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Accepted Manuscript

Title: Thiol Modified Moringa Gum – A Potential


Bioadhesive Polymer

Authors: Pankaj Grewal, Jyoti Mundlia, Munish Ahuja

PII: S0144-8617(18)31560-1
DOI: https://doi.org/10.1016/j.carbpol.2018.12.100
Reference: CARP 14454

To appear in:

Received date: 31 May 2018


Revised date: 20 December 2018
Accepted date: 31 December 2018

Please cite this article as: Grewal P, Mundlia J, Ahuja M, Thiol Modified
Moringa Gum – A Potential Bioadhesive Polymer, Carbohydrate Polymers (2019),
https://doi.org/10.1016/j.carbpol.2018.12.100

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Thiol Modified Moringa Gum – A Potential Bioadhesive Polymer

Pankaj Grewal, Jyoti Mundlia, Munish Ahuja*

Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Guru

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Jambheshwar University of Science and Technology, Hisar-125001, India

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*Address for correspondence

Prof. Munish Ahuja,

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Drug Delivery Research Laboratory,

Department of Pharmaceutical Sciences, N


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Guru Jambheshwar University of Science and Technology,
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Hisar, Haryana- 125001 (India)

E-mail: munishahuja17@yahoo.co.in
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Phone: +91-01662263515
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Highlights
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 Thiolation of Moringa gum was carried out.


 Thiolation imparts bioadhesive property.

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Metronidazole loaded buccal tablets were formulated and characterized.


 Thiolated gum sustained the in vitro release of metronidazole benzoate following
First order kinetics.

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Abstract

In the present study Moringa gum was modified by thiolation to enhance its mucoadhesive
potential. Thiolation was accomplished by esterification with thioglycolic acid. Thiolated
Moringa gum was characterized by FT-IR, TGA, DSC, XRD and SEM-EDX studies. Modified
gum was found to have 0.956±0.024 mM of thiol groups/g as determined by Ellman’s method.

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The greater force of detachment of polymer compacts of modified gum from the mucin discs as
compared to native gum indicates its enhanced mucoadhesive potential. The thiolated Moringa

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gum was formulated into buccal tablets using metronidazole benzoate as the test drug. A
comparative evaluation of buccal tablets revealed 1.5-fold higher ex vivo bioadhesion time of

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buccal tablets of modified gum than the native gum. The results of in vitro release studies
showed that modified gum tablets provided a sustained release of metronidazole over 24 h of the
study following First-order kinetics with diffusion mechanism of release.

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Keywords: Moringa gum; thiolation; mucoadhesion; buccal tablet
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1.0 Introduction
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Natural polymers and its modified derivatives have been extensively used in many applications
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in the food, construction, electronics (Pandey, 2016; Pandey & Ramontja, 2016a), biomedical
(Pandey & Nanda, 2016), pharmaceutical (Pandey & Ramontja, 2016b), water purification
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(Pandey, 2017) and engineering industries (Pandey & Tiwari, 2015). In view of its potential
advantage of biocompatibility, biodegradability and easy availability, they are widely accepted in
pharmaceutical industry (Pawar, Kamat & Choudhary, 2015). The utility of these polymers can
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further be enhanced by physically or chemically modifying them (Makhado, Pandey,


Nomngongo & Ramontja, 2017; Makhado, Pandey & Ramontja, 2018). A number of approaches
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such as grafting (Bhattacharya & Mishra, 2004), cross-linking (Banegas, Zornio, Borges, Porto
& Soldi, 2013), thiolation (Sharma & Ahuja, 2011), carboxymethylation (Rimpy, Abhishek &
Ahuja, 2017) etc. have been explored to meet tailor made specifications. Amongst these
thiolation is a promising approach which imparts mucoadhesive characteristics on the modified
derivatives. A number of mucoadhesive approaches are based on non-covalent bonding such as

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Van der’s Waal forces, hydrogen bonds and ionic interaction which are weak in nature and thus
doesn’t provide sufficient residence time in the alimentary tract (Bernkop-Schnurch, 2005).
However, modification of polymers by introduction of thiol groups to the polymer provides
higher cohesive properties. The thiol groups form disulfide linkage between the polymer and
mucus layer improving its bioadhesion (Bernkop-Schnurch, Kast & Richter 2001). During earlier
studies thiol modification of chitosan (Mahmood et al, 2017), tamarind seed polysaccharide

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(Kaur, Yadav, Ahuja & Dilbaghi, 2012), pectin (Sharma & Ahuja, 2011), alginate, hyaluronic
acid (Kafedjiiski et al., 2007), xanthan gum (Bhatia, Ahuja & Mehta, 2015), gellan gum (Yadav,

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Ahuja, Kumar & Kaur, 2014) etc., have been carried out to improve their mucoadhesive
characteristics.

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Moringa gum is a natural gum obtained from Moringa oleifera (family: Moringaceae). Moringa

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gum comprises of L-arabinose, L-glucoronic acid, L-galactose and L-rhamnose. The gum has
actually been investigated as binder, disintegrant, release retardant for pharmaceutical
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applications (Panda, Choudhury, Yedukondalu, Si & Gupta, 2008). Moringa gum has hydroxyl
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groups to the side chain which are able to interact with mucus contributing to its mucoadhesion
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ability. Grafting of Moringa gum with (N-vinyl-2-pyrrolidone) has already been investigated in
order to improve its bioadhesion (Abhishek, Rimpy & Ahuja, 2018). The present study is the
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first ever report on thiolation of Moringa gum which has been carried out with the objective to
enhance its mucoadhesive properties.
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The thiolation of Moringa gum was carried out by esterification of the gum with thioglycolic
acid. The thiolated Moringa gum was characterized by FT-IR, TGA, DSC, XRD, SEM studies
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and for its compression nature. The thiol substitution on the Moringa gum was estimated by
Ellman’s method. The mucoadhesive property of the thiolated Moringa gum was comparatively
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evaluated with native gum using texture analyzer. Thiolated Moringa gum was formulated into
buccal tablets using metronidazole benzoate as the test drug. Further, the buccal tablets were
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evaluated for friability, thickness, content uniformity, average weight, ex vivo bioadhesion and in
vitro release behaviour.

2. Experimental

2.1 Materials

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Moringa oleifera gum (mol wt. 190 kDa) was procured from Nutramine Life Sciences (Delhi,
India). Metronidazole benzoate was received as gift sample from Ranbaxy Laboratories Ltd.
(Gurugram, India). Thioglycolic acid, citric acid and hydrochloric acid were purchased from SD
Fine-Chem Ltd. (Mumbai, India). L-Cysteine and Ellman’s reagent (5’5’-dithiobis-(2-
nitrobenzoic acid) or (DTNB), mucin, acetone, disodium hydrogen phosphate and methanol were
procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Polyethylene glycol-4000 was

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obtained from Merck Ltd. (Mumbai, India).

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2.2Thiol functionalization of Moringa oleifera gum

Thiol modification of Moringa gum was carried out by the esterification of Moringa gum with

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thioglycolic acid under acidic condition (Dicharry et al., 2006). Briefly, 7.59 g of thioglycolic
acid (80%, v/v) and 4 ml of hydrochloric acid (7 N) were added to the aqueous dispersion of

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Moringa gum (2%, w/v) in hot water (50 ml). The temperature of the reaction mixture was raised

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to 80 °C, and the reaction was continued till 150 min. The reaction mixture was then cooled and
precipitated by adding acetone (500 ml). The precipitated thiol functionalized Moringa gum was
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further washed with2×200 ml of acetone and dried in oven at 50 ±2 °C for 1 h.
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2.3 Characterization of thiolated Moringa oleifera gum


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2.3.1 Estimation of thiol substitution

Ellman’s method was employed to determine the number of thiol groups/g of the modified gum
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(Bernkop-Schnurch et al., 2003). Solutions of Moringa gum and modified Moringa gum (50 mg)
prepared in 1 N NaOH (25 ml) were diluted with an equal volume of 0.5 M phosphate buffer (pH
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8.0). The above reaction mixture was allowed to react with 5 ml of Ellman’s reagent (DTNB,
0.03% w/v) in 0.5 M phosphate buffer (pH 8.0) at room temperature for 2 h. The thiol group
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substitution was determined by measuring the absorbance of the reaction mixture at 450 nm,
calculated using the calibration curve of L-cysteine solution prepared in the same manner.
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2.3.2Fourier Transform Infra-Red Spectroscopy (FT-IR)

Fourier-transform infrared (FT-IR) spectrum of samples were recorded by FT-IR


spectrophotometer (IR-Affinity-I, Shimadzu, Japan) using KBr disc method in the range of 4000-
500cm-1. The samples of Moringa gum and thiolated Moringa gum in powder form were
triturated with KBr using agate pestle and mortar, and the blend was then compressed into disc

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using IR hydraulic press (IR Hydraulic Press CAP-15T, PCI Analytics, Mumbai, India) at a
pressure of 75 kg/cm2 for 30 s.

2.3.3Thermogravimetric Analysis

Moringa gum and thiolated Moringa gum samples were analysed thermogravimetrically
employing Mettler Toledo TGA, DSC 3 PLUS (California, USA) instrument under nitrogen

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atmosphere from 25 oC to 400 oC at a heating rate of 10 oC/min.

2.3.4Differential Scanning Calorimetry Analysis

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DSC thermogram of Moringa gum and thiolated Moringa gum were recorded using differential

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scanning calorimeter (TA instruments, DSC25, California, USA). Finely powdered sample of
Moringa gum or thiolated Moringa gum (5 mg) was sealed by crimping in standard aluminium

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pan. The pan was heated at a rate of 10 °C/min over a temperature range of 40-300 °C under the
nitrogen purge of 50 ml/min.
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2.3.5Scanning Electron Microscopy Analysis (SEM)

The shape and surface morphology of Moringa gum and thiolated Moringa gum samples were
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investigated using scanning electron microscope (ZEISS, EVO18, China). The samples were
coated with gold and mounted on a sample holder and electron micrographs were captured at an
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accelerating voltage of 20 kV.


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2.3.6 Energy-dispersive X-ray microanalysis (EDX)

The samples of Moringa gum and thiolated Moringa gum were analysed for presence of sulphur
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by SEM-EDX (ApreoLoVac, FEI). The images of Moringa gum and thiolated Moringa gum
were captured after placing the samples on a sample stub and coating them with carbon.
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2.3.7 Compression nature

The compressibility characteristics of Moringa gum and modified Moringa gum were assessed
by determining the crushing strength of powder blends of Moringa gum or thiolated Moringa
gum with magnesium stearate as reported earlier (Verma & Ahuja, 2017) using portable digital
hardness tester (VTHT 500, Vin Digital Tester, Mumbai, India). In brief, blends of Moringa gum
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or thiolated Moringa gum containing magnesium stearate (1%, w/w) were prepared. In first and
second blends Moringa gum or thiolated Moringa gum were blended with magnesium stearate
for, while in the case of third blend mixing was carried out for 30min. The powder blends so
obtained were compressed under the pressure of 75 kg/cm2 into tablets using hydraulic press
(PCI Analytics, Mumbai, India) keeping a dwelling time of 2 s for powder blends first and third
or 30s for blend second. The crushing strength of tablets was determined after 24 h storage in a

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dessicator.

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2.3.8Biocompatibility studies

Biocompatibility of thiolated Moringa gum was evaluated comparatively with Moringa gum by

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assessing their thrombogenic and haemolytic potential. Thrombogenic potential was determined
by gravimetric method as reported earlier (Singh & Kumar, 2018). Briefly, samples of Moringa

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gum or thiolated Moringa gum (0.5 g) were allowed to swell in phosphate buffer saline (pH 7.2)

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at 37 OC for 24 h. The hydrated samples were removed and placed in contact with citrated blood
(0.2 ml), to which 0.2 ml of 0.1 M calcium chloride was added. After 45 min, 5 ml of distilled
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water was added to stop the clotting. The clots so formed were fixed by adding 38%
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formaldehyde (5 ml), followed by drying and weighing. The results were calculated as follows-

𝑊𝑠−𝑊𝑛
𝑇ℎ𝑟𝑜𝑚𝑏𝑜𝑠𝑒 (%) = × 100
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- - - - - - - - - - - - - - - - - - (1)
𝑊𝑝−𝑊𝑛
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Where Ws, Wp and Wn are the weights of sample, positive and negative controls respectively. The
positive controls consisted of weight of the clots without sample while negative control consisted
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of weight of residue without blood and samples.

Haemolytic potential was evaluated using the method reported earlier (Singh & Kumar, 2018).
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For determining haemolytic potential, samples (0.5 g) of Moringa or thiolated Moringa gum
were placed in phosphate buffer saline (PBS) for 24 h at 37 oC. The swollen samples were
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incubated with 5 ml of PBS and 2 ml of citrated blood at 37 oC for 3 h. The positive and negative
controls comprised of 2 ml of citrated blood with 5 ml of distilled water and PBS respectively.
After incubation, the samples were centrifuged and the supernatant fluid was analysed or
haemolysis by detecting the absorbance at λmax 540 nm. The haemolytic index was calculated as
follows-

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𝐴𝑠−𝐴𝑛
𝐻𝑎𝑒𝑚𝑜𝑙𝑦𝑡𝑖𝑐 𝑖𝑛𝑑𝑒𝑥 (%) = × 100 - - - - - - - - - - - - (2)
𝐴𝑝−𝐴𝑛

where As, Apand An is the absorbance of sample, positive and negative controls respectively.

2.4Formulation of metronidazole benzoate loaded buccal tablets of Moringa and thiolated


Moringa gum

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Moringa gum and thiolated Moringa gum were tested as mucoadhesive polymeric carrier by
formulating buccal tablets of metronidazole benzoate. Table 1 shows the composition of various

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batches of buccal tablets prepared using Moringa gum or thiol modified Moringa gum. Powder
blends of Moringa gum or thiolated Moringa gum, metronidazole benzoate (16 mg), PEG-4000

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(25 mg) and lactose were prepared and compressed into tablets using IR hydraulic press (PCI
Analytics, Mumbai, India) under pressure of 75 kg/cm2 for 30 s.

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2.5Evaluation of buccal tablet
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Metronidazole benzoate loaded Moringa gum or thiolated Moringa gum buccal tablets were
evaluated for uniformity of weight, thickness, friability, in vitro release behaviour and ex vivo
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bioadhesion time.
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2.5.1Tablet friability

Six buccal tablets of each batch were weighed (Wpre) and placed in the tablet friabilator
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(RemiEquipments, Mumbai, India). The drum of the friability test machine was rotated for 100
revolutions at the rate of 25 rpm. After this, the buccal tablets were again weighed (Wpost). The
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friability (%) was calculated as follows-

𝑊𝑝𝑟𝑒−𝑊𝑝𝑜𝑠𝑡
𝐹= × 100 - - - - - - - - - - - - - - - - - - - - - - (3)
𝑊𝑝𝑟𝑒
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2.5.2Thickness

The thickness of buccal tablets was determined by using digital vernier caliper (YAMAYO
Digital Caliper, Pune, India). Ten buccal tablets of each batch were selected randomly and
thickness of individual tablets was determined and the average was calculated.

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2.5.3Uniformity of weight

The weight uniformity of buccal tablets was determined by weighing individually ten buccal
tablets of each batch selected randomly and calculating the average weight and standard
deviation.

2.5.4 Drug content uniformity

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Uniformity of drug contents in buccal tablets was determined by measuring the contents of
metronidazole in three tablets of each batch. The tablets were dissolved in Mcllvaine buffer (pH

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6.8), filtered and diluted appropriately. The content of metronidazole benzoate in the filtrate was
determined spectrophotometrically by measuring the absorbance at 231 nm.

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2.5.5Mucoadhesion test

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The mucoadhesive nature of Moringa gum and thiolated Moringa gum tablets was investigated

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by determining the force of adhesion between the tablets of mucin and polymer using texture
profile analyzer (Universal texture analyzer) (Jones, Woolfson & Brown, 1997). Mucin (250 mg)
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was compressed into tablet in an IR hydraulic press by applying the compression force of 35
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kg/cm2 for a dwell time of 10 s. Mucin tablets were adhered employing double-sided adhesive
tape to the lower and upper probes of the texture analyzer. Polymer tablets were placed in
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contact with simulated gastric fluid (SGF, pH 1.2) for 5min and allowed to hydrate. The hydrated
tablets were placed on the lower mucin tablet and the upper probe with adhered mucin tablet was
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lowered to bring in contact with the surface of hydrated tablets and a downward force (0.1 N)
was applied for 1 min to provide intimate contact between the polymer and mucin tablet. After
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60 s, the upper probe was withdrawn by moving up at a constant speed of 0.5 mm/s, and the
force of detachment of the mucin tablet from the surface of the polymer tablet was inferred from
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the resultant force-time plots.

2.5.6Ex-vivo bioadhesion time


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The ex-vivo bioadhesion test was carried out to evaluate the mucoadhesive capability using chick
buccal pouch as biological membrane. Chick buccal pouch, procured within half an hour from
slaughter was washed and cleaned to remove loose tissues and other debris using Mcllvaine
buffer (pH 6.8) at 37 oC. The washed buccal pouch was tied to the side of the paddle of USP

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type-II dissolution apparatus (TDL-08L, Electrolab, India). The buccal tablet was then pasted on
the inner side of mucosal membrane of pouch using light force for about 20 s. The paddle was
then rotated at 50 rpm in dissolution basket containing 250 ml of Mcllvaine buffer (pH 6.8),
maintained at 37 oC (Jangra, Ahuja & Kumar 2013). The bioadhesion time was then assessed by
finding the time required for the buccal tablets to detach or erode from the surface of mucosa.
The results were reported as average.

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2.5.7In vitro release studies

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In vitro release rate of metronidazole benzoate from buccal tablets of Moringa gum was carried
out in USP type 2 dissolution apparatus at 37±0.5 °C. Buccal tablets were attached to the bottom

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of the beaker (250 ml) using cyanoacrylate glue. The beaker was filled with 250 ml of Mcllvaine
buffer (pH 6.8) and rpm was maintained at 50. At appropriate time intervals, aliquot of 5 ml of

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sample were withdrawn and then replaced with 5 ml of Mcllvaine buffer (Yehia, El-Gazayerly &

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Basalions, 2008). The contents of metronidazole benzoate in sample were analysed by measuring
the absorbance in UV-Visible spectrophotometer at 231 nm.
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3.Results and discussion
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3.1 Thiolation of Moringa gum


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Moringa gum is reported to consist of arabinogalactan moiety (Raja, Bera & Ray, 2016). In the
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present study, the reaction between the hydroxyl groups of Moringa arabinogalactan and
carboxyl group of thioglycolic acid was carried out under acidic conditions to achieve thiol
derivatization of Moringa gum. Fig. 1 provides the schematic representation of the thiol
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modification of Moringa gum. The product obtained was an off-white ordorless powder with an
average yield of 83%. It was found to be soluble in 1 N NaOH but insoluble in water. The thiol
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group content in thiolated Moringa gum as determined by Ellman’s method was found to be
0.956±0.024 mM of thiol groups/g of Moringa gum.
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3.2 Characterization of modified Moringa gum

Fig. 2 exhibits the FT-IR spectrum of Moringa gum and thiolated Moringa gum. The IR
spectrum of Moringa gum shows a broad peak at 3421 cm-1 which is attributed to –OH
stretching. The C-H stretching of alkane shows peak at 2939 cm-1 and the glucoronic acid entity

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shows -COOH stretching at 1620 cm-1. In thiolated Moringa gum the OH band of aliphatic
alcohol appears at 3420 cm-1, while the –SH stretch appears as a weak shoulder at 2554 cm-1.
The carboxylic groups of thiolated Moringa gum shows on stretch at 2938 cm-1 and C-O stretch
at 1253 cm-1. The stretch at 1074 cm-1 can be due to C-O stretch of primary alcohol. In a recent
study it was also reported that thiol bands are weakly detectable in FTIR spectroscopy
(Fernanda, Borsagli, Carvalho & Mansur, 2018).

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Fig. 3(A) compares the weight loss of thiolated Moringa gum and Moringa gum samples as a

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function of temperature. TGA curve of Moringa gum presents 3 stage of degradation. The first
stage of degradation which begins around 50 °C and end at 225 °C is characterized by weight

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loss of 20%. This weight loss may be ascribed to desorption of water bound to the saccharide
structure and dehydration due to the loss of –OH groups on the polysaccharide backbone (Cozic,

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Picton, Garda, Marlhoux & Cerf, 2009). The second stage from 225 oC to 303 oC is characterized
by a very fast weight loss of 37%. The third stage is from 303 oC to 400 oC show a relatively
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small loss of 8%. The degradation at such high temperature can be attributed to the
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depolymerization and pyrolitic decomposition stages which result in the evolution of CO, CO2,
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CH4 etc. (Zohuriaan & Shokrolahi, 2004). On the other hand thiolated Moringa gum degrades
with 12% weight loss during first stage from 50 oC to 133 oC, followed by loss of 38% from 133
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C to 270 oC and then 17% from 270 oC to 400 oC. At the end of 400 oC, the residual mass of
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35% and 33% of Moringa gum and thiolated Moringa gum respectively was left. The results
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show a faster thermal degradation of thiolated Moringa gum at temperature less than 270 oC but
above 270 oC there is not much difference in the thermal degradation rate of Moringa gum and
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thiolated Moringa gum.

The DSC thermogram (Fig. 3B) of Moringa gum showed a broad endothermic peak followed by
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two exothermic peaks. The endothermic peak appeared at 128.84 oC with an onset of 67.89 oC
and enthalpy of fusion 313.20 J/g. The two exothermic peaks appeared at 256.09 oC and 284.62
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C with enthalpy of fusion of 8.7857 J/g and 20.878 J/g, respectively. Thermal curve of thiolated
Moringa gum presented four endothermic peaks at 108.90 oC, 144.16 oC, 150.42 oC and 156.87
o
C with respective heat flow values of 21.518 J/g, 2.6399 J/g, 0.4006 J/g and 1.9382 J/g. Further
thiolated Moringa gum curve shows one exothermic peak at 242 oC with heat flow of 31.220 J/g.

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Fig. 4 shows the scanning electron micrographs of Moringa gum (a, b) thiolated Moringa gum
(c, d). The particles of Moringa gum are polyhedral while that the images thiolated Moringa gum
shows the presence of polyhedral plate shape flakes. It can be seen that surface of Moringa gum
is rough and granular and that of thiolated Moringa gum is smooth and porous. The smooth
surface of thiolated Moringa gum is expected to provide the greater area of contact with the
mucus layer as compared to the Moringa gum.

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The EDX spectrum of Moringa gum (Fig. 5a) and thiolated Moringa gum (Fig. 5b) confirms the

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successful thiolation of Moringa gum. The new X-ray intensity was observed at around 2.5 keV
in thiolated Moringa gum due to the presence of sulphur in thiolated Moringa gum. The EDS

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layered images of Moringa and thiolated Moringa gum are presented as supplementary material
(Fig S1 and S2).

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3.3 Compression behaviour

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Thiol modified Moringa gum was further characterized for its compression behaviour studies.
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The results of compressibility characterization study revealed that the tablets of Moringa gum
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prepared using blends first, second, and third had the crushing strength of 73.3 N, 81.3 N and
19.1 N respectively. On the other hand tablets of thiolated Moringa gum showed crushing
strength of 120.3 N, 138.4 N and 114.8 N. It can be observed from the results that increasing the
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dwell time increase the bending strength while the increase in blending time reduces bonding
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which indicate that both Moringa gum and thiolated Moringa gum show plastic behaviour.
However, thiol modification results in increase in bond strength of particles.
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3.4 Biocompatibility studies


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Moringa gum is biocompatible and has been used in traditional medicines in the management of
diarrhoea and for its diuretic effects. During earlier studies, radiation, cross linked Moringa gum-
polyacrylic acid hydrogels were evaluated for biocompatibility by blood compatibility tests
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(Singh & Kumar, 2018). The comparative blood compatibility tests conducted on Moringa gum
and thiolated Moringa gum revealed that the amount of clot formed in case of Moringa gum
(0.1258 g/ 2 ml citrated blood) and thiolated Moringa gum (0.1939 g/ 2ml of citrated blood) was
less that the clot formed in case of positive control (0.2471 g/ 2ml of citrated blood). Thus, the
percentage thrombose of Moringa gum and thiolated Moringa gum was found to be 50.91% and

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78.47% respectively. As the thrombus formed was lower in case of Moringa gum and thiolated
Moringa gum as compared to control, Moringa gum and thiolated Moringa gum can be
categorized as non-thrombogenic. The results of haemolytic study revealed that the haemolytic
index of Moringa gum and thiolated Moringa gum was 3.86% and 1.20% respectively. Since the
haemolytic index value is less than 5%, they can be regarded as safe and suitable for drug
delivery applications.

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3.5Mucoadhesion studies

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Thiolation of polymers imparts the mucoadhesive characteristics on the polymer. So, thiolated
Moringa gum was tested for mucoadhesive properties using texture analyzer. Fig. 6 displays the

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force time curve of Moringa gum and thiolated Moringa gum compacts. The force of detachment
of polymer compacts of Moringa gum and thiolated Moringa gum from mucin tablets was found

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to be 804 mN and 1070 mN respectively. The greater force of detachment of thiolated Moringa
gum indicates its mucoadhesive nature. N
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3.6Formulation and characterization of buccal tablets
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Considering the mucoadhesive potential of thiolated Moringa gum, it was decided to formulate
mucoadhesive buccal tablets. Metronidazole benzoate a commonly used anti gingivitis drug
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(Mitchell, 1984) was used as the model drug. Buccal tablets were formulated using lactose as the
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diluent, PEG-4000 as the plasticizer. The results of the characterization of buccal tablets indicate
that the average weight, drug content and thickness were within the range. However, as the
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buccal tablets prepared using lower and higher levels of Moringa gum or thiolated Moringa gum
showed higher friability while the buccal tablet containing equal proportion of polymer and
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lactose passed the friability test. The ex vivo bioadhesion time study reveal about 1.5-fold higher
bioadhesion time of buccal tablets of modified gum compared to that of native gum.
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3.7 In vitro release of buccal tablets

Fig. 7 compares the results of in vitro release study carried out in Mcllvaine buffer (pH 6.8). It
can be observed that almost the entire drug was released within 6 h from the buccal tablets
formulated using Moringa gum while buccal tablets of thiolated Moringa gum were able to
sustain the release till 24 h. Slower release can be attributed to the disulfide bond formation

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between glycoproteins of mucus layer and polymer which are much stronger (Bernkop-
Schnurch, Kast & Richter 2001). Further the release data was subjected to model fitting, the
results (Table 2) of which unveil that the release of metronidazole benzoate from Moringa gum
buccal tablet followed Higuchi’s square root kinetics with the anomalous release mechanism i.e.
combination of diffusion and erosion. On the other hand release from thiolated Moringa gum
buccal tablet followed first order kinetics with the primary mechanism of release being diffusion

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through the matrix. Metronidazole is commonly used in the treatment of intestinal infections. To
explore the possibility of use of metronidazole loaded thiolated Moringa gum tablets in such

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case, the in vitro release study was also conducted using Mcllvaine buffer (pH 1.2). It was
observed that at acidic pH, the release of drug from gum followed Higuchi’s square root kinetics

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with diffusion through the matrix as primary mechanism of release, except batch F4 which
showed anomalous transport. The results of which are presented as supplementary material

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(Fig.S3-S15).

Conclusions
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Moringa gum was modified by esterification of its hydroxyl groups with thioglycolic acid to
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synthesize thiolated Moringa gum. The modified Moringa gum was characterized by FTIR,
TGA, DSC, XRD and SEM studies. The thiolation of Moringa gum improved its mucoadhesion
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ability as evaluated by tensile studies using texture analyzer. The thiolated Moringa gum was
further explored for pharmaceutical applications by formulating metronidazole benzoate loaded
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tablets. The thiolated Moringa gum containing tablets showed higher bioadhesion time as
compared to the unmodified gum. The in vitro release studies indicate that thiolation of Moringa
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gum imparts sustained release characterization. Thus, it can be inferred that thiolation of
Moringa gum is a feasible approach to improve its mucoadhesion and release retardant property.
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However, further in vivo studies in animals are needed to exploit its potential for use in buccal
drug delivery in human beings.
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Acknowledgements

The authors express gratitude to Department of Science & Technology, Govt. of India for
providing financial assistance to Jyoti Mundlia under DST-PURSE programme. The authors also
express gratitude to the Department of Textile Technology, IIT, Delhi and Materials Science

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Laboratory, Department of Physics, GJUS&T, Hisar for providing scanning electron microscopy
and X-ray diffraction facility, respectively.

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Fig. 1 Scheme for thiolation of Moringa polysaccharide


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Fig. 2 FTIR spectra of Moringa gum and thiolated Moringa gum


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Fig. 3(A) TGA curves (B) DSC thermograms of Moringa gum and thiolated Moringa gum.

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(a) (b)

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(c) (d)
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Fig.4 Scanning electron micrographs showing (a) surface (b) shape of Moringa gum and (c)
surface (d) shape of thiolated Moringa gum.
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Fig. 5 EDX-ray analysis of (a) Moringa gum (b) thiolated Moringa gum
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Fig.6 Texture profile analysis of Moringa gum and thiolated Moringa gum tablet.

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Fig.7In vitro release profile of metronidazole benzoate from thiolated Moringa gum buccal tablet
and Moringa gum buccal tablet.
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Table 1: Composition and evaluation of various batches of buccal tablets.

Batch Quantity (mg/tablet) Average Thickness Drug Friability Ex vivo


code MG TMG Lactose weight (mm) content (%) bioadhe
(mg) (%) sion
time (h)
F1 50 100 187.2 ± 0.36 1.31 ± 0.01 98 ± 1.40 3.7 12.1
F2 75 75 187.1 ± 0.38 1.10 ± 0.01 99 ± 0.42 0.79 11.8

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F3 100 50 187.3 ± 0.37 1.31 ±0.03 98 ± 0.20 20.96 12.2
F4 50 100 188.1 ± 0.40 1.13 ±0.03 98 ± 0.80 5.83 7.9
F5 75 75 187.3 ± 0.21 1.11 ±0.01 99 ± 0.84 0.86 8.0

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F6 100 50 187.6 ± 0.39 1.17 ±0.02 98 ± 0.72 6.36 8.3
MG: Moringa gum; TMG: ThiolatedMoringagum

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Table.2 Modeling and release kinetics of metronidazole benzoate tablet formulations.

Batch R2 n
Code
Zero-order First-order Higuchi Korsmeyer-Peppas
square- root
F1 0.717 0.943 0.901 0.976 0.256
F2 0.628 0.940 0.901 0.959 0.334

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F3 0.749 0.886 0.800 0.967 0.191
F4 0.807 0.948 0.939 0.963 0.777
F5 0.964 0.923 0.949 0.983 0.691

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F6 0.965 0.960 0.968 0.994 0.799

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