PART II EXPERIMENTAL STUDIES

"Start, Wherever you are, with Whatever you have and Never be satisfied"

– Anonymous –

 

 

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II: EXPERIMENTAL STUDIES:

4. Aim

The aim of the current study undertaken was

1. To review herbal drug interaction information.

2. To examine effects of simultaneous administration of selected herbal extracts and

synthetic drugs on antidepressant models and delineate the possible mechanisms of

interactions and safety parameters.

The following interactions between the herbal extracts and conventional antidepressants

were studied on animals in laboratory.

1. The potential interaction between Bacopa monniera and fluoxetine.

2. The potential interaction between Centella asiatica and imipramine.

3. The Potential interaction between Curcuma longa and reboxetine.

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5. Materials and methods

5.1. Herbal extracts

Following standardized, aqueous extracts were purchased from commercial supplier;

Ansar Industries, Surat Gujarat, India.

1. Bacopa monniera extracts (BME) containing 20% of bacosides.

2. Centella asiatica extracts (CAE) containing 10% of asiaticoside.

3. Curcuma longa extracts (CLE) containing 0.5% of curcumin.

5.2. Identification and authentication of the herbal extracts

The three extracts aquired commercially, mentioned above were identified and

authenticated at Bapalal Vaidh Institute, Botanical Research Center, VNSGU, Surat and

the voucher specimens were deposited at department of pharmacology, Institute of

Pharmacy, Nirma University, Ahmedabad, Gujarat, India. The extracts were evaluated

for their prescribed quality as per the standard specifications given in the text compared

to that with the actual results obtained. The TLC and HPTLC identifications for the

respective phytoconstituents in the herbs were carried out in the laboratory.

TLC identity test for the three extracts was done as per the following method (Rajpal,

2002). The stationary phase employed was TLC aluminium sheets pre-coated with

silica gel 60 F254, layer thickness 0.2 mm: 20 x 20 cm, E. Merck, Germany for all the

TLC identifications.

TLC method for Bacopa monniera dry extracts

Test solution:

1.0 g of sample was refluxed with 25 ml pure methanol at 80 oC for 1 hour and filtered.

The filtrate was concentrated under vacuum and dissolved in 1 ml of methanol.

Solvent system: Chloroform: methanol: (9:1 v/v)

Spray reagent : 20% Sulphuric acid in methanol

Evaluation: The plate was observed in day light and in UV light chamber.

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TLC method for Centella asiatica dry extracts

Test solution

1.0g of the sample was extracted by refluxing it with 25 ml of methanol, for 1 hour at

60oC and filtered. The concentrate was then filtered in vacuum and dissolved in 1 ml of

methanol. Following solvent system was employed for the separation.

Solvent system: Chloroform: Glacial acetic acid: Methanol: Water (60:32:12:8 v/v/v).

Spray reagent: Anisaldehyde sulphuric acid reagent

Visualization: The plate was heated at 100 oC and observed in day light as well as in

UV light.

TLC method for Curcuma longa dry extracts

Test solution: 1.0g of the sample was extracted by refluxing it with 25 ml pure

methanol, for 1 hour at 60 oC and filtered. The concentrate was filtered in vacuum and

dissolved in 1 ml of methanol.

Solvent system: Chloroform: Ethanol: Acetic acid (94:5:1 v/v/v)

Spray reagent : Vanillin-sulphuric Acid

Visualization: The plate was observed in day light as well as in UV light.

HPTLC determination of active constituents

Instrumentation

A Camag’s HPTLC system with an automated TLC sampler Linomat V (Camag,

Muttenz, Switzerland) controlled by WinCATS software, with twin-trough glass

chambers, and a TLC scanner III was used for the densitometry.

Preparation of standards

A 0.5 mg/ml of solution of pure bacoside A (purity 98.5%), asiaticoside (98%) and

curcumin (95%) were prepared in methanol. The reference standard markers for

phytoconstituents were procured on request as gift sample from Avance’

Phytotherapies Pvt. Ltd., Ahmedabad, Gujarat, India.

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Preparation of sample: BME

10 gm of BME sample was accurately weighed and extracted exhaustively with 90%

methanol. The extract was concentrated and successively partitioned with petroleum

ether, chloroform, diethyl ether and finally with n-butanol. The extracts were filtered,

pooled and transferred to a 100 ml volumetric flask and volume was made up to 100 ml

with methanol.

Preparation of sample: CAE

1.0 g of sample was accurately weighed in 100 ml round bottom flask and 15 ml of

aqueous methanol (1:1) was added to it. It was refluxed for 20 minutes on water bath

and then filtered through a Whatman no. 41 filter paper in a separate conical flask. The

extraction was repeated three times again as above using 15 ml of solvent each time.

The filtered extracts were pooled and transferred in to 100 ml volumetric flask and

made up to mark with methanol.

Preparation of sample: CLE

0.1 gm of sample was extracted with acetone (3X10 ml), filtered and concentrated

under vacuum, dissolved in methanol and made up to 5 ml in volumetric flask for

HPTLC analysis.

Procedure

Five microliters from the above sample solution was used for spotting in HPTLC

analysis. Sample solutions were applied onto the plates with an automated TLC sampler

Linomat V (Camag, Muttenz, Switzerland) controlled by WinCATS software. The

plates were pre-washed by dipping in methanol and the solvent was allowed to overrun

the plate. The plates were activated at 60oC for 5 min prior to the chromatography. A

constant application rate of 0.1 micro litres was used with a bandwidth of 6 mm for the

Linomat. The slit dimension was kept at 6.0 x 0.45 mm, a 10 mm/s scanning speed and

the monochromatic bandwidth was set at 20 nm for the scanner.

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Plate development

Plates were developed in 10 x 10 cm twin-trough glass chambers. The mobile phase for

BME consisted of chloroform: methanol: water (18: 09: 06 v/v/v) in a twin trough

chamber to a distance of 93 mm; that for CAE was, ethyl acetate: formic acid: acetic

acid: water (100: 11: 11: 27 v/v/v) (upper layer) up to 80 mm under chamber saturation

condition and that for CLE was, chloroform: methanol (98: 2 v/v) up to the distance of

80 mm. The optimized chamber saturation time for mobile phase was 15 min at room

temperature (25o ± 2oC) at relative humidity (60 ±5%). The length of chromatogram

was run up to the solvent front.

Scanning

After development, chromatographic plates were removed from the chamber and were

dried in a current of air with the help of a hair dryer, normal mode for 5 min. The plates

were then sprayed with Liebermann Burchard reagent (LB Reagent) using Camag TLC

spray tank. The plates were then dried in air for 15 min and were scanned at 540 nm

(for BME), at 550 nm (for CAE) and at 366 nm in adsorption reflection mode (for

CLE), using a Camag TLC scanner III. The peaks were recorded for each of the active

phytoconstituents.

5.3. Conventional drugs

The following conventional antidepressant drugs in form of active pharmaceutical

ingredients (API) were obtined as gift samples on request from the pharma industries.

1. Fluoxetine HCl (Sun Pharma Pvt. Ltd, Mumbai, Silvassa plant).

2. Imipramine HCl (Sun Pharma Pvt. Ltd, Mumbai, Silvassa plant).

3. Reboxetine (Cadila health care, Ahmedabad, Gujarat, India.)

The analytical profiles for each of the above conventional drugs were maintained in the

records.

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5.4. Animals

Healthy, male, Wistar rats (150-250g) and Swiss, albino mice (25–30 g) were obtained

from Jai Research Foundation (JRF), Vapi, Gujarat, India. On arrival, the animals were

housed six per cage and acclimatized to a departmental animal house with controlled

ambient temperature (25 ± 2oC), humidity (50-60%) and a 12 h light/dark cycle. They

were fed with standard diet in form of pellets obtained from JRF, Vapi and provided

with water ad libitum throughout the experimental period except on the day of

experiment. The animals were allowed to acclimate 15 days before commencing the

experiments. All The experiments were performed between 8:00 and 16:00 h.

The procedures were conducted in accordance with the CPCSEA while handling

experimental animals and the research protocol were approved by the institutional

animal ethical committee (IAEC).

5.5. Preclinical toxicity tests: dose range finding

The pre clinical tests for toxicity were conducted according to the WHO guidelines

(WHO, 2002). In the test, 2 male rats and 2 male mice were divided in to 2 groups of

each for evaluation of three extracts. One group was dosed 1000 mg/kg and another

group was dosed 2000 mg/kg of BME, CAE and CLE respectively, in each of the three

combinations of the herb and drug evaluated. The mortality was observed for 24 hours

after the dosing. Hence, additional 4 + 4 male rats and mice were selected for the study

and dosed as mentioned above and observed for 14 days for any mortality. At no sign

of mortality, the main study was conducted. Healthy, male Swiss, albino mice and

Wistar rats were divided randomly in to eight groups each. Animals were allowed food

and water ad libitum. The aqueous BME, CAE and CLE were administered orally in

doses of 0.25, 0.5, 1.0, 1.5 and 2.0 g/kg body weight were administered once for

experimental groups in three combinations, respectively. The control group received 1

ml of distilled water. The volume of each administered dose did not exceed 1 ml. The

animals were observed for 24-hour period and mortality was observed, if any.

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Simultaneously, visual observations were made for external appearance of animal

including skin colour, texture changes, mobility, aggression and sensitivity to the sound

and pain and other bizarre behaviour, if any.

Sub acute toxicity tests

A similar dosing was performed for three herb extracts, mentioned above in same

fashion for sub acute test and animals were observed for every 24 hours up to 28 days.

Simultaneously weight changes were noted, once weekly starting from 0 week up to 1

and 2- week of the experiments and visual observation were noted for gross behaviour

of the animal.

Additional observations include changes in skin, fur, eyes and mucous membrane,

incidence of secretions or excretions, if any. The ANS activities like lacrimation,

piloerection, pupil size and unusual respiration pattern, changes in gait, posture and

response to handlings as well as presence of clonus and tonic movements stereotypy

(e.g. Excessive grooming, repetitive circling movements) or bizarre behaviour (e.g. Self

mutilation, walking backwards) were also observed, if any. Also, reaction to various

stimuli of different types (IPCS, 1986) (e.g. auditory, visual and propioception) (Moser

et al., 1991; Gad, 1982; Tupper and Wallace, 1980), assessment of grip strength (Meyer

et al., 1979) and motor activity (Crofton et al., 1991) were scrutinized. Attention was

also directed to the observation of tremor, convulsion, salivation, diarrhoea, lethargy,

sleep and coma, if any of it appeared.

5.6. Preparation of extracts and dosage administrations

Each of the extracts was refluxed with methanol for 1 hour at 60oC. The dried extracts

dissolved in distilled water were used for the three combinations of herbs and drugs

evaluated. The animals were randomly divided into control and experimental groups of

eight consisting of 06 animals each.

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Three sets of administrations were carried out in rodents for three combinations of herb

extracts and drug evaluated respectively, as mentioned below.

Dosage administrations

I: For 2-week administration of Bacopa monniera extracts (BME) and fluoxetine in

rats.

Group I: Control: Dist. Water

Group II: Fluoxetine (20 mg/kg)

Group III: BME 20 mg/kg

Group IV: BME 40 mg/kg

Group V: BME: 80 mg/kg

Group VI: BME-20 mg/kg + fluoxetine (20 mg/kg)

Group VII: BME-40 mg/kg + fluoxetine (20 mg/kg)

Group VIII: BME-80 mg/kg + fluoxetine (20 mg/kg)

II. For 2-week administration Centella asiatica extracts (CAE) and imipramine in mice

Group I: Control: Dist. Water

Group II: Imipramine 20 mg/kg

Group III: CAE 100 mg/kg

Group IV: CAE 200 mg/kg

Group V: CAE: 300 mg/kg

Group VI: CAE-100 mg/kg + imipramine (20 mg/kg)

Group VII: CAE-200 mg/kg + imipramine (20 mg/kg)

Group VIII: CAE-300 mg/kg + imipramine (20 mg/kg)

III. For 2-week administration of Curcuma longa extracts (CLE) and reboxetine in

mice.

Group I: Control: Dist. Water

Group II: Reboxetine 20 mg/kg

Group III: CLE 140 mg/kg

Group IV: CLE 280 mg/kg

Group V: CLE 560 mg/kg

Group VI: CLE 140 mg/kg + reboxetine (20 mg/kg)

Group VII: CLE 280 mg/kg + reboxetine (20 mg/kg)

Group VIII: CLE 560 mg/kg + reboxetine (20 mg/kg)

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The BME, CAE and CLE in three different doses, and the respective synthetic

antidepressants, fluoxetine, imipramine and reboxetine in single, fixed dose were

dissolved in distilled water for administration to serve as positive controls. All the

solutions were prepared fresh, daily and were administered in animals orally for 2

weeks by intragastric gavages in morning between 8-00-10-00 hours. The

administrations were continued further for one more week at the time of chronic fatigue

test. In the groups receiving combinations, the herbs were administered orally, closely

followed by the respective drug in the doses mentioned above.

Dose calculations

The doses of the drugs were calculated by extrapolating the therapeutic dose to rat dose

on the basis of body surface area ratio (conversion factor 0.18 for rats) by referring to

the table of “Paget & Barns" (Paget and Barnes, 1964).

i.e. For rats, Humans dose x 0.018 = X g/200g. of rat.

X x 5 - Yg. /kg. of rat.

For example; human dose × 0.018 for rat weighing 200g.

i.e. 2000 mg × 0.018 = 36 mg / 200g / day rounded to 40 mg/200 g rat; Conversion to

dose/kg body wt. = 40 mg × 5 = 200 mg / kg.

For mice, human dose x 0.0026 - Xg/10g. of mice.

X x 50 = Yg. /kg. of mice.

Human dose × 0.0026 for mouse weighing 20g.

i.e. 2000 mg× 0.0026 = 5.2 mg/ 20g/ day rounded to 5.5 mg/20 g mouse; Conversion

to dose/kg body wt = 5.5 mg × 50 = 275 mg/kg.

5.7. Procedures: behavioural assessments

All the experiments were performed between 9-00 am- 18-00 pm hours. On the test

days, animals were transported to the dimly lit laboratory and left undisturbed for 2 h

prior to the testing. Neuropsychopharmacological studies were carried out on animal

models of depression for the behavioural assessments.

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1. Gross Behaviour

The test was performed as described earlier (Morpugo, 1971). The procedure involved

assignment of the scores on 0-3 point scale as per the average intensity of the

phenomenon observed. The test drug was administered one hour before the experiment.

There after observations were made at every hour, at 1, 2, 3 and 4 hours. The mice were

placed one by one in the centre of three concentric circles drawn by chalk on a rubber

sheet diameter 7cm, 9cm and 13cm. The profile measured was checked in to the

following heads.

CNS DEPRESSION

Hypo activity

Passivity

Relaxation

Narcosis

Ataxia

ANS EFFECTS

Ptosis

Exophthalmos

CNS STIMULATION

Hyper activity

Irritability

Stereotypy

Tremors

Straub tail

Analgesia

Any other behavioral changes,

Mortality if any.

The test is depicted in figure 2.1. ( Figure 2.1. Gross behaviour in mice).

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2. Locomotor activity

The locomotor activity was assessed using a photoactometer (Medicraft, Inco, India) as

previously reported (Kulkarni, 1999). Each animal was placed individually in activity

cage for 10 minutes at an interval of 30 minutes till maximum activity is recorded (up

to 3 hours). When the beam of light falling on photocell was cut off by the movement

of animal inside activity cag, the counts were automatically recorded as the cumulative

total counts of spontaneous motor activity of the animal. (Refer, figure 2.2. Locomotor

activity in rat) and (Figure 2.3. Actophotometer).

Open field Behaviour

This test was carried out in mice using the open field apparatus described earlier

(Bhattacharya et al., 1993). The apparatus used was a square box of 96x96 cm. and

about 15cm. high sidewall. The floor was divided into 36 equal squares. The apparatus

was kept in a dimly lit and quite area during the experiment. Each mouse was placed at

the same bottom left hand corner, an hour after drug administration and allowed to

explore the arena for 5 minutes. The test is displayed in figure 2.4. (Figure 2.4. Open

field behaviour in mice). The following parameters were noted.

The number of rearing.

Number of fecal pellets expelled.

Number of squares crossed.

Duration of immobility (freezing time).

Time of initiation.

3. Motor coordination

The test for motor coordination was carried out on each group of mice as described

previously (Dunham and Mija, 1957), using rota rod apparatus (Medicraft, Inco, India).

Each animal was placed on a horizontal rotating rod having a diameter of 32 mm.

revolving at the rate of 25 revolutions per minute (rpm). Animals were trained to

remain on the rod for 5 minutes in four trials carried out in two sessions divided into

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morning and evening. The four animals at a time were placed separately in four

compartments of rotating rod for 5 minutes, an hour after the administration of drugs

and the “Fall off time” was noted digitally, when the animals loose the grip and fall

from the rotating rod. Refer, figures (Figure 2.5. Motor coordination in mice by rota

rod) and (Figure 2.6. Motor coordination in rats by rota rod).

4. Forced swimming test

The test was carried out as described formerly (Porsolt et. al 1977a; 1977b). In a

standard protocol, one hour after administration of a respective agents on the day

preceding the test, each of the rats were placed individually for 15 minutes in a narrow

plexiglass cylinder (45× 40 × 30) with 20 to 25 cm water level maintained at 250 C ±

20C in a way that the rat could not touch the bottom of the cylinder with its hind paws

or tail or climb over the edge of apparatus. Following the swim, the animals were

removed from the cylinder and allowed to dry in a warm enclosure. 24 hours later they

were made to swim again for 10 minutes, during which they make an initial frenzied

struggle to escape, swim vigorously and thereafter exhibited immobile posture,

showing complete cessation of all movements with just the head floating above the

water level, which was noted in last 6 minutes of total 8 minutes of the test period by a

single trained observer. The protocol was the same in mice except that the vessel

dimension and water level were different (25 x 12 x 25 cm.) containing fresh water up

to a height of 15 cm at room temperature (22o C ± 3o C). Two animals were evaluated

simultaneously by an observer in two separate identical cylinders with the card board

placed in between, in order to avoid a visual interaction between the two animals. The

animals were subjected to the test on 1st, 7th and 14th day. Refer, figure (Figure 2.7.

forced swimming test in mice).

5. Tail suspension test

The test was employed as described previously (Steru et al., 1985). The mice were

individually suspended 50 cm above the floor by means of an adhesive tape, placed

approximately 1 cm from the tip of the tail.

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Testing was carried out in a darkened room with minimal background noise. The time

during which mice remained immobile was quantified during last 4 minutes of a total

test period of 6 minutes. Mice were considered immobile only when they hung

passively and completely motionless. Refer, figure (Figure 2.8. Tail suspension test in

mice).

6. Chronic fatigue test

A modified behavioral despair test has been employed (Kaur and Kulkarni, 2000) to

assess chronic fatigue in each group of animals. The animals were forced to swim

individually in glass jar (25 x 12 x 25 cm.) containing water at room temperature (25oC

± 3o C). The height of water level was adjusted to 15 cm. and kept constant throughout

the experiments. After an initial period of vigorous activity, each animal assumed an

immobile posture. The animals were judged immobile when they ceased struggling and

made minimal movement of their limbs to keep the head above water level. The

duration of immobility was noted in last 4 minutes of total test period of 6 minutes. The

initial 2 minutes was given to let the animal get acclimatized to the stressed situation in

jar. This procedure was followed for 7 days. The drugs were administered according to

groupings, 1 hour prior to the exposure to stressful stimuli, daily for 7 days. This

chronic forced swimming produced depression and fatigue resembling chronic fatigue

syndrome (CFS). Refer, figure (Figure 2.9. Chronic fatigue test).

Three combinations of herbal extracts along with the conventional antidepressants were

assessed. The first six behavior paradigms mentioned above, were studied in the same

sets of animal after a 15-day wash out period, in each combination checked, (n=48 X 3-

repeated 4 times for each three combinations of herb and drug at 2-week interval) And

the new set of animals were employed (n=48 X 3) for the 7th paradigm chronic fatigue

test, in each three combinations, after completion of which (21 days), the animals were

sacrificed for the biochemical estimations.

Figures 2.1-2.10 shows the photos of animal models and procedures utilized in

experiments in the present study.

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Procedures: Behaviour models

Figure 2.1. Gross behaviour in mice. (Morpugo, 1971).

Figure 2.2. Locomotor activity in rat (Kulkarni, 1999).

Figure 2.3. Actophotometer (Kulkarni, 1999).

Figure 2.4. Open field activity in mice (Bhattacharya, 1993).

Figure 2.5. Motor coordination in mice by rota rod :

(Dunham and Mija, 1957).

Figure 2.6. Motor coordination in rats by rota rod:

(Dunham and Mija, 1957).

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Figure 2.7. Forced Swimming Test (FST) in mice: (Porsolt et al., 1977a; 1977b).

Figure 2.8. Tail Suspension Test (TST) in mice (Steru et al., 1985).

Figure 2.9. Chronic Fatigue Test (CFT) (Porsolt et al., 1978; 1977).

Figure 2.10. Rat brains isolated for biochemical estimations.

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5.8. Biochemical estimations

The animals were sacrificed and their brains were rapidly removed on dry ice at -20o C,

washed with isotonic saline, weighed and were preserved separately as per groups at -

80o C until biochemical studies, to be commenced later. (Refer, figure 2.10. Rat brains

isolated for biochemical estimations).

Preparation of brain tissue

At the time of testing, the brains were thawed, rinsed with isotonic saline, weighed

again and homogenized (Homogenizer, REMI, India). The 10% homogenates were

prepared in 10 vol. of cold phosphate buffer (10mM, pH 7.4), mingled at 4o C for 20

minutes. The mixture was centrifuged (Centrifuge, REMI, India) at 10,000 rpm for 30

minutes at 0o C and the pellets were re suspended in the same buffer. Ad and NA were

measured by enzyme radioimmunoassay and serotonin was estimated by direct

estimation method as described previously (Weissback, et al, 1958; Udenfriend, 1955a;

1955b).

Determination of adrenalin (Ad) and noradrenalin (NA)

1. Introduction and principle of the test

Adrenalin (Ad) and noradrenalin (NA) estimation was carried out by Elisa Reader (Bio-

Tek instruments) using a kit (2 CAT EIA (BA-10-1500) (LDN, Germany).

The assay kit provides materials for the quantitative measurement of Ad and NA in

urine, plasma or tissue homogenates. Ad and NA were extracted using a cis-diolspecific

affinity gel followed by acylation to N-acyladrenaline and N-acylnoradrenaline which

was then get converted enzymaticaly during the detection procedure into N-

acylmetanephrine and N-acylnormetanephrine, respectively. This competitive EIA kit

used the microtiter plate format. Ad and NA respectively, are bound to the solid phase

of the microtiter plate. Acylated Ad and NA from the sample and solid phase bound Ad

and NA compete for a fixed number of antiserum binding sites. When the system is in

equilibrium, free antigen and free antigen-antiserum complexes were removed by

washing. The antibody bound to the solid phase catecholamine is detected by an anti-

rabbit IgG-peroxidase conjugate using TMB as a substrate.

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The reaction was monitored at 450nm with the amount of antibody bound to the solid

phases catecholamine being inversely proportional to the catecholamine concentration

in the sample.

1. Contents of the kit:

The 2 CAT EIA (BA-10-1500) contains materials for 96 quantitative determinations

(including the standards) Ad and NA.

Table 1. Reagents for the extraction and acylation

BA 10-1601 Standard A Standard A, 0 ng/ml (adrenalin); 0 ng/ml (Noradrenalin); 1x1 ml, ready for use.

BA 10-1602 Standard B Standard B, 1 ng/ml (adrenalin); 4 ng/ml (Noradrenalin); 1x1 ml, ready for use.

BA 10-1603 Standard C Standard C, 4 ng/ml (adrenalin); 16 ng/ml (Noradrenalin); 1x1 ml, ready for use.

BA 10-1604 Standard D Standard D, 16 ng/ml (adrenalin); 64 ng/ml (Noradrenalin); 1x1 ml, ready for use.

BA 10-1605 Standard E Standard E, 64 ng/ml (adrenalin); 256 ng/ml (Noradrenalin); 1x1 ml, ready for use.

BA 10-1606 Standard F Standard F, 256 ng/ml (adrenalin); 1,024 ng/ml (Noradrenalin) 1x1 ml, ready for use.

BA 10-1651 Control 1 Control 1, 1x1 ml, ready for use.

BA 10-1652 Control 2 Control 2, 1x1 ml, ready for use.

BA 1611 ACYL-BUFF Acylation Buffer, 1x20 ml, ready for use.

BA 1612 ACYL-REAG Acylation Reagent, 2x1.5 ml, ready for use.

BA 1613 ASSAY-BUFF Assay Buffer, 2x4 ml, ready for use,contain 1M HCl.

BA 1617 EXTRA-BUFF Extraction Buffer, 2x4 ml, ready for use.

BA 1618 EXTRACT PLATE 48

Macrotiter plate, 2x48 wells,coated with boronate affinity gel

BA 1619 HCL Hydrochloric acid, 1x20 ml, ready for use (Yellow color),Contains 0.025M HCl.

Table 2. Reagents for the enzyme immunoassay

BA 1615 ENZYME Enzyme, 4x1ml /vial, lyophilized, contains the enzyme catechol-o-methyltransferase.

BA 1614 COENZYME Coenzyme, 2 x 0.75ml, ready for use, S-adenosyl-L-methionine.

BA 1616 ENZYME-BUFF

Enzyme-Buffer, 1x4ml, ready for use.

BA 10-0110 ADR-AS Adrenalin Antiserum, 1x 6ml, from rabbit ready for use, Blue colored.

BA 10-0210 NAD-AS Noradrenalin Antiserum, 1x 6ml, from rabbit ready for use, Yellow colored.

BA 10-0131 ADR-MIT-STRIPS

Adrenalin microtiters strips, 12 strips, 8 well each, break apart, precoated with derivatized adrenalin (Blue Colored).

BA 10-0231 NAD-MIT-STRIPS

Noradrenalin microtiters strips, 12 strips, 8 well each, break apart, precoated with derivatized Noradrenalin (Yellow Colored).

BA 10-0030 WASH-BUFF Wash buffer concentrate, 3x50ml, concentrate, dilute content with dist. Water to final volume of 500ml.

BA 10-0040 CONJUGATE Enzyme Conjugate, 2x11ml, ready for use, anti-rabbit IgG Conjugate with peroxidase.

BA 10-0055 SUBSTRATE Substrate, 2x11ml, ready for use, containing TMB.

BA 10-0080 STOP-SOLN Stop solution, 2x11ml, ready for use, containing 0.25M Sulphuric acid.

BA 10-0090 FOILS Adhesive Foils 2 x 4foils.

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6. Additional materials and equipment required but not provided in the kit

Automatic pipettes (10,20,25,50,100,125,150,300,500,600,and 1000 l)

Microtiter plate washing device

EIA Reader capable of reading absorbance at 450 nm

Orbital Shaker (Capable of shaking between 400-900rpm: to an orbital shaker with shaking

orbit

Distilled water

Vortex mixer

Test procedures

A list of the agents required for the assay as provided in the kit are given in table 1 and

2. (Refer Table 1. Reagents for the extraction and acylation) and (Table 2. Reagents for

the enzyme immunoassay) in this section. Reagents and samples were allowed to reach

room temperature before use in estimations.

Preparation of wash buffer

A 50 ml wash buffer concentrate was diluted with distilled water to a final volume of

500 ml. the diluted wash buffer concentrate was stored at 2-8C.

Preparation of enzyme solution

The enzyme solution was prepared freshly prior to the assay (not longer than 10-15

minutes in advance). The content of the vial labelled ‘Enzyme’ with 1 ml were

reconstituted with distilled water and mixed thoroughly. A 0.3 ml of coenzyme was

added followed by 0.7 ml of enzyme buffer. The total volume of the enzyme solution

was made up to 2.0 ml.

Steps for Sample preparation, extraction and acylation

Each 10l of standards, control 1 and 2 and the samples, respectively were extracted as

follows.

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1. A 10l of standard A-F, 10l of control 1 and 2 and 10l of urine samples were

pipetted into the respective wells of the microtiter plate. A 250 ml l of distilled

water was added to those wells to correct for volume. A 300 l of plasma sample

was pipetted into the respective wells for extracting Ad and NA.

2. A 50 l of assay buffer was pipetted into all wells.

3. A 50 l of extraction buffer was pipetted into all wells.

4. The plate was covered with adhesive foil and incubated for 30 min at room temp on

orbital shaker (600-900 rpm).

5. The foil was removed and discarded. Immediately the plate was decanted and

residual liquid was removed by tapping the inverted plate on a paper towel.

6. 1 ml of diluted wash buffer was pipetted and concentrated in to all wells. It was

covered with adhesive foil and incubated for 5 min at room temperature on an

orbital shaker (600-900 rpm).

7. The foil was removed and discarded. Immediately the plate was decanted and

residual liquid was removed by tapping the inverted plate on a paper towel.

8. Step 6 was repeated, discarded and residual liquid was removed by tapping the

inverted plate on a paper towel.

9. A 150 l of acylation buffer was pipetted in to all wells.

10. A 25 l of acylation regent was pipetted in to all wells.

11. The plate was incubated without foil for 15 minutes at room temperature on an

orbital shaker (600-900 rpm).

12. The plate was decanted immediately and residual liquid was removed (as in step 5).

13. 1 ml of diluted wash buffer was pipetted and concentrated in to all wells.

14. The plate was incubated with foil for 10 ml at room temp on an orbital shaker (600-

900 rpm).

15. The plate was decanted immediately and the residual liquid was removed (see step

5).

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16. A 150 l of HCl was pipetted in to all wells to elute Ad and NA.

17. The plate was covered with adhesive foil and incubated for 10 min at room temp on

an orbital shaker (600-900 rpm). Precaution was exercised, not to decant the

supernatant thereafter.

The following volume of elute were needed for EIA

Adrenaline Noradrenaline

100 l 20 l

Adrenalin enzyme immunoassay

1. A 25 l of the freshly prepared enzyme solution was pipetted in to all wells.

2. A 100 l of the extracted standards, controls and samples were pipetted to the

appropriate wells.

3. The above were incubated for 30 min at room temp on a shaker at 400-500 rpm.

4. A 50 l of adrenalin antiserum was pipetted in to all wells.

5. The above was incubated for 2 hours at room temperature on orbital shaker at 400-

500 rpm.

6. The contents of the well were discarded or aspirated and washed thoroughly each

with 100 l diluted wash buffer concentrate (the washing procedure was repeated 2

times) and blow dried by inverting the plate on absorbent material.

7. A 100 l of enzyme conjugate was pipetted in to all wells.

8. The above was incubated for 30 minute at room temperature on an orbital shaker

(400-500 rpm).

9. Each well was aspirated, discarded and washed 3 times and blow dried by inverting

the plate on absorbent material.

10. A 100 l of substrate was pipetted in to all wells.

11. The above was incubated for 20-30 minute at room temperature on orbital shaker

(400-500 rpm) direct exposure to sunlight was avoided.

12. A 100 l of stop solution was added to each well and microtiter plate was shaken to

ensure a homogeneous distribution.

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13. The absorbance of the solutions in the well was read within 10 minutes, using a

microplate reader set to 450 nm keeping the reference wavelength between 620 nm

and 650 nm.

Noradrenalin enzyme immunoassay

1. A 25 l of the freshly prepared enzyme solution was pipetted in to all wells.

2. 20 l of the extracted standards, controls and samples were pipetted to the

appropriate wells.

3. The above were incubated for 30 minutes at room temp on a shaker at 400-500 rpm.

4. A 50 l of noradrenalin antiserum was pipetted in to all wells.

5. The above was incubated for 2 hours at room temperature on orbital shaker at 400-

500 rpm.

6. The contents of the well were discarded or aspirated and washed thoroughly each

with 100 l diluted wash buffer concentrate (the washing procedure was repeated 2

times) and blow dried by inverting the plate on absorbent material.

7. A 100 l of enzyme conjugate was pipetted in to all wells.

8. The above was incubated for 30 minutes at room temperature on an orbital shaker

(400-500 rpm).

9. Each well was aspirated, discarded and washed 3 times and blow dried by inverting

the plate on absorbent material.

10. A 100 l of substrate was pipetted in to all wells.

11. The above was incubated for 20-30 minutes at room temperature on orbital shaker

(400-500 rpm) direct exposure to sunlight was avoided.

12. A 100 l of stop solution was added to each well and microtiter plate was shaken

to ensure a homogeneous distribution.

13. The absorbance of the solutions in the well was read within 10 minutes using a

microplate reader set to 450 nm keeping the reference wavelength between 620 nm

and 650 nm.

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Calculation of the results

The mean absorbance for each of the standards, control and samples were calculated.

The linear mean absorbance readings of the standards were plotted along y-axis versus

log of the standard concentrations in pg/ml along the x-axis using a linear curve fit.

The analyte concentrations of the controls and samples were determined from the

standard curve by matching their mean absorbance readings with the corresponding

analyte concentrations which were directly read from the standard curve.

Estimation of serotonin

Introduction and principle

The unique fluorescence characteristics of 5-HT (Udenfriend et al., 1955a; 1955b) have

made it possible to determine it directly in many tissues extracts after precipitation of

the proteins (Weissbach et al., 1958) This method is applicable to most tissues

containing more than 1g of 5-HT per gram and to blood containing over 1g per ml.

Although, it is a method of choice for its simplicity, it lacks specificity since all 5-

hydroxyindoles have the same fluorescence characteristics. However, 5-HT is usually

the only hydroxyindole present in tissues. When the amounts of 5-HT present are small

and interfering materials are present, it is necessary to purify and concentrate the tissue

extracts before assaying 5-HT. This is done by extraction of the 5-HT in to n-butanol

from a salt-saturated alkaline solution. The 5-HT can then be re-extracted from the

butanol in to a small volume of dilute HCl (Udenfriend et al., 1955a; Bogdansky et al.,

1956). The 5-HT in the final acid is then assayed spectrofluorimetrically.

Reagents and extraction procedure

1. A 10% zinc sulphate H2O and 1N NaOH.

2. Borate buffer: To 94.2g. of boric acid in 3 ml of water, 165 ml of 10 N NaOH was

added. The buffer solution was then saturated with purified n-butanol and NaCl by

adding these substances in excess and shaking. Excess n-butanol was removed by

aspiration and excess salt was allowed to settle. The final pH was made about 10.0.

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3. n-butanol: Reagent grade n-butanol was purified by first shaking with an equal

volume of 0.1 N NaOH and then with equal volume of 0.1 N HCl and finally twice

with distilled water.

Procedure

The brain tissues are thawed, weighed and homogenized in at least 2 volumes of 0.1 N

HCl and an aliquot, corresponding to 1g was diluted to 8.5 ml with water. The proteins

were then precipitated by the addition of 1 ml of 10% zinc sulphate followed by 0.5 ml

of 1 n NaOH. After 5 minutes, the tubes were shaken and centrifuged at 2500 rpm for

20 minutes. 1 ml of the clear supernatant was transferred to a quartz cuvette containing

0.3 ml of 12 N HCl. The fluorescence was measured in the spectrophotofluorometer

(Perkin-Elmer, LS 50B) (activation wavelength 295 m; detecting fluorescence

wavelength at 550 m).

Calculation of the results

The mean fluorescence intensity for each of the standards, control and samples were

calculated. The linear mean fluorescence intensity readings of the standards were

plotted along y-axis versus the standard concentrations in ng/ml along the x-axis using

a linear curve fit. The analyte concentrations of the controls and samples were

determined from the standard curve by matching their mean fluorescence intensity

readings with the corresponding analyte concentrations which were directly read from

the standard curve.

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5.9. Statistical analysis of the data

The statistical analysis were performed using a statistical software SPSS (Statistical

package for social science, SPSS 15.0 for Windows Evaluation version).

In study, parametric tests were used for the majority of the data, but in case of an

absence of normal distribution, non-parametric statistics were used. The significance of

differences in group means at base line and after treatment, means between groups and

within groups were determined using one-way ANOVA followed by post hoc Dunnet

T3 or Tukey HSD multiple comparisons for dependent and independent variables in

paired and grouped data.

The One-Way ANOVA procedure produced a one-way analysis of variance for a

quantitative dependent variable by a single factor (independent) variable to find the

significance among the different groups. The test was used to inspect the hypothesis

that several means were equal (homogeneity of variance). The Levene’s statistics

rejected the null hypothesis that the group variances were equal; however, when the

data which did not follow the normality, we have taken the log transformation to get the

normality of the data.

In addition, for comparing the means, post hoc tests were run to determine that

differences existed among the means. For equal variance, the Tukey test, the

Studentized range statistics was used to make all of the pair wise comparisons between

groups. In case of the data, where equal variances were not assumed, a Dunnett's T3

multi comparisons was used which is a pair wise comparison test based on the

studentized maximum modulus.

For all data in each groups, mean, standard deviation, standard error of the mean,

minimum, maximum, and 95%-confidence interval were calculated. Levene's test for

homogeneity of variance, analysis-of-variance table and robust tests of the equality of

means for each dependent variable, and post hoc range tests analysed were recorded.

All values are presented as mean ± S.E.M (n=6) and for all the data, a probability of

less than 0.05 (P <0.05) was considered statistically significant.