Khadayat et al.

Clinical Phytoscience (2020) 6:34

https://doi.org/10.1186/s40816-020-00179-8

ORIGINAL CONTRIBUTION Open Access

Evaluation of the alpha-amylase inhibitory

activity of Nepalese medicinal plants used
in the treatment of diabetes mellitus
Karan Khadayat1†, Bishnu P. Marasini1†, Hira Gautam1, Sajani Ghaju1 and Niranjan Parajuli2*

Abstract
Background: α-Amylase catalyses the hydrolysis of starch and ultimately producing glucose. Controlling the
catalytic activity of this enzyme reduces glucose production in the postprandial stage, which could be a therapeutic
benefit for people with diabetes. This study was conducted to evaluate α-amylase inhibition for utilizing the crude
extracts of some medicinal plants traditionally used in Nepal for the treatment of diabetes and its related
complications.
Methods: Microtiter plate approach has been used to assess inhibitory activities of in vitro α-amylase of methanolic
extracts of thirty-two medicinal plants. A starch tolerance test was used in rats to investigate the in vivo study of
the methanolic extract concerning glibenclamide as the positive control.
Results: Acacia catechu, Dioscorea bulbifera, and Swertia chirata exhibited inhibitory activity against α-amylase and
with IC50 values; 49.9, 296.1, and 413.5 μg/mL, respectively. Kinetics study revealed that all the extracts displayed a
mixed type of inhibition pattern, with Ki values ranging from 26.6–204.2 μg/mL. Free radical scavenging activity was
again re-examined and found prominent in extracts of A. catechu. Likewise, A. catechu and S. chirata showed
significant reduction of blood glucose concentration up to 30 min after oral dose of 250 mg/kg (F (4, 20) = 4.1,
p = .048), and (F (4, 20) = 4.1, p = .036), respectively.
Conclusions: Enzymatic assay for α-amylase inhibition using extracts was successfully evaluated. Also, the in-vitro
and in-vivo study model revealed that medicinal plants could be a potent source of α-amylase inhibition. So, they
could serve as potential candidates for future drug development strategies for curing diabetes with minimal or no
adverse side effects.
Keywords: Diabetes, α-Amylase, Acacia catechu, Oral starch tolerance test

Background diabetes in Nepalese adults was observed to be 4% and

In Diabetes, the body cannot produce enough insulin or accounted for approximately 696,900 sufferings and 11,
cannot use insulin and is diagnosed by observing raised 700 adult deaths due to diabetes [1]. In the medical
levels of glucose in the blood. Among 7.7 billion total term, diabetes is the endocrine-metabolic disorder. It is
populations (2019), around 463 million adult people defined as fasting blood glucose equals to or higher than
have diabetes with a global prevalence of 9.3% and may 7 mmol/L or on medication for raised blood glucose [2].
rise to 10.9% by 2045. In 2019, the prevalence rate of It is a complex disease delineate by disarray in carbohy-
drate, protein, and fat metabolism that aftermath in mi-
* Correspondence: nparajuli@cdctu.edu.np cro- and macro-vascular changes causing secondary
†
2
Karan Khadayat and Bishnu P. Marasini contributed equally to this work. complications [3]. These secondary complications in-
Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu,
clude heart attack, stroke, kidney failure, leg amputation,
Nepal
Full list of author information is available at the end of the article vision loss, and nerve damages [4]. Maintaining stable
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Khadayat et al. Clinical Phytoscience (2020) 6:34 Page 2 of 8

and lower blood glucose can be achieved by delaying foundation for the development of functional foods or
glucose absorption through inhibition of carbohydrate- the identification of lead bioactive compounds or active
hydrolyzing enzymes, such as α-glucosidase and α- ingredients against type 2 diabetes.
amylase in the digestive tract [5].
Among the different classes of the enzyme, α-amylase Materials and methods
(α-1, 4 glucan-4-glucanohydrolase; EC 3.2.1.1) belongs Chemicals and materials
to hydrolases class and can be found in microbes, plants, Acarbose, porcine pancreatic α-amylase (PPA), and 2-
and animals [6]. It is a metalloenzyme requiring at least chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) were
one Ca2+ ion per enzyme molecule crucial for their ac- obtained from Sigma-Aldrich (Germany). 2,2-Diphenyl-
tivity and stability [7]. Porcine pancreatic α-amylase 1-picrylhydrazyl (DPPH) was purchased from Hi-media
shares 83% similarity with human pancreatic amylase (India). Quercetin, dimethyl sulphoxide, sodium dihy-
and composed of 496 amino acid residues [8]. The α- drogen orthophosphate, starch, and other chemicals
amylase is found in saliva and pancreatic juice [9], that were purchased from Fisher Scientific (India).
hydrolyzes alpha-linked polysaccharide’s alpha bonds
like in starch and glycogen, resulting in glucose and mal-
Collection of plant materials
tose that could quickly enter the bloodstream [10]. It is
The leaves, fruit part, stem, bark, and roots of different
the primary amylase type present in humans and other
plants were collected from different areas of Nepal dur-
mammals. Inhibition of α-amylase delays the digestion
ing May–December, 2016. The plant samples were col-
process by hampering the breakdown of starch in the in-
lected based on a review, such as the ethnomedicinal
testine and hence can be utilized as an effective strategy
uses of Nepalese medicinal plants. None of these plants
for regulating hyperglycemic conditions [11].
are endangered in Nepal. Collected plant samples were
For the regulation of blood glucose levels, different
identified by the Department of Plant Resources, Na-
classes of drugs such as sulfonylureas, non-sulfonylureas
tional Herbarium, Godawari, Ministry of Forests and Soil
secretagogues, biguanides, meglitinide, dipeptidyl peptid-
Conservation, Nepal.
ase 4 (DPP-4) inhibitors, sodium-glucose cotransporter
(SGLT2) inhibitors, α-glucosidase inhibitors, and thiazo-
lidinediones (glitazones) are employed [10]. However, Preparation of extracts
these drugs are associated with the lack of specificity The collected plants were shade dried at room
and hence have been reported to cause several gastro- temperature by avoiding direct sunlight and pulverized.
intestinal side effects like cramping, flatulence, abdom- The powder of different plants was soaked in methanol
inal distention, and diarrhea [12]. In recent years, for 24 h, filtered by muslin cloth, and collected in a conical
research on medicinal plants for managing diabetes has flask; the same process was repeated for three successive
attracted scientists [13]. There are several advantages of days with fresh methanol. Then the filtrates were again fil-
natural herbal drugs such as reduction of risk of side ef- tered through Whatman filter paper (No. 1) and were
fects, effectiveness with chronic conditions, widespread concentrated in a vacuum in a rotary evaporator at 50 °C.
availability, and low cost [14]. Therefore, inhibitors of
the α-amylase enzyme, which are extracted from plants, In vitro antioxidant assay
could be emerging candidates to control hyperglycemia The antioxidant activity was determined using DPPH as
in diabetic patients [15, 16]. described previously [20] with slight modification.
Azadirachta indica, Carum carvi, Tinospora sinensis, Briefly, 100 μL of various concentrations of the extracts
Vitex negundo, Acacia catechu, Ficus religiosa, Cirsium in 50% DMSO was added to 100 μL of a 0.1 mM sample
verutum, Mirabilis jalapa, Myrica esculenta, Nephrolep- of DPPH in methanol. After 30 min of incubation at
sis cordifolia, Rubus ellipticus, Zea mays, Asparagus room temperature in the dark, the absorbance was read
racemosus, and Aloe vera are the most common and po- at 517 nm. The percentage of inhibition on the DPPH
tent antidiabetic medicinal plants traditionally used in radical was calculated by using the following expression:
Nepal [17, 18]. This research is focused mainly on type 2
diabetes, which is a chronic metabolic disease character-  
OD of extract
ized primarily by insulin resistance in target tissue lead- %Inhibition ¼ 100−  100
OD of control
ing to hyperglycemia and, ultimately, malfunctions of
the β-cells [19]. The aim of this study was in vitro
screening the crude extracts of selected Nepalese medi- Where optical density (OD) is the absorbance. The
cinal plants for inhibitory activities against α-amylase by IC50 value was calculated using the EZ-Fit enzyme
microtiter plate-based assay as well as in vivo screening kinetics program (Perellela Scientific, Inc., Amherst,
in the rat. The results of this study would provide a Mars, USA).
Khadayat et al. Clinical Phytoscience (2020) 6:34 Page 3 of 8

Assay for α-amylase Resources, Kathmandu, Nepal. Oral starch tolerance

The α-amylase inhibition activity was evaluated in 50 tests in non-diabetic rats were done according to the
mM phosphate buffer pH 7.0 with 0.9% NaCl. The PPA method described previously [26] with slight modifica-
at the final concentration of 1.5 units/mL (prepared in tion. The rats were kept for acclimatization at an ambi-
buffer mentioned above) with the various concentrations ent temperature of 25 ± 2 °C and 45–55% relative
of test compounds (prepared in DMSO) was incubated humidity, with 12 h alternation of dark and light cycles
at 37 °C for 15 min. The reaction was started by the and were fed pelleted diet and tap water with free excess
addition of the substrate, CNPG3, at 375 μM final con- [26]. The protocol for this test was certified by the De-
centration prepared in the buffer as above. The change partment of Plant Resources, Animal Ethics Committee,
in absorbance by released p-nitroaniline was continu- Kathmandu, Nepal (DPR), with the ethical certificate
ously monitored at 405 nm [21]. The final concentration number - 92/074/75.
of DMSO was not more than 5% and was taken as con- In this test, 25 overnight-fasted (12 h) non-diabetic
trol. All the experiments were performed in triplicate in rats were divided into five groups of five rats each (n =
a final volume of 200 μL, by using a microplate reader 5), oral doses of 250 mg/kg extracts dissolved in distilled
(Epoch2, BioTek, Instruments, Inc., USA). water (group I, II and III), glibenclamide with an oral
The results were processed by using Gen5 Microplate dose of 5 mg/kg (group IV) and distilled water as control
Data Collection & Analysis Software and then by MS (group V) were administered, respectively. After 30 min,
Excel. The percentage inhibition based upon initial vel- the rats were administered starch orally at a dose of 2 g/
ocity and was calculated as: kg body weight, and blood was collected via tail punc-
  ture for blood glucose estimation before (0 min) and at
OD= minof extract 30, 60, and 120 min following starch treatment. Indeed,
%Inhibition ¼ 100−  100
OD= minof control no rats during the experiment are purposely harmed,
and handling of rats was carried out according to “the
IC50 (Inhibition of enzymatic hydrolysis of the sub- ethical guidelines based laboratory animals for research
strate CNPG3 by 50%) value was calculated using the 1994” (National Regulations of Nepal).
EZ-Fit enzyme kinetics program.
Statistical analysis
α-Amylase inhibition kinetics study The results are present as the mean ± standard error of
The change in optical density per minute (OD/min) was the mean of triplicate experiments. All data obtained
determined by incorporating various concentrations of were analyzed using analysis of variance (one-way
compounds over a range of substrate (CNPG3) concen- ANOVA) and further analyzed by Dunnett’s one side
trations between 0.187 mM and 1.5 mM. Reciprocal of (less than control) comparison by SPSS software (version
the rate of the reactions against the reciprocal of the 19).
substrate concentration as Lineweaver–Burk plot (and
its secondary plot; slope versus compound concentra- Results
tion); the Dixon plot (and its secondary plot; slope ver- Screening of plant extracts for α-amylase inhibition
sus reciprocal of compound concentration) [22–25]. A total of 32 plant extracts were selected for the study,
Graphs were plotted using GraFit 4 (Erithacus Software and their crude extracts (500 μg/mL) were evaluated for
Limited, Surrey, UK) and GraphPad Prism 5 (GraphPad the inhibitory activity against PPA (Table 1). The ex-
Software, California, USA). The types of inhibition were tracts, which showed over 50% inhibition, were consid-
determined by the comparison of Km and Vmax values ered for further study to maintain hit to lead rate at
of the substrate and their ratios (Km/Vmax) in the pres- approximately 5%.
ence and absence of the inhibitors. The types of inhib-
ition were also determined by graphical views of Dixon In vitro antioxidant assay
plots, Lineweaver-Burk plots, and their secondary plots. DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scaven-
The Ki values obtained directly from the software (Gra- ging assay was done to analyze the antioxidant capacities
Fit 4 and GraphPad Prism 5), were also cross-checked of the potent amylase inhibitor plants. Therefore, DPPH
and tallied in these graphs. The final concentration of radical scavenging potential of the three extracts, which
DMSO was maintained at 5%. had exhibited strong α-amylase inhibitory potential, was
evaluated (Table 2). The results of DPPH radical scaven-
In vivo oral starch tolerance test in non-diabetic rats ging were reported as IC50, which shows the concentra-
(NDRs) tions of the samples, which scavenge 50% DPPH radical.
Healthy Swiss albino rats (8 weeks old) weighing about Table 2 reveals the IC50 value of three extracts and quer-
150–200 g were obtained from the Department of Plant cetin as a standard.
Khadayat et al. Clinical Phytoscience (2020) 6:34 Page 4 of 8

Table 1 List of plants selected for the study of α-amylase inhibitory potentials of their methanolic extracts
S. N. Scientific Name Local Name Family Inhibitiona(%) Ethnobotanical use
1 Abrus precatorius L. Ratigedi Fabaceae < 50 [27]
2 Acacia catechu (L.f.) Willd. Khayar Fabaceae 89.9 [17, 28]
3 Acorus calamus L. Bojho Araceae < 50 [29]
4 Aegle marmelos (L.) Correa Bel Rutaceae < 50 [27, 30]
5 Aloe vera (L.) Burm.f. Gheukumari Asphodelaceae < 50 [27, 31]
6 Amomum subulatum Roxb. Alachi Zingiberaceae < 50 [29]
7 Azadirachta indica A. Juss. Neem Meliaceae < 50 [17, 27]
8 Bauhinia variegata L. Koiralo Fabaceae < 50 [28]
9 Berberis asiatica Roxb. ex DC. Chutro Berberidaceae < 50 [30]
10 Catharanthus roseus (L.) G. Don Barmase Apocynaceae < 50 [31]
11 Cinnamomum tamala (Buch. -Ham.) T. Nees & Eberm. Tejpat Lauraceae < 50 [29, 30]
12 Cleistocalyx operculatus (Roxb.) Merr. & L.M.Perry Kyaamuna Myrtaceae < 50 [28]
13 Coccinia grandis (L.) Voigt Kundru Cucurbitaceae < 50 [32]
14 Dioscorea bulbifera L. Bhyakur Discoreaceae 58.7 [33]
15 Ficus benghalensis L. Bar Moraceae < 50 [27, 34]
16 Ficus racemosa L. Gular Moraceae < 50 [27, 34]
17 Ficus religiosa L. Peepal Moraceae < 50 [27]
18 Girardinia diversifolia (Link) Friis Chalne Sisno Urticaceae < 50 [35]
19 Holoptelea integrifolia Roxb. Planch Kanju, Sano Pangro Ulmaceae < 50 [36]
20 Justicia adhatoda L. Asuro Acanthaceae < 50 [27]
21 Momordica charantia L. Tite Karela Cucurbitaceae < 50 [27, 32]
22 Murraya koenigii L. Spreng. Mitho Neem, Mechiya Saag Rutaceae < 50 [30]
23 Nyctanthes arbortristis L. Parijat Oleaceae < 50 [37]
24 Ocimum tenuiflorum L. Kalo tulsi Lamiaceae < 50 [27]
25 Punica granatum L. Anar Punicaceae < 50 [27]
26 Saraca asoca (Roxb.) Willd. Ashok Fabaceae < 50 [36]
27 Shorea robusta Gaertn. Sal Dipterocarpaceae < 50 [28]
28 Swertia chirata Buch.-Ham.ex Wall. Chiraito Gentianaceae 63.5 [28, 38]
29 Syzygium cumini (L.) Skeels Fadel Myrtaceae < 50 [27]
30 Tinospora sinensis (Lour.) Merr. Gurjo Menispermaceae < 50 [17]
31 Urtica parviflora Roxb. Sisnoo Urticaceae < 50 [27]
32 Zanthoxylum armatum DC. Timur Rutaceae < 50 [30]
At 500 μg/mL
a

Table 2 α-Amylase inhibition (IC50) and binding affinities of methanolic extracts (Ki) and their DPPH radical scavenging (antioxidant)
activities
α-Amylase inhibition (μg/mL) Antioxidant activity (μg/mL)
Plant IC50 value Ki value Type of inhibition IC50 value
Acacia catechu 49.9 ± 0.4 26.6 ± 6.7 Mixed 3.9 ± 0.3
Dioscorea bulbifera 413.5 ± 11.1 384.6 ± 36.8 Mixed 49.3 ± 3.6
Swertia chirata 296.1 ± 8.7 204.2 ± 17.9 Mixed 53.6 ± 2.7
Acarbosea 6.1 ± 0.1 – – –
Quercetin b
– – – 2.3 ± 0.1
Acarbosea was used as the standard for α-amylase inhibition
Quercetinb was used as the standard for radical scavenging activity
Khadayat et al. Clinical Phytoscience (2020) 6:34 Page 5 of 8

In vitro and kinetic study of α-amylase enzyme uniformity and reliability are some advantages of micro-
Table 2 reveals the IC50 value of potent three extracts, plate reader assays [39–41]. The microtiter plate-based
their inhibitor constant (Ki), and mode of inhibition. assay using CNPG3 is easy, rapid, and reliable [42].
The active extracts were investigated to find binding In this work, 32 crude extracts were examined on
affinity and mechanism of inhibition of α-amylase by PPA-catalyzed hydrolysis of CNPG3; among them, A.
kinetics study (Fig. 1 and Figure S2 (supporting data). catechu, S. chirata and D. bulbifera had potent IC50
The initial rate was measured at the different substrate values ranging from 49.9 to 413.5 μg/mL. In the previous
and inhibitor concentrations in order to determine in- study, the IC50 value of A. catechu ethanolic seed extract
hibition constants (Ki) and the mode of reversible inhib- was noted to be 341.2 ± 15.3 μg/mL [43], while the
ition of PPA by methanolic extracts. Then the quest for present study of methanolic extract was found to be
a type of inhibition was done from the generated data 49.9 μg/mL. Previously, the IC50 value of the methanolic
among competitive, noncompetitive, or uncompetitive extract of S. chirata was reported 52.4 ± 0.4 μg/mL [44]
models - the Lineweaver-Burk plot, which revealed as compared to 296.1 ± 8.7 μg/mL in our study. Numer-
mixed-type inhibition. The kinetic behavior of CNPG3 ous factors such as degree of ripeness at the time of har-
at different concentrations of CNPG3 and methanolic vest, environmental factors, processing, and storage
extracts is shown in Fig. 1. The extracts act as a mixed- affect the polyphenol content of plants, which might be
type inhibitor, and the values of inhibitor constant (Ki) responsible for variation in the IC50 value of plants [45].
varied from 26.6 to 384.6 μg/mL, respectively. The IC50 value of the positive control acarbose (IC50 =
6.1 μg/mL) is less than plant extracts, but it is not sur-
In vivo assay in a mouse model prising that the IC50 of extracts is high. The plant ex-
A. catechu and S. chirata showed the reduction of blood tracts are mixtures of numerous compounds as
glucose levels up to 30 min in in vivo study. Their effica- compared to single compound acarbose.
cies were tested in rats as well for the glucose level re- In plant extracts, compounds like dioscorin, sapoge-
duction. The methanolic extracts of A. catechu and S. nins, choline, L-arginine, polysaccharides, and proteins
chirata reduce the glucose level up to 30 min [(F (5, might be the source of antidiabetic behaviors [46]. Cat-
25) = 3.8, p = .048), and (F (5, 25) = 3.8, p = .029)], re- echin and epicatechin are the active ingredients in the A.
spectively. The crude extract of D. bulbifera failed to catechu [47, 48], which have been identified as a potent
show its effectiveness in the rat (Supplementary file). inhibitor of α-amylase [49, 50]. Swerchirin [51] could be
the active ingredient in the S. chirata that plays the role
Discussion of the inhibition of α-amylase [52]. Diosgenin, the con-
The amylase activity monitoring methods such as starch- stituent of D. bulbifera might be responsible for the in-
iodine and dinitrosalicylic acid assay are labor-intensive, hibition of PPA [33]. Hydrogen bonding between amino
inconsistent, and include heating process, which makes acids has been elucidated by molecular docking of cat-
the formation of insoluble clumps. With the help of a mi- echin (Glu233, Arg195, His201, and Ile235 and Asp197)
croplate reader, large samples can be evaluated simultan- epicatechin (Glu233, Arg 195, His201, and Ile235 and
eously in the minimal interval; besides this accuracy, Asp197) and diosgenin (Asp300) with α-amylase. In

Fig. 1 α-Amylase inhibition kinetics study. a Line-weaver Burk plot of extracts of A. catechu. b The secondary plot of Line-weaver Burk plot
Khadayat et al. Clinical Phytoscience (2020) 6:34 Page 6 of 8

addition to these, some amino acids also participated in of Nepal [67]. A total of 1158 tons (worth 690 Million
hydrophobic bonding. In sum, hydrogen bonding and NPR) of Khair Kathha extracted from A. catechu was
hydrophobic interactions could contribute to inhibit the exported from Nepal in the year 2017 [68].
activity of α-amylase [33, 53]. Swertia chirata was one of the most exported plants
From the Lineweaver-Burk Plot, Dixon plot, and their from a District of Nepal [69]. D. bulbifera is one of the
secondary plot, it is confirmed that the mode of inhib- major food in some of the ethnic groups of Nepal and
ition is a mixed type. The inhibition constant (Ki) was has been cultivating widely [70].
calculated by GraFit 4 software and was confirmed by The crude extract contains several bioactive com-
the secondary plot of Lineweaver-Burk and Dixon plot pounds; therefore, we need to investigate for the fully
[22]. Kinetic study reveals that both EI and ESI com- characterized molecule, which may be a potent enzyme
plexes are formed in mixed-type inhibition, where all inhibitor.
lines are intersected at the second quadrant on the
Lineweaver-Burk plot and Dixon plot as shown in Fig. Conclusion
1a and Figure S2(a) [54]. In mixed-type inhibition, the The microtiter plate-based assay using extracts of the
inhibitor can bind to both active site and non-active sites medicinal plant is successfully evaluated for α-amylase
of the enzyme. The catalytic barrel and non-catalytic C- inhibition assays. The present study demonstrated that
terminal domains of barley α-amylase contain two sec- the traditionally used medicinal plants by local people to
ondary carbohydrate-binding sites which have been as- manage diabetes might contain certain ingredients that
sumed to exist on other amylases [55, 56]. inhibit α-amylase. The kinetic analysis demonstrates that
Glibenclamide releases endogenous insulin by cytosolic inhibition is that of mixed type for plants extract with
depolarization caused due to selective blockade of ATP porcine α-amylase, and shows that extracts might serve
sensitive K+ channels in the plasma membrane of β-cells as potential candidates for future drug development
of the pancreas showing antihyperglycemic activity in dia- strategies. This study aimed to provide a scientific basis
betic mice [57]. Similarly, plant extracts also act either by for the pre-existing ethnobotanical use of medicinal
tonic liberation of insulin by β-cells of islets of Langerhans plants by selecting potential candidate plants with higher
or by impeding certain enzymes responsible for increasing antidiabetic properties.
blood glucose levels in mice [58]. The allantoin compound
isolated from Dioscorea spp. significantly reduce plasma Supplementary information
glucose in streptozotocin-induced diabetic rats [59]. The Supplementary information accompanies this paper at https://doi.org/10.
previous study has shown that diosgenin failed to show its 1186/s40816-020-00179-8.
effectiveness in either normal or streptozocin induced dia-
betic mice [60]. Therefore, D. bulbifera may contain dios- Additional file 1.

genin but may not contain allantoin, which might be the

reason for its failure in mice. Abbreviations
PPA: Porcine pancreatic α-amylase; CNPG3: 2-chloro-4-nitrophenyl-α-D-
Clinical and experimental evidence support that cellu- maltotrioside; DPPH: 2,2-diphenyl-1-picrylhydrazyl; OD: Optical density
lar oxidative stress occurred as a result of the generation
of reactive oxygen species and reduced antioxidant po- Acknowledgements
tential could enhance diabetic complications [61–63]. We thank National Herbarium and Plant Laboratory, Ministry of Forest and
Soil Conservation, Nepal, for the identification of plant species. We are
The number of studies has shown that oxidative stress is grateful to Mr. Rajeshwor Ranjitkar (Department of Plant Resources, Nepal)
assumed as a major cause of pancreatic cell death and for surveillance and assisting us with all the animal experiments.
its relationship in β-cell loss or apoptosis [64]. Recently,
Authors’ contributions
antioxidant therapy is employed for the remedy and con- N.P. and B.P.M. designed research; K.K., S.G., and H.G. performed research;
trol of diabetes [65]. B.P.M. contributed analytical tools; N.P. and B.P.M. analyzed data, and B.P.M.
All three plants, which were the most potential inhibi- and K.K. wrote the paper. The author(s) read and approved the final
manuscript.
tor of amylase enzyme in our study, i.e., Acacia catechu,
Dioscorea bulbifera, and Swertia chirata are easily avail- Funding
able and gaining attention for commercial production This work was financially supported by TWAS (The World Academy of
with high yield rate. Science-15-227 RG/CHE/AS_G – FR3240287027), and the higher education re-
form project, Nepal to Niranjan Parajuli.
A. catechu grows well in the degraded lands and popu-
lar in choice of community plantations in Nepal because Availability of data and materials
it is used as fuelwood, small timber and fodder as well as Herbaria of plant specimens and the identification information sheets are
kathha and other pharmacological uses [66]. A. catechu stored in the laboratory and can be retrieved when necessary. Data
supporting this manuscript are protected in laboratory information systems
was found a second-best plant for commercial produc- at our institution and are available from the corresponding author on
tion in a research conducted in a district of Terai region reasonable request.
Khadayat et al. Clinical Phytoscience (2020) 6:34 Page 7 of 8

Ethics approval and consent to participate 19. American Diabetes Association. Classification and diagnosis of diabetes. Sec.
Experiment related to animals was executed at the Department of Plant 2. In standards of medical care in diabetes −2015. Diabetes Care. 2015;38:
Resources, Natural Products Research Laboratory, Government of Nepal, S8–16.
based on the ethical guidelines provided by the Department for Nepal. 20. Brand-Williams W, Cuvelier ME, Berset CL. Use of a free radical method to
evaluate antioxidant activity. LWT-Food Sci Technol. 1995;28(1):25–30.
21. Senger MR, Gomes LD, Ferreira SB, Kaiser CR, Ferreira VF, Silva FP Jr. Kinetics
Consent for publication
studies on the inhibition mechanism of pancreatic α-amylase by
The consent to publish the results of animal experiments was provided by
Glycoconjugated 1H-1, 2, 3-Triazoles: a new class of inhibitors with
the Department of Plant Resources, Natural Products Research Laboratory
Hypoglycemiant activity. ChemBioChem. 2012;13(11):1584–93.
Government of Nepal. We also declared that there no conflict of interest
22. Marasini BP, Rahim F, Perveen S, Karim A, Khan KM, Choudhary MI.
among authors to publish these research findings.
Synthesis, structure-activity relationships studies of benzoxazinone
derivatives as α-chymotrypsin inhibitors. Bioorg Chem. 2017;70:210–21.
Competing interests 23. Lineweaver H, Burk D. The determination of enzyme dissociation constants.
The authors declare that they have no competing interests. J Am Chem Soc. 1934;56(3):658–66.
24. Dixon M. The determination of enzyme inhibitor constants. Biochem J.
Author details 1953;55(1):170.
1
Department of Biotechnology, National College, Tribhuvan University, Naya 25. Segel IH. Enzyme kinetics: behavior and analysis of rapid equilibrium and
Bazar, Kathmandu, Nepal. 2Central Department of Chemistry, Tribhuvan steady state enzyme systems. Hoboken: Wiley; 1993.
University, Kirtipur, Kathmandu, Nepal. 26. Ali RB, Atangwho IJ, Kuar N, Ahmad M, Mahmud R, Asmawi MZ. In vitro and
in vivo effects of standardized extract and fractions of Phaleria macrocarpa
Received: 24 December 2019 Accepted: 20 May 2020 fruits pericarp on lead carbohydrate digesting enzymes. BMC Complement
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