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MATERIALS AND METHODS

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4. 4.1 PROCUREMENT AND STANDARDIZATION OF PLANTS

4.1.1 PROCUREMENT OF THE PLANT MATERIAL

The plant materials (leaves of Adhatoda vasica Nees., roots of Clerodendrum

serratum Linn., rhizomes of Curcuma longa Linn., fruits of Solanum

xanthocarpum Schrad & Wendl. and fruits of Piper longum Linn.) were obtained

from Government Ayurvedic Udhyan (Gandhinagar, Gujarat, India). They were

identified and authenticated by the Department of Pharmacognosy, K.B. Institute

of Pharmaceutical Education and Research, Gandhinagar, India. Voucher

specimens as well as herbarium were deposited at the Department of

pharmacognosy, K.B. Institute of Pharmaceutical Education and Research,

Gandhinagar, India. Herbarium no. was given as per Table-4.1.1.

Table 4.1.1: Herbarium number of plants

Plants Herbarium Number

Adhatoda vasica Nees. PH/508/002

Clerodendrum serratum Linn. PH/508/003

Curcuma longa Linn. PH/508/004

Solanum xanthocarpum Schrad & Wendl. PH/508/005

Piper longum Linn. PH/508/006

4.1.2 MONOGRAPHIC ANALYSIS OF PLANT MATERIAL

The individual plants were evaluated with regard to their standard specifications

according to ‘The Ayurvedic Pharmacopoeia of India (2001)’. Determination of

various Extractive-values and Ash-values were carried out as per method

described in ‘The Ayurvedic Pharmacopoeia of India (2001)’.


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4.1.2.1 Determination of Extractive-values

(A) Determination of water-soluble extractive value

Five g of the air-dried powdered drug was macerated with 100 ml of distilled

water in a closed flask for 24 hrs. The flask was shaken intermittently during 6

hrs and allowed to stand for 18 hrs. The extract was filtered rapidly, taking

precautions against loss of solvent. 25 ml of the filtrate was evaporated to

dryness in a porcelain dish and dried at 105C, to a constant weight. The

percentage of water-soluble extractive value was calculated with reference to the

air dried drug.

(B) Determination of ethanol-soluble extractive value

Five g of the air-dried powdered drug was macerated with 100 ml of ethanol (95

%) in a closed flask for 24 hrs. The flask was shaken intermittently during 6 hrs

and allowed to stand for 18 hrs. The extract was filtered rapidly, taking

precautions against loss of solvent. 25 ml of the filtrate was evaporated to

dryness in a porcelain dish and dried at 105C, to a constant weight. The

percentage of ethanol-soluble extractive value was calculated with reference to

the air dried drug.

4.1.2.2 Determination of Ash-values

(A) Determination of total ash

Accurately weighed 2 g of the powdered drug was incinerated in a crucible at a

temperature not exceeding 450C until carbon free ash was obtained. It was

cooled, weighed and percentage of ash was calculated with reference to the air-

dried drug.

(B) Determination of acid-insoluble ash

The ash obtained in above was boiled for 5 minutes with 25 ml of 2 M

hydrochloric acid and filtered using an ash less filter paper to collect insoluble

matter. The ash obtained was washed with hot water and filter paper was burnt

to constant weight. The percentage of acid-insoluble ash was calculated with

reference to the air-dried drug.


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(C) Determination of water-soluble ash

Total ash obtained was boiled for 5 minutes with 25 ml of water and insoluble

matter was collected on an ash less filter paper, washed with hot water and

ignited for 15 minutes at a temperature not exceeding 450C. Difference in

weight of ash and weight of water insoluble matter gave the weight of water-

soluble ash. The percentage of ash was calculated with reference to the air-dried

drug.

4.1.3 PRELIMINARY PHYTOCHEMICAL SCREENING

The powder of all the plants were subjected to the following tests individually for

the presence of various phytoconstituents like alkaloids, steroids & terpenoids,

flavonoids, tannins, saponins, anthraquinones and carbohydrates.

Alkaloids (Sim, 1970)

One g of drug powder was extracted with 20 ml alcohol (95%) by refluxing for 15

minutes. The extract was filtered and filtrate was evaporated to dryness. The

residue was dissolved in 15 ml of H2SO4 (2 N) and filtered. After making alkaline,

the filtrate was extracted with chloroform. The residue left after evaporation was

tested with dragondroff’s reagent. Appearance of orange coloured precipitates

indicating the presence of alkaloids.

Sterols and Triterpenoids (Freudenberg & Weinger, 1962; Robinson, 1963)

Liberman Buchardt test

One g of powder drug moistened with 1 ml of acetic anhydride on a clean tile. To

the above mixture, 1 to 2 drops of concentrated H2SO4 was added. Presence of

dark green coloration of the solution indicated the presence of steroids and dark

pink or red coloration of the solution indicated the presence of terpenoids.

Salkowski reaction

To the 2 g of powder, 2 ml chloroform and 2 ml concentrated H2SO4 were added

and shake well. Chloroform layer was observed for appearance of red and acid

layer was observed for appearance of greenish yellow fluorescence.


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Flavonoids (Kokate et al., 1999)

Shinoda test

One g of powder was extracted with 10 ml of ethanol (95%) for 15 minutes in a

boiling water-bath and filtered. To the filtrate, a piece of magnesium ribbon and 3

to 4 drops of concentrated H2SO4 was added. Appearance of pink red or red

coloration of the solution indicated the presence of flavonoids in the drug.

Fluorescence test

One g of power drug was extracted with 15 ml of methanol for 2 minutes on a

boiling water-bath, filtered while hot and evaporated to dryness. To the residue,

0.3 ml boric acid solution (3% w/v) and 1 ml oxalic acid solution (10% w/v) were

added. The mixture was evaporated to dryness and the residue was dissolved in

10 ml ether. The ethereal layer was observed for greenish fluorescence under

the UV indicating the presence of flavonoids.

Tannins (Kokate et al., 1999)

Aqueous extract of the drug was prepared by refluxing 10 g of drug powder with

50 ml of water for about 1 hr and used for the following test.

Gelatin test

To 2-3 ml of aqueous extract, 1% gelatin solution containing NaCl (10% w/v) was

added. Appearance of heavy white precipitates indicated presence of tannins.

Reaction with FeCl3

Two ml of aqueous extract was treated with 0.5 ml 5% FeCl3. Appearance of dark

blue or greenish grey coloration of the solution indicated the presence of tannins

in the drug.

Saponins (Kokate et al., 1999)

Froth test

0.1 g powder was vigrously shaken with 5 ml of distilled water in a test tube for

30 secs and was left undisturbed for 20 minutes. Presence of persistent froth

indicated presence of saponins in the drug.


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Carbohydrates (Kokate et al., 1999)

Molisch test

One g of powder was extracted with 10 ml ethanol for 15 minutes on a boiling

water bath and filter. On addition of α-naphthol and concentrated H2SO4 to the

filtrate, purple color was observed if carbohydrates were present in the drug.

Fehling test

One ml Fehling A and Fehling B solutions were mixed, boiled for 1 min. and

equal volume of test solution was added. Then, it was heated on boiling water

bath for 5-10 minutes. First yellow and then brick red precipitates were observed

indicating the presence of carbohydrates.

Anthraquinone glycoside (Kokate et al., 1999)

Borntrager’s test

The drug powder was taken and extracted with ether or any other water

immiscible organic solvent. To the filtered ethereal extract, dilute ammonia was

added. The aqeous layer was observed for the appearance of pink red or violet

color after shaking.

Modified borntrager’s test

To the aqeous solution of drug, FeCl3 and dilute HCl were added, heat it, cool it

and filtered. Filtrate was shaken with ether or any other organic solvent. The

ethereal extract was shaken with dilute ammonia. The aqeous layer was

observed for the appearance of rose pink or cherry red color after shaking.

4.1.4 ESTIMATION OF VASICINE, PIPERINE, CURCUMIN AND SOLASODINE

BY HPTLC METHOD

The powders of all the plants were subjected to HPTLC analysis to quantify some

known active constituents according to procedures described in the literature

(Gawas and Grampurohit, 1998; Singh et al., 2007b; Paramasivam et al., 2008;

Pawar et al., 2008).

Principle

The High Performance Thin Layer Chromatography (HPTLC) – now also called

planar chromatography – is, like all chromatographic techniques, based on a


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multistage distribution process. This process involves: a suitable adsorbent (the

stationary phase), solvents or solvent mixtures (the mobile phase or eluent), and

the sample molecules. For HPTLC, the adsorbent is coated as a thin layer onto a

suitable support (e.g. glass plate, polyester or aluminum sheet). On this layer the

substance mixture is separated by elution with a suitable solvent.

Instrument

Camag Linomat V (Automatic spotting device)

Hemilton 100 μl HPTLC syringe

Camag Twin trough chambers (20x10 cm)

Camag TLC scanner 3

Win CATS integration software

Chromatographic conditions

Separation technique: Ascending

Developing character: Twin trough chamber

Stationary phase: Precoated TLC plate of silica gel 60 F254 (methanol

washed, 20x10 cm with 250 μm thickness)

Chamber saturation time: 30 mins

Temperature: 25±2C

Relative humidity: 60±5 %

Migration distance: 7 cm

Spotting parameters

Start position: 10 mm from bottom edge

Band width: 8 mm

Space between two bands: 4 mm

HPTLC Analysis

(A) Standard solution and calibration curve of vasicine (Table-4.1.2):

Test solution

10 mg of crude powder was dissolved in methanol and volume was made up to

10 ml with methanol.


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Standard solution

A stock solution of vasicine (1 mg/ml) was prepared by dissolving 10 mg of

vasicine in 10 ml of methanol in a volumetric flask. From this stock solution, 1 ml

solution was pipetted out and diluted to 10 ml (100 ng/μl). Graded concentrations

of standard solution (100 ng/μl) in 4, 6, 8, 12 and 16 μl volume were applied on a

precoated silica gel 60 F254 TLC plate. The concentration of vasicine was 400,

600, 800, 1200 and 1600 ng/μl respectively.

Calibration curve

The plate was developed in the solvent system [Dioxane: ammonia (9:1)] in twin

trough chamber to a distance of 7 cm and was scanned densitometrically at 285

nm. The peak area was recorded and the calibration curve was prepared by

plotting peak area vs concentration of vasicine applied.

Estimation of vasicine in the drug

10 l of the test solution was applied on a precoated silica gel 60 F254 TLC plate.

The plate was developed in the solvent system [Dioxane: ammonia (9:1)] and the

chromatogram was recorded as described above for the calibration curve. The

amount of vasicine present in the sample was calculated from the calibration

curve of vasicine.

(B) Standard solution and calibration curve of piperine (Table-4.1.2):

Test solution

10 mg of crude powder was dissolved in methanol and volume was made up to

the volume of 10 ml with methanol. Pipette out 1 ml from this and diluted to 10 ml

with methanol in a volumetric flask.

Standard solution

A stock solution of piperine (1 mg/ml) was prepared by dissolving 10 mg of

piperine in 10 ml of methanol in a volumetric flask. From this stock solution, 1 ml

solution was pipetted out and diluted to 10 ml (100 μg/ml). From this, 0.5 ml

solution was pipetted out and diluted to 10 ml (5 ng/μl). Graded concentrations of

standard solution (5 ng/μl) in 5, 8, 12, 15 and 18 μl volume were applied on a

precoated silica gel 60 F254 TLC plate. The concentration of piperine was 25, 40,

60, 75 and 90 ng/μl respectively.


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Calibration curve

The plate was developed in the solvent system [Benzene: ethyl acetate:

ammonia (9:0.5:0.5)] in twin trough chamber to a distance of 7 cm and scanned

densitometrically at 333 nm. The peak area was recorded and the calibration

curve was prepared by plotting peak area vs concentration of piperine applied.

Estimation of piperine in the drug


The plate was developed in the solvent system [Benzene: ethyl acetate:

ammonia (9:0.5:0.5)] and the chromatogram was recorded as described above

for the calibration curve. The amount of piperine present in the sample was

calculated from the calibration curve of piperine.

(C) Standard solution and calibration curve of curcumin (Table-4.1.2):

Test solution

10 mg of crude powder was dissolved in methanol and the volume was made up

to 10 ml with methanol.

Standard solution

A stock solution of curcumin (1 mg/ml) was prepared by dissolving 10 mg of

curcumin in 10 ml of methanol in a volumetric flask. From this stock solution, 1 ml

solution was pipetted out and diluted 10 ml (100 ng/μl). Graded concentrations of

standard solution (100 ng/μl) in 2, 4, 6, 8 and 10 μl volume were applied on a

precoated silica gel 60 F254 TLC plate. The concentration of curcumin was 200,

400, 600, 800 and 1000 ng/μl respectively.

Calibration curve

The plate was developed in the solvent system [Chloroform: ethanol: formic acid

(96:3.5:0.5)] in twin trough chamber to a distance of 7 cm and scanned

densitometrically at 365 nm. The peak area was recorded and the calibration

curve was prepared by plotting peak area vs concentration of curcumin applied.

Estimation of curcumin in the drug


The plate was developed in the solvent system [Chloroform: ethanol: formic acid

(96:3.5:0.5)] and the chromatogram was recorded as described above for the


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calibration curve. The amount of curcumin present in the sample was calculated

from the calibration curve of curcumin.

(D) Standard solution and calibration curve of solasodine (Table-4.1.2):

25 g of powdered drug was defatted with 150 ml of n-hexane. The marc was

hydrolyzed with 250 ml of 5N aqueous hydrochloric acid under reflux on a water

bath for 5 hrs. It was filtered through Whatman 1 filter paper and washed with

distilled water twice and made the marc alkaline with 10% sodium carbonate

solution. The marc was washed with distilled water till the pH reaches neutral.

The marc was dried at 50C in a hot air oven. The dried marc was transferred to

a reflux flask and was extracted with chloroform (4 x 200 ml) under reflux on a

water bath for 2 hrs. The chloroform extract was filtered, dried over sodium

sulphate and concentrated. 10 mg of chloroform extract was dissolved in

chloroform and the volume was made up to 10 ml with chloroform.

Standard solution

A stock solution of solasodine (0.5 mg/ml) was prepared by dissolving 5 mg of

solasodine in 10 ml of methanol in a volumetric flask. From this stock solution, 1

ml solution was pipetted out and diluted to 10 ml (50 ng/μl). Graded

concentrations of standard solution (50 ng/μl) in 2, 4, 6, 8 and 10 μl volume were

applied on a precoated silica gel 60 F254 TLC plate. The concentration of

solasodine was 100, 200, 300, 400 and 500 ng/μl respectively.

Calibration curve

The plate was developed in the solvent system [Chloroform: methanol (9.5:1.2)]

in twin trough chamber to a distance of 7 cm. The plate was derivatized with

anisaldehyde-sulphuric acid reagent and was scanned densitometrically at 620

nm. The peak area was recorded and prepared the calibration curve by plotting

peak area vs concentration of solasodine applied.

Estimation of solasodine in the drug


The plate was developed in the solvent system [Chloroform: methanol (9.5:1.2)]

and the chromatogram recorded as described above for the calibration curve.


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The amount of solasodine present in the sample was calculated from the

calibration curve of solasodine.

Table 4.1.2: HPTLC chromatographic conditions

Compound Spotting volume Amount/band (For standard

curve)

Mobile phase Densitometric

scanning

For calibration curve

For test solution

Vasicine 4 – 16 μl 10 μl 400 – 1600 ng/μl

Dioxane: ammonia (9:1) 285 nm

Piperine 5 – 18 μl 10 μl 25 – 90 ng/μl Benzene:ethyl acetate: ammonia (9:0.5:0.5)

333 nm

Curcumin 2 – 10 μl 10 μl 200 –1000 ng/μl

Chloroform:ethanol:formic acid (96:3.5:0.5)

365 nm

Solasodine 2 – 10 μl 10 μl 100 – 500 ng/μl

Chloroform:methanol (9.5:1.2) 620 nm

4.1.5 THIN LAYER CHROMATOGRAPHY (TLC) FINGERPRINT ANALYSIS OF

ETHANOLIC EXTRACT, PHE AND LM-02

The TLC analysis of ethanolic extract, PHE and LM-02 was performed as per

method described in section-4.1.4.

4.2 PREPARATION OF PLANT EXTRACTS

The plant materials were dried, comminuted and passed through a # 60 mesh

screen. The dried powder of each plant was defatted using petrol ether and then

each was exhaustively extracted with ethanol using soxhlet extractor at 60C.

The extracts were concentrated under reduced pressure and stored in air-tight

borosil glass containers in cool place until usage. The extractive values of plants

were as per Table-4.2.1.

Table 4.2.1: Extractive value of ethanol extracts of Plants

Plants Extractive value

Adhatoda vasica Nees 0.5%w/w

Clerodendrum serratum Linn. 1%w/w

Curcuma longa Linn. 0.7%w/w

Solanum xanthocarpum Schrad & Wendl. 0.8%w/w

Piper longum Linn. 0.4%w/w


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4.3 PREPARATION OF POLYHERBAL FORMULATIONS

(A) PREPARATION OF MIXTURE OF POLYHERBAL EXTRACTS (PHE)

For preparation of mixture of polyherbal extracts, the plant extracts were mixed

as per Table-4.3.1 and was packed in small airtight container.

Table 4.3.1: Proportion of plant extract in Polyherbal mixture

Plants Proportion of ethanolic extract

Adhatoda vasica Nees. 40%

Clerodendrum serratum Linn. 30%

Curcuma longa Linn. 10%

Solanum xanthocarpum Schrad & Wechdle.

10%

Piper longum Linn. 10%

(B) PREPARATION OF CAPSULE (LM-01)

The PHE and 1% cabosil (as glidant) were filled in the capsule (GEM Capsule

filing machine, India). The amount of PHE in each capsule was 500 mg. The

capsules were polished, packed in small airtight containers and properly labeled.

(C) PREPARATION OF ORAL AEROSOL SPRAY (LM-02)

Each oral aerosol spray container was of 300 metered doses (10 mg/dose). The

PHE and 0.1 ml (1 % w/v) peppermint solution were dissolved in ethanol. The

ethanolic solution (40%) and Hydrocarbon as aerosol propellant (60%) were filled

in aerosol container with the help of Terco Inc, Germany.

PHE (dissolved in 1% Tween-80 solution) and LM-02 were subjected to pre-

clinical study. Further, LM-01 and LM-02 were used for clinical study.


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4.4 PRE-CLINICAL STUDIES

4.4.1 SELECTION OF ANIMALS

All animals were housed at ambient temperature (22±1C), relative humidity

(55±5%) and 12h/12h light dark cycle. Animals had free access to standard pellet

diet and water given ad libitum. The protocol of the experiment was approved by

the institutional animal ethical committee as per the guidance of the Committee

for the Purpose of Control and Supervision of Experiments on Animals

(CPCSEA), Ministry of Social Justice and Empowerment, Government of India

(KBIPER/0891, KBIPER/08103 and KBIPER/08/116).

4.4.2 PHARMACOLOGICAL EVALUATION

4.4.2.1 Anti-Asthmatic Activity

(A) Effect of PHE (Polyherbal extracts) and LM-02 on Histamine and

Acetylcholine induced bronchospasm in guinea pigs

The method used in the present study was similar to that described by Sheth et

al., 1972.

Hartley strain guinea pigs of either sex weighing 350-500 g were selected and

randomly divided into seven groups, each containing six animals. The PHE and

LM-02 were administered as per following schedule:

Group I Ketotifen (1 mg/kg, per oral route) (Standard)

Group II PHE (200 mg/kg, per oral route)

Group III PHE (400 mg/kg, per oral route)

Group IV PHE (800 mg/kg, per oral route)

Group V LM-02 (3 mg/kg, per inhalation route)

Group VI LM-02 (6 mg/kg, per inhalation route)

Group VII LM-02 (12 mg/kg, per inhalation route)

The animals were kept in a closed chamber and exposed to an aerosol (Voyage

Mist, Shoney Medical Technology, USA) of 0.25% histamine hydrochloride (1

ml/min) and time for preconvulsion dyspnoea (PCD) (The time of aerosol

exposure to the onset of dyspnoea leading to the appearance of convulsions)

was noted. As soon as PCD commenced, animals were removed from the


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chamber and placed in fresh air to recover. This time for PCD was taken as basal

value. The PHE, LM-02 and ketotifen were administered for 7 days. On the 7th

day, 2 hrs later of the drug administration, the animals were exposed to

histamine aerosol and time for PCD was noted. The effect of drug was calculated

by the following formula:

% increase in PCD time = [1-T1/T2] x 100

where, T1= time for PCD onset on day 0

T2= time for PCD onset on day 7

Similar procedure was repeated by exposure of aerosol of 0.5% acetylcholine (1

ml/min) in another seven groups of animals and PCD time was noted down. In

Group I, atropine (2 mg/kg, per oral route) was used as standard drug instead of

ketotifen.

(B) Effect of PHE on Trypsin and Egg albumin-induced bronchospasm in

conscious mice

Here, trypsin and egg albumin were used as sensitizers (antigens). Albino mice

of either sex weighing 25-30 g were selected and randomly divided into four

groups, each containing six animals.

Group I 1% Tween 80 solution orally (Control group)

Group II Asthmatic control (Positive Control group)

Group III Dexamethasone (5 mg/kg, per oral route) (Standard group)


On the first day before starting exposure of antigens, all animals were examined

for following parameters:

pO2

Serum bicarbonate level

Tidal volume

Respiratory rate

Air flow rate

The animals of Group I were exposed once daily to aerosol (Voyage Mist,

Shoney Medical Technology, USA) of saline for 5 min. Animals of Group II, III

and IV were exposed once daily to aerosol of trypsin (1 mg/ml, 1 ml/min) for 5


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mins followed by a rest of 2 hours and then exposed to egg albumin (1%

solution, 1 ml/min) for 5 mins. This procedure was repeated for 10 days. Later

on, egg albumin aerosol was discontinued where as trypsin exposure was

continued till 21st day. On 22nd day, the animals were examined for the above

mentioned parameters. From day 22nd to 35th, dexamethasone and PHE were

administered (per oral route) once a day to animals of Group III and IV

respectively. On 35th day, 2 hrs after the drug treatment, animals of Group II, III,

and IV were exposed to egg albumin (1% solution, 1 ml/min) for 3 mins and were

examined for the above mentioned parameters.

Measurement of pO2

The method described by Apps, 1992 and Fabbri et al., 1997a was used.

The measurement of arterial O2 tension (pO2) was done with the help of Pulse

Oxymeter instrument (Nasan Medical Electronics Pvt Ltd, India). The animals

from each group were taken and pO2 of each animal was measured on 35th day.

Measurement of serum bicarbonate level

The method used in present study to measure serum bicarbonate level was

slightly modified, from that described by Godkar, 1994.

The serum bicarbonate level was measured in both sensitized and unsensitized

mice on 35th day of study.

Specimen collection

About 1-2 ml of blood was collected from each animal after 1 hr of the exposure

to egg albumin. The serum was separated from blood by minimum exposure to

air and it was stored in a sealed tube till bicarbonate level was estimated.

Procedure

Ten ml of 1 g/dl saline was pipetted out in 100 ml beaker. To this, 0.1 ml of the

specimen was added. Then 2 drops of phenol red indicator was added and

mixed well. In above mixture, drop by drop NaOH (0.01N) was added till the end

point was achieved (7.35 pH or color changed from yellow to pink). The volume

of NaOH required was noted down and considered as control reading (X ml). In

another set, 9.0 ml of 1 g/dl saline & 1 ml HCl (0.01N) was added. The above

experiment was repeated and volume of NaOH required was noted (Y ml).


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Now R = Y ml – X ml

Calculation

Serum bicarbonate level mEq/ltr = (1-R) x 100

Standard serum bicarbonate level = 21-28 mEq/ltr

Measurement of tidal volume

The method used in the present study was described by Khandpur, 1997.

Definition

It is volume of air inspired or expired per breathe during normal breathe.

Procedure

The measurement of tidal volume was done with the help of ‘Respiratory volume

transducer’ that was used with ‘Strain gage coupler’ and student physiograph.

The physiograph was calibrated with the help of standard 0.1cc volume air

supplier. The tidal volume was measured in terms of height of response

obtained. Then, the height was converted into volume.

Measurement of respiratory rate

The method used in present study was described by Apps, 1992 and Fabbri et

al., 1997a.

Definition

It is no. of breathes per unit time

Unit: Breathes per min (BPM)

Measurement of air flow rate

The method used in present study was described by Guyton and Hall, 2006a.

Definition

It is volume of air inspired or expired per unit time during normal breathing.

Unit: Volume per unit time (ml/min).

Calculation

Air flow rate = Respiratory rate x Tidal volume

BAL Fluid study: differential leukocyte count

The method described by Natiello et al., 2009 was used.

On 35th day, the tracheobronchial tree was lavaged with 1 ml of saline and the

fluid so collected was centrifuged at 2000 rpm for 5 mins and then pellet was


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resuspended in 0.5 ml saline. 0.2 ml Geimsa stain in buffered saline (pH 6.8) was

added to it. After 5 mins, the number of each type of leukocyte in 0.5 ml fluid was

determined under the microscope at 450x magnification.

Histopathology of lungs

The method for histological studies was as descried by Garg et al., 1991.

On 35th day, the mice were sacrificed and histological studies of lungs were

carried out. Briefly, the procedure used included fixation of the tissue with

formalin, embedding in paraffin blocks, sectioning with microtome (0.7 µm

thicknesses) and finally staining by Haematoxylin and Eosin stain technique.

Principle

Haematoxylin stains nucleus light blue which turns red in presence of acid. The

cell differentiation is achieved by treating the tissue with acid solution. The

counterstaining is performed by using eosin which imparts pink color to the

cytoplasm.

(C) Effect of PHE on Triple antigen-induced anaphylaxis in rats

The method described by Gupta et al., 1968 was used.

Twelve Wistar albino rats of either sex, weighing 200-250 g were used for the

study. All the rats were sensitized by single subcutaneous injection of 0.5 ml

horse serum along with 0.5 ml of triple antigen containing 20 million Bordetella

pertusis organisms. After sensitization, the rats were divided into two groups of 6

animals in each. Rats of Group-I received vehicle (1% Tween 80) and served as

control. Rats of Group-II received PHE (200 mg/kg, p.o.) once a day for 10 days.

On 10th day, 2 hrs after treatment, the rats were challenged with intravenous (i.v)

injection of 0.25 ml horse serum. The rats were observed for: onset of symptoms

such as dyspnoea and cyanosis (mins); duration of persistence of symptoms

(mins) and death.

With respect to symptoms, the severity score was graded as 2 – increased

respiratory rate, 4 – dyspnoea for 10 mins, 8 – dyspnoea and cyanosis for 10

mins, 12 – collapse and were recorded for both the groups.


8

(D) Effect of PHE on Compound 48/80 induced rat mesentric mast cell

degranulation

The method used in present study was described by Nortan, 1954.

The Wistar albino rat was sacrificed and the pieces of mesentry were collected in

petri dish containing Ringer Locke solution and then subjected to the following

treatment schedules.

Petri dish no.1 - Ringer Locke solution (Vehicle control)

Petri dish no. 2 - Ringer Locke solution (Positive control)

Petri dish no. 3 - 0.1ml of Ketotifen (10 g/ml)

Petri dish no. 4 - 0.1ml of PHE in Tween-80 (500 g/ml)



Each petri dish was incubated for 15 mins at 37C. Later Compound 48/80 (0.1

ml, 10 g/ml) was added to each petri dish except petri dish no.1 and again

incubated for 10 mins at 37C. After that, all pieces were transferred to 4%

formaldehyde solution containing 0.1% Toluidine blue and kept aside for 20 to 25

mins. After staining and fixation of mast cells, mesentry pieces were transferred

through acetone and xylene two times and mounted on slides. All the pieces

were examined under the high power of light microscope. Percent protection of

the mast cells in the control group and the treated groups were calculated by

counting the number of degranulated mast cells from total of at least 100 mast

cells counted. Percent inhibition of mast cell degranulation for each treatment

was calculated by following formula:

% inhibition of MCD = 1 – Number of degranulated mast cell X 100

Total number of mast cells


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4.4.2.2 Anti Inflammatory Study

(A) Effect of PHE on Carrageenan-induced rat paw edema

The method used in present study was described by Winter et al., 1962.

Wistar albino rats of either sex, weighing 200-250 g were divided in five groups of

6 animals each. The single dose treatments were given for 7 days. The following

schedule of treatment was administered:

Group I 1% Tween 80 solution orally (Control)

Group II Indomethacin (10 mg/kg, per oral route) (Standard)



Group V PHE (800 mg/kg, per oral route)

On 7th day, subsequently 1 hr after treatment; 0.1 ml of 1 % carrageenan was

injected subcutaneously into the planter region of right hind paw to induce

edema. The paw volume was measured initially and at 1, 3 and 5 hr after

carrageenan injection using plethysmographic method. Increase in paw volume

and per cent inhibition was calculated as follow:

Increase in paw thickness in control/treatment PC/PT = Pt – P0.

Per cent inhibition = (PC – PT ) x100

PC

Where Pt is paw thickness at time t, P0 is initial paw thickness, PC is increase in

paw thickness of the control group and PT is the increase in paw thickness of the

treatment groups.

(B) Effect of PHE on Acetic acid-induced increase in capillary permeability

Effect of the test samples on the increased vascular permeability induced by

acetic acid in mice was determined according to Whittle (1964) method with

some modifications.

Albino mice of either sex, weighing 25-30 g were divided in five groups of 6

animals each.


Group II Indomethacin (10 mg/kg, per oral route) (Standard)



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Group V PHE (800 mg/kg, per oral route)

Each test sample was administered orally for seven day. On 7th day, 30 minutes

after the drug administration, each mouse was injected with 0.1 ml of 4% Evans

blue in saline solution (i.v.) at the tail. Then, 10 min after the i.v. injection of the

dye solution, 0.4 ml of 0.5% (v/v) acetic acid was injected intraperitonealy (i.p.).

After 20 mins, the mice were sacrificed, and the viscera were exposed and

irrigated with distilled water, which was then poured into 10 ml volumetric flasks

through glass wool. Each flask was made up to 10 ml with distilled water, 0.1 ml

of 0.1N NaOH solution was added to the flask and the absorption of the final

solution was measured at 590 nm using normal saline as blank with a Shimadzu

UV-160 spectrophotometer.

4.4.2.3 Immunomodulatory Activity

(A) Effect of PHE on Immunological responses to Sheep Red Blood Cells

(SRBC)

Sheep blood was collected aseptically from the city slaughter house in a

sterilized bottle containing Alsever’s solution (2% dextrose, 0.8% sodium citrate,

0.05% citric acid and 0.42% sodium chloride). SRBC was thoroughly washed

with sterile normal saline and stored in Alsever’s solution in refrigerator till

experimentation.

Wistar albino rats of either sex, weighing 200-250 g were selected for the study.

The animals were randomly divided into following groups of 6 animals each.


Group II Dexamethasone (5 mg/kg, per oral route) (Standard)


The animals of Group-II and Group-III received respective drug treatment orally

once daily, for 22 days. Besides above treatment, each group received SRBC

(0.5×109 cells/100 g, i.p.) on Day 7 and 13, as the antigenic material to sensitize

them for immunological studies.


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Humoral immune response

The humoral immunity was assayed as per the method of Joharapurkar et al.,

2003.

On the Day 13 and 20, blood was collected from all antigenically challenged rats

and serum was separated. Twenty-five µl of serum was serially diluted with 25 µl

of phosphate-buffered-saline. SRBC (0.025×109 cells) were added to each of

these dilutions and incubated at 37C for one hour. The rank of minimum dilution

that exhibited haemagglutination was considered as an antibody titre. The level

of antibody titre on Day 13 of the experiment was considered as the primary

humoral immune response and the antibody titre on Day 20 of the experiment

was considered as the secondary humoral immune response.

Cellular immune response

The cell mediated immunity was assayed as per the method of Joharapurkar et

al., 2003.

This was assayed by the footpad reaction method. The edema was induced in

the right paw of rats by injecting SRBC (0.025×109 cells) in the subplantar region

on Day 20. The increase in the paw volume in 48 hours i.e. on Day 22 was

assessed by plethysmographic method. The mean % increase in paw edema

was considered as delayed type hypersensitivity and index of cell mediated

immunity. The volume of left hind paw, injected similarly with phosphate buffered

saline, served as a control.

Histopathology of spleen

The method for histological studies was as descried by Garg et al., 1991.

After completion of 22 days drug treatment, rats were sacrificed; spleens were

dissected out and were transferred to 10% formalin solution. The

histopathological study was carried out as per method described in Section-

4.4.2.1B.


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(B) Effect of PHE on Neutrophil Adhesion Test

The method described by Wilkinson, 1978 was used.

Wistar albino rats of either sex, weighing 200-250 g were selected for the study.

The animals were randomly divided into following groups of 6 animals each.


Group II Dexamethasone (5 mg/kg, per oral route) (Standard)


The animals received respective drug treatment orally once daily, for 14 days. On

the 14th day after the drug treatment, blood samples were collected into EDTA

vials and analyzed for total leukocytes counts (TLC) and differential leukocytes

counts (DLC) by Sysmax 3000, Japan. After initial counts, blood samples were

incubated with 80 mg/ml of nylon fibers for 15 mins at 37C. The incubated blood

samples were again analyzed for TLC and DLC. The product of TLC and %

neutrophil gives neutrophil index (NI) of blood sample [NI= (TLC × % neutrophil)].

Percentage neutrophil adhesion was calculated as shown below:

Neutrophil adhesion (%) = (NIu- NIt) × 100.

NIu

Where,

NIu = NI of untreated blood sample.

NIt = NI of fiber-treated blood sample.

4.4.2.4 Acute Toxicity Study of PHE

Acute oral toxicity study was performed as per OECD – 423 guidelines (acute

toxic class method) (2002). Wistar albino rats (female) were selected. The

animals were kept fasting for overnight and provided only with water, after which

the PHE was administered orally at 300 mg/kg body weight in three rats and

observed for 14 days. The rats were observed individually for any sign of acute

toxicity, morbidity or mortality after dosing at least once during the first 30

minutes, periodically during the first 24 hrs and daily thereafter, for a total of 14

days. If mortality was observed in two out of three animals, then the dose

administered was assigned as toxic dose and the procedure was repeated with


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lower dose i.e. 50 mg/kg body weight. If mortality was observed in one animal,

then the same dose was repeated again to confirm the toxic dose. If mortality

was not observed, the procedure was repeated for higher dose i.e. 2000 mg/kg

body weight.

4.4.3 STATISTICAL ANALYSIS

The values were expressed as Mean SEM. Statistical significance of difference

in parameters was determined by One way ANOVA, followed by Tukey’s multiple

range test and Unpaired student ‘t test’. P<0.05 was considered to be significant.


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4.5 CLINICAL STUDIES

An open label, randomized, non-comparative, multicenter clinical study was

carried out of LM-01 on 36 patients and LM-02 on 27 patients of either sex,

having mild to moderate bronchial asthma at Government Referral Hospital,

Mansa, India as well as Ayukalp Clinic, Ahmedabad, India. The Independent

ethics committee has approved the protocol for carrying out the clinical study. All

patients were made fully aware of the investigative protocol and provided

informed consent before they were enrolled in the study (Annexure-I).

4.5.1 SUBJECT SELECTION CRITERIA

The patients were evaluated for eligibility to participate in the study based on

inclusion and exclusion criteria.

Inclusion Criteria

The following were the inclusion criteria.

Male and female patients between 20 to 80 years of age.

Patients having mild to moderate bronchial asthma and with duration ≥6

months (diagnosis was made based on complete clinical history, commonly

observed symptoms, physical and hematological examinations, and pulmonary

function test).

Exclusion Criteria

The following were the exclusion criteria.

Patients having very severe bronchial asthma (PEFR < 20%, FEV1 < 20% of

predicted value).

Patients with steroid-dependant asthma.

Asthma requiring hospitalization once or emergency treatment more than once

within the 6 months before or during the study.

Pregnant and breast feeding women.

Present smokers.

Patients with symptoms of respiratory tract infection.

Patients with diabetes, cardiovascular disorders, hepatic & renal diseases,

pulmonary tuberculosis etc.


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Those patients who fulfill all above mentioned criteria were enrolled in the study

(Annexure-II).

4.5.2 TREATMENTS

(1) LM-01(Capsule)

Patients were advised to take 2 capsules twice daily for 12 weeks.

(2) LM-02 (Oral aerosol spray)

Patients were advised to take 2 puffs of oral aerosol spray twice daily for 4

weeks.

Follow ups:

Patients were advised to visit the hospital daily during the 1st week. Subsequently

they visited every week for follow-up and collection of medications. At weekly

visit, patients were asked for any untoward effect and improvement in the

symptoms observed.

4.5.3 ASSESSMENT

Comprehensive assessment; comprising of baseline characteristics, clinical

assessment, laboratory investigations and pulmonary function tests was

recorded at the beginning of study and at the end of study (Annexure-II).

Clinical assessment

Patients were examined weekly for heart rate, blood pressure and respiratory

rate during the study period.

Hematological examinations

Blood samples were analyzed for hematological examinations include

Hemoglobin (Hb) estimation, Total leukocyte count (TLC), Differential leukocyte

count (DLC) and Erythrocyte sedimentation rate (ESR).

Total serum IgE measurement (Bassion, 1989; Halpern, 1989)

Toatl IgE was measured in the serum of patient before and at the end of the

treatment. Total serum IgE was determined by ACS:180 total IgE assay. The

ACS:180 total IgE assay is two-site sandwich immunoassay using direct

chemiluminometric technology, which uses constant amounts of two antibodies


Page 186

to IgE. The first antibody, in the Lite reagent, is a goat anti-human IgE antibody

labeled with acridinium ester. The second antibody, in the solid phase, is a

mouse anti-human IgE antibody which is covalently coupled to paramagnetic

particles. Briefly, 30 μl of standard and sample were dispensed into cuvettes.

Hundred (100) μl of Lite reagent and 450 μl of solid phase were dispensed and

incubated for 7.5 minutes at 37°C. Cuvettes were separated, aspirated and

washed with reagent water. Three hundred (300) μl each of chemiluminescence

substrate solutions were dispensed to initiate chemiluminescent reaction. Read

wells with a chemiluminescence microwell reader. Calculate the average read

Relative light units (RLU) for each set of reference standards and samples.

Construct a standard curve by plotting the mean RLU obtained for each

reference standard vs IgE concentration in IU/ml on linear graph paper, with RLU

on the vertical (y) axis and concentration on the horizontal (x) axis. Using the

mean absorbance value for each sample, determine the corresponding

concentration of IgE in IU/ml from the standard curve.

Sputum test

The test involves % Eosinophils count in the sputum of patient before and at the

end of the treatment. Sputum was induced through the method described by

Keatings et al., 1997. The patients were instructed to wash their mouths

thoroughly with water. They then inhaled nebulized 3.5% saline at room

temperature from an ultrasonic nebulizer (Voyage Mist, Shoney Medical

Technology, USA) at the maximum saline output (4 ml/min). The total period of

sputum induction was 15 mins. Subjects were encouraged to cough deeply.

Mouth washing before each cough was encouraged in order to minimize salivary

contamination. The initial sample from the first cough was discarded. Sputum

was collected into a 50-ml polypropylene tube, kept at 4°C, and processed within

2 hrs. For sputum processing, 1 ml Hanks’ balanced salt solution (HBSS)

containing 1% dithiothreitol (DTT) was added to the sputum. The mixture was

vortexed and repeatedly aspirated at room temperature until the sputum was

homogenized. Samples were left at room temperature for 5 mins. Sputum

volume was then recorded, the sputum was further diluted with HBSS to a


Page 187

volume of 5 ml, and the preparation was vortexed briefly and centrifuged at 400

rpm for 10 min at 4°C. The cell pellets from the sputum centrifugation were

resuspended. Slides were prepared and were stained with May–Grunwald–

Giemsa stain for differential cell counts.

Symptom Score

The symptom score was measured for all commonly observed symptoms of

bronchial asthma i.e. dyspnoea, wheezing, rhonchi, cough and accessory muscle

use (AMU)-sternocleidomastoid before start of the treatment and at the end of

the treatment. Score was graded as 3, 2, 1 and 0 for presence of severe,

moderate, mild and absence of any symptom respectively (Govindan et al.,

2004).

Pulmonary Score (PS)

The pulmonary score was measured before start of the treatment and at the end

of the treatment. The PS is the aggregate of three parameters: respiratory rate,

wheezing and accessory muscle use, each scored on a 0-3 scale (Table-4.5.1).

The PS ranges from 0 (no or very mild exacerbation) to 9 (severe exacerbation)

(Smith et al., 2002).

Table 4.5.1: Pulmonary score

Score Respiratory rate (breathes/min)

Wheezing Accessory muscle use

0 <20 None No apparent increase

1 21-35 Terminal expiration Mild increase

2 36-50 Entire expiration with stethoscope Increased

3 >50 Inspiration & expiration without stethoscope

Maximal activity

Pulmonary function Tests (Govindan et al., 1999)

Pulmonary function tests (PFT) were carried out with the help of Spirometer

(Spirobank, France) before treatment and at the end of the treatment. Patient

was asked to keep mouthpiece of spirometer in mouth and hold it in the mouth

firmly with the help of lips. Patient was asked to breathe normally a few times.

Then the patients were asked to take a deep inspiration followed by forcible

expiration in to the spirometer. The spirogram and the values of lung volumes

and lung flow rates obtained were recorded. The whole process was repeated


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three times and the best results were recorded. Parameters assessed were

Forced vital capacity (FVC), Forced expiratory volume in 1 second (FEV1), Peak

expiratory flow rate (PEFR) and Forced expiratory flow rate (FEF25-75%).

Maximum ventilatory volume (MVV) was determined by asking the patient to

breathe as hard and fast as possible for 1 minute.

Frequency of asthmatic attacks/day

Frequency of asthmatic attacks were noted before and every week during their

visit to hospital.

4.5.4 STATISTICAL ANALYSIS

The values were expressed as Median (IQR) or Mean SD. Statistical

significance of difference in parameters before and after treatment was

determined by Wilcoxon Signed Rank test and Student’s paired t-test. P<0.05

was considered to be significant.

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