Tóm tắt luận án tiến sĩ study on chemical constituents and inhibitory activities of enzymes α glucosidase and α amylase of gymnema sylvestre (RETZ ) r BR EX SM and gymnema latifolium wall EX wight

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY --- DUONG THI HAI YEN STUDY ON CHEMICAL CONSTITUENTS AND INH

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

-

DUONG THI HAI YEN

STUDY ON CHEMICAL CONSTITUENTS AND INHIBITORY ACTIVITIES OF

ENZYMES α-GLUCOSIDASE AND α-AMYLASE OF

Major: Organic chemistry Code: 9.44.01.14

SUMMARY OF CHEMISTRY DOCTORAL THESIS

Hanoi - 2021

This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science

and Technology

Advisor 1: Prof Dr Phan Văn Kiem

Advisor 2: Prof Dr Nguyen Xuan Nhiem

The thesis can be found in

- The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology

- The National Library of Vietnam

INTRODUCTION

Diabetes is a group of disorders of carbohydrate, fat and protein metabolism when the insulin hormone

of the gland is lacking or reduced in action in the body, manifested by high blood sugar levels In the early stages, the patient often urinates a lot, urinates at night and causes thirst, which in turn damage many of the body's systems, in particular the vessels and nerves

Over time, diabetes can lead to blindness, kidney failure, and nerve damage Diabetes is also one of the important factors that cause the formation of atherosclerosis, leading to stroke and cardiovascular disease Chronic complications whether severe or mild, occur sooner or later vayring from patient to patient However, with good blood sugar control we can prevent, slow down or lessen the chronic complications of diabetes [1]

The trend of using natural medicinal sources in general, especially natural compounds derived from plants to treat some of tropical diseases and serious diseases has been a concern not only in our country's pharmaceutical industry but also many countries in the region as well as countries in the world Many indigenous medicinal plants have been found to have hypoglycemic effects This is an advantage because of the ability to provide raw materials for treatment as well as less side effects According to statistics, there are about 800 species that are believed to have the ability to treat diabetes in the world [2] Typical among them

are the species: fenugreek (G sylvestre), bitter melon (Momordica charantia), mustard greens (Brassica

Juncea), dandelion (Elephantopus scaber), fairy hair (Liriope spicata), dandelion oil (Ricinus communis),

ginseng (Smallanthus sonchifolius), zebra orchid (Vernonia anthelmintica),

Species of the Gymnema genus of the family Apocynaceae have received a lot of attention from

scientists around the world They are considered as rich sources of active ingredients and exhibit biological

activities (anti-hyperglycemic, anti-cancer, anti-oxidant, anti-inflammatory, ) In particular, the species G

sylvestre has been widely used as an herbal remedy for diabetes for over 2000 years in India This species is

also used to treat asthma, eye pain, inflammation and snakebite Besides, G sylvestre has antibacterial and

hepatoprotective properties However, in Vietnam, there are only a few studies on the chemical composition

and pharmacology of Gymnema sp Therefore, we propose the topic "Study on chemical constituents and inhibitory activity of α-glucosidase and α-amylase enzymes of Gymnema sylvestre (Retz.) R.Br ex Sm and

Gymnema latifolium Wall ex Wight

The aim of the thesis:

Study on main chemical constituents of two Gymnema species including Gymnema sylvestre and

enzymes of isolated compounds

The main contents of the thesis:

1 Isolate compounds from the leaves of G sylvestre và G latifolium in Vietnam

2 Elucidate chemical structures of the isolated compounds by physical and chemical methods

3 Evaluate the inhibitory effect of α-glucosidase and α-amylase enzymes of isolated compounds from G

sylvestre and G latifolium

CHAPTER 1: OVERVIEW

1.1 Introduction to Gymnema genus

1.1.1 Botanical features of the genus gymnema

The Gymnema belongs to the family Apocynaceae, order Gentianales, subclass Mint (Lamiidae), class

Magnoliopsida, phylum Magnoliophyta

According to the statistics of the world association of botanists "http://www.theplantlist.org", the online library of the catalog of botanical species in the world, the genus Gymnema currently has 52 species

were accepted

According to the list of plant species in Vietnam [3], the genus Gymnema in Vietnam currently has 8

species: G albiflorum Cost, G alternifolium (Lour.) Merr., G foetidum Tsiang, G griffithii Craib, G

inodorum (Lour.) Decne, G latifolium Wall ex Wight, G reticulatum (Moon) Alston and G sylvestre (Retz)

R Br Ex Sm

Table 1.2 List species of the genus Gymnema in Vietnam

Thanh Hóa, Kon Tum

1.1.2 Introduction to Gymnema sylvestre and Gymnema latifolium

1.1.3 Overview of the chemical composition of the genus gymnema

Species of the Gymnema genus have been studied by many scientists in the world In particular, scientists mainly focused on the species G Sylvestre, whereas the rest are less studied (mainly G tingens, G

griffithii, G montanum, G inodorum and G alternifolium) Studies on the chemical composition of this species

have shown the presence of steroid, terpenoid, phenolic, and flavonoid compounds These compounds exhibit anti-hyperglycemic, anti-obesity, antibacterial, hepatoprotective, and antioxidant activities

1.1.3.1 Steroid compounds

Steroids are specific substances belonging to the Gymnema genus, they exist mainly as pregnane

skeleton with chains of pseudo sugars, including cymarose (cym), 6-deoxy-3-oxymethylallopyranose (all), D- thevetopyranose (thv), oleandropyranose (ole) and canaropyranose (can) and digitoxose (dig)

1.1.3.2 Triterpenoid compounds

Triterpennoids are the major class of metabolites and account for the largest number of substances

found in the Gymnema genus, occurring as oleanane saponins, dammarane and lupane skeleton derivatives

1.3.2.3 Phenolic and other compounds

1.1.4 Overview the biological activity of the genus Gymnema

1.1.4.1 Anti-diabetes activity

The hypoglycemic effect of a saponin extract with 5 triterpene saponins gymnemic acid I (52)-IV (55)

and gymnemasaponin V (95) from G sylvestre has been reported The compound gymnemic acid IV (3.4/13.4

mg/kgP) was found to have an anti-hypoglycemic of 14-60% within 6 h in comparison with glibenclamide In addition, this compound also increased insulin levels when given to diabetic rats at a concentration of 13.4 mg/kgP [33]

Another study on the hypoglycemic effect of G sylvestre leaves via controlling blood glucose and

lipid levels in Wistar rats at the concentration of 200 mg/kgP showed that the extract of this species significantly reduced blood sugar, plasma and blood lipids (VLDL, LDL cholesterol parameters) [34]

The compound dihydroxy gymnemic triacetate (148) was isolated from the leaves of G sylvestre by

Pitchai Daisy et al (20 mg/kgP orally for 45 days) with positive effects on all biochemical parameters in diabetic rats [30] Evaluation parameters including blood glucose, insulin, glycated hemoglobin (HbA1c), tissue glycogen, lipid parameters such as triglycerides, total cholesterol, LDL-cholesterol, HDL-cholesterol, and activity of marker liver enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and acid phosphatase (ACP) In addition, gymnemic acids from leaves of

1.1.4.1 Antiobesity activity

1.1.4.2 Antioxidant activity

1.1.4.3 Hepatoprotective effect

1.1.4.4 Antimicrobial activity

1.5 Overview the genus Gymnema in Vietnam

In 2017, Master Hoang Minh Chau isolated 13 compounds from G sylvestre species, of which 9 new

compounds are gymnemoside ND1-ND9 (170-178) and 4 known compounds (179-182) Compounds 176-178

were tested in vitro for 2-NBDG uptake in 3T3-L1 adipose tissue The results showed that at the concentration

of 20 µM, the glucose absorption of these solutions was as effective as that of 10 nM insulin [53]

Conclusion: In Vietnam, study on chemical constituents as well as biological activities on species of

Gymnema genus are quite limited Based on the existed results, we have selected G sylvestre and G latifolium

for chemical constituents and biological activities with the expectation to contributing to the elucidation of

chemical components, accouting for the thge use this medicinal plant in folk and orientating for further medical and pharmaceutical research

1.2 Overview of diabetes

Diabetes is a group of disorders of carbohydrate, fat and protein metabolism when the insulin hormone

of the gland is lacking or reduced in action in the body, manifested by high blood sugar levels Diabetes is divided into three main types:

There are three main types of diabetes:

- Type 1 diabetes: (also known as insulin-dependent diabetes): the body stops producing insulin Common in children or adolescents Patients with type 1 diabetes need to be treated with insulin every day to stay alive

- Type 2 diabetes: A chronic disease that develops when the pancreas does not produce enough insulin

or when the tissues in the body cannot use insulin properly Type 2 diabetes is very common (accounting for more than 90% of cases), usually in people over 40 years old, in which people with diabetes are often prone to some comorbidities such as hypertension, angina, heart attack, cataract and often have a shorter life expectancy than others

- Gestational diabetes: This is a disorder of glucose tolerance during pregnancy This disease increases blood sugar in the fetus leading to the risk of miscarriage, stillbirth, malformations

1.3 About the role of α-glucosidase and α-amylase enzymes

1.3.1 The concept of enzymes

1.3.2 Enzyme α-amylase (EC 3.2.1.1)

Amylase is a common enzyme in living organisms with the systematic name of 1,4-α-D-glucan glucanohydrolase [57] These enzymes belong to the group of hydrolysis enzymes [57, 58], which catalyze the breakdown of intramolecular bonds in the polysaccharide group with the participation of water

RR’ + H-OH →RH + R’OH Amylase hydrolyzes starch, glycogen and dextrin to low molecular weight glucose, maltose and dextrin Amylase enzymes are present in saliva (also known as ptyalin), in the digestive juices of humans and animals, in germinating seeds, filamentous fungi, actinomycetes, yeasts and bacteria [59] Ptyalin initiates starch hydrolysis from the mouth, and this process is completed in the small intestine by pancreatic amylase

1.3.3 Enzyme α-glucosidase (EC 3.2.1.20)

α-Glucosidase is an enzyme that acts by exohydrolysis, catalyzing the hydrolysis of α-1,4-glycoside bonds to release α-D-glucose molecules [60] The specific substrates of the enzyme α-glucosidase are disaccharides, oligosaccharides, and other aryl- and akyl-α-glucopyranosides [57], [61]

CHAPTER 2 EXPERIMENTS AND RESULTS

2.1 Plant materials

2.1.1 Gymnema sylvestre (Retz.) R.Br ex Sm

The stems and leaves of Gymnema sylvestre (Retz) R Br ex Sm) were collected in Hai Loc , Hai Hau,

Nam Dinh in November 2015 The scientific name is given by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources The NCCT-P20 specimen is kept at the Structure Research Department, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology

Figure 2.1 Gymnema sylvestre

2.1.2 Gymnema latifolium Wall ex Wight

The stems and leaves of G latifolium Wall ex Wight., were collected at the Hanoi Research Center

for Medicinal Plants Cultivation and Processing in April 2017 The scientific name is given by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources The NCCT-P76 specimen is kept at the Structure Research Department, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology

Figure 2.2 Gymnema latifolium

2.2 Methods

2.2.1 Methods for isolation of secondary metabolites

2.2.1.1 Thin layer chromatography

2.2.1.2 Column chromatography (CC)

2.2.1.3 High performance liquid chromatography (HPLC)

2.2.2 Methods for determination of chemical structure of compounds

2.2.2.1 High resolution electrospray ionization mass spectrometry (HR-ESI-MS)

2.2.2.2 Nuclear magnetic resonance spectra (NMR)

2.2.2.3 Optical rotation

2.2.2.4 Method for determining sugar

● Sugar type identification by nuclear magnetic resonance spectroscopy (NMR)

● Determination of D and L sugars: Firstly, hydrolysis to break the sugar chains into monosaccharides, and then purification by a combined chromatographic methods to obtain monosaccharides Secondly, measuring the optical rotation of monosaccharide and comparing the specific optical rotation with reported data Finally, defining the D/L configuration of the monosaccharide

2.2.3 Methods to evaluate biological activity

2.2.3.1 Evaluate the inhibitory activity of the enzyme α-glucosidase

4-nitrophenyl-D-glucopyranoside (pNPG) to glucose and p-nitrophenol, a yellow compound, catalysed by

α-glucosidase enzyme When the test sample shows the α-α-glucosidase inhibitory activity, the formation of

p-nitrophenol compounds will decrease The optical density (OD) of p-p-nitrophenol was compared with that of the control, uninhibited sample will decrease accordingly The optical density (OD) of p-nitrophenol produced after the reaction was measured at 405 nm and was used to evaluate the enzyme inhibitory activity of the sample Positive control: Acarbose

Procedure: Using a 96-well plate, each well contained the sample (20 µL of sample dissolved in

DMSO), then the α-glucosidase enzyme (40 µL) and 120 µL of phosphate buffer were added After 5 min, 40

µL 4-nitrophenyl-α-D-glucopyranoside 5 mM was added and then incubated for 30 min at 37°C The optical absorbance OD was recorded at 405 nm [67]

- The results were calculated according to the following formula:

% Inhibitor =

In which:

ODc+: Optical density of positive control (no sample, with α-amylase; in this case, the inhibitory value is 0%);

ODc-: Optical density of negative control (no sample and α-amylase; This case is considered as 100% inhibitory value)

ODs: Average optical density of the test specimen

ODb: Average optical density of white sample (có mẫu thử, không có α-amylase)

Half maximal inhibitory concentration (IC50) was built on 5 test concentrations

IC50 value was determined by linear regression on Graphpad Prism 8.0

2.2.3.2 Evaluate the inhibitory activity of the enzyme α-amylase

Principle: The α-amylase inhibitory activity was performed based on the hydrolysis of potato starch in water

in the presence of α-amylase enzyme The starch solution catalyzed by the α-amylase produces a blue reaction

mixture with iodine, then color reduction was measured at 650 nm to evaluate the enzyme inhibitory activity

of the studied sample Positive control: Acarbose

GS2G2

H:A 2,5:1

Silica c,c H:A gradident 40:1→0:1

HPLC 40% ACN HPLC

Dichloromethane

GS2F1E GS2G1

C:M 20:1

GS4C4 GS4C3

RP-18 c.c, M:W 2:1

C:M 1:1 C:M 5:1 C:M 10:1

GS4C1 GS4C2

GS4C4A GS4C4B GS4C4C GS4C4D GS4C4E

RP-18 c.c A:W 0,8:1

: săc ký lỏng hiệu năng cao

: săc ký cột pha đảo Wt R

: water : thời gian lưu (phút)

t R 44,7

t R 31,5 t R 33,4

Silica c,c, C:M gradident 20:1→1:1

t R 38,5 t R 41,1 t R 42,1 t R 34,3 t R 35,7 t R 37,8

Figure 2.3 Isolation of compounds from G sylvestre

2.3.2 Compounds isolated from G latifolium

GL2D3A

RP 18 c, c M:W 4:1 RP-18 c,c

GL2D1D GL2D1C

GL2D1B

GL-4C4 GL-4C3

Silica gel c, c, CMW 4/1/0,1

H:A 10:1

C:M 1:1 C:M 5:1 C:M 10:1

GL4C1 GL-4C2

Diaion HP-20 M:W, 0%→100% Silica gel c.c

HPLC 48%ACN

HPLC, 48%ACN

GL8

(20,3 mg)

HPLC 35%ACN

GL7

(15,0 mg)

GL2F

Acetone H:A 40:1

: săc ký lỏng hiệu năng cao

: săc ký cột pha đảo WtR

: water : thời gian lưu (phút)

t R 54,0 tR39,1

GL2E4

Figure 2.4 Isolation of compounds from G latifolium

2.4 Physical properties and spectroscopic data of the isolated compounds

2.4.1 Physical properties and spectroscopic data of the isolated compounds from G sylvestre

This section presents physical properties and spectroscopic data of 16 compounds from G sylvestre

2.4.2 Physical properties and spectroscopic data of the isolated compounds from G latifolium

This section presents physical properties and spectroscopic data of 8 compounds from G latifolium

2.5 Results on biological activities of isolated compounds

All compounds isolated from G sylvestre and G latifolium were tested for α-glucosidase inhibitory activity in vitro (Table 2.2) Acarbose, a common diabetes drug, was used as a positive control The results of

α-glucosidase inhibitory activity of 23 compounds (GS1-GS16 and GL1-GL8) are shown in the table below

In which the pure compounds were tested at the concentration of 200 µM, the extracted residue at the concentration of 500 µg/ml and the positive control at the concentration of 500 µg/ml

Compounds with inhibition percentage more than 50% were further tested to find the IC50 value

Accordingly, GS8 show the half maximal inhibitory concentration IC50 = 173,8 ± 0,7 µM, acarbose has IC50

= 580,3 ± 0,5 µM

Table 2.3 The α-glucosidase inhibitory activity of isolated compounds from G latifolium

2.5.2 Results on α-amylase inhibitory activity of compounds

All compounds isolated from G sylvestre and G latifolium were tested for α-amylase inhibitory

activity in vitro The results of the α-amylase inhibitory activity of 23 compounds at the concentration of 200

µM are shown in the table below Acarbose was used as a test positive control at the concentration of 100 µg/ml

Compound GS2, GS3 with inhibitory percentage greater than 50% were further tested to find the IC50

value These compounds were further tested to find IC50 values were175,8 ± 2,3, 162,2 ± 2,7 and 113,0 ± 0,7

μM respectively and acarbose positive control with IC50 = 72,4 ± 0,8 µM

Table 2.5 The α-amylase inhibitory activity of isolated compounds from G latifolium

Compound % Inhibition Compound % Inhibition

3.1 Chemical structure of isolated compounds

This section presents the detailed results of spectral analysis and structure determination of 23 isolated

compounds from G sylvestre and G latifolium

* 16 compounds from G sylvestre

From G sylvestre , sixteen compounds were isolated, including seven new compounds and eight

known compounds Seven new compounds: gymsyloside A (GS1), gymsyloside B, (GS2), gymsyloside C (GS3), gymsyloside D (GS4), gymsyloside E (GS5), gymnepregoside R (GS6), and gymnepregoside T (GS7)

Nine known compounds: verticilloside M (GS8), verticilloside D (GS9), 12-O-(E)-cinnamoylgymnepregoside

F (GS10), gymnepregoside F (GS11), stephanoside I (GS12), 3

β-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-O- β-D-glucopyranosyl ester (GS13), gymnemoside-W1 (GS14),

3β-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-O-

β-D-glucopyranosyl ester (GS15) and alternoside XIX (GS16)

Figure 3.1 Chemical structures of compounds isolated from G sylvestre

Figure 3.2 Chemical structures of compounds isolated from G latifolium

* 8 compounds from G latifolium

From G latifolium, eight compounds were isolated including four new compounds and four known

compounds Four new compounds: gymlatifoside A (GL1), gymlatifoside B (GL2), gymlatifoside C (GL3), and gymlatifoside D (GL4) Four known compounds: verticilloside J (GL5), lucyoside H (GL6), 3β-O-β-D-

glucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (GL7 coincides with GS13), and 3-O- β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-β-D-glucopyranosyl-

(1→6)-β-D-glucopyranosyl ester (GL8)

3.1.1 Some featured points of pregnane glycoside compounds isolated from G sylvestre and G latifolium

The pregnane glycosides isolated from G sylvestre and G latifolium are steroids with 21-carbon

aglycone (C21), fit in with the published pregnane glycoside compounds for the genus Gymnema

The structure of these pregnane glycosides consists of 2 parts:

- Pregnane skeleton: 10β,13β-dimethyl-17β-ethyl derivative of the gonane ring system (formed by the

characteristic arrangement of four cycloalkane rings, including three 6 (A, B, and C) and 1 5 (D) The pregnane glycosides appear double bonded at the C-5 position and are usually hydroxylated at the C-3, C-8, C-14, C-17

and C-20 positions The C-3 position is always the β - configuration of natural steroids The relative position

of the A/B ring is always trans and the C/D ring is cis [68]

Figure 3.3 Pregnane skeleton

- Sugar component: Sugars are linked at C-3 via an acetal bond Pregnane glycosides typically contain one to six sugar units occurring in a linear form, made up of the simple sugars deoxy (deoxy sugar) and β-D-glucopyranose The sugars discovered from the two species mentioned above are similar to those commonly

found in the Gymnema genus, namely cymarose (cym), 6-deoxy-3-oxymethylallopyranose (all), Dthevetopyranose (thv) and D-oleandropyranose (ole)