Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

A molecular connection of Pterocarpus marsupium,

Eugenia jambolana and Gymnema sylvestre with
dipeptidyl peptidase-4 in the treatment of
diabetes

Jayasankar Kosaraju, Anil Dubala, Santhivardhan Chinni, Rizwan Basha

Khatwal, M. N. Satish Kumar & Duraiswamy Basavan

To cite this article: Jayasankar Kosaraju, Anil Dubala, Santhivardhan Chinni, Rizwan Basha
Khatwal, M. N. Satish Kumar & Duraiswamy Basavan (2014) A molecular connection
of Pterocarpus marsupium, Eugenia jambolana and Gymnema sylvestre with dipeptidyl
peptidase-4 in the treatment of diabetes, Pharmaceutical Biology, 52:2, 268-271, DOI:
10.3109/13880209.2013.823550

To link to this article: https://doi.org/10.3109/13880209.2013.823550

Published online: 30 Sep 2013. Submit your article to this journal

Article views: 1446 View related articles

View Crossmark data Citing articles: 6 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=iphb20
 http://informahealthcare.com/phb
ISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. Pezzuto
Pharm Biol, 2014; 52(2): 268–271
! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.823550

SHORT COMMUNICATION

A molecular connection of Pterocarpus marsupium, Eugenia jambolana

and Gymnema sylvestre with dipeptidyl peptidase-4 in the treatment
of diabetes
Jayasankar Kosaraju1, Anil Dubala2, Santhivardhan Chinni3, Rizwan Basha Khatwal2, M. N. Satish Kumar3, and
Duraiswamy Basavan1
1
Department of Pharmacognosy, 2Department of Pharmaceutical Biotechnology, and 3Department of Pharmacology, JSS College of Pharmacy,
Udhagamandalam, Tamil Nadu, India

Abstract Keywords
Context: Pterocarpus marsupium (PM) (Leguminosae), Eugenia jambolana (EJ) (Myrtaceae) and Dipeptidyl peptidase-4, dissociation kinetics,
Gymnema sylvestre (GS) (Asclepiadaceae) are the most important medicinal plants in the Indian enzyme inhibitory half-lives, glucagon-like
system of traditional medicine for the treatment of hyperglycemia. peptide-1
Objectives: Dipeptidyl peptidase-4 (DPP-4) inhibitors are the emerging class of anti-diabetic
agents. However, only few compounds are commercially available. Therefore, in the present History
study we tried to explore the naturally occurring PM, EJ and GS semi-standardized extracts for
their potential DPP-4 inhibition in vitro and in vivo. Received 4 February 2013
Materials and methods: DPP-4 inhibition was evaluated by in vitro inhibitory assay, and enzyme Revised 29 April 2013
kinetics were calculated using one-phase exponential decay equation. Glucose load (2 g/kg) Accepted 5 July 2013
was administered to control and diabetic rats 30 min following extract administration (100, 200 Published online 25 September 2013
and 400 mg/kg) orally once, and blood samples were withdrawn at 0, 0.5, 1, 1.5, 2 and 3 h to
measure plasma active glucagon-like peptide-1 (GLP-1) levels.
Results: PM and EJ inhibit DPP-4 potently with IC50 values of 273.73  2.96 and 278.94  6.73 mg/
mL, respectively, compared to GS (773.22  9.21 mg/mL). PM, EJ and GS exhibit long duration
of action with enzyme inhibitory half-lives of 462.3, 317.2 and 153.8 min, respectively. Extracts
significantly increase GLP-1 levels compared to negative control groups and peak GLP-1 level
was observed at 2 h for PM and EJ, whereas for GS it was at 1.5 h
Discussion and conclusion: Taken together, results suggest the extracts may have potent DPP-4
inhibitory action, and their hypoglycemic action attributed through an increase in plasma active
GLP-1 levels.

Introduction GLP-1 is inhibited by an enzyme called dipeptidyl pepti-

dase-4 (DPP-4).
Diabetes is a chronic metabolic disorder present in almost all
DPP-4 is a prolyl peptidase expressed in various types
countries with an estimated number of 366 million in 2011
of cells including intestinal epithelial, liver, lung, placenta,
and may increase up to 552 million by 2030 (Nigel et al.,
2011). Currently available agents for diabetes target either
kidney and renal proximal tubules. DPP-4 is involved in 13
20
various functions including signal transduction, regulation
relative insulin deficiency or the insulin resistance, and have
of immunological and inflammatory response (Kikkawa et al.,
limitations such as increased risk of hypoglycemia, weight
2005). DPP-4 is known for transforming GLP-1 into an
gain, gastrointestinal side effects and edema (Inzucchi, 2002).
inactive metabolite, resulting in very short half-life of intact
Discovery of incretin hormone, glucagon-like peptide-1
hormone (Drucker, 2003) and the GLP-1 effect will be
(GLP-1) lead a breakthrough in diabetes research by
renewed by inhibiting DPP-4 (Michel et al., 2008). Inhibition
regulating postprandial glucose levels. The risk of hypogly-
of DPP-4 leads to improved beta cell survival with increased
cemia will be minimized due to insulinotropic actions of
neogenesis and reduced apoptosis (Rosenstock & Zinman,
GLP-1 (Thomas et al., 2008). Other than insulin secretion,
2007). Researchers are active in the search for DPP-4
GLP-1 is also responsible for beta cell regeneration with
inhibitors that are available for the treatment of diabetes.
reduced apoptosis (Drucker, 2003). Despite these facts,
Metformin is such a drug proved as a DPP-4 inhibitor
(Lindsay et al., 2005).
The practice of herbal medicine in the field of metabolic
disorders, especially diabetes, is growing. Herbs are a
Correspondence: Jayasankar Kosaraju, Research Scholar, Department of
Pharmacognosy, JSS College of Pharmacy, Udhagamandalam 643001, promising therapeutic approach in the treatment of diabetes
Tamil Nadu, India. E-mail: jayasankar87@hotmail.com as complementary and alternative medicine. In the present
DOI: 10.3109/13880209.2013.823550
study we explored naturally occurring Pterocarpus marsu-
pium (PM) (Leguminosae), Eugenia jambolana (EJ)
(Myrtaceae) and Gymnema sylvestre (GS) (Asclepiadaceae)
semi-standardized extracts for their potential of DPP-4
inhibition in vitro and in vivo. The hypoglycemic action of
selected plants has been proven well enough and the
constituents responsible for hypoglycemic action are also
recognized (Kanetkar et al., 2007; Manish et al., 2009;
Manjeshwar et al., 2011). Though the selected plant extracts
are available in various marketed ayurvedic formulations such
as Bio-Gymnema, Diabecon and Diapal, and being used
extensively, their molecular mechanism of hypoglycemic
action is not explored clearly. So, present study was designed
to explore the precise hypoglycemic mechanism of PM, EJ
and GS.
Materials and methods
Chemical, reagents and herbals used
DPP-4 inhibitor screening assay kit was obtained from
Cayman Chemicals (Ann Arbor, MI; Catalog no. 700210)
and stored at 80  C. Active GLP-1 ELISA kit was purchased
from Millipore (India; Catalog no. EGLP-35K). PM heart
wood extract containing 5% of pterostilbene, EJ fruit extract
containing 20% of total polyphenols and 2% of ellagic acid,
and GS leaves extract containing 25% total gymnemic acids
was supplied by Natural Remedies Pvt Ltd (Bangalore, India).
All other chemicals and reagents used in the study were of
analytical grade and obtained from Sigma-Aldrich
(Bangalore, India).
Animals and treatment
Male Wistar rats weighing 150–180 g were procured from the
central animal house facility of JSS College of Pharmacy,
Ooty, Tamil Nadu, India. Animals were housed in large
spacious, hygienic polypropylene cages, well-ventilated and
maintained at temperature 25  3  C with relative humidity
44–56% along with light and dark cycles at 12 h. Animals
were fed with rat-fed pellet supplied by Hindustan Unilever
Ltd, Bangalore, India, and water ad libitum. All experiments
were performed after obtaining prior approval from
Committee for the Purpose of Control and Supervision on
Experiments on Animals and Institutional Animal Ethical
Committee (JSSCP/IAEC/PH.D/PH.COG/02/2011-2012).
Animals were allowed an acclimatization period up to a
week and fasted overnight prior to the experiment. Eleven
groups were divided consisting of six in each. Group I served as
vehicle control, Groups II–XI were induced with diabetes by
intraperitoneal injection of streptozotocin at a dose of 55 mg/kg
body weight (Ravi et al., 2005) and assigned as follows: Group
II served as negative control, Groups III–V treated with PM,
Groups VI–VIII treated with EJ and Groups IX–XI treated with
GS. Extracts were administered orally to their respective
groups at a dose of 100, 200 or 400 mg/kg body weight.
DPP-4 inhibition assay
The assay was carried out using a DPP-4 inhibitor screening
assay kit as per the manufacturer’s instructions. Briefly, the
experiment was performed triplicate in a white half-volume
Herbal dipeptidyl peptidase-4 inhibitors 269
96-well plate. Thirty mL of assay buffer, 10 mL of DPP-4
enzyme, 10 mL of dimethoxysulphoxide (DMSO) and 50 mL
of DPP substrate were added to 100% initial activity wells.
Fourty mL of assay buffer, 10 mL of DMSO and 50 mL of DPP
substrate were added to background wells. Thirty mL of assay
buffer, 10 mL of DPP-4 enzyme, 10 mL of extract (sample) and
50 mL of DPP substrate were added to inhibitor wells. The
plate was incubated for 30 min at 37  C, and the fluorescence
was recorded using a multimode microplate reader (Varioskan
Flash, v.4.00.53) at excitation and emission wavelengths of
355 and 455 nm, respectively.
Dissociation kinetics of DPP-4
The dissociation kinetics was performed after 30 min pre-
incubation of DPP-4 with 1000 mg/mL of each extract
dissolved in assay buffer. The enzyme reaction was then
initiated by adding DPP substrate and fluorescence intensities
were measured every 5 min up to 10 h. Dissociation rate
constants (Koff) and enzyme inhibitory half-lives (EI half-
lives) were calculated from one-phase exponential decay
equation fitted to the data points.
Estimation of GLP-1 levels
Glucose load (2 g/kg) was administered orally 30 min follow-
ing extracts or vehicle administration, and blood samples
were withdrawn at 0, 0.5, 1, 1.5, 2 and 3 h to measure plasma
active GLP-1 levels. Plasma active GLP-1 level was estimated
using commercially available ELISA kit according to manu-
facturer’s instructions. Briefly the assay was carried out in
triplicate in a 96-well plate, 300 mL diluted wash buffer was
added to each well, incubated for 5 min at room temperature
and decanted. Two-hundred mL of assay buffer were added to
blank wells and 100 mL of assay buffer were added to the
remaining wells. One-hundred mL standards (GLP-1) were
added in ascending order at concentrations of 2, 5, 10, 20, 50
and 100 pM. Next 100 mL quality control samples were added
(QC1 and QC2) each and 100 mL samples were added in
remaining wells. The plate was shaken gently for proper
mixing and covered with plate sealer for an overnight
incubation (24 h) at 4  C. After the incubation liquid was
decanted, washed 5-times with 300 mL of wash buffer with
5 min incubation at each wash and finally the excess buffer
was tapped out after the fifth wash. Two-hundred mL of
detection conjugate was added to each well and decanted after
2 h incubation at room temperature. Then the plate was
washed with 300 mL diluted wash buffer for 3-times and
excess buffer was tapped out. Two-hundred mL of diluted
substrate was added to each well and incubated for 20 min at
room temperature in dark. Then 50 mL stop solution was
added to each well in the same order as the substrate was
added and incubated for 5 min at room temperature in the
dark. Finally, fluorescence was recorded using multimode
micro plate reader (Varioskan Flash, v.4.00.53) at excitation
and emission wavelength of 355 and 460 nm, respectively.
Statistical analysis
The IC50 values were calculated using regression analysis
and expressed as mean  SD. Koff and EI half-lives were
calculated using Graph Pad Prism program (v.5.01) (La Jolla,
270 J. Kosaraju et al.
CA). ANOVA followed by the Bonferroni post-test was
applied for GLP-1 levels and values were said to be
significant when p50.01.
Results and discussion
GLP-1 is an endogenous peptide, synthesized and secreted
from L-cells of the gastrointestinal tract (Perry & Greig,
2002). Unfortunately, GLP-1 has very short half-life (2 min)
due to rapid degradation by DPP-4 (Yamazaki et al., 2006).
Preclinical and clinical studies on DPP-4 inhibitors indicate
their potential utility in the treatment of diabetes. Currently
available DPP-4 inhibitors including sitagliptin, vildagliptin,
saxagliptin, and several others are in the development phase.
In the course of development, we found DPP-4 inhibitors
from herbal origin such as PM, EJ and GS. The present study
demonstrates the molecular mechanism of PM, EJ and GS at
their enzymatic level and characterization of their effect
in vitro and in vivo.
Extracts showed concentration-dependent inhibition of
DPP-4; potent inhibition was observed for PM and EJ with
IC50 values of 273.73  2.96 and 278.94  6.73 mg/mL,
respectively, when compared to GS (773.22  9.21 mg/mL)
(Table 1). A clear time-dependent steady state approach was
observed from the dissociation kinetic curves of the extracts
(Figure 1). The initial reaction rates of DPP-4 with different
concentrations of extracts were analyzed using one-phase
exponential decay equation and data indicates that the
selected extracts inhibit DPP-4 in competition to substrate
binding sites. Inhibitors that bind tightly to the target are
important for the pharmacological activity, as they inhibit
enzyme function even if the circulatory free drug is cleared
(Thomas et al., 2008). Koff and EI half-lives of PM, EJ and GS
were found to be 1.49, 2.18 and 4.50  103/s and, 462.3,
317.2 and 153.8 min, respectively. Compared to GS, PM
Table 1. Effect of extracts on DPP-4 inhibition and kinetics.
Extract IC50 Koff EI half-life
P. marsupium 273.73  12 1.49 462.3
E. jambolana 278.94  16 2.18 317.2
G. sylvestre 773.22  19 4.50 153.8
IC50 expressed in mg/mL. Koff – dissociation constant and expressed as
103/s. EI half-life – Enzyme inhibitory half-life and expressed in
minute.
Figure 2. Effect of extracts on plasma active glucagon-like peptide-1 levels in streptozotocin-induced diabetic rats. Graph represented as mean  SD
(n ¼ 6). Two way ANOVA followed by Bonferroni post test was applied. ***p50.001, **p50.01 vs negative control. (a) P. marsupium (PM), (b) E.
jambolana (EJ) and (c) G. sylvestre (GS).
Pharm Biol, 2014; 52(2): 268–271
exhibited a 3-fold slower rate of decline with DPP-4 whereas
EJ exhibited a 2-fold slower rate of decline. Irreversible
binding was not observed with any of these extracts, yet PM
and EJ were potent and longer acting DPP-4 inhibitors than
GS.
The potency and duration of action of these extracts were
further confirmed in an animal model of diabetes. A dose-
dependent and significant (p50.01) increase in plasma active
GLP-1 levels were observed following glucose load in extract-
treated groups when compared to vehicle and negative control
groups (Figure 2). The area under the curve (AUC) of PM and
EJ shows peak GLP-1 levels at 2 h (Figure 3a and b), whereas
GS at 1.5 h (Figure 3c). The insulin-secreting property of PM,
EJ and GS (Ahmad et al., 1991; Ahmed et al., 2010; Sharma
et al., 2008) was consistent with the elevated GLP-1 levels.
Increase in plasma active GLP-1 levels might enhance insulin
secretion due to DPP-4 inhibition by the extracts; indeed this
pattern was not observed in vehicle and negative control
groups. The elevated levels of GLP-1 following glucose load
indicate that the secretion was glucose dependent and this
potent long lasting in vivo action reflects in vitro EI half-lives.
Postprandial glucose level is regulated by DPP-4 inhibitors
and free from side effects due to its glucose-dependent action
(Ng & Kong, 2007). PM/EJ/GS administration before the
Figure 1. Dissociation kinetics of P. marsupium (PM), E. jambolana
(EJ) and G. sylvestre (GS) with dipeptidyl peptidase-4 (DPP-4). DPP-4
was pre-incubated with each extract (1000 mg/mL), and the enzyme
reaction was initiated by adding substrate. Fluorescence intensities were
measured every 5 min for a total of 10 h. The values were the mean of
triplicate studies.
DOI: 10.3109/13880209.2013.823550
Figure 3. Effect of extracts on the area under the plasma active glucacon-like peptide-1 concentration time curve. The graph is represented as
mean  SD (n ¼ 6). One way ANOVA followed by Bonferroni multiple comparison test. **p50.01 versus negative control. (a) P. marsupium at 2 h, (b)
E. jambolana at 2 h and (c) G. sylvestre at 1.5 h.
meal may lead to an increase in GLP-1 levels by inhibiting
DPP-4 which further controls post prandial glucose levels in
diabetic patients. To our knowledge, for the first demonstra-
tion that PM, EJ and GS inhibit DPP-4 and increases GLP-1
levels, and lead to control glucose levels by secreting insulin
from beta cells, the possible hypoglycemic mechanism.
Conclusion
In conclusion, with the aim of better understanding the
biological role of herbal extracts, we showed that PM, EJ and
GS inhibit DPP-4 and at the same time the data offers new
perspectives for further development of herbal DPP-4 inhibi-
tors. Finally, the hypoglycemic action of PM, EJ and GS
might be attributed through DPP-4 inhibition. Further
molecular docking studies on the constituents of PM, EJ
and GS on DPP-4 (PDB id: 3C45) using the Schrodinger
molecular modeling package is ongoing and will be reported
in due course.
Declaration of interest
This work was supported by the Council of Scientific and
Industrial Research (CSIR, New Delhi, India). The authors
wish to thank Dr M. Ramanathan for his valuable suggestions
and providing necessary facilities to carry out the study.
References
Ahmad F, Khalid P, Khan MM, et al. (1991). Hypoglycemic activity
of Pterocarpus marsupium wood. J Ethnopharmacol 35:71–5.
Ahmed AB, Rao AS, Rao MV. (2010). In vitro callus and in vivo leaf
extract of Gymnema sylvestre stimulate beta cells regeneration and
anti-diabetic activity in Wistar rats. Phytomedicine 17:1033–9.
Drucker DJ. (2003). Glucagon-like peptides: Regulators of cell prolif-
eration, differentiation and apoptosis. Mol Endocrinol 17:161–71.
Inzucchi SE. (2002). Oral antihyperglycemic therapy for type 2 diabetes:
Scientific review. J Am Med Assoc 287:360–72.
Herbal dipeptidyl peptidase-4 inhibitors 271
Kanetkar P, Singhal R, Kamat M. (2007). Gymnema sylvestra:
A memoir. J Clin Biochem Nutr 41:77–81.
Kikkawa F, Kajiyama H, Shibata K, et al. (2005). Dipeptidyl peptidase
IV in tumor progression. Biochim Biophys Acta 1751:45–51.
Lindsay JR, Duffy NA, McKillop AM, et al. (2005). Inhibition of
dipeptidyl peptidase IV activity by oral metformin in type 2 diabetes.
Diabet Med 22:654–7.
Manish D, Arun N, Ansari SH. (2009). Pterocarpus marsupium Roxb. –
A comprehensive review. Phcog Rev 3:359–63.
Manjeshwar SB, Harshith PB, Bantwal RVB, et al. (2011).
Phytochemistry, traditional uses and pharmacology of Eugenia
jambolana Lam. (black plum): A review. Food Res Int 44:1776–89.
Michel MC, Fliers E, Van Noorden CJ. (2008). Dipeptidyl peptidase IV
inhibitors in diabetes: More than inhibition of glucagon-like peptide-1
metabolism? Naunyn Schmiedebergs Arch Pharmacol 377:205–7.
Ng VWS, Kong APS. (2007). Dipeptidyl peptidase (DPP)-IV inhibitor:
A novel class of oral anti-hyperglycemic agents. Drug Rev 12:33–4.
Nigel U, David W, Leonor G, et al. (2011). The IDF Diabetes Atlas, 5th
ed. Brussels, Belgium: International Diabetes Federation.
Perry T, Greig NH. (2002). The glucagon-like peptides: A new genre
in therapeutic targets for intervention in Alzheimer’s disease.
J Alzheimers Dis 4:487–96.
Ravi K, Rajasekaran S, Subramanian S. (2005). Antihyperlipidemic
effect of Eugenia jambolana seed kernel on streptozotocin-induced
diabetes in rats. Food Chem Toxicol 43:1433–9.
Rosenstock J, Zinman B. (2007). Dipeptidyl peptidase-4 inhibitors and
the management of type 2 diabetes mellitus. Curr Opin Endocrinol
Diabetes Obes 14:98–107.
Sharma B, Balomajumder C, Roy P. (2008). Hypoglycemic and
hypolipidemic effects of flavonoid rich extract from Eugenia
jambolana seeds on streptozotocin induced diabetic rats. Food
Chem Toxicol 46:2376–83.
Thomas L, Eckhardt M, Langkopf E, et al. (2008). (R)-8-(3-
Amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-
2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel
xanthine-based dipeptidyl peptidase 4 inhibitor, has a superior
potency and longer duration of action compared with other dipeptidyl
peptidase-4 inhibitors. J Pharmacol Exp Ther 325:175–82.
Yamazaki K, Yasuda N, Inoue T, et al. (2006). 7-But-2-ynyl-9-
(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one is
a novel competitive and selective inhibitor of dipeptidyl peptidase IV
with an antihyperglycemic activity. J Pharmacol Exp Ther 319:
1253–7.