Just Accepted by Nanotoxicology

Physicochemical characterization and toxicological evaluation of plant based anionic polymers and their nanoparticulated system for ocular delivery

Deepa Pathak, prashant kumar, Gowthamarajan Kuppusamy, Ankur Gupta, Bhagyashree Kamble, Ashish Wadhwani

doi:10.3109/17435390.2013.834996

Abstract

The water soluble fractions of mucilages and gum from the seeds of fenugreek, isphagula and mango bark exudate were isolated, purified and characterized using X-ray diffraction spectrometry (XRD), fourier transform infrared spectroscopy (FT-IR), maldi/GC-MS, elemental analysis, 1D (1H and 13C) and 2D (HMQC, COSY) nuclear magnetic resonance spectroscopy (NMR). The fenugreek mucilage was identified to be a galactomannan chain consisting of 4 units of galactose attached to the backbone of 6 mannose units in 1:1.5 ratio. The isphagula mucilage was identified to be an arabinoxylan polysaccharide chain consisting of 4 units of arabinofuranose attached to the backbone of 9 xylopyrannose units in 1:3 ratio. The mango gum showed the presence of amylose, α-arabinofuranosyl and β-galactopyranosyl respectively.The characterterized mucilages and gum were individually formulated into nanoparticulate system using their complementarily charged polymer chitosan. The particles were observed to be spherical in shape in the range of 61.5-90 nm having zetapotential between 31-34 mV and PDI of 0.097-0.241. The prepared nanoparticles were observed to be non-irritant and non-toxic in vitro and in vivo upto 2000µg/ml .Therefore, these mucilages and gum can be the alternatives of anionic polymers for the ocular drug delivery system.

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Title Page:

Physicochemical characterization and toxicological evaluation of plant based anionic polymers and

their nanoparticulated system for ocular delivery

Deepa Pathak1, Prashant Kumar1, Gowthamarajan Kuppusamy 1* Ankur Gupta2, Bhagyashree Kamble3,

Ashish Wadhwani4,

1. Department of Pharmaceutics, J.S.S. College of Pharmacy, Udhagamandalam, T.N.-643 001 (Off

campus J.S.S. University, Mysore), India.

2. Department of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Udhagamandalam, T.N.-643 001

(Off campus J.S.S. University, Mysore), India.

3. Department of Pharmacognosy, J.S.S. College of Pharmacy, Udhagamandalam, T.N.-643 001 (Off

campus J.S.S. University, Mysore), India.

4. Department of Pharmaceutical Biotechnology, J.S.S. College of Pharmacy, Udhagamandalam, T.N.-643

001 (Off campus J.S.S. University, Mysore), India.

* Author for Correspondence

Gowthamarajan Kuppusamy

Prof. & Head

Department of Pharmaceutics

J.S.S. College of Pharmacy

Udhagamandalam, T.N.-643 001

(J.S.S. University, Mysore), India.

Phone number: +91-9443089812

Email address: gowthamsang@gmail.com, deepap16@rediffmail.com

Abstract

The water soluble fractions of mucilages and gum from the seeds of fenugreek, isphagula and mango bark

exudate were isolated, purified and characterized using X-ray diffraction spectrometry (XRD), fourier

transform infrared spectroscopy (FT-IR), maldi/GC-MS, elemental analysis, 1D (1H and 13C) and 2D

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(HMQC, COSY) nuclear magnetic resonance spectroscopy (NMR). The fenugreek mucilage was

identified to be a galactomannan chain consisting of 4 units of galactose attached to the backbone of 6

mannose units in 1:1.5 ratio. The isphagula mucilage was identified to be an arabinoxylan polysaccharide

chain consisting of 4 units of arabinofuranose attached to the backbone of 9 xylopyrannose units in 1:3

ratio. The mango gum showed the presence of amylose, α-arabinofuranosyl and β-galactopyranosyl

respectively.The characterterized mucilages and gum were individually formulated into nanoparticulate

system using their complementarily charged polymer chitosan. The particles were observed to be spherical

in shape in the range of 61.5-90 nm having zetapotential between 31-34 mV and PDI of 0.097-0.241. The

prepared nanoparticles were observed to be non-irritant and non-toxic in vitro and in vivo upto 2000µg/ml

.Therefore, these mucilages and gum can be the alternatives of anionic polymers for the ocular drug

delivery system.

Keywords: Anionic polymers isolation, Charcterization, Nanoparticle formulation, Ocular toxicity studies.

1. Introduction

Eyes are sensitive, delicate and vital organs of the human body. The most common diseases

associated with the eyes are conjunctivitis, cataract, glaucoma, diabetic retinopathy and age-related

macular degeneration. Various formulations such as eye drops, occuserts, intravitreal injection, ointment,

aqueous gel and routes either systemic or local route have been applied for the treatment of these

complications. Systemic administration requires higher dosage and frequent administration that results in

severe adverse effects. Local injections, particularly intravitreal and subconjunctival injections are

alternate strategies to achieve therapeutic concentration in the vitreo-retinal disorders (Pathak et al., 2012).

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However, to maintain the effective concentration repeated injections are required, which causes clinical

complications or patient discomfort (Kimura and Ogura, 2001).

The application of noninvasive polymeric drug delivery systems such as micro and nanoparticles,

microspheres, liposomes, hydrogels and ocular implants have permitted a targeted delivery of drugs to

specific sites in controlled manner. Apart from various advantageous effects of polymeric systems, these

systematic approach as may lead to high cost (Prow, 2010) and ocular toxicity due to size (Koster et al.,

1996, Sohaebuddin et al., 2010), charge (Hillegass et al., 2010) and metallic nature (Park, 2007, Theodore

et al., 2005).

Further, the polymers of natural and synthetic origin are widely employed for the preparation of

polymeric systems. The synthetic polymers are frequently used for its ability and usefulness in controlled

delivery of drugs and biological products. The disadvantages of synthetic polymers are their cost,

availability and sometimes toxic effect. Bejjani et al., 2005 and Tamboli et al., 2011 have reported non-

cytoxicity of poly (lactic) acid nanoparticles (PLA) for gene delivery in human and bovine retinal pigment

epithelial cells up to 4 mg/mL concentration but beyond this concentration they are toxic for eyes. Some

drugs show incompatibilities with different range of excipients. Marini et al., 2003 has reported atenolol-

PVP, atenolol-mg-stearate showed drug-excipient incompatibilities due to reaction between aldehydic

sugars and primary/secondary amines, leading to the formation of Schiff bases.

Polysaccharides of natural origin have recently been used as polymers for the preparation of

nanoparticles to overcome the problems associated with metallic and synthetic polymer based

nanoparticles. The natural polymers such as chitosan, sodium alginate, bovine serum albumin and gelatin

are widely employed in preparation of nanoparticles for ocular delivery due to their biodegradability,

mucoadhesive property, therapeutic potential, stability in the biological fluids and during storage.

(Motwani et al., 2008). However, with the mucoadhesive and biodegradability properties, these polymers

sometimes not suitable for ocular delivery due to adverse reaction in the eye for example albumin

microspheres (Hui and Robinson, 1985). The other example is of gelatin based aqueous delivery system

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which acts as a thermoreversible hydrogel with low mechanical strength which breaks at 30 °C and results

in drug loss. Glutaraldehyde is used as a crosslinking agent to provide stability toward thermal

degradation (Natu et al 2007) but it causes eye irritation. To avoid the use of toxic chemicals and high

cost polymers plant based polymers which are abundantly available in nature may be evaluated for their

composition, structure and toxicity.

Amelia M. Avachat et al., 2010 described various plant based mucilages and gums like fenugreek

mucilage, isphagula mucilage and mango gum etc. for their mucoadhesive and binding property for oral

drug delivery. The isolation and characterization of mucilages and gum from these sources are available

as mixture of water insoluble and soluble fraction (Nitalikar et al., 2003, Murthy et al., 2010, Singh et al.,

2010). Gowthamarajan et al., 2012 recently reported the use of natural gums in the preparation of

mucoadhesive tablet. Mehra et al., 2010, reported the use of tamarind gum based gel for ocular drug

delivery system.

To the best of our knowledge no reports are available on water soluble polymeric fractions of

mucilages and gum and their nanoformulation for ocular drug delivery. There are various advantages of

using water soluble factions for prepation of nanoparticles as, it excludes the use of organic solvents

(Chellat et al., 2000), it produces smaller particle size less than 350 nm which is required to cross blood

retinal barrier (Gaudana et al., 2010) and reach to posterior region of the eye and it reduces the production

cost.

Therefore, the current study has been carried out with the focus on isolation, purification,

characterization and toxicity evaluation of individual water soluble fractions from fenugreek, isphagula

seeds and mango bark exudates and their nanoformulation with chitosan for safe, feasible and non

invasive ocular drug delivery system.

2. Materials and methods

2.1. Materials

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The polymer chitosan (CS) (M.W:100,000-300,000 daltons, Deacetylation>80%) was procured

from Sigma-Aldrich (St. Louis, MO, USA). Fenugreek and Isphagula seeds were purchased from

Udhagamandalam local market (India). The mango bark exudate was purchased from Hyderabad local

market. The herbal samples were authenticated by Department of Pharmacognosy, J.S.S. College of

Pharmacy, Udhagamandalam, T.N.

2.2. Methods

2.2.1. Isolation, purification and preliminary tests of water soluble fractions

2.2.2.1. Extraction and purification of mucilage from fenugreek and isphagula seed

The seeds were collected and washed with water to remove the dirt and debris. The seeds (250 g)

were soaked in double distilled water (500 ml) overnight and then heated at 50 °C for 2h. The solution was

filtered through muslin cloth and to the filtrate equi-volume ratio of 90% v/v alcohol was added. The

obtained precipitate was filtered and dried in a hot air oven at 45ºC to obtain ≈150 g of powder. The

obtained powder was re-dissolved in 100 ml of water, filtered and centrifuged for 10 min at 3000 rpm. The

supernatant clear solution was collected, evaporated and dried. This process of purification was repeated

thrice. The purified solid mass was dried under reduced pressure at 40 °C, grounded and passed through

sieve no. 80 and stored in an airtight container.

2.2.2.2. Extraction and purification of mango gum

Crude plant exudates were collected in season of March to June. Mango gum was obtained from

the incised trunk of Mangifera indica. The Mango gum resin (250 g) was extracted by distilled water 500

ml on a water bath maintaining at 50 °C for 45 min with intermittent stirring, extraneous materials were

removed by straining through a several folds of muslin cloth. The gum was then precipitated by using 90%

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v/v alcohol followed by centrifugation at 3000 rpm. The extracted gum was filtered and dried in oven at

45 °C. The obtained powder was re-dissolved in 100 ml of water, filtered and centrifuged for 10 min at

3000 rpm ((Remi instruments ltd., Mumbai, India)). The supernatant clear solution was collected,

evaporated and dried. This process of purification was repeated thrice. The purified solid mass was dried

under reduced pressure at 40 °C, grounded and passed through sieve no. 80 and stored in an airtight glass

container.

2.2.2.3. Biochemical tests of isolated mucilages and gum

The purified samples of mucilages and gum were subjected to various biochemical tests for

carbohydrates, proteins, tannins, alkaloids, saponins, phenol and flavonoids as per official methods (Treas

& Evans, 1996).

2.2.2. Physico-chemical and structural characterization of mucilages and gum

2.2.2.1. X-Ray diffraction analysis (XRD)

In X-ray studies, an automatic X-ray diffractometer (Bruker, AXS/8, Berlin, Germany) equipped

with a PW R30 X-ray generator was used. The dry sample powder was pressed into pellets and X-ray

diffraction spectra were recorded using nickel-filtered Cu kα1 radiation having a wavelength of 1.5106 Å,

operating at 35 kW and 20 mA. X-ray diffractograms were obtained at a scanning rate of 1 degree (2θ) per

minute.

2.2.2.2. Elemental analysis

Elemental compositions of C, H and N were analyzed by using an elemental analyzer (Perkin

Elmer, 2400 Series, CHNSO Analyser, U.K). Accurately weighed 0.5 mg sample was heated to 1150 ºC

seperately and the corresponding element was determined by using a thermal conductivity detector.

2.2.2.3. Atomic absorbtion spectroscpic (AAS) analysis

The AAS instrumental parameters ((Shimadzu AA-6300, Serial No-A305245) for the estimation of

major, trace and heavy metals were set which include the lamp current, wavelength, slit width, lamp mode,

fuel flow rate, support gas flow rate, flame type and burner height were optimized. Deionized water was

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used for all the dilutions. All the plastic materials and glass wares were cleaned by soaking in dilute nitric

acid solution for 24 h and rinsed with distilled water followed by deionized water prior to use. The

calibration curves for the analyte ions were drawn after setting various parameters. All measurements were

performed in triplicate (n=3) and the standard deviation (SD) was recorded.

2.2.2.4. Fourier Transform Infrared spectroscopy (FT-IR)

The FT-IR spectrum (Shimadzu FTIR 84000 S, Japan) of samples were recorded on FT-IR

spectrophotometer. The dry sample (10 mg) was ground into fine powder using mortar and pestel then

pressed using potassium bromide (KBr 100 mg) disc technique under a hydraulic press at 10,000 psi. Each

KBr disc was scanned at 4 mm/s a resolution of 2 cm over a wave number region of 4000 – 400 cm-1.

2.2.2.5. Mass Spectroscopic analysis (MS)

The average molecular weight of sample was determined by matrix assisted laser

desorption/ionization-time of flight (Bruker Daltonik, Bremen, Germany, Reflex II MALDI-TOF

instrument) analyzer in the negative ionization mode. For the ionization, a nitrogen laser (337 nm, 3 ns

pulse width, 3 Hz) was used. Sample (5 mg) was dissolved in D2O solvent. 0.5 mL of the sample was

applied to the target followed by the addition of 1 mL of matrix solution (2,5-dihydroxybenzoic acid), and

dried under a gentle stream of air. All spectra were measured in the reflector mode using external

calibration.

2.2.2.6. 1D and 2D NMR spectroscopy

NMR spectra of 1H and 13C of sample were recorded in NMR spectrometer (Bruker, Bremen,

Germany). 100 mg of sample was dissolved in D2O and chemical shifts were reported in ppm relative to an

internal standard TMS (tetramethylsilane). NMR spectrum was obtained at a base frequency of 400MHz,

with 16 transitions and delay time 1.5 s using D2O as solvent. The chemical shifts were expressed in δ

(ppm) relative to the resonance of TMS.

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2D NMR spectrum was applied using double-quantum filtered correlated spectroscopy (DQF

COSY), hetero nuclear single-quantum coherence (HMQC) with a NMR spectrometer (Bruker, Bremen,

Germany).

2.2.2.7. Acid-base hydrolysis and FTIR, GC/MS analysis of mucilages and gum

2.2.2.7.1. Acid-base hydrolysis

The pH of the sample solution (300 mg/ml) was adjusted to 2.0 by adding 0.5 M sulfuric acid. The

solution was heated to 100 ºC for 24 h. After cooling, the pH of solution was adjusted to 10 by the addition

of 0.5 M NaOH and heated to 100 ºC for 24 h. After cooling the solution was neutralized with barium

carbonate and filtered. The solution was then dialyzed for 24 h against de-ionized water; freeze dried (De

Paula et al., 1998). The obtained hydrolysed dry samples were subjected for FTIR and Gas

chromatography-/mass spectrometry (GC-MS) studies.

2.2.2.7.2. FTIR and GC-MS analysis of hydrolysed fraction

The freezed dried hydrolysed fraction of mucilages and gum were subjected for FT-IR (method

described in section 2.2.2.4) and GC-MS analysis (Agilent Technologies, Palo Alto, USA). The GC-MS

was performed using HP-5 capillary column (0.25 mm x 30 m) linked to model TD800 Finningam ion trap

mass spectrometer (MS) operated at 70 eV. The columns were programmed from 50-220 ºC at 40 ºC min-1.

2.2.3. Pre-formulation studies of mucilages and gum

2.2.3.1. Determination of surface charge and pH

1% w/v solution of sample was prepared in distilled water and determined surface charge by

potentiometric analysis using potentiometer. The pH of same solutions (1% w/v) were determined by using

digital pH meter (pH meter pH/ /ion/MV meter, ion 510 bench pH, Eutech Instruments, Mumbai). All

measurements were performed in triplicate (n=3) and the standard deviation (SD) was recorded.

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2.2.3.2. In vitro cell viability assay

Cytotoxicity of the sample was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assay using L929 mouse fibroblast cell line. 1x 105cells/ml were plated in

each well of a 96 well microtitre plate in 100 µL of medium and incubated at 37 °C in CO2 incubator.

In the first six wells cells were left without any treatment as a positive control. Stock solutions of the

samples of different concentrations (0.05mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL and 5mg/mL) were made

in 1% DMSO and diluted with minimal essential medium (MEM) to a final concentration of 50, 500, 1000,

2000, 5000 µg/mL in the plate. Once the confluent monlolayer was ready, concentrations of the test

samples of 50 µL was added to each well in triplicate and incubated at 37 oC in a CO2 incubator for 72 h.

After incubation, 50 µL of MTT dye (2mg/mL) was added to each well and again incubated at 37 oC

for another 4 h. Formed formazan was washed and dissolved using 50 µL of isopropanol and optical

density was recorded with a microtitre plate reader at 550 nm (Scudiero et al., 1988). All the

measurements were performed in triplicate (n=3) and the standard deviation (SD) was recorded. Cell

viability was calculated by the formula:

………………………………………………………..Eq. 1

Where Isample is the absorbance of samples treated well and Icontrol is the absorbance of control wells

without samples.

2.2.3.3. Compatibility studies

The isolated mucilage-chitosan and isolated gum-chitosan (physical mixture 1:1 ratio) were

subjected for compatibility studies carried out by Differential scanning calorimetry (DSC, Water Q 200,

Bangalore) analysis to characterize the thermal behavior of the individual mucilages, polymer, gum and

their nanoparticles. Samples were crimped in standard aluminium pans and heated from 20 to 400 ºC at a

heating rate of 10 ºC/min under constant purging of dry nitrogen at 30mL/min. An empty pan, sealed in the

same way as the sample, was used as a reference.

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2.2.4. Formulation and Evaluation of nanoparticles

2.2.4.1. Preparation of polymeric nanoparticles

The purified mucilages and gum solution of 0.04% w/v was prepared by dissolving the individual

mucilages and gum in double distilled water. The pH of the solution was adjusted to 5.5 using 1 N

hydrochloric acid. The chitosan of 0.02% w/v was dissolved in a solution of 0.1% v/v acetic acid, volume

adjusted using distilled water and pH modified to 5.5 using 0.1 M NaOH. The solutions were filtered under

vacuum and were further taken for the preparation of nanoparticulate suspension by a modified

coacervation method at 3000 rpm for 2h using Remi stirrer (Douglas and Tabrizian, 2005). The formed

suspension was freeze dried using 0.5% w/v mannitol as lyoprotectant and stored in desiccator under

vacuum.

2.2.4.2. Characterization of nanoparticles

2.2.4.2.1. Transmission electron microscopic (TEM)

TEM (TEM, Philips CM-10, USA) was used to study the shape of nanoparticles. Samples of the

nanoparticles suspension (5-10 µl) were dropped onto formvar-coated copper grids. After complete drying,

the samples were stained using 2% w/v phosphotungstic acid. Digital Micrograph and soft imaging viewer

software were used to perform the image capture.

2.2.4.2.2. Particle size and zeta potential

Nanoparticles size distribution and zeta potential were determined using zetasizer photon

correlation spectroscopy (Malvern, Model No. 3000 HF, Malvern (U.K.). The size distribution analysis

was performed at a scattering angle of 90° and at a temperature of 25 °C using samples appropriately

diluted with filtered water (0.2 µm filter). Zeta potential was measured using a disposable zeta cuvette. For

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each sample, the mean diameter/zeta potential ± standard deviation of six determinations was calculated

applying multimodal analysis.

2.2.4.2.3. Sterility testing

The sterility testing was performed according to Indian Pharmacopoeia (IP), 2007 method on four

different media namely, fluid thioglycolate media (FTM), nutrient broth medium (NB), soyabean casein

digests medium (SCDM) and sorbitol-dextrose broth (SDB) to investigate the pesence or absence of

aerobic bacteria, anaerobic bacteria and fungi. FTM media was incubated at 37 ± 0.5 °C under aerobic

condition, NB at 30 ± 2.5 °C under anaerobic condition in a bacteriological incubator while SCDM and

SDB were incubated at 25 ± 0.5 °C under aerobic condition in a fungal incubator for 14 days. The

experiment was performed in triplicate.

2.2.4.2.4. In-vitro ocular irritation studies of nanoparticle (HET-CAM Test)

White Leghorn chicken eggs were obtained from the Poultry Research Centre, Kerala. White

Leghorn chicken eggs were selected for the study because they have no hereditary defects and yields

reproducible results. Eggs were incubated in the incubator for 9-10 days at 37 ºC. On day 9, eggs were

tested with candle light to ensure that all were viable. On day 10, the air cell was marked with a pencil and

removed the shell by tapered forceps. The membrane was carefully moistened with 0.9% NaCl solution

and the membrane was carefully removed without injuring any underlying blood vessels. 0.1 N NaOH and

sterile distilled water were used as positive and negative control/solvent control. The chorioallantoic

membrane (CAM) of each egg was applied directly with 0.3 mL of the positive/negative control and

different concentration of pure gum/mucilage and nanoparticles (50 µg/mL -5000 µg/mL). Two eggs for

controls and three for test substance were used for each assay. The reactions of hemorrhage, coagulation

and lysis on the CAM were observed over a period of 6h. The time for each reaction was recorded in 5 min

and an irritation score (IS) was calculated according to the formula given below. Each test was done in

triplicate and the mean score of the three eggs was determined. At the end of each assay the embryos were

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killed quickly by placing the eggs into a freezer at -20 ºC (Kishore et al., 2009). All measurements were

performed in triplicate (n=3) and the standard deviation (SD) was recorded.

H= hemorrhage; L = vessel lysis; C = coagulation; Min= 5 min. After treating for 6 h the main reaction

(hemorrhage, lysis or coagulation) was scored as follows: 0=no reaction; 1=slight reaction; 2=moderate

reaction; 3=severe reaction.

2.2.4.2.5. Haemocompatibility studies

Blood samples of healthy human volunteers were obtained from blood bank of government

hospital, Ooty in evacuated siliconized glass tube containing sodium citrate. Red blood cells were

separated by centrifugation at 1500 rpm for 10 min and then washed 3 times with phosphate buffer saline

pH 7.4. Stock solution of erythrocytes in PBS was prepared such that the cell count was 1×108 cells/ml.

Equal volumes of RBC suspension and nanoparticles dispersion were suspended in a microcentrifuge tube

such that the final concentrations of pure mucilages/gum and nanoparticle dispersion were 50 µg/mL -5000

µg/mL and incubated at 37 ºC for 1 h. 1% v/v Triton X and PBS were used as positive and negative

controls respectively. After 1 h the tubes were centrifuged at 1500 rpm for 10 min and the hemoglobin

released in the supernatant was detected by UV absorbance at 540 nm. All measurements were performed

in triplicate (n=3) and the standard deviation (SD) was recorded. The percent haemolysis was calculated by

(Gulatia et al., 2010)

Where AbSsample is the absorbance of supernatant of erythrocyte and nanoparticles suspension. Abs0% is the

absorbance of supernatant of erythrocyte and PBS suspension. Abs100% is the absorbance of supernatant of

erythrocyte and Triton X suspension.

2.2.4.2.6. In-vivo ocular irritation studies

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Experimental animals were obtained from in house animal facility of JSSCP, Udhagamandalam.

All procedures using animals were reviewed and approved by the Institutional Animal Ethics Committee

(Proposal No.-JSSCP/IAEC/PH.D/PH-CEUTICS/01/2012-13).

Animals: The female New Zealand white rabbits, in the range of 2–3 kg body weight were

nulliparous and non-pregnant. The acute ocular irritation potential of gum nanoparticles were determined

according to OECD 404 and 405 test guidelines. The experiment was divided into groups (Positive control,

pure mucilages and gum, nanoparticles in 50 µg/mL-2000 µg/mL) and 3 animals were included in each

group.

Experiment: Both eyes of the experimental animals selected for testing were examined with an

ophthalmoscope 24 h prior to starting experiment. None of the animal’s selected for the study showed any

ocular defects or pre-existing corneal injury. Different concentrations of pure gum/mucilage and

nanoparticle solution (50 µg/mL-2000 µg/mL) were applied into the conjunctival sac of left eye of each

animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for

about 1 s in order to prevent loss of the material. The right eye which remained untreated served as

negative control. The eyes of the test animals were washed with distilled water at 24 h following the

application of test materials to remove the presence of residual test substance if any. The conjunctiva, iris,

and cornea of both treated and control eyes were evaluated at the end of 24, 48, and 72 h checked for the

sign of redness, lacrimation and inflammation by Draize’s scoring approach (Vinardell et al., 1994).

3. Results

3.1. Characterization of isolated mucilages and gum

The mucilages of Trigonella foenum graecum and Plantago ovata were isolated from the seeds

using water as solvent and alcohol (90% v/v) as non-solvent. After precipitation the water soluble fraction

of mucilages were obtained and purified by using water. The yield of water soluble fraction was found to

be 44% w/w and 42.5% w/w respectively.

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The gum from Magnifera indica was purified from crude mango gum exudates using water as

solvent and alcohol (90% v/v) as non-solvent. After precipitation of gum the water soluble faction was

obtained and purified using water. The yield of water soluble fraction of gum was found to be 43.8% w/w.

Mucilages and gum showed pink colour upon treatment with ruthenium red and a gelatinous mass

was obtained by heating and cooling. Various biochemical tests were performed to confirm the presence of

carbohydrates by Molisch’s test, polysaccharide iodine test, test for enzyme, tannins, alkaloids, glycoside,

alkaloids, saponins, flavonoids and various functional group tests.

No characteristic peaks in the spectrum were observed in powder XRD analysis. The elemental (C,

H, N, O % w/w) composition of fenugreek mucilage (FM), isphagula mucilage (IM) and mango gums

(MG), were found to be (FM) 41.12, 6.32, 0.19, 51.46; (IM) 38.29, 5.43, 0.40, 55.88, and (MG) 38.46,

6.11, 0.15, 55.28. The experimental results of mineral profile indicated that the fenugreek mucilage and

mango gum have high content of sodium and potassium where as isphagula mucilage contains high

concentration of potassium. The mucilages and gum were found to be consisted of low concentration of

calcium and absence of lead, cadmium, copper, mercury, iron and arsenic.

The major functional groups present in the FT-IR spectrum of mucilages and gum (FM) 3429.83

cm-1, (IM) 3433.72 cm-1, (MG) 3427.96 cm-1 indicate the stretching vibrations of free –OH groups; (FM)

2924.52, (IM) 2923.80, (MG) 2925.52/cm, corresponding to C-H stretching band; (FM) 1633.57 cm-1,

(IM) 1633.34 cm-1, (MG) 1633.57 cm-1 represents the carboxylate groups and (FM) 1161.45 cm-1, (IM)

1124.55 cm-1, (MG) 1051 cm-1 suggested the presence of C-O-C and –OH in the pyran structure (Yang et

al., 2006).

The MS analysis of FM, in the negative ion mode, showed the molecular ion peak at m/z 1699.5

daltons (M-H+Na+K), base peak at 1541.821 daltons (M-2H-Mannose+ Na+K) and a daughter ion peak at

704.014 daltons (M-4 mannose-2 galactose) (Figure 1a). Results of both 1H and 13C NMR studies gave a

complete and reliable assignment of 1H and 13C signals. The chemical shift in the 1H/13C NMR spectrum of

the fenugreek gum shows δH/C values for galactose, H-1/C-1 at 4.94/100.1, H-2/C-2 at 3.82/69.89, H-3/C-3

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at 3.93/70.66, H-4/C-4 at 4.00/69.89, H-5/C-5 at 3.90/72.19 and H-6/C-6 at 3.755/62.64. The δ values for

mannosyl residue, H-1/C-1 at 4.80/102.5, H-2/C-2 at 4.3/71.57, H-3/C-3 at 3.794/73.28, H-4/C-4 at

3.874/78.45, H-5/C-5 at 3.748/75.96 and C-6 at 68.39 indicating its O-6 substitution (Figure 1b,1c). The

assignment of the 1H resonances of mannose and galactose as being either due to α or β was also

compared with available data (Ramesh et al., 2001 and Andrews et al., 1952). The FTIR spectrum of the

acid-base hydrolysed fenugreek mucilage showed a prominent peak at 1710.31 cm-1 indicating the opening

of pyran ring and the presence of ester and –CHO group. The GC-MS analysis of the hydrolysed fraction

showed four peaks at retention time (RT) 10.12min, 11.02min, 12.15min and 13.68min with molecular ion

peaks of m/z ratio, 350.75, 424.23, 951.26 and 930.08 respectively consisted of 1 to 5 units of open

galactomannan chain (Figure 1d).

The MS analysis of IM, in the negative ion mode, showed the molecular ion peak at m/z 1967.972

daltons (M-H+6K) (Figure 2a). Results of 1H, 13C NMR and HMQC (Figure 2b) studies gave a complete

and reliable assignment of 1H and 13C signals. The chemical shift in the 1H/13C NMR spectrum of the

isphagula gum shows δH/C values for xylosepyranose unit, H-1/C-1 at 4.6/107.6, H-2/C-2 at 3.4/73.2, H-

3/C-3 at 3.5/76.16, H-4/C-4 at 3.65/69.86, H-5/C-5 at 3.91/64.8 and H-6/C-6 at 3.27/64.8. The δ values for

arabinofuranosyl residue, H-1/C-1 at 4.5/97.8, H-2/C-2 at 3.65/72.1, H-3/C-3 at 3.76/83.2, H-4/C-4 at

4.13/68.86, H-5/C-5 at 3.79/67.06 and H-6/C-6 at 3.67/67.06. The assignment of the 1H resonances of

xylopyrannose and arabinofuranose as being either due to α or β was also compared with available data

(Fischer et. al., 2004). The FTIR spectrum of the acid-base hydrolysed isphagula gum showed a prominent

peak at 1713.02 cm-1 indicating the opening of pyran ring and the presence of ester and –CHO group. The

GC-MS analysis of the hydrolysed fraction showed two peaks at retention time (RT) 10.57min, 11.60 min

with molecular ion peaks of m/z ratio, 884.23 and 850.17 respectively consisted of 4 units of open

xylopyrannose and 2 units of open arabinofuranose chain (Figure 2c).

The MS analysis of MG, in the negative ion mode, showed the molecular ion peak at m/z 1927.6

daltons (M-H+Na+K) (Figure 3a). The evidence for amylose in mango gum was characteristic signals in

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its 13C NMR spectrum at 100.6 ppm (C-1), 71.3 ppm (C-2), 74.1 ppm (C-3), 79.2 ppm (C-4), 72.3 ppm (C-

5) and 61.6 ppm (C-6) (Figure 3c). Signals at δ109.7 ppm and 104.1 ppm corresponding to C-1 of α-

arabinofuranosyl (A-1) and β-galactopyranosyl (GA-1) respectively (Iagher et. al., 2002). The FTIR

spectrum of the acid-base hydrolysed mango gum showed a prominent peak at 1707.12 cm-1 indicating the

opening of pyran ring and the presence of ester and –CHO group. The GC-MS analysis of the hydrolysed

fraction showed two peaks at retention time (RT) 9.35min, 10.05min with molecular ion peaks of m/z

ratio, 940.47 and 932.45 respectively consisted of 5 units of open amylose chain (Figure 3d).

3.2. Preformulation evaluation

The preformulation studies were carried out for the characterized mucilages and gum (FM, IM and

MG). Potentiometric analysis showed that these mucilages and gum at 1% w/v solution carry negative

surface charge of potential -25 mV (pH 5.68), -22 mV (pH 6.37), and -32 mV (pH 6.48) respectively.

Preliminary toxicity profile of the characterized mucilages and gum was confirmed by the in vitro cell

viability studies on L929 mouse fibroblast cell culture using MTT assay.

DSC thermograms of mucilages and gum were showed their respective endothermic peaks

according to their melting points ranging from 95-100 °C, 75-80 °C and 90-95 °C respectively.

3.3. Characterization of nanoparticles

The prepared nanoparticles were evaluated for their particle size, zeta potential, polydispersity

index (PdI) and surface morphology. The size of the particles formed with chitosan-fenugreek mucilage

complex (FM+C) was 90.01 nm, chitosan-isphagula mucilage (IM+C) complex was 81.80 nm and

chitosan-mango gum complex (MG+C) was 61.50 nm with the zeta potential/PdI 31.4 mV/0.139, 34.7

mV/0.097 and 39.4 mV/0.241 respectively. The shape of prepared nanoparticles was shown in Figure 4.

The amorphous powders of the prepared nanoparticles were re-dispersed in water for injection (5000

µg/mL) and sterilized using membrane filtration (0.2 µm). The sterility testing of the different suspensions

was carried out as per Indian Pharmacopoeia 2007 and no growth of bacteria and fungi were found after 14

days under aerobic and anaerobic condition.

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3.4 In-vitro and in-vivo ocular irritation studies

The in-vitro ocular irritation studies were carried out using HET-CAM method and results were

shown in Figure 5.

The RBC haemolysis testing is widely used as an important in-vitro tool for determining the

haemocompatibility of the nanoparticles depicted in table 1.

The in-vivo occular irritation studies were carried out using rabbit eye for the concentration 50

µg/mL-2000 µg/mL (selected on the basis of in-vitro studies) and determined for upto 72 h. The ocular

safety observation in the rabbit eyes adopting Draize’s scoring approach. The ocular safety score for the

mucilages and gum were found to be 0.23-0.25 and their naoparticles in the range of 0.27-0.31 (Table 2).

4. Discussion The preliminary biochemical tests of mucilages and gum confirmed the presence of carbohydrates,

reducing sugars and positive test for mucilage. All the mucilages and gum was found to be absence of

proteins, flavonoids, tannins, alkaloids, glycoside, saponins and flavonoids. XRD analysis indicated that

the isolated mucilages and gum were completely amorphous in nature.

The elemental analysis of indicated the composition of C, H, N, O. The experimental results of

mineral profile indicated that the FM and MG have high content of sodium and potassium where as IM

contains high concentration of potassium. The mucilages and gum were found to be consisted of low

concentration of calcium and absence of lead, cadmium, copper, mercury, iron and arsenic.

The major functional groups present in the FT-IR spectrum of mucilages and gum are free –OH

group, C-H stretching band, carboxylate groups and suggested the presence of C-O-C and –OH in the

pyran structure.

The FM was identified to be a galactomannan chain consisting of 4 units of galactose attached to

the backbone of 6 mannose units in 1:1.5 ratio (Brummer et al., 2003). The structure of the obtained

fenugreek mucilage has been proposed from the combined interpretation of elemental (C,H,N,O) analysis,

mineral element composition analysis, FT-IR, MALDI, 1H and 13C NMR (Figure 1).

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The IM was identified to be an arabinoxylan polysaccharide chain consisting of 4 units of

arabinofuranose attached to the backbone of 9 xylopyrannose units in 1:3 ratio. The structure of the

obtained isphagula mucilage has been proposed from the combined interpretation of elemental (C,H,N,O)

analysis, mineral element composition analysis, FT-IR, MALDI, 1H, 13C NMR and HMQC (Figure 2).

MG had 11 units of open amylose chain. The structure of the obtained MG has been proposed from

the combined interpretation of elemental (C,H,N,O) analysis, mineral element composition analysis, FT-

IR, MALDI, 1H, 13C NMR and HMQC (Figure 3).

The mucilages and gum were found to be negative surface charge and non toxic up to 5000 µg/mL

solution and therefore taken ahead for further studies.

Chitosan being a cationic polymer has been used for the production of nanoparticulate system by

modified coacervation method with negatively charged mucilages and gum (Calvo et al., 1997). The

Chitosan-mucilages/gum polyionic complexes are formed through the ionic gelation via interactions

between the –NH2 groups of chitosan and –OH groups of mucilages and gum which in DSC thermogram

showed merged endothermic peaks with chitosan (83.5 °C) ranging between 80-100 °C.

The shape of prepared nanoparticles was observed to be spherical (Figure 4). The amorphous

powders of the prepared nanoparticles were re-dispersed in water for injection (5000 µg/mL) and sterilized

using membrane filtration (0.2 µm). The sterility testing of the different suspensions was carried out as per

Indian Pharmacopoeia 2007 and was found to be sterile.

HET-CAM analysis the pure mucilages/gum and prepared nanoformulations were found to be

non-irritant and safe on blood vessels up to 2000 µg/mL solution with the irritation score of zero (Figure

5). Above this concentration dilation of blood vessels takes place. The irritation score for positive and

negative control was found to be 16.85 and zero indicated severe irritant and non-irritant respectively.

The RBC haemolysis testing is widely used as an important in-vitro tool for determining the

haemocompatibility of the nanoparticles. This method has been widely encouraged by various regulatory

agencies including OECD guidelines for determining biocompatibility to chemicals. The values of RBC

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haemolysis testing are shown in table 1. In this study less than 5% haemolysis is considered as an

acceptable limit for biocompatibility. The result indicated that mucilages/gum and prepared nanoparticle

were haemocompatible with insignificant toxicity up to 2000 µg/mL and above this concentration the

percentage haemolysis was found to be similar with nagative control causing haemolysis of blood vessels.

The in-vivo occular irritation studies were carried out using rabbit eye for the concentration 50

µg/mL-2000 µg/mL (selected on the basis of in-vitro studies) and determined for upto 72 h. The ocular

safety observation in the rabbit eyes adopting Draize’s scoring approach. The ocular safety score for the

mucilages and gum were found to be 0.23-0.25 and their naoparticles in the range of 0.27-0.31 considered

as practically non-irritating to rabbit eyes. (Table 2). These safety score were very insignificant when

compared to the maximum score of 110 and no symptoms of ocular irritation such as redness, tearing,

inflammation or swelling. Furthermore fluoresceine staining did not indicate corneal or conjunctival

epithelial defects. Thus, the developed ocular delivery systems were apparently free from any ocular

irritation and can be safely administered to human beings.

Conclusion

The nanoparticulated system of the characterized anionic mucilages and gum were formulated

using chitosan as a cationic polymer and were found to be non-irritant and non-toxic up to 2000µg/mL.

Therefore, these mucilages and gum can be used as an alternative of other anionic polymers for novel

ocular drug delivery system.

Acknowledgement

We would like to thank Department of Science and Technology, New Delhi, India for providing

financial assistance for carrying out this project. We also thank J.S.S. College of Pharmacy,

Udhagamandalm, India for providing facilities to carry out this research.

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Figure 1. (a) MALDI, (b) 1H, (c) 13C NMR of fenugreek mucilage and (d) GC-MS of hydrolysed fenugreek mucilage

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Figure 2. (a) MALDI, (b) HMQC analysis of isphagula mucilage and (c) GC-MS of hydrolysed isphagula mucilage

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Figure 3. (a) MALDI, (b) 1H, (c) 13C NMR of mango gum and (d) GC-MS of hydrolysed mango gum

Figure 4. TEM morphology of prepared nanoparticles

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HET-CAM analysis of following mucilages, gum and their nanoformulations

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Table 1: Haemocompatibility analysis of mucilages, gum and their nanoformulations

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Ocular safety scores for mucilages, gum and their nanoparticles Reviewer 1: Comment 1: Introduction need to be broken in paragraphs. Clarification: Accepted and corrected. Comment 2: Specify the units of strength of alcohol used. Clarification: It was 90% v/v. Comment 3: Was the powder re-dissolved/re-suspended in water. Clarification: Obtained powder was re-dissolved. Comment 4: Mention the centrifuge used? Clarification: Remi centrifuge C-24 BL at 3000 rpm for 10 min. (Remi instruments ltd., Mumbai, India). Comment 5: Detail the setting parameters of atomic absorption spectroscopy. Clarification: The setting parameters of AAS are shown in table no 3. Table 3. Setting parameters of AAS

Elements Instrument Parameter Ca Na K Cu Pb As Hg Cd Al

Lamp current (mA)

10 12 10 6 10 12 4 8 10

Wavelength (nm)

422.7 589.0 768.0 324.8 283.3 193.7 243.7 228.8 309.3

Slit weidth (nm)

0.7 0.2 0.7 0.7 0.7 0.7 0.7 0.7 0.7

Lamp mode BGC-D2

Non-BCG

Non-BCG

BGC-D2 BGC-D2 BGC-D2 BGC-D2 BGC-D2 BGC-D2

Fuel gas flow rate (L/min)

2.0 1.8 2.0 1.8 2.0 2.0 Nil 1.8 7.0

Support gas flow rate (L/min)

15 15 15 15 15 15 Nil 15 15

Flame type Air-C2H2

Air-C2H2

Air-C2H2

Air-C2H2

Air-C2H2

HVG (Air-C2H2)

HVG cold Vaporizer technique

Air-C2H2

N2O-C2H2

Burner height (mm)

7 7 7 7 7 7 Nil 7 11

Linearity range (ppm)

0.5-2.5 2-10 1-5 0.5-2.5 2-10 0.010-0.050

0.010-0.050

0.10-0.50

5-25

Regression equation

Y=0.0137X-0.0015

Y=0.0921X+0.0261

Y=0.0142X+0.0028

Y=0.0712X-0.0064

Y=0.0021X+0.00092

Y=0.0238X+0.2108

Y=0.010X+0.0038

Y=0.000067X-0.00006

Y=0.0007X+0.0009

Correlation coefficient

0.9999 0.9989 0.9989 0.9999 0.9994 0.9945 0.9989 0.9998 0.9994

Comment 6: Correct HE-CAM test for HET-CAM test. Clarification: Accepted and corrected.

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Comment 7: Label the equations sequentially. Match the equation terms in their explanation. Clarification: Accepted and corrected. Comment 8: While stating potentiometric analysis are the authors referring the zeta size analysis. Clarification: No. Potentiometric analysis was performed for the isolated gum and mucilages to determine the surface charge. This is an important observation to select a suitable complimentary charged polymer for precipitation method for preparing the nano particles. Once the nanoparticles prepared, the zeta potential of the nanoparticles was determined which was already described in section 3.3. Comment 9: The TEM images are not describing morphology but just revealing its shape. Present better image that reveal legible toolbar. Clarification: Accepted and corrected. The scale was 100 nm and operating at 80 kV (TEM, Philips CM-10, USA). Comment 10: Stastical analysis is missing. Clarification: Where required all measurements were performed in triplicate (n=3) and the standard deviation (SD) was recorded. Comment 11: The discussion lacks references to previously cited literature. It appears to be compilation of result. Clarification: In each and every place whatever the references available we cited. Comment 12: Explain the acronym OECD guidelines. Clarification: Organisation for Economic Co-operation and Development. Comment 13: Check the manuscript for grammatical errors. Clarification: Accepted and corrected. Comment 14: The resolution of fig 1, 2, and 3 is poor. Clarification: Accepted and corrected. Comment 15: Table number and table captions are missing. Clarification: Accepted and included. Table 1: Haemocompatibility analysis of mucilages, gum and their nanoformulations.

Table 2. Ocular safety scores for mucilages, gum and their nanoparticles.

Reviewer 2: Comment 1: The introduction consists of a single, very long paragraph. It needs to be broken up into separate paragraphs by topic. Clarification: Accepted and corrected. Comment 2: pg 3 line 27-38 this text seems to compare natural and synthetic polymers but is confusing as written. Clarification: Accepted and corrected. Comment 3: The raw materials for the study were obtained from a local market. An issue common to all studies using natural products is standardization of materials. Would material would be the same composition and characteristics be obtained elsewhere or at a different time of years? Clarification: In case of all material there are lots of literatures available for their composition and it is not going to vary according to zone. Comment 4: Pg 5 ….subjected to various biochemical tests is vague. A little more detail is needed. Clarification: We performed the test for carbohydrates, proteins, tannins, alkaloids, saponins, phenol and flavonoids and found the presence of carbohydrate and reducing sugar and absence of others. The results of tests are shown in table 4. Table 4. Various physicochemical tests for isolated mucilages and gum

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SL NO.

TEST

INFERENCE

OBSER

VATIO

N

1. Test for carbohydrates (Molisch’s test)

Dissolve a little amount of the powder in water , add 1 ml

of Molisch’s reagent and shake

A violet ring is

formed at the

junction of two

liquids

+

2. Iodine test

100 mg dried mucilage powder + 1 ml 0.2 N iodine

solution.

No color observed in solution

Polysaccharides present (starch is absent)

3 Protein test Dissolve 100 mg dried mucilage

powder in 20 ml-distilled water;

add 0.5 ml of benzidine in alcohol

(90%). Shake and allow to stand

for few minutes

No blue color produced

Enzyme absent

2. Test for Tannins (Ferric chloride test)

To a solution add Ferric chloride reagent

No white

precipitate is

observed

_

3. Test for Glycosides ( Keller - Killiani test)

Add one drop of 90% alcohol and 2 drops of 2 % 3, 5 –

dinitro benzoic acid. Make alkaline with 20 % NaOH

solution

No purple

color is

obtained

_

4. Test for Alkaloids (Wagner’s Test)

Wagner’s reagent (Iodine-potassium iodide solution) was

added to test solution

No precipitate

obtained

_

5. Test for Steroids (Salkowaski Test)

Treat the extract with few drops of acetic anhydride, boil

and cool. Then add concentrated sulphuric acid from the

side of test tube

No brown ring

is formed at

the junction

_

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6. Test for flavanoids (Shinoda Test)

To the solution add few magnesium turnings and

concentrated hydrochloric acid drop wise

No color is

obtained

_

7. Test for Mucilage (Ruthenium Test)

Treat the solution with Ruthenium red solution

Pink color is

obtained

+

8. Test for chlorides(Silver nitrate test)

To the test solution add dil.HNO3 and few drops of silver

nitrate

No precipitate _

9. Test for Sulphate (Barium chloride test)

To the test solution add barium sulfate solution

No precipitate _

10. Test for Carbonyl compounds

Add few drops of unknown to a solution of 2,4 – dinitro

phenol

Yellow color +

11. Test for Aldehyde group

Add 1 ml of dil H2SO4 1 ml of Potassium dichromate. Add

1 ml of unknown and heat to 500C on water bath

No change in

color

No reduction

of dichromate

_

12. Test for Aldehyde group

Tollen’s Reagent (Silver mirror test)

Add few drops of aqueous NaOH to 1 ml of silver nitrate

to produce precipitate of silver oxide. Add just enough

aqueous ammonia to redissolve the precipitate and form

the colorless solution. Add 1 ml of the unknown and heat

in water bath at 500C for few minutes

No change _

13. Test for ketone group (IodoformTest)

Four drops of unknown in a test tube .Add 0.5 ml of

distilled water. Add 0.25 ml of 6 M NaOH and 0.25 ml of

water, add five drops of KI3

Yellow cloudy

precipitate

+

14.

Test for –COOH group

Add 1 ml of ethanol in a test tube; add 1 ml of the

unknown followed by few drops of con Sulphuric acid.

Heat to 500C in water bath for 5 min .Pour the product into

a beaker containing 20 ml of Sodium carbonate

Fruity odor +

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Comment 5: The description of the instrument conditions for AAS analysis simply as being optimized is not description of the standard used. Clarification: Accepted and included. Comment 6: It should be HET-CAM test. Clarification: Accepted and corrected. Comment 7: For an original article, the result and discussion must be separate. Clarification: Accepted and corrected. Comment 8: Are these the results of low level screening tests or more sophisticated analyses? Clarification: The experiments are analysed and validated by using sophisticate equipment at a maximum extended as described in methods. All the experiments were performed in triplicate for reproducibility as shown in the respective methods and tables. Comment 9: No data are shown for inorganic in the products. Clarification: The traces amount of various inorganic substances is shown in table 5. Table 5. Mineral profile of isolated mucilages and gum

Concentration (mcg/g) S.N. Element

FM IM MG

1 Pb 0.00±0.00 0.0007±0.0001 0.00±0.00

2 Cd 0.0002±0.0001 0.0007±0.0001 0.00±0.00

3 Cu 0.0057±0.001 0.0032±0.001 0.0038±0.001

4 Hg 0.00±0.00 0.00±0.00 0.00±0.00

5 As 0.0012±0.0001 0.0004±0.001 0.0001±0.0001

6 Fe 0.00±0.00 0.0002±0.0001 0.0021±0.001

7 Na 432.901± 14 7.988± 3 350.500± 12

8 K 146.92± 7 6398.46± 110 381.538± 13

9 Ca 8.785± 7 4.223± 3 9.261± 8

Comment 10: Information on the distribution of particle sizes should be provided. Clarification: We found uniform distribution in all cases which was confirmed by the intensity vs size distribution graph. Comment 11: A reference for OECD guidelines and why is less than 5% haemolysis considered acceptable for this study. Clarification: For both we already mention reference in method section. Comment 12: Pg 17 the sentence should end with ……..were observed. Clarification: Accepted and corrected.

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Setting parameters of AAS

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Various physicochemical tests for isolated mucilages and gum

Mineral profile of isolated mucilages and gum Figure 1. (a) MALDI, (b) 1H, (c) 13C NMR of fenugreek mucilage and (d) GC-MS of hydrolysed fenugreek mucilage Figure 2. (a) MALDI, (b) HMQC analysis of isphagula mucilage and (c) GC-MS of hydrolysed isphagula mucilage Figure 3. (a) MALDI, (b) 1H, (c) 13C NMR of mango gum and (d) GC-MS of hydrolysed mango gum Figure 4. TEM morphology of prepared nanoparticles

Figure 5. HET-CAM analysis of following mucilages, gum and their nanoformulations

Table 1: Haemocompatibility analysis of mucilages, gum and their nanoformulations

Table 2. Ocular safety scores for mucilages, gum and their nanoparticles

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