thành phần hóa học của cây lá xuân hoa mạng pseuderanthemum reticulatum radlk họ ô rô (acanthaceae)

viii Table 1: The content of some major and trace minerals in the Table 2: The NMR data of RE-EA1 and comparison of its ones Table 3: The NMR data of RE-EA3 and comparison of its ones

ĐẠI HỌC KHOA HỌC TỰ NHIÊN



NGUY ỄN PHI LINH

Pseuderanthemum reticulatum Radlk

LU ẬN VĂN THẠC SĨ KHOA HỌC HÓA HỌC

NGƯỜI HƯỚNG DẪN KHOA HỌC

GS TS NGUY ỄN KIM PHI PHỤNG

Tp Hồ Chí Minh, 2011

1.1.1.3.2 P carruthersii var ovatifolium (Brem.) Brem 4

v

2.4 Analysis of fatty acid of two glycoglycerolipids 13

3.5 Structure elucidation of two diastereomers RE-EA7 and RE-EA8,

3.5.1 Structure elucidation of compound RE-EA7 21

3.5.2 Structure elucidation of compound RE-EA8 23

APPENDICES

vi

1D/2D-NMR : One/Two Dimensional – Nuclear Magnetic Resonance

AchE : Acetylcholinesterase

ALT : Alanine aminotransferase

AST : Aspartate aminotransferase

DMSO : Dimethyl sulfoxide

DEPT : Distortionless Enhancement by Polarization Transfer

EA : Ethyl acetate

HIV : Human Immunodeficiency Virus

HSQC : Heteronuclear Single Quantum Correlation

HMBC : Heteronuclear Multiple Bond Coherence

HPLC : High Performance Liquid Chromatography

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

J : Coupling constant

GC-MS : Gas Chromatography - Mass Spectrometry

LC : Liquid Chromatography

viii

Table 1: The content of some major and trace minerals in the

Table 2: The NMR data of RE-EA1 and comparison of its ones

Table 3: The NMR data of RE-EA3 and comparison of its ones

Table 4: The NMR data of RE-EA4 and comparison of its ones with

those of stigmasterol, β-sitosterol and β-D-glucopyranose 20

Table 6: The NMR data of RE-EA8 and RE-EA7 and comparison

of their ones with those of (6S,9S)-vomifoliol 24 Table 7: The comparison of 13C-NMR data of RE-EA7, RE-EA8 and the

aglycon of RE-EA6 with those of the aglycons of four roseoside

Table 10: The NMR data of RE-Bu4 and comparison of its ones with

Table 11: The NMR data of RE-PE1 and comparison of its ones with

those of 1-linolenoyl-2-palmitoyl-3-galactosylglycerol 36

ix

Figure 1: Xuân Hoa Nhọn, P acuminatissimum (Miq.) Kuntze 2 Figure 2: Xuân hoa lá-hoa, P bracteatum Imlay 2 Figure 3: Xuân hoa đỏ, P carruthersii (Seem.) Guill var

Figure 4: Nấp vũm, P carruthersii var ovatifolium (Brem) Brem 2

Figure 7: Xuân hoa, P palatiferum (Nees) Radlk 2

Scheme 1: Extraction procedure of Pseuderanthemum

Scheme 2: Isolation procedure of compounds from

x

Scheme 4: Isolation procedure of compounds from

Chapter 1 LITERATURE REVIEW & AIM OF STUDY

1.1 General information of Pseuderanthemum

Pseuderanthemum or Eranthemum[1] is a member of Acanthaceae The genus

contains about 196 species, including perennial shurbs or herbs The characteristics of

the genus are beautiful flowers and foliage, high humidity need, flowering in spring

and summer

acuminatissimum (Miq.) Kuntze (figure 1), P bracteatum Imlay (figure 2), P

carruthersii (Seem.) Guill var atropurpureum (Bull.) Fosb (figure 3), P carruthersii

var ovatifolium (Brem.) (figure 4), P crenulatum (Lindl.) R Ben (figure 5), P

eberhardtii R Ben (figure 6), P palatiferum (Nees) Radlk (figure 7), P poilanei R

Ben.Z (figure 8), P reticulatum Radlk (figure 9) and P tonkinense R Ben (figure 10)

In addition, we collected pictures of some species of Pseuderanthemum from

laxiflorum (figure 13)[34], P sinuatum (figure 14)[35], P tuberculatum (Hook f.) Radlk

1.1.1 Generic description

In this section, the botanic morphology of some Vietnamese Pseuderanthemum

species was described

Vietnamese name: Xuân hoa nhọn (figure 1)

Perennial shrub Stems terete in older positions, glabrous and tetragonal in

younger positions Leaves 10-20 cm long, 5-9 cm wide; costa and lateral veins 6 pairs,

petioles 4-5 cm long; peduncle 20 cm long Flowers in cymes, bisexual, often

zygomorphic; calyx 3.5 cm long, glabrous; corolla tube 3.5 cm long Capsule 3.5 cm

long, glabrous

The herb, a species of Java Island (Indonesia), has been planted in Singapore and

Vietnam (Bảo Lộc, Sài Gòn)

Figure 1: Xuân hoa nhọn,

P acuminatissimum (Miq.) Kuntze Figure 2: Xuân hoa lá-hoa, P bracteatum Imlay

Figure 3: Xuân hoa đỏ,

P carruthersii (Seem.) Guill var

atropurpureum (Bull.) Fosb

Figure 4: Nấp vũm,

P carruthersii var ovatifolium (Brem)

Brem

Figure 5: P crenulatum (Lindl.) R Ben Figure 6: P eberhardtii R Ben

Figure 7: Xuân hoa, P palatiferum (Nees) Radlk Figure 8: P poilanei R Ben.Z

Figure 9: Xuân hoa mạng, P reticulatum Radlk Figure 10: P tonkinense R Ben

Figure 11: Eranthemum pulchellum Figure 12: P alatum

Figure 15: P tuberculatum (Hook f.) Radlk Figure 16: P variabile

1.1.1.2 P bracteatum Imlay.[1]

Vietnamese name: Xuân hoa lá-hoa, Hoàn ngọc đỏ, Hồng ngọc (figure 2)

Herb, 50-60 cm tall Stems quadrangular and ciliate in younger positions Leaves

5-9 cm long, 3-5 cm wide; costa and lateral veins 5-6 pairs; petioles 2-3 cm long

wide, ciliate; lobes 6-7 mm long, stamens 2 Capsule 2 cm long, glabrous

The herb has wildly growed on Dinh mountain, Bà Rịa-Vũng Tàu province

This species has two varieties consisting of P carruthersii (Seem.) Guill var

atropurpureum (Bull.) Fosb and P carruthersii var ovatifolium (Brem.) Brem

Vietnamese name: Xuân hoa đỏ, Ô rô đỏ, Nhớt tím (figure 3)

Shrub, 1-2 m tall Leaves, elliptic-ovate, smooth, violet-red, mottled

yellow-green, short petioles Flowers in cymes, bisexual; corolla tube rosy-purple, 1.3 cm

long; stamens 2

The herb has been cultivated for ornamentation in Ho Chi Minh city

Vietnamese name: Nấp Vũm (figure 4)

Herb, different variety of P carruthersii (Seem.) Guill var atropurpureum

(Bull.) Fosb due to leaves oval, 8.5 cm long, 4.5 cm wide, costa and lateral veins 7-8

pairs

The herb has wildly growed in Sông Bé provine and Sài Gòn

Vietnamese name: Xuân hoa (figure 7)

Perennial shrub, 1-2 m tall Leaves 12-17 cm long, 3.5-5 cm wide, opposite,

decussate, lanceolate, petioles 1.5-2.5 cm long Flowers in cymes, bisexual, often

zygomorphic; peduncles 10-16 cm long; pedicles 0-5 mm long; bracts

linear-triangular, 2.5mm long; calyx 4-5 lobed, 5-7 mm; corolla tube 25 mm long, pubescent,

5-lobed but often 2-lipped with lower lip 3-lobed and upper lip; stamens 2, attached to

corolla Capsule woody, clavate with long, glabrous

1.1.1.5 P reticulatum Radlk.[1]

Vietnamese name: Xuân hoa mạng (figure 9)

Perennial shrub, 0.5-2.5 m tall Stems quadrangular and ciliate in younger

positions Leaves opposite and decussate, 8-15 cm long, 5-10 cm wide, oval, glabrous;

the costa and lateral veins 9-10 pairs; petioles 1 cm long Flowers in cymes, bisexual,

zygomorphic; corolla tube 1.2 cm long, 5-lobed but often 2-lipped with lower lip

3-lobed and upper lip; stamens 2; calyx 2 mm

The shrub has wildly growed in Bà Nà mountain, Đà Nẵng province

1.1.2 Pharmaceutical studies of Pseuderanthemum

Until now, there has only been the pharmaceutical information on three species of

Pseuderanthemum including P carruthersii (Seem.) Guill var atropurpureum (Bull.)

Fosb., P latifolim and P palatiferum (Nees) Radlk However, P palatiferum (Nees)

Radlk has merely been studied on pharmaceutical by modern methods

P carruthersii (Seem.) Guill var atropurpureum (Bull.) Fosb.: leaves, roots and

P latifolim: in Yunnan (China), it has been used as a medicinal herb, and has

been a potential herb in the group of species that were screened for HIV healing by

P palatiferum (Nees) Radlk: it was found in Vietnam since 1980s and has been

used as a medicinal herb Now this plant has been studying on chemical constituents

and pharmaceutical by using modern methods

In Vietnam, P palatiferum (Nees) Radlk has been planted in many places as a

be used as folk remedy for haemostatic, restoring of health for sick and exhausted

people, treating digestive disorder, diarrhea, dysentery, constipation, gastritis,

gastroduodenal ulcer, internal haemorrhoid, nephritis, haematuria, prostate ulcer,

regulation of hypertension

of crude extract of leaves of P palatiferum The results indicated that the crude extract

had inhibitory activity against Gram-negative bacteria (Escherichia coli, Pseudomonas

Streptococcus pyogenes), molds (Aspergillus niger, Fusarium oxysporum, Pyricularia

oryzae, Rhezoctonia solanii) and yeasts (Saccharomyces cerevisiae, Candida

albicans)

antifungal activity of ethyl acetate and n-butanol extracts of P palatiferum at the

concentration of 10 mg/ml by using disk diffusion technique The results showed that

both extracts had inhibitory activity against Gram-positive bacteria (Bacillus subtilis,

Staphylococcus aureus), Gram-negative bacteria (Escherichia coli) and molds

(Candida albicans, Candida stellatoides); and the activity of the ethyl acetate extract

was higher than the n-butanol one The study also exhibited that the ethyl acetate

extract had high inhibitory activity against Salmonella typhi 158 (the diameter of zone

of inhibition of 21 mm) They also studied the antioxidant activity of the extracts by

investigating the effect of these extracts on peroxydase enzyme in blood (Savron’s

method) and the results revealed that both extracts had antioxidant activity

P palatiferum had antibacterial activity against Staphylococcus aureus (IC50 174.9

g/ml)

leaves of P palatiferum on Siamese Fighting fish The results indicated that the

extracts had no toxic effect on Siamese Fighting fish The same results were obtained

from the studies on mice and rabits

effect of crude extract of P palatiferum on mice The results of acute toxicity tests

indicated that the crude extract at doses of 0.83, 1.67, 3.13, 5.56, 9.19 and 11.5 g/kg

body weight did not have the acute toxicity effect on mice The hepatoprotective effect

cells The results of hepatoprotective effect test exhibited that at the toxic dose of 1 ml

aminotransferase) and ALT (alanine aminotransferase) were very high, but the MDA

(malondialdehyde) content remarkably reduced from 14.12 nM to 10.71 nM and the

the MDA level significantly reduced from 11.57 nM to 6.25 nM, the AST, ALT levels

decreased a little, and the liver cells recovered

diarrhea of piglets The results of treatment with P palatiferum powder was compared

with two prevalent effective antibiotics against piglets’ diarrhea: Coli-norgen and

Cotrimxazol The results showed that the recovered rate after three days of treatment

of P palatiferum powder, Coli-norgen and Cotrimxazol were 92.86, 90.48 and 83.33

%, respectively; the relapsed rate were 7.14, 9.52 and 14.29 %; the duration of

diarrhea were 2.16, 2.24 and 2.03 days, respectively

inhibitory effect of the aqueous extract of leaves of P palatiferum on albino rats The

results showed that only AChE activity in the hippocampus was significantly inhibited

by the extract at the dose of 0.7 and 1.0 g/kg body weight However, AChE activity in

the serum and red blood cells of treated rats showed no significant differences from

that of controls These results suggested that the extract could reduce the synthesis of

AchE in the rats’ brain; and the remarkably inhibitory effect of the extract indicated

the potential of this plant in the treatment of Alzheimer’s disease

1.1.3 Chemical studies of Pseuderanthemum

Up to now, there have only been the chemical studies on four species of

Pseuderanthemum including Eranthemum pulchellum, P carruthersii atropurpureum,

P latifolim and P palatiferum

1.1.3.1 Eranthemum pulchellum

glucoside eranthemoside (24) from the peduncle of Eranthemum pulchellum

1.1.3.2 P carruthersii atropurpureum

(8) and stigmasterol (9), a mixture of β-sitosterol 3-O-β-D-glucopyranoside (10) and

stigmasterol 3-O-β-D-glucopyranoside, a mixture of oleanolic acid (14) and ursolic

acid (15), indole-3-carboxaldehyde (35), uracil (34), adenine (27) and betaine (33)

from the leaves of P carruthersii atropurpureum

1.1.3.3 P latifolim

P latifolim including stigmasterol (9), stigmasterol 3-O-β-D-glucopyranoside (11), an

ester of β-sitosterol 3-O-β-D-glucopyranoside sitoindoside I (13), an iridoid

anthirrinoside (25), three flavonoids 7,4'-dihydroxyflavone (28),

5,4'-dihydroxy-7-methoxyflavone (29) and 5,6-dihydroxy-3',4',7,8-tetra5,4'-dihydroxy-7-methoxyflavone (30), a purine

skeleton compound allantoin (26)

1.1.3.4 P palatiferum

leaves of P palatiferum The results (see table 1) showed that the content of mineral

elements including Na, K, Ca, Mg, Al and Fe in the leaves of P palatiferum were very

high (40-900 mg/100 g fresh leaves)

Table 1: The content of some major and trace minerals in the leaves of P palatiferum

Major minerals Content

(mg/100g fresh leaves) Trace minerals

Content (mg/100g fresh leave)

of P palatiferum including phytol (22), β-sitosterol (8), β-sitosterol

3-O-β-D-glucopyranosid (10) and a mixture of stigmasterol (9) and poriferasterol (12)

Four compounds and two mixtures were isolated from the dried leaves of

P palatiferum by Phan Minh Giang et al.[13] (2003) including 1-pentacosanol (2),

palmitic acid (5), β-sitosterol (7), stigmasterol (8), a mixture of β-sitosterol

O-β-D-glucopyranoside (10) and stigmasterol O-β-D-O-β-D-glucopyranoside (11), a mixture of

3-methoxykaempferol glucopyranoside (31) and apigenin

7-O-β-D-glucopyranoside (32)

Võ Hoài Bắc et al.[10] (2003) anlysed the amino acids content of P palatiferum

leaves, collected at the different times of year The results revealed that the total

content of free and linked amino acids in the leaves, which were collected between

January and February or between September and October, was relatively high (751-1365 mg%); especially, the isoleucine, leucine and valine contents were very

high (25-150, 46-85 and 20-1001 mg%, respectively) The best time for collecting was

Septemper and October because of the extremely high content of free amino acids

(1347 mg%)

1-hexadecanoate (5), salicylic acid (6), palmitic acid (4) from the leaves of P

palatiferum

β-sitosterol (8) and stigmasterol (9), a mixture of β-β-sitosterol 3-O-β-D-glucopyranoside

(10) and stigmasterol 3-O-β-D-glucopyranoside (11)

phytol (22), palmitic acid (4), β-sitosterol (8), stigmasterol (9),

24-methylenecycloartanol (20), loliolide (7), a mixture of β-sitosterol

3-O-β-D-glucopyranoside (10) and stigmasterol 3-O-β-D-3-O-β-D-glucopyranoside (11) from the leaves

of P palatiferum

Six compounds including lupeol (18), lupenone (19), betulin (21), pomolic acid

(16), palmitic acid (4) and asperglaucide (36) were isolated from the roots of P

palatiferum by Trần Công Khánh et al.[7] (2007)

Ursolic acid (15)

HO

COOH HO

R

HO

COOH HO

1.2 Aim of study

Nowadays, the advance of sciences and technologies, as well as those of

chemistry, significantly contribute to improving the quality of human life, and

especially treating diseases A great number of medicines have been synthesized for

restoring health and curing diseases, but some of them are expensive and have side

effects Hence, people have a tendency to return to traditonal medicine, and used

medicinal herbs for treatments The phytochemical and pharmaceutical studies of

medicinal herbs are very important because they not only clarify the activities of

medicinal herbs, but also contribute to confirm the value of medicinal herbs and

discover new bioactive compounds for treating diseases

In Vietnam, some Pseuderanthemum species have been used as medicinal herb

for treating digestive disorder, liver disease, restoring health, ect However, there have

been a few studies about the phytochemistry and bioactivity of Pseuderanthemum and,

above all, no studies on P reticulatum Radlk Therefore, the thesis on contribution to

the chemical study of leaves of P reticulatum Radlk has been carried out

Chapter 2 EXPERIMENTAL

2.1 Materials

The solutions of different extracts were evaporated by using rotary evaporator

Buchi-111 1H-NMR, 13C-NMR and 2D-NMR spectra were acquired on Bruker

Avance 500III (500 MHz for 1H-NMR and 125 MHz for 13C-NMR) HR-MS were

recorded on Bruker microOTOF Q-II Preparative HPLC were carried out on LC-8A

Shimadzu preparative liquid chromatograph with Sulpeco C18 column (10 µm, 25 cm x

21.2 mm) and UV-Vis Spd-20A detector at 245 nm GC-MS was carried out on

Agilent technologies 7890A GC System, HP-5 MS capillary column (5% phenyl

methyl silox, 325 °C, 30 m x 250 µm x 0.25 µm), Agilent technologies 5975C VL

MSD with triple-Axis detector Optical rotations were measured on a Kruss (German)

polarimeter All instruments are in the Center Analysis of the University of Science,

National University- Ho Chi Minh City

Solvents: Petroleum ether (60-90 °C), chloroform, ethyl acetate, n-butanol,

methanol, acetic acid

Thin-layer chromatography (TLC) and preparative TLC was performed on silica

gel GF254 (Merck), chemicals visualizing TLC plates: 30% aqueous solution of H2SO4

Column chromatography was performed on silica gel (Merck) type 100

(70–230 Mesh ASTM)

2.2 Plant material

The leaves of P reticulatum Radlk were collected in Vũng Tàu province, and the

scientific name was identified by Pharmacist Phan Đức Bình, Assistant Editor of

Medicine and Health Semimonthly A voucher specimen (No US-A009) was deposited

in the herbarium of the Department of Organic Chemistry, Univeristy of Science

2.3 Extraction and isolation procedures

The clean, air-dried and ground material (3.2 kg) was extracted by ethanol at

ambient temperature, and the filtrated solution was concentrated under reduced

pressure to afford ethanol residue (450.0 g) The residue was dissolved in solvent

systems of methanol: water (1: 9); then was partitioned against petroleum ether, ethyl

acetate and n-butanol, respectively; and the obtained solutions were evaporated to

afford corresponding residues: petroleum ether (PE) residue (120.0 g), ethyl acetate

(EA) residue (20.5 g), n-butanol (Bu) residue (30.3 g) and water residue (278.1 g)

The PE residue (120.0 g) was subjected to silica gel column chromatography,

eluted with a gradient solvent system of petroleum ether: ethyl acetate (10: 0 to 0: 10)

and then ethyl acetate: methanol (10: 0 to 0: 10) to yield five fractions: PE-A (30.5 g),

PE-B (20.8 g), PE-C (22.4 g), PE-D (8.3 g), PE-E (10.7 g)

The EA residue (20.5 g) was applied to silica gel column chromatography, eluted

with gradient solvent systems of ethyl acetate and then ethyl acetate: methanol from 1

to 100 % methanol to yield three fractions: EA-A (1.2 g), EA-B (5.5 g) and EA-C

(3.2 g)

The Bu residue (30.3 g) was subjected to silica gel column chromatography,

eluted with a gradient solvent system of ethyl acetate and methanol (10: 0 to 0:10) to

yield five fractions: Bu-A (1.2 g), Bu-B (4.1 g), Bu-C (1.0 g), Bu-D (4.0 g) and Bu-E

(4.3 g)

The EA-B residue (5.5 g) was rechromatographed many times and applied to

preparative TLC to afford a mixture of RE-EA2 (20.0 mg), a mixture of RE-EA4

(209.5 mg) and three compounds: RE-EA1 (20.0 mg), RE-EA3 (6.7 mg), RE-EA6

(5.0 mg)

The mixture RE-EA2 (20.0 mg) was subjected to preparative HPLC [Sulpeco C18

(10 µm, 25 cm x 21.2 mm), 16% MeOH aq., 8 ml/min, UV detection (254 nm)] to

yield two compounds RE-EA7 (1.0 mg), RE-EA8 (2.0 mg)

The Bu-B residue (4.1 g) was rechromatographed many times and applied to

preparative TLC to yield three compounds RE-Bu2 (5.0 mg), RE-Bu3 (20.0 mg) and

RE-Bu4 (2.5 mg)

The PE-D residue (8.3 g) was rechromatographed many times to yield two

compounds RE-PE1 (60.0 mg) and RE-PE2 (8.9 mg)

2.4 Analysis of fatty acid of two glycoglycerolipids[29]

Purified glycoglycerolipids ( 2.0 mg of each) was methanolysed by refluxing

with 4.0 ml solution of 5% HCl in methanol, at 95 °C for 4.5 hours Afterward, the

reaction solution was partitioned against n-hexane The n-hexane layer were applied to

°C/min), then held constant for 4 mins in order to determine the fatty acid moities of

two glycoglycerolipids

Scheme 1: Extraction procedure of Pseuderanthemum reticulatum Radlk

Scheme 2: Isolation procedure of compounds from the PE residue of scheme 1

Ethanol residue (450.0 g)

Water residue (278.1 g)

Ground material (3.2 kg)

- Macerate with ethanol at ambient temperature

- Concentrate in reduced pressure

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

- Partition against petroleum ether

Scheme 3: Isolation procedure of compounds from the EA residue of scheme 1

Scheme 4: Isolation procedure of compounds from the Bu residue of scheme 1

Chapter 3 RESULTS & DISCUSSION

3.1 Structure elucidation of compound RE-EA1

 Compound RE-EA1 was isolated as colorless oil

system of petroleum ether: ethyl acetate (1: 1) and visualized by 30% aqueous

Discussion on the chemical structure determination

4-(2-hydroxyethyl)phenol

OH HO

Tyrosol

1 2

3 4 5 6

1' 2'

3.2 Structure elucidation of compound RE-EA3

 Compound RE-EA3 was isolated as yellowish amorphous powder

system of chloroform: methanol (9.5: 0.5) and visualized by UV lamp

Discussion on the chemical structure determination

cis-coupled protons at  7.69 (1H, d, 7.5 Hz, H-6), 5.44 (1H, d, 7.5 Hz, H-5)

13C-NMR spectrum revealed two lactam carbons at  164.4 (C-4), 151.6 (C-2); two

Table 2: The NMR data of RE-EA1 and comparison of its ones

3.3 Structure elucidation of compound RE-EA4

 Compound RE-EA4 was isolated as white amorphous powder

solvent system of chloroform: methanol (85: 15), visualized by 30% aqueous

Discussion on the chemical structure determination

dd, 15.0 Hz; 8.5 Hz, H-22a), 5.01 (1H, dd, 15.0; 9.0 Hz, H-23a) were characteristic of

protons attached to olefinic carbons of stigmasterol and -sitosterol The 1H-NMR also

3-O-β-D-glucopyranoside with the ratio of 3: 7, respectively - because the ratio of integral

values of H-6a and H-6b was 3: 7

Table 3: The NMR data of RE-EA3 and comparison of its ones

3.4 Structure elucidation of compound RE-EA6

 Compound RE-EA6 was isolated as colorless oil

system of ethyl acetate: methanol: water (8: 1: 1) and visualized by 30%

 Mass spectrum (Appendix 9): HR-ESI-MS (positive mode): m/z 409.1894

 1H, 13C and DEPT-NMR spectra (CD3OD) (Appendix 10-14): see Table 5

 COSY, HSQC and HMBC spectra (Appendix 15-17)

Discussion on the chemical structure determination

s, H-12)

C-1 and C-6 indicated that all C-2, C-6, C-11 and C-12 connected with saturated

quaternary carbon C-1 In HMBC spectrum, observed correlations from H-13 to C-4

together respectively as well as C-13 connected to C-5 The olefinic proton H-4

O

OH

1 2 3 4 5 6

11 12

13

O

7 8 9 10

1' 2' 3' 4'

5' 6'

O

OH OH

OH HO

Figure 17: The HMBC correlations of RE-EA6

Table 4: The NMR data of RE-EA4 and comparison of its ones with those

C-7, C-8, C-9 and C-10 connected together, respectively The HMBC correlation of

to the aglycon of RE-EA6 at C-9

The LC/HR-ESI-MS (positive mode) of RE-EA6 gave a pseudomolecular ion

roseoside

Vomifoliol has two chiral centers at C-6 and C-9, therefore it has two pairs of

Table 7) indicated that RE-EA6 was (6S,9R)- or (6R,9R)-roseoside Unfortunately, the