Accepted Manuscript

Nano encapsulation of catechin in starch nanoparticles: Characterization; re-

lease behavior and bioactivity retention during in-vitro digestion

Mudasir Ahmad, Priti Mudgil, Adil Gani, Fathalla Hamed, Farooq A. Masoodi,
Sajid Maqsood

PII: S0308-8146(18)31163-4
DOI: https://doi.org/10.1016/j.foodchem.2018.07.024
Reference: FOCH 23139

To appear in: Food Chemistry

Received Date: 6 April 2018

Revised Date: 28 June 2018
Accepted Date: 3 July 2018

Please cite this article as: Ahmad, M., Mudgil, P., Gani, A., Hamed, F., Masoodi, F.A., Maqsood, S., Nano
encapsulation of catechin in starch nanoparticles: Characterization; release behavior and bioactivity retention during
in-vitro digestion, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.07.024

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 Running title: Novel starch-catechin nanocapsules

Nano encapsulation of catechin in starch nanoparticles: Characterization; release behavior

and bioactivity retention during in-vitro digestion

Mudasir Ahmad1,2, Priti Mudgil1, Adil Gani2*, Fathalla Hamed3, Farooq. A. Masoodi2, Sajid
Maqsood1*
1
Department of Food Science, College of Food and Agriculture, United Arab Emirates
University, 15551 Al Ain, United Arab Emirates
2
Department of Food Science and Technology, University of Kashmir, Srinagar, India, 190006.
3
Department of Physics, College of Science, United Arab Emirates University, 15551 Al Ain,
United Arab Emirates

Corresponding authors:

Sajid Maqsood, 1Department of Food Science, College of Food and Agriculture, United Arab
Emirates University, 15551 Al Ain, United Arab Emirates

Email: sajid.m@uaeu.ac.ae

Tel: +971 37134519

Fax: +971 37675336
Adil Gani
Email: adil.gani@gmail.com

1
Abstract
Novel starch-based nanoparticles from three sources: horse chestnut (HSC), water chestnut

(WSC) and lotus stem (LSC) were prepared for nano-encapsulation of catechin. Average particle

size of HSC, WSC and LSC based nano-particles were 322.7, 559.2 and 615.6 nm with

encapsulation efficiency of 59.09, 48.30, and 55.00% and negative zeta potential of -18.05, -21.5

and -18.05 mv, respectively. Structural, physical and thermal properties were characterized by

fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray

diffraction (XRD) and differential scanning calorimetry (DSC). SEM revealed capsule formation

with entrapped catechin, while broad characteristic peaks at 3475, 1650, 1383, 1148, 1083 and
-1
790 cm depicts encapsulation of catechin in starch nanoparticles without any evident

interaction. XRD showed loss of crystallinity after encapsulation. Higher content of catechin in

intestinal juice ensured controlled release in intestine. Bioactive properties were retained at

higher level in encapsulated catechin compared to free catechin upon in-vitro digestion.

Key words: Nano-encapsulation; starch; catechin; bioactivity; in-vitro digestion.

2
1. Introduction
Catechins are flavonoid compounds found in a variety of plant-based fruits, vegetables

and beverages. Fruits like grapes, apple, pear, cherries and green tea are some major sources of

catechins (Grzesik, Naparło, Bartosz, Sadowska-Bartosz, 2018). Even though catechins are not

essential to human nutrition but they play an important role in improving human health by

preventing various diseases like inhibiting carcinogenesis of the skin, lung, esophagus, stomach,

liver, small intestine, colon, bladder, prostate and mammary glands demonstrated in various

animal studies (Chung & Liu, 2010). Catechin is also known to have better antioxidant properties

than ά- tocopherol, butylated hydroxyanisole or butylated hydroxytoluene (Chun et al., 2001).

However, the major limitation for the catechin is their low bioavailability, less permeability and

degradation during human digestion, which poses the restrictions for gaining the maximum

health benefits from it. The major portion of catechin degradation is most likely to occur under

intestinal conditions where elevated pH and presence of reactive oxygen species provide

favorable conditions for catechin auto-oxidative reactions (Neilson et al., 2007). To some extent,

the researchers have overcome this problem by using microencapsulation techniques, which

involves the physical entrapment of sensitive bioactive compounds in the matrix of

macromolecules that leads to safe delivery of catechins through deleterious environments, until

assimilated in the proper human organs (Galland, 2013). But the large particle size in

microencapsulation method could not solve the problem related to low bio-accessibility of

catechins after digestion and release process. In this context, nano-encapsulation could be an

effective approach to increase their bioactivity and bio-accessibility as well as to increase their

stability under digestive conditions and protecting them from interacting with other components

of digestion and premature degradation before reaching the target site (Neilson et al., 2007). In

nano-encapsulation, the average particle diameter at nano level enhances the cell permeability

3
and solubility during the digestion process that results in proper absorption and bioavailability of

nano-encapsulated compounds (Hu et al., 2002). The most widely used materials used for

encapsulation in food or pharmaceutical applications are polysaccharides including starch and

starch derivatives, pectin, glucans, cellulose and protein based materials such as polypeptone,

soy protein, milk proteins and gelatin derivatives (Ahmad, Qureshi, Maqsood, Gani, & Masoodi,

2017; Gani, Shah, Ahmad, Ashwar, & Masoodi, 2018; Arif, Batool, Khalid, Ahmed & Janjua,

2017; Wani et al., 2016; Ahmad, Ashraf, Gani, & Gani, 2018). Researchers are always interested

in exploration of cheap and efficient source of wall material due to economic considerations of

final product. In the present study, we are utilizing a novel source of starch as wall material

obtained from horse chestnut, lotus stem and water chestnut. These crops are naturally cultivated

at large scale, but they generally go waste due to no or less consumption in many parts of the

world, even though they contain high quality and quantity of starch as per our previous studies

(Gani, Haq, Masoodi, Broadway, & Gani, 2010; Wani et al., 2014). Thus, the present research is

carried out to investigate the efficacy of water chestnut, lotus stem and horse chestnut starch for

nano-encapsulation of catechin as well as to prevent the degradation of catechin from harsh

conditions during digestion process. The release behavior of catechin from nano-capsules and

the fate of nano-encapsulation to retain the bioactive properties of catechin during simulated

gastric and intestinal conditions were studied. The catechin nano-capsules may find wide scope

in functional food industry for improving the nutraceutical properties of food products,

especially of functional beverages fortified with nano-encapsulated catechin.

2. Materials and Methods

4
2.1 Materials

Catechin hydrate and all other chemicals used were of analytical grade and purchased

from Sigma Aldrich (USA). Lotus stem and water chestnut was procured from the local market

of Srinagar, India, while as seeds of horse chestnut were collected from the trees located in the

main campus of University of Kashmir, Srinagar, India at their maturity stage. Different batches

of seeds were collected and pooled together and were de-hulled and stored at 5 °C until further

use. All starch samples were prepared, and encapsulation experiments were conducted at Food

Science Department, United Arab Emirates University, Al Ain, UAE.

2.2 Starch extraction

The samples of horse chestnut, water chestnut, and lotus stem were de-hulled/peeled and

then cut into small pieces. The pieces were pulverized along with water for 5 min in a domestic

blender and then starch was extracted using the alkaline steeping method as described by

(Ahmad et al., 2017)

2.3. Synthesis of starch nano-particles and nano encapsulation of catechin

Starch solutions (1.5%) was pretreated in 0.1M NaOH solution for 1 hour, then

centrifuged at 5000 rpm for 15 minutes, supernatant was collected and replaced by equal volume

of distilled water. This hydrolyzed starch solution was then preheated under continuous magnetic

stirring for 30 minutes and catechin hydrate (1.5%) dissolved in ethanol was then added

dropwise at the ratio of 1:10 on the slurry of this hot starch and stirred continuously for another

30 min using a magnetic stirrer, simultaneously the ethanol was removed during heating process.

To increase encapsulation efficiency of catechin hydrate and to reduce the size of the starch

particle to nano-scale, the solution was subjected to ultrasonication treatment at 40 KHz for 30

min using the fixed probe sonicator (Q55-Qsonica, USA). The sonication time of 30 min was

5
carried out at intervals of 5 min to avoid excessive heating. The resulting solution was frozen at -

18 °C then lyophilized using freeze dryer (Telstar-Cryodos, Spain) in order to make fine the

powder of catechin encapsulated nanoparticles. The prepared nano-capsules were stored at 4 °C

for further analysis.

2.4. Encapsulation efficiency

The encapsulation efficiency of the starch encapsulated catechin nanoparticles was

calculated following the method described by (Ahmad et al., 2018) with modifications. The

encapsulated nanoparticles (200 mg) were washed with 10 ml of water to remove the surface

catechin, the suspension thus obtained was centrifuged at 3000 rpm for 5 min, the supernatant

was discarded and the residue left was re-suspended in 10 ml of water and sonicated for 30 min

to extracted the entrapped catechins from nano-starch capsules. The suspension was then again

centrifuged at 5000 rpm for 10 min and the supernatant was collected. Catechin concentration

was determined by measuring the absorbance of supernatant at 290 nm using monochromatic

ELISA reader (Tecan Epoch 2, USA) and catechin content was calculated by using the following

calibration curve equation prepared by using different concentrations of catechin as standard.

Y=0.0834X+1.3955, R² = 0.9954

Where, Y is the absorbance of catechin solution, and X is the concentration of catechin in mg/ml

The encapsulation efficiency (EE) was calculated using the following equation (Eq. 1)

(Eq. 1)

2.5 Zeta potential and particle size analysis of the starch based nano-encapsulated catechin

6
 Particle size and zeta potential of nano-encapsulated catechin molecules were determined

with a Zeta-sizer (Nano S, Malvern Instruments, Worcestershire, U.K.). For the particle size

measurements, the sample (0.01%, w/v) was suspended in milli-Q water (Elix-10, Millipore,

Molsheim, France) sonicated for 30 min at 40 KHz in sonicator bath (Model) to fully disperse

the polymer. For zeta potential measurement, the sample (0.01%) was suspended in 0.1mM

KCl, pH was adjusted to 6 and samples were allowed to equilibrate overnight before

measurement.

2.6 Color values native starch and nano-encapsulated catechin in starch

A hunter lab digital colorimeter (Hunter Color Lab Elex EZ, USA) was used to measure

the color of starch wall material and starch encapsulated catechin. The samples were taken in a

hunter lab cup and uniformly spread on the base of the cup. The hunter values L*, a*, and b*

values were recorded, where L* denotes (lightness, 0-100), a* denotes the redness (-) to

greenness (+) and b* denotes the blueness (-) to yellowness (+).

2.7 Thermal characterization by Differential Scanning Calorimeter (DSC)

The thermal behavior of nano-encapsulated catechin samples were studied using DSC

(TA Instruments, DSC Q100, USA). All samples (2-3 mg) were placed in hermetically sealed

aluminum pans and loaded into the equipment chamber at room temperature. Nitrogen gas at a

flow rate of 40 mL/min was employed in the purge line to control the local environment around

the sample. Temperature was set in 20°C to 200 °C range at the flow rate of 10 °C/ min. Data

were analyzed with the universal analysis software provided along with DSC instrument.

Characteristic temperatures of transitions were defined as T o (Onset), Tp (Peak of Gelatinization),

Tc (Conclusion) and enthalpy of gelatinization (Δ H) were recorded.

7
2.8 Structure elucidation of starch nano-encapsulated catechin molecules by fourier

transform Infrared (FTIR) spectroscopy

Infrared spectra of the nano-encapsulated catechin molecules were measured using FTIR

spectrometer (Nicolet, Thermo Electron, USA). The samples were mixed well with potassium

bromide and pressed to transform into pellets. The pellets were scanned in the range of 4000 to

650 cm- 1 and spectra of all samples were collected and analyzed by using the system software.

2.9. X-ray diffraction (XRD) analysis

The crystallographic structural analysis was carried out by X-ray diffractometer

(Shimadzu Lab X–XRD–6100). The operating conditions were X-ray line (λ= 1.5418Å), voltage

40 kV and current 30 mA. The powdered samples were loaded onto an aluminum plate and X-

ray diffraction profiles were obtained for 2θ ranging from 10º to 50º with scanning rate of

0.02/min. The relative crystallinity (RC) of starch was calculated as described by (Rabek, 1980)

using the equation (Eq. 2):

Where Ac is the crystalline area; Aa is the amorphous area on the X-ray diffractogram.

2.10. Scanning electron microscopy (SEM)

Microstructural features of the starch nano-encapsulated catechin were monitored by JEOL

JSM-6010LA scanning electron microscope (SEM, Akishima, Tokyo, Japan). The samples were

placed on an aluminum SEM stub with the help of a double-sided adhesive carbon tape,

sputtered with gold up to 15 nm thickness using a JEOL Neo Coater (model MP-19020NCTR).

Fields of the samples were inspected under a high-vacuum (Neo- Scope JCM-5000 benchtop

SEM JEOL Ltd., Tokyo, Japan) and micrographs of the samples were recorded.

8
2.11. Release behavior under simulated gastro-intestinal conditions

A static model that simulates digestion in stomach and intestine was adapted. The

simulated gastric juice (SGJ) was prepared by dissolving 3 g/L pepsin in sterile NaCl solution (9

g/L) and the pH was adjusted to 2.0 with 1.0 mol/L HCl. The simulated intestinal juice (SIJ) was

prepared by dissolving 10 g/L bile salts and 3 g/L pancreatin in phosphate-buffered saline, pH

was maintained at 8 with 0.1M NaOH solution. The simulated gastric juice (SGJ) and simulated

intestinal juice (SIJ) was prepared according to the method of Gani et al. (2018). Powder samples

(200 mg) were placed in glass test tubes and incubated at 37°C under constant stirring. The

samples were first digested in simulated stomach condition by addition of 10 ml of SGJ and after

constant stirring, aliquot (1 ml) was collected after 30 min & 1h of incubation. The mixture was

centrifuged at 3000 rpm for 5 min, the supernatant was collected and 10 ml of SIJ was added to

the residue and incubated again at 37°C after constant stirring/mixing, aliquots (1 ml) was

collected after 2h and 4h. All the aliquots were centrifuged at 5000 rpm for 10 min and

supernatant liquid was collected and stored at -20°C for further analysis which were done within

a week time. The amount of catechin released was measured and calculated as mentioned in

sections 2.4.

2.12. Bioactivity retention under simulated gastrointestinal digestion conditions

Free and nano-encapsulated catechins were analysed for bioactivities (antidiabetic and

anti-obesity) to explore the effectiveness of nano-encapsulation on retention of bioactive

properties of catechin after their transit through in-vitro digestion. The 100 mg of nano-

encapsulated powder was dissolved in SGJ (5ml) and stirred gently in shaking water bath at 37

°C for 1h. The mixture was centrifuged at 3000 rpm for 5 min, the supernatant was collected and

9
5 ml of SIJ was added to the solution and kept for 2h incubation. After subjecting to SGID

conditions, the samples were centrifuged at 5000 rpm for 10 min and supernatant was collected

and stored at refrigeration temperature for further analysis. The free catechin (1mg/ml)

corresponding to the amount of catechin present in nano-encapsulated powder per ml of digesta

was also subjected to SGID conditions and prepared in a similar way as described above.

2.12.1Antidiabetic properties of nano-encapsulated catechin

2.12.1.1 α-glucosidase inhibition assay

α-glucosidase inhibitory activity was determined according to a method described by

(Zhang et al., 2015) with slight modifications. In brief, 50 µl of sample was mixed with 50 µl of

4 mM 4-nitrophenyl b-D-glucuronide (pNPG) solution (dissolved in 0.1 M phosphate buffer, pH

6.8), and 50 µl of 0.2U/ml α-glucosidase enzyme solution from yeast and mixed well into a 96

well plate. The plates were incubated at 37ºC for 30 min and release of p-nitrophenol from pNPG

was measured at 405 nm. The percentage of inhibition was calculated as showin in (Eq. 3)

Where,

A (Control) was the absorbance in the presence of enzyme and without test sample.

B (Control blank) was the absorbance without enzyme and test sample.

C (Reaction) was the absorbance with enzyme and test sample.

D (Reaction blank) was the absorbance with test sample but without enzyme.

2.12.1.2 DPP-IV inhibition assay

DPP-IV inhibitory activity of free catechins and encapsulated chatechins after transit

through simulated gastric and intestinal digestion conditions were assessed according to the

10
method described by (Nongonierma, Paolella, Mudgil, Maqsood, & FitzGerald, 2018) with some

modifications. Briefly, 50 µL Gly-Pro-p-Nitroanilide (1.6 mM, SigmaAldrich, St Louis, MO,

USA) and 25 µL sample (or 25 µl PBS as a control) were pre-incubated at 37 °C for 10 min

Then the reactions were initiated by adding 50 µL DPP-IV (8 U/L, from porcine kidney, ≥10 U

mg−1 protein, Sigma-Aldrich) and mixtures were incubated at 37°C for 90 min. Reaction was

terminated by adding 100µl of 1M sodium acetate buffer (pH 4.0). The release of p-nitrophenol

was measured by taking absorbance at 405 nm by Multiskan MK3 ELISA plate reader (Thermo

Labsystems, Franklin, MA, USA). Each sample was analyzed in triplicate, and the absorbance

values were normalized to sample blanks in which DPPIV was replaced with Tris–HCl buffer

(100 mM, pH 8.0). The percent inhibition was calculated using same Eq. 3 as mentioned above.

2.12.2 Anti-obesity activity of nano-encapsulated catechin

2.12.2.1 Pancreatic lipase (PL) inhibition assay

The lipase inhibitory activity was performed using a colorimetric method as described by

(Badmaev et al., 2015) with some modifications. In the assay p-nitrophenol butyrate (pNPB)

was used as substrate. The total assay mixture composed of 25 µL of pNPB [(10 mM) and 50 µL

of samples. After a preincubation of 10 min 25 µL of the PPL enzyme solution (50 mg/mL) in

sodium phosphate buffer (0.2 M; pH 7.2) was added to initiate the enzymatic reaction and

incubated for 30 min at 37℃. The released p-nitrophenol was monitored at 405 nm enzyme

inhibition percentage was calculated as above in Eq. 3.

2.12.2.2 Cholesterol esterase (CE) inhibition assay

Cholesterol Esterase (CE) inhibitory activity was performed using a colorimetric method of

(Shirai & Jackson, 1982) with some modifications. The total assay mixture composed of 25µL of

11
pNPB (10 mM), 50 µL of samples and 25 µL of the cholesterol esterase enzyme solution (10

µg/mL) in sodium phosphate buffer (0.2 M; pH 7.2) and incubated for 30 min at 37℃. The

released p-nitrophenol was monitored at 405 nm and the enzyme inhibition percentage was

calculated was above in Eq. 3.

2.13. Statistical analysis

Nano-encapsulation of the catechin molecules in nano-particle starch matrix was carried in three

different batches which served as three replicates for each starch wall material. Statistical

analysis was performed using commercial statistical package SPSS.10.1 (USA) and the data

were assessed by analysis of variance (ANOVA) using Duncan’s multiple range test at 5%

significance level. The results are presented as means ± standard deviation (n=3). All

experiments were performed in triplicate.

3. Results and Discussion

3.1 Encapsulation efficiency

The encapsulation efficiency determines the retention capability of wall material to hold

the small bioactive molecules inside its hollow structure. The encapsulation efficiency of nano-

starch particles is shown in Table 1 (a). The encapsulation efficiency of horse chestnut starch

(HSC), lotus stem starch (LSC) and water chestnut starch (WSC) was found to be 57.09 %,

55.00% and 48.30%, respectively. The encapsulation efficiency for the HSC and LSC showed no

significant difference in the retention capability of catechin, while it was significantly higher

than that of WSC. The difference in the encapsulation efficiency could be due to the differences

in starch type and extent of interactions occurring between starch and catechin molecules, which

could facilitate the incorporation of catechin in starch network (Ceborska, Zimnicka, Wszelaka-

12
Rylik, & Troć, 2016). The encapsulation efficiency also depends upon other several factors like

the choice of reaction, synthesis process, particle size and core material concentration (Chin,

Yazid, Akmar, & Pang, 2014). Higher encapsulation efficiency of HSC could also be due its

least particle size diameter (322 nm) because small particles have better encapsulation efficiency,

due to their ability to form a better film around the core material, which could help in better

retention of encapsulated molecules (Zhu, 2017).

3.2 Particle size, polydispersity index and zeta potential

The molecular size distribution of polymers in solution is important for understanding

their structure function relationship. However, it must be used with care to avoid artifacts arising

from incomplete dissolution of the substrate, band broadening, size calibration and shear scission

during separation as described in several previous literatures (Gidley et al., 2010; Lu et al.,

2016). Hence, the dissolution of starch was carried in deionized water at approximately neutral

pH. The average particle size of catechin nano-encapsulated starch particles are shown in Table

1(b) and a representative figure showing average zeta potential and particle size of horse

chestnut starch encapsulated catechin is shown in Figure 1a and 1b, respectively. The average

particle size diameter of HSC, LSC and WSC was found to be 322.6 nm, 615.5 nm and 559 nm,

with polydispersity index of 0.2, 0.59 and 0.62, respectively. The polydispersity index less than

0.4 is the indication of narrow size distribution of particles (Hasanvand, Fathi, Bassiri,

Javanmard, & Abbaszadeh, 2015). The lowest particle size of HSC may be attributed to the

difference in branching pattern of amylopectin within the starch molecules. Wani et al. (2014)

observed A type starch in horse chestnut. The A type starch possesses unbranched amylopectin

chains (Gidley, 1987), which would have been easily broken down by the effect of sonication

process during nano particle preparation, leading to smaller particles size of the molecules. The

13
zeta potential of all nano encapsulated catechin samples was found to possess negative values.

The WSC displayed significantly higher negative zeta potential (-20.1 mv) than HSC and LSC

which had same zeta potential (-18.05 mv). The higher zeta potential is responsible for

electrostatic repulsions and it is desirable for the stability of the nanoparticles, which decreases

the vander walls force between the particles. The vander walls forces is responsible for the

particle agglomeration that eventually results into bigger particles (Schäfer, Hecht, Harting, &

Nirschl, 2010). The negative zeta potential could be due to the interaction of starch and catechin,

which imparts negative charge on the starch-catechin nanoparticles.

3.3 Color analysis of native and encapsulated starch –catechin nano-particles

The color values of native and encapsulated starch –catechin nano-particles are shown in

Figure 1 (c). All the native starch molecules had lower a* and b* values compared to starch

encapsulated catechin molecules (P<0.05).The a* and b* values increases dramatically after

encapsulation of catechin (P<0.05). This indicated that starch- catechin particles were more

reddish/yellowish in color. The native starches were whiter in color as indicated by highest L*

value which also decreases significantly after catechin encapsulation (P<0.05). This indicated

that catechin after encapsulation has imparted reddish and yellowish color to the starch based

nano-capsules. Ahmad et al. (2017) also reported that folic acid after encapsulation has imparted

yellow and reddish color to the horse chestnut and ß-cyclodextrin microcapsules. Among the

starch-catechin nano-particles, the WSC was found to be more yellow in color (higher b* values)

followed by LSC and HSC (P<0.05). This was also reflected in the highest L* value for WSC

and least encapsulation efficiency of catechin which could have resulted in light colored nano-

encapsulated starch. The HSC was more reddish in color, which can be also explained by the fact

that it had less lightness (L*) values and highest encapsulation efficiency. These results are in

14
agreement with the previous study reported by Ahmad et al. (2015) who also observed increase

in a* and b* values after fortification of cookies with green tea which is the predominant source

of catechins.

3.4 Thermal characterization by Differential Scanning Calorimeter (DSC)

The thermographs of catechin and encapsulated starch-catechin nanoparticles are

depicted in Figure 1 (d). The catechin hydrate showed the several endothermic peaks with on set

temperatures at 27.68 °C, 116.18 °C and 139.84 °C, peak temperatures at 34.17 °C, 123.20 °C, &

152.63 °C and end set temperatures at 77.87 °C, 135.79 °C & 159.49 °C, respectively, their

respective heat of enthalpies was found to be 65.71 J/g, 12.46 J/g and 59.16 J/g. Upon

encapsulation and interaction with starch nanoparticles, the HSC and LSC showed small

exothermic peaks at 108.05 °C and 114.72 °C, which demonstrates the little crystalline nature of

particles. The ∆ H (heat of enthalpy) of HSC and LSC for exothermic peaks was found to be

11.75J/g and 12.46J/g respectively, which is very small and indicates that crystallized behavior

was not so prominent, the same was observed from the XRD analysis. In case of WSC, the

broader endothermic peak was observed from 95°C- 161°C, with the peak temperature at 122°C.

Such type of broader endothermic peak was not observed in HSC and LSC which may be

because of higher encapsulation efficiency than WSC or more likely due to variation in thermal

properties of coating material. Apart from above, we observed the shift of broad endothermic

peaks similar to catechin hydrate to higher temperatures after encapsulation in all the samples

that reveals that encapsulation offers thermal protection of catechin, similar observation was

concluded by Ahmad et al. (2017) during encapsulation of folic acid in horse chestnut starch.

3.5. Structural characterization by Fourier transform infrared spectroscopy (FTIR)

15
 The FTIR spectra of catechin, native starches and starch nano-encapsulated catechin are

depicted in Figure 3, which explains possible interaction between catechin and starch matrix. In
-1
the native starch, the broad peak at wavelength around 3300 cm represents OH stretching
-1
vibrations (Ahmad, Gani, Shah, Gani, & Masoodi, 2016), and regions between 927 cm and
-1
1148 cm are usually associated with stretching or bending vibrations of C=C, C=O and C=H

(Yu & Huang, 2010). The finger print regions in case of catechin were observed at 3479 cm -1,

1653 cm -1, 1283 cm -1, 1144 cm -1, and 1070 cm -1,

which corresponds to the presence of

aromatic ring quadrant, OH deformation of aromatic alcohol, CO stretching of aromatic alcohol

and aliphatic secondary alcohol, and CO stretch, respectively (Painter et al., 1981). Catechin-

starch matrix exhibited the characteristic peaks of catechin and starch. The similar peaks with

broad range were observed in catechin- starch nanoparticles, which depicts the encapsulation of

catechin in starch nanoparticles without any evident interaction. The absorption bands between

1070 cm-1 in catechin hydrate and native starch samples represent C–O–H bending in polymers

(García‐ Rosas et al., 2009). This C–O–H bending is mostly related to hydrogen bond forming.

The slight shift of this wavenumber to lower frequency (1022 cm-1, 1000 cm-1) was observed in

catechin-starch nanoparticles (LSC, HSC and WSC), which depicts network formation between

catechin and starch. Previous studies have reported that hydrogen bonding is considered as the

major interaction between phenolic compounds and the starch-based wall materials (Liu,

Chaudhary, Yusa, & Tadé, 2011). In the present study, OH groups of catechin and starch

molecules may play an important role in the formation of hydrogen bonding, which lead to

change in the spectrum of starch after encapsulating catechin. Furthermore, the presence of

benzene ring was also observed in the catechin hydrate nanoparticle which corresponds to

16
wavenumber at 860 cm-1. This band was found to broader and less sharp in starch–catechin nano-

particles that again confirms the encapsulation of catechin in starch nanoparticles (Jacox, 2003).

3.6. Crystallography analysis by XRD

The crystallographic patterns of native and starch-catechin nanoparticles are depicted in

Figure 3. In the native starch, diffraction peaks at 15 -17° and 23° on top of the amorphous

hump are clearly seen. This indicates that portion of the sample is in the crystalline state. The

observed diffraction peaks are identified with type A crystal structure usually found in starch

(Imberty, 1988). The broadness of the crystalline diffraction peaks indicates the existence of fine

crystallites. The size of these crystallite could be estimated from the Scherrer equation, D = K λ

/ (βcos θ), where D is the mean crystallite size, λ is the wavelength of the Cu X-ray line, K is

the shape factor (= 0.9), β is full width of the diffraction peak at half maximum and θ is the

Bragg's diffraction angle. The average crystallite sizes range from 4 to 20 nm. These nano-

crystallites are assumed to be dispersed within the amorphous starch nanoparticles. The relative

crystallinity was evaluated in accordance with the method described by Lopez-Rubio, Flanagan,

Gilbert, and Gidley (2008). The area under the diffraction peaks and the amorphous hump was

obtained from Gaussian profile fits. The relative crystallinity was estimated to be 13% for HS,

26% for WS and 22% for LS. Most of these crystallinities diminished upon nano-encapsulation

treatment which led to nanoparticles with highly amorphous character. The decrease in relative

crystallinity is due to destruction of long range ordering linked to the ordered structure of

crystalline and amorphous regions in starch granules (Chung & Liu, 2010) as was observed by

Boufi et al., (2018) after ultrasonic treatment. This is most likely the reason for the disappearance

of the diffraction peaks in the present case. Boufi et al. (2018) also observed the complete loss of

the diffraction peaks indicating that the crystalline structure of starch was destroyed after

17
ultrasonic treatment. It follows that the ultrasonic treatment strongly disrupted the crystalline

structure of starch and led to nanoparticles with highly amorphous character.

3.7. Microstructural features of the nano-encapsulated catechin by scanning electron

microscopy

The morphology and distribution of the starch-catechin nano-capsules were observed by the

scanning electron microscopy (SEM). The micrographs as shown in Figure 4 reveals the

changes in starch that occur during synthesis of starch nanoparticles and during nano-

encapsulation of catechin. All the starch-catechin nano-capsules had different morphological

features than the free catechin. HSC had highly porous and void structures with empty or filled

channels and starch granular structure was found to be disrupted to some large extent. The WSC

possessed some embedded particles within granules or capsules and gel-like paste was also

found which possibly could be due to starch gelatinization. In LSC, a gel like network with void

spaces in between and few granular structures were also observed. On comparing, the

morphological features of catechin hydrate, we conclude that HSC, LSC and WSC are embedded

with catechin particles, however it is not so clear as the size and shape of encapsulated catechin

would not be so similar to native catechin due to the preparation process of nano-capsules. The

morphological features however clearly show the transformation of granular structure to

formation of capsules and extent of this transformation is also correlated with size of particles as

HSC has least granular structure and also smallest average size as observed by zeta particle sizer.

Ahmad et al. (2017) also observed similar morphological characteristics in encapsulation of folic

acid in horse chestnut starch.

3.8. Release behavior of catechin during simulated gastric and intestinal digestion

18
 The nano-encapsulated catechins were subjected to simulated gastric and intestinal

digestion (SGID) conditions and their release behavior from nano-starch matrices were studied.

It is important to understand, how catechin is released from the nano-encapsulated structures

during the process of digestion along the gastrointestinal (GI) tract, when exposed to different

pH conditions & in the presence of gastric and intestinal enzymes. The purpose of the

encapsulation is to safeguard the catechin from degradation from harsh gastric conditions and

interaction with other components until they reach to their targeted absorption site. The amount

of catechin released from per 100 mg of powder sample upon their transit through SGID is

depicted in Table 1. It was observed that initial content of catechin released in the first 30 min of

gastric condition was significantly higher (P<0.05) compared to the released catechin during

intestinal digestion condition. This immediate release in the gastric condition is attributable due

to presence of free catechin on the surface of wall material called as surface or free catechin.

The amount of catehin released remained approximately constant even after 1h of gastric

digestion which indicates that entrapped or encapsulated catechin was not released from the

inner core of capsules as the values were insignificant compared to 30 min of digestion (P>0.05).

After 2h of intestinal conditions, the entrapped catechins were released from the nano-

encapsulated structures. The amount of catechin released after 2h and 4h of intestinal digestion

was found to be 3.87, 3.17, 4.06 and 4.8, 3.39, 5.35 mg/100 mg of powder, respectively from

HSC, WSC, and LSC nano-particles. The significantly higher content of catechin release was

found after 4h intestinal digestion in LSC and HSC. However, in WSC the values were non-

significant compared to the released catechin after 2h of intestinal digestion (P>0.05), which

might be due to the enhanced swelling of the nano-encapsulates in the simulated gastrointestinal

fluid that might have increased the diffusion path length within the nanoparticles of WSC

19
(Wongsasulak, Pathumban, & Yoovidhya, 2014). Therefore, it can conclude that WSC has a

better retention capacity as it allowed slow release of catechin from its core. Thus, encapsulation

of catechin using nano-starch particles could serve as an effective protection for slow release of

catechin at the absorption site of the GI tract when incorporated in the potential functional food.

3.9 Retention of bioactive properties of nano-encapsulated catechin under simulated

gastrointestinal digestion (SGID) conditions

The effect of nano-encapsulation on bioactivity retention of catechin before and after in-vitro

digestion has been summarized in Table 2. Anti-obesity activities via inhibition of pancreatic

lipase (PL) and cholesterol esterase (CE) and antidiabetic activities via DPP-IV and α-

glucosidase were determined for the free catechin and nano-encapsulated catechin upon

simulated gastric and intestinal digestion (SGID). The results revealed that upon in-vitro

digestion (SGID), bioactive properties for catechin showed a significant decrease for all the

bioactivities tested (P<0.05). PL and CE inhibitory activity was decreased by 81% and DPP-IV

inhibitory and α-glucosidase activity were decreased by 87.7 and 9.1%, respectively. However,

interestingly upon nano-encapsulation of catechin molecule in different starch matrix and

subjecting them to SGID conditions, the bioactive properties were significantly retained when

compared to the free catechin molecule subjected to SGID conditions. The values of PL and CE

inhibition from SGID nano-encapsulated catechin viz HSC, WSC and LSC were found to be

53.57%, 64.67%, 50.22% and 65.96%, 63.86%, 59.06%, respectively, when compared to 36.45

and 26.51% inhibition demonstrated by free catechin upon SGID, respectively (P<0.05).

Juhel et al., (2000) reported that green tea extract (AR25) containing 25% catechin

inhibited gastric lipase completely when used at a level of 40 mg AR25/g with tributyrin as

substrate. Whereas PL inhibition was maximum (78.8 ± 0.7%) with 80 mg AR25/g and tributyrin

20
(as substrate). It was reported that when compared to the control, lipolysis of triolein (lipid

substrate) under the successive action of the two digestive lipases was reduced by 37 ± 0.6% in

the presence of AR25. Moreover, in vitro fat digestion was significantly inhibited by 60 mg of

AR25/g triolein. Gastric as well as pancreatic lipase inhibition by the green tea extract (AR25)

could be related to altered lipid emulsification in gastric or duodenal media. Thus, SGID

subjected catechin upon nano-encapsulation could exhibiting marked inhibition of digestive

lipases in vitro which is likely to reduce fat digestion in humans. Therefore, WSC and HSC

encapsulated catechin were able to retain anti-obesity activity to a higher level as displayed by

higher inhibition values for pancreatic lipase and cholesterol esterase (CE) compared to the

inhibitory values displayed by LSC encapsulated catechin molecules.

Inhibitors of the dipeptidyl-peptidase IV (DPP-IV) are among the newest glucose-

lowering drugs available in the market for the management of type 2 diabetes. Inhibition of α-

glucosidase, a carbohydrate-hydrolyzing enzyme, is a way to control type 2 diabetes by delaying

the digestion and absorption of carbohydrates and suppress the postprandial hyperglycemia

(Zhang et al., 2015). Nano-encapsulated catechins subjected to SGID conditions showed higher

DPP-IV inhibitory activities compared to the than free catechin molecules upon in-vitro

digestion (SGID) (P<0.05). Previous reports have illustrated that catechin was tested in glucose-

induced insulin secretion in HIT-T15 cells and it was found that catechin (0-10 µM) enhance the

insulin secretion in the dose-dependent manner. Catechin was also reported to regulate blood

glucose level and improve postprandial hyperglycemia in diabetic induced mice (Huang et al.,

2011). Free catechin before (WSGID) and after SGID displayed very high α-glucosidase

inhibitory potential of 92.4 and 88.79%, respectively. Similar results were reported by Yilmazer-

Musa, Griffith, Michels, Schneider and Frei (2012) who reported that green and white tea extract

21
and catechin 3-gallates were potent inhibitors of α-glucosidase with IC50 values of more than

300 times than that of acarbose, a commercial α-glucosidase inhibitor. However, upon nano

encapsulation of the catechin molecules, lower inhibition of α-glucosidase activity was

demonstrated when compared to free catechin subject to SGID (P<0.05). This could be due to

the action of enzyme on starch wall material which release glucose molecule that eventually

reverse the process of inhibition. The lower value of α-glucosidase inhibition was observed in

WSC followed by HSC and LSC, this difference could be due the presence the higher resistant

starch content in horse chestnut and lotus stem that controls the action of enzyme. Finally, we

conclude that nano encapsulation offers protection to the catechin from gastric environment

which helps in retention of its bioactivity during digestion process.

4. Conclusion

Catechins are well known for their health-related bioactivities and most widely consumed

among all polyphenols but they face limitation for their incomplete bioavailability at the target

absorption site due to degradation during digestion process and limited cell permeability. This

study concluded that nano-encapsulation could be an efficient approach to overcome this

limitation using the novel sources of starch-based nanoparticles. The nano-particles have better

bio-accessibility and cell permeability and can protect the catechin during the digestion process

when incorporated in the fortified functional foods. Among three encapsulating materials, we

have observed horse chestnut starch (HSC) had highest encapsulation efficiency and produced

lower particle size. On the other hand, water chestnut starch (WSC) had highest stability and also

allowed slower release of catechin from its core. Nano-encapsulation using starch based nano-

particles provided protection to the catechin against harsh gastric environment and helped in

retaining its bioactive properties during in-vitro digestion process. Hence, we suggest HSC and

22
WSC to be more suitable for nanoparticle preparations and encapsulation process of catechin.

Future research should be carried out to check the bio-accessibility and bioactivity retention of

starch nanoparticles encapsulated catechin molecules when fortified as bioactive ingredient in

the functional foods.

Acknowledgement

Authors are thankful to United Arab Emirates University for the funding this research through a

research grants (UPAR-31F094) awarded to PI, Sajid Maqsood.

23
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26
Figure Legends
Figure 1. Zeta-potential (a) and particle size distribution (b) for a representative sample of nano-

encapsulated catechin in horse chestnut (HSC), color values (c) and Differential scanning

calorimetry thermographs (d) of native starch molecules and starch nano-encapsulated catechin.

For color analysis, values reported are mean ± standard deviation (n=3). Bars with different

letters within each color parameter are significantly different (p˂0.05).

Keynote: HSC, WSC and LSC represent nano capsule starch from horse chestnut, water

chestnut and lotus stem, while HS, LS and WS represent color of native starch from horse

chestnut, lotus stem, and water chestnut, respectively.

Figure 2. FTIR Spectra of native starch samples, catechin and starch nano-encapsulated catechin

molecules. (a) Lotus starch (LSC), (b) horse chestnut starch (HSC), (c) water chestnut starch

(WSC)

Figure 3. Effect catechin nano-encapsulation on crystallography of starch; (a) horse chestnut

starch (b) Water chestnut starch (c) Lotus starch.

Figure 4. Micrographs of nano-encapsulated catechin in starch nanoparticles. Keynotes: HSC,

WSC and LSC represent nano-encapsulated catechin in horse chestnut, water chestnut and lotus

stem starch nanoparticles, and C represent free catechin.

27
Highlights

 Novel starch based nano-particles were used for encapsulating catechin

 Horse chestnut starch (HSC) had highest encapsulation efficiency and produced lower particle

size

 Water chestnut starch (WSC) had highest stability and displayed slower release of catechin

 Nano-encapsulation helped in retaining bioactive properties of catechin during digestion process.

28
Table 1 (a): Encapsulation efficiency and release behavior of catechin from starch
nanoparticles during gastric condition
Conditions Duration (Hour) HSC* WSC* LSC*

SGJ 0.5 5.94 ± 0.06Aa 5.53 ±0.11Aa 5.57 ±0.33Aa

1 5.82 ± 0.03Aa 5.45 ±0.42Aa 6.04 ±0.11Aa

SIJ 2 3.87 ± 0.61ABb 3.17 ±0.48Bb 4.06 ±0.96Ab

4 4.8 ± 0.35Aab 3.39 ±0.09Ab 5.35 ±0.84Aa

Encapsulation Efficiency % 59.09 ±1.33A 48.30 ± 1.97B 55.00± 5.8A

Entrapped catechins (mg)** 5.92 ± 0.71A 4.83 ± 0.55B 5.5 ±0.65A

Values reported are mean ± standard deviation of the analysis performed in triplicates. Data with different
superscript small letter in the same column and capital letters in the same row are significantly different (p˂0.05).
*Content of catechin (mg) released from 100 mg of powered samples
**Content of catechin entrapped in 100 mg of sample

Table 1 (b). Particle size, polydispersity index and zeta potential of catechin encapsulated

in horse chestnut, water chestnut and lotus stem starch nano-particles.

Sample Z-average (nm) Poly dispersity index Zeta potential (mv)

HSC 322.7±78.3 0.20 -18.05± 1.59

WSC 559.2±130.4 0.62 -20. 1 ± 0.81

LSC 615.6± 95.7 0.59 -18.05 ± 1.26

HSC, WSC and LSC represent nano-encapsulated catechin in horse chestnut, water chestnut and
lotus stem starch nanoparticles.

29
Table 2: Effect of nano-encapsulation on bioactivity retention of catechin during in-vitro
digestion.

Bioactivity Tests Catechin Catechin HSC WSC LSC

(WSGID) (SGID)

Lipase Inhibition % 83.5 ± 5.11a 36.45± 3.21 d 53.57 ± 4.62 c 64.67 ± 5.34 b 50.22 ± 2.15 c

Cholesterol esterase 72.51 ± 4.32 a 26.51 ± 4.17 d 65.96 ± 5.13 bc 63.86 ± 3.78 b 59.06 ± 3.65 c

Inhibition %

DPP-IV inhibition % 70.00 ± 3.76 a 17.44 ±5.89 d 46.16 ± 3.98 b 47.96 ± 4.75 b 33.98 ± 3.12 c

Glucosidase 92.4± 6.61 a 88.79 ± 1.12 b 82.54 ± 1.32 c 42.24 ±4.27 e 68.31 ± 3.56d

Inhibition%

Values reported are mean ± standard deviation of the analysis performed in triplicates. Data with different superscript
small letter in the same column and capital letters in the same row are significantly different (p˂0.05).
HSC, WSC and LSC represent nano encapsulated catechin in horse chestnut, water chestnut and lotus stem starch
nanoparticles. Catechin WSGID means native free catechin before the process of simulated gastro-intestinal conditions
and Catechin SGID means native free catechin after the process of simulated gastro-intestinal conditions.

30
a) b)

45 HSC LSC WSC

e f HS LS WS c)
40
d d)
35
30
Intensity

25 c
20 b
a
15
f
10 de
5 e dc bc
aba a
0
L* a* b*
Color parameters
Figure 1

31
 Catechin LS LSC
180
160
140 7909 1083 1650
1383 2925
3300
Transmittance %

120 1022

100 1360 1673 2930

927 1144
80 3300
1022
60
40 2925
1283
20 860 1144 3479
1070 1653
0
400 900 1400 1900 2400 2900 3400 3900
Wavelength (cm-1 )
180
Catechin HS HSC
160

140 1590
Transmittance %

790 1383 1699

120
1021 1083 2845
3475
100 1654
1148 1360 2929
80
750 959 1000 3273
60

40

20

0
400 900 1400 1900 2400 2900 3400 3900
Wavelength (cm-1 )

Catechin WS WSC
180
160
140
790 1006 1156 1384 1663 2845
120
Transmittance %

3475
100
1148 1360 1645
2929
80 1075
762 959 1000
60 3300

40
20
0
400 900 1400 1900 2400 2900 3400 3900
Wavelength (cm-1 )
Figure 2

32
 a) b)

c)

Figure 3.

HSC WSC

33
Figure 4.

34