New natural protein tyrosine phosphatase 1B inhibitors from Gynostemma pentaphyllum

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RESEARCH ARTICLE

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2024, VOL. 39, NO. 1, 2360063

New natural protein tyrosine phosphatase 1B inhibitors from

Gynostemma pentaphyllum

Xianting Wanga*, Yidan Denga*, Jianmei Wanga*, Lin Qina,b, Yimei Dua,b, Qianru Zhanga,b, Di Wua,b, Xingdong

Wua,b, Jian Xiea,b, Yuqi Hea,b and Daopeng Tana,b

aGuizhou Engineering Research Center of Industrial Key-technology for Dendrobium Nobile, Zunyi Medical University, Zunyi, Guizhou, China; bJoint

International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou, China

ABSTRACT

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease mainly caused by insulin resistance, which

can lead to a series of complications such as cardiovascular disease, retinopathy, and its typical clinical

symptom is hyperglycaemia. Glucosidase inhibitors, including Acarbose, Miglitol, are commonly used in the

clinical treatment of hypoglycaemia. In addition, Protein tyrosine phosphatase 1B (PTP1B) is also an

important promising target for the treatment of T2DM. Gynostemma pentaphyllum is a well-known oriental

traditional medicinal herbal plant, and has many beneficial effects on glucose and lipid metabolism. In the

present study, three new and nine known dammarane triterpenoids isolated from G. pentaphyllum, and

their structures were elucidated by spectroscopic methods including HR-ESI-MS,1H and 13C NMR and X-ray

crystallography. All these compounds were evaluated for inhibitory activity against α-glucosidase, α-amylase

and PTP1B. The results suggested that compounds 7~10 were potential antidiabetic agents with significantly

inhibition activity against PTP1B in a dose-dependent manner.

Abbreviations:NASH: non-alcoholic steatohepatitis; T2DM: Type 2 diabetes mellitus; PTP1B: Protein tyrosine

phosphatase 1B; G. pentaphyllum: Gynostemma pentaphyllum; HR-ESI-MS: High resolution electrospray

ionisation mass spectroscopy; NMR: Nuclear magnetic resonance spectroscopy; NASH: Non-alcoholic

steatohepatitis; TLC: Thin layer chromatography; UPLC: Ultra performance liquid chromatography; Q/TOF-MS:

Quadrupole-time of flight mass spectrometry; UV: Ultraviolet and visible spectrum; EtOAc: Ethyl acetate; PE:

Petroleum ether; DMSO: Dimethyl sulfoxide; EDTA: Ethylene diamine tetraacetic acid; DTT: Dithiothreitol;

pNPP: Disodium-4-nitrophenyl phosphate; p-NP: p-Nitrophenol; PNPG: 4-Nitrophenyl α-D-glucopyranoside;

DNS: 3,5-Dinitrosalicylic acid; PBS: Phosphate buffer solution; HPLC: High performance liquid chromatography;

EE: Encapsulation efficiency; DL: Drug loading; FBS: Foetal bovine serum; HepG2: Human hepatocellular

carcinomas; CCK-8: Cell counting kit-8; PCR: Polymerase chain reaction; RT-qPCR: Reverse transcription

quantitative real-time polymerase chain reaction; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase;

HMBC: Heteronuclear multiple bond correlation; HSQC: Heteronuclear singular quantum correlation; COSY:

Correlation spectroscopy; NOESY: Nuclear overhauser effect spectroscopy; IC50: Half maximal inhibitory

concentration; PDI: Polydispersity index; IR: Insulin receptor

Introduction

Gynostemma pentaphyllum (Thunb.) Makino, named “Jiaogulan” in

China, is also widely distributed in Korea, Japan, Vietnam and

other Asian countries.1–3 In China, G. pentaphyllum was used as

vegetable and functional tea from Ming Dynasty (1368–1644

AD).4,5 It has also been prescribed as traditional Chinese medicine

to treat various diseases, such as non-alcoholic steatohepatitis

(NASH) and diabetes.6 Previous phytochemical investigations

showed that the dammarane triterpenoid saponins, sterols and fla-

vonoids were the major chemical constituents in G. pentaphyl-

lum.7,8 To date, more than 180 dammarane triterpenoid saponins

were reported from G. pentaphyllum with various bioactivities

including antioxidant, antidiabetic, anti-inflammatory, hepatopro-

tective and antitumor effects.9–13 The structure–activity relationship

studies showed that dammarane triterpenoid aglycones, saponins

removed sugar moieties, were more effective than the prototypical

saponins.14,15 However, the active ingredients in G. pentaphyllum

and their mechanism of pharmacodynamic effects are unclear.

In our previous study,16 five new dammarane triterpenoid agly-

cones were isolated from the hydrolyzate of total G. pentaphyllum

saponins which showed significant inhibitory activity against

protein-tyrosine phosphatase 1B (PTP1B). PTP1B is an important

negative regulator and plays a very important role in the process

of insulin signalling.17 Inhibition of PTP1B activity can increase

insulin sensitivity of target cells.18 Therefore, inhibition of PTP1B is

CONTACT Yuqi He yqhe.pharm@foxmail.comDaopeng Tan 1474755772@qq.com Guizhou Engineering Research Center of Industrial Key-technology for

Dendrobium Nobile, Zunyi Medical University, Zunyi, Guizhou, China

*Xianting Wang, Yidan Deng and Jianmei Wang are the rst authors of this paper.

https://doi.org/10.1080/14756366.2024.2360063

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits

unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the

posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ARTICLE HISTORY

Received 31 January 2024

Revised 8 May 2024

Accepted 17 May 2024

KEYWORDS

Gynostemma

pentaphyllum; protein

tyrosine phosphatase 1B;

α-glucosidase; α-amylase;

phytochemistry

2X. WANG ETAL.

considered as an effective treatment for type-2 diabetes melli-

tus(T2DM) and obesity.19–22 T2DM is a chronic metabolic disease

mainly caused by insulin resistance, which can lead to a series of

complications such as cardiovascular disease, retinopathy, and its

typical clinical symptom is hyperglycaemia.23 In addition to PTP1B

inhibitors, α-glucosidase and α-amylase inhibitors are also com-

monly used for hypoglycaemia in clinic, mainly including Acarbose,

Miglitol, Voglibose and Etogliflozin. These drugs can reduce post-

prandial blood glucose and improve insulin levels.24

In the present work, to further investigate the potential hypo-

glycaemic active constituents in G. pentaphyllum, a hydrolyzate of

total G. pentaphyllum saponins was isolated by column chromatog-

raphy to obtained three undescribed dammarane triterpene deriv-

ative 1–3, together with nine known analogues 4–12. Their

structures were elucidated by spectroscopic methods including

HR-ESI-MS,1H and 13C NMR and X-ray crystallography. In addition,

their inhibitory activity against PTP1B, α-glucosidase and α-amylase

were also evaluated in vitro.

Materials and methods

General experimental procedures

In the separation process, 100–200 or 300–400 mesh silica-gel

(Qingdao Marine Chemical Co., Ltd., China) and Sephadex LH-20

(GE Healthcare, Marlborough, MA) were employed for the column

chromatography. The TLC detection was performed by heating sil-

ica gel plate (Qingdao Marine Chemical Co. Ltd., China) after spay-

ing with 10% H2SO4 in ethanol. HR-ESI-MS data were recorded by

a UPLC-Q/TOF-MS system including a 1290 Infinity II UPLC system

and an Agilent 6545 Q/TOF-MS system (Agilent, MA, USA). The 1H

(400 MHz) and 13C NMR (100 MHz) spectra were detected by Varian

INOVA AS 400 (Agilent, Santa Clara, USA). CDCl3 solvent for NMR

analysis was purchased from Sigma Aldrich. The X-ray single crys-

tal diffraction data were obtained by SuperNova, Dual, AtlasS2 dif-

fractometer (Rigaku, Japan). PTP1B (from human, ab51277) was

purchased from Abcam Trading Co., Ltd. α-Glucosidase (from yeast,

G8821) was purchased from Beijing Solarbio Co., Ltd., and

α-amylase (from porcine pancreas, A3176, type VI-B) was pur-

chased from Sigma–Aldrich (Shanghai) Co., Ltd. HepG2 cells was

obtained from Cell Bank of the Chinese Academy of Sciences,

Shanghai, China. Besides, other analytical grade chemical solvents

were purchased from Jinhuada Chemical Co., Ltd., China.

Plant material

The commercialised total saponins extract of G. pentaphyllum was

the same batch (No. 20190501) as the sample used in our previous

study16 and purchased from Shaanxi Zhongxin Biotech Co. Ltd.

(Unified Social Credit Code: 91610000677914226 R), in 2019. A

voucher specimen was authenticated by Professor Daopeng Tan

(Pharmacognosy, Zunyi Medical University) and deposited in the

Herbarium of Zunyi Medical University.

Acid hydrolysis and isolation

The total G. pentaphyllum saponins (>80%, by UV) was purchased

from Shaanxi Zhongxin biotech Co., Ltd. According to the litera-

ture,16 the G. pentaphyllum saponins (500 g) was dissolved in

1000 ml of methanol and then hydrolysed by adding 500 ml of

10% HCl for 8 h under 50 °C.5 The dammarane triterpenoid

aglycones residue (150 g) was obtained after decompression evap-

oration and extraction by ethyl acetate (EtOAc). The residue was

separated by silica gel column chromatography and gradient

eluted with petroleum ether (PE)/EtOAc (50:1–1:1) or CH2Cl2/MeOH

(50:1–1:1) to afford 10 fractions (Fr. A-J). The Fr. A (5.0 g) was sep-

arated again by silica gel column chromatography with n-hexane/

EtOAc (50:1) to afford compound 1 (25 mg), 2 (20 mg), 3 (40 mg)

and 4 (25 mg). Fr. B (2.5 g) was purified over Sephadex LH-20 col-

umn chromatography with CH2Cl2/MeOH (1:1) to obtain compound

5 (30 mg), 6 (35 mg) and 7 (40 mg). Fr. C (5.3 g) was separated by

silica gel column chromatography with n-hexane/acetone (30:1) to

yield compound 8 (35 mg), 9 (20 mg), 10 (15 mg), 11 (15 mg) and

12 (50 mg).

Gypensapogenin VI (1): White amorphous powder; The 1H NMR

(400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectral data was

shown in Table 1; HR-ESI-MS: m/z 443.3897 [M + H-H2O]+, (calcd. for

443.3950, for C30H52O3).

Gypensapogenin VII (2): White amorphous powder; The 1H NMR

(400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectral data was

shown in Table 1; HR-ESI-MS: m/z 453.3363 [M + H]+, (calcd. for

453.3324, for C30H44O3).

Gypensapogenin VIII (3): White amorphous powder; The 1H NMR

(400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectral data were

shown in Table 1; HR-ESI-MS: m/z 497.3633 [M + H]+ (calcd for

497.3586, for C32H48O4).

Molecular docking

The PTP1B, α-glucosidase and α-amylase protein crystal structures

were obtained from PDB Database (https://rcsb.org) and modified by

Autodock tools.25,26 In this protocol, missing hydrogens were added,

bond orders were assigned, and all water molecules and heteroat-

oms were deleted except the native ligand. After the optimisation

step addressing overlapping hydrogens, a restrained minimisation

was applied using the OPLS4 force field.27 Before molecular docking,

dammarane triterpenoid aglycones isolated from G. pentaphyllum

were selected as ligands for the molecular docking analysis. Their 3D

structures were built by ChemBioDraw Ultra14.0 and converted to

PDBQT coordinated by AutoDockTools. The rotatable bonds of ligand

were assigned using AutoDock Tools, and the molecular docking

was performed through the AutoDock Vina. The protein-ligand

interactions 2D maps constructed by LigPlot software was used to

analyse the interaction forces between proteins and ligands. The

protein–ligand interactions 3D maps constructed by PyMOL software

was used to show the binding sites between proteins and ligands.

Protein tyrosine phosphatase 1B inhibition assay

The PTP1B inhibitory activities of dammarane triterpenoid agly-

cones isolated from G. pentaphyllum was determined by the micro-

plate method.16 Each dammarane triterpenoid aglycones and

Na3VO4 (positive control) were separately dissolved in dimethyl sulf-

oxide (DMSO) and then diluted to gradient concentrations by MOPS

solution [pH = 7.0, 25 mM MOPS, 1 mM EDTA, 2 mM dl-dithiothreitol

(DTT), 0.1 M NaCl]. The reaction system was 100 L: PTP1B (25 nM),

Table 1. Primers used for qRT-PCR.

Gene Forward primer (5′–3′)Reverse primer (5′–3′)

PTP1B AGCCAGTGACTTCCCATGTAG TGTTGAGCATGACGACACCC

GAPDH CAGCCTCAAGATCATCAGCA ATGATGTTCTGGAGAGCCC

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