly, in 2010, Olaleye et al.[56] also confirmed the

hepatoprotective activity of leaf extracts of B diffusa against

acetaminophen-induced liver damage

In 2000, Hiruma-Lima et al.[22] reported the remarkably analgesic property

of juice of fresh leaves of B diffusa L

The alkaloidal component of B diffusa was proved to possess various biological activities Mungantiwar et al (1999)[52] communicated that the

alkaloidal fraction exhibited immunomodulatory effect in vitro by inhibiting SRBC-induced delayed hypersensitivity reactions in mice Leyon et al (2005)[40]communicated that aqueous methanolic 70% extract of B diffusa inhibited the

metastasis of B16F-10 melanoma cells in C57BL/6 mice Manu and Kuttan (2009)[42] continuously reported that punanarvine isolated from B diffusa

possessed antimetastatic property using B16F-10 melanoma cells in C57BL/6 mice due to the abilitity to suppress MMP-2, MMP-9, ERK-1, ERK-2, and VEGF in the lung tissue of metastasis-induced animals Manu and Kuttan[43] also reported that punanarvine induced apoptosis in B16F-10 melanoma cells by inhibiting NF-kB signaling

In 2002, Mehrotra et al.[47] reported immunomodulatory effect of the

ethanolic extract of B diffusa The study showed that the extract exhibited cytotoxic effect against human NK cell line in vitro, production of NO in mouse macrophage cells, IL-2 and TNF-α human PBMCs In addition, Mehrotra et

al.[48]noted that the extract also exerted the antiproliferative effect against several

lympho cell lines of mouse and human origin, i.e mouse macrophage cells

(RAW 264.7), human macrophage cells (U937), human monocytic cells 1), mouse fibroblast cells (L929), human embryonic kidney cells (HEK293), mouse liver cells (BNLCL.2), African green monkey kidney cells (COS-1), mouse lymphoma cells (EL-4), human erythroleukemic cells (K562) and human

(THP-T cells (Jurkat).Meanwhile, the methanolic extract of whole plant B diffusa was

reported possessing antiproliferative effect against breast cancer cell lines 7).[70] Bharali et al (2003)[10] reported that B diffusa possessed DMBA-induced

(MCF-cancer chemopreventive activity

8

1.2.3 BOERHAAVIA ERECTA L

There have been few publications on the pharmacological activities of

Boerhaavia erecta L to the best of our knowledge Stintzing et al.[72] reported the

qualitative and quantitative analysis of the chemical composition of B erecta

containing betacyanins and phenolic compounds which might be responsible for its medicinal use in West Africa

Hilou et al (2006)[21] noted that the extract of B erecta possessed in vivo

antimalarial activity The plant extract showed significant antimalarial activity in 4-day suppressive antimalarial assay in mice inoculated with red blood cells

paratisized with Plasmodium berghei berghei While the obtained value for ED50

was 564 mg/kg, the toxicity of the plant extract was very low with the value of

LD50 ca 2150 mg/kg

In 2012, Suriyavathana et al.[74] reported that leaf extract of B erecta

exerted positive antimicrobial and antifungal activities against several bacterial and fungal species

1.3 BOERHAAVIA GENUS – CHEMICAL STUDIES

1.3.1 BOERHAAVIA COCCINEA M

According to the best of our knowledge, there were three publications

reported for B coccinea

In 1986, Messana et al.[50] reported the presence of two

12a-hydroxyrotenoids from roots of B coccinea, but the authors did not offer their

chemical structures

In 1991, Ferrari and Messana[18] reported the presence of coccineone A (19)

and B (20) in B coccinea roots

In 1998, Santos et al.[62] reported the isolation from the whole plant three

rotenoids coccineone C (21), coccineone D (23) and coccineone E (24)

1.3.2 BOERHAAVIA DIFFUSA L

Up to now, although there have been many reports on Boehaavia genus, these reports concentrated mostly on Boehaavia diffusa species

There were several reports on the chemical constituents of B diffusa

collected in India Generally, to the best of our knowledge, the secondary

metabolites of B diffusa mainly consisted of flavonoids including flavonols,

isoflavones, and rotenones as the major composition aside from the minor component of steroids, triterpenoids, lignans and betanins The very first study

on B diffusa dates back to 1971 when Misra and Tiwari[51]reported the isolation

of β-sitosterol (1), ursolic acid (7), D-glucose (50), D-galactose (51), D-xylose (52), fructose (53) from roots of B diffusa Eleven years later, Suri et al

(1982)[73] reported the identification of β-ecdysone (3), stigmasterol 3-O-

-D-glucopyranoside (6) from roots of B diffusa In 1986, Seth et al.[64] reported the

isolation of punarnavoside (46) from B diffusa possesing antifibrinolytic activity

In 1989, two other rotenoids boeravinone A (9) and B (10) were isolated

and identified also from roots of B diffusa by Kadota et al [33] And in the same

year, Kadota et al.[32] reported for the first time the isolation and identification

of a rotenoid, namely boeravinone C (11), from B diffusa roots Since

boeravinone C belonged to a subgroup of rotenoids which was designated as hydroxyrotenoid, this resulted in the necessity in the determination of the

12a-stereochemistry of C-6a and C-12a Accordingly, Lami et al (1990)[36] published

a detailed report on the stereochemistry of boeravinone C suggesting that the B/C

ring junction oriented in trans-geometry The presence of boeravinone D (12), E (13), and F (14) from roots of B diffusa were subsequently reported in 1991 by

Lami et al [37] Additionally, Lami et al (1991)[38] aslo reported the isolation of

liriodendrin (48) and syringaresinol mono-β-D-glucoside (47) in which

liriodendrin posessed significant calcium (Ca2+) channel antagonistic effect

A xanthone named borhavine (49) was identified in roots of B diffusa by

Ahmed et al in 1992.[6] It was not until 2005 that two other rotenoids were

reported by Borrelli et al.[12] when they isolated boeravinone G (15) and H (16)

along with boeravinone D and E from roots of B diffusa In the following year, Borrelli et al.[13] continued to publish a report on the identification of 10-

demethylboeravinone C or coccineone C (21), 6-O-demethylboeravinone H (25),

10

coccineone B (20), 9-O-methyl-10-hydroxycoccineone B (22), coccineone E (24) and 2′-O-methylabronisoflavone (38) in roots of B diffusa In 2007, while

Belkacem et al.[9] reported the presence of boeravinone I (17) and J (18) in which

the latter was a coumaronochromone instead of a rotenone as in the previous series of boeravinones Some other authors[20][42] also reported the presence of

some compounds from the whole plant of B diffusa: stigmasterol (2),

boerhavisterol (4), -sitosterol 3-O--D-glucopyranoside (5), boerhavilanostenyl benzoate (8), diffusarotenoid (26), eupalitin (28), kaempferol (29) and eupalitin

3-O-β-D-galactopyranoside (34)

1.3.3 BOERHAAVIA ERECTA L

There were two publications on the chemical constituents of Boerhaavia

erecta L to the best of our knowledge In 2003, Stintzing et al.[72] reported the

qualitative and quantitative analysis of the chemical composition of B erecta

5,7-dihydroxy-3’,4’-dimethoxyl-6,8-dimethylflavone (27), quercetin 3-O-glucoside (31), isorhamnetin 3-O-rutinoside (32), quercetin 3-O-rutinoside (33) (predominant compound), (+)-catechin (39),

(-)-epicatechin (40), procyanidin B1 (41), procyanidin B2 (42) and betalains such

as betanin (43), neobetanin (44) and amaranthine (45)

1.3.4 BOERHAAVIA REPENS L

There was only one publication on the chemical constituents of Boerhaavia

repens Jianxin Li et al.[31] reported the presence of eupalitin 3-O-

-D-galactopyranoside (34), eupalitin 3-O-rutinoside (35), kaempferol 3-O-rutinoside (36), and 6-methoxylkaempferol 3-O-rutinoside (37) in the methanolic extract of

the whole plant of this species

12

14

AMINO ACID: Alanine, aspartic acid, glutamic acid, glycine, histidine, leucine,

methionine, proline, serine, threonine, tyrosine, valine

FATTY ACID: Palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid

CH3–(CH2)14–COOH : Hexadecanoic acid (Palmitic acid)

CH3–(CH2)16–COOH : Octadecanoic acid (Stearic acid)

CH3–(CH2)18–COOH : Eicosanoic acid (Arachidic acid)

CH3–(CH2)20–COOH : Docosanoic acid (Behenic acid)

CH3–(CH2)7–CH=CH–(CH2)7–COOH : Octadecenoic acid (Oleic acid)

1.4 AIM OF THE STUDY

In the view of the importance of the plants as remedy to diseases, we

chosed green stem-leaves Boerhaavia diffusa, red-purple stem-leaves Boerhaavia

diffusa and Boerhaavia erecta for chemical investigation

The aim of this research is to isolate and structurally elucidate the compounds and to test biological activities of the compounds from these three

species of the genus Boerhaavia, Nyctaginaceae which are grown in Vietnam

16

CHAPTER 2: EXPERIMENTAL

2.1 INSTRUMENTATION AND MATERIALS

NMR spectra were recorded on a Bruker Avance III spectrometer 500 MHz

using residual solvent signals as internal references: chloroform-d H 7.24, C

77.23; methanol-d4 H 3.31, C 49.15; DMSO-d6 H 2.50, C 39.51; acetone-d6 H

2.09, C 206.31, 30.6; and pyridine-d5 at H 8.74, 7.58 and 7.22, C 123.87, 135.91, 150.35

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 microscope hot stage, Kofler, Polytherm A and was uncorrected The specific rotation was measured by

a polarimeter ‒ A Krüss Optronic, Germany These instruments are in the Laboratory of the Department of Organic Chemistry, University of Science, National University - Ho Chi Minh City

UV absorption and CD spectra were measured on a JASCO V-570 spectrophotometer and on a JASCO J-820E spectropolarimeter, respectively These instruments are in the Laboratory of Analytical Chemistry, Osaka University, Japan

TLC was carried out on precoated Silica gel 60 F254 or Silica gel 60 RP–18

F254S (Merck) Spots were visualized by spraying with 20% aqueous H2SO4 or 5% ferric chloride solutions 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

Some instruments are at the Central Analysis Laboratory of the University

of Science, National University-Ho Chi Minh City and some are at the

Laboratory of the Department of Organic Chemistry, University of Science, National University - Ho Chi Minh City

Solvents used as n-hexane, chloroform, ethyl acetate, methanol, acetic acid

for TLC and CC were purchased from Chemsol Acetic acid, water, and acetonitrile used for HPLC analysis were purchased from Merck

 Chemical used in the acetylcholinesterase biological activity

 Acetylthiocholine iodide (ATCI) (21.67 mg/ml) dissolved in deionized water (Sigma)

 Acetylcholinesterase (AChE) dissolved in 0.1 % bovine serum albumin (BSA)

 Bovine serum albumin (Sigma)

 5,5’-Dithio-bis-nitrobenzoic acid (DTNB) (Sigma) 0.01 MDTNB is dissolved

in buffer containing 0.1 M NaCl and 0.02 M MgCl2

 Galanthamine (Sigma)

 Buffer of solution Tris HCl 1M pH 8.0 (Invitrogen)

 Enzyme AChE, ATCI, galanthamine are stored at – 80 0C

 BSA, DTNB, Tris HCl are stored at 4 0C

Composition of 1 well

 25 µl of the solution 15 mM acetylthiocholine iodide (ATCI)

 125 µl of the solution 5’-dithio-bis-nitrobenzoic acid (DTNB) (Sigma) 3

mM in the buffer C (50 mM tris–HCl pH 8.0, 0.1 M NaCl, 0.02 M MgCl2)

 50 µl buffer solution B (50 mM Tris–HCl pH8, 0.1% bovine serum albumin (BSA)

 25 µl sample of extract or pure compound is dissolved in buffer solution containing the maximum of 10% MeOH

Samples were tested at 3 concentrations: 1.0 mg/mL, 0.5 mg/ mL and 0.25 mg/ mL

 25 µl Acetylcholinesterase (AChE) 0.22 U/ml (in buffer solution B)

 Pure Galanthamine (Sigma) was used as positive control, at 3 corresponding concentrations (ten time dilution): 0.1 mg/ mL, 0.05 mg/ mL and 0.025 mg/ mL

18

Figure 2.1 Green stem-leaves Boerhaavia diffusa

Stem and leaves (left), flower (middle), root (right)

Figure 2.2 Red-purple stem-leaves Boerhaavia diffusa

Figure 2.3 Stem-leaves Boerhaavia erecta

2.2 PLANT MATERIAL

2.2.1 PLANT MATERIAL OF THE GREEN STEM – LEAVES BOERHAAVIA

DIFFUSA L (Figure 2.1)

Roots of green stem-leaves Boerhaavia diffusa L were collected on the

coastal zone of Phu Hai beach, Phu Thuy Ward, Phan Thiet City, Binh Thuan Province, Vietnam in July, 2010 and the plant was identified by deceased pharmacist Phan Duc Binh, assistant editor of Medicine and Health Semimonthly and by the botanist Vo Van Chi

A voucher specimen of the green B diffusa (No US–A001) was deposited in the herbarium of the Department of Organic Chemistry, Ho Chi Minh City University of Science, National University-HCMC

2.2.2 PLANT MATERIAL OF THE RED-PURPLE STEM-LEAVES

At the same place of Phu Hai beach, Phu Thuy Ward, Phan Thiet City, Binh

Thuan Province, besides the Boerhaavia diffusa L with green leaves and stems,

we found another species whose generic description resembled to B diffusa L

but its leaves and stems were red-purple The botanists Vo Van Chi and Phan

Duc Binh determined that it was also B diffusa

A voucher specimen of whole red-purple B diffusa (No US–A003) was deposited in the herbarium of the Department of Organic Chemistry, Ho Chi Minh City University of Science, National University-HCMC

Our preliminary observation by thin layer chromatography (Figure 2.4) and HPLC (Figure 2.5) showed that they were somewhat different

The HPLC fingerprints of the methanolic extracts of green and red-purple

B diffusa showed a slight distinction in their chemical composition More

importantly, while red-purple B diffusa showed the presence of eupalitin (34) and eupalitin 3-O-β-D-galactopyranoside (35) in considerable amount which

were characteristic compounds in the chemical constituent of B diffusa, such those two compounds were not detected in green B diffusa L

Figure 2.5 HPLC chromatograms of (a) red-purple and (b) green B diffusa

Note: Sample of methanolic extract of green or red-purple B diffusa was dissolved in DMSO and filtered

through an ultra membrance filter (pore size 0.45 μm, Agilent, USA) prior to injection in the sample loop The mobile phases were (A) aqueous acetic acid 0.2% solution and (B) acetonitrile Gradient elution was used from 5% to 10% B in A for six minutes followed by 17%, 25%, 50% 60%, 100% B in A in the subsequent 12, 25, 35, 45, 55 minutes, respectively The UV-Vis detector monitored at the wavelength of

254 nm and 10 μL injection volume for all samples and standards was applied The column was equilibrated

with 100% A for ten minutes prior to each analysis Eupalitin (34) and eupalitin 3-O-β-D-galactopyranoside

(35) were used as internal and external standards under the same running condition.

34

35

Figure 2.4 Comparison of TLC the four methanolic extracts of green

stem-leaves (GS), purple stem-leaves (RS), green root (GR) and

red-purple root (RR) of Boerhaavia diffusa

(a): Elution of chloroform: methanol: H2O (8:2:0.5) TLC spots were developed by 30% H2SO4 solution followed by heating at 100  C

(b): Elution of chloroform: methanol: acetic acid (7:3:0.5) TLC spots were seen by UV254 lamp

(c): Elution of chloroform: methanol: acetic acid (7:3:0.5) TLC spots were developed by ferric chloride 5% in EtOH followed by heating at 100  C

(d): Elution of chloroform: methanol: acetic acid (6:4:0.5) TLC spots were developed by 30% H2SO4 solution followed by heating at 100  C

Therefore, we decided to study on the chemical constituents of the purple leaves Our study later showed that these two plants afforded different organic compounds

red-2.2.3 PLANT MATERIAL OF BOERHAAVIA ERECTA L (Figure 2.3)

The aerial parts of the Boerhaavia erecta (Figure 2.3) were collected at

Linh Trung Ward, Thu Đuc District, Ho Chi Minh City, Vietnam in September,

2010 The scientific name of the plant was determined by the botanist Vo Van

Chi A voucher specimen of B erecta (No US–A002) ) was deposited in the herbarium of the Department of Organic Chemistry, Ho Chi Minh City University of Science, National University-HCMC

2.3 EXTRACTION AND ISOLATION PROCEDURE

DIFFUSA L.

The dried, ground roots (2.90 kg) were macerated with methanol at room temperature (6 x 5 L) The filtrated solution was evaporated to dryness under reduced pressure to afford a methanolic extract (180 g) This residue was partitioned against chloroform then butanol The extracted solutions were separately evaporated under reduced pressure to afford three types of extract: the chloroform residue (28.50 g), butanol residue (29.20 g) and the remaining aqueous residue (80.00 g)

The chloroform residue (28.50 g) was silica gel column chromatographed, eluted with petroleum ether–ethyl acetate (9:1 to 0:10), then ethyl acetate–methanol (10:0 to 1:1), to afford 5 fractions: C1 (4.50 g), C2 (2.50 g), C3 (1.00 g), C4 (1.60 g) and C5 (16.60 g) Silica gel column chromatography on C2,

eluted with petroleum ether–ethyl acetate (9:1) to give 83 (300.0 mg) and 55 (6.0

mg) The same manner was applied on C3 eluted with petroleum ether–ethyl acetate (9:1 to 1:1), then on C-18 and eluted with water:methanol (6:4) to afford

11 (47.0 mg), 21 (54.0 mg) and 65 (6.0 mg) The same manner was applied on

Silica gel column chromatography on M2 eluted with ethyl acetate–

methanol–water (95:5:0.1) to afford 88 (27.0 mg), 89 (18.0 mg) and 90 (15.0

mg) The same manner was applied on M3 eluted with ethyl acetate–methanol–

water (9:1:0.1) to give 91 (35,000.0 mg)

The butanolic residue as well as some other extracts, along with fractions C1, C5, M1, M4, have not been chemically studied because their TLC showed a very low resolution between individual spots

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

the green Boerhaavia diffusa was presented in Scheme 1

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

2.3.2 EXTRACTION AND ISOLATION OF THE AERIAL PARTS OF

RED-PURPLE BOERHAAVIA DIFFUSA L

The dried, ground whole plants (6.00 kg) were macerated with methanol at room temperature (6 x 10 L) The filtrated solution was evaporated to dryness under reduced pressure to afford a methanolic extract (330 g) This residue was partitioned against petroleum ether and ethyl acetate The extracted solutions were separately evaporated under reduced pressure to afford three types of extract: the petroleum ether residue (20.80 g), ethyl acetate residue (25.00 g) and the remaining methanolic residue (260.00 g)

The ethyl acetate residue (25.00 g) was silica gel column chromatographed, eluted with hexane–ethyl acetate (9:1 to 0:10), then ethyl acetate–methanol (10:0

to 1:1), to afford 6 fractions: EA.A (0.90 g), EA.B (2.50 g), EA.C (2.30 g), EA.D (1.80 g), EA.E (1.40 g), EA.F (1.50 g) and four other fractions (12.50 g)

Silica gel column chromatography on EA.A (0.90 g), eluted with hexane–chloroform (9:1) to give three sub-fractions: A1 (0.37 g), A2 (0.23 g) and A3 (0.21 g) Silica gel column chromatography on A2, eluted with hexane–

chloroform (9:1), gave 87 (15.80 mg) The same manner was applied on EA.B

(2.50 g), eluted with hexane–chloroform (1:1) to afford four sub-fractions: B1 (1.20 g), B2 (0.20 g), B3 (0.53 g) and B4 (0.12 g) Sub-fraction B2 was silica gel

column chromatographed to afford 62 (18.8 mg) and 11 (11.0 mg) The same manner was applied on B3 to obtain 57 (5.7 mg), 15 (8.0 mg), 9 (12.0 mg), 10 (13.0 mg), and on B4 to give 56 (11.0 mg) and 6 (25.4 mg)

Silica gel chromatography on EA.C (2.3 g), eluted with chloroform–

methanol (95:5), afforded 86 (2.4 mg), 85 (2.1 mg), 80 (12.3 mg) and 75 (17.4 mg); on EA.D (1.8 g), eluted with chloroform–methanol (9:1) yielded 59 (4.2 mg), 76 (21.7 mg) and 60 (870.0 mg) Reverse phase column chromatography on EA.E (1.4 g), eluted with water–methanol (6:4) obtained 77 (8.9 mg) and 78 (6.2

mg) The same manner was applied on EA.F (1.5 g), eluted with water–methanol

(6:4 to 75:25) to afford 79 (34.7 mg), 82 (2.2 mg) and 71 (11.8 mg)

The petroleum ether residue and the remaining residues have not been chemically studied because their TLC showed a very low resolution between individual spots

The procedure of extraction and isolation of compounds from the

red-purple Boerhaavia diffusa was presented in Scheme 2

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

2.3.3 EXTRACTION AND ISOLATION OF THE AERIAL PARTS OF

BOERHAAVIA ERECTA L

24

The dried, ground aerial parts (3.8 kg) were macerated with methanol at room temperature (6 x 10 L) The filtrated solution was evaporated to dryness under reduced pressure to afford a methanolic extract (245 g) This residue was partitioned against hexane and ethyl acetate The fractionated solutions were separately evaporated under reduced pressure to afford three types of residue: the hexane residue (38.1 g), ethyl acetate residue (25.1 g) and the remaining methanolic residue (160.0 g)

The ethyl acetate residue (25.1 g) was silica gel column chromatographed, eluted with hexane–ethyl acetate (8:2 to 0:10), followed by ethyl acetate–methanol (10:0 to 1:1), to afford 6 fractions: EA.A (3.9 g), EA.B (4.5 g), EA.C (2.3 g), EA.D (7.1 g), EA.E (2.4 g) and EA.F (3.5 g)

Silica gel column chromatography on EA.B (4.5 g), eluted with hexane–chloroform (1:1) to give four sub-fractions: EA.B1 (0.93 g), EA.B2 (0.74 g), EA.B3 (0.85 g) and EA.B4 (1.5 g) Silica gel column chromatography on EA.B2

(0.74 g), eluted with chloroform–methanol (98:2) to afford 15 (20.0 mg), 93 (4.7 mg) and 72 (3.0 mg) Column chromatography on C-18 silica gel applied on EA.B3 (0.85 g), eluted with water–methanol (6:4) to afford 11 (72.0 mg), 21 (154.0 mg), 16 (14.0 mg), 54 (80.0 mg) and 39 (5.0 mg)

Column chromatography on EA.D (7.1 g), eluted with chloroform–methanol (10:0 to 9:1) to afford four sub-fractions: EA.D1 (0.35 g), EA.D2 (1.25 g), EA.D3 (1.30 g) and EA.D4 (4.1 g) Reverse phase column chromatography

on EA.D1 (0.35 g), eluted with water–methanol (6:4) gave 63 (76.0 mg), 81 (34.0 mg), 73 (28.3 mg) and 74 (18.4 mg) The same manner was applied on EA.D3 (1.3 g), eluted with water–methanol (7:3) to give 18 (22.0 mg), 58 (65.0 mg), 84 (60.0 mg), 69 (12.5 mg) and 70 (13.0 mg), and on EA.D4 (4.1 g), eluted with water–methanol (75:25) to obtain 56 (8.7 mg), 64 (5.0 mg), 66 (20.0 mg),

75 (13.0 mg), 60 (170.0 mg), 61 (184.0 mg) and 47 (44.0 mg) Silica gel column

chromatography on EA.F (3.5 g), eluted with chloroform–methanol–water

(8:2:0.1) afforded 67 (85.0 mg), 31 (11.4 mg), 32 (4.4 mg), 36 (24.0 mg), 33 (1,100.0 mg), 30 (9.0 mg), 71 (10.0 mg), 68 (9.4 mg) and 92 (8.0 mg)

The hexane residue and the remaining residue were not chemically studied because their TLC showed a very low resolution between individual spots

The procedure of extraction and isolation of compounds from the aerial

parts of Boerhaavia erecta was presented in Scheme 3

The chemical structures of the isolated compounds was studied by spectroscopic methods such as MS, 1D and 2D-NMR as well as comparison their data with the ones in the literatures

2.4 BIOLOGICAL ASSAYS

2.4.1 CYTOTOXIC ACTIVITY AGAINST THREE CANCER CELL LINES

Determination of cytotoxic activities against the HeLa (human epithelial carcinoma), MCF–7 (human breast cancer) and NCI-H460 (human lung cancer) cell lines 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[57][82]

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, 784355 Ho Chi Minh City, Vietnam The responsible for the unit: 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 oC

in a 5% CO2 atmosphere with 95% humidity Viable cells were counted and inoculated in 96-well plate with density of 104 cells/100 µL/ well After 24 h the 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

in an atmosphere of 5% CO2, 95% humidity at 37 oC for 48 h Adherent cell cultures were fixed by adding 50 µL of cold 50% (w/v) trichloroacetic acid per well and incubated at 4 oC for 1 h The plates were washed five times with

% Inhibitive activity = ODc – ODs / ODc x 100

With OD = OD’ tested sample – OD’ blanck

OD’ = OD492 – OD620 ODc = OD of 0.25% DMSO ODs = OD tested sample Evaluation of the result based on the I% (at the concentration of 100

g/mL): 0–49% (inactive), 50–70% (active), 70–90% (good), 90–100% (very

Samples were sent to be in vitro tested at the Department of Pharmacy,

University of Medicine and Pharmacy, 41 Dinh Tien Hoang Str., Dist 1, Ho Chi Minh City, Vietnam The responsible for the unit: Assoc Prof Vo Phung Nguyen

The principle of the method is the enzyme acetylcholinesterase (AChE) hydrolyzes the substrate (Acetylthiocholine is used as the substrate) to give a compound This compound will further react with Ellman reagent, 5,5’-dithio-bis-2-nitrobenzoate ion (or DTNB) to give 2-nitrobenzoate-5-mecaptothiocholine and 5-thio-2-nitrobenzoate The later possesses a yellow color and therefore, the rate of color production is measured at 405 nm in a spectrophotometer All samples were tested in triplicate

The 250 µl of the mixed solutions (*) were kept in 15 minutes at 25 ºC, then the optical absorbance (OA) of the mixture was measured by ELISA reader at

405 nm The percent inhibitive activity was determined by using the following formula:

% Inhibition = [(OA1-OA2)-(OA3-OA4)]/(OA1-OA2)

With:

OA1: the OA of the mixed solution (*) with replacement 25µl of buffer B,

instead of 25µl of samples

OA2: the OA of the mixed solution (*) with replacement 50µl of buffer B,

instead of 25µl of samples and 25µl of 0.22 U/ml AChE

OA3: the OA of the mixed solution (*)

OA4: the OA of the mixed solution (*) with replacement 25µl of buffer B,

instead of 25µl of 0.22 U/ml AChE

Evaluation of the result based on the I% (at the concentration of 1.0 mg/mL): 0–30% (very weak), 30–50% (weak), 50–70% (average), 70–90% (good), 90–100% (very good)

The result of acetylcholinesterase inhibition was presented in Table 3.25

28

Scheme 1: Extraction and isolation procedure for green Boerhaavia diffusa

Ground material of roots of

green Boerhaavia diffusa

(2,900.0 g)

Methanolic extract (180.0 g)

Chloroform residue (28.5 g)

Butanolic residue (29.2 g)

Aqueous residue (80.0 g)

C2 (2.48 g)

C3 (1.0 g)

C4 (1.6 g)

- Macerated with methanol at room temperature (6x5 L)

- Filtrated, evaporated in the reduced pressure

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

- Partitioned against chloroform and then butanol

- Evaporated in the reduced pressure

CC PE:EA (9:1 – 1:1)

CC C:M (95:5) C:M (9:1)

- Dissolved in MeOH

- Filtrate

Methanolic residue (54.4 g)

Residue (22.1 g)

CC EA:M (10:0 – 1:1)

M2 (1.5 g)

M3 (42.0 g)

CC EA:M:W (95: 5: 0.1)

M1 (1.2 g)

M4 (12.0 g)

C5 (16.6 g)

C1

(4.5 g)

RP W: M (6: 4)

Scheme 2: Extraction and isolation procedure for red purple Boerhaavia diffusa

Ground material of whole plants

of red-purple Boerhaavia diffusa

(6,000.0 g)

Methanolic extract (330 g)

Petroleum ether residue

(20.8 g)

Ethyl acetate residue (25.0 g)

Remaining residue (260.0 g)

EA.A

(0.9 g)

EA.B (2.5 g)

EA.C (2.3 g)

- Macerated with methanol at room temperature (6x10 L)

- Filtrated, evaporated in the reduced pressure

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

- Partitioned against petroleum ether and ethyl acetate

- Evaporated in the reduced pressure

EA.E (1.4 g)

B1 (0.90 g)

B2 (0.20 g)

B3 (0.53 g)

B4 (0.12 g)

C1 (0.43 g)

C3 (0.24 g)

C4 (1.26 g)

C2 (0.05 g)

D1 (0.5 g)

D3 (0.21 g)

D4 (1.02 g)

D2 (0.07 g)

E1 (0.60 g)

F1 (0.87 g)

F2 (0.27 g)

E2 (0.40 g)

F3 (0.12 g)

F4 (0.11 g)

EA.A

(3.9 g)

EA.B (4.5 g)

EA.C (2.3 g)

- Macerate with methanol at room temperature (6x10 L)

- Filtrate, evaporate in the reduced pressure

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

- Partition against petroleum ether and ethyl acetate

EA.E (2.4 g)

C:M:W (8:2:0.1)

EA.D1 (0.35 g)

EA.D2 (1.25 g)

EA.D4 (4.10 g)

EA.B1

(0.93 g)

EA.B3 (0.85 g)

RP C 18 W:M (6:4) H:C (1:1)

RP C 18 W:M (7:3)

RP C 18 W:M (75:25)

EA.B2

(0.74 g)

EA.B4 (1.50 g)

EA.D3 (1.30 g)

CHAPTER 3 RESULTS AND DISCUSSION

3.1 CHEMICAL STRUCTURE ELUCIDATIONS

From three species of the genus Boerhaavia, 55 compounds were isolated

including 16 new compounds along with 39 known ones The new compounds were verified by checking with Scifinder software at Leuven University, Belgium in Jannuary 2013 The structures of all compounds were elucidated on the basis of NMR and MS spectroscopic experiments The compounds were divided into 8 groups as listed below:

Group A: 12a-Hydroxyrotenones (five compounds, three news and two knowns) Group B: Dehydrorotenones (ten compounds, five news and five knowns) Group C: Coumaronochromones (four compounds, two news and two knowns)

Group E: Nitrogen compounds (three known compounds)

Group F: Phenolic compounds (twelve compounds, two news and ten knowns) Group G: Terpenoid and steroid (six compounds, one news and five knowns)

3.1.1 CHEMICAL STRUCTURE OF 12a-HYDROXYROTENOIDS (GROUP A)

3.1.1.1 Structure elucidation of Boeravinone C (11)

Compound 11 was obtained from the fraction C3 of Scheme 1, from the

fraction EA.B2 of Scheme 2 and from the fraction EA.B3 of Scheme 3 with essential physical data as described below

 Pale yellow needle, mp 247 – 249 oC (methanol)

1H and 13C spectra (Acetone-d6) (Appendix 1 and 2): see Table 3.1 and Table 3.2

 HSQC, HMBC spectra (Acetone-d6) (Appendix 3 and 4)

Discussion of chemical structure

Its 1H-NMR spectrum indicated the presence of four aromatic methines at H

7.81, 6.86, 6.85 and 6.20, one methine at H 4.78, one methylene at H 4.49 and 4.45, one methoxyl at H 3.93 and one methyl at H 1.97 The 13C-NMR spectrum of

32

11 showed eighteen signals including one methoxyl, one methyl, one methine, one

methylene, four aromatic methine carbons, five oxygenated quaternary carbons, four quaternary carbons and one carbonyl carbon at C 195.2

The substitution pattern was confirmed by HMBC correlations The proton at

H 4.78 (H-6a) showed interactions with C-12a (C 66.8) and C-6 (C 62.3), whereas the methylene proton at H 4.49 (H-6) correlated with C-4a (C 143.9), C-12a (C

66.9) and C-6a (C 77.0) These correlations of HMBC spectrum confirmed the

skeleton of 12a-hydroxyrotenone of 11 The presence of methoxyl group at C-9 and

the methyl group at C-10 also be inferred through HMBC correlations Therefore,

compound 11 was elucidated as a typical rotenoid, boeravinone C.[36]

3.1.1.2 Structure elucidation of 10-Demethylboeravinone C (21)

Compound 21 was obtained from the fraction C3 of Scheme 1, from the

fraction EA.B2 of Scheme 2, and from the fraction EA.B3 of Scheme 3 with essential physical data as described below

 White needle, mp 232 – 234 oC (methanol)

1H and 13C spectra (Acetone-d6) (Appendix 5 and 6): see Table 3.1 and Table 3.2

 COSY, HSQC, HMBC spectra (Acetone-d6) (Appendix 7, 8 and 9)

Discussion of chemical structure

The spectral features of compound 21 resembled those of boeravinone C The differences were that the signal for the quaternary carbon C-10 in 21 was replaced

by a methine carbon in 21 and the disappearance of the methyl group The structure

of 21 was further confirmed by an HMBC experiment The methoxyl group at H

3.88 was correlated with C-9 (C168.8)

4,11,12a-Trihydroxy-9-methoxyl-10-methylrotenone

or Boeravinone C (11)