CH

Stre

Che

is p

sali

citri

aliq

Woc

stud

che

wat

AN

The

Wis

app

age

anim

24±

and

fed

Fee

Mah

Pun

ad

150

gen

(4-8

with

The

mod

inst

EXP

EMICALS

eptozotoc

emicals Co.

prepared by

ne and adj

ic acid. Th

uots and

ckhardt Ltd

dy were o

micals use

ter was em

IMALS

e study was

star alb

proximately

e-group, ho

mal rooms

±2°C with

d 60±5% h

with Amru

d, manuf

harashtra C

ne, India. W

libitum.

0±20 g (2

ntamicin in

8 week old

h diabetic n

e study of

dels was ca

titutional et

PERIME

S

cin, glucose

(St. Louis,

y dissolving

justing the

he citrate

stored at

d., India, w

of Span D

ed were o

mployed for

s performed

bino ra

y the

oused in v

at a tempe

12h light/d

humidity. T

t Laborato

factured

Chakan Oil

Water was

Animals

-3 week o

nduced AR

) were use

nephropath

the phytoc

arried out i

thical comm

 

ENTAL D

e-6-phosph

, USA). Sod

g 147 mg of

e pH to 4.5

buffer sho

-20°C. Ge

were used in

iagnostics

f analytica

r preparing

d on male

ats of

same

ventilated

erature of

dark cycle

They were

ry Animal

by Nav

Mills Ltd,

s provided

weighing

old) were

RF while

ed to perfo

y.

chemicals

in SN med

mittee.

‐46‐ 

 

DESIGN

hate (G-6-P

dium citra

f tri-sodium

5 with appr

ould be us

entamicin

n the study

Ltd., India

al grade (A

the reagen

used to te

animals

rm anti ren

as therape

ical college

Figure

Male wis

N & MET

P) was pro

ate buffer

m citrate in

roximately

ed fresh o

injections

y. All Assay

a. All ot

AR). Only

nts.

est protect

of body

nal failure

eutic agen

e, Agra hav

e 3.1

star rat

THODOL

cured from

(0.01 M, p

49.5 ml of

0.5 ml of

or frozen i

manufactu

y Kits use

her reagen

double d

tive effect

weight 2

studies in

ts in exper

ving approv

 

LOGY

m Sigma

pH 4.5)

f normal

1 mol/l

in 1 ml

ured by

d in the

nts and

distilled

against

50±50g

animals

rimental

val from

SEL

Plan

stud

o

o

o

Afo

on t

EXT

Plan

pre

No.

plan

Plan

and

part

extr

petr

LECTION

nt Androgra

dy on basis

It is abun

Plant havi

Preliminar

in the plan

resaid char

this plant.

TRACTIO

nts were c

liminary st

AP-D02, H

nts were w

nt material

d root parts

ts (2 kg)

raction suc

roleum eth

A

OF PLAN

aphis panic

s of the follo

dantly foun

ing bitter ta

ry studies

nt.

racteristics,

N

collected f

udy, while

Herbarium V

ashed quic

was dried

s. Aerial pa

and roots

ccessively

er, chlorofo

 

FigAndrograp

NT

culata (Bur

owing poin

nd in all par

aste, is sup

showed th

, motivated

from the

it was pro

Voucher No

ckly with th

d under sha

arts and ro

s (5 kg) w

with the

orm and m

‐47‐ 

 

ure 3.2 phis panicu

rm. f.) Nee

ts:

rts of the c

pposed to h

e existence

d us to car

botanical g

ocured from

o. 445785)

he water to

ade. Plants

oots were

were separ

solvents w

ethanol. Ea

ulata

es has bee

ountry.

ave medici

e of renop

rry out det

garden of

m NBRI, Lu

for the bul

o remove a

s were part

powdered,

rately subj

with increa

ach extract

en selected

inal propert

rotective p

tailed inves

our instit

ucknow (Ac

lk study. Th

ny foreign

titioned int

separately

jected for

asing polar

t was conce

 

for the

ties.

potential

stigation

tute for

ccession

he fresh

matter.

to aerial

y. Aerial

soxhlet

rity viz.

entrated

und

pur

g),

aeri

and

Crude

der reduced

ging nitrog

root (96.7

ial (199.89

d when requ

e extract b

d pressure

en and fina

798 g); CH

96 g), root

uired.

 

Figurbeing conc

(Rota vap

ally weighe

HCl3: aerial

(260.000

‐48‐ 

 

re 3.3 centrated

pour vacuu

ed as follow

l (95.889 g

g). Extract

using rota

um distillat

ws: Pt. eth

g), root (4

ion process

a vapor

ion) and d

her: aerial

47.739 g);

s was repe

 

dried by

(12.223

MeOH:

eated as

PR

The

met

sus

ethe

2-3

IND

Gen

Anim

thre

Gro

rece

(0.5

and

wer

man

Ind

con

wel

nep

et a

min

EPARATI

e dried ext

thanol; ro

pended in

er extract c

drops of e

DUCTION

ntamicin in

mals were

ee groups

oup I was k

eiving

5 ml, i.p.) f

d animals

re admin

nufactured

ia (80 mg

secutive

l-known to

phrotoxicity

al. , 1994).

nimize the c

ON OF TE

tracts viz.

ots: petro

1 % gum

containing

mulsifier Tw

N OF RENA

nduced Re

randomly

of six an

kept as no

isotonic

for 8 conse

of groups

nistered

by Woc

g/kg/day,

days,

o produce

y in rats (Ab

Injections

circadian va

 

EST EXTR

aerial pa

oleum eth

acacia, se

wax was p

ween-80 (0

AL FAILU

enotoxicit

divided int

nimals each

rmal contr

salin

ecutive day

II and I

gentamicin

khardt Ltd

i.p.) for

which

significa

bdel Gayou

of gentam

ariation in n

‐49‐ 

 

Intrap

RACTS

rts: petrol

er, chloro

eparately. T

prepared in

0.1%).

URE

ty

to

h.

ol

ne

s,

II

n,

d,

8

is

ant

um

micin were m

nephrotoxic

Figureperitoneal

weighing

eum ether

form and

The suspen

gum acac

made daily

city (LeBrun

e 3.5 injection g <200 g

r, chlorofo

methano

nsion of pe

ia with the

at 08:00 h

n et al., 19

FigurDried ext

 

to rat

rm and

l, were

etroleum

help of

hours to

999).

re 3.4 tracts

Dia

Dia

dep

follo

inje

mg/

in

citra

and

to d

wee

inje

dete

from

wer

con

diab

nep

BLO

Bloo

cen

trea

sinu

allo

tem

was

rpm

mic

unt

sam

by

cag

urin

crea

abetic Nep

betes wa

prived o

owed by

ection of s

/ kg) (Sing

freshly pr

ate buff

d Allen, 200

develop dia

ek. A w

ection, blo

ermined in

m the tail v

re consider

centrations

betic rats,

phropathy if

OOD & UR

od samples

trifuge tu

atment, aft

us under

wed to c

mperature (

s separated

m for 2

cro-centrifu

il the anal

mples were

placing the

es as and

ne samples

atinine, ure

hropathy

as induc

f water

a single

streptozoto

gh et al., 2

repared 0.

fer, pH

07) and the

abetic neph

week aft

ood glucos

n blood sa

vein, using

red to be

s > 250

referred to

f their bloo

RINE SAM

s were colle

ubes at t

er 8h fastin

light ether

clot for 3

(Anand et a

d by centrif

2 min

ge) and

ysis was c

collected o

e rats in in

d when re

s were an

ea and uric

 

ced in

r for

intraperito

ocin (STZ,

2006) disso

01 M sod

4.5 (T

e rats were

hropathy f

ter the

se levels

amples coll

Glucomete

diabetic

mg/dl (Ch

o as STZ r

d urea and

MPLE COL

ected in cle

the end

ng by retro

r anesthes

30 min at

al., 2010).

fugation at

(REMI

stored at

carried out

over a 24 h

dividual m

quired. Se

alyzed for

acid.

‐50‐ 

 

In

rats

24h

oneal

50

olved

dium

Tesh

e left

for a

STZ

were

lected

r (Accu-Ch

if they ha

hen and Q

rats, were

creatinine

LLECTION

ean dry

of the

o-orbital

sia and

t room

Serum

t 12000

RM-12C

-20°C

t. Urine

h period

etabolic

erum and

glucose,

Figtraperitonrat weig

FiMetabolic

c

ek, one tou

ad elevated

Quilley, 20

considered

values we

N AND AN

ure 3.6 neal injecthing >200

igure 3.7 c cages fo

collection

uch ultra).

d plasma

008). Then

d to show

re elevated

NALYSES

 

tion to 0 g

or urine

Animals

glucose

, these

diabetic

d.

   

‐51‐ 

 

 

BIOCHEMICAL ANALYSES

All biochemical analyses were performed on an automated analyzer ‘Erba

Chem-5 Chemistry Analyzer’ using standard assay kits (Span

Diagnostics Ltd., India).

GLUCOSE (Trinder, 1969; Sacks, 1999)

Glucose (C6H12O6), a monosaccharide (or simple sugar), is a ubiquitous

fuel molecule in biology. It is oxidized though a series of

enzyme-catalyzed reactions to form carbon dioxide and water, yielding the

universal energy molecule ATP. Due to its importance in metabolism,

glucose level is a key diagnostic parameter for many metabolic disorders.

Increased glucose levels have been associated with diabetes mellitus,

hyperactivity of thyroid, pituitary and adrenal glands. Decreased levels are

found in insulin secreting tumors, myxedema, hypopituitarism and

hypoadrenalism.

Principle

Glucose Oxidase (GOD) oxidizes Glucose to Gluconic acid and Hydrogen

peroxide. In presence of enzyme Peroxidase, released Hydrogen Peroxide

is coupled with Phenol and 4-Aminoantipyrine (4-AAP) to form

Quinoneimine dye. Absorbance of coloured dye is measured at 505 nm

and is directly proportional to glucose concentration in the sample.

Glucose + O2 + H2O Glucose Oxidase Gluconic Acid + H2O2

H2O2 + Phenol + 4-AAP Peroxidase Quinoneimine

dye + H2O Pink

   

‐52‐ 

 

 

Reagents

Glucose Reagent (R1) Phosphate Buffer 100 mM/l

Glucose Oxidase ≥ 15000 U/l

Peroxidase ≥ 1600 U/l

4-AAP 0.28 mM/l

Glucose Diluent (R2) Phenol 10mM/l

Glucose Standard (R3) Dextrose 100 mg/dl

Sample Preparation

Plasma

- Collect blood using an anticoagulant such as heparin, EDTA or citrate.

- Centrifuge the blood at 700-1000 x g for 10 minutes at 4 °C. Pipette

off the top yellow plasma layer without disturbing the white buffy

layer. Store plasma on ice, if not assaying the same day, freeze at

–80 °C. The plasma sample will be stable for one month while stored

at –80 °C.

Serum

- Collect blood without using an anticoagulant.

- Allow blood to clot for 30 minutes at 25 °C.

- Centrifuge the blood at 2000 x g for 15 minutes at 4°C. Pipette off the

top yellow serum layer without disturbing the white buffy layer. Store

serum on ice, if not assaying the same day, freeze at –80 °C. The

serum sample will be stable for one month while stored at

-80°C.

- Serum does not need to be diluted before assaying.

Urine

- Urine does not require any special treatments.

- If not to assay on the same day, freeze at -80 °C.

Working Reagent Preparation

Working reagent is prepared by adding reagent 2 to reagent 1

Note: The working reagent is light sensitive; it should be stored at 2-8 °C

(do not freeze) in dark coloured bottle, to protect from sunlight.

   

‐53‐ 

 

 

Assay Parameters

Wavelength 505 nm (490-550 nm)

Flow cell temperature 37 °C

Optical path length 1 cm

Sample volume 10 µl

Working Reagent Volume 1000 µl

Incubation 10 min. at 37 °C/ 30 min. at room

temperature (15-30 °C)

Concentration of standard 100 mg/dl

Stability of colour 1 hour

Units mg/dl

Procedure

Tube Working Glucose Re-

agent (µl)

Glucose Standard

(µl)

Sample (µl)

Blank 1000 - -

Standard 1000 10 -

Sample 1000 - 10

Mix well. Incubate at 37 °C for 10 minutes or at room temperature

(15-30 °C) for 30 minutes.

Program the analyzer as per assay parameters.

1. Blank the analyzer with reagent blank.

2. Measure absorbance of standard followed by the test.

3. Calculate results as per given calculation formula.

Calculation

Glucose (mg/dl) = ABSORBANCE OF TEST X 100

ABSORBANCE OF STANDARD

   

‐54‐ 

 

 

CREATININE (Bowers, 1980)

Creatinine, an anhydride of creatine, is a waste product formed by the

spontaneous dehydration of kidneys. Most of the creatinine is found in

muscle tissue where it is present as creatine phosphate and serves as a

high energy storage reservoir for conversion to ATP. In the blood,

creatinine is removed by filtration though the glomeruli of the kidney and

is secreted into urine. In healthy individuals, creatinine secretion is

independent of diet and is fairly constant. Serum creatinine concentrations

depend almost entirely upon its excretion rate by the kidneys. For this

reason, its elevation is highly specific for kidney diseases. In kidney

disease, creatinine levels in the blood are elevated, whereas the creatinine

clearance rate or GFR drops and hence the urine levels are diminished.

Principle

The assay of creatinine has been based on the reaction of creatinine with

alkaline picrate as described by Jaffe. The improved Jaffe method

utilizes picrate that forms a red colored complex with creatinine. The

intensity of the color, measured at 510nm, is directly proportional to

creatinine concentration in the sample. The optimized formulation

substantially reduces interference by substances in the raw sample.

Creatinine + Picric acid Alkaline Solution Creatinine - Picric acid

complex

(Orange colour)

Reagents

Picrate Reagent (R1) Picric acid 40 mM/l

Sodium Hydroxide (R2) Sodium Hydroxide 200 mM/l

Creatinine Standard (R3) Creatinine 2.0 mg/dl

   

‐55‐ 

 

 

Working Solution

Working reagent is prepared by mixing equal volume of R1 (Picrate

reagent) with R2 (Sodium hydroxide) to make up the desired volume. Mix

gently for 2 minutes.

Sample Preparation

Specimen Storage at Stability Remarks Serum/ Plasma

Room Temperature (15 -30 °C)

1 day Freshly separated unhaemolysed serum/ plasma should be used. For plasma, fluoride anticoagulant should not be used. 2-8 °C 1 week

-20 °C 6 months Urine (24h collection)

Room Temperature (15-30 °C)

4 hours Dilute the urine sample 1:10 with purified water. With ‘Thymol’ as preservative, the Urine sample is stable for 4-7 days at room temperature (15-30°C).

-20 °C 6 months

Assay Parameters

Wavelength 505 nm (490-530 nm) Blank Purified Water Optical path length 1 cm Sample volume 100 µl Working Reagent Volume 1000 µl Concentration of standard 2 mg/dl Delay 30 seconds Interval 120 seconds Units mg/dl

Procedure

Tubes Standard Test

Sample - 100 µl

R3 100 µl -

Working reagent 1000 µl 1000 µl

Mix well.

   

‐56‐ 

 

 

Program the analyzer as per assay parameters.

1. Blank the analyzer with purified water.

2. Measure the initial absorbance of the standard i.e. A1 after 30

seconds and final absorbance A2 after an interval of another 120

seconds.

3. After standard reading are noted, take the reading of test i.e. A1

and A2 accordingly ( follow same procedure as for standard)

Calculation

∆A = A2 – A1

Serum Creatinine (mg/dl) = (∆A of Test / ∆A of Standard) X

concentration of standard (mg/dl)

Urine Creatinine = ∆ATEST X Conc. of Std. X dilution factor X

(mg/day) ∆ASTANDARD 24 hour urine volume in dl

UREA (Berthelot, 1859)

Urea is primarily produced in the liver and secreted by the kidneys. Urea is

the major end product of protein catabolism in animals. It is the primary

vehicle for removal of toxic ammonia from the body. Urea determination is

very useful for the medical clinician to assess kidney function of patients.

In general., increased serum urea levels are associated with nephritis,

renal ischemia, urinary tract obstruction, and certain extra-renal diseases,

e.g., congestive heart failure, liver diseases and diabetes. Decreased

   

‐57‐ 

 

 

levels indicate acute hepatic insufficiency or may results from over

vigorous parenteral fluid therapy.

Principle

Urea is hydrolyzed in the presence of urease and water to yield

ammonium ions and carbon dioxide. The ammonium ions formed react

with salicylate and hypochlorite to give a green dye (2, 2- dicarboxyl

indophenol) which is measured at 580±10 nm.

Urease NH2 - CO - NH2 + H2O 2NH3 + CO2

NH3 + Salicylate + Hypochlorite 2, 2-dicarboxy indophenols Green Dye

Reagents

Buffer (R1): 2x250 ml Phosphate buffer, pH 6.7 50 mmol/l

EDTA 2 mmol/l

Sodium Salicylate 60 mmol/l

Sodium Nitroprusside 3.2 mmol/l

Enzyme Reagent (R2):2x250 ml Urease 30000U/l

Hypochlorite Solution (R3):2x250 ml Sodium Hypochloride 140 mmol/l

Sodium Hydroxide 150 mmol/l

Standard (R4):1x5 ml Urea 50 mg/dl (8.325 mmol/l)

Preparation and Stability

Buffer/R1 is ready to use. Add 1 vial enzyme reagent/R2 to 1 bottle of

buffer/R1. The working solution is stable for 4 weeks at +2 to + 8°C and 6

days at + 20 to + 25°C. Reagent/R3 and standard/R4 are ready to use

and stable up to the expiry date specified when stored at +2 to +8°C.

Protect all reagents against direct light.

   

‐58‐ 

 

 

Samples Unhaemolysed serum/ plasma should be used. For plasma, EDTA or

Heparin may be used as anticoagulant. Urine should be diluted 1:50 with

distilled water. Procedure

Wavelength: 580 nm

Temperature: 25 - 37°C

Cuvette: 1 cm light path

Zero adjustment: Against reagent blank

Reagent Blank Standard Sample Working Solution

R1+R2 1000 µl 1000 µl 1000 µl

Standard / R4 - 10 µl - Sample - - 10 µl

Mix and incubate at + 37 °C for 5 minutes or for 10minutes at +15 to

+ 25 °C. Then add:

Reagent/ R3: 1000 µl 1000 µl 1000 µl

Mix and incubate at + 37 °C for 5 minutes or at +15 to + 25 °C for 10

minutes.

Calculation

Serum Urea Concentration (mg/dl) = ASAMPLE X Standard Concentration

ASTANDARD

Urine Urea(mg/day) = ∆ASAMPLE X Conc. of Std. X dilution factor X ∆ASTANDARD 24 hour urine volume in dl

   

‐59‐ 

 

 

TOTAL PROTEIN (Bradford, 1976; Stoscheck, 1990)

Proteins are polypeptides made up of amino acids. All proteins contain a large

number of peptide bonds and Cu2+ ions in a moderately alkaline medium, a

colored chelate complex of unknown composition is formed between the Cu++

ions and carbonyl (-C=O) and imine (=NH) groups of the peptide bonds.

Analogous reaction takes place between the cupric ion and organic compound

biuret (-NH2) and therefore, the reaction is termed as the Biuret reaction. Principle The reaction takes place between the cupric ion and compound at least two

NH2CO-, NH2CH2-, NH2CS-, and similar groups joined together directly, or

though a carbon or nitrogen atom. Amino acids and dipeptides cannot give the

reaction, but tri- and polypeptides and proteins react to give pink/

reddish-violet products. In this reaction, one copper ion is linked between four

and six nearby peptide linkages by coordinate bonds; more protein present,

more peptide bonds available for reaction. The intensity of colour produced is

proportional to the number of peptide bonds undergoing reaction. Thus Biuret reaction is used for a simple and rapid colorimetric method for determining

protein.

Procedure

Reagent preparation 1. Dissolve following reagents in about 900 ml of water one after the other as

given: Sodium-Potassium Tartarate (9.0 g), Copper Sulphate (CuSO4.5H2O) (3.0 g), Potassium Iodide (KI) (5.0 g), Sodium Hydroxide (8.0 g). Make up to 1000 ml with water. 2. Protein standard (8.0 g of bovine albumin in 100 ml of isotonic

sodium chloride). Protocol

Blank Standard Test

Biuret reagent 5.0 ml 5.0 ml 5.0 ml Protein standard - 0.1 ml - Serum - - 0.1 ml

Mix well and let stand for 15-20 minutes at room temperature and read. Set zero with blank.

Cal

HIS

At t

rem

(0.9

wer

ana

197

The

emb

hae

Ecli

was

nor

a sc

tub

++

nec

culation:

Total Prot

STOPATHO

the end of

moved quic

9% w/v; N

re weighed

alyzed for s

71. The en

e kidneys w

bedded in

ematoxylin

pse E400)

s done acc

mal structu

core descri

ules; ++,

+, severe

crosis/sclero

s

tein (g %)

OLOGY

treatment

ckly, wash

aCl). Post

and their

significance

tire kidney

were section

paraffin w

and eosin

. The light

cording to

ure were re

bed as follo

moderate,

necrosis in

osis.

 

FigPreser

specimen

= (ODTEST/

t, animals

hed thorou

mortem ex

ratios to b

e using stu

y specimen

ned and fix

ax; 5-6 µm

(H&E) and

t microscop

Houghton

egistered. R

ows: +, m

more tha

n almost ev

‐60‐ 

 

gure 3.8 rved kidnefor exami

/ OD STANDAR

were sacrif

ughly in i

xamination

body weight

udent's T-te

s were exa

xed in 10%

m sections

d assessed

pic examin

et al., 197

Renal tubu

ild usually

n one cell

very power

ey nation

RD) x Conce

ficed and b

ce-cold ph

was perfor

t were calc

est accordi

amined by

% formalin

were rout

in a light m

ation of th

78. All alt

lar necrosis

single-cell

involved i

r field; ++

ntration of

both kidney

hysiological

rmed. The

culated. Da

ng to Men

y light micr

for 24h, an

tinely stain

microscope

he kidney s

terations fr

s was asse

necrosis in

in sparse t

++, massi

 

f STD

ys were

l saline

kidneys

ata were

denhall,

roscopy.

nd were

ed with

e (Nikon

sections

rom the

essed on

n sparse

tubules;

ve total

BIO

Eac

nal

nep

to

adm

stre

sus

adm

(ve

atta

wer

exp

pote

pote

nep

(Sc

OASSAY (R

ch extract w

failure m

phrotoxicity

a dose

ministered

eptozotocin

pension w

ministration

hicle) only.

ached to a

re perform

perimental p

ent extract

ent agains

phropathy,

heme 3.2).

O

RENAL PRO

was examin

models viz

y and diabe

of 200

to the anim

induced

was thorou

n. Control

. The extra

syringe), i

med as sh

protocol (S

t was trace

st gentam

and therefo

.

 

FOral admin

usin

OTECTIVE

ned for the

z. male a

etic nephro

mg/kg a

mals with

d diabetic

ughly mixe

groups o

acts were f

nserted do

hown in t

Scheme 3.1

ed. Methan

micin induc

ore selected

‐61‐ 

 

Figure 3.9nistration g oral gav

E ACTIVITY

renoprotec

albino rats

opathy. Tes

nd 250

gentamicin

c nephro

ed to ens

of animals

fed, using a

own the eso

the schem

). Based o

olic root ex

ced acute

d for the de

9 of extractvage

Y) OF THE

ctive activit

s with ge

st extracts

mg/kg bo

n induced n

pathy, re

ure homo

received

an oral gav

ophagus. A

matic repre

n the obse

xtract was

renal fail

etailed phy

t

E EXTRACT

ty against b

entamicin

(1 ml) eq

ody weigh

nephrotoxic

espectively

ogeneity, p

1% gum

vage (narro

All the expe

esentation

rvations, th

found to b

ure and

ytochemical

 

TS

both re-

induced

uivalent

ht was

city and

y. The

prior to

acacia

ow tube

eriments

of the

he most

be most

diabetic

studies

   

‐62‐ 

 

 

Scheme 3.1

IDENTIFICATION OF THE MOST EFFECTIVE SOLVENT EXTRACT

EXHIBITING RENOPROTECTIVE ACTIVITY

PLANT MATERIAL

Separation

AERIAL PARTS ROOTS

Shade Dried and Powdered

SOXHALATION

Separately/Successively

Pt. ETHER ARIAL EXTRACT Pt. ETHER ROOT EXTRACT

CHCl3 AERIAL EXTRACT CHCl3 ROOT EXTRACT

MeOH AERIAL EXTRACT MeOH ROOT EXTRACT

RENOPROTECTIVE ACTIVITY AGAINST GENTAMICIN

INDUCED ACUTE RENAL FAILURE

RENOPROTECTIVE ACTIVITY AGAINST STREPTOZOTOCIN

INDUCED DIABETIC NEPHOPATHY

Bioassay of each extract separately

Identification of most effective extract

MeOH ROOT EXTRACT

Evaluation of Renal Profile

   

‐63‐ 

 

 

Scheme 3.2

ACTIVITY GUIDED CHROMATOGRAPHIC FRACTIONATION AND

CHARACTERIZATION OF BIOACTIVE PRINCIPLES

MeOH ROOT EXTRACT

Column Chromatographic Separation MeOH: CHCl3 (84:16)

RECOVERED SUBSTANCE (BROWN MASS)

Re-Column Chromatographic Separation

MeOH: CHCl3 [GRADIENT ELUTION]

FMEA FMEB FMEC FMED

Trace Amount

RENOPROTECTIVE BIOASSAY

MOST EFFECTIVE FMEB + FMEC

STRUCTURAL ELUCIDATION

FMEB QUALITATIVE TESTS: Diterpene Lactone

QUALITATIVE TESTS: Diterpene Glycoside

ELEMENTAL ANALYSIS IR

NMR MASS

GLUCOSE DERIVATIVE OF ANDROGRAPHOLIDE ANDROGRAPHOLIDE

BIOACTIVE PRINCIPLES

FMEC

ACT

ROO

Met

sub

usin

4.0

mes

(84

brow

mon

(TLC

[CH

chro

dev

brow

diam

elut

RE-

Diff

and

ens

(CH

6:2

valu

FME

Rf =

frac

(thi

Rf =

MeO

Rem

(FM

trac

TIVITY GU

OT EXTRA

thanolic r

bjected to

ng a colum

cm, statio

sh; 125 g)

:16). After

wn semi-s

nitored by

C) us

HCl3:Toluen

omatogram

veloped in

wn mass

meter: 3.0

ted with CH

-COLUMN

ferent fract

d subjected

ure thei

HCl3):Toluen

.5:1.5) sol

ues, fractio

EA (first

= 0.78125

ctions 78-1

rd fracti

= 0.9678 la

OH ex

moval of so

MEC) and pa

ce amount

UIDED CH

ACT

oot extra

chromat

mn (length:

onary phas

) and elute

r the remov

solid mass

y thin lay

sing

e:MeOH

m showed

an iodine

was re-ch

cm, statio

HCl3:MeOH

CHOMATO

tions of 25

d to thin l

ir purity

ne (C6H5C

vent syste

on 29-47;

fraction),

labeled as

106, Rf = 0

on) and

abeled as F

xtract

olvent furn

ale yellow (

and not con

 

HOMATOGR

act (10.0

ographic

: 120 cm,

se: silica g

ed with M

val of solve

s was obt

yer chrom

solvent

(6:2.5:1.5

four sp

e chamber

romatograp

onary phas

mixture in

OGRAPHY

5 ml each

ayer chrom

y using

CH3):Metha

em. Fractio

Rf = 0.718

fractions

FMEB (sec

0.9375 lab

fraction

FMED (fourt

of A.

nished a lig

(FMED) com

nsidered fo

‐64‐ 

 

RAPHIC F

g) was

separation

diameter:

el, 60-120

eOH:CHCl3ent, a light

ained and

matography

system

5)]. The

pots whe

r. The ligh

phed using

se: silica ge

varying pro

OF MeOH

were collec

matography

(Chlorof

nol (CH3O

ns of same

875 labeled

48 -

cond fractio

beled as FM

ns 113-1

th fraction)

panic

ght brown

mpounds. C

or bioassay

Sepaof Me

FPrepar

RACTIONA

n

ht

g column

el, 60-120

oportions.

FRACTION

cted

y to

orm

OH);

e Rf

d as

65,

on),

MEC

19,

) of

culata

(FMEA), gre

Compound F

.

Figure 3.arated fraeOH root e

Figure 3.1ration of C

ATION OF

(length: 1

mesh; 80

NS

were

reen (FMEB)

FMED was f

 

11 ctions extract

0 Column

F MeOH

150 cm,

g) and

mixed.

), white

found in

   

‐65‐ 

 

 

RENAL PROTECTIVE BIOASSAY OF MeOH FRACTIONS [FMEA, FMEB

and FMEC]

The compounds (FMEA, FMEB and FMEC) recovered from methanol extract

were screened for renoprotective activity against gentamicin induced ne-

phrotoxicity and diabetic nephropathy. The different fractions were

taken in concentrations ranging from 5-100 µg/ml. No noticeable activity

could be observed in the compound labeled as FMEA at any concentration.

Compounds FMEB and FMEC showed significant renoprotective activity at a

concentration of 20 µg/ml, in gentamicin induced rats and 90 µg/ml in

streptozotocin induced rats, and therefore, subjected for their chemical

characterization.

STATISTICAL ANALYSES

All results are expressed as mean ± standard error (SE). Statistical

differences between correlated samples were evaluated using Student’s -t

test and noted to be significantly different where p < 0.05. Student t test

calculations were assessed on the online scientific calculators (GraphPad

Quick Calcs, 2009). Composite treatments were compared using

one-way analysis of variances (ANOVA) and considered significantly

different where probability values were found to be equal to or less than

0.05. All ANOVA tests, as well as mean and standard error of mean

calculations, were performed using GraphPad Prism (GraphPad Software,

Inc., San Diego, USA).

STRUCTURAL ELUCIDATION OF BIOACTIVE PRINCIPLE

The compounds FMEB and FMEC being bioactive in nature were considered

for structure elucidation using combination of various techniques including

Infrared, Mass, Nuclear Magnetic Resonance Spectrometry.