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American Journal of  
Chemistry and Pharmacy (AJCP)

Synthesis and Characterization of  Alpinia calcarata Loaded 
Nanoparticles to Control Hyperglycemia

B.S. Wanniarachchi1, P.G.U. Chathuranga1, P.H.U.W. De Silva1, B.M. Jayawardena1*

Volume 2 Issue 1, Year 2023
ISSN: 2834-0116 (Online)

DOI: https://doi.org/10.54536/ajcp.v2i1.1293
https://journals.e-palli.com/home/index.php/ajcp

Article Information ABSTRACT

Received: February 12, 2023

Accepted: March 02, 2023

Published: March 10, 2023

Bovine Serum Albumin (BSA) nanoparticles loaded with the bioactive compounds of  A. 
calcarata, which is known to exert its antidiabetic activity through the inhibition of  pancre-
atic enzymes, are a good form of  an antidiabetic nutraceutical as they have reduced side 
effects, protection of  active compounds from environmental agents, specific delivery to 
target sites and prolonged shelf-life. The objective of  the present study was to synthesize 
and characterize A. calcarata loaded nanoparticles (ALNP) to be used as a powder form 
nutraceutical with higher antidiabetic activity. In this study an aqueous A. calcarata extract 
(4.00 mL) was added to BSA (20 mg/mL, 4.00 mL, pH 9) in the presence of  citric acid as the 
cross-linking agent. The ALNP gave an IC50 value of  147 µg/mL, a glucose (5 mM) uptake 
percentage of  73.09% at a 0.5 mg/mL concentration, a solubility value of  64%, A. calcarata 
loading percentage of  6.66% and A. calcarata entrapment efficiency of  87.71%. They had 
a spherical morphology and uniform size with a particle size of  1030.70 nm, PDI of  0.199 
and a zeta potential of  2.57 mV. The UV-Visible absorbance spectra and FT-IR spectra 
showed that citric acid had caused conformational changes in the protein structure of  BSA 
and that the active compounds were successfully loaded into the synthesized nanoparticles 
which interacted with the protein matrix via covalent bonds. Therefore, it can be concluded 
that the synthesized nanoparticles have an antidiabetic effect and the antidiabetic activity of  
bioactive compounds of  the aqueous A. calcarata extract become enhanced when loaded 
onto the nanocarriers.

Keywords
A.Calcarata, Antidiabetic, 
BSA Nanoparticles, Citric 
Acid, Nutraceutical 

1 Department of  Chemistry, University of  Kelaniya, Sri Lanka
* Corresponding author’s e-mail: bimali@kln.ac.lk

INTRODUCTION
Diabetes mellitus, the most common non-communicable 
disease in the world is coined from the Greek word 
diabetes, meaning to pass through a large discharge of  
urine and the Latin word mellitus, meaning sweet. It is 
a general term used to describe a variety of  metabolic 
disturbances, the primary cause of  which is chronic 
hyperglycemia. (Sapra & Bhandari, 2022; Kerner & 
Brückel, 2014) Type 1 diabetes mellitus (T1DM), type 2 
diabetes mellitus (T2DM), ‘other’ and gestational diabetes 
mellitus (GDM) are the classifications of  diabetes based 
on etiology and pathology. (Guthrie & Guthrie, 2004) 
According to the World Health Organization (WHO), 
in 2019, diabetes was the ninth leading cause of  death 
and is also responsible for the largest rise in male 
deaths with an 80% increase since 2000. It is the No.1 
cause of  kidney failure, adult blindness and lower-limb 
amputations. Also in 2019, among the US population, 
283,000 children and adolescents younger than 20 years 
suffered from diagnosed diabetes. The 2022 National 
Diabetes Statistics Report states that currently about 382 
million (8.3%) people are affected by diabetes and this 
number is projected to increase to 552 million (53%) by 
2035. (Kharroubi & Darwish, 2015)
Oral antidiabetic drugs that are used in the treatment of  
diabetes have serious side effects such as hypoglycemia, 
weight gain, anemia and congestive heart failure. Also, 
due to their high cost and these side effects, scientists are 
searching for more effective and safer antidiabetic drugs. 

Therefore, recently attention has been focused on natural 
products including food plants, as possible sources of  
more potent and safer antidiabetic therapy. (Kazeem & 
Davies, 2016)
Alpinia calcarata Roscoe which belongs to the family 
Zingiberaceae is a rhizomatous plant that is widely used 
as a medicinal source in Sri Lanka. Rhizomes of  this plant 
are known as Heen araththa or Katu kikiriya in Sinhala 
and snap ginger in English. The mature rhizomes are 
dense and branched with a light to dark brown colour 
and they are the most important part of  this plant, as they 
are a major part of  indigenous medicinal formulation 
for the treatment of  blood impurities, indigestion, throat 
inflammation, voice improvement and to marinate 
youthful vigor. The decoction of  Alpinia calcarata rhizome 
is widely used to treat respiratory ailments, cough, 
bronchitis, asthma and arthritis. The ethanolic as well 
as aqueous extracts of  Alpinia calcarata rhizomes show 
antibacterial, antifungal, anthelminthic, anti-inflammatory, 
antioxidant, anticancer, antinociceptive, gastroprotective, 
aphrodisiac and antidiabetic effects. This herb is also 
used as a traditional medicine for stomachache, fever and 
rheumatism. (Rahman & Islam, 2015)
Analysis of  A.calcarata Rosc. grown in Sri Lanka has 
revealed the presence of  quercetin, protocatechuic acid, 
1,8-cineole, β-pinene, vanillic acid, 4-O-methyl-syringic 
acid and methyl cinnamate as well as several terpenes 
and diterpenes as constituents. Novel bis-labdanic 
diterpenoids such as calcaratarin D and calcaratarin 

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E have been isolated and identified from its rhizomes, 
which are known to be a pair of  stereoisomers according 
to spectral evidence. Qualitative phytochemical analysis, 
of  the hot water extract and hot ethanol extract, also 
revealed the presence of  alkaloids, flavonoids, steroid 
glycosides, polyphenols and tannins in its rhizomes. The 
relative percentages of  water extractable matter and 
ethanol extractable matter are 18.6-20.5 and 22.6-24.8, 
respectively. (Rahman & Islam, 2015) The essential oils of  
rhizomes, roots and leaves when analyzed using capillary 
GC and GC/MS showed the presence of  18 compounds. 
The major compound in the leaf  and rhizome oils was 
1,8-cineole and in the root oil it was α-fenchyl acetate. 
A. calcarata, having such a huge range of  phytochemical 
diversity, has been ensured non-toxic and safe in animal 
studies. (Arambewela et al., 2005)
Scientific studies carried out with the oral administration 
of  the hot ethanolic and hot water extracts of  the 
rhizomes of  A. calcarata to normoglycemic and 
streptozotocin-induced diabetic rats have shown 
hypoglycemic and antihyperglycemic effects, respectively. 
Both hot water and hot ethanol extracts significantly 
reduce the blood glucose level, body weight gain, plasma 
triglyceride and total cholesterol levels as well as improve 
the glucose tolerance. Alpinia calcarata extract exerts its 
potent antidiabetic activity through the inhibition of  
intestinal glucose absorption by inhibiting the activity 
of  pancreatic α-amylase and α-glucosidase enzymes. 
However, the hypoglycemic effect of  hot ethanol extract 
is usually higher than that of  hot water extract. Phenolic 
compounds such as flavonoids, polyphenols and 
tannins that are present in its rhizomes are known to be 
responsible for its antidiabetic activity. (Rahman & Islam, 
2015; Wasana et al., 2021)
Extraction procedures are carried out to separate the 
medicinally active portions of  plant or animal tissues 
from the inactive or inert components by using selective 
solvents. (Handa et al., 2008) Some of  the extraction 
techniques that can be used to extract the active 
compounds of  the bark of  A.calcarata are microwave 
digestion, pressurized water extraction, solvent extraction, 
decoction water extraction and infusion water extraction. 
Out of  these techniques, the pressurized water extraction 
has been proven to result in an aqueous extract that is 
more active than the other. (Wariyapperuma et al., 2018)
Unlike the traditional extraction methods that require 
large volumes of  non-environmental friendly organic 
solvents and is time consuming with low extraction 
efficiency, pressurized water extraction, which is a green 
solvent extraction method, is environmentally friendly 
as it uses minimum or no organic solvent, cheap with 
shorter extraction time and high extraction efficiency. 
The analytes extracted using this method are also safe for 
human consumption, testing and processing as organic 
solvents are not involved. As water is easily available, 
non-toxic and can be recycled or disposed with minimal 
environmental problems, pressurized water extraction 
has steadily become an efficient and low-cost method of  

extraction for less-polar organic components. (Teo et al., 
2010; Jayawardena & Smith, 2010) 
“Pressurized hot water” is used to denote the region 
of  condensed phase of  water from 100 ℃ to 374 ℃, 
which is the critical point of  water. The density of  water 
remains almost constant over this temperature range 
so that the pressure effect on the properties of  water 
is minimal. During extraction, moderate pressures such 
as 15 bar at 200 ℃ and 85 bar at 300 ℃ are needed to 
keep a condensed phase of  water. Under these conditions 
the dielectric constant of  water decreases, and it starts 
behaving as an organic solvent such as ethanol and 
methanol, which can then extract organic and non-polar 
compounds from numerous kinds of  matrices. However, 
if  water was used at room temperature and atmospheric 
pressure, it will not be suitable for the extraction of  
organic and non-polar compounds, because of  its high 
polarity and high dielectric constant due to its hydrogen 
bonded structure. (Teo et al., 2010)
With the advance of  nanotechnology, scientists have 
developed nanoencapsulation techniques for the targeted 
release and protection of  pharmaceuticals and food 
bioactive components, so that they can be safely added 
into formulations and result in maximum bioavailability. 
Conventional microencapsulation technologies include 
physical and chemical processes such as spray drying, 
freeze drying, extrusion, coacervation, liposomes 
and conventional emulsions. Unlike these methods, 
nanocarriers could provide more bioavailability through 
increased surface-to-volume ratios and therefore higher 
muco-adhesive possibility within the small intestine and 
higher feasibility of  interacting with enzymes and metabolic 
factors, and also these tiny particles could easily pass 
through the cell membranes and penetrate into the target 
cells and release their encapsulated material. (Assadpour 
& Mahdi Jafari, 2019)  In addition, these nanocarriers can 
also improve the solubility of  hydrophobic compounds, 
such as the active compounds of  cinnamon, protect the 
chemical structure of  nutraceuticals from environmental 
agents such as light, temperature, pH, radicals and 
oxygen, allow specific delivery to target sites, allow a 
controlled release of  the encapsulated compound, result 
in reduction of  side effects and prolonged shelf-life, and 
also, they have minimum influence on the appearance of  
final food products. (Assadpour & Mahdi Jafari, 2019; 
Paolino et al., 2021)  
A nanocarrier is a nano-sized system and is scientifically 
defined in the pharmaceutical area as a particle with a 
size of  a few nm to just below 1000 nm. (Assadpour & 
Mahdi Jafari, 2019) Nanoparticles fall into the category 
of  colloidal drug delivery system as they behave as a 
whole unit with respect to its properties and transport 
mechanism. Several types of  nanoparticle systems have 
been identified such as polymeric nanoparticles, polymeric 
micelles, solid nanoparticles, lipid-based nanoparticles for 
example, solid lipid nanoparticles (SLN), nanostructured 
lipid carriers (NLC) and lipid drug conjugate (LDC), 
liposomes, inorganic nanoparticles, dendrimers, magnetic 

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nanoparticles, nanocrystals and nanotubes. In recent 
years, biopolymer-based nanoparticles including protein 
nanoparticles are actively used in pharmaceuticals due 
to their low toxicity and biodegradability. They also have 
better biocompatibilities and the possibility for surface 
modification. These nanocarriers can be synthesized 
using proteins like albumin, gelatin, whey protein, gliadin, 
legumin, elastin, zein, soy protein and milk protein. These 
protein nanoparticles are an ideal material for delivery of  
bioactive compounds as their amphiphilic nature allows 
them to interact with both the bioactive compounds and 
the solvent. (Verma et al., 2018)
Albumin is an attractive macromolecule carrier due to its 
high binding capacity and is obtained from a variety of  
sources such as BSA, egg white (ovalbumin) and human 
serum albumin (HSA). It has a molecular weight of  66.5 
kDa and is a water-soluble protein which is therefore 
used in maintaining the osmotic pressure, binding and 
transport of  nutrients to the cells. Albumin also dissolves 
in ethanol (40% w/v), it is stable in the pH range of  4-9 
and can be heated at 60 ℃ for 10 hours without showing 
any kind of  denaturation effects. It is widely used in the 
preparation of  nanospheres and nanocapsules as these 
albumin nanocarriers are non-toxic, biocompatible, 
biodegradable, easy to prepare, non-immunogenic, have 
well defined sizes and also carry some reactive groups 
such as thiols, amines and carboxyl. These groups can 
work as surface modifiers during the cross-linking process. 
Also, the bioactive compounds encapsulating albumin 
nanoparticles can be easily digested by the enzyme 
protease, releasing the entrapped material. Achieving the 
required particle size of  albumin nanoparticles is easy and 
reproducible. (Verma et al., 2018; Amighi et al., 2020)
Albumin nanoparticles can be prepared by various 
techniques such as desolvation, emulsification, 
Nanoparticle Albumin-Bound (NAB) technology 
and nano-spray drying. (Amighi, et al., 2020).  Among 
these methods, the most commonly used methods 
are desolvation and emulsification. (Niknejad & 
Mahmoudzadeh, 2015)
Under the desolvation method, a desolvation agent 
which is an organic solvent such as ethanol or acetone or 
even a natural salt is added into the aqueous solution of  
albumin. By adding desolvation agents, albumin starts to 
change its tertiary structure slowly. Then at a certain level, 
protein clumps (aggregates) are made and finally these 
unstable aggregates are hardened by the cross-linking 
agent. (Niknejad & Mahmoudzadeh, 2015) In order to 
separate the particles, the turbidity of  the system should 
be increased. In the emulsification method, an aqueous 
phase of  albumin is prepared with distilled water, which 
is added to an organic phase plant oil such as cotton seed 
oil under mechanical homogenizer until an oil-water 
emulsion is prepared.  This emulsion is then added into 
preheated oil over 120 ∘C drop by drop. This results 
in evaporation of  water and irreversible destruction of  
albumin which leads to the formation of  nanoparticles. 
The resulting particles are then suspended in an ice-cold 

bath. (Verma et al., 2018)
BSA nanoparticles can be synthesized using the 
desolvation method, where ethanol can be used as the 
desolvation agent to obtain nanoparticles with narrower 
size distribution and controllable particle size. Cross-
linking is an essential step in nanoparticle synthesis, as 
it influences bio-decomposability of  the loaded bioactive 
compounds and their release from the nanocarrier 
system. A cross-linker is added to stabilize the newly 
formed nanoparticles. The addition of  a cross-linking 
agent changes the surface charge of  nanoparticles. Also, it 
modifies the colloidal stability and electrostatic potential 
of  the BSA nanoparticles in solution. Recently, natural 
origin cross-linkers such as citric acid which guarantees 
human health have started to become very popular. 
During the cross-linking process of  citric acid, more than 
one carboxyl group reacts with proteins. Also, citric acid 
starts its cross-linking process in alkaline pH values. At 
higher pH values, amino groups are deprotonated, so the 
free amino groups attack the partially positively charged 
carbonyl carbons of  citric acid resulting in a nucleophilic 
substitution. (Amighi et al., 2020; Aniesrani Delfiya et al., 
2016))
The objective of  the present study was to synthesize 
and characterize ALNP, so that it could be used as an 
antidiabetic nutraceutical to treat hyperglycemia. 

MATERIALS AND METHODS
Raw Materials
Alpinia calcarata rhizomes

Chemicals
BSA, Ethanol, Citric acid, Sodium hydroxide, Alpha 
amylase, Starch, Dinitrosallicylic acid reagent (DNS), 
Sodium hydrogen phosphate, Sodium dihydrogen 
phosphate, Dimethyl sulfoxide (DMSO), Anhydrous 
KBr, Baker’s yeast and Anhydrous dextrose.

Instruments
Electric grinder (Sumeet, India), Analytical balance 
(Kern ALJ 120-4 Germany), Pressure cooker (Prestige 
India), Centrifuge machine, Magnetic stirrer, pH meter, 
Thermometer, Electric oven, Microplate reader (Spectra 
Max M5, Molecular Devices, CA, USA), Malvern Zetasizer 
Nano ZS apparatus (Malvern Instruments Ltd., Malvern, 
UK), Field emission scanning electron microscope 
(Hitachi SU6600 FE-SEM), UV-VIS spectrophotometer 
(Agilent Technologies, Germany. Cary 60), FT-IR 
(Fourier transform infrared) spectrometer (PerkinElmer, 
L 1600300 Spectrum TWO LITA, Liantrisant, UK).

Preparation of  Alpinia calcarata powder
Dried Alpinia calcarata rhizomes grown in Sri Lanka were 
obtained and ground into fine powder using an electric 
grinder and refrigerated at -10 ℃ until further use.

Preparation of  aqueous Alpinia calcarata extract
A. calcarata powder (10.00 g, < 0.5 mm) was digested using 

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distilled water (100.00 mL) under a pressure of  15 psi 
(121 ℃) for 15 minutes in medium pressure. The extract 
was filtered using a muslin cloth and the filtrate was 
centrifuged at 3000 rpm for 15 minutes. The supernatant 
obtained was then stored at -20 ℃ until further analysis. 

Preparation of  ALNP
For the synthesis of  ALNP, an aqueous extract of  A. 
calcarata (4.00 mL) was mixed with ethanol (16.00 mL) 
and added at a rate of  1.00 mL/min to a BSA solution (20 
mg/mL, 4.00 mL, pH 9) while the solution was constantly 
stirring at 600 rpm at 4 ℃. Then citric acid (8% w/v, 
230 µL) was added as the cross-linking agent, and the 
mixture was allowed to stir for another 2-3 hours, at 4 
℃. The mixture was then stored at 4 ℃ for 24 hours, for 
the formation of  stable nanoparticles, by facilitating the 
cross-linking process. The nanoparticle bearing solution 
was centrifuged at 3000 rpm for 30 minutes and the pellet 
containing the nanoparticles was purified by five cycles 
of  centrifugation (15000 g, 10 min) using Milli-Q water. 
The obtained particles were then dried at 50 ℃ until a 
constant weight was observed and then stored at 4 ℃ 
until further analysis. 

Determining the yield of  ALNP
Weight of  the obtained product was measured. Total 
concentration of  all compounds in A.calcarata extract was 
determined by evaporating A.calcarata extract (1.00 mL) 
on an evaporating glass and weighing the remaining. Using 
the obtained data, the yield was calculated according to 
the following equation.
Yield = (Weight of  the product (g) /Weight of  used BSA 
+ Weight of  A.calcarata extract)×100 

Determining antidiabetic activity of  ALNP
Antidiabetic activity of  the aqueous Alpinia calcarata 
extract and ALNP were determined by carrying out in-
vitro alpha amylase inhibition assay.

Alpha amylase inhibition assay
The alpha amylase inhibitory activity of  aqueous A.calarata 
extract and ALNP were determined by following the 
method specified in Oyedemi, et al., 2017 with slight 
modifications.
A concentration series (1 – 8 mg/mL) of  the aqueous 
A.calarata extract was prepared. Each solution (500 µL) 
was placed in a separate Eppendorf  tube. Alpha amylase 
enzyme (0.05 mg/mL, 250 µL) in sodium phosphate 
buffer (0.02 M, pH 6.9) was added to each Eppendorf  
tube. The mixtures were then incubated at room 
temperature (28 ± 2 ℃) for 15 minutes. Starch solution 
(1%, 250 µL) in sodium phosphate buffer (0.02 M, pH 
6.9) was added. The reaction mixtures were incubated 
at room temperature (28 ± 2 ℃) for 15 minutes. DNS 
reagent (250 µL) was added to each Eppendorf  tube, and 
all the tubes were boiled for 5 minutes. Absorbance was 
measured at 540 nm using the microplate reader. This 
procedure was triplicated. 

Three more separate series were carried out, one where 
the enzyme was replaced with sodium phosphate buffer 
(0.02 M, pH 6.9, 250 µL), another where the aqueous 
A.calcarata extract was replaced with sodium phosphate 
buffer (0.02 M, pH 6.9, 500 µL) and another series where 
both the aqueous A.calcarata extract and the enzyme were 
replaced by sodium phosphate buffer (0.02 M, pH 6.9, 
750 µL). Each of  these series were also performed in 
triplicates. 
Percentage inhibition of  the alpha amylase enzyme was 
calculated using the following formula.

A = Absorbance at 540 nm without inhibitor and with enzyme
B = Absorbance at 540 nm without inhibitor and enzyme
C = Absorbance at 540 nm with inhibitor and enzyme
D = Absorbance at 540 nm with inhibitor and without enzyme
Based on the percentage inhibition values, the IC50 
value of  the aqueous A.calcarata extract on alpha amylase 
enzyme was calculated using the GraphPad Prism 9.2.0 
software. 
Concentration series of  ALNP (0.13 – 1.00 mg/mL) was 
prepared using DMSO as the solvent. The percentage 
inhibition and IC50 value of  ALNP on alpha amylase 
enzyme was calculated using the same procedure that was 
followed for the aqueous A.calcarata extract.
 
Yeast glucose uptake assay
Glucose uptake by yeast cells in the presence of  A. 
calcarata aqueous extract and ALNP were determined 
by following the method described by Cirillo, et al., 
1962 with slight modifications. A 1% yeast solution was 
prepared by dissolving commercial baker’s yeast (0.25 
g) in distilled water (0.25 mL) and was left overnight at 
room temperature. The following day, the solution was 
centrifuged at 4200 rpm for 5 min repeatedly until a 
clear supernatant was obtained. 10% (v/v) suspension 
was prepared in distilled water. Glucose solution (25 
mM) was prepared by dissolving anhydrous glucose 
(0.45 g) in distilled water (100.0 mL). Then using the 
prepared glucose solution, 10 mM and 5 mM glucose 
concentrations were prepared by diluting with distilled 
water. A concentration series of  A. calcarata aqueous 
extract (0.48 – 7.60 mg/mL) as well as a concentration 
series of  ALNP (0.03 – 0.54 mg/mL) were prepared by 
using distilled water and DMSO as solvents, respectively. 
The extract (1.00 mL) and nanoparticle suspension (1.00 
mL) were added separately to glucose solution (5 mM, 10 
mM, and 25 mM, 1.00 mL) and incubated at 37 °C for 
10 min. Then the yeast suspension (100 μl) was added 
to start the reaction, and the mixtures were vortexed 
and further incubated at 37 ℃ for 60 min. The tubes 
were then centrifuged (2500 g, 5 min) and glucose in 
the supernatant was estimated by adding DNS reagent 
(250 μl) to supernatant (1.00 mL) and the mixtures were 
boiled for 5 min. Absorbance was measured at 540 nm 
by microplate reader. Blank was prepared by adding 
distilled water (300 μl) instead of  the sample solution. 

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The procedure was triplicated. The percentage increase 
in glucose uptake by yeast cells was calculated using the 
following formula.

Characterization of  ALNP
Solubility
ALNP (5.0 mg) was added to distilled water (5.00 mL) and 
stirred for 30 minutes at 600 rpm at room temperature. 
After that the mixture was centrifuged at 10 000 rpm for 
15 minutes. Pellet was dried at 100 ◦C in an oven until 
a constant weight was observed. The procedure was 
triplicated. The solubility was calculated according to the 
following formula. 

A.calcarata loading and entrapment efficiency
A concentration series from the pressured water 
extract of  A.calcarata was prepared. Absorbance of  the 
concentration series was measured at 310 nm. A graph 
of  absorbance at 310 nm vs concentration of  A.calcarata 
extract was plotted (Attieh, et al., 2015). Absorbance of  
the supernatants which remained after the separation of  
the product containing pellet, was measured at 310 nm. 
By comparing the absorbance value of  the supernatant 
with the standard curve, the concentration of  remaining 
A.calcarata compounds in the supernatant was found. 
The procedure was triplicated. A.calcarata loading and 
entrapment efficiencies were determined by using 
following formulae.
Weight of  A.calcarata loaded = Weight of  A.calcarata 
added - Weight of  A.calcarata in supernatant

Determination of  particle size and zeta potential
The particle size, PDI and zeta potential of  the ALNP 
were determined with the Malvern Zetasizer Nano ZS 
apparatus. An aqueous suspension of  ALNP was diluted 
1:100 with ultrapure water and the solution was placed 
in a disposable polystyrene cuvette and the particle 
size measurement was obtained. The solution was also 
placed in a folded capillary zeta cell and the zeta potential 
measurement was obtained. Both procedures were 
triplicated. 

Morphological observations
Field emission scanning electron microscope was used to 
visualize the morphology and shape of  the synthesized 
ALNP. The sample was mounted onto the sample stub 

using carbon tapes and the images were taken after gold 
sputter coating for 15 seconds.
UV-visible absorbance spectra
The UV-visible absorption spectra were analyzed using 
a UV-visible spectrophotometer from 200 to 500 nm 
within a 1 cm quartz cell. A 0.8 mg/mL synthesized 
ALNP sample was prepared after dissolving it in DMSO 
and its absorbance spectrum was obtained. It was then 
compared with the absorbance spectra of  1 mg/mL pure 
BSA, 0.07 mg/mL aqueous A.calcarata extract and 0.8% 
w/v citric acid. The absorbance spectrum of  distilled 
water was subtracted from all sample spectra.

FT-IR spectroscopy
The molecular characteristics of  the ALNP were 
examined and compared with that of  pure BSA, oven 
dried aqueous A.calcarata extract and pure cross-linking 
agent, citric acid using an FT-IR spectrometer. Each 
sample was mixed with anhydrous KBr in a 1:10 ratio 
and ground using a motor and pestle until a fine powder 
was obtained. A small portion of  the powder was placed 
in the pellet forming mold and pressed under pressure. 
Then the pellet was placed in the FT-IR spectrometer and 
scanned in the wavenumber range of  750-4000 cm-1.

Statistical Analysis
The obtained data were statistically analyzed by one-way 
analysis of  variance (ANOVA) using Minitab software 
package. The results were expressed in the form of  mean 
± standard deviation of  triplicate determinants. The level 
of  significance was taken at 5% confidence interval (p < 
0.05). 

RESULTS 
Physical appearance of  ALNP

Table 1: Yield and morphology of  ALNP
Yield (%) Morphology
94.11 Light brown coloured powder

Table 2: IC50 values of  alpha amylase enzyme
Sample IC50 value on alpha amylase 

enzyme (µg/mL)
Aqueous A.calcarata 
extract

152.10 (±0.10) a

ALNP 147.00 (±0.97) b

Means followed by different letters are significantly different (p 
< 0.05)

Antidiabetic activity
Alpha amylase inhibition assay

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Figure 1 : The graph of  percentage inhibition (%) of  
aqueous A.calcarata extract on alpha amylase enzyme vs. 
concentration

Figure 2 : The graph of  percentage inhibition (%) of  
ALNP on alpha amylase enzyme vs. concentration 

Table 3 : Percentage increase in glucose uptake by yeast cells at different glucose concentrations in the presence of  
aqueous A.calcarata extract
Concentration(mg/mL) Glucose uptake percentage (%) at different glucose concentrations

5 mM 10 mM 25 mM
7.6 68.66 (±0.02) a 43.57 (±0.26) a 24.20 (±2.02) a

3.8 64.61 (±0.29) b 32.28 (±0.26) b 22.22 (±0.13) ab

1.9 58.45 (±0.34) c 20.68 (±0.91) c 20.20 (±0.39) b

0.95 50.78 (±0.07) d 17.14 (±0.47) d 16.69 (±0.16) c

0.475 45.30 (±0.97) e 15.23 (±0.40) e 14.16 (±0.30) d

Means followed by different letters are significantly different (p < 0.05)

Table 4 : Percentage increase in glucose uptake by yeast cells at different glucose concentrations in the presence of  
ALNP
Concentration(mg/mL) Glucose uptake percentage (%) at different glucose concentrations
0.54 73.09 (±0.06) a 49.81 (±0.09) a 28.89 (±0.56) a
0.27 71.28 (±0.52) b 38.41 (±0.28) b 26.82 (±0.88) b
0.135 68.97 (±0.80) c 31.66 (±0.40) c 24.82 (±0.76) c
0.0675 64.17 (±0.16) d 25.38 (±0.72) d 20.97 (±0.87) d
0.03375 60.80 (±0.70) e 18.03 (±0.82) e 16.63 (±0.35) e

Means followed by different letters are significantly different (p < 0.05)

Yeast glucose uptake assay

Characterization of  ALNP

Table 5 : Solubility, A.calcarata loading percentage and A.calcarata entrapment efficiency of  ALNP
Solubility in water (%) A.calcarata loading percentage (%) A.calcarata entrapment efficiency (%)
64.00 (±1.00)  6.66 (±0.01) 87.71 (±0.07) 

Table 6 : Particle size, PDI and zeta potential of  ALNP
Parameter Mean ± Standard deviation
Particle size 1030.70 (±75.3) nm
PDI 0.199 (± 0.003)
Zeta potential 2.57 (±0.32) mV

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Figure 3: SEM image of  ALNP

Figure 4 : UV-Visible absorbance spectra of  pure BSA, aqueous A.calcarata extract, pure citric acid and ALNP

Figure 5 : FT-IR spectra of  pure BSA, aqueous A.calcarata extract, pure citric acid and ALNP

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DISCUSSION
As the rhizome extract of  Sri Lankan Alpinia calcarata has 
shown potential antidiabetic effects, its pressured water 
extract was used to synthesize nanoparticles, in order to be 
used as an antidiabetic nutraceutical. (Samarasinghe, et al., 
2020) Nanoparticles were synthesized using its extract in 
order to increase the solubility of  its bioactive compounds 
while allowing specific delivery and controlled release to 
target sites with reduced side effects and prolonged shelf-
life. (Assadpour & Mahdi Jafari, 2019; Paolino et al., 2021)
The synthesized ALNP appeared in powder form (Table 1). 
When compared to the conventional microencapsulated 
products obtained from techniques such as spray drying 
and freeze drying, these powder form nanocarriers provide 
more bioavailability through increased surface-to-volume 
ratio providing them the ability to easily pass through the 
cell membranes and penetrate into target cells releasing 
their encapsulated compounds. (Assadpour & Mahdi 
Jafari, 2019) The obtained colour of  the ALNP should 
be due to the entrapment of  the bioactive compounds of  
the extract within the synthesized nanocarriers. As a high 
concentration of  BSA was used during the nanoparticle 
synthesis, a high yield of  94.11% of  ALNP was recorded. 
(Aniesrani et al., 2016)
IC50 values of  aqueous A .calcarata extract and ALNP 
are given in in table 2. When comparing the IC50 values, 
it can be seen that the synthesized ALNP showed a 
higher alpha amylase inhibitory activity than the crude A. 
calcarata extract. This proves that the antidiabetic activity 
of  bioactive compounds becomes much higher when 
loaded onto nanocarriers probably due to the increased 
surface area to volume ratio resulting in a higher feasibility 
of  interacting with the alpha amylase enzyme. 
The percentage increase in glucose uptake by yeast 
cells in the presence of  aqueous A.calcarata extract and 
ALNP are given in table 3 and table 4 respectively. The 
glucose uptake by yeast cells is indicated by the change 
in glucose concentration in the medium after a particular 
time interval. The aqueous A.calcarata extract as well as 
ALNP have promoted the uptake of  glucose across the 
plasma membrane of  yeast cells according to tables 3 and 
table 4 respectively. However, the glucose uptake capacity 
at approximately 0.5 mg/mL aqueous extract was 45.30 
(±0.97) %, and for that of  synthesized ALNP was 73.09 
(±0.06) %, for the 5mM glucose concentration. Similarly, 
the ALNP exhibited a higher activity than the aqueous 
extract at all glucose concentrations used in the study. 
Therefore, this once again proves that the antidiabetic 
activity of  the aqueous extract becomes further 
enhanced when going into the nanoscale. Moreover, 
the glucose uptake capacity at 0.03 mg/mL of  ALNP 
was 60.80 (±0.70) % and that has reached up to 73.09 
(±0.06) % when 0.5 mg/mL of  ALNP was used, in the 
presence of  a 5 mM glucose concentration. This means 
that by increasing the nanoparticle concentration, the 
capability of  yeast cells to uptake more glucose from the 
environment can be increased. A similar increment in the 
glucose uptake capacity was observed when 10 mM and 

25 mM glucose concentrations were used. However, an 
inverse relationship to the molar concentration of  glucose 
was observed with the percent increase in glucose uptake 
by yeast cells, when 5 mM, 10 mM and 25 mM glucose 
concentrations were compared for the same amount of  
ALNP. From the results it is clear that lower the glucose 
concentration, higher the uptake by yeast cells. This 
observation is in good agreement with the earlier work 
of  Rehman, et al., 2018 and Bhutkar, et al., 2016. 
The solubility of  ALNP in water is given in table 5. 
Solubility of  the ALNP is determined by the pH of  the 
BSA solution as it affects the size of  the nanoparticles 
formed. The isoelectric point of  BSA is around 4.7. 
Hence, at pH 9 the BSA protein molecules are ionized 
and therefore they tend to repulse each other. This causes 
the synthesized ALNP to become smaller and smaller, 
increasing their surface area and thereby resulting in 
greater solubility in water. Table 5 also shows the A. 
calcarata loading percentage and A. calcarata entrapment 
efficiency of  the ALNP. During nanoparticle synthesis, 
as the BSA concentration that was used was high (20 mg/
mL), it may have increased the number of  nanoparticles 
that were formed. When the quantity of  nanoparticles is 
higher, it will decrease the amount of  A. calcarata that gets 
loaded with constant addition. Also, the high pH of  the 
BSA solution (pH 9) will also decrease the amount of  
A. calcarata that gets loaded due to the high electrostatic 
repulsion between BSA and A.calcarata. This explains the 
small value for the A. calcarata loading percentage of  the 
synthesized ALNP. Also, a high BSA concentration will 
increase the entrapment efficiency as larger particles have 
larger volumes which increases the quantity of  A. calcarata 
that can be held by the synthesized ALNP. This explains 
the high value obtained for the A.calcarata entrapment 
efficiency of  ALNP. (Aniesrani et al., 2016; Ranasinghe, 
et al., 2013)
ALNP showed a mean diameter of  1030.70 (±75.3) 
nm (Table 6). According to Amighi, et al., 2020, the 
nanoparticle size that is obtained under the conditions 
that were used in ALNP synthesis with citric acid as the 
cross-linking agent is 1201.00 (±58.4) nm. As the mean 
particle size obtained for ALNP is very close to this value, 
it is clear that these nanoparticles have been formed 
effectively. 
According to Danaei, et al., 2018, the PDI of  a nanoparticle 
sample with effective particle size distribution should be 
between 0.05-0.7. As the PDI value of  ALNP falls within 
this range, it can be stated that a homogenous population 
of  nanocarriers have been synthesized, which can act 
as safe, stable and efficient nanocarriers of  A. calcarata. 
Therefore, the tendency of  this antidiabetic nutraceutical 
to accumulate in the target tissue, which depends on the 
particle size distribution will be minimal. 
Nanoparticles with a zeta potential between -10 and +10 
mV are considered neutral, while nanoparticles with zeta 
potentials greater than +30 mV are considered strongly 
cationic and those with zeta potentials less than -30 mV are 
considered strongly anionic. Since most cell membranes 

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are negatively charged, zeta potential can strongly affect 
a nanoparticle’s tendency to penetrate membranes, with 
cationic particles displaying toxicity due to cell membrane 
disruption. (Clogston & Patri, 2011) As the zeta potential 
of  ALNP is 2.57 (±0.32) mV, these nanoparticles 
are neutral and will be able to easily penetrate the cell 
membranes without causing any toxicological effects. 
This further ensures that the synthesized ALNP can be 
effectively used as an antidiabetic nutraceutical. 
The morphological analysis of  ALNP was carried out 
with FE-SEM, and the obtained image is shown in figure 
3. The SEM micrograph obtained revealed morphological 
aspects of  ALNP with a spherical shape and uniform size. 
When comparing the UV-visible absorbance spectrum of  
ALNP with that of  the aqueous A.calcarata extract, peaks 
at 210 nm and 280 nm were observed in both spectra 
(Figure 4). This means that the active compounds of  
A. calcarata have been successfully loaded into the BSA 
nanoparticles. Therefore, this proves that the active 
compounds of  the aqueous Alpinia calcarata extract have 
been successfully loaded into the BSA nanoparticles. 
However, a peak corresponding to citric acid was not 
observed in the UV absorbance spectrum of  the A. 
calcarata loaded nanoparticles. This means that citric 
acid was completely washed away during the washing 
step performed in the purification of  nanoparticles. 
The pure BSA spectrum showed a characteristic peak at 
280 nm. Therefore, the peak observed at 280 nm in the 
ALNP spectrum may be due to both BSA and the active 
compounds of  A. calcarata.
When considering the FT-IR spectrum of  ALNP, it too 
showed peaks that could also be observed in the FT-
IR spectrum of  the aqueous Alpinia extract (Figure 5). 
Characteristic peaks including =C-H stretch at 3300 cm-
1, C=C benzene stretch of  two peaks between 1400-1600 
cm-1 and OH stretch by a broad peak between 3000-
3600 cm-1 were observed in both spectra. Therefore, this 
gives further evidence for the successful entrapment of  
the A.calcarata compounds within the synthesized ALNP. 
According to Xu, et al., 2015, one to two carboxyl groups 
of  one citric acid molecule can react with BSA, resulting 
in an increase in the total amount of  carboxyl groups and 
a decrease in the total amount of  amine groups in the 
cross-linked nanoparticles. This explains the presence 
of  a peak corresponding to the OH stretch and decrease 
in intensity of  the peak at 3400 cm-1 corresponding to 
the N-H stretch in the spectrum of  ALNP. This means 
that interactions between BSA and citric acid have caused 
conformational changes in the protein structure of  the 
synthesized nanoparticles. A similar observation has been 
made by Amighi, et al., 2020 during the synthesis of  BSA 
nanoparticles using citric acid as the cross-linking agent. 
Furthermore, two peaks at 1665 cm-1 and 1537 cm-1 
were observed in the pure BSA spectrum corresponding 
to the Amide I and Amide II stretches respectively. 
However, in the ALNP spectrum the peak corresponding 
to the Amide I stretch was absent and a noticeable shift 
was seen in the peak corresponding to the Amide II 

stretch. Hence, loading of  A. calcarata compounds have 
also induced conformational changes in the structure of  
the BSA protein. This means that the active compounds 
of  A. calcarata have interacted with the protein matrix of  
the synthesized nanoparticles via covalent bonds. This 
result is similar to the results obtained by Rani, 2016.

CONCLUSION
The present study has proven that the antidiabetic activity 
of  bioactive compounds of  Alpinia calcarata becomes 
enhanced when loaded onto nanocarriers. A high yield 
of  94.11% of  ALNP were synthesized which appeared 
in powder form, had a spherical morphology, uniform 
size, with effective particle size distribution and a neutral 
surface charge. As the BSA solution used had a pH 
value of  9, which was further away from its isoelectric 
point, ALNP reported a solubility percentage of  64%. 
A high A. calcarata entrapment efficiency of  87.71% and 
an A.calcarata loading percentage of  6.66% were also 
recorded for the synthesized nanoparticles. The mean 
particle size of  the ALNP was 1030.70 nm. The FT-IR 
spectrum of  nanoparticles showed that the cross-linking 
agent, citric acid had caused conformational changes 
in the protein structure of  BSA and that the active 
compounds were successfully loaded into the synthesized 
nanoparticles which interacted with the protein matrix via 
covalent bonds. The UV-visible absorbance spectrum of  
ALNP further proved the successful entrapment of  the 
active A. calcarata compounds within the nanoparticles and 
also showed that citric acid was completely washed away 
during the washing step. Therefore, it can be concluded 
that ALNP have been synthesized effectively, which can 
be used as a powder form antidiabetic nutraceutical. A 
limitation of  this study was the high particle size obtained 
for the synthesized ALNP and therefore future work 
can be carried out to reduce it by testing various other 
desolvation agents and cross-linking agents. Also, in-vivo 
antidiabetic analysis can be carried out to test the effect 
of  the synthesized ALNP within living organisms in the 
future. 

Acknowledgements 
This study was funded by the University Grant 
RP/03/02/06/01/2021 which is gratefully acknowledged. 

Conflict of  interest
There is no conflict of  interest.

REFERENCES 
Amighi, F., Emam-Djomeh, Z., & Labbafi-Mazraeh-

Shahi, M. (2020). Effect of  different cross-linking 
agents on the preparation of  bovine serum albumin 
nanoparticles. Journal of  the Iranian Chemical Society, 
17(5), 1223–1235. https://doi.org/10.1007/s13738-
019-01850-9

Aniesrani Delfiya, D. S., Thangavel, K., & Amirtham, 
D. (2016). Preparation of  Curcumin Loaded 
Egg Albumin Nanoparticles Using Acetone and 

https://journals.e-palli.com/home/index.php/ajcp


Pa
ge

 
30

https://journals.e-palli.com/home/index.php/ajcp

Am. J. Chem. Pharm. 2(1) 21-31, 2023

Optimization of  Desolvation Process. The Protein 
Journal, 35(2), 124–135. https://doi.org/10.1007/
s10930-016-9652-3

Arambewela, L. S. R., Kumaratunge, A., Arawwawela, M., 
Owen, N. L., & Du, L. (2005). Volatile Oils of  Alpinia 
calcarata Rosc. Grown in Sri Lanka. Journal of  Essential 
Oil Research, 17(2), 124–125. https://doi.org/10.1080
/10412905.2005.9698850

Assadpour, E., & Mahdi Jafari, S. (2019). A systematic 
review on nanoencapsulation of  food bioactive 
ingredients and nutraceuticals by various nanocarriers. 
Critical Reviews in Food Science and Nutrition, 59(19), 
3129–3151. https://doi.org/10.1080/10408398.201
8.1484687

Assadpour, E., & Mahdi Jafari, S. (2019). A systematic 
review on nanoencapsulation of  food bioactive 
ingredients and nutraceuticals by various nanocarriers. 
Critical Reviews in Food Science and Nutrition, 59(19), 
3129–3151. https://doi.org/10.1080/10408398.201
8.1484687

Attieh, H. A., Abu Lafi, S., Jaber, S., Abu-Remeleh, 
Q., Lutgen, P., & Akkawi, M. (2015). Cinnamon 
bark water-infusion as an in-vitro inhibitor of  
β-hematin formation. https://dspace.alquds.edu/
handle/20.500.12213/1023

Bhutkar, M. A., Bhinge, S. D., Randive, D. S., & Wadkar, G. 
H. (2017). Hypoglycemic effects of  Berberis aristata 
and Tamarindus indica extracts in vitro. Bulletin of  
Faculty of  Pharmacy, Cairo University, 55(1), 91–94. 
https://doi.org/10.1016/j.bfopcu.2016.09.001

Cirillo, V. P. (1962). Mechanism of  glucose transport 
across the yeast cell membrane. Journal of  Bacteriology, 
84(3), 485–491. https://www.ncbi.nlm.nih.gov/
pmc/articles/PMC277903/

Clogston, J. D., & Patri, A. K. (2011). Zeta potential 
measurement. Methods in Molecular Biology (Clifton, 
N.J.), 697, 63–70. https://doi.org/10.1007/978-1-
60327-198-1_6

Danaei, M., Dehghankhold, M., Ataei, S., Hasanzadeh 
Davarani, F., Javanmard, R., Dokhani, A., Khorasani, 
S., & Mozafari, M. R. (2018). Impact of  Particle Size 
and Polydispersity Index on the Clinical Applications 
of  Lipidic Nanocarrier Systems. Pharmaceutics, 10(2), 
57. https://doi.org/10.3390/pharmaceutics10020057

Fourier Transform Infrared Spectroscopy (FTIR) 
Spectral Analysis of  BSA Nanoparticles (BSA NPs) 
and Egg Albumin Nanoparticles (EA NPs). (2016). 
Research Journal of  Chemical Sciences, 6(2), 29–36. 
http://www.isca.in/rjcs/Archives/v6/i2/5.ISCA-
RJCS-2016-006.php

Guthrie, R. A., & Guthrie, D. W. (2004). Pathophysiology 
of  diabetes mellitus. Critical Care Nursing Quarterly, 
27(2), 113–125. https://doi.org/10.1097/00002727-
200404000-00003

Hanadi, A. A., Saleh, A. L., Suhair, J., Qassem, A. R., 
Pierre, L., & Mutaz, A. (2015). Cinnamon bark 
water-infusion as an in-vitro inhibitor of  -hematin 
formation. Journal of  Medicinal Plants Research, 9(38), 

998–1005. https://doi.org/10.5897/JMPR2015.5931
Jayawardena, B., & Smith, R. M. (2010). Superheated 

water extraction of  essential oils from Cinnamomum 
zeylanicum (L.). Phytochemical Analysis: PCA, 21(5), 
470–472. https://doi.org/10.1002/pca.1221

Kazeem, M. I., & Davies, T. C. (2016). Anti-diabetic 
functional foods as sources of  insulin secreting, 
insulin sensitizing and insulin mimetic agents. 
Journal of  Functional Foods, 20, 122–138. https://doi.
org/10.1016/j.jff.2015.10.013

Kerner, W., Brückel, J., & German Diabetes 
Association. (2014). Definition, classification 
and diagnosis of  diabetes mellitus. Experimental 
and Clinical Endocrinology & Diabetes: Official 
Journal, German Society of  Endocrinology German 
Diabetes Association, 122(7), 384–386. https://doi.
org/10.1055/s-0034-1366278

Kharroubi, A. T., & Darwish, H. M. (2015). Diabetes 
mellitus: The epidemic of  the century. World Journal 
of  Diabetes, 6(6), 850–867. https://doi.org/10.4239/
wjd.v6.i6.850

Niknejad, H., & Mahmoudzadeh, R. (2015). Comparison 
of  Different Crosslinking Methods for Preparation 
of  Docetaxel-loaded Albumin Nanoparticles. Iranian 
Journal of  Pharmaceutical Research : IJPR, 14(2), 385–
394. https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC4403054/

Oyedemi, S. O., Oyedemi, B. O., Ijeh, I. I., Ohanyerem, 
P. E., Coopoosamy, R. M., & Aiyegoro, O. A. 
(2017). Alpha-Amylase Inhibition and Antioxidative 
Capacity of  Some Antidiabetic Plants Used by the 
Traditional Healers in Southeastern Nigeria. The 
Scientific World Journal, 2017, 3592491. https://doi.
org/10.1155/2017/3592491

Paolino, D., Mancuso, A., Cristiano, M. C., Froiio, 
F., Lammari, N., Celia, C., & Fresta, M. (2021). 
Nanonutraceuticals: The New Frontier of  
Supplementary Food. Nanomaterials (Basel, Switzerland), 
11(3), 792. https://doi.org/10.3390/nano11030792

Rahman, M. A., & Islam, M. S. (2015). Alpinia calcarata 
Roscoe: A potential phytopharmacological source of  
natural medicine. Pharmacognosy Reviews, 9(17), 55–62. 
https://doi.org/10.4103/0973-7847.156350

Rahman, M. A., & Islam, M. S. (2015). Alpinia calcarata 
Roscoe: A potential phytopharmacological source of  
natural medicine. Pharmacognosy Reviews, 9(17), 55–62. 
https://doi.org/10.4103/0973-7847.156350

Ranasinghe, P., Pigera, S., Premakumara, G. A. S., 
Galappaththy, P., Constantine, G. R., & Katulanda, 
P. (2013). Medicinal properties of  “true” cinnamon 
(Cinnamomum zeylanicum): A systematic review. 
BMC Complementary and Alternative Medicine, 13, 275. 
https://doi.org/10.1186/1472-6882-13-275

Rehman, G., Hamayun, M., Iqbal, A., Ul Islam, S., Arshad, 
S., Zaman, K., Ahmad, A., Shehzad, A., Hussain, A., 
& Lee, I. (2018). In Vitro Antidiabetic Effects and 
Antioxidant Potential of  Cassia nemophila Pods. 
BioMed Research International, 2018, e1824790. https://

https://journals.e-palli.com/home/index.php/ajcp


Pa
ge

 
31

https://journals.e-palli.com/home/index.php/ajcp

Am. J. Chem. Pharm. 2(1) 21-31, 2023

doi.org/10.1155/2018/1824790
Samarasinghe, B., Kaliyadasa, E., & Marasinghe, P. (2020). 

Physicochemical Properties and Bioactivities of  Six 
Alpinia Species in Sri Lanka. International Journal of  
Ayurvedic Medicine, 11(4). https://doi.org/10.47552/
ijam.v11i4.1717

Sapra, A., & Bhandari, P. (2022). Diabetes Mellitus. In 
StatPearls. StatPearls Publishing. http://www.ncbi.
nlm.nih.gov/books/NBK551501/

Teo, C. C., Tan, S. N., Yong, J. W. H., Hew, C. S., & Ong, E. 
S. (2010). Pressurized hot water extraction (PHWE). 
Journal of  Chromatography A, 1217(16), 2484–2494. 
https://doi.org/10.1016/j.chroma.2009.12.050

Verma, D., Gulati, N., Kaul, S., Mukherjee, S., & Nagaich, 
U. (2018). Protein Based Nanostructures for Drug 
Delivery. Journal of  Pharmaceutics, 2018, 9285854. 
https://doi.org/10.1155/2018/9285854

Wariyapperuma, W. A. N. M., Kannangara, S., 
Wijayasinghe, Y., Subramanium, S., & Jayawardena, 
B. (2018). Pressured water extraction and solvent 
extraction of  Cinnamomum zeylanicum (L.) bark and 
evaluation of  anti-diabetic properties.

Wasana, K. G. P., Attanayake, A. P., Jayatilaka, K. A. 
P. W., & Weerarathna, T. P. (2021). Antidiabetic 
Activity of  Widely Used Medicinal Plants in the Sri 
Lankan Traditional Healthcare System: New Insight 
to Medicinal Flora in Sri Lanka. Evidence-Based 
Complementary and Alternative Medicine, 2021, e6644004. 
https://doi.org/10.1155/2021/6644004

Xu, H., Shen, L., Xu, L., & Yang, Y. (2015). Controlled 
delivery of  hollow corn protein nanoparticles via 
non-toxic crosslinking: In vivo and drug loading 
study. Biomedical Microdevices, 17(1), 8. https://doi.
org/10.1007/s10544-014-9926-5

https://journals.e-palli.com/home/index.php/ajcp

