4. materials and methods - information and...

Materials and methods

43 | P a g e Department of Pharmacognosy, JSSCP (JSS University, Mysore), Udhagamandalam-643001

4. MATERIALS AND METHODS

This chapter describes the equipments, materials, methods and procedures used in the

experiments and throughout the study. The chapter is divided into following sections;

Section 4.1. Quality control and standardization of G. sylvestre extract : Quantitative

estimation of GMG from various G. sylvestre formulations and extract using HPLC-

ESI-MS/MS as a quality control tool.

Section 4.2. Isolation, purification and characterization of GMG from G. sylvestre

extract.

Section 4.3. Evaluation of acute oral toxicity of G. sylvestre extract and GMG as per

OECD 423 guidelines.

Section 4.4. Determination of absolute bioavailability of GMG after oral and iv

administration in normal rats.

Section 4.5. Development, optimization & characterization of G. sylvestre extract and

GMG loaded polymeric nanoparticles.

Section 4.6. Evaluation of comparative bioavailability of developed polymeric

nanoformulations with GMG and G. Sylvestre extract.

Section 4.7. Preparation & characterization of sodium and potassium salts of isolated

GMG.

Section 4.8. Evaluation of comparative anti-hyperglycemic potential of GMG, its

sodium, potassium salts, and GMG nanoparticles with G. sylvestre extract in

streptozotocin induced diabetic rats.

Section 4.9. In vitro cytotoxicity evaluation of GMG using MTT assay.

Section 4.10. Herb-drug interaction studies of G. sylvestre extract with selected

conventional drug, glimepiride.



4.1. QUALITY CONTROL AND STANDARDIZATION OF GYMNEMA

SYLVESTRE EXTRACT: Quantitative estimation of gymnemagenin (GMG)

from various Gymnema sylvestre formulations and G. sylvestre extract using

HPLC-ESI-MS/MS as a quality control tool

4.1.1. Plant extract, reference standard, marketed formulations, chemicals and

solvents

Alcoholic extract of G. sylvestre leaves (GYM-1) was kindly provided as a gift

sample with certificate of analysis [(ANNEXURE I (a)] by M/s Natural Remedies

Pvt. Ltd, Bangalore, India. Various marketed formulations such as, Diabecon tablets

(GYM-2; Batch no.:A036012B), Himalaya Drug Company, Bangalore, India;

Meshashringi capsules (GYM-3; Batch no.: F247005G), Himalaya Drug Company,

Bangalore, India; Mersina capsules (GYM-4) J & J Dechane Labs Pvt. Ltd,

Hyderabad, India; Gudmar Ghana (GYM-5; Batch no.: P0811), Chaitanya

Pharmaceuticals Pvt. Ltd, Nasik, India; and Gudmar Churna (GYM-6), Ayurvedic

Pharmacy Store, Pune, India were purchased from the local ayurvedic pharmacy

market, Pune, India. Standard gymnemagenin (GMG) was purchased from Natural

Remedies Pvt. Ltd, Bangalore, India [(ANNEXURE I (b))]. Withaferin A was

purchased from ChromaDex (Laguna Hills, CA, USA,) and used as internal standard

(IS). HPLC grade water (J. T. Baker, Mumbai, India), acetonitrile, methanol, ethanol,

potassium hydroxide (KOH) and hydrochloric acid (HCl) were procured from Merck.

All other chemicals and solvents otherwise specified were of analytical grade.

4.1.2. Preparation of reference standard

A reference solution of GMG was prepared by dissolving 5 mg in 5 mL of methanol.

Calibration standards for GMG ranging from 5.280 to 305.920 ng/mL were

subsequently prepared diluting with water:methanol (50:50). The withaferin A (IS)

was prepared by dissolving 5 mg in 5 mL of methanol. Further, withaferin A solution

of 15 ng/mL was prepared and used for the study.

4.1.3. Preparation of samples

Samples (GYM-1 to GYM-6) were prepared using acid–base hydrolysis of gymnemic

acids. 75 mg each of G. Sylvestre extract and marketed formulations (weight

equivalent to G. Sylvestre) were dissolved in 5 mL of 50% v/v ethanol. To 1 mL of



each of these solutions, 0.5 mL of 4N HCl was added and heated on a boiling water

bath for 1 h. After cooling, pH was adjusted between 7.5-8.5 with 11% w/v KOH

solution. The final volume of the solution was adjusted to 10 mL with 50% v/v

ethanol. The solution was filtered through a membrane filter (0.22 mm) and diluted

with methanol (1:1)214

. Withaferin A (IS) solution (15 ng/mL) was added and

subjected to HPLC–ESI–MS/MS analysis.

4.1.4. HPLC Instrument

The HPLC-MS system consists of liquid chromatography (Shimadzu Prominence,

Japan), a binary gradient pump (LC-20 AD), an autosampler (SIL HTC) and column

oven (CTO-10ASVP). Compounds were separated on a Luna C-18 column (150 mm

x 4.6 mm; 5 mm particle size; Phenomenex, Torrance, CA, USA). The detailed

optimized HPLC conditions are given in (Table 6).

Table 6. Optimized HPLC conditions for GMG

Parameter Conditions

Flow Rate 0.8 mL/min

Total run time 7 min

Mobile phase composition Solvent A: Water (with 0.1% formic acid and 0.3%

ammonia)

Solvent B: Methanol (with 0.1% formic acid and 0.3%

ammonia)

Gradient conditions Time (min) Solvent A

(%)

Solvent B

(%)

0 to 1 40 60

1 to 4.5 5 95

4.5 to 5.5 40 60

5.5 to 7 Equilibration period

Detector wavelength 210 nm

Application volume 10μL

Column oven temperature 40°C

Autosampler temperature 4°C

4.1.5. MASS (MS) Instrument

For HPLC–ESI–MS/MS analysis, a triple quadrupole mass spectrometer (API 4000;

Applied Biosystems/MDS SCIEX, CA, USA) in multiple reaction monitoring (MRM)

mode was used. The quantification of the analyte was performed using MRM

acquisition mode due to the high selectivity and sensitivity. Dual switch mode such as



negative and positive was used for GMG and withaferin A respectively. The detailed

optimized MS parameters are given in (Table 7).

Table 7. Optimized MS parameters for GMG and Withaferin A

Parameter Values

Gymnemagenin Withaferin A

Curtain gas (psi) 20 20

Ion source (V) -4500 5500

Ion source temperature (°C) 600 600

DP (V) -144 84

EP (V) -9 7

CE (V) -54 24

CXP (V) -22 5

MRM (m/z) transitions 505.40→455.4 471.40→281.4

DP: Declustering potential; EP: entrance potential; MRM: Multireaction monitoring

CE: Collision cell energy; CXP: Collision exit cell

4.1.5.1. Method validation

Method validation including linearity, limit of detection (LOD), limit of quantitation

(LOQ), accuracy and precision, was carried out as per ICH-Q2B guidelines (ICH,

1996)215

. The validation parameters used were as follows:

4.1.5.1.1. Linearity

Eight different concentrations from 5.280 to 305.920 ng/mL of standard solution were

run in triplicate and peak area of each concentration was determined. Linearity of the

method was determined by plotting calibration curve of mean peak area versus

concentration of the standard marker compound, GMG. Linear equation of line was

used for quantification of marker compound in G. sylvestre extract and its marketed

formulations. The correlation coefficient (r2) and slope of the line were calculated.

The marker content was expressed in % w/w.

4.1.5.1.2. Limit of detection and quantitation

Limit of detection (LOD) and limit of quantitation (LOQ) were calculated on the basis

of signal to noise ratios of 3:1 and 10:1 respectively. LOD and LOQ estimations were

based on three determinations for blank (methanol) and marker compound.



4.1.5.1.3. Intermediate precision (Intra and Inter-day) and repeatability

Intermediate precision (intra and inter-day) and repeatability were carried out on the

GYM-1 sample. Percentage relative standard deviation (% RSD) was considered as a

measure of precision and repeatability. The GYM-1 sample was prepared and

analysed for GMG concentration on the same day (n = 15) and on three consecutive

days (n = 5) for intra and inter-day precision respectively. For repeatability, the

GYM-1 sample was prepared on three different days and analysed for GMG content

(n =5).

4.1.5.1.4. Accuracy

Accuracy of the method was studied using a standard addition method. The GMG (at

100% concentration level) was added to GYM-1 and the sample was processed as per

sample preparation method described in the experimental section 4.1.3. Mean

percentage (%) recovery of GMG was used as a measure of accuracy and was

calculated using the formula;

4.1.6. Applicability studies

The validated method was applied for quantitative estimation of GMG in G. sylvestre

extract and its marketed formulations. The details of procured marketed formulations

are mentioned in section 4.1.1.

4.2. Isolation, purification and characterization of GMG from Gymnema sylvestre

extract

4.2.1. STEP 1: Hydrolysis of Gymnema sylvestre extract

Hydrolysis of G. sylvestre extract was carried out using the process of hydrolysis

mentioned in the monograph of G. sylvestre by Natural Remedies Pvt. Ltd, Bangalore

India. 100 g of G. sylvestre extract was dissolved in 1 L of 50% v/v ethanol and then

200 mL of 11%w/v KOH was added. The mixture was heated under reflux on heating

mantle for 1 h and cooled. To this solution conc. HCl was added to adjust the pH up

to 1 and heated under reflux for 1 h. After cooling, the pH was adjusted between 7.5-

8.5 using 11%w/v KOH solution (Figure 11).



Figure 11. Structure of reference standard gymnemagenin obtained after hydrolysis of gymnemic acids

4.2.1. STEP 2: HPTLC Finger printing: The HPTLC finger printing was developed

and optimized for identification of GMG in hydrolysed fraction of G. sylvestre

extract. HPTLC was performed on 10 cm × 10 cm aluminium backed plates coated

with silica gel GF 254 (Merck, Mumbai, India). Standard solution of GMG and

hydrolyzed extract were applied to the plates as bands of 8.0 mm wide, 30.0 mm

apart, and 10.0 mm from the bottom edge of the same chromatographic plate by using

Camag (Muttenz, Switzerland) Linomat IV sample applicator equipped with a 100-µL

Hamilton (USA) syringe. Ascending development to a distance of 80 mm was

performed at room temperature in a Camag glass twin-trough chamber previously

saturated with mobile phase vapour for 20 min. After development, the plates were

dried and scanned at 254 nm using a Camag TLC scanner with WINCAT software,

using the deuterium lamp. The optimized chromatographic conditions used are given

in (Table 8).

Table 8. Optimized HPTLC chromatographic conditions

Sr .no. Parameters Chromatographic conditions

1 HPTLC plate Silica gel GF 254 (10 x 10 cm with 0.2 mm

thickness

2 Mobile phase toluene: chloroform: methanol; 5:8:3 v/v/v

3 Temperature 28 ± 2°C

4 Detection wavelength 254 nm

5 Application volume 10 µl

4.2.2. Isolation and Purification of GMG from hydrolyzed fraction

The solution from step 1 was evaporated under reduced pressure and filtered to obtain

the sticky dark green solid residue. The solid residue was dried and defatted with 1 L

of toluene under reflux for 30 min and filtered. The obtained residue was dried and



powdered. The powdered dry mass was further refluxed in 1 L of toluene under

vigorous stirring for 30 min and cooled. The green suspension was filtered and the

residue was dried to obtain solid mass. The process was repeated 5-6 times until the

filterate becomes colorless. The solid mass obtained was subjected to reflux in 500

mL of 5% methanol (MeOH) in dichloromethane (DCM) under vigorous stirring for

30 min and filtered hot. The process was repeated with the remaining solid mass 5-6

times until no trace of aglycone moiety in the solid was visible on TLC. All the

filtrates were combined and evaporated to dryness to obtain a pale green color

powder. The obtained powder was further refluxed with toluene under vigorous

stirring for 45 min, cooled, filtered and dried. The dried powder was re-dissolved in

50% MeOH:DCM and activated charcoal was added to it. The charcoal suspension

was boiled and filtered over a celite bed. The obtained filtrate was further subjected to

decolourization and finally concentrated to dryness to obtain a buff color solid mass.

The obtained solid was recrystalized using 5% MeOH:DCM. The obtained solid mass

was subjected to melting point (m.p), log P value determination, and characterization

using analytical methods like U.V., FTIR, HPTLC, elemental (C,H,N) analysis, 1D,

2D-NMR, Mass, HPLC-MS/MS.

4.2.3. Characterization of isolated GMG

4.2.3.1. Melting Point was determined in open capillaries using Veego VMP-1

melting point apparatus and expressed in °C.

4.2.3.2. Ultraviolet-Visible (UV) spectrum: The UV-visible spectrum was recorded

for the determination of λmax in MeOH (1mg/mL) using Shimadzu UV-1700 model

(version 1.7) with 1cm quartz cuvettes.

4.2.3.3. Determination of partition coefficient (Log P) value of GMG in

octanol:water by U.V. spectrophotometer216

4.2.3.3.1. Pre-saturation of solvents

The two solvents, n-octanol (AR Grade, Merck, India) and double distilled water were

mutually saturated by shaking with each other at room temperature on a mechanical

shaker for 24 h and were allowed to stand enough to achieve a saturation state.



4.2.3.3.2. Preparation of stock solution and dilution

The stock solution of GMG was prepared by dissolving 10 mg in 10 mL of AR grade

DMSO. 0.5 mL of this stock solution was further diluted to 50 mL with presaturated

solution of n-octanol to get the concentration of 10 μg/mL of GMG.

4.2.3.3.3. Partition co-efficient (Log P) determination

The λmax of GMG was determined in n-octanol and the absorbance at that wavelength

was noted (BE-absorbance before extraction). 20 mL of the GMG solution (10

μg/mL) in n-octanol and 20 mL of double distilled water were added to 250 mL

iodine flask which was then kept on mechanical shaker for 24 h to create endogenous

environment. After 24 h, the mixture was transferred to a separating funnel and was

kept without disturbance for separation of phases. The absorbance of the separated n-

octanol phase was then measured at the λmax specific for GMG (AE-absorbance after

extraction). The experiment was performed in triplicate. Log P value of GMG was

calculated by using following formulae;

Log P= BE/BE-AE

4.2.3.4. HPTLC of isolated GMG: Carried out as per the procedure mentioned in

section 4.2.2.

4.2.3.5. Fourier Transform Infrared spectroscopy (FTIR): FTIR spectrum of

GMG was recorded on Perkin Elmer Spectrum RX1 FTIR spectrophotometer using

KBr pellet method.

4.2.3.6. Elemental Analysis: Elemental composition of C, H and N were analysed by

using elemental analyser (Perkin Elmer, 2400 series, CHNOS analyser) at the

Department of Organic Chemistry, IISc, Bangalore, India. Accurately weighed

sample was heated to 1150°C and the corresponding C:H:N ratio was determined by

using a thermal conductivity detector.

4.2.3.7. Mass spectroscopy (MS): Isolated compound was characterized using Time

of Flight (TOF) analyser assisted mass spectrometer in positive ionization mode.

4.2.3.8. HPLC-ESI MS/MS analysis for purity determination: Percentage purity of

isolated compound was determined by comparing the peak area with that of reference



standard in HPLC-ESI-MS/MS analysis. HPLC-ESI-MS/MS was carried out as

described in section 4.1.5.

4.2.3.9. Nuclear Magnetic Resonance spectroscopy: 1H,

13C (1D) and COSY (2D)

NMR analysis were carried out for isolated GMG. NMR spectra of the sample was

recorded on a Bruker DPX 300 NMR spectrometer using tetramethylsilane (TMS) as

an internal standard and DMSO-d6 as solvent at 25°C in the Department of Organic

Chemistry, University of Pune, India.

4.3. Evaluation of acute oral toxicity of Gymnema sylvestre extract and GMG as

per OECD 423 guidelines217

The study was conducted as per OECD guidelines after necessary permission from

institutional ethics committee (JSSCP/IAEC/PH.COG/PH.D/04/2011-12). Wistar

(male) rats in weight range 180-200 gm were used in this study. The animals were

housed in standard conditions of temperature (22 ± 5°C), humidity (55 ± 15%) and in

12 h light-dark cycles. They were fed on conventional laboratory pelleted diet and

water ad libitum. The experiment was performed to evaluate the toxic effect, if any,

produced by G. sylvestre extract and GMG. Rats were fasted for 16-20 h and

randomly divided into 5 groups, each group containing five rats (Table 9).

Table 9. Grouping of animals for acute oral toxicity study

Sr. no Name of the substance Dose (mg/kg) Group

1. G. sylvestre extract 2000 Group I

2. G. sylvestre extract 5000 Group II

3. GMG 2000 Group III

4. GMG 5000 Group IV

5. Control 0.5%w/v CMC Group V

5 animals in each group

4.3.1. Preparation and administration of GMG and G. sylvestre extract

G. sylvestre extract and GMG suspension, prepared in 0.5%w/v solution of carboxy

methyl cellulose (CMC), were administered orally to the respective groups. The

control group was administered only 0.5%w/v CMC (1 mL/kg b.w). The test doses

were administered orally via gastric intubation with ball-tipped oral dosing needles

that were affixed to the appropriate size syringes. Animals were returned to feed after

3-4 h of dosing. The animals were observed for the following toxicity parameters:



4.3.1.1. Mortality

All animals in safety study were observed for their mortality at 30 min, 1, 3, 4 h post

administration of test agent dose on day 0. Animals were observed twice daily

(morning as well as afternoon) thereafter for 14 days.

4.3.1.2. Cage side observations

Animals were observed at approximately 30 min, 1, 3, 4 h post dose on day 0 and

twice daily (morning and afternoon) thereafter for 14 days. The parameters studied

for cage side observations were effect on body condition, locomotor activity, posture,

presence of any discharge through natural orifice/eyes, ears etc, effect on mucous

membranes and excretion activity (change in urine color and consistency of stools).

4.3.1.3. Body weight

Body weight of animals were recorded on 30 min, 1, 2, 3, 4 h post administration of

test material on day 1 and later on day 7 and 14.

4.3.1.4. Haematological investigations

At the day of termination, all animals in the safety study were bleeded for

hematological investigations; hemoglobin (Hb), white blood cells (WBCs), red blood

cells (RBCs), platelet percentage of neutrophils (N), eosinophil (E), basophils (B),

lymphocytes (L) and monocytes (M) were counted.

4.3.1.5. Histopathological findings

Histopathology was carried out under the guidance of consultant pathologist (Dr.

Amol Harshe, Plus Laboratories, Nobel Hospital, Pune, India). Upon termination, all

animals in the safety study were sacrificed and organ systems such as liver, kidney,

and pancreas were isolated, weighed and all the organs were immediately fixed in

10% formalin and were sent for histopathological studies.

4.4. Determination of absolute bioavailability of gymnemagenin (GMG) after

oral and iv administration

4.4.1. Experimental conditions

Wistar (male) rats in weight range 220-250 gm were used in this study. The animals

were housed in standard conditions of temperature (22 ± 5°C), humidity (55 ± 15%)

and in 12 h light-dark cycles. They were fed on conventional laboratory pelleted diet



and water ad libitum. The animals were randomly divided into two different groups

containing five animals in each group (Table 10). All the procedures were performed

as per the guidelines of the Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Animal Welfare Division,

Government of India, New Delhi, and was approved by the Institutional Animal

Ethics Committee of J.S.S College of Pharmacy, Udhagamandalam

(JSSCP/IAEC/PH.COG/PH.D/04/2011-12).

Table 10. Different study groups used in bioavailability experiment

Group Name of the

substance

Dose

(mg/kg)

Route

Group I GMG 100 Oral

Group II GMG 100 Intravenous


4.4.2. Collection of blood samples

Animals were fasted overnight (16 h fasting with free access to water) prior to the

administration of GMG (dissolved in 1 mL of 1% DMSO) to the respective groups.

Blood samples were collected at predosing 0 min and after oral administration at 0.5,

1, 2, 4, 6, 8, 12 and 24 h. and for iv, samples were collected at 0, 0.167, 0.333, 0.667,

1, 2, 3, 4, 5 and 6 h respectively. Blood samples were withdrawn from the animals by

retro orbital puncture under light ether anaesthesia (0.4 - 0.5 ml) in EDTA vaccutainer

(3 mL, BD Biosciences).

4.4.3. Separation of plasma

Blood samples were centrifuged at 7000 g for 15 min at 4°C to obtain the plasma and

were stored at -70 °C until analysis.

4.4.4. Sample preparation

An aliquot of 150 µL of plasma and 20µL of internal standard (150 ng/mL of

Withaferin-A) were mixed and vortexed for 30 sec to which 2 ml of Tetra-Butyl

Methyl Ether (TBME) was added and vortexed for another 6 minutes at high speed.

The solution was centrifuged for 6 minutes at 6500 rpm. The organic layer was

transferred and evaporated to dryness under nitrogen vacuum concentrator. The

residue obtained was reconstituted with 150uL of mobile phase (MeoH:Water, 60:40



v/v) and transferred to the injection vials. 20 µL of each sample was injected into

HPLC-MS/MS system.

4.4.5. Analysis of samples

HPLC-ESI-MS/MS method was used for quantitative analysis of GMG in various

plasma samples using multiple reactions monitoring mode (MRM). The method was

validated as per USFDA guidelines on bio-analytical method validation218

. Withaferin

A was used as internal standard.

4.4.6. Study parameters

Pharmacokinetic parameters such as area under the plasma concentration-time curve

(AUC0-t), terminal elimination half-life (t1/2) and systemic clearance (CL) were

calculated using a non-compartmental analysis with the help of software WinNonlin

version 3.0 (Pharmasight corporation, Mountain view, CA). The maximum plasma

concentration (Cmax) and the time (Tmax) to reach Cmax were directly read from each

plasma concentration-time plot.

4.4.7. Method validation

The developed method was then validated for selectivity, linearity, accuracy,

precision, recovery and stability.

4.4.7.1. Selectivity

Selectivity was ascertained by analyzing six blank rat plasma samples without adding

IS to determine the interference with the analyte.

4.4.7.2. Linearity

Three sets of calibration curve ranging from 5.820 - 305.920 ng/ml of GMG was

constructed by plotting the peak area ratio of the analyte/IS versus analyte

concentration in blank rat plasma. Lower limit of quantification (LLOQ) was

measured for GMG as the lowest analyte concentration (S/N=10), which can be

determined with an accuracy and precision <20%.



4.4.7.3. Precision and accuracy

For the evaluation of precision (intra and inter-day) and accuracy of the method, five

replicates of four different QC samples (18.32, 64.76, 163.33 and 214.90 ng/mL) were

analyzed on three different days. Accuracy was calculated using the following

equation: [(mean measured concentration / nominal concentration)] ×100. Percentage

coefficient of variation (% CV) was used as measure of precision of the method. CV

less than 15% and accuracy within ±15% were accepted.

4.4.7.4. Recovery

Extraction recovery (relative recovery) of analytes from the rat plasma after the

extraction procedure was assessed in three quality control (QC) samples (18.32,

163.33 and 214.90 ng/mL). The detector response (peak area) of processed QC

samples (R2) was compared with the response of directly injected aqueous QC

samples (R1). Extraction recovery was calculated as follows;

recovery (%) = (R2/R1) x 100.

4.4.7.5. Stability study

Two different QC samples in triplicate at low (18.32 ng/mL) and high (214.90 ng/mL)

concentration level were prepared. The prepared samples were subjected for the

evaluation of three freeze/thaw cycle stability and room temperature storage stability

(6 h). Post preparative stability was evaluated by re-injection of the QC samples after

12 h of storage under auto-sampler conditions.

The validated method was used for the determination of absolute bioavailability using

the following formula.

D: Dose administered through oral and iv route

Section4.5. Development, optimization and characterization of Gymnema

sylvestre extract and GMG loaded polymeric nanoparticles

4.5.1. Preformulation studies: These studies have been performed to understand the

underlying variables and their relationship to performance.



a. Melting point (m.p) determination: The melting points of all the components

used in this study were measured by the use of open capillary tube in Veego VMP-1

melting point apparatus expressed in °C.

b. Solubility study: Solubility of G. sylvestre extract was determined using orbitary

shaker (IKA, KS4000, icontrol, Chennai, India) at 37 ± 0.5 °C for 48 h in various

organic solvents such as chloroform, methanol, ethanol, di-ethyl ether, and DMSO.

Aqueous phase solubility study was carried out in phosphate buffer solution of pH

1.2, 4.0, 6.8, and 7.4.

c. Selection of analytical wavelength: G. sylvestre extract solution (100 μg/mL) was

prepared in phosphate buffer solution (PBS) of pH 7.4. The solution of G. sylvestre

extract was scanned from 200 to 400 nm in an UV visible spectrophotometer and the

spectrum was recorded. The λmax of G. sylvestre extract was measured against

phosphate buffer solution of pH 7.4 as a blank. The analytical wavelength (λmax)

determination of GMG in methanol has been already discussed in section 4.2.3.2.

d. Development of calibration curve: Stock solutions of 1 mg/mL of GMG and G.

sylvestre extract were prepared by dissolving 10 mg of GMG and G. sylvestre extract

in 10 mL of methanol and phosphate buffer solution (pH 7.4) respectively. From the

stock solution, a range of 10 to 100 μg /mL dilutions were prepared. The U.V.

absorbance of all the samples was measured at their respective λmax and standard

calibration curves were developed for GMG and G. sylvestre extract. The slope (k)

and the intercept (B) values were calculated from the standard plot.

e. Compatibility study of G. sylvestre extract and GMG with other excipients by

the FTIR and Differential Scanning Calorimeter (DSC)

i. FTIR: Infrared (IR) spectral matching approach was employed to detect any

possible chemical interaction of GMG and G. sylvestre extract individually with other

formulation excipients. The physical mixtures under study were; GMG: chitosan(1:1),

GMG:PLGA(1:1), G. sylvestre extract:chitosan (1:1) and G.sylvestre extract:PLGA

(1:1). The samples of physical mixtures were scanned from 4000 to 400 cm-1

using

Shimadzu FTIR spectrophotometer 8400S. Similarly, the individual IR spectra of G.



sylvestre and the other excipients were also recorded. I.R analysis of GMG has

already been discussed in section 4.2.3.5.

ii. Differential scanning calorimetric (DSC): DSC (Water Q200, Bangalore, India)

analysis was performed to study the thermal behaviour of GMG, G. sylvestre extract,

polymers (chitosan and PLGA) and their physical mixtures. Samples were crimped in

standard aluminium pans, sealed and heated from 20-400°C at a heating rate of

10°C/min under constant purging of dry nitrogen at a rate of 30 mL/min. An empty

pan sealed in the same way was used as a reference. DSC thermograms were obtained

using an automatic thermal analyzer system. The temperature calibration was

performed using Indium calibration reference standard.

4.5.2. Nanoformulation Studies

Chemicals and Solvents

Chitosan (viscosity average molecular weight 20 kDa, degree of N-deacetylation (75–

80%) was received as gift sample from Ranbaxy Research Laboratory, Gurgaon,

Harayana, India [(ANNEXURE I (c)]. Tripolyphosphate (TPP), Polyvinyl alcohol

(PVA), Poly(dl-lactide-co-glycolide) (PLGA), were procured from Sigma-Aldrich,

Bangalore, India. All other chemicals were of analytical grade.

Nanoparticles of GMG and G. sylvestre extract were prepared using two

different methods which are described below;

4.5.2.1. Method I

Nanoparticles were prepared by ionic cross-linking method using TPP219

. GMG

(dissolved in methanol) and chitosan (dissolved in 0.1% acetic acid) were mixed and

vigorously stirred for 30 min at 1500 rpm. Further, the TPP solution was added drop

wise to the mixture and was stirred for 3 h at 6000 rpm. The formed nanoparticle

suspension was centrifuged at 12,000 rpm for 45 min and the collected residue was

re-dispersed in double distilled water. G. sylvestre extract nanoparticles were prepared

using the same technique by dissolving it in phosphate buffer solution pH of 7.4.

Multiple batches were prepared by various drug: polymer:TPP ratios (Table 11 and

12) in order to optimize the process.



Table 11. Different ratios of GMG:chitosan:TPP evaluated for nano-formulation

Table 12. Different ratios of G. sylveste extract:chitosan:TPP evaluated for nano-formulation

4.5.2.2. Method II

Nanoprecipitation technique was used to prepare the nanoparticles of GMG and G.

sylvestre extract220

. Different ratios of GMG and G. sylvestre extract with polymer

were tried to prepare the nanoparticles (Table 13 and 14). Each of GMG in PLGA

and G. sylvestre extract in PLGA in different ratios were dissolved in 5 mL of acetone

and 5mL methanol, respectively, at room temperature. The resulting solution was

continuously added into 20 mL of water containing 0.5-1.5 % w/v polyvinyl alcohol

(PVA) with continuous magnetic stirring at 5000 rpm. The organic solvent was

evaporated and the final volume of suspension was collected. The formed

nanoparticle suspension was centrifuged at 12,000 rpm for 45 min and the collected

residue was re-dispersed in double distilled water. 0.5% w/v mannitol was added as a

cryoprotectant and lyophilized at -70 °C to obtain free flowing powder.

Table 13. Different ratios of GMG: PLGA evaluated for nano-formulation

Table 14. Different ratios of G. sylvestre:PLGA evaluated for nano-formulation

Sr. No GMG: Polymer ratio Quantity of TPP added Batch Label

1. 1:1 100 µL Sample A

2. 1:2 150 µL Sample B

3. 2:1 200 µL Sample C

Sr.

No

G. sylvestre extract :

Polymer ratio

Quantity of TPP added Batch Label

1. 1:1 100 µL Sample 1

2. 1:2 150 µL Sample 2

3. 2:1 300 µL Sample 3

4. 2:4 200 µL Sample 4

5. 1:5 300 µL Sample 5

Sr. No GMG: Polymer ratio Quantity of PVA added Batch Label

1. 1:5 1.5% w/v GMG Nano A

2. 1:3 1.0% w/v GMG Nano B

3. 1:2 0.5% w/v GMG Nano C

Sr.

No

G.sylvestre extract:

Polymer ratio

Quantity of PVA added Batch Label

1. 1:5 1.5% w/v EXT Nano 1

2. 1:3 1.0% w/v EXT Nano 2

3. 1:2 0.5% w/v EXT Nano 3



4.5.3. Physicochemical evaluation of nanoparticles

The particle size, zeta potential, SEM and TEM studies were carried out at Dr. Bhat’s

“Polymer characterization Research Laboratory” at National Chemical Laboratory

(NCL), Pune, Maharashtra, India.

4.5.3.1. Particle size and zeta potential

Particle size and zeta potential of the nanoparticles were measured by Dynamic Light

scattering system spectroscopy using Brookhaven Instruments Corp. PALS Zeta

Potential Analyzer Ver. 3.54.

4.5.3.2. Surface morphology of nanoparticles using SEM & TEM analysis

External morphology of nano particles was determined using Scanning Electron

Microscopy (SEM) and Transmission Electron Microscopy (TEM). The lyophillized

nanoparticle samples were subjected to SEM analysis. The samples were spread on a

sample holder and dried using vacuum. They were subsequently coated with gold

(JFC 1200 fine coater, Japan) and examined by a Scanning Electron Microscope

(SEM). The TEM (Philips CM-10, USA) samples were dropped on to formvar-coated

copper grids. After drying, the samples were stained using 2% w/v phosphotungistic

acid. Digital micrograph and soft imaging viewer software was used to perform the

image capture.

4.5.3.3. Entrapment efficiency

Entrapment efficiency of nanoparticles was determined by centrifugation method.

Prepared nanoparticles of GMG and G. sylvestre extract were dispersed in double

distilled water and were subjected to centrifugation on cryocentrifugation (Remi R4C)

at 13000 rpm for a period of 45 min. The clear supernatant was removed carefully to

separate non-entrapped drug and absorbance was recorded. The sediment in the

centrifugation tube was diluted to 100 ml with suitable solvent and the absorbance of

this solution was recorded at their respective λmax. Entrapment efficiency was

calculated using the following formula;



4.5.3.4. In vitro release studies

The in vitro release of developed nanoparticles was carried out by dialysis membrane

method (Figure 12). GMG and G.sylvestre extract loaded PLGA nanoparticles

(equivalent to 5 mg of GMG, G.sylvestre extract) were re-dispersed in 5 mL of

demineralized water in dialysis bags (Sigma aldrich) with a molecular cut off of 12

kDa. The bag was suspended in 100 mL of release medium (phosphate buffer solution

pH 7.4 for G. sylvestre extract and 50% v/v methanol for GMG) at 37.5 °C). The

medium was stirred by using the magnetic stirrer at 40±10 rpm. At each suitable

time interval 5 mL of sample was withdrawn and the same volume of medium was

replaced to maintain the sink condition. Finally, the samples were analyzed by U.V.

spectrophotometer using PBS pH 7.4 as blank for G. sylvestre extract formulation and

methanol for GMG formulation respectively. Methanol was used in release medium to

provide sink conditions as GMG is poorly soluble in water221

.

Figure 12. Schematic representation of Dialysis membrane apparatus for in vitro release

4.5.3.5. Stability studies

A stability study of nanoparticle formulations was carried out as per ICH guidelines.

The nanoparticle formulations were stored in refrigerator, at 4°C and 25°C

respectively for 3 months. The change in the appearance, color and drug content were

monitored at predetermined time intervals of 0, 30, 60, 90th

day.



4.6. Evaluation of comparative bioavailability of developed polymeric

nanoformulations with GMG and G. Sylvestre extract

4.6.1. Animals

Wistar (male) rats in the weight range 220-250 g were used in the present study. The

animals were housed in standard conditions of temperature (22 ± 50C) and humidity

(55 ± 15%) and 12 hrs light–dark cycles. They were fed on conventional laboratory

pelleted diet and water ad libitum. The animals were randomly divided into two

groups (Table 15). All the procedures were performed as per the guidelines of the

Committee for the Purpose of Control and Supervision of Experiments on Animals

(CPCSEA), Ministry of Animal Welfare Division, Government of India, New Delhi,

and was approved by the Institutional Animal Ethical Committee of J.S.S College of

Pharmacy (JSSCP/IAEC/PH.COG/PH.D/04/2011-12), Udhagamandalam.

Table 15. Grouping of animals for BA studies of GMG and G. sylvestre extract nano-formulation

5 animals in each group; NP: Nanoparticles

4.6.2. Dosing of animals and blood sample collection

Animals were fasted overnight (16 h fasting with free access to water) prior to

administration of developed formulations in their respective groups. After oral

administration of the formulation, blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8,

12 and 24 h time intervals. Blood samples were withdrawn from the animals by retro

orbital puncture under light ether anasthesia (0.4-0.5 mL) in EDTA vaccutainer (3

mL, BD Biosciences).

4.6.3. Separation and collection of plasma

Blood samples were centrifuged at 7000 g for 15 min at 4°C to obtain the plasma

which were stored at -70 °C until analysis.

Sr. No Group Coding Oral dose (mg/kg)

1. I NP GMG-100 100

2. II G. sylvestre-400 400

3. III NP G. sylvestre-400 400



4.6.4. Statistical analysis

Pharmacokinetic parameters such as area under the curve from time zero to last

interval (AUC0-t), clearance (CL) and terminal elimination half life (t1/2) were

calculated from mean plasma concentration versus time graph. Peak plasma

concentration (Cmax) and Tmax were directly read from each plasma concentration-time

plot. All the values were expressed as mean ± SDs. Pharmacokinetic parameters after

parent extracts/GMG administration were compared with pharmacokinetic parameters

obtained after administration of their nanoformulations using paired t-test. P value

≤0.05 was considered as level of significance.

Section 4.7. Preparation and characterization of sodium and potassium salts of

isolated GMG222

A two neck round bottom flask (RBF) attached to a reflux condenser was charged

with ethanol (20 mL), GMG (1 mmol) and NaOH and/or KOH (6 mmol). The

reaction mixture was kept under vigorous, constant stirring at 85 °C for 1 h and the

solvent was evaporated under reduced pressure to obtain the salts of GMG (Figure

13). The obtained salts were evaluated for their melting point and characterized using

FTIR, NMR, Mass, atomic absorption spectrometry (AAS) and water solubility.

FTIR, NMR and Mass were carried out as per the section 4.2.3.

HO

HO

H

H

OHH

OH

OH

OH

6m/e NaOH

ethanolreflux, 1-2hr

O

O

H

H

OH

O

ONa

O

Na

Na

Na

Na

Na

HO

HO

H

H

OHH

OH

OH

OH

6m/e KOH

ethanolreflux, 1-2hr

O

O

H

H

OKH

O

OK

O

K

K

K

K

Na-GMG

K-GMG

Figure 13. Synthesis of Na-GMG and K-GMG salts



4.7.1. Atomic absorption spectrometer for determination of sodium and

potassium223

Reagents and Instrument

The ultrapure water collected from milliQ system was used to carry out AAS. Nitric

acid (HNO3) and hydrogen peroxide (H2O2) were of analytical grade (Merck). Metal

standard solutions [Sigma Aldrich] were prepared by appropriate dilutions. The

atomic absorption measurements were performed with a Shimadzu model AA 6300

flame atomic absorption spectrometer (Tokyo, Japan) equipped with a deuterium

background corrector.

Procedure

The number of Na+ and K

+ atoms corresponding to each form of salt was determined

taking into account the molecular mass (MM) of each GMG salt derivative (638.3g

and 740.36g respectively). Then, 25 mg of each salt was separately added to 4 mL of

conc. HNO3 and subjected to microwave irradiation [900 W, 100°C] for 90 sec. The

samples were cooled and 3 mL of 30 % v/v hydrogen peroxide was added for further

oxidation of the digested organic material and subjected to microwave irradiation for

90 sec. The samples were cooled and further 3 mL of 30 % v/v hydrogen peroxide

was added to ensure complete oxidation and irradiated for 90 sec. The digested

samples were cooled, filtered, made up the volume to 25 mL with ultrapure water and

subjected to analysis.

The number of Na or K atoms in individual salts was then calculated as per the under

mentioned formulae;

1.

2.

3.



4.8. In vivo anti-hyperglycemic evaluation of isolated GMG, its water soluble

salts and developed nano-formulations in comparison with G. sylvestre extract224

4.8.1. Animals

Wistar rats (male) in the weight range 220-250 g were used in this study. The animals

were housed in standard conditions of temperature (22 ± 5 °C) and humidity (55 ±

15%) and 12 h light–dark cycles. They were fed on conventional laboratory pelleted

diet and water ad libitum. The animals were randomly divided into different groups

(Table 16), each group containing five animals. All the procedures were performed as

per the guidelines of the Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Animal Welfare Division,

Government of India, New Delhi, and was approved by the Institutional Animal

Ethics Committee of JSS College of Pharmacy. (JSSCP/IAEC/PH.COG/PH.D/04/

2011/12),Udhagamandalam.

Table 16. Different study groups used in antidiabetic experiment

Sr. No Group no Coding Oral dose

(mg/kg)

1. I Normal control (NC) Vehicle

2. II Diabetic control (DC) Vehicle

3. III MET-200 200

4. IV GMG-50 50

5. V GMG-100 100

6. VI G. sylvestre ext. 400 400

7. VII G. sylvestre extract Nano 400 Eq. to 400

8. VIII GMG Nano Eq. to 100

9. IX Na-GMG Eq. to 100

10. X K-GMG Eq. to 100


4.8.2. Experimental induction of Diabetes

Streptozotocin (STZ) was purchased from S.R.L. Ltd., Mumbai, India. All other

chemicals used in this study were of analytical grade. Metformin (MET) along with

certificate of analysis was provided as gift samples from Franco Indian

Pharmaceuticals Pvt Ltd. MumbaI India [(ANNEXURE 1(d))]. Diagnostic kits for



cholesterol and triglyceride estimation were purchased from Biolab diagnostic (I) Pvt.

Ltd, Mumbai, India.

Diabetes mellitus was induced by single intraperitoneal injection of freshly prepared

STZ (40 mg/kg bw) in 0.1M citrate buffer (pH 4.5) in a volume of 1 mL/kg bw.

Diabetes was established in these STZ treated rats over a period of 4 days. After 4

days, the plasma glucose level of each rat was determined by tail vein puncture

method using one touch simple select glucometer (Johnson & Johnson, Mumbai,

India). Rats with a fasting plasma glucose range between 200-300 mg/dL were

considered as diabetic.

4.8.3. Dosing of animals

All the groups were treated with their respective drug, dosing orally once in a day in

the morning.

4.8.4. Parameters studied

Physical Parameters

Body weight of all experimental animals were recorded from each group on day 0, 7,

14, 21, 28th

day using digital weighing scale.

4.8.4.1. Biochemical parameters

Every week, following overnight fasting (12 h fasting with free access to water) the

blood samples were withdrawn from the animals by tail vein puncture method. Blood

glucose concentration was measured by Glucometer (One touch simple select,

Johnson & Johnson Mumbai India).

4.8.4.2. Estimation of plasma total cholesterol (TC)

The estimation of plasma TC was carried out using cholesterol reagent diagnostic kit

by UV spectrophotometer method.

Principle: In hot acidic medium, cholesterol oxidizes ferric ion to a brown colored

complex which absorbs at 530 nm and its intensity is directly proportional to

cholesterol concentration.

The plasma concentration of TC (mg/dL) was calculated using following formula;



Cu = Au/As x Cs

Cu = Conc. of cholesterol in plasma sample; Au = Absorbance of sample; As = Absorbance of

standard; Cs = Conc. of standard (200 mg/dL)

4.8.4.3. Estimation of plasma triglycerides (TG)

The estimation of plasma triglycerides was carried out using triglyceride cholesterol

reagent diagnostic kit by UV spectrophotometer method.

Principle: The assay carried out using the GPO-Trinder method, end point meant for

in vitro diagnostic use only.

The reaction is as follows:

Triglyceride + H2O2 Lipase

Glycerol + Free fatty acids

Glycerol + ATP GK

Glycerol-3-Phosphate + ADP

Glycerol-3-Phosphate + O2 GPO

DAP + H2O2

H2O2 + 4AAP + 3,5-DHBS Peroxidase

Quinoneimine dye + 2H2O

(GK – Glycerol kinase, GPO – Glycerol phosphate oxidase, DAP – Dihydroxyacetone phosphate, ATP

– Adenosine triphosphate, 4-AAP – 4 Aminoantipyrine, DHBS – 3, 5- Dichloro-2-hydroxybenzene

sulfonate)

The intensity of chromogen (Quinoneimine) formed is proportional to the

triglycerides concentration in the sample when measured at 510 nm (500-540 nm).

The plasma concentration of TG (mg/dL) was calculated using following formula;

Cu = Au/As x Cs

Cu = Conc. of triglycerides in plasma sample; Au = Absorbance of sample; As = Absorbance of

standard; Cs = Conc. of standard (200 mg/dL)

4.8.4.4. Estimation of glycosylated haemoglobin (HbA1c)

Serum samples were sent to Plus pathology laboratory of Nobel hospital Pune, India

for estimation glycosylated haemoglobin (HbA1c) by ion exchange resin method.

4.8.4.5. Histopathology of pancreas

Pancreas histopathology was carried out on selected study groups on 14th

and 28th

day

under the guidance of Dr. Amol Harshe from Plus pathology laboratory of Nobel

Hospital Pune, India. Pancreas of rats were removed immediately from the animals

after sacrificing and rinsed with ice-cold saline. The tissue samples were fixed with

10% formaldehyde, dehydrated in a graded series of ethanol and embedded in paraffin



wax before sectioning. Then the paraffin sections were cut into sections (about 5μm

thickness) dewaxed and rehydrated. The sections were then stained in haematoxylin

and eosin (H & E). The photomicrographs of the each tissue section were observed

using imaging software for laboratory microscopy (Olympus, Tokyo, Japan).

Pathological grading was done on the basis of extent of necrosis of islet cells

(ANNEXURE IIb).

4.8.4.6. Statistical analysis

Study data was expressed as mean SEM. Statistical analysis was carried out by

One-way ANOVA with post hoc Tukey’s test using Graph Pad Instat software

(version3, San Diego, California, USA). P < 0.05 was considered as level of

significance.

Section 4.9. In vitro cytotoxicity evaluation of GMG using MTT assay225

4.9.1. Principle

Measurement of cell viability and proliferation forms the basis for numerous in vitro

anticancer assays of cell population’s response to external factors. Reduction of

tetrazolium salts is now widely accepted as a reliable way to examine cell

proliferation. The yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) is reduced by metabolically active cells, in part by the action of

dehydrogenase enzymes, with the use of the reducing equivalents such as NADH and

NADPH. The resulting intracellular purple formazan can be solubilised and quantified

by spectrophotometric means (Figure 14). The formazan product has a low aqueous

solubility and is present as purple crystals. Dissolving the resulting formazan with

proper detergents such as DMSO permits the convenient quantification of product

formation. The intensity of product color, measured at 550-590 nm, is directly

proportional to the number of living cells in the culture.

N N

N

N

S

N

NN

NH

N

S N

mitochondrial reductase

MTT Formazan

Figure 14. Reduction of MTT into formazan crystals by mitochondrial dehydrogenase



4.9.2. Maintenance of cell lines

The cell lines were maintained using Eagle’s minimum essential medium (MEM)

supplemented with 10% Fetal bovine serum (FBS) and 50 μg/mL gentamycin

sulphate at 37°C in CO2 incubator in an atmosphere of humidified 5% CO2. The cells

were maintained by routine sub culturing in 25 cm2 tissue culture flasks.

4.9.3. Sub culturing process of cell lines

The culture media from the flasks containing monolayer culture was aspirated

and washed with sterile phosphate buffered saline (PBS).

To the flasks, 2 mL of 0.1% trypsin-EDTA solution was added and after few

seconds it was aspirated and flask was kept in incubator for 2-3 minutes for

cell detachment.

The flasks were removed from the incubator and the cell detachment was

confirmed by observing under an inverted microscope (Olympus).

Once the cells were completely detached from the flasks, 2-3 mL of MEM

media containing 10% FBS was added and mixed well.

Cell viability was checked with a small sample of the suspension by tryptan

blue dye exclusion test.

From the stock cell suspension, 1x104 viable cells/mL suspended in media

were seeded in 25 cm2 tissue culture flask containing about 4 mL of fresh

media and incubated until the flasks attained 60-70% confluence.

4.9.4. Preservation of the cells

Tumor cells from the first and second passage of transplantation were stored in a

liquid nitrogen in cryovials, at a concentration of 1x106 cells/mL containing

respective media supplemented with 20% serum and 10% DMSO as a preservative.

This constitutes the tumor bank. After every ten passages, the tumor cells were

discarded and new passage was started using the original tumor cells from the tumor

bank.

4.9.5. Trypsinisation

To obtain a single cell suspension from monolayer culture, cells were dislodged from

culture flasks by trypsinisation.



From a 60-70% confluent flask, the culture media was aspirated out using

micropipette and transferred to the culture flask.

Cells were washed with 3 mL of PBS to remove trace amount of media.

To each culture flask 2 mL of trypsin-EDTA was added and after 20 sec it was

aspirated and the flask was kept in the incubator for 3-4 min for cell detachment.

Culture flasks were observed under an inverted microscope to ensure that the

cells were completely dislodged.

Trypsin activity was stopped by adding 2-3 mL media containing 10% FBS.

4.9.6. In vitro cell proliferation by MTT assay method

10 mg of GMG was dissolved in 1 mL of distilled dimethyl sulphoxide (DMSO) and

volume was made up to 10 mL with maintenance medium to obtain a stock solution

of 1 mg/mL concentration, sterilized by filtration and further dilutions from 50-1000

μg/mL were made from the stock. The cytotoxicity assays were carried out using 0.1

mL of cell suspension, containing 10,000 cells/well of a 96 well microtitre plate.

Fresh medium containing different concentrations of the test compound was added

after 24 h of partial monolayer. Control cells were incubated without test compound

and with MEM. The microtitre plates were incubated at 37ºC in a humidified

incubator with 5% CO2 for a period of 72h. Four wells were used for each

concentration of the compound. The morphology of the cells was inspected daily and

observed for microscopically detectable alterations. The 50 percent cytotoxic

concentration (CTC50) was determined by the standard MTT assay. 20 µL of MTT

solution (2 mg/mL in PBS) was added to the plates and was incubated for 4 h at 37

ºC. MTT-formazon crystals formed were dissolved in 100 µL of DMSO and optical

density was read with a microtitre plate reader (Biorad) at 570 nm. The percentage

cytotoxicity of GMG at various concentrations was calculated as follows;

% Cytotoxicity =

(Control – Blank) – (Test – Blank)

X 100 (Control – Blank)



4.10. Herb-drug interaction study of G. sylvestre extract with selected

conventional drug, Glimepiride

4.10.1. In silico studies and computer aided molecular modelling226

The in silico molecular modelling studies were carried out on Windows 7 workstation

(J.S.S College of Pharmacy, Udhagamandalam, India) using Glide, version 5.7,

Schrodinger suit 2011, LLC, NewYork, on a Maestro graphical user interface.

4.10.1.1. Ligand Preparation

The structures of the ligands (Gymnemic acids I-V, Gymnemoside A, B, GMG and

Glimepiride) were generated in the CDX format using the tool Chem Draw ultra

version 8.0. These ligands were then converted to the mol2 format and subjected to

LigPrep module of Maestro in the Schrödinger suite of tools. They were converted

from 2D to 3D structures by including stereochemical, ionization, tautomeric

variations, as well as energy minimization and optimized for their geometry, desalted

and corrected for their chiralities and missing hydrogen atoms. The bond orders of

these ligands were fixed, and the charged groups were neutralized. The ionization and

tautomeric states were generated between pH 6.8 to 7.2 using Epik module. In the

final stage of LigPrep, compounds were minimized using Optimized Potentials for

Liquid Simulations-2005 (OPLS-2005) force field in Impact package of Schrodinger

until a root mean square deviation of 1.8Ǻ was achieved. Steepest descent algorithm

was used for minimization, followed by conjugate gradient method. A single low

energy ring confirmation per ligand was generated and the optimized ligands were

used for docking studies.

4.10.1.2. Protein Preparation

The X-ray crystal structure of proteins, DPP-4227

(PDB ID: 3NOX), PTP-1B (PDB

ID: 1C83), NaKATPase228

(PDB ID: 3A3Y), AR229

(PDB ID: 3G5E) and GSK-3ß

(PDB ID: 3F7Z) were obtained from RCSB Protein Data Bank (PDB)

(http://www.rcsb.org/pdb). The proteins were prepared using protein preparation

wizard of Schrödinger suit. The proteins were pre-processed separately by deleting

the substrate used in order to model the protein structure in this study. In general, the

protein structures are refined for their bond orders, formal charges and missing

hydrogen atoms, topologies, incomplete and missing residues and terminal amide



groups. The water molecules beyond 5 Å of the heteroatom were removed. The

possible ionization states were generated for the heteroatom present in the protein

structure and the most stable state was chosen. The hydrogen bonds were assigned

and orientations of the retained water molecules were corrected. Finally, a restrained

minimization of the protein structure was carried out using OPLS 2005 force field to

reorient side-chain hydroxyl groups and alleviate potential steric clashes. The

minimization is restrained to the input protein coordinates by a predefined Root Mean

Square Deviation (RMSD) tolerance of 0.3 Å.

4.10.1.3. Receptor grid generation

The co-crystallized ligands in the crystal structure were retained in the structure of the

prepared protein for receptor grid construction. The binding box dimensions (within

which the centroid of a docked pose is confined) of the protein was set to 14 Å x 14 Å

x14 Å.

4.10.1.4. Validation of the docking programme

The accuracy of the docking procedure was determined by finding how closely the

lowest energy pose (binding conformation) of the co-crystallized ligand predicted by

the object scoring function, Glide score (G Score), resembles an experimental binding

mode as determined by X-ray crystallography. Extra precision Glide docking

procedure was validated by removing the co-crystallized ligand from the binding site

of the protein and re-docking the ligand with its binding site. The hydrogen bonding

interactions and the root mean square deviation (RMSD) between the predicted

conformation and the observed X-ray crystallographic conformation were used for

analyzing the results.

4.10.1.5. Glide Ligand docking

The glide docking of the synthesized molecules was carried out using the previously

prepared receptor grid and the ligand molecules. The favorable interactions between

ligand molecules and the receptor were scored using Glide ligand docking program.

All the docking calculations were performed using Extra precision (XP) mode and

OPLS-2005 force field. The above docking process was run in a flexible docking

mode which automatically generates conformations for each input ligand. The ligand

poses generated were passed through a series of hierarchal filters that evaluate the



ligand’s interaction with the receptor. The initial filter test the spatial fit of the ligand

to the defined active site, and examines the complementarity of the ligand-receptor

interactions using grid-based method patterned after the empirical ChemScore

function. This algorithm recognizes favorable hydrophobic, hydrogen-bonding and

metal-ligation interactions, and penalizes steric clashes. Poses that pass these initial

screens enter the final stage of the algorithm, which involves evaluation and

minimization of a grid approximation OPLS nonbonded ligand-receptor interaction

energy. Finally, the minimized poses were re-scored using GlideScore scoring

function. GlideScore is based on ChemScore, but includes a steric-clash term, adds

buried polar terms to penalize electrostatic mismatches, and has modifications to other

terms.

GScore = 0.065*vdW + 0.130*Coul + Lipo + Hbond + Metal + BuryP + RotB + Site

(vdW: Vander Waals energy, Coul: Coulomb energy, Lipo: Lipophilic term, Hbond: Hydrogen-

bonding term, Metal: Metal-binding term, BuryP: Penalty for buried polar groups, RotB: Penalty for

freezing rotatable bonds, Site: Polar interactions in the active site).

4.10.2. Effect of concurrent administration of extract of Gymnema sylvestre on

pharmacodynamic and pharmacokinetics of Glimepiride in STZ induced

diabetic animals230

, 231

4.10.2.1. Experimental Conditions

The study was carried out on optimized dose (400 mg/kg body weight) of Gymnema

sylvestre extract administered concurrently with glimepiride 0.8 mg/kg (Glimepiride

was provided as gift sample from Ranbaxy reasearch laboratories India

[Annexure I (e)] and evaluated for its effects on the pharmacokinetic and

pharmacodynamic of glimepiride using Wistar (male) rats, weighing 250 ± 25 gm.

Animals were randomly divided into different groups (Table 17) for the study. The

induction of diabetes was carried out as per section 4.8.2.

Table 17. Grouping of animals for invivo herb drug interaction study

Group no Test material Dose (mg/kg bw; po)

in 5% DMSO

Group I Normal Control vehicle

Group II Diabetic Control vehicle

Group III Diabetes + G. sylvestre extract 400

Group IV Diabetes + GLM 0.8

Group V Diabetes + GLM + G.sylvestre extract 400+0.8




4.10.2.2. Study design

G. sylvestre extract and glimepiride (GLM) were administered in STZ induced

diabetic animals from day 0 to day 28 to the respective groups. Fasting blood glucose

level (FBGL) was monitored on day 0, 7, 14, 21 and 28. Other parameters such as

glycosylated hemoglobin (HbA1c), total plasma cholesterol (TC), triglyceride level

(TG) were measured on 28th

day. Separate group of animals were bleed at suitable

intervals for the evaluation of pharmacokinetic interactions of G. sylvestre and GLM

in concurrently administered STZ induced diabetic animals on 28th

day (Figure 15).

Figure 15. Representation of pharmacokinetic and pharmacodynamic interaction study

4.10.2.3 Blood sample collection

Animals were anaesthetized with light ether anesthesia and blood was collected from

retro-orbital route (0.4-0.5mL) in EDTA vaccutainer (3 mL, BD Biosciences).

Sampling interval was 0, 0.5, 1, 2, 3, 5, 10, 24, 48 and 72 h after dose administration.

Blood samples were centrifuged at 7000 g for 15 min at 4°C to obtain the plasma

which were stored at -70°C until analysis using HPLC-ESI-MS/MS method.

4.10.2.4. HPLC-ESI-MS/MS method for simultaneous estimation of GMG and

GLM 232

4.10.2.4.1. Sample preparation

100 µL of the aliquot and 25µL of internal standard (150 ng/mL of Withaferin-A)

were mixed and vortexed for 30 seconds to which 1 mL of ethyl acetate was added

and vortexed it for another 2 mins at 2500 rpm. The solution was centrifuged for 5

min at 4500 rpm. The aliquot supernatant was separated and evaporated. The

evaporated aliquot supernatant was reconstituted with 100 µL of mobile phase

(MeOH:Water, 60:40 v/v). The solution was vortexed for 2 min and 20 µL was

injected into the HPLC system.



4.10.2.4.2. Analysis of samples

Simultaneous estimations of GMG and GLM in drug interaction study samples were

carried out using HPLC-ESI-MS/MS method. The method was validated as per

USFDA guidelines on bio-analytical method validation. Withaferin A was used as

internal standard.

4.10.2.4.3. Method validation

The developed method was then validated for selectivity, linearity, accuracy,

precision, recovery and stability.

4.10.2.4.3.1. Selectivity

Selectivity was ascertained by analyzing six blank rat plasma samples without adding

IS to determine the interference with the analytes.

4.10.2.4.3.2. Linearity

Three sets of calibration curves ranging from 5.00-306.00 ng/mL for GMG and from

0.5-50.00 ng/mL for GLM were constructed by plotting the peak area ratio of the

analyte/IS versus analyte concentration in blank rat plasma. Lower limit of

quantification (LLOQ) was measured for GMG and GLM as the lowest analyte

concentration (S/N=10), which can be determined with an accuracy and precision

<20%.

4.10.2.4.3.3. Precision and accuracy

For the evaluation of precision (intra and inter-day) and accuracy of the method five

replicates of four different QC samples for GMG (11.00, 58.00, 177.00 and 239.00

ng/mL) and for GLM (0.50, 1.25, 25.07 and 37.69 ng/mL) were analyzed on three

different days. Accuracy was calculated using the following equation: [mean

measured concentration/nominal concentration] ×100. Percentage coefficient of

variation (% CV) was used as measure of precision of the method. CV less than 15%

and accuracy within ±15% were accepted.

4.10.2.4.3.4. Recovery

Extraction recovery (relative recovery) of analytes from the rat plasma after the

extraction procedure was assessed in three QC samples for GMG (11.00, 177.00 and

239.00 ng/mL) and for GLM (0.50, 25.07 and 37.69 ng/mL). The detector response



(peak area) of processed QC samples (R2) was compared with the response of directly

injected aqueous QC sample (R1). Extraction recovery was calculated as follows:

recovery (%) = (R2/R1) x 100.

4.10.2.4.4. Stability study

Two different QC samples in triplicate for GMG (11.00 and 239.00 ng/mL) and for

GLM (0.50 and 37.69 ng/mL) were prepared and subjected for the evaluation of the

three freeze/thaw cycle stability and room temperature storage stability (6 h). Post

preparative stability was evaluated by re-injection of the QC samples after 12 h of

storage under auto-sampler conditions.

4.10.2.4.5. Study parameters

4.10.2.4.5.1. Pharmacokinetic parameters such as area under the curve from time

zero to last interval (AUC0-t), clearance (CL) and terminal elimination half life (t1/2)

were calculated from mean plasma concentration versus time graphs. Peak plasma

concentration (Cmax) and Tmax were directly read from each plasma concentration-time

plot. All the values were expressed as mean ± SDs. Pharmacokinetic parameters of

GLM after administration of GLM alone and co-comitant administration with extract

were compared using paired t-test. Also pharmacokinetic parameters of GMG after

administration of extract alone and con-comitant administration with GLM were

compared using above statistical tests. P value ≤ 0.05 was considered as level of

significance

4.10.2.4.5.2. Pharmacodynamic parameters Effect of G. sylvestre extract,

Glimepiride (GLM) and their concomitant administration on fasting plasma glucose

level (FPGL), glycosylated hemoglobin (HbA1c), serum insulin, total body weight,

plasma total cholesterol (TC), triglycerides (TG) levels and pancreatic tissue

histopathology were evaluated in STZ induced diabetic rats. Study data was expressed

as mean SEM. Statistical analysis was carried out by One-way ANOVA with post

hoc Tukey’s test was performed using Graph Pad Instat software (version3, San

Diego, California, USA). P < 0.05 was considered as level of significance.

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