UNIVERSITY OF SCIENCE AND TECHNOLOGY OF HANOI

DEPARTMENT OF CHEMISTRY

Research and Development

BACHELOR THESIS
By
Nguyen Hai Dang
Title:
Investigation on chemical constituents from the stem of Piper betle
Linn.

Supervisor(s): Ph.D. Bui Huu Tai

Department of Structure Determination, Institute of Marine
Biochemistry, Vietnam Academy of Science and Technology

Hanoi, July 2020

 UNIVERSITE DES SCIENCES ET DES TECHNOLOGIES DE HANOI
UNIVERSITY OF SCIENCE & TECHNOLOGY OF HANOI
TRƯỜNG ĐẠI HỌC KHOA HỌC & CÔNG NGHỆ HÀ NỘI

To whom it may concern,

I, Bui Huu Tai, certify that the thesis/ internship report of Mr. Nguyen Hai Dang is
qualified to be presented in the Internship Jury 2020-2021.

Hanoi, ……………………………,.........

Supervisor’s signature

ii
TABLE OF CONTENT
ACKNOWLEDGEMENTS…………………………………….…………………….iv
LIST OF ABBREVIATIONS………………………………………….……………...v
LIST OF TABLES…………………………………………………………………….vi
LIST OF FIGURES………………………………………….……….………………vii
ABSTRACT……………………………………………………………………………1
CHAPTER 1: INTRODUCTION……………………………..………….…………..2
CHAPTER 2: OVERVIEW…………………………………………………………...4
CHAPTER 3: MATERIALS AND METHODS…………………………………….13
CHAPTER 4: EXPERIMENTALS………………………………………………….15
CHAPTER 5: RESULTS AND DISCUSSION……………………………………...18
CHAPTER 6: CONCLUSION AND PERSPECTIVE……………………….…….25
REFERENCES…………………………………………………………..………..….26

iii
ACKNOWLEDGEMENTS

The completion of this thesis took place mainly at the Department of Structural
Determination of the Institute of Marine Biochemistry in Vietnam Academy of Science
and Technology. During my research experience, I was fortunate enough to get to meet
and know all the staff and researchers of the department. I would like to express my
gratitude to all those who have helped me throughout the internship process.

I would like to thank Assoc. Prof. Phan Van Kiem and Assoc. Prof. Nguyen Xuan
Nhiem for letting me join their research laboratory. I would especially like to thank M.Sc.
Do Thi Trang and Ph.D. Bui Huu Tai for their direct supervision and guidance during
the experimentation process as well as all the members of the Department for their
overall generosity and hospitality.

I want to say a great thank you to all the staff of the Chemistry department of the
University of Science and Technology of Hanoi, especially Assoc. Prof. Tran Dinh
Phong for continually looking after our class during the three years of higher studies and
providing us the best conditions to do our research. I’ve gained a new perspective of
real-life work world now am comfortable with what I choose to pursue in my career path.

To my class, I want to say that we did it! We may not have the same starting point,
nor do we have the same outlooks in life, but we have, in our own way, survived together.
I want to say thank you for the things that you’ve shown me in the last two years. I wish
you guys all the best and may you all find the satisfaction and success you look for in
whatever it is that you choose to do in life.

Lastly, I would like to acknowledge my friends in ASMN, FST and ICT

departments. Thank you for standing by my side throughout my whole college
experience. Thank you for showing me interesting points of view, and that I was not so
alone. For that, I salute you!

iv
LIST OF ABBREVIATIONS

13
C-NMR Carbon-13 Nuclear Magnetic Resonance
1
H-NMR Proton Nuclear Magnetic Resonance
ACN Acetonitrile
CC Column Chromatography
DCM Dichloromethane
DMSO Dimethyl Sulfoxide
EtOAc Ethyl Acetate
HMBC Heteronuclear Multiple Bond Correlation
HPLC High Performance Liquid Chromatography
HSQC Heteronuclear Single Quantum Coherence
IMBC Institute of Marine Biochemistry
MeOH Methanol
MS Mass Spectroscopy
TMS Tetramethyl Silane
VAST Vietnam Academy of Science and Technology

v
LIST OF TABLES
Table 1. Alkaloids and polyphenols of the genus Piper…………………………….…6
Table 2. Phenolics of the genus Piper……………………………….………………....8
Table 3. Measured and reference NMR data of 16C2A……………..……………...19
Table 4. Measured and reference NMR data of 17D2…………..……………..……23

vi
LIST OF FIGURES
Fig. 1. Stem and leaves of P. betle………..……….…………...……………………….5
Fig. 2. Alkaloids and polyphenols of the genus Piper……………………………........8
Fig. 3. Phenolics of the genus Piper…………………………….……………………...9
Fig. 4. Extraction scheme of compounds from the stem of P. betle………….……...16
Fig. 5. Chemical composition and HMBC correlations of compound 16C2A..……18
Fig. 6. 1H-NMR spectrum of 16C2A…………………………………………………20
Fig. 7. 13C-NMR spectrum of 16C2A……………………………………………..….20
Fig. 8. HSQC spectrum of 16C2A………………………………………….……..….21
Fig. 9. 13C-NMR spectrum of 16C2A……………………………………………..….21
Fig. 10. Chemical composition of compound 17D2………………………………….22
Fig. 11. 1H-NMR spectrum of 17D2………..……………...………………..…....…..24
Fig. 12. 13C-NMR spectrum of 17D2…………………………………………………24

vii
ABSTRACT

Piper betle Linn. (Piperaceae) is a pantropical plant known for its antioxidant,
antimicrobial, anti-inflammatory and hepato-protective activities. With the means of CC
and HPLC, two compounds were isolated from the water extract of its stem. Structural
elucidation by means of NMR techniques revealed that they are 2-methoxyphenyl O-β-
D-glucopyranoside and 1-hydroxyl-2-O-β-D-glucopyranosyl-4-allylbenzene, both
aromatic glucosides with O-glycosidic bonds.

Keywords: genus Piper, Piper betle Linn, anti-microbial, aromatic glucoside, NMR

1
 INTRODUCTION

Vietnam, along with other South East Asian (SEA) countries such as Indonesia
and India, is geographically located in the tropical monsoon climate region, whose
terrain is divided by mountainous landscapes. Therefore, there exist many diverse
climate conditions, with each sub-region possessing its own characteristic micro-
ecosystem. These factors have created various ecological conditions with dense, moist,
evergreen climate suitable for many types of vegetation to grow and develop. It is
estimated that there are over 12 000 phyto-species here, amongst which up to one third
have been used as ethnological medicine by our ancestors for centuries. The more
recognisable names include Eurycoma longifolia, Momordica charantia, Angelica
sinesis, Morinda oficinalis, Platago major, Panax vietnamensis, Panax pseudoginseng,
Curcuma longa, etc, which serve as local traditional drugs to be used in many aspects of
daily life. Up until now, hundreds of medicinal plants have been examined and evaluated
by modern pharmacological studies, and their values for treatment have been proven.

In these modern times, the prospect of understanding the medicinal properties of

herbal plants by analysing their chemical components, then discerning the behaviour of
each component with respect to the human body has become more widely accepted in
the scientific community as it helps to gain a deeper perspective. These findings provide
the basis for further drug developments such as docking and drug targeting to take place
in the future. One common family of plant that is extensively distributed in this
pantropical region is called the “Pepper,” scientifically named the genus Piper. Not only
is it a well-known spice, but it also carries important secondary metabolites that have
advantageous effects for the human health. This thesis focuses on one plant belonging to
this genus, the Piper betle Linn.

Although its effects are generally well-known, not much studies have been done
to acquire an insight to these pharmacological behaviours, evidenced by the fact that
there have not been many scientific papers written specifically on this plant but rather
2
more on the genus Piper as a whole. Some notable effects include anti-inflammatory
effects in the central nervous system (CNS); defence against microglia activity;
antimicrobial activity. The aim of this thesis therefore is:

1. Give an overview of the genus as well as the plant, including the

characteristics, chemical components, and bioactivities.
2. Extract the chemical components from the stem of P. betle.
3. Elucidate the structures of the isolated compounds using NMR
spectroscopic methods.

3
 OVERVIEW
2.1. Botanical characteristics of P. betle.
The taxonomic classification of Piper betle Linn. is as follows:

Kingdom: Plantae

Clade: Tracheophytes

Clade: Angiosperms

Clade: Magnoliids

Order: Piperales

Family: Piperaceae

Subfamily: Piperoidaea

Genus: Piper

P. betle is a plant indigenous to regions of India, but over the years it has seen an
extensive spread to tropical and subtropical ecological systems of the world ranging from
high elevation points to diverse climatic places. The genus Piper is more commonly
known as “Pepper,” and it consists of two main species: black and white amongst many
others that have not yet been thoroughly studied. It is estimated that there are well over
700 species, presenting themselves as either erect, in form of herbs, shrubs, but less
frequently as trees [1, 2]. It is used as a medicinal plant primarily in countries that border
the Indian Ocean, from South Africa and extending up to Papua New Guinea in Oceania,
even extending to South America as these regions provide the suitable climate, i.e., high
temperatures with very low level of humidity [3].

P. betle is a long-stemmed, woody vine plant. Its branches are cylindrical, smooth,
longitudinally notched, and are rooted at nodes. The leaves are 10 - 13 cm long, 4.5 - 9
cm wide, heart-shaped, smooth on both sides with dark glossy upper surface, pointy tips,
prominent veins on lower surface, petiole-elongated sheath, alternating growth
4
positioning and their bases diverge in trajectories. Flowers grow in clusters of squirrel
tails hanging in the upper leaves in clusters with male and female flowers. Male flowers
are long with hairy peduncles, female flowers are about 5 cm long, peduncles covered
with thick hairs, gourds are hairy at the top. Fruits are round, containing light yellow
essential oil and strong aroma, hot and spicy when tasted [4, 5].

Fig. 1. Stem and

Fig.leaves
1. of P. betle

Although a holistic research of the of the plant as a whole has yet to be published,
one area has received particular attention over the past decade: the leaf - this is because
ancient people had the practice of masticating the leaves with the quid (nut of either areca
or betle) inside both for therapeutic as well as hygienic purposes. Along with the sliced
quid wrapped inside the leaf, they also daubed lime in the form of calcium hydroxide
and chewed as a way to facilitate the sublingual absorption of the betle chemicals into
the bloodstream. The main chemical components of the leaves are phenols and terpenes,
which give them their distinctive aroma. The leaves provide biological defence against
fungi, larvae, protozoa, filaria, ulcers as well as possessing effects on the digestive
system such as hepatoprotective, gastrointestinal, thyroid, cardioprotective amongst
many others [6].

5
2.2. Chemical components
It is best to look at the constituents of genus Piper, as few fewer studies have been
done on P. betle. Main compounds include polyphenols, alkaloids and terpenes, along
with other secondary metabolites such as neolignans, lignans, lactones, hydroquinones,
steroids and flavonoids that are heterogeneous distributed amongst Piper species. The
tables and figures below provide the main compounds that extracted from the genus.

2.2.1. Alkaloids and polyphenols

Alkaloids and polyphenols are more abundantly found in the two species of P.
betle and Piper nigrum Linn. Some prominent components that have been reported
include the following:

Table 1. Alkaloids and polyphenols of the genus Piper

Number Name of compound Species Reference

1 Stigmast-4-en-3,6-dione Piper betle Linn [7]
2 4-allyl resorcinol Piper betle Linn [7]
3 Aristololactam A-II Piper betle Linn [7]
4 1-n-dodecanyloxy resorcinol Piper betle Linn [8]
desmethylenesqualenyl deoxy-
5 Piper betle Linn [8]
cepharadione-A
6 Nigramide A Piper nigrum [9]
7 Nigramide B Piper nigrum [9]
8 Nigramide C Piper nigrum [9]
9 Nigramide D Piper nigrum [9]
10 Nigramide E Piper nigrum [9]
11 Nigramide F Piper nigrum [9]
12 Nigramide G Piper nigrum [9]
13 Nigramide H Piper nigrum [9]
14 Nigramide I Piper nigrum [9]
15 Nigramide J Piper nigrum [9]
16 Nigramide K Piper nigrum [9]
17 Nigramide L Piper nigrum [9]
18 Nigramide M Piper nigrum [9]
19 Nigramide N Piper nigrum [9]

6
20 Nigramide O Piper nigrum [9]
21 Nigramide P Piper nigrum [9]
22 Nigramide Q Piper nigrum [9]
23 Nigramide R Piper nigrum [9]
24 Nigramide S Piper nigrum [9]

7
 Fig. 2. Alkaloids and polyphenols of the genus Piper
2.2.2. Phenolics
From the species Piper umbellata, Piper methysticum and Piper Solmsianum,
phenolic compounds appear to be the major component. The table and figure below
demonstrate these compounds:

Table 2. Phenolics of the genus Piper

Number Name of compound Species Reference

1 2’’-O-β-D-glucopyranoside Piper umbellata [10]
2 Apygenin-8-C-β-D-glucopyranoside Piper umbellata [10]
3 Orientin 8-C-β-glucopyranoside Piper umbellate [10]
4 5-hydroxy-7,3’,4’-trimetoxyflavone Piper umbellate [10]
5 Velutin Piper umbellate [10]
6 Sesamin Piper umbellate [10]
7 Diidrocubebin Piper umbellate [10]
8 4-nerolidilcatecol Piper umbellata [10]
9 7,8-dihydrokavain Piper methysticum [11]
10 5,6,7,8-tetrahydroxyyagonin Piper methysticum [11]
11-hydroxy-12-
11 Piper methysticum [11]
methoxydihydrokavain
12 Dihydromethysticin Piper methysticum [11]
5 Methysticin Piper methysticum [11]

8
6 Hydroxykavain Piper methysticum [11]
7 Kavain Piper methysticum [11]
8 Yagonin Piper methysticum [11]
9 Desmethoxyyagonin Piper methysticum [11]
10 Isolemicin Piper solmsianum [12]
11 Syringaldehyde Piper solmsianum [12]
12 3,4,5-trimethoxy-benzoic acid Piper solmsianum [12]
14 Grandisin Piper solmsianum [12]
Rel-(7R,8R,7’R,8’R)-3’,4’-
15 methylenedioxy-3,4,5,5’- Piper solmsianum [12]
tetramethoxy-7,7’-epoxylignan
Rel-(7R,8R,7’R,8’R)-3,4,3’,4’-
16 dimethylenedioxy-5,5’-dimethoxy- Piper solmsianum [12]
7,7’-epoxylignan

Fig. 3. Phenolics of the genus Piper

9
2.3. Bioactivities
2.3.1. Antiallergic activity
Allergies or topical infections against microbes such as Streptococcus aureus, β-
hemolytic streptococcus group A and dermatophytes have seen their reduction under the
usage of Piper betle Linn ointment. The feeling is caused by histamine and granulocyte
macrophage-colony-stimulating factor (GM-CSF) mediators produced by the bone
marrow mast cells (BMMCs) and eotaxin and interleukin-8 (IL-8) secreted in the
pulmonary epithelium. The EtOH extract have shown significant inhibitory effects
against these mediators, particularly thanks to the phenolic compounds chavibetol and
allyl pyrocatechol, which also selectively hinder their effects thereby reducing the signal
transmitted to sensitive sensory nerfs in the brain [13].

2.3.2. Anti-atherogenic and cardiovascular activity

Contributors of hypercholesterolemia and atherosclerosis include high level of
cholesterol, triglycerides (TG), low-density lipoprotein (LDL) and very low-density
lipoprotein (VLDL) and the liver marker enzymes consisting of aspartate amino-
transferase, alanine amino-transferase, alkaline phosphatase and gamma glutamyl
transpeptidase. Other inducing factor of atherogenesis is reactive oxygen species (ROS)
that causes atherosclerotic lesions and the deactivation of endothelium. Venkadeswaran
et al. have demonstrated using Wistar rats that, the cholesterol and enzyme levels in the
liver cells amongst Piper-regulated rats were much lower than the with the ones only
given regulatory salt, which greatly helps reduce the risk of cardiovascular disease and
oxidative stress [14]. It has also been shown that immediate effects of the plant after
mastication include increase in heart rate, stamina, blood pressure and glucose level in
the blood stream. This is because the endothelium-dependent cardio-relaxants in the
body are mediated by nitric oxide of the plant [15].

10
2.3.3. Antibacterial activity
Aqueous, MeOH, EtOAc and petroleum ether extracts have demonstrated notable
inhibitory effects against fungi, yeast and bacteria such as Staphylococcus aureus and
Escherichia coli [16, 17].

2.3.4. Anticancerogenic activity

Three main mechanisms to prevent the carcinogenetic growth include the
induction of selective apoptosis, anti-tumour promoting activity and antimutagenic
effect. Betle vine presents all these activities that are anticancerogenic, particularly
thanks in large part to the phenolic compound hydroxychavicol (HC) [18]. The work by
Chang et al. further presents advantages of HC as it greatly reduces tumours induced by
acetoxymethyl nitrosamine, displaying scavenging effect on hydrogen peroxide (H2O2)
as well as being a superoxide radical scavenger [19].

2.3.5. Antioxidant activity

It has been demonstrated by Manigauha et al. that in the MeOH extract of the
plant presents great reducing power, significant scavenging activity for 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radicals and anions of superoxide. Furthermore, it also aids the
increase hepatic enzyme level, which in part helps to protective the liver from toxic
substances [20].

2.3.6. Hepatic activity and hepatotoxicity

The human liver can be damaged in many ways, for example excessive alcohol
consumption, oxidative stress, or accumulated amount of D-galactosamine (D-GalN).
Mechanisms that help combat these damages include the increase in reactive oxygen
intermediates, antioxidant activity and regulation of hepatic marker enzymes. The
MeOH extract of Piper betle Linn shows significant effect in both the regulation as well
as a decrease in these enzymes thanks to its antioxidant activity [21]. It also induces lipid
peroxidation, demoting the hepatotoxic substance D-GalN [22].

11
2.3.7. Insecticidal activity
It has been reported that the alkaloids of the genus Piper are mainly responsible
for the toxicity that gives the plant its insecticidal activity. Extracts from the seed of
Piper nigrum containing piperamides, pipercide, pellitorine and piperine present toxicity
ranging from 0.15 to 20 µg/dose, which is sufficient to kill insects but not harm humans.
Furthermore, there is a 4-day window of repellence after spraying. These factors
constitute a good alternative as a biological insecticide to more toxic artificial ones [23].

2.3.8. Oral hygiene and anticariogenic activity

During mastication, Piper betle Linn releases chemicals that aid the salivation
process, which in turn promotes the discharge of enzymes such as peroxidase, lysozyme,
and secreted antibodies, all of which are beneficial for the inhibition of resist bacterial
growth. The plant provides a positive feedback on peroxidase, greatly ameliorating the
antibacterial processes and directly hinders the cellular development procedure of
Streptococcus mutans ATCC 25175 and Actinomyces viscosus ATCC 15987 in the oral
cavity. Results have also shown that the phenolic compounds in the plant is effective
against Staphylococcus aureus and Streptococcus aureus, bacteria which are the main
cause of foul oral odour [3, 24].

12
 MATERIALS AND METHODS
3.1. Plant material
The stem of the P. betle was collected in Thai Binh province, Vietnam in May
2020. A voucher specimen (No. PB052020) is kept at Department of Structural
Determination, IMBC, VAST. After collection, fresh samples were cut into small pieces
(2-5 cm) and dried at 50°C. The dried samples were pulverised and stored at in open air
before investigation.

3.2. Investigation methods

3.2.1. Compound separation methods
 Thin layer chromatography (TLC)

Normal-phase and reversed-phase TLC were performed on precoated plates DC-

Alufolien F254 (Merck 1,05715) and/or RP18 F254S (Merck 1,15389) respectively. The
eluents were then observed either by illuminating the plate under UV-light at the
wavelengths of 254nm and 365nm or by spraying with 10% H2SO4, drying and gently
heating until revelation of colours.

 Preparative TLC

Preparative TLC was performed on silica gel 60G F254 pre-coated plate (Merck
1,05875). Revelation of the compound by UV irradiation or cutting the edge of the plate,
spraying with10% H2SO4, drying and gently heating until revelation of colours, locating
the area of appearance of the substance on the original plate, scraping the part of silica
gel containing the compound, desorbing and re-crystallising in the suitable solvent.

 Column chromatography (CC)

Silica gel was used as the stationary phase for normal-phase CC and octadecylsilyl
(ODS) for reversed-phase CC. Silica gel size varies from 0.040 to 0.063 mm (240-430
mesh). Reversed-phase CC uses RP18 particles of size 120 to 150 µm (YMC Chemical
Ltd.). Ion exchange resins of Diaion HP20 (Misubishi Chemical Industries Co., Ltd.).

13
  High performance liquid chromatography (HPLC)

Preparative HPLC technique was used for compound purification at the last step. Pre-
HPLC was acquired on an Agilent 1100 or 1260 system including high pressure pump,
autosampler, and DAD detector, and equipped with J’sphere H-80 (column size 20 x
250mm, particle size 4µm) column. Depending on the compound needing to be purified,
the isocratic composition of solvent mixture between distilled water and ACN will be
injected accordingly at a flow rate of 3 ml/min.

3.2.2. Structure elucidation methods

The common method to elucidate the chemical structure of a compound is a
combination of determining its physical specifications along with modern spectroscopic
techniques, at the same time consulting reference documents to analyse the data. These
techniques include:

 Nuclear magnetic resonance (NMR) spectroscopy

NMR spectroscopy was measured on Bruker Avance III 500MHz spectrometer at the
Institute of Chemistry, VAST. Internal standard reference is TMS. Notable employed
NMR techniques include:

_One-dimensional NMR: 1H-NMR, 13C-NMR and DEPT.

_Two-dimensional NMR: HSQC and HMBC.

Solvents typically used are CDCl3, CD3OD and DMSO, all deuterated in order to not
interfere with the magnetic field of the proton present in the compounds. The choice of
solvent depends on the characteristics of each compound, according to the principle that
the sample needs to dissolve completely in it.

 Polarimeter [α] D

The optical rotation of the compound was measured on JASCO P2000 Polarimeter
at the IMBC, VAST.

14
 EXPERIMENTALS
4.1. Raw sample processing
The collected sample containing the bark, stem and leaves were firstly chopped
into smaller pieces, then separated into respective parts and dried at 50°C. The stem was
then taken and milled in order for the essential oil to be squeezed out, ready to be
separated.

4.2. Extraction and isolation

The air-dried sample of P. betle of 7kg was ultrasonically extracted 3 times with
8L MeOH in Elmasonic S 120H (50oC, 10h). The extract was then filtered, gathered and
put under rotavapor to eliminate excess solvent, obtaining 290g of MeOH extract. This
extract was then suspended homogeneously in water, then successively partitioned with
n-Hexane, DCM and EtOAc. After that, each extract was dissolved in its respective
solvent of n-Hexane (55g), DCM (173g), EtOAc (7g) and H2O. The H2O extract then
underwent Dianion HP20 CC in order to eliminate inorganic substances and glycosidic
groups present in the extract with increasing MeOH to H2O (M/W) ratio of 25, 50, 75
and 100% to obtain 2 the extracts of 25% MeOH (9.4g) and 100% MeOH (30g). The
latter was further separated using gradient silica gel fraction with varying D/M ratios of
increasing polarity from 20/1, 10/1, 5/1, 2.5,1 to 1/1 to obtain 11A, 11B (3.4g), 11C
(91.g), 11D and 11E fractions. After performing TLC to verify the presence of
compounds, fractions 11B and 11C were mixed together to further be chromatographed
under Dianion CC, then was diluted with a minimum amount of MeOH, mixed with 47g
of silica gel, evaporated until loose, dried then submitted under rever-sephase silica gel
with eluting solvent mixture M/W of 1/3, giving 10 further fractions from 15A to 15K.
Through further TLC analysis, two fractions were to be chromatographed from 15C and
15D of eluting solvent mixture E/M of 15/1 and 15K of further Dianion cleaning and
then eluting solvent mixture acetone to H2O (A/W) of 1/3. The process is summarised in
Fig. 4. below:

15
Fig. 4. Extraction scheme of compounds from the stem of Piper betle
Linn

16
 The fraction 16C obtained from the mixture of 15C and 15D underwent HLPC
two times, firstly of 16% ACN to give 7 fractions, of which the fraction 16C2 was then
further purified with 14% ACN to give 16C2A (5mg). Lastly, fraction 15K gave 5
fractions, of which 17D was to undergo HPLC of 25% ACN to be separated into 2
fractions of 17D1 and 17D2. In this thesis, two of the isolated compounds will be
analysed: 16C2A and 17D1.

4.3. Physical specifications of the separated compounds

4.3.1. Compound 16C2A: 2-methoxyphenyl O-β-D-glucopyranoside
White amorphous powder

Angle of rotation: [𝛼]23

𝐷 – 38 (c = 0.5, MeOH)
o

Chemical formula: C13H18O7

HR-ESI-MS: m/z 287 [M+H]+

1
H and 13C-NMR (CD3OD) data can be found in Table 3.

4.3.2. Compound 17D2: 1-hydroxyl-2-O-β-D-glucopyranosyl-4-allylbenzene

White amorphous powder

Angle of rotation: [𝛼]23

𝐷 – 56.8 (c = 0.44, MeOH)
o

Chemical formula: C15H20O7

1
H and 13C-NMR (CD3OD) data can be found in Table 4.

17
 RESULTS AND DISCUSSION
5.1. Structure elucidation of the isolated compounds
5.1.1. Compound 16C2A: 2-methoxyphenyl O-β-D-glucopyranoside

Fig. 5. Chemical composition and HMBC correlations of compound 16C2A

Compound 16C2A was obtained as white amorphous powder. The 1H-NMR

spectrum of 16C2A reveals the presence of four aromatic protons at δH 7.19 (1H, d, J =
8.5 Hz), 7.02 (2H, overlapped signals), 6.91 (1H, ddd, J = 8.5, 8.5, 2.0) a methoxy group
proton at δH 3.88 (3H, s), an anomeric proton at δH 4.92 (1H, d, J= 7.5 Hz) and other
proton signals of sugar moiety in the range of δH 3.42 - 3.89 ppm. The 13
C-NMR
spectrum of compound 16C2A displays signals for thirteen carbon atoms including six
downfield carbon signals at δC 113.9, 118.2, 122.3, 124.1, 148.1 and 150.9, suggesting
the presence of a benzene ring, a methoxy group signal at δC 56.72 and six signals for
carbon in sugar moiety at δC 62.5, 71.4, 74.9, 77.9, 78.2, and 102.9. Assignment of
carbons and their bearing protons were then performed by HSQC spectra as in Table 3.
From the evidence gathered from 1H and 13
C-NMR spectra, we can propose that this
compound is an ortho-disubstituted benzene derivative. The carbon chemical shifts of
the sugar moiety, as mentioned above, demonstrate the presence of a glucopyranosyl
group. The J-coupling constant for the glycosyl anomeric proton at J = 7.5 Hz suggests
aβ-glycosidic linkage. Detailed 1H and 13C-NMR spectral assignments were done with
the aid of HMBC spectrum. The HMBC correlations between Glc H-1′ (δH 4.92) and C-
2 (δC 148.1), methoxy protons (δH 3.88) and C-1 (δC 150.9) indicate that both the O-β-
D-glucopyranosyl group and methoxy group are linked to benzene ring. A doublet

18
aromatic proton signal (δH 7.19) is assigned to H-3, which is further corroborated by its
HMBC correlation to C-1 (δC 150.9). Then, carbon signal at δC 124.1 was assigned to C-
5 by HMBC correlation between H-3 and C-5 (δC 124.1). Aromatic proton signal at δH
6.91 is assigned to H-4 by its HMBC correlation to C-2 (δC 148.1) and the HMBC
correlation between H-4 and C-6 (δC 113.9) supports the assignment of the last carbon
(C-6) of the benzene ring. Similarly, 1H and 13
C-NMR spectral assignment of sugar
moiety were done as shown in the Fig. 5. Consequently, compound 16C2A was
determined to be 2-methoxy-1-O-β-D-glucopyranosyl benzene. All 1D-NMR spectral
data of the compound match the data previously reported for this compound (Table 3)
[25], but detailed NMR assignments for each carbon and proton have been provided in
the above explanation.

Table 3. Measured and reference NMR data of 16C2A

C δC* δCa,b δHa,c (mult., J = Hz)

1 150.18 150.9 -
2 148.09 148.1 -
3 113.18 113.9 7.19 (d, 8.5)
4 122.55 124.1 7.02*
5 121.48 122.3 6.91(ddd, 8.0, 8.0, 2.0)
6 116.51 118.2 7.02*
1-OCH3 55.93 56.7 3.88 (s)
3-O-β-D-Glc
1’ 102.24 102.9
4.92 (d, 7.5)
2’ 74.90 74.9
3.51 (dd, 9.0, 7.5)
3’ 78.54 78.2
3.42 (m)
4’ 71.26 71.4
3.42 (m)
5’ 78.85 77.9
3.48 (m)
3.71 (dd, 5.5, 12.0)
6’ 62.35 62.5
3.89 (dd, 2.0, 12.0)
a
recorded in CD3OD, 125 MHz, 500 MHz, *overlapped signals
b c

19
Fig. 6. 1H-NMR spectrum of 16C2A

Fig. 7. 13C-NMR spectrum of 16C2A

20
Fig. 8. HSQC spectrum of 16C2A

Fig. 9. HMBC spectrum of 16C2A

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5.1.2. Compound 17D2: 1-hydroxyl-2-O-β-D-glucopyranosyl-4-allylbenzene

Fig. 10. Chemical composition of compound 17D2

Compound 17D2 was obtained as a white amorphous powder. The 1H-NMR
spectrum of 17D2 revealed the presence of three aromatic protons in an ABX coupled
system at δH 6.70 (1H, d, J = 2.0 Hz), 6.61 (1H, dd, J = 8.0 and 2.0 Hz), and 7.11 (1H,
d, J = 8.0 Hz); an methylene group at δH 3.26 (2H, d, J = 7.0 Hz); three olefinic proton
signals of a vinyl group at δH 5.93 (1H, ddd, J = 16.0, 10.0, 7.0), 5.06 (1H, d, J = 16.0
Hz), 5.02 (1H, d, J = 10.0 Hz); one signal of glycosyl anomeric proton at δH 4.75 (1H, d,
J = 7.5 Hz) and other signals of carbinol protons in the range of δH 3.45 – 3.51. The
doublet multiplicity of methylene protons (δH 3.26) and doublet of doublet of doublet
signal of a vinylic proton (δH 5.93) indicates that the vinyl group is linked to the
methylene group to form an allyl group. The 13C-NMR spectrum of 17D2 indicates the
resonance signals for fifteen carbon atoms. Of these, eight downfield carbon signals at
δC 115.7, 117.3, 119.0, 120.9, 137.2, 138.9, 145.0 and 148.2 are assigned to a benzene
ring and a vinyl alkene group. Additionally, two downfield carbon signals at δC 145.0
and 148.2 indicates that they are bonded to an oxygen atom. An upfield carbon signal at
δC 40.5 is assigned to an aliphatic methylene group. The remaining six oxygenated sp3
hybridised carbons at δC 62.3, 71.2, 74.8, 77.5, 78.1, and 104.5 are assigned to a
glucopyranosyl moiety. Moreover, the J-coupling constants (J = 7.5 Hz) of the anomeric
protons suggest that the sugar moiety is β-D-glucopyranosyl group. The above analysis
reveals that compound 17D2 is composed of an 1,2,4-trisubstituted benzene ring, an allyl
group, an O-β-D-glucopyranosyl group and a hydroxy group. Finally, comparison of

22
NMR spectral data of 17D2 with literature [26] indicates that the structure of 17D2 is 1-
hydroxyl-2-O-β-D-glucopyranosyl-4-allylbenzene (Table 4).

Table 4. Measured and reference NMR data of 17D2

C δC# δCa,b δHa,c (mult., J = Hz)

1 145.2 145.0 -
2 148.4 148.2 -
3 119.1 119.0 6.70 (d, 2.0)
4 137.7 137.2 -
5 120.0 120.9 6.61 (dd, 8.0, 2.0)
6 117.4 117.3 7.11 (d, 8.0)
7 40.6 40.5 3.26 (d, 7.0)
8 139.1 138.9 5.93 (ddd, 16.0, 10.0, 7.0)
5.06 (d, 16.0)
9 115.8 115.7
5.02 (d, 10.0)
2-O-β-D-Glc
1’ 104.7 104.5 4.75 (d, 7.5)
2’ 74.9 74.8 3.45-3.51
3’ 78.3 78.1 3.45-3.51
4’ 71.3 71.2 3.45-3.51
5’ 77.5 77.5 3.45-3.51
3.91 (dd, 12.0, 2.0)
6’ 62.5 62.3
3.74 (dd, 12.0, 6.0)
a
recorded in CD3OD, 125 MHz, 500 MHz, #Data previously
b c

reported in [26]

23
Fig. 11. 1H-NMR spectrum of 17D2

Fig. 12. 13C-NMR spectrum of 17D2

24
 CONCLUSION AND PERSPECTIVE

By CC and HPLC methods, two compounds (16C2A and 17D2) from the stem of
Piper betle Linn were isolated. Their chemical structures were determined to be 2-
methoxyphenyl O-β-D-glucopyranoside and 1-hydroxyl-2-O-β-D-glucopyranosyl-4-
allylbenzene, respectively, using 1D- and 2D-NMR spectroscopic methods. Both of
them are phenolic glucosides which are representative chemical composition of Piper
genus. Compound 16C2A have been reported to belong to other Piper species but 17D2
is a new component of the genus. Future outlooks include investigating the
phytochemistry of other fractions and other parts of the plant and then by testing
bioactivities of obtained compounds.

25
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